Animals and methods

The present invention relates to animals exhibiting characteristics of substance addiction, identification of genetic loci involved in substance addiction and methods for obtaining and using such animals and genetic information.

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

40 to 60% of the risk for developing alcohol dependence is considered to be determined by genetic factors (Schuckit (1994) A clinical model of genetic influences in alcohol dependence J Stud Alcohol 55, 5-17). Identification of the genes involved may yield targets for therapeutic intervention, for example to reduce alcohol consumption. To date, progress in identifying genes related to the risk for alcoholism in man has been limited. Indeed, the feasibility of cloning susceptibility loci in humans is unclear due to the complexities of gene- environment interactions and the diversity of alcoholic families (Buck (1998) Recent progress toward the identification of genes related to risk for alcoholism. Mammalian Genome 9, 927-928).

Genetic analysis of animal models has been tried instead. Large differences exist in the degree to which animals will voluntarily ingest substances such as alcohol, morphine and cocaine which can lead to substance abuse and addiction in humans (see, for example, George & Goldberg (1989) Trends Pharmacol Sd 10, 78-83). Inbred mice strains consume different, strain dependent, levels of alcohol in the presence of competing water and food (McClearn & Rodgers (1959) Differences in alcohol preference among inbred strains of mice Quart J Stud Alcoh 20, 691- 695; MeIo et al (1996) Identification of sex-specific quantitative trait loci controlling alcohol preference in C57BL/6 mice Nature Genet 13, 147-153). Quantitative trait loci (QTL) mapping of inbred mice strains has been the method of choice for identifying potentially relevant candidate loci, mediating alcohol consumption/preference, withdrawal and sensitivity. QTL mapping has yielded

candidate loci mediating alcohol consumption/preference (chromosomes 1, 2 and 9; see Buck et al (1997) Quantitative trait loci involved in predisposition to acute alcohol withdrawal in mice J Neurosci 1117, 3946-3955)). In addition, approximately 70% of the genetic variance that mediates acute alcohol withdrawal has been mapped to three QTLs on mouse chromosomes 1, 4 and 11 (Buck et al (1997).

However, identification and testing of candidate genes underlying the QTLs may take several years (Rikke & Johnson (1998) Towards the cloning of genes underlying murine QTLs Mamm Genome 9, 963-968). Further, the differences in alcohol or other substance preference between different inbred strains are likely to have polygenic origins. This can make it difficult to identify a particular gene as a useful target for diagnosis or intervention.

Molecular biology techniques have also been used in direct methods of investigating the effects of candidate genes in modulating the response to alcohol. Alcoholism is a polygenic trait (Browman & Crabbe (1999) Alcohol and genetics: new animal models MoI Med Today 5, 310-318) but data from mice with targeted gene deletions affecting neurotransmitter pathways indicate that deletion of single candidate susceptibility genes can alter the response to alcohol in a reproducible and measurable way. For example, mutation of Ala291 of the GAB A _A a 1 subunit of the GABA _A receptor results in the abolition of alcohol sensitivity in mice (Mihic et al (1997) Sites of alcohol and volatile anaesthetic action on GABA (A) and glycine receptors Nature 389, 385-389). In addition, mice with a deletion of the gene encoding fyn, a non-receptor tyrosine kinase, have increased sensitivity to the sedative effects of .alcohol (Miyawaka et al (1997) Fyn-kinase as a determinant of ethanol sensitivity: relation to NMDA-receptor function. Science 278, 698-701). However, one limitation in the use of knockout and transgenic animals in alcohol research is that they are limited to the investigation of known genes. Moreover, in knockout and transgenic animals the target gene, and hence the encoded protein, is often truncated or entirely absent.

Assays of gene expression following alcohol exposure have also been used to suggest candidate genes (Miles et al (1994) Ethanol-responsive genes in neural cells include the 78-ldlodalton glucose-regulated protein (GRP78) and 94- kilodalton glucose-regulated protein (GRP94) molecular chaperones MoJ Pharmacol 46, 873-879; Schafer et al (1998) Identification of neuroendocrine- specific protein as an ethanol-regulated gene using mRNA differential display Mamm Genome 9, 979-982).

We have used a different, non-gene driven approach. We have subjected mice with a defined (low to intermediate) alcohol preference to chemical (ENU) mutagenesis and conducted a genome wide screen of 1,500 mice over 3 years for mutations which alter the phenotype of alcohol (in the first instance) preference. By using such an approach we aimed to identify mutations with a dominant single-gene effect on substance, for example alcohol, preference.

We have identified such a mutation. Animals with such a mutation display a pronounced preference for addictive substances such as alcohol. Animals with such a mutation are useful in screening for potential therapeutic agents for preventing or treating addiction, and in investigating other factors that may lead an individual towards or away from addiction.

The mutation occurs in the GABA _A receptor βl gene. The mutation is a single T to G base substitution at position 94 of exon 8 in the GABAA receptor β 1 gene, resulting in a leucine to arginine change at amino acid position 310 of the protein (L310R; numbering including the signal peptide: L285R of the mature protein ie after cleavage of the signal peptide). This change is not found in any other mouse strains for which sequence data is available. The GABAA receptor β 1 gene is extremely well conserved between species. Only 5 amino acid changes are to be found between mouse and human, indicating that most regions of this subunit of the GABA receptor are of high functional significance.

GABAA receptors belong to a family of transmembrane ligand-gated ion channels that includes the nicotinic acetylcholine, glycine and 5-HT3 receptors. These

receptors are responsible for rapid neuronal transmission in the mammalian CNS. GABA _A receptors primarily occur in the postsynaptic membrane where they are clustered at synapses, and also expressed at extrasynaptic locations. There is evidence that certain GABA _A receptor subtypes are preferentially expressed at extrasynaptic loci. Classically, GABAA receptors are the site of action of a number of drugs, including barbiturates, benzodiazepines and anesthetics.

GABA _A receptors are pentameric hetero-oligomers with a high degree of homology with nicotinic acetylcholine receptors. GABA _A receptors have numerous subunit families, many with multiple members, including: 6α, 4β, 3γ, 3p, δ, ε, θ and π. Each subunit has an extracellular N-terminal domain that typically contains ligand-recognition sites and 4 membrane-spanning domains, of which the second (TM2) is thought to line the ion channel, and a large intracellular loop that contains consensus sites for phosphorylation by various kinases. The subunits are arranged in a radial fashion such that they surround a central ion pore that opens in the presence of ligand. Once the ion channel opens, net ion flux follows the electrochemical gradient that is established across the neuronal membrane. In the case of GABAA receptors, the ion pore conducts chloride and bicarbonate ions.

GABA is classified as an inhibitory amino acid neurotransmitter because the influx of chloride ions into the postsynaptic cell after the activation of these receptors moves the postsynaptic membrane potential further away from its firing threshold and/or provides a conductance shunt for excitation. The discrete distribution of receptor subtypes suggests that each has a specific function within the CNS. In mammalian tissue, the most common receptor subtype contains αl, β2 and γ2 subunits. Within each subunit is a large N-terminal domain, which includes clusters or "loops" of amino acids thought to form the neurotransmitter binding domain(s). Interestingly, these receptors not only bind their neurotransmitter ligands, but can also interact with a number of compounds that bind at distant sites on the protein and allosterically modulate the actions of the neurotransmitter.

These modulatory agents include benzodiazepines, barbiturates, neurosteroids,

Zn ⁺ and anesthetics.

Davis (2003) J Psychiatry Neurosci 28(4), 263-27 '4 reviews the role of GABA _A receptors in mediating the effects of alcohol in the central nervous system. The document does not suggest that the GABA _A subunit βl may have a role in mediating a preference for any addictive substances, including alcohol.

Boehm et al (Biochemical Pharmacology 2004, 68, 1582-1602) review the role of GABA _A subunits in mediating ethanol action. Again, the document does not suggest that the GABA _A subunit βl may have a role in mediating a preference for any addictive substances, including alcohol.

Greenfield et al (2002) Neuropharmacology 42, 505-521 reports the effect of a mutation in GABA _A receptor βl subunit (in the TMl region of the polypeptide) in HEK293 cells. But the authors do not comment on whether the GABA _A subunit βl may have a role in mediating a preference for any addictive substances, including alcohol.

Ueno et al (1999) Br J Pharmacol 127, 377-382 reports the effect of mutations in the GABA _A receptor α ₂ , βi and γ _2L subunits on ethanol actions on GABA _A receptors expressed hi Xenopus oocytes, but does not suggest that the GABAA subunit βl may have a role in mediating a preference for any addictive substances, including alcohol.

US 6,066,726 reports 5'-flanking regions and core regulatory domains that underlie neuronal specific expression of the human GABA _A receptor βi subunit gene, but does not suggest that the GABA _A subunit βl may have a role in mediating a preference for any addictive substances, including alcohol.

Porjesz et al (Proc Natl Acad Sci USA 2002, 99, 3729-3733) report linkage disequilibrium between the β frequency of human EEG and a GABA _A receptor

gene locus encoding α2, σ.4 and βl subunits. EEG β activity is increased in alcoholics, consistent with alterations in neurotransmission. However the authors conclude that the GABA _A α2 subunit may be the most likely candidate for the observed linkage disequilibrium findings.

In light of this, it is surprising that a mutation of a βl subunit of the GABA _A receptor can cause animals to have a pronounced preference for addictive substances such as alcohol. Hence it may be the case that the normal βl subunit of the GABAA receptor is particularly sensitive or insensitive to addictive substances such as alcohol.

A first aspect of the invention provides a non-human animal having an altered preference for an addictive substance, wherein the altered preference results from an altered amount and/or function of GABAA subunit βl polypeptide.

We have used ENU mutagenesis to establish a mouse line with a heritable alcohol preference and have identified a single mutation in the GABAA receptor βl subunit. Functional analysis of the GABA _A receptor incorporating the mutated GABA _A receptor βl subunit indicates that the mutation reduces activation of the GABAA receptor. The mutation causes a single leucine to arginine substitution at position 310 (L310R) of the GABAA receptor βl subunit polypeptide (numbering including the signal peptide; L285R of the mature protein ie after cleavage of the signal peptide).

Therefore, a preferred embodiment of the invention is wherein the altered amount and/or function of GABAA subunit βl polypeptide causes a reduction in the activation of the GAB AA receptor.

Identification of the location of the mutation also has diagnostic implications, for example in the testing and possibly subsequent treatment and/or counselling of members of a family with a history of substance abuse.

Animals with such a mutation display a pronounced preference for addictive substances such as alcohol. Animals with such a mutation may therefore be useful in screening for potential therapeutic agents for preventing or treating addiction, and in investigating other factors that may lead an individual towards or away from addiction. The preference may be heritable as a dosage-dependent effect: thus, the alcohol (or other substance) preference may be higher in a homozygote than in a heterozygote and higher in a heterozygote than in the homozygote for an allele that is not linked with a high preference for ethanol (or other substance).

Such animals do not develop liver disease but, because they imbibe alcohol whereas normal mice do not, they are useful in identifying additional host and environmental factors which act synergistically or additively with alcohol in producing liver disease. In this setting they are useful in investigating, or screening for potential therapeutic agents for treating or preventing, diseases or conditions arising from or promoted by substance abuse, for example arising from or promoted by excessive alcohol consumption. In particular, such animals may be useful in investigating or screening for potential therapeutic agents for treating or preventing liver disease which can be promoted by alcohol consumption (for example fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Examples of such environmental factors include a high fat diet or presence of the Leiden Factor V mutation which synergise with alcohol, or hepatitis C or NASH, to produce liver diseases.

Therefore the invention provides a non-human animal having an altered amount and/or function of GABAA subunit βl polypeptide. Such an animal may be used for screening for potential therapeutic agents for preventing or treating addiction, as discussed above. Preferably the altered amount and/or function of GABAA subunit βl polypeptide results in the claimed non-human animal having an altered preference for an addictive substance.

The non-human animal may be any non-human animal, including non-human primates such as baboons, chimpanzees and gorillas, new and old world monkeys

as well as other mammals such as cats, dogs, rodents, pigs or sheep, or other animals such as poultry, for example chickens, fish such as zebrafish, or amphibians such as frogs. However, it is preferred that the animal is a rodent such as a mouse, rat, hamster, guinea pig or squirrel. Preferably the animal is mouse.

The non-human animal may also be a progeny or descendant (for example a further generation or generations removed) of an animal or mouse according to the invention. Such a progeny or descendant may share the preference for an addictive substance, for example, alcohol, of the parent animal or mouse. For example, the progeny or descendant may have a high preference for alcohol consumption. A progeny or descendant that is selected as having the degree of preference for an addictive substance of the parent animal or mouse (for example a high preference for alcohol consumption) may be particularly useful.

By "altered preference" we include that an animal of the first aspect of the invention, when given a choice, shows an altered preference to the consumption of an addictive substance when compared to an animal not having an altered amount and/or function of GABAA subunit βl polypeptide.

Preferably the animal of the first aspect of the invention has a high preference for the addictive substance. By "high preference" we include that an animal of the first aspect of the invention, when given a choice, chooses to consume the addictive substance on more than 50% of occasions, preferably more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. Such an animal on 95 % of the occasions consumes 60-80 % of total liquid as 10 % ethanol. For example, a mouse having a L310R (L285R using the numbering of the mature polypeptide) mutation of GABAA receptor βl subunit may take 70% to 80% of their total fluid intake, as measured over two 10 day periods, as 10% ethanol, rather than as water without ethanol.

By "addictive substance" we include that an animal of the first aspect of the invention has an altered preference for consumption of alcohol, preferably ethanol; opiates such as heroin, cocaine, morphine and methadone; nicotine;

benzodiazepines such as temazepam, flumazenil, diazepam; sucrose; and cannabis or marijuana. Preferably the animal has a high preference for alcohol consumption.

5 We have shown that an altered amount and/or function of GABAA subunit βl polypeptide causes an animal having an altered preference for addictive substances. As discussed above, the βl subunit is one of a number of different GABA _A receptor subunits.

10 By "altered amount and/or function" we include that, in comparison to a normal animal of the same species or strain, the animal of the first aspect of the invention has a reduced or elevated amount of GABA _A receptor βl subunit polypeptide and/or a reduced or elevated amount of the GABA _A receptor βl subunit polypeptide can function in the way that the same polypeptide operates in the

15 comparative animal.

For example, the animal of this aspect of the invention may have the same amount of polypeptide per se, but the polypeptide is in a non-functional state. Preferably the animal of the first aspect of the invention has a reduced amount and/or 20 function of GABA _A subunit βl polypeptide.

Alternatively, the altered amount of GABA _A subunit βl polypeptide may be due to an altered amount of nucleic acid encoding the GABA _A receptor βl subunit polypeptide.

25

Methods of determining the amount and/or function of polypeptide or the amount of nucleic acid are provided below. Preferably, "altered amount and/or function of includes where the animal has a reduced amount, i.e. 50%, 25%, 10%, 5%, 1%, 0.1% or 0% of the amount and/or function of the polypeptide, or nucleic acid,

30 _. in the normal animal. Preferably the non-human animal has no functional GABAA receptor βl subunit polypeptide.

Alternatively, the animal may have an elevated amount, i.e. 150%, 200%, 250%, 500%, 1000%, or 10000% of the amount and/or function of the polypeptide, or nucleic acid, in the normal animal.

The non-human animal of the first aspect of the invention may have an altered amount and/or function of GABA _A receptor βl subunit polypeptide due to an agent which can modify said polypeptide function being supplied to the animal, for example a compound which acts to prevent GABA _A receptor βl subunit function or sub-cellular localisation, or a peptide or antibody which can bind to the GABA _A receptor βl subunit and prevent function or sub-cellular localisation. The non-human animal of the first aspect of the invention may have an altered amount of nucleic acid encoding GAB A _A receptor βl subunit polypeptide due to an agent which can cause or induce degradation of said nucleic acid, for example a ribozyme which can target the nucleic acid, or an antisense molecule which can bind to the nucleic acid. By "antisense" we include RNA interference (RNAi) technologies.

Alternatively, the animal may be genetically modified in such a manner as to alter the amount and/or function of GABAA receptor βl subunit, or the amount of nucleic acid encoding said polypeptide. A genetic modification may alter the intrinsic function of the GABA _A receptor. Preferably, the animal is genetically modified.

The term "genetically modified" is well known to those skilled in the art. The term includes animals having introduced native or foreign nucleic acid. The animal may have had a modification made to its genome or may have been supplied with a nucleic acid which can act without modifying the genome.

There are a number of different methods that can be employed to generate a non- human animal according to the first aspect of the invention. These will be discussed in turn below. Preferred methods include those in which the gene encoding the said polypeptide is altered or removed so as to produce little or none

of said polypeptide. Other methods include inhibiting the transcription of the said gene or preventing any mRNA encoded by said gene from being translated.

Preferably, the methods set out below are employed to generate a non-human animal according to the first aspect of the invention in which the function of the GABA _A subunit βl polypeptide altered.

"Homologous recombination" is a technique well known to those skilled in the art. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as "knockout" animals. Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat. No. 5,557,032, incorporated herein by reference. Hence this aspect of the invention includes wherein the amount and/or function of GABA _A subunit βl polypeptide is altered by mutated one or more gene(s) encoding GABAA subunit βl polypeptide by homologous recombination. As a result the animal will no longer be able to synthesise GABAA subunit βl polypeptide, i.e. there will be a reduction in the amount of this polypeptide(s).

FLP/FRT or CRE/LOX recombination systems can also be used to mutate one or more gene(s) encoding GABA _A subunit βl polypeptide, as would be appreciated by a person skilled in the art.

Homologous recombination can be used to modify specific regions of a gene(s) encoding GABAA subunit βl polypeptide. For example, an introduced GABA _A subunit βl gene lacking certain protein motifs, for example the TM3 region or M2-M3 linker, may replace a native gene encoding GABA _A subunit βl polypeptide.

"Insertional mutagenesis" is also a term well known to those skilled in the art. Examples of such mutagenesis include transpo son-tagging, homing endonuclease genes (HEGs). In such methods a region of DNA is introduced into a gene such that the controlling or coding region of the gene is disrupted. Such methods can be

used to disrupt one or more genes encoding GABA _A subunit βl polypeptide. As a result the animal will no longer be able to synthesise GABA _A subunit βl polypeptide, i.e. there will be a reduction in the amount of this polypeptide.

Chemical or physical mutagenesis can also be used in the method of this aspect of the invention. Here, a gene is mutated by exposing the genome to a chemical mutagen, for example ethyl methylsulphate (EMS) or ethyl Nitrosurea (ENU), or a physical mutagen, for example X-rays. Such agents can act to alter the nucleotide sequence of a gene or, in the case of some physical mutagens, can rearrange the order of sequences in a gene. Practical methods of using chemical or physical mutagenesis in animals are well known to those skilled in the art. Such methods can be used to disrupt one or more genes encoding GABA _A subunit βl polypeptide. As a result the animal may no longer be able to synthesise GABAA subunit βl polypeptide, i.e. there will be a reduction in the amount and/or function of this polypeptide; alternatively the mutation may cause overactivity of the mutated polypeptide, i.e. there will be a increase in the amount and/or function of GABAA subunit βl polypeptide; alternatively, the mutation may cause an altered function of the mutated polypeptide.

Homologous recombination, insertional mutagenesis and chemical or physical mutagenesis can be used to generate a non-human animal which is heterozygous for the target gene, e.g. GABAA subunit βl gene ( ⁴ ^). Such animals may be of particular use if the homozygous non-human animal has too severe a phenotype.

Antisense oligonucleotides are single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-KNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are often termed "antisense" because they are complementary to the sense or coding strand of the gene. Recently, formation of a triple helix has proven possible where the oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby. This

suggests that it is possible to synthesise sequence-specific molecules which specifically bind double-stranded DNA via appropriate formation of major groove hydrogen bonds.

By binding to the target nucleic acid, the above oligonucleotides can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking the transcription, processing, poly(A)addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradations.

Antisense oligonucleotides are prepared in the laboratory and then introduced into cells, for example by microinjection or uptake from- the cell culture medium into the cells, or they are expressed in cells after transfection with plasmids or retroviruses or other vectors carrying an antisense gene. Antisense oligonucleotides were first discovered to inhibit viral replication or expression in cell culture for Rous sarcoma virus, vesicular stomatitis virus, herpes simplex virus type 1, simian virus and influenza virus. Since then, inhibition of mRNA translation by antisense oligonucleotides has been studied extensively in cell-free systems including rabbit reticulocyte lysates and wheat germ extracts. Inhibition of viral function by antisense oligonucleotides has been demonstrated in vitro using oligonucleotides which were complementary to the AIDS HIV retrovirus RNA (Goodchild, J. 1988 "Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides", Proc. Natl. Acad. Sd. (USA) 85(15), 5507-11). The Goodchild study showed that oligonucleotides that were most effective were complementary to the poly(A) signal; also effective were those targeted at the 5' end of the RNA, particularly the cap and 5' untranslated region, next to the primer binding site and at the primer binding site. The cap, 5' untranslated region, and poly(A) signal lie within the sequence repeated at the ends of retrovirus RNA (R region) and the oligonucleotides complementary to these may bind twice to the RNA.

Typically, antisense oligonucleotides are 15 to 35 bases in length. For example, 20-mer oligonucleotides have been ^" shown to inhibit the expression of the epidermal growth factor receptor mRNA (Witters et al, Breast Cancer Res Treat 53:41-50 (1999)) and 25-mer oligonucleotides have been shown to decrease the expression of adrenocorticotropic hormone by greater than 90% (Frankel et al, J Neurosurg 91:261-7 (1999)). However, it is appreciated that it may be desirable to use oligonucleotides with lengths outside this range, for example 10, 11, 12, 13, or 14 bases, or 36, 37, 38, 39 or 40 bases.

By "antisense" we also include all methods of RNA interference, which are regarded for the purposes of this invention as a type of antisense technology.

A further method of generating a non-human animal of this aspect of the invention is wherein the amount of GABAA subunit βl polypeptide is altered by supplying the animal with a ribozyme capable of cleaving RNA or DNA encoding GABA _A subunit βl polypeptide. A gene expressing said ribozyme may be administered in substantially the same and using substantially the same vehicles as for antisense molecules.

Ribozymes which may be encoded in the genomes of the viruses or virus-like particles herein disclosed are described in Cech and Herschlag "Site-specific cleavage of single stranded DNA" US 5,180,818; Altman et al "Cleavage of targeted RNA by RNAse P" US 5,168,053; Cantin et al "Ribozyme cleavage of HIV-I RNA" US 5,149,796; Cech et al "RNA ribozyme restriction endoribonucleases and methods", US 5,116,742; Been et al "RNA ribozyme polymerases, dephosphorylases, restriction endonucleases and methods", US 5,093,246; and Been et al "RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods; cleaves single-stranded RNA at specific site by transesterification", US 4,987,071, all incorporated herein by reference.

It will be appreciated that it may be desirable that the antisense molecule or ribozyme may be expressed from a cell-specific promoter element, or a regulatable promoter.

The genetic constructs of the invention can be prepared using methods well known in the art.

A further method for altering the amount and/or function of GABA _A subunit βl polypeptide in a non-human animal is by supplying the animal with one or more agents that act as antagonists.

The term "antagonist" is well known to those skilled in the art. By "antagonist" we include in this definition any agent that acts to alter the level and/or functional ability of GABA _A subunit βl polypeptide. An example of an antagonist would include a chemical ligand that binds to and affects βl subunit function, and in broader terms this could also include an antibody, or antibody fragment, that binds to one of the said polypeptides such that the polypeptide cannot effect its normal function. The antagonist may also alter the sub-cellular localisation of GABAA subunit βl polypeptide. In this way, the amount of functional polypeptide is reduced.

In a further method of generating a non-human animal of this aspect of the invention, the amount of nucleic acid encoding GABA _A subunit βl polypeptide is altered by modifying the chromatin structure at or adjacent to gene(s) encoding a GABAA subunit βl polypeptide. This may be achieved using, for example, targeted DNA methylation. Such methods are known to those skilled in the art.

In a further method of generating a non-human animal of this aspect of the invention, the amount and/or function of GABA _A subunit βl polypeptide is altered by supplying an animal with a dominant inactive form of a GABAA subunit βl polypeptide. For example a polypeptide may be modified so as to generate a dominant inactive form of a GABA _A subunit βl polypeptide that can bind to the

same polypeptides as GABAA subunit βl polypeptide in, for example, a GABAA receptor, but cannot function as GABAA subunit βl polypeptide. Alternatively the dominant inactive form may not be correctly trafficked within the cell, e.g. it may not be targeted to the plasma membrane. Hence overexpression of a dominant inactive form of a GABA _A subunit βl polypeptide in a non-human animal may act to block (i.e. reduce) the function of the native GABA _A subunit βl polypeptide.

As discussed above, we have shown that an alteration in the amount and/or function of GABAA receptor βl subunit can lead to an animal having an altered preference for an addictive substance, preferably alcohol. GABA _A receptor βl subunit is a component of the GABA _A receptor, a ligand-gated ion channel which regulates the influx of chloride ions into a postsynaptic cell. Preferably, the altered amount and/or function of GABA _A subunit βl polypeptide may result from disrupted folding of the polypeptide, as discussed in the accompanying example.

Preferably the altered amount and/or function of GABAA subunit βl polypeptide results from a mutation, preferably at or vicinal to TM3 or the TM2-TM3 linker. It is preferred that the altered amount and/or function of GABA _A subunit βl polypeptide results from a substitution of the leucine amino acid residue at position 310 (position 285 using the numbering of the mature polypeptide ie not including the signal peptide), preferably the substitution is with an arginine residue.

To date human and mouse GABAA subunit betal polypeptides and nucleotide sequences have been characterized. The polypeptide and nucleotide sequences for the GABA _A subunit betal are given in GenBank Accession Numbers NP_000803 and MIM:137190 respectively for human and NP_032095 and MGL95619 respectively for mouse, and are also provided in Figure 1.

By "GABAA subunit βl polypeptides" we include the human and mouse GABAA subunit βl polypeptides as well as further homologues, orthologues or paralogies of GABAA subunit βl polypeptides.

Further GABA receptor β subunit polypeptides are disclosed in GenBank. The following internet link lists some of these:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein& amp;cmd=search&term=G ABA+Beta+subunit

Methods by which homologues, orthologues or paralogues of polypeptides can be identified are well known to those skilled in the art: for example, in silico screening or database mining. Preferably, such polypeptides have at least 40% sequence identity, preferably at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the polypeptide sequence of GABA _A subunit βl polypeptide.

Methods of determining the percent sequence identity between two polypeptides are well known in the art. For example, the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.

The alignment may alternatively be carried out using the Clustal W program (Thompson et ah, (1994) Nucleic Acids Res 22, 4673-80). The parameters used may be as follows:

Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM.

The term "nucleic acid encoding GABAA subunit βl polypeptide" includes both DNA and RNA molecules, including mRNA. By encode we mean that the

sequence of bases in the nucleic acid molecule is such that, on transcription and/or translation, it encodes a polypeptide having the sequence of a GABA _A subunit βl polypeptide. The term also includes single-stranded or double-stranded molecules, or those nucleic acids that are the complement of nucleic acids encoding said polypeptide, as would be appreciated by those skilled in the art. Therefore, we include in this term all nucleic acid molecules that encode a GABA _A subunit βl polypeptide as defined above, including homologues, orthologues and paralogues as identified from the in silico screening and database mining methods discussed above.

A second aspect of the invention provides a method for providing an animal or mouse having an altered preference for an addictive substance, the method comprising the steps of:

(i) breeding an animal or mouse according to the first aspect of the invention with a second animal or mouse respectively;

(ii) testing the progeny or descendant of step 1 for preference for an addictive substance; and,

(iii) selecting an animal or mouse exhibiting an altered preference for an addictive substance.

Such a method can be used to cross an animal having an altered preference for an addictive substance with an animal which may have, for example, a tendency to develop specific disorders associated with a abuse of such an addictive substance. Preferably the addictive substance is alcohol. Preferably the animal has a high preference for the addictive substance.

For example, an animal having a high preference for alcohol consumption may be crossed with an animal which may be predisposed to develop liver disorders. Since coagulation pathways may be of importance in liver injury, such an animal may be an animal which has a coagulation-promoting genotype, for example a mutation in a coagulation factor, for example Factor V, for example the Factor V

Leiden mutation. Hence the progeny or descendents of breeding an animal having a high preference for alcohol consumption with an animal which may be predisposed to develop liver disorders may be of use in screening for compounds which may be of use in preventing alcohol induced liver injury.

Such animals do not develop liver disease but, because they imbibe alcohol whereas normal mice do not, they are useful in identifying additional host (Leiden Factor V Mutation) and environmental factors (moderate or high fat diet or choline deficient diet) which act synergistically or additively with alcohol in producing liver disease. In this setting they are useful in investigating, or screening for potential therapeutic agents for treating or preventing, diseases or conditions arising from or promoted by substance abuse, for example arising from or promoted by excessive alcohol consumption. In particular, such animals may be useful in investigating or screening for potential therapeutic agents for treating or preventing liver disease which can be promoted by alcohol consumption (for example fibrosis, cirrhosis and hepatocellular carcinoma. Examples will be hybrids of an alcohol preferring mouse with mouse exhibiting:

- Leiden Factor V mutation or other procoagulant mutations; Expression of hepatitis C nucleocapsid or other viral proteins that cause hepatitis steatosis

- Non alcohol related steato-hepatitis (NASH) (IGT/6) to produce liver disease or liver cancer.

The terms "high preference" and "addictive substance" have been previously discussed above.

The step of breeding animal or mouse according to the first aspect of the invention with a second animal or mouse will be well known to those skilled in the art.

The step of selecting an animal exhibiting a high preference for an addictive substance necessarily is dependent upon the animal used.

The method of the second aspect of the invention can also be used to cross an animal having a high preference for an addictive substance with an animal which may also have a high preference for the same addictive substance. For example, an animal having a high preference for alcohol consumption may be crossed with a second animal which has a high preference for alcohol consumption.

By high ethanol preference is included consumption of 10% ethanol as at least about 65%, 70% or 75% of total liquid intake. By low ethanol preference/consumption is included consumption of 10% ethanol as at least about 15% to 25%. Intake between about 25% and 65% may be classed as moderate ethanol preference or consumption.

An embodiment of this aspect of the invention is wherein the second mouse or animal is of a strain exhibiting an aversion to alcohol, for example mouse strain DBA/2J. This strain is obtainable from The Jackson Laboratory, 600 Main Street, Bar Harbor, MAINE 04609 USA (www.jax.org).

Alternatively, the second animal or mouse may be of a strain exhibiting a low or moderate alcohol consumption, for example strain C3H or strain BALB/c or strain 129 or strain A/J. These strains are obtainable from The Jackson Laboratory, 600 Main Street, Bar Harbor, MAINE 04609 USA (www.jax.org).

Alternatively, the second animal or mouse may be of a strain exhibiting a high preference for alcohol consumption, for example of strain C57BL/6. This strain is obtainable from The Jackson Laboratory, 600 Main Street, Bar Harbor, MAINE 04609 USA (www.jax.org).

The second mouse or animal may be a mouse or animal of the invention as defined in the first aspect of the invention.

A third aspect of the invention provides a method for identifying an animal with a- dominant mutation affecting preference for an addictive substance comprising the steps of:

(i) providing a randomly-mutated animal;

(ii) testing the animal or progeny of descendant of such an animal for preference for an addictive substance; and, (iii) selecting an animal displaying a selected preference for an addictive substance; and, optionally,

(iv) breeding the selected animal.

An example of such a method is described in Example 1. The animal is preferably one that is convenient to house, breed and analyse for substance preference, and preferably also amenable to genetic analysis. For example, the animal may be a rodent, for example a mouse. Techniques useful for generating random mutations will be well known to those skilled in the art and include transposon tagging, chemical mutagenesis, for example using ENU, as described in Example 1, radiation induced mutagenesis from exposure of parental mice to ionising radiation, and virally induced mutagenesis which is induced by exposure of the embryo to an infectious agent. A further method of generating random mutations is by mutagenising mouse embryonic stem cells using one of the methods set out above, followed by injection of the stem cells into host blastocysts and transfer into foster mothers. Preferably the randomly-mutated animal is an ENU-mutated mouse.

By "dominant mutation" we mean that an animal which is heterozygous for the mutated gene will nonetheless exhibit some or all of the phenotypes associated with the mutation, as would be appreciated by a person skilled in the art. Hence the method can identify an animal which has an altered preference for an addictive substance when only harbouring one copy of the mutated gene.

An advantage of a screen to identify dominant mutations is that the first generation of mutated animals can be screened to identify a desired trait, whereas a mutagenesis project to screen for recessive or modifier loci would require a

much more extensive breeding program since it is necessary to breed the mutated animals and screen the descendents.

Techniques for assessing preference for an addictive substance are also well known to those skilled in the art, and include the "two bottle" test system discussed in Example 1 and cited references. Preferably the addictive substance is alcohol.

A fourth aspect of the invention provides an animal or mouse obtained or obtainable by the method of the second and third aspects of the invention.

A fifth aspect of the invention provides a cell comprising an altered amount and/or function of GABA _A subunit βl polypeptide.

As discussed above, we have shown that an altered amount and/or function of GABA _A subunit βl polypeptide causes an animal having an altered preference for addictive substances. Hence, such a cell may be used for screening potential therapeutic agents for preventing or treating addiction., as discussed further in the Examples.

By "altered amount and/or function" we include that, in comparison to a normal cell, the cell of this aspect of the invention has a reduced or elevated amount of GAB A _A receptor βl subunit polypeptide and/or a reduced or elevated amount of the GABAA receptor βl subunit polypeptide can function in the way that the same polypeptide operates in a comparative cell. Methods of determining the amount and/or function of polypeptide or the amount of nucleic acid are provided below. An embodiment of this aspect of the invention is wherein the altered amount and/or function of GABAA subunit βl polypeptide causes a reduction in the activation of the GAB AA receptor.

As discussed above, we have shown that an alteration in the amount and/or function of GABAA receptor βl subunit can lead to an animal having an altered

preference for an addictive substance, preferably alcohol. GABA _A receptor βl subunit is a component of the GABA _A receptor, a ligand-gated ion channel which regulates the influx of chloride ions into a postsynaptic cell.

Preferably, the altered amount and/or function of GABA _A subunit βl polypeptide in the cell of this aspects of the invention results from disrupted folding of the polypeptide, as discussed in the accompanying example. Preferably the altered amount and/or function of GABAA subunit βl polypeptide results from a mutation, preferably at or vicinal to TM3 or the TM2-TM3 linker. It is preferred that the altered amount and/or function of GABA _A subunit βl polypeptide results from a substitution of the leucine amino acid residue at position 310 (position 285 of the mature polypeptide ie without the signal peptide); preferably the substitution is with an arginine residue.

Methods of preparing a cell according to this aspect of the invention include mutating the gene encoding the GABA _A subunit βl polypeptide, or using antisense technology, as discussed above in relation to the first aspect of the invention. Also, a cell can be prepared by introducing into a suitable cell type nucleic acid encoding an altered amount and/or function of GABAA subunit βl polypeptide. An example of such a nucleic acid is where a polynucleotide encoding a GABAA subunit βl polypeptide is altered so that the leucine amino acid residue at position 310 (285 using the numbering of the mature polypeptide ie without the signal peptide), is substituted with an arginine residue. Examples of polynucleotide encoding a GABA _A subunit βl polypeptide are provided above. Methods of altering such polynucleotides so as to encode an altered amount of functional of GABAA subunit βl polypeptide are well known in the art. Methods of introducing such a polynucleotide into cells are also well known to those skilled in the art.

The cell of this aspect of the invention can be either prokaryotic or eukaryotic, though are typically eukaryotic, for example mammalian, typically human or mouse. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli

such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories hie, Bethesda, MD, USA, and RRl available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343). Preferred eukaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NTH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-I cells available from the ATCC as CRL 1650. Further preferred mammalian host cells and cells of the invention are discussed in the Examples. The cell of the invention may be derived from an animal of the invention. Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.

Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Set USA 69, 2110 and Sambrook et al (2001) Molecular Cloning, A Laboratory Manual, 3 ^rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.

In addition to the cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.

A sixth aspect of the invention provides a method of screening for compounds of use in preventing or treating substance abuse wherein a non-human animal is administered a test compound and the effect of the test compound on the amount and/or function of GABA _A subunit βl polypeptide is assessed.

A seventh aspect of the invention provides a method of screening for compounds of use in preventing or treating substance abuse wherein a cell having functional GABAA subunit βl polypeptide is treated with a test compound and the effect of the test compound on the amount and/or function of GABA _A subunit βl polypeptide is assessed.

An eighth aspect of the invention provides a method of screening for compounds of use in preventing or treating substance abuse wherein a cell having an altered amount and/or function of GABA _A subunit βl polypeptide is treated with a test compound and the effect of the test compound on the amount and/or function of GABA _A subunit βl polypeptide is assessed. An example of such a cell of use in this method of the invention is provided above in the fifth aspect of the invention.

An embodiment of the sixth, seventh and eighth aspects of the invention is wherein the methods further comprise the step of selecting a compound that increases the amount and/or function of GABA _A subunit βl polypeptide.

An embodiment of the sixth, seventh and eighth aspects of the invention is wherein the addictive substance is alcohol.

The methods of the sixth, seventh and eighth aspects of the invention relate to screening methods for drugs or lead compounds. The test compound may be a drug-like compound or lead compound for the development of a drug-like compound.

The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it

suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water-soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential.

The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, nonselective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.

The methods of the sixth, seventh and eighth aspects of the invention include a step of assessing the effect of a test compound on the amount and/or function of GABAA subunit βl polypeptide.

In common with all these methods is the need for a "reference sample", i.e. a sample of protein or nucleic acid taken from an animal or cell which has not been exposed to the test compound. By comparing the amount and/or function of GABA _A subunit βl polypeptide in a sample of protein or nucleic acid taken from an animal or cell which has not been exposed to the test compound, to the amount and/or function of GABAA subunit βl polypeptide in a sample of protein or nucleic acid taken from an animal or cell which has been exposed to the test compound it is possible to determine the effect of the test compound on the amount and/or function of GABA _A subunit βl polypeptide. This will show the test comρound(s) to produce an elevation, reduction or no effect on expressed levels of the βl subunit, or a potentiation, inhibition or no effect on the function of receptors containing the βl subunit.

The step of assessing the amount and/or function of GABA _A subunit βl polypeptide may be performed using a number of different methods.

Firstly, the effect of the test compound in the sixth, seventh and eighth aspects of the invention can be determined by quantifying the amount of nucleic acid, preferably mRNA, encoding the GABA _A subunit βl polypeptide.

Levels of mRNA encoding the GABA _A subunit βl polypeptide may be assayed using the RT-PCR method described in Makino et al, Technique 2:295-301 (1990). By this method, the radioactivities of the "amplicons" in the polyacrylamide gel bands are linearly related to the initial concentration of the target mRNA. Briefly, this method involves adding total RNA isolated from a biological sample in a reaction mixture containing a RT primer and appropriate buffer. After incubating for primer annealing, the mixture can be supplemented with a RT buffer, dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubation to achieve reverse transcription of the RNA, the RT products are then subject to PCR using labeled primers. Alternatively, rather than labelling the primers, a labeled dNTP can be included in the PCR reaction mixture. PCR amplification can be performed in a DNA thermal cycler according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction mixture is electrophoresed on a polyacrylamide gel. After drying the gel, the radioactivity of the appropriate bands (corresponding to the mRNA encoding the GABA _A subunit βl polypeptide) is quantified using an imaging analyzer. RT and PCR reaction ingredients and conditions, reagent and gel concentrations, and labeling methods are well known in the art. Variations on the RT-PCR method will be apparent to the skilled artisan. Any set of oligonucleotide primers which will amplify reverse transcribed target mRNA can be used and can be designed as will be well known to those skilled in the art. Alternative techniques by which to measure mRNA levels include incorporation of SybrGreen or other fluorophores into primers or probes as part of RealTime PCR experiments.

Levels of mRNA encoding the GABAA subunit βl polypeptide can also be assayed using northern blotting, a method well known to those skilled in the art and described further in Sambrook et al, Molecular Cloning. A laboratory manual. 1989. Cold Spring Harbour publications.

Further methods which may be of use in measuring mRNA levels include in situ hybridisation (In Situ Hybridization Protocols. Methods in Molecular Biology Volume 33. Edited by K H A Choo. 1994, Humana Press Inc (Totowa, NJ, USA) pp 48Op and In Situ Hybridization: A Practical Approach. Edited by D G Wilkinson. 1992, Oxford University Press, Oxford, pp 163), in situ amplification, nuclease protection, probe arrays, and amplification based systems.

Such methods can also be used to determine if there is any variation in the location of the mRNA encoding the GABA _A subunit βl polypeptide. In addition, microarray analysis, a technique well known to those skilled in the art, may also be used to assess both the amount and/or location of mRNA encoding the GABA _A subunit βl polypeptide.

A further method of assessing the effect of the test compound on the amount of GABA _A subunit βl polypeptide is to quantify the amount of said polypeptide.

Assaying the amount of GABAA subunit βl polypeptide in a biological sample can be performed using any art-known method. Preferred for assaying GABAA subunit βl polypeptide levels in a biological sample are antibody-based techniques. For example, GABAA subunit βl polypeptide expression can be studied with classical immunohistological methods. In these, the specific recognition is provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system can utilize fluorescent, enzyme, or other conjugated secondary antibodies. As a result, an immunohistological staining of tissue section for pathological examination is obtained. Tissues can also be extracted, e.g., with urea and neutral detergent, for the liberation of GABAA

subunit βl polypeptide for Western-blot or dot/slot assay (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)). In this technique, which is based on the use of cationic solid phases, quantitation of GABA _A subunit βl polypeptide can be accomplished using isolated GABAA subunit βl polypeptide as a standard.

Other antibody-based methods useful for detecting GABA _A subunit βl polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example, a GABA _A subunit βl polypeptide -specific monoclonal antibody can be used both as an immunoadsorbent and as an enzyme-labelled probe to detect and quantify the GABA _A subunit βl polypeptide. The amount of GABAA subunit βl polypeptide present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm. Such an ELISA for detecting a tumour antigen is described in Iacobelli et al., Breast Cancer Research and Treatment 11: 19-30 (1988).

In addition to assaying GABA _A subunit βl polypeptide levels in a biological sample obtained from an animal or a cell, GABAA subunit βl polypeptide can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of GABA _A subunit βl polypeptide include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or caesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labelling of nutrients for the relevant hybridoma.

GABA _A subunit βl polypeptide-specific antibodies for use in the screening methods of the present invention can be raised against the intact GABA _A subunit βl polypeptide or an antigenic polypeptide fragment thereof, which may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids),

without a carrier.

Such methods can also be used to determine if there is any variation in the location of GABA _A subunit βl polypeptide. In addition, methods of determining the location of GABA _A subunit βl polypeptide include in situ imrnuno- histochemistry analysis using an antibody that recognises said polypeptide, as would be appreciated by a person skilled in the art; or cell fractionation followed by an immunoassay for the presence of GABAA subunit βl polypeptide, again as would be appreciated by a person skilled in the art.

As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to GABA _A subunit βl polypeptide. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less nonspecific tissue binding of an intact antibody (WaH et al, J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.

The antibodies as used herein also include humanised antibodies capable of specifically binding to GABA _A subunit βl polypeptide.

Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non- human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al, Nature, 321:522-525 (1986); Riechmarm et al, Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Pat. No. 5,225,539). In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise

residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et ah, 1986; Riechmann et al, 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

The antibodies as used herein also include humanised antibodies capable of specifically binding to GABAA subunit βl polypeptide. Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et ah, 1983 Immunol Today 4: 72) and the ^' EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et ah, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may also be produced by using human hybridomas (see Cote, et ah, 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et ah, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. MoI. Biol., 227:381 (1991); Marks et ah, J. MoI. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all

respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al {Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, {Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).

Human antibodies may additionally be produced using transgenic nonhuman animals that are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells that secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.

A further method of assessing the effect of the test compound in the sixth, seventh and eighth aspects of the invention is to assess the effect of the test compound on the function of GABA _A subunit βl polypeptide.

As discussed above, GABA _A subunit βl polypeptide is a component of the GABA _A receptor family. An assay for determining the function of the GABA _A receptor is provided in the accompanying example. In essence, the effect of a test compound on a cell having GABA _A receptor with a GABA _A subunit βl polypeptide can be compared to a cell having GABA _A receptor without a GABA _A subunit βl polypeptide. The effect of the test compound can thus be measured as a function of any change in the activation, deactivation, desensitisation and single channel properties of the GABA _A receptor, accomplished using electrophysiological methods. Essentially, cells expressing pure populations of recombinant GABA _A receptors are recorded from using the technique of patch clamp recording which is well known to those in the field. The receptors are activated by suitable agonist, e.g., GABA, and the activation of the receptor and how it behaves thereafter (deactivation and desensitsation) in terms of current flow through its integral ion channel is studied using electrophysiology. This technique can also be applied to the study of a single GABA _A receptor protein in a cell membrane (vis, single channel recording). The technique is capable of analysing the concentration response relationship of activation and how the test compound may interfere with this either by interacting with one or more binding sites on the GABA _A receptor. The test compound can be analysed in this way using either wild-type GABA _A receptors, or by using such receptors that are carrying selected mutations in one or more subunits. These procedures are well known to those in the field.

Details of methods that may be used in the screening methods of the invention are provided in Example 2.

An example of a screening method according to the eighth aspect of the invention is also provided in the examples.

It is preferred that the non-human animal or cell of the sixth or eighths aspects of the invention have a substitution of the leucine amino acid at position 310 (position 285 using the numbering of the mature polypeptide without signal sequence), for example a leucine to argmine substitution, in the GABA _A receptor subunit βl polypeptide. As set out above, such a mutation can lead to altered preference to addictive substances, for example alcohol.

A ninth aspect of the invention provides a method for screening for compounds of use in preventing or treating addiction to an addictive substance comprising the steps of:

(i) providing a test animal according to the first or fourth aspects of the invention; (ii) exposing the test animal to a test compound;

(iii) determining whether the compound affects an addictive substance preference of the test animal; and

(iv) selecting a compound that reduces an addictive substance preference of the test animal.

An embodiment of the screening methods of the invention is wherein the substance is alcohol.

It is preferred that the test animal of the ninth aspect of the invention has a substitution of the leucine amino acid at position 310 (position 285 using the numbering of the mature polypeptide without signal sequence), for example a leucine to arginine substitution, in the GABAA receptor subunit βl polypeptide.

As set out above, such a mutation can lead to altered preference to addictive substances, for example alcohol.

The method of the ninth aspect of the invention relate to screening methods for drugs or lead compounds. The test compound may be a drug-like compound or lead compound for the development of a drug-like compound.

Again there is the need for a "reference sample", i.e. an animal which has not been exposed to the test compound. By comparing the addiction of a test animal exposed to the test compound to a test animal not exposed to the test compound to it may be possible to determine the effect of the test compound on addiction.

The method used to measure of addiction of a test animal to an addictive substance necessarily varies according to the addictive substance. An example of assaying addiction of a test animal to alcohol is provided in the accompanying examples.

It has been previously shown that the compound SCS (Salicylidene salicylhydrazide) specifically binds to GABA _A receptors containing a βl subunit and, in this case, specifically inhibits a GABA _A receptor containing the βl subunit (Thompson et al (2004) Br J Pharmacol 142, 97-106). The structure of SCS is shown in Figure 5. SCS is commercially available from, for example, Tocris.com; catalogue number 2143.

Therefore SCS, or an analogue or derivative thereof, is an example of a compound which can be tested using the screening methods of the sixth, seventh, eighth and ninth aspects of the invention. By "analogue or derivative" of SCS we include structurally related compounds able to selectively inhibit GABAA receptors containing a βl subunit, for example those compounds mentioned in Thompson et al (2004) Br J Pharmacol 142, 97-106.

Opioid receptors (Maldonado et al (2002) J Neucrosci 22, 3326-3331) are involved in the rewarding (reinforcing) effect of addictive drugs, including alcohol. It is known that opioid antagonists reduce consumption and self- administration of ethanol in animals (Weiss et al (2002) J Neurosci 22, 3332-

3337), and the opioid receptor antagonist Naltrexone has been shown to reduce the alcohol intake in the high ethanol preferring C57/BL6 mice, from 60% to 40-50% (Middaugh et al (2000) Psychopharmacology (Berl) 151, 321-327). By contrast, μ-receptor knockout mice cannot be induced to self-administer alcohol (Spanagel R. Behavioural and molecular aspects of alcohol craving and relapse. In : Maldonando R, ed Molecular Biology of drug addiction. Totowa NJ: Humana Press, 2002: 295-313).

Therefore Naltrexone, or an analogue or derivative thereof, is an example of a compound which can be tested using the screening methods of the sixth, seventh, eighth and ninth aspects of the invention. By "analogue or derivative" of Naltrexone we include structurally related compounds able to act as an opioid receptor antagonist to reduce alcohol intake.

Furthermore, the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor (reviewed in Davies et al (2003) J Psychiatry Neurosci 28, 263-274) is also involved in the rewarding (reinforcing) effect of addictive drugs, including alcohol. The NMDA receptor antagonist Acamprosate has been shown to reduce the ethanol intake of high drinking mice (Chester et al (2001) Behav Pharmacol 12, 535-543; McGeehan et al (2003) Br J Pharmacol 138, 9-12).

Therefore Acamprosate, or an analogue or derivative thereof, is a further example of a compound which can be tested using the screening methods of the sixth, seventh, eighth and ninth aspects of the invention. By "analogue or derivative" of Acamprosate we include structurally related compounds able to act as NMDA receptor antagonist to reduce the ethanol intake.

As discussed above, some of the actions of ethanol are mediated by GABAA receptors. Hodge et al (1999) Nat Neurosci 2, 997-1002 demonstrated that mutant mice lacking protein kinase C epsilon (PKCepsilon) were more sensitive than wild-type littermates to the acute behavioural effects of ethanol and other drugs that allosterically activate GABAA receptors. GABA _A receptors in membranes

isolated from the frontal cortex of PKCepsilon null mice were also supersensitive to allosteric activation by ethanol and flunitrazepam. In addition, these mutant mice showed markedly reduced ethanol self-administration. These findings indicate that inhibition of PKCepsilon increases sensitivity of GABA _A receptors to ethanol and allosteric modulators.

Therefore, pharmacological agents that inhibit PKCepsilon may be useful for treatment of alcoholism and may provide a non-sedating alternative for enhancing GABA _A receptor function to treat other disorders such as anxiety and epilepsy (Hodge et al supra). Hence such pharmacological agents are examples of compounds which can be tested using the screening methods of the sixth, seventh, eighth and ninth aspects of the invention. An example of a PKCε inhibitor is: myristoylated Vl -2 peptide. This peptide must be delivered intracellulary as it will not penetrate cell membranes. We also include analogues or derivatives of myristoylated Vl -2 peptide, i.e. structurally related peptides able to act as a PKCε inhibitor.

A tenth aspect of the invention is a method for screening for compounds of use in preventing or treating a disease or condition arising from or promoted by a addiction to an addictive substance, comprising the steps of:

(i) providing a test animal according to the first or fourth aspects of the invention;

(ii) exposing the test animal to a test compound and, optionally, the addictive substance;

(iii) determining whether the compound affects the development of the disease or condition in the test animal; and

(iv) selecting a compound that reduces the incidence, severity and/or rate of progression of the disease or condition.

An embodiment of the tenth aspect of the invention is wherein the disease or condition is one arising from or promoted by addiction to alcohol.

It is preferred that the test animal of the tenth aspect of the invention has a substitution of the leucine amino acid at position 310 (position 285 using the numbering of the mature polypeptide without signal sequence), for example a leucine to arginine substitution, in the GABA _A receptor subunit βl polypeptide. As set out above, such a mutation can lead to altered preference to addictive substances, for example alcohol.

An embodiment of the tenth aspect of the invention is wherein the disease or condition is liver fibrosis, cirrhosis or hepatocellular carcinoma. Further embodiments of this aspect of the invention is wherein the disease or condition is obesity, anorexia nervosa, pancreatitis, foetal alcohol syndrome, cerebral atrophy, cardiomyopathy, alcohol related vitamin deficiency states and DT.

Addiction to an addictive substance can lead to disease or condition arising the addition. For example, addiction to alcohol can lead to liver fibrosis, cirrhosis or hepatocellular carcinoma. The tenth aspect of the invention provides a method of identifying a compound which may be of use in treating such a disease or condition.

As with the other screening methods of the invention there may be the need for a "reference sample", i.e. an animal which also addicted to the addictive substance but which is not exposed to the test compound.

The screening methods of the invention can be used in "library screening" methods, a term well known to those skilled in the art. Thus, for example, the methods of the invention may be used to detect (and optionally identify) a test compound capable of affecting the amount and/or function of GABAA subunit βl polypeptide, preventing or treating an addiction to an addictive substance, or preventing or treating a disease or condition arising from or promoted by an addiction to an addictive substance. Aliquots of a library may be tested for the ability to give the required result.

A further embodiment of the method of the sixth, seventh, eighth, ninth and tenth aspects of the invention is wherein the selected compound is formulated into a pharmaceutically acceptable composition.

An eleventh aspect of the invention provides a method of making a pharmaceutical composition comprising the method of the screening methods of the invention and .the step of mixing the selected compound (or a derivative or analogue thereof) with a pharmaceutically acceptable carrier.

Preferably, the formulation is a unit dosage containing a daily dose or unit, daily sub- dose or an appropriate fraction thereof, of the active ingredient.

The compounds will normally be administered orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.

In human therapy, the compounds can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the compounds can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The compounds may also be administered via intracavernosal injection.

The compounds can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrastemally, intracranially, intra-muscularly or subcutaneously, or they may be administered by

infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a physiologically relevant pH of from 3 to 9, preferably to a pH close to 7.4), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

A twelfth aspect of the invention provides a method for determining the likelihood that a person is, or may become, addicted to addictive substances comprising the steps of:

(i) obtaining a sample containing nucleic acid and/or protein from the person; (ii) assessing the amount and/or function of GABAA subunit βl polypeptide and/or determining the genotype of the person's GABA _A subunit βl gene.

This method may be useful in the diagnosis of addiction or as a basis of genetic counselling.

Methods of determining the amount and/or function of GABAA subunit βl polypeptide are presented above in relation to the screening methods of the invention. We also include the methods for determining the amount of nucleic acid encoding GABAA subunit βl polypeptide as disclosed herein, including northern blotting and RT-PCR.

This method also includes determining whether there is any change in the excitability of GABAA receptor comprising a GABAA subunit βl polypeptide. Methods of assessing the excitability of GABAA receptor are provided in the accompanying example and discussed above.

Furthermore, it is possible that genomic rearrangements can lead to an increase in the copy number of gene(s) encoding GABA _A subunit βl polypeptide, i.e. nucleic acids encoding said polypeptide. Methods of determining gene copy number include Southern blotting (essentially as performed as set out in Sambrook et al (1989). Molecular cloning, a laboratory manual, 2 ^nd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York) or quantitative PCR.

Methods of determining the person's genotype of GABA _A subunit βl gene include determining whether the person has one or more mutation(s) in the gene, or complete absence of the gene(s) encoding GABA _A subunit βl polypeptide, leading to altered expression of the polypeptide(s) or expression of functionally inactive versions of the polypeptide(s). By "gene" we include the coding region and the controlling region, e.g. the promoter, of the gene. Such genetic assay methods include the standard techniques of restriction fragment length polymorphism assays and PCR-based assays, as well as DNA sequencing.

The assay may involve any suitable method for identifying such polymorphisms, such as: sequencing of the DNA at one or more of the relevant positions; differential hybridisation of an oligonucleotide probe designed to hybridise at the relevant positions of either the wild-type or mutant sequence; denaturing gel electrophoresis following digestion with an appropriate restriction enzyme, preferably following amplification of the relevant DNA regions; Sl nuclease sequence analysis; non-. denaturing gel electrophoresis, preferably following amplification of the relevant DNA regions; conventional RFLP (restriction fragment length polymorphism) assays; selective DNA amplification using oligonucleotides which are matched for the wild-type sequence and unmatched for the mutant sequence or vice versa; or the selective introduction of a restriction site using a PCR (or similar) primer matched for the wild-type or mutant genotype, followed by a restriction digest. The assay may be indirect, ie capable of detecting a mutation at another position or gene which is known to be linked to one or more of the mutant positions. The probes and primers may be fragments of DNA isolated from nature or may be synthetic. The methods used to determine genotype(s) are well known to those skilled in the art.

An embodiment of the twelfth aspect of the invention is wherein a reduced amount of functional GABA _A subunit βl polypeptide, or if the person's GABAA subunit βl gene has one or more deleterious mutations, then the person is considered to be addicted to, or at risk of becoming addicted to, addictive substances. Preferably the addictive substance is alcohol. For example, if the person has a substitution of the leucine amino acid at position 310 (position 285 using the numbering of the mature polypeptide without signal sequence), for example a leucine to arginine substitution, in the GABA _A receptor subunit βl polypeptide, then the person may be considered to be addicted to, or at risk of becoming addicted to, alcohol.

Suitable samples that may be used in the methods of the twelfth aspect of the invention include those which contain representative samples of the patient's polypeptide and/or nucleic acid.

A thirteenth aspect of the invention provides a kit of parts useful for determining the likelihood that a person is, or may become, addicted to addictive substances comprising one or more agents useful in determining the amount and/or function of GABA _A subunit βl polypeptide and/or determining the genotype of the person's GABA _A subunit βl gene and, optionally, a positive and/or negative control.

It is preferred that the kit of parts comprises agents which can be used to determine whether the person has a substitution of the leucine amino acid at position 310 (position 285 using the numbering of the mature polypeptide without signal sequence), for example a leucine to arginine substitution, in the GABAA receptor subunit βl polypeptide.

Agents that can be used in this aspect of the invention to determine the amount and/or function of GABAA subunit βl polypeptide include antibodies or peptide or compounds that can bind to said polypeptides, as discussed above in relation to the screening methods of the invention.

Agents that can be used in this aspect of the invention to determine the amount of nucleic acid encoding GABAA subunit βl polypeptide include primers, oligonucleotides, or other nucleic acid molecules useful in PCR-based methods, northern blotting and in situ hybridisation methods set out above in relation to the screening methods of the invention.

Agents that can be used in this aspect of the invention to determine the genotype of GABA _A subunit βl gene include primers, oligonucleotides, or other nucleic acid molecules, as discussed above in relation to the tenth method of the invention.

A further embodiment of the thirteenth aspect of the invention comprises means for isolating protein and/or nucleic acid from a sample.

A fourteenth aspect of the invention provides the use of SCS or a salt or analogue or derivative thereof in the manufacture of a medicament for preventing or treating substance abuse.

A fifteenth aspect of the invention provides a method of preventing or treating substance abuse comprising administering to a patient an appropriate quantity of SCS or a salt or analogue or derivative thereof.

As discussed above, SCS (Salicylidene salicylhydrazide) specifically binds to GAB A _A receptors containing a βl subunit and, in this case, specifically inhibits a GABAA receptor containing the βl subunit (Thompson et al (2004) Br J Pharmacol 142, 97-106). Therefore the compound may be of use in preventing or treating substance abuse in a patient.

Salts which may be conveniently used in therapy include physiologically acceptable base salts, for example, derived from an appropriate base, such as an alkali metal (eg sodium), alkaline earth metal (eg magnesium) salts, ammonium and NX ₄ ⁺ (wherein

X is C] _-4 alkyl) salts. Physiologically acceptable acid salts include hydrochloride, sulphate, mesylate, besylate, phosphate and glutamate.

Salts of SCS may be prepared in conventional manner, for example by reaction of the parent compound with an appropriate base to form the corresponding base salt, or with an appropriate acid to form the corresponding acid salt.

By "analogue or derivative" of SCS we include structurally related compounds able to selectively inhibit GAB A _A receptors containing a βl subunit, for example those compounds mentioned in Thompson et al (2004) Br J Pharmacol 142, 97- 106.

SCS may also be supplied to a patient in the form of a prodrug. The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumour cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form (see, for example, D.E.V. Wilman "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions 14, 375-382 (615th Meeting, Belfast 1986) and VJ. Stella et al "Prodrugs: A Chemical Approach to Targeted Drug Delivery" Directed Drug Delivery R. Borchardt et al (ed.) pages 247-267 (Humana Press 1985)).

Methods of formulating and administering a compound are provided above in relation to the eleventh aspect of the invention.

By "substance abuse" we mean that the patient has an altered preference for an addictive substance, as mentioned above in relation to the first aspect of the invention. Preferably the addictive substance is alcohol.

A sixteenth aspect of the invention provides an antibody which binds to the GABAA receptor βl subunit and prevents the GABAA receptor binding alcohol. Such an antibody may be of use in the prevention or treatment of substance abuse.

An GABA _A receptor βl subunit polypeptide may be used as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.

Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a polypeptide, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference), and are well known to those skilled in the art.

Screening assays to determine binding specificity of such an antibody are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.

Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications" , J G R Hurrell (CRC Press, 1982). Such methods include the use of hybridomas, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

The antibody of this aspect of the invention may be humanised or human. Methods of producing humanised or human antibodies are provided above in relation to the screening methods of the invention.

A further aspect of the invention provides a recombinant nucleic acid encoding a GABA _A receptor βl subunit in which the leucine amino acid residue at position 310 (position 285 of the mature polypeptide ie without the signal peptide) of the GABA _A subunit βl polypeptide is substituted with an arginine residue. Preferences for preceding aspects of the invention apply as appropriate to the recombinant nucleic acid of the invention.

The invention will now be described in more detail, for the purposes of illustration only, in the following Examples and Figures.

Figure 1: The polypeptide and polynucleotide sequences of human and mouse GABA _A subunit βl polypeptides.

Figure 2: Consumption of 10% Ethanol in mutant GABA _A beta 1 L31 OR (L285R using the numbering of the mature polypeptide ie without the signal peptide) mutant and wild type of ALCO/22 line, in females of 3 ^rd backcross generation.

Figure 3: Maximal current induced by GABA after transfection of HEK293 cells with cDNAs for α2, βl and γ2S subunits or α2, βl ^L310R and γ2S subunits, together with the reporter gene. The L310R (L285R using the numbering of the mature polypeptide ie without the signal peptide) mutation caused a large reduction in the excitability compared to cells expressing wild-type.

Figure 4: Expression of GABA _A receptor subunit protein production at the surface of HEK 293 cells compared between βl ^L310R mutant (L285R using the numbering of the mature polypeptide) and wild type in relationship to co- expressed nicotinic acetylcholine receptor β4 subunit, used as a control. 1: 50 dilution.

Figure 5: Structure of SCS (Salicylidene salicylhydrazide).

Figure 6: Sucrose preference

Figure 7: Saccharin preference

Figure 8: Saccharin consumption a) before and b) after correction for body weight differences between wt and mutant ALCO/22 mice (see section 3, Fig 9)

Figure 9: Body weight of female (left) and male littermates (right) (G4 generation) .Bodyweight of ALCO/22 heterozygotes and wild-type littermates. The bodyweight of heterozygous female mice (n = 26) averages 16.1 g (S.D. = 1.6), compared to 23.7 g(S.D. = 2.6) for wild-type female littermates (n = 27), pO.0001. The bodyweight of heterozygous male mice (n = 34) averages 18.6 g (S.D. = 1.9), and 26.9 g (S.D. = 2.9)for wild- type male littermates (n=32), pO.0001. Data are taken from the fourth backcross generation. The variable "generation" did not have any effect on either preference or ethanol consumption.

Figure 10: Effect of Naltrexone treatment on relative ethanol preference. Groups are: 1 = before naltrexone 6 mg/kg; 2 = day 1 naltrexone 6 mg/kg; 3 = day 2 naltrexone 6 mg/kg; and 4 = after naltrexone treatment.

Figure 11: Effect of Naltrexone treatment on relative ethanol preference - increasing Naltrexone doses. Groups are: 1 = ethanol preference before naltrexone treatment; and 2 = ethanol preference after naltrexone treatment 1-25 mg/kg, 5 i.p. injections.

Figure 12: Effect of Naltrexone treatment on relative ethanol preference - Naltrexone administered after ethanol-free break. Phases are: 1 = ethanol preference (days 1-10) before Naltrexone treatment; 2 = day 16, after 1st Naltrexone treatment; 3 = day 17,after 2nd Naltrexone treatment; 4 = day 18, after 3rd Naltrexone treatment; 5 = day 19, after 4th Naltrexone treatment; 6 = day 19- 22, (Fri-Mon) after last Naltrexone treatment; and 7 = days 22-24 (Mon-Wed) after last Naltrexone treatment. Remark: between phase 1 and 2 (days 10-15, Wed-Mon) no ethanol.

Figure 13: Effect of Acamprosate (250 mg/kg) on Ethanol preference. Phases are: 1 = EtOH preference (10 days) before Acamprosate/saline treatment; 2 = EtOH preference during 4 days of daily i.p. injections of Acamprosate/saline (Mo- Fr); and 3 = EtOH preference after discontinuation of Acamprosate/saline i.p. (Fr- Wed).

Figure 14:Ethanol consumption influenced by 250 mg/kg Acamprosate. Phases are: 0 = before Acamprosate (10 days); 1 = after day 1 of acamprosate injection; 2 = after day 2 of acamprosate injection; 3 = after day 3 of acamprosate injection; 4 = after day 4 of acamprosate injection; 5 = days 5-8, no acamprosate injection; and 6 = days 8-10, no acamprosate injection.

Figure 15: Effect of Acamprosate and saline injection on absolute daily EtOH consumption of drinking ALCO/22 mutants and B6 mice. Statistical analysis of Effect of Acamprosate (after first injection) ethanol consumption of drinking ALCO22 and B6 mice. Result of two t-tests. ALCO/22 before A+S vs Acamprosate (effect of first injection): mean diff 9.17,

p<0.01 B6 before A vs B6+Acamprosate (effect of first injection) : mean diff , p< 0.01. Other comparisons: p>0.05.

Figure 16: Ethanol consumption (g/kg) and blood ethanol levels in C57BL/6 mice. Voluntary ethanol consumption (g/kg) and blood ethanol levels (mg/lOOml) for non-food-deprived mice (C57BL/6) with free access to ethanol at 2, 4, and 6 hr into the active (dark) phase of the circadian cycle.

Figure 17: Heterozygous ALCO/22 (+/Gabrbl ^L285R ) mice (numbering of mature polypeptide) show increased (a) ethanol preference and (b) absolute daily ethanol consumption (g ethanol per kg bodyweight) compared to wild-type (+/+). (c)

Although bodyweight was decreased, mice bearing the βi ^L285R mutation and exhibiting alcohol preference showed the same preference ofr sucrose (d) as their wild-type littermates. Data for a, b and c obtained from the fourth backcross generation.

Figure 18: Effects of βl ^L285R on GAGA _A R function in HEK cells, (a) The βi ^L2S5R mutation (•) increased the potency of low GABA concentrations but reduced the GABA response amplitude (Inset, black traces) compared to that for wild-type (□). The partial activation of mutant receptors in the absence of GABA was reversed by lOOμM CTB (grey traces; broken line shows zero current level), (b) Single channel currents recorded from outside-out patches of HEK293 cells expressing wild-type or mutant receptors at =70mV. The spontaneous activation of mutant receptors in the absence of GABA was blocked by CTB. Mutant channels opened less frequently in 10 μM GABA than wild-type, (c) Membrane trafficking of GABA _A R in HEK cells. The individual channels for the EGFP transfection marker (FITC- green) and βl ^myc (TRITC - red) are shown for each cell. Surface fluorescence appeared equal for wild-type and mutant, (d) No significant differences were observed in the sensitivities of wild-type and mutant receptors to 200 mM ethanol (EtOH) or lμM allopregnanolone (ALLOP) on ECi ₀ GABA responses (control responses = 100%).

Figure 19: (a) Mapping of the ENU-induced mutation to a region of mouse chromosome 5. The mutation was located between markers D5MU304 and D5MU356 (68.78 and 71.84 Mb), which includes the GABA _A receptor gene cluster α2, α4, βl and γ2, as well as the non-receptor tyrosine kinases, Tec and Txk. The GABAA receptor α2 and γl genes were exluded as sites for the mutation responsible for the altered phenotype, because of the absence of BAB/c DNA (the strain of the original ENU exposed male mouse) in one of the informative recombinants, at microsatellite marker D5MUSK2 (69.66 Mb). The single T to G mutation occurs in exon 8 of the β 1 subunit gene, (b) Electropherogram of the mutation T>G in exon 8 of the GABAA receptor βl subunit gene. The point mutations segregated with the phenotype (95% penetrance).

Figure 20: Amino acid sequence of βl subunit polypeptide in different species. The leucine at position 310 (position 285 of the mature protein, i.e. lacking the signal peptide).

Example 1: Mutation in the GABAA receptor beta 1 gene in an ENU mutagenised mouse line with significantly raised alcohol preference.

Abstract

Alcoholism is often familial: genetic factors account for 40-60 % of the risk ¹ . Inbred mouse strains consume different amounts of alcohol ² and complex trait studies have concluded that this is determined by several genes. We have used ENU mutagenesis to establish a mouse line with a heritable alcohol preference and have identified a single mutation in the GAB A _A receptor βl subunit, which is the cause of the changed phenotype. Functional analysis of the mutant GABAA receptor indicates that the mutation drastically reduces excitability of the receptor.

These experiments establish a causative role of altered GABA receptor function, in increased alcohol intake and establish the utility of ENU Mutagenesis in the dissection of the genetic components of this important and complex behavioral

phenotype ³ . The mouse mutant will be a valuable tool to study the role of GABAergic signaling in addiction.

Introduction

It is estimated that 14 million individuals (7.4 %) in the US abuse alcohol or are alcoholic ⁴ . In 1995 over 200,000 people in US died as a result of alcohol related diseases. In the UK, the Chief Medical Officer in his annual report of 2003, indicated that death from alcohol-related liver disease had increased 4-fold in young adults and is now the commonest cause of death in young men. Heavy drinking is also known to be a causal factor of various cancers, foetal damage during pregnancy, a range of other debilitating conditions and imparts a heavy social and economic burden. Current therapies appear to be only limited in their effectiveness.

The role of genetic factors in alcoholism is now well established ^l . Identification of the genes involved will be important in achieving a better understanding of the mechanisms resulting in alcohol addiction, allowing the identification of new targets for therapeutic intervention. To date, progress in identifying genes related to the risk for alcoholism in man, has been slow, due to the complexities of gene- environment interactions and the diversity of alcoholic families ⁵ .

Detailed genetic analysis of animal models is proving more fruitful. Mice from different inbred strains consume strain specific levels of alcohol in the presence of competing water and food ⁶ ' ⁷ . Most inbred lines dislike ethanol and will not voluntarily consume it. However, C57BL/6 mice have, when given a choice of water and alcohol, a preference for alcohol and for other addictive substances, including morphine ^8"10 and cocaine ^π . Preliminary mapping studies suggest that these individual preferences are independently inherited, indicating that the dependence phenotype in these animals is polygenic.

One approach to trying to identify the underlying genes in complex traits in mice utilizes Quantitative Trait Locus (QTL) mapping, and such mapping in inbred strains is now yielding candidate loci mediating alcohol consumption/preference, withdrawal and sensitivity in mice. QTLs for alcohol consumption/preference have been independently confirmed for regions of mouse chromosomes 1 , 2 and 9 ⁵ . hi addition, approximately 70% of the genetic variance that mediates acute alcohol withdrawal has been mapped to three QTLs on mouse chromosomes 1, 4 and 11 ¹² . However, identification and testing of candidate genes underlying these QTLs may take several years ¹³ , as the step from QTL mapping to QTL identification is far from routine.

Recently, experimental animal models and novel genetic manipulations have provided clues leading to several genes involved in alcoholism ^14'16 . Moreover, the effect on the alcohol related behaviour of a few genes (GABAA receptor subunits αl, β2 ¹⁷ and α2, α5, α6, β3, γ2S and γ2L, δ ¹⁸ , protein kinase C ¹⁹ and the μ- opioid receptor ) ²⁰ has been demonstrated in knock-out mice, and the cannabinoid receptor system has to be shown to play an important role in regulating the positive reinforcing properties of alcohol .

While these knockout (KO) lines highlight the potential target genes for the development of new drugs in the therapy of alcohol abuse/addiction, they are less suitable to serve as a model to test new drugs designed to reduce alcohol intake, since the effect of the KO is to reduce alcohol intake in the high ethanol intake C57BL/6 strain, where other multiple QTL loci may still contribute to the phenotype. In addition, KO have the disadvantage of giving little information about the nature of the molecular interactions involved in the signaling pathway.

In contrast, the N-ethyl-N-nitrosurea (ENU) mutagenesis program developed at the MRC Mammalian Genetics Unit (MGU) ²² is a non-hypothesis driven approach in which ENU, at a calculated dose, introduces random point mutations into the mouse genome, at a low density, allowing the identification of mice with a heritable preference for alcohol. Mouse genomics are then used in back-cross

experiments to identify the mutations causing the altered phenotype. As part of this program, a mouse line, derived from BALB/cAnN and C3H/HeJ matings, both of which usually exhibit a preference for water rather than alcohol, has been identified that displays a high preference for alcohol. In this line, which we called ALCO/22, 50% of the mice (heterozygote offspring) take around 70-80 % of their total fluid intake, which is measured over two 10 day periods, as 10 % ethanol (Figure 2). This phenotype is inherited in an autosomal dominant pattern with high penetrance (80-90%), and has been followed so far over 7 generations of back- crosses.

Further details concerning identification and characterisation of the ALCO/22 mice are also provided in Examples 3 and 4.

Results

Identified mutant and phenotype

Identified mutant ALCO22 (EMRC184.2e; IVF268(M) (Fl original mutant) consumes by choice around 35% of its fluid intake from the bottle containing 10% ethanol. G2 females first backcross to mouse strain C3H in general consume either between about 65% and 80% of their fluid intake from the bottle containing 10% ethanol in the heterozygote mutant, or between 20 and 30% of their fluid intake from the bottle containing 10% ethanol in the wild type. The mutation is apparently dominant as there is an "addiction" phenotype in heterozygous mutant animals.

Animals of the ALCO/22 line have a 20 % lower weight and size compared to their littermates. Histology has revealed that the females have underdeveloped ovaries, indicating further consequences of the mutation in the GABAergic system, which also involves the synthesis of hormones in the hypothalamus.

No histological or biochemical effect (raised ALT) after 3 months continuous consumption of 80 % of (10%) ethanol.

Genotype ofALCO/22

BALB/cAnN x C3H backcross, ENU mutation T to G at nucleotide 95 of exon 8 of the GABAA receptor βl subunit gene, resulting in a leucine to arginine change at amino acid position 285 of the mature protein (aa 310 in protein including signal peptide: NP_032095; GL6679907).

Cloning of the ALCO/22 mutation

We have mapped the site of the ENU-induced mutation to a region of mouse chromosome 5 (human ortholog: chromosome 4) between markers D5Mit304 and D5Mit356 (69.58 and 73.17 Mb). It is in this interval that the genes for the 4 GABA receptor subunits γl, α2, α4 and βl, reside. After excluding the GABA _A receptor γl and α2 gene as candidate genes because of the absence of BALB/cAnN DNA in one of the two informative recombinants, we subsequently sequenced the exons and exon/intron borders of GABAA receptor βl and α4 ₅ of ALCO/22, in comparison with the parental mouse strains BALB/cAnN and C3H/HeJ. This revealed a single T to G base mutation at position 94 of exon 8 in the GABAA receptor βl gene, resulting in a leucine to arginine change at amino acid position 310 of the protein (L310R), including the signal peptide (L285R excluding the signal peptide). This change is not found in any other mouse strains for which sequence data is available. The GABA _A receptor βl gene is extremely well conserved between species. Only 5 amino acid changes are to be found between mouse and human, indicating that most regions of this subunit of the GABAA receptor are either intolerant of change or of high functional/structural significance.

Functional analysis

In order to study the functional consequences of the L310R (L285R using the numbering of the mature polypeptide) mutation in GABA _A receptor βl, HEK293 cells were transfected with cDNAs for α2, βl and γ2S subunits or α2, βi ^L310R and γ2S subunits, together with the reporter gene, enhanced GFP. Whole-cell patch clamp recordings were made under standard conditions. The L310R mutation

caused a large reduction in the maximal current induced by GABA compared to cells expressing wild-type receptors (Figure 3).

Further preliminary observations using confocal microscopy, suggest that the reduction in current amplitude is unlikely to be due to a reduction in the amount of GABAA receptor subunit protein production at the cell surface: the level of cell surface expression in HEK cells of the βl ^L310R mutant (Figure 4) was indistinguishable from the expression of βl wild-type subunits when compared with co-expressed nicotinic acetylcholine receptor β4 subunit, used as a control.

Therefore, the reduced responses to GABA are likely to reflect one or more of the following:

- a reduced sensitivity of the GAB AA receptor to GABA; - a disruption of subunit folding and aberrant receptor assembly insufficient to prevent trafficking;

- impaired/altered ion channel gating.

Discussion

Mutation in GABAA receptors

GABA _A receptors are Cl ^' permeable ligand-gated ion channels that mediate the majority of fast synaptic inhibition in the CNS (Moss and Smart (2001) Nat Rev Neurosci 2, 240-250; Sieghart, (1995) Pharmacological Reviews 47, 181-234). These receptors are also targets for several clinically important drug classes, including: benzodiazepines, barbiturates, general anaesthetics as Well as ethanol (Sieghart, supra; Korpi et al (2002) Prog Neurobiol 67, 113-159). GABA _A receptors are believed to be pentameric hetero -oligomers constructed from a number of subunit classes, including: α (1-6), β (1-3), γ (1-3), δ, ε, θ and π; with the p 1-3 subunits forming homomers often referred to as GABAc receptors (Sieghart, supra; Rabow et al (1996) Synapse 21, 189-274; Davies et al (1997) Nature 385, 820-823; Hedblom et al (1997) J Biol Chem 272, 15346-15350;

Bonnert et al (1999) Proc Natl Acad Sci USA, 96, 9891-9896). However, in vivo, the majority of receptor subtypes are believed to be composed of α, β and γ subunits in a 2:2:1 ratio (Changet al (1996) Journal of Neuroscience 16: 5415-24; Tretter et al ^' (1997) Journal of Neuroscience 17: 2728-37; Farrar et al (1999) J.Biol.Chem 274: 10100-04). Each subunit is composed of external N- and C- termini encompassing 4 TMs (TM 1-4) of which TM2 lines the ion channel. One external linker region exists between TM2-3 - near the site of our mutation - and two internal domains connect TM 1-2 and TM3-4, respectively (Sieghart, supra).

Using in vitro studies, the consensus view is that acute exposure to alcohol can potentiate GABA _A receptor function over the concentration range 10-20OmM (Davies et al (2003) J Psychiatry Neurosci 28, 263-274; MiMc et al (1994) EurJ.Pharmacol 268: 209-14; Grobin et al (1998) Psychopharmacology (Berl) 139: 2-19; Aguayo et al (2002) Curr.Top.Med.Chem 2: 869-85). Although still controversial, this action of ethanol has not been shown to be clearly dependent upon the receptor subunit composition (Mihic supra; Harris et al (1998) J.Pharmacol.Exp.Ther 284: 180-88; Kurata et al (1993) Brain Res. 631: 143-46; Marszalec et al (1994) J.Pharmacol.Exp.Ther. 269: 157-63); however, more recently, α4β2δ, α4β3δ and α6β3δ have been demonstrated to be the most sensitive (l-5mM) GABA _A receptors to ethanol (Wallner et al (2003) Proc.Natl.Acad.Sci.U.S.A; 100: 15218-23; Sundstrom-Poromaa et al (2002) NatNeurosci. 5: 721-22). The potentiation is achieved by ethanol increasing the frequency and mean duration of GABA channel opening (Tatebayashi et al (1998) NeuroReport 9, 1769-1775) whilst the phosphorylation status (possibly via protein kinase C) of the γ2L subunit may also be influential to the effect of ethanol (Homanics et al (1999) Neuropharmacology 38: 253-65; Wafford et al (1991) Neuron 7: 27-33; Wafford et al (1992) FEBS Lett. 313: 113-17; Sigel et al (1993) FEBS Lett.324: 140-42; Harris et al (1995) Proc.Natl.Acad.Sci.U.S.A 92: 3658- 62).

To date, several residues have been identified on GABAA receptors as being vital for the action of ethanol. These include: S265 on βl and S270 and A291 on α

subunits; residues that are located on TM2 and TM3. Currently, S270 is thought to form part of the alcohol binding site following cysteine mutagenesis and covalent labelling with sulphydryl-based chemical probes (Mascia et al (2000) Proc Natl Acad Sci USA 97, 9305-9310) with the other residues possibly participating in signal transduction. Particular GABA _A receptor subunits that mediate the effect of ethanol are not well defined. Using subunit knock-out strategies, both γ2L and ct6 subunits appear not to be very important (Homanics 1997, 1999 supra), whereas, the δ subunit knock-out caused mice to imbibe less ethanol (Mihalek et al (2001) Alcohol Clin Exp Res 25, 1708-1718). Although the TM2 and TM3 domains appear important for alcohol and anaesthetic action on the GABAA receptor, the role of the TM2-TM3 linker has not been considered as a vital domain for ethanol induced potentiation of GABA-activated currents, making the position of the ENU mutation at the top of TM3, of considerable interest.

The L310 (L285R using the numbering of the mature polypeptide) mutation in the gene encoding the GABA _A receptor βl subunit of the ALCO/22 strain of mice is vicinal to residues in TM3 which have been found to be essential for GABA activation of the receptor (Hosie, Thomas & Smart, unpublished observations). However, L310R also lies close to the TM2-TM3 loop, which is the sole point of contact between the transmembrane domains and the N-terminal GABA-binding region and also key to receptor activation ²³ . Mutations at this point of contact in the closely related nicotinic acetylcholine receptor may also adversely affect gating, protein folding and surface expression ⁴ . In preliminary studies the GABA sensitivity of the β subunit mutant GABA _A receptors appeared unchanged when compared to their wild-type counterparts, suggesting a possible disruption of protein folding rather than signal transduction.

Non-obvious GABAA βl association:

Twin and adoption studies indicate that alcohol dependence is, at least in part, genetically determined. In one micro-satellite linkage study, using over 300 markers, linkage was established with loci on chromosomes 1 and 4. When alcoholism as a quantitative trait was analysed, evidence for genetic linkage with a

region on chromosome 4 was revealed (Williams et al. (1999) Am. J. Hum. Genet. 65: 1148-60; Reich et al.(1998) Am. J. Med. Genet. 81 : 207-15). In subsequent studies, people who need to consume large amounts of alcohol to obtain a euphoric state, were found to be at high risk of becoming alcoholic and twin studies of these individuals demonstrated that this trait is in linkage with a region on chromosome 1 (Schuckit et al. (2001) Alcohol Clin. Exp. Res. 25: 323-29). Alcohol-dependent people suffering from depression have again been shown in twin studies to exhibit a linkage on chromosome 1 (Nurnberger et al. (2001) Am. J. Psychiatry 158: 718-24). The analysis in those individuals using significant amounts of alcohol without developing alcohol-related problems, indicated linkage disequilibrium with a marker on chromosome 4, close to the gene for alcohol dehydrogenase, an enzyme involved in alcohol metabolism (Reich, 1999 supra). Finally the alcoholism trait defined by the tendency to take a large amount of alcohol within a 24-hour period, was linked to a separate locus on chromosome 4 (Saccone et al (2000) Am. J. Med. Genet. 96: 632-37).

Several electrophysiological measures are altered in people with alcoholism (Porjesz et al (1998) J. Clin. Neurophysiol. 15: 44-57) and have been used to define a phenotype. The ERP brain wave (P300) is smaller in size and the EEG beta activity is increased in alcoholics, consistent with alterations to neurotransmission. These phenotypes are again genetically determined with linkage to a region on chromosome 4 (Williams et al 1999, supra; Almasy et al. (2001) Am. J. Hum. Genet. 68: 128-35), and linked to a microsatellite marker in the GABA _A receptor βl gene in another study (Porjesz et al. (2002) Proc. Natl. Acad. Sci. U.S.A 99: 3729-33). However the authors of Porjesz et al. concluded that the GABAA α2 subunit is the most likely candidate for the observed linkage disequilibrium findings.

Taken overall, these studies suggest the involvement of genetic loci on chromosomes 1 and 4 in the aetiology of some types of alcohol dependency.

Chromosome 4 is of particular interest since several genes encoding for the

GABA _A receptor subunits, α2, α4, βl and γl, are located there (Whiting et al

(1995) International Review of Neurobiology 38: 95-138). Allele association studies in the COGA cohort of patients, have also recently suggested a role for altered transcription of the α2 subunit in alcohol addiction (Edenberg HJ et al. (2004) Am. J. Hum, Genet. 74: 705-14).

However, none of these documents suggest that the GABA _A subunit β 1 may have a role in mediating a preference for any addictive substances, including alcohol.

In addition, these allele association studies might indicate altered regulation of other genes within the region. It has been demonstrated that chronic exposure of cortical neurons (in culture) to GABA decreases αl, βl, β2, and γ2 subunit mRNA levels as well as βl promoter activity (Russek et al (2000) see below). Whilst no suggestion is made herein as to how far this finding can be applied to alcohol dependence /alcoholism, it is possible that the assumed regulatory functions within the GABA _A receptor α2 subunit might impose a regulatory effect on the regulation of the promoter region of the GABA _A receptor betal subunit. Alternatively, altered regulation of GABAA receptor β 1 subunit might have a regulatory effect of the GABAA receptor cc2 subunit. In fact, in the association study with positive association for the GABA _A receptor subunit α2, did not look for associations with variations with the promoter region of the GABAA receptor β 1 subunit. This might be the region which will give a positive association with alcoholism in human studies. Alternatively, the intergenic region might contain regulatory elements that directly influence the transcriptional level of both loci (Steiger et al (2004) see below). The fact that the genes encoding the GABA _A receptor subunit α2 and βl are oriented in opposite directions on the chromosome might be in favour of this hypothesis.

Distribution of GABA _A receptor subunits

The distribution of the 13 major subunits of the GABA _A receptor complex have been studied in the rat brain (Schwarzer et al (2001) J Comp Neurol 433, 526- 549). The GABA _A βl subunit is not as widely distributed as the, αl, α2 and β2

subunits, but observed in most hypothalamic areas (amygdala, thalamic nucleus, hippocampus) (Pirker et al (2000) Neuroscience 101, 815-850); it might be possible to specifically target those areas of the brain expressing the βl subunit in order to treat alcohol dependence/alcoholism. Furthermore, the mutation in the GABA _A βl subunit at position 310 (285 of the mature polypeptide) might have consequences for the distribution of the βl subunit and its associations with other . subunits.

Li et al (1997) J Biol Chem 272, 16564-16569 report the coexistence of two β subunit isoforms in the same GABA _A receptor. Using quantitative immunoprecipitation of solubilized GABA receptors from various rat brain regions, they showed that βl is present in a very small proportion (3%) of cerebellar GABA _A receptors, and at a high proportion (49%) of hippocarnpal and

(32%) cerebral cortex GABAA receptors. However, these numbers may not be that reliable.

The authors also show that 33% of GABA receptors may have both β2 and β3 subunits; 19% of GABA receptors may have both βl and β3 subunits. Co- localization of β subunit isoforms seems to be highest in the hippocampus and lowest in the cerebellum.

Pirker et al. (2000) Neuroscience 101, 815-850 report the immunocytochemical distribution of 13 subunits of GABAA receptors in the adult rat brain. They show that βl immunoreactivity is found throughout brain, although there are differences in tissue distribution in hypothalamic areas, for example this subunit is more concentrated in neostriatum than in pallidum and entopeduncular nucleus. Moreover, βl is highly concentrated in the dendrites of principal cells, in intemeurons, the CA2 region of the hippocampus and the granulular layer of the cerebellum. There is thus a differential immunocytological distribution.

Schwarzer et al (2001) J Comparative Neurology 433, 526-549 reported the distribution of the major GABAA receptor subunits in the basal ganglia and

associated limbic brain areas of the adult rat. They found a heterogeneous distribution of individual GABA _A receptor subunits, with a predominance of βl subunits in the striatum and nucleus accumbens, olfactory tubercle and globus pallidus. They stated that the highly heterogeneous distribution of individual GABA _A receptor subunits suggested the existence of differently assembled, and possibly functionally different, GABAA receptors within individual nuclei of the basal ganglia and associated limbic brain areas.

Malatynska et al (2001) Alcohol and Alcoholism 36, 309-313 reported that the subunit composition of GABAA receptors in some neurons in the cerebral cortex is αl β2 γ2; while in some neurons of the hippocampus, α2,3,5 βl γ2 predominate.

Mohler H, et al (2004) Biochemical Pharmacology 68, 1685-1690 report specific GABA _A receptor based neuronal circuits in brain development and therapy. They show that a α3(5)βl,3 γ2 receptor is a minor subtype (10-15%) in the cerebral cortex, hippocampus and olfactory bulb, possibly residing extrasynaptically.

Alcohol addiction

There is a limited pharmacopoeia for the treatment of alcoholism. The leading anti-addiction drug is calcium acetylhomotaurine (Acamprosate; Campral). It has a chemical structure similar to that of the amino acid neurotransmitters, GABA and glutamate, and evidence suggests that it acts by stabilising the 'network imbalance', which is seen in alcohol dependency. In three trials, Acamprosate has been shown to be significantly more effective than placebo at maintaining abstinence, increasing the time to first relapse and increasing the total cumulative period of abstinence in alcohol dependent patients over a one year treatment period ²⁵ . Among many other potential targets for alcohol, its interaction with the opioid system is considered indirect, resulting in the eventual activation of the opioid system ²⁶ . This is associated with reinforcing effects (probably via μ opioid receptors) and aversive effects (most likely via K receptors) ²⁷ . The opioid system is also involved in the craving for alcohol, and opioid ^• antagonists, such as naltrexone, block the rewarding effects and craving for alcohol. However there is

increasing evidence that this antagonist has only limited efficacy. Apart from this, the older and more unpleasant treatment with the aldehyde dehydrogenase inhibitor, disulfiram, remains.

Drug addiction

Alcoholics and illicit drug users often consume a wide variety of drugs (Hubbard et al (1989) Drug Abuse Treatment: a national study of effectiveness (North Carolina, University of North Carolina Press; Hammersley et al (1990) Br J Addict 85, 1583-1594; Ball & Ross (1991) The Effectiveness of Methadone Maintenance Treatment (New York, Springer- Verlag). For example, estimates indicate that 50, 33, 47 and 69% of heroin addicts applying for methadone treatment in the United States are regular users of alcohol, benzodiazepines, cocaine and marijuana, respectively (Ball & Ross, supra). Prevalence of marijuana use among cocaine and alcohol abusers ranges from 25 to 70% (Miller, Gold & Pottash, 1989; Hubbard, 1990; Higgins et al, 1991; Schmitz et al, 1991). Polydrug abuse presents a range of problems to treatment and public health initiatives.

• One possibility is that these various addictions are caused by a relatively large number of genes, such that it is extremely difficult to identify the individual genes involved. The ALCO/22 mouse model with its single point mutation in the GABAA receptor βl subunit is the ideal tool to test whether or not this mutation is also responsible in mediating preference for other drugs like cocaine or opiates (morphine).

Since the activation of the opioid system (Weiss supra) is associated with reinforcing effects (probably via μ opioid receptors) and aversive effects (most likely via K receptors) (Laviolette et al (2004) Nat Neurosci 7, 160-169), and the craving for alcohol, opioid antagonists, such as naltrexone, block the rewarding effects and craving for alcohol. Because of the interaction of the opioid system, GABA and ethanol, it is not surprising that a mutation in one of the genes

involved in this pathway might result in preference for alcohol and opiates (such as morphine) at the same time.

Lack of mouse models The data obtained from allele association studies in man have already focused attention on the role of the GABAergic system in alcoholism.

The crucial role of the two predominant (αl and β2) subunits of GABA _A receptor in hypnotic drug action has been established by studying mice lacking these two subunits. Results show that removal of either αl or β2 subunits of GABA _A receptors produce strong and selective decreases in hypnotic effects of different drugs. Overall, these data confirm the crucial role of the GABA _A receptor in mechanisms mediating sedative/hypnotic effects ^2S .

However, the complete lack of entire GABA _A receptor subunits in the models available so far does not allow the study of the specific mechanism which results in the GABA mediated effect on ethanol consumption, and therefore is not sufficient to identify possible new drug targets for the treatment of alcohol addiction.

In contrast, the monogenic mouse model of 'alcoholism' described here is the first illustration that a single mutation in the GABA receptor is sufficient to alter the alcohol preference of an animal. This model deserves further study to determine the mechanism that has produced this altered phenotype, and might be a valuable tool to identify new drug targets.

Moreover, it has been demonstrated that GABA _A receptor βl subunit could be used as a specific target for drugs, as the compound SCS (Salicylidene salicylhydrazide) specifically binds to the beta subunit and, in this case, specifically inhibits the function of a GABAA receptor containing the βl subunit (Thompson et al (2004) Br J Pharmacol 142, 97-106).

Summary

These studies will lead to the identification of new genes and pathways involved in addiction and improve our understanding of the genetic components of alcohol dependence/abuse and addiction. The results may suggest novel diagnostic and therapeutic targets in man to cure genetically based alcohol dependence/addiction. Since the currently available drugs are predominantly effective in preventing relapse, and naltrexone does not reduce the rate of relapse or alcohol consumption in patients with alcoholism (Krystal et al (2001) N Eng J Med 345, 1734-1739) and have also some remarkable side effects (Oscar et al (2003) Therapie 58, 371- 374) there is a need to develop new and better drugs to cure humans from alcohol abuse and addiction. The above described new mouse mutant might contribute to this aim.

Further observations

1. GABAA βl subunit as a potential drug target in influencing alcohol intake On the basis of the above described findings, and the fact that the GABAA-P 1 subunit has not previously been shown as influencing alcohol related behaviour, we propose the GABA _A -P 1 subunit as a potential new drug target in influencing alcohol intake. Whereas the influence of other GABA _A - subunits αl, αl, α5, α6, β2, β2, γ2S, γ2L and δ, has been shown on alcohol related behaviour in knock-out and/or transgenic mouse models , there is currently no animal model showing the influence of the βl subunit on alcohol related behaviour.

2, Functional consequences of the L310R mutation in the GABAA-P 1 subunit Electrophysiological data show the diminished membrane conductance of HEK293 cells expressing the mutated GABA _A -βl subunit in combination with α2, and γ2S subunits. This may mean:

a) A possible disruption of protein folding rather than signal transduction

b) A change in the ability of the βl subunit to associate with the usual subunits (most likely α2 and γ2), interrupting the allosteric interactions between subunits, leading to altered composition of the GABA _A receptor in the brain. c) The distribution of the 13 major subunits of the GABA _A receptor complex have been studied in the rat brain ²⁹ . The GABA _A βl subunit is not as widely distributed as the, ocl, oc2 and β2 subunits, but observed in most hypothalamic areas ³⁰ . Therefore it might be possible to specifically target those areas of the brain expressing the βl subunit in order to treat alcohol dependence/alcoholism. Furthermore, the mutation in the GABA _A βl subunit at position 310 (285 of the mature polypeptide ie without the signal sequence) might have developmental consequences for the distribution of the βl subunit and its associations with other subunits (see ³¹ ). Those effects can be studied using, e.g. immunocytochemistry and in vitro electrophysiology with selective pharmacological probes.

3. ENU mutagenesis generated mouse line with significantly increased alcohol preference

The ALCO/22 mouse line generated by ENU mutagenesis as described here is unique. No one before has used this method to establish a mouse line with a heritable alcohol preference with an identified single mutation in the GABA _A receptor βl subunit, which is the cause of the changed alcohol preference phenotype.

4. Drug selectivity for the GABA _A -βl subunit

The compound salicylidene salicylhydrazide (SCS) binds selectively to the GABA _A -betal subunit ⁷⁵ . Therefore it is expected that analogs of the compound SCS will also bind specifically to the GABAA βl subunit and because the role of the GABAA βl in mediating alcohol preference, such GABA _A βl specific compounds will influence alcohol intake.

5. Mouse as in vivo model to test drugs for treatment of alcohol related behaviour

Opioid receptors ³² and the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor (for review ref ³³ ) are involved in the rewarding (reinforcing) effect of addictive drugs, including alcohol. Opioid antagonists reduce consumption and self-administration of ethanol in animals , and the opioid receptor antagonist Naltrexone has been shown to reduce the alcohol intake in the high ethanol preferring C57/BL6 mice, from 60% to 40-50% ³⁴ . By contrast, μ-receptor knockout mice cannot be induced to self-administer alcohol (Spanagel R, supra).

The NMDA receptor antagonist Acamprosate has been shown to reduce the ethanol intake of high drinking mice ³⁵ ' ³⁶ .

The effect of Acamprosate and Naltrexone on the volume of 10% alcohol consumed by Alco22 mice will be determined in cross-over experiments, over a period of 4 weeks.

It is expected that the ethanol intake of the ALCO/22 high drinking mutants will be reduced by drugs as Naltrexone and Acamprosate, and that the ALCO/22 mouse line can be used as a model to test the effectiveness of other new drugs for the treatment of alcoholism.

6. Opiates, cocaine, and benzodiazepines

Alcoholics and illicit drug users often consume a wide variety of drugs (Hubbard et ω/. ,1989; ³⁷ ; Ball & Ross, 1991). For example, estimates indicate that 50, 33, 47 and 69% of heroin addicts applying for methadone treatment in the United States are regular users of alcohol, benzodiazepines, cocaine and marijuana, respectively (Ball & Ross, 1991). Prevalence of marijuana use among cocaine and alcohol abusers ranges from 25 to 70% (Miller, Gold & Pottash, 1989; Hubbard, 1990; Higgins et al, 1991; Schmitz et α/.,1991). Polydrug abuse presents a range of problems to treatment and public health initiatives.

One possibility is that these various addictions are caused by a relatively large number of genes, such that it is extremely difficult to identify the individual genes involved.

The activation of the opioid system ²⁶ is associated with reinforcing effects (probably via μ opioid receptors) and aversive effects (most likely via K receptors) ²⁷ , and the craving for alcohol, opioid antagonists, such as Naltrexone, block the rewarding effects and craving for alcohol. Because of the interaction of the opioid and GABA systems with ethanol, it is not surprising that a mutation in one of the genes involved in this pathway might result in a preference for alcohol and opiates (such as morphine) at the same time.

The ALCO/22 mouse model with its single point mutation in the GABAA receptor βl subunit is the ideal tool to test whether or not this mutation is also responsible in mediating preference for other drugs like cocaine or opiates (morphine). The inventors predict that the single point mutation in the GABAA receptor βl subunit in ALCO/22 also will have an effect on the preference for other drugs like cocaine or opiates (morphine), and benzodiazepines.

7. Change (LSlOR) in the GABAA-βl receptor could alter sensitivity to anaesthetics

The mutation (L310R; L285R using the numbering of the mature polypeptide ie without the signal peptide) in the GABA _A -βl receptor, lies close to a residue (asparagine 289 in β2) in the β2 and β3 subunits that influences the sensitivity of GABAA receptors to intravenous general anaesthetics. Only those GABA _A receptors containing β2 or β3 subunits are modulated by I/V anaesthetics such as etomidate (Belelli et al 1997, PNAS ₅ 94, 11031-11036). βl subunit-containing receptors require replacement of the homologous serine residue with an asparagine to exhibit sensitivity to I/V anaesthetics. How this will be affected by L31 OR, if at all, remains to be seen.

8. HEK293 cell line

The HEK293 cell line transfected with different combination of subunits of the GABA _A receptor can be used to study further effects of the function of GABA _A receptor as response to the L310R (L285R using the numbering of the mature polypeptide) mutation. This cell line is used widely for such purposes.

9. EEG β activity (in human alcoholism) linked to GABA _A -betal receptor gene

Several electrophysiological measures are altered in people with alcoholism ³ and have been used to define a phenotype. The ERP brain wave (P300) is smaller in size and the EEG beta activity is increased in alcoholics, consistent with alterations to neurotransmission. These phenotypes are again genetically determined with linkage to a region on chromosome 4 ³⁹ ' °, and linked to a micro satellite marker in the human GABA _A receptor β 1 gene in another study ⁴¹ .

In rats, the EEG beta activity has been shown to increase after exposure to ethanol, as compared to EEG-changes in abstinent alcoholics (Bergschicker et al, Biomed Biochim Acta 1988).

The EEG beta activity in the ALCO22 mutant mouse brain can be compared to the wild type brain. It is expected that the EEG beta activity will be altered in mice with the mutant L310R (L285R using the numbering of the mature polypeptide ie without the signal peptide) mutation, reflecting the changes characteristic for human alcoholics.

10. Mutations in the human GABA _A -βl receptor gene

Because of the effect of the point mutation in the GABAA receptor βl gene on the alcohol drinking behaviour of the mutant mouse, variations in the transcription of the human GABA _A receptor βl gene, its regulatory regions or its sensitivity to ligands, are expected to increase the risk for alcohol dependence in humans.

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Methods

Mice. BALB/cAnN mice were exposed twice weekly to 100mg/kg of the mutagen N-ethyl-N-nitrosurea (ENU) and backcrossed to female C3H/HeJ mice (http://www.mut.har.mrc.ac.uk). Mice were given free access to food and water and kept under 12 hrs light/12 hrs dark with artificial light from 7 a.m to 7 p.m, and standard conditions and procedures as approved by the local home office.

Phenotyping. 7-10 week old mice from first generation backcrosses (BALB/c AnN mut x C3H/HeJ), or subsequent backcrosses, were housed in single cages and offered a choice of water and 10 % v/v ethanol in water, from two bottles, randomly positioned in the cage. The consumption of water and ethanol was measured over two periods of 10 days and the ethanol consumption calculated as the ratio of ethanol over the total of liquid consumed. The weights of the animals were recorded at the start of each 10 day period.

Mapping. For the initial mapping DNA was extracted from tail tips of 13 affected mice with high ethanol preference (2 G2, 11 G3), originating from the same founder with the desired phenotype, using a nucleon kit, and tested in an initial Genome Scan Mapping procedure using 86 fluorescently labeled microsatellite markers which are polymorphic between BALB/cAnN and C3H/HeJ. The initial mapping revealed a region of common BALB/cAnN DNA between D5Mitl5 (64.45Mb) and D5MitlO (101.3 Mb) on mouse chromosome 5. Subsequently, in order to narrow down the region of BALB/cAnN DNA originating from the ENU mutagenesis event and carrying the point mutation which possibly leads to the altered phonotype, we tested a further 169 animals/ 82 high drinking mice for the presence of BALB/cAnN DNA in this region, using eleven further polymorphic markers between D5Mit394 and D5MitlO. Amongst those we found 8 recombinants in the interval between D5Mitl5 and D5MitlO, two of them eventually narrowing down the region of common BALB/cAnN DNA to an interval of 69.23 Mb and 72.28 Mb ₅ between markers D5Mit304 and D5Mit356. For fine mapping and identification of informative recombinants, DNA was extracted from ear biopsies by incubation in a proteinase K containing lysis buffer (5OmM TrisHCL, pH 8.5, ImM EDTA, 0.5%Tween 20, 150-300 μg/ml proteinase K ) for 4-12 hours at 55 ⁰ C, and denatured for 12 min at 95 ⁰ C. In addition extra polymorphic markers were placed between D5Mit304 and D5Mit355 and between D5Mitl34 and D5Mit356 in order to narrow down the region of candidate genes on mouse chromosome 5. Size differences for the fragments of the additional polymorphic markers were between 4-20 bp and separated on 4% agarose gels (AGTC).

Sequencing. The two main candidate genes in the mutant were eventually sequenced in comparison with the two parental strains. Using intronic primers (Sigma Genosys) we amplified the 10 coding exons including intron/exon borders from genomic DNA of GABAA receptor beta 1 (and also the 10 coding exons of gene of GABAA receptor alpha4) from the ALCO/22 mutant and control strains BALB/cAnN and C3F£/HeJ. PCR products were subjected to gel purification using the Wizard kit (Promega). Products were sequenced on a 3700 ABI Sequencer

using "Big Dye" chemistry (Applied Biosystems) and analyzed with BioEdit software. Primer sequences are available on request. Sequences were derived from the information of ENSMUST00000031122 in "Ensembl", version 32.

Functional Studies. Calcium phosphate co -precipitation was used to transfect HEK293 cells with human α2, murine βl and γ2S subunits and EGFP. Whole-cell patch clamp recordings were made under standard conditions At present, no functional differences between human and murine GABAA receptors have been reported, so we feel a combination of human and murine subunits is justifiable for a pilot study. Whole-cell patch clamp recordings were made under standard conditions (membrane potential clamped at -40 mV). Modulators were co-applied with a GABA concentration that elicits 10% of the maximal GABA response (EC ₁₀ ).

Confocal microscopy. HEK293 cells were fixed in phosphate-buffered saline (PBS) with 4% paraformaldehyde before being quenched with 5OmM NH ₄ Cl in PBS. Cells were permeabilised using 0.5% Triton X in PBS containing 10% foetal calf serum (FCS), 0.4% bovine serum albumin (BSA). Cells were incubated at room temperature with primary anti-βl/3 subunit antibody for 45 min, washed in FCS/BSA in PBS solution before incubation for 45 min with a TPJTC-coηjugated secondary antibody conjugate or an antibody conjugate mixture. Cells were thoroughly washed and mounted in glycerol. Mounted slides were viewed using an upright Zeiss Axiophot microscope and confocal images were obtained from a Zeiss LSM 510 Meta laser-scanning confocal microscope.

Antibodies. A rabbit polyclonal anti βl/3 antibody was used and was obtained as a gift from Dr Werner Sighart, Univ Vienna (described in J. Neurosci 17, (1997) 2728-2737).

GenBank accession numbers. Mouse GABAA receptor βl protein ^' NP_032095; GL6679907.

Example 2: A screen for compounds which can modulate GABAA receptor βl subunit activity

Thompson et al (2004) Br J Pharmacol 142, 97-106 describe a screen of a large library of compounds to identify novel GABA _A subtype-selective compounds. A similar approach may be taken to identify compounds capable of modulating GABAA receptor βl subunit activity.

Primary screening data can be generated using a Voltage/Ion Probe Reader assay (VIPR _T M; Aurora Biosciences, CA, U.S.A.) as described in (Adkins et al., 2001 J Biol Chem 276, 38934-38939; Smith & Simpson, 2003 Anal Bioanal Chem 377, 843-851). Briefly, cells stably expressing GABA _A receptor βl subunit are seeded into 96-well plates and receptor expression induced. Cells are washed in low-Cl_ buffer (in mM sodium-D gluconate 160, potassium-D-gluconate 4.5, CaCl ₂ 2, MgCl ₂ 1, D-glucose 10, HEPES 10, pH 7.4) and dye-loaded for 30 min to give final concentrations of 4 μM chlorocoumarin-2-dimyristoyl phosphatidylethanolamine (CC2-DMPE; FRET donor) and 1 μM bis(l,3-diethyl-2- thiobarbiturate)trimethineoxonol (DiSBAG>(3); FRET acceptor), with 0.5mM tartrazine present extracellularly.

Plates are then placed in a VIPR which performs automated additions using a Hamilton 2200 pipettor and records fluorescence emissions at 460 and 580 nm simultaneously from eight wells. A 400DF 15 filter is used in the excitation pathway, and460DF45 and 580DF60 filters in the respective emission pathways. Rapid ratiometric FRET measurements are made of GABA _A -evoked depolarizations in low-CTbuffer as previously described (Adkins et al., 2001) and the ability of compounds to modulate an EC ₅₀ response to GABA examined.

For each time point at each fluorescence emission wavelength, background fluorescence are subtracted (recorded from wells without cells in the same plate) and the ratio of fluorescence at 460-580πm calculated. GABA-evoked depolarizations are then expressed as a fractional change in this ratio.

Whole cell patch clamp experiments on mammalian cells can also be performed to screen for compounds capable of modulating GABA _A receptor βl subunit activity using methods described in Brown et al., (2002) Br J Pharmacol 136, 965-974.

Briefly, cells are patch clamped using a pipette with a tip diameter of approximately 1-2 μm and a resistance of around 1-5 Mω. The intracellular solution contained (in mM): CsCl 130, HEPES 10, BAPTA.Cs 10, ATP.Mg 5, leupeptin 0.1, MgCl ₂ 1, NaVO ₃ 0.1 (pH adjusted to 7.3) with CsOH and 320-340 mOsm by adding sucrose. Cells are voltage -clamped at -2OmV via an Axon 200B amplifier (Axon Instruments, Foster City, CA, U.S.A.) and perfused with artificial cerebrospinal fluid (aCSF) consisting of (in mM) NaCl 149, KCl 3.25, HEPES 10, MgCl ₂ 2, CaCl ₂ 2, D-glucose 11, sucrose 22, at pH 7.4. Drug solutions are applied to cells via a multi-barrel drug-delivery system, which could pivot the barrels into place using a stepping motor. This can ensure rapid application and washout of the screening compound.

The measured agonist exchange time using this system is approximately 20-30 ms. GABA (1 mMand 1 μM) are applied to the cell (5 s on, 30 s washout) and the amplitude of the currents used to calculate an approximate EC ₂₀ concentration (individually determined for each cell and ranging from 8 to 35% of the response to ImM GABA). Non-cumulative concentration— response curves examining the modulatory effects of SCS can be constructed with SCS being applied for 30 s prior to coapplication with the GABA EC _2O . Data are recorded and analysed using P-clamp (Version 8, Axon Instruments, Foster City, CA, U.S.A.).

Using such methods, a large number of compounds can be screened from structurally diverse screening library.

Moreover, such methods can be used in conjunction with exposing the cells to addictive substances, for example alcohol or opiates, and observing the effects of the tested compounds on GABAA receptor activity in this environment.

The screening methods described above can be used to identify compounds that modulate the activity of normal GABA _A receptor βl subunit activity. However, it is also possible to generate cells expressing one or more mutations of the GABA _A receptor βl subunit and to use such cells in the screening methods set out above. Methods of generating such cell lines are described herein.

The data present in the application show that mutations in a GABA _A receptor subunit polypeptide can lead to increased alcohol intake in mice. Mutations in other GABA _A receptor subunit polypeptide may also lead to altered alcohol intake.

In Examples 2 and 4 we show that the important electrophysiological changes caused by a L285R mutation in the βl subunit polypeptide (numbering according to the mature polypeptide) are the spontaneous firing of a GABAA receptor having the mutant βl polypeptide, which results in increased tonic inhibition and reduced response to synaptically released GABA.

The cellular screening assays described herein can be used to screen for compounds that reverse the electrophysiological changes caused by a mutation in a GABAA receptor subunit polypeptide, for example a L285R mutation in the βl polypeptide. Any compounds which reverse these electrophysiological changes can then be supplied to mice having such a mutation, for example having a L285R βl mutant subunit polypeptide, and screened to establish whether they can change alcohol intake in those mice.

Example 3: Further characterisation of the ENU mutagenised mouse line with significantly raised alcohol preference

In this Example, the following are assessed:

1. SUCROSE PREFERENCE and CONSUMPTION of heterozygote ALCO/22 mutants

2. SACCHARIN PREFERENCE and CONSUMPTION of heterozygote ALCO/22 mutants

3. FOOD CONSUMPTION of heterozygote ALCO/22 mutants

4. Effect of anti-relapse drug NALTREXONE on Ethanol preference of heterozygote high drinking ALCO/22 mutants

5. Effect of anti-relapse drug ACAMPROS ATE on Ethanol preference of heterozygote high drinking ALCO/22 mutants

6. BLOOD ALCOHOL LEVELS

1. SUCROSE PREFERENCE:

Background:

The interpretation of sucrose preference data in models of alcohol preferring mouse models is somewhat controversial. Some authors describe a higher sucrose preference to the alcohol preferring C57BL mouse strains compared to other low drinking strains, e.g. 129/J. The difference of sucrose intake and preference between those two strains allowed the identification of two genetic loci on mouse chromosome 4 which are responsible for over 50% of the genetic variability for sucrose intake (Bachmanov et al, 1997 ¹ ). On the other hand some researchers use the fact that there is no difference in sucrose consumption between a wild-type and a mutant strain with ethanol preference, in order to show that there is no caloric motivation involved (Spanagel et al 2005, ² ).

In the case of ALCO/22, which is on a BALBc/C3H background, the inbred strains BALB/c and C3H are not high drinking and also not known for increased sucrose preference. Therefore we wanted to test if the high ethanol preference and

intake in the ALCO/22 mutant was associated with higher sucrose preference compared to wild-type littermates. This then could have been interpreted as either a general hedonic enforcing mechanism, or alternatively as a caloric motivation as preference for higher caloric solutions. This latter interpretation was even more relevant in the case of ALCO/22 heterozygote mutants because of their lower body weight and size compared to their wild-type littermates.

Method:

Mice were offered a 4 % (0.12M) solution of sucrose, which was the concentration of sucrose which detected a significant difference in sucrose consumption between the inbred strain B6 and 129 (Bachmanov et al, 1997 ¹ ), which led to the identification of two loci on chromosome 4 contributing to 50% of the genetic effect for sucrose consumption. The solution was offered for 10 days (as the ethanol preference test, which the bottles being weighed on day 1, 3, 5, 8 and 10), and the sucrose preference calculated as amount of sucrose solution compared to the total amount of liquid consumed (water plus 4% sucrose), and sucrose consumption calculated as grams of sucrose per kg body weight.

Statistical analysis: Two group comparisons for the characterization of the phenotype of the mice were performed with two-sided exact Wilcoxon rank sum tests. The means are represented in the graphs of Figure 6 and S. D. (standard deviations) are reported. P-values of < 0.05 were considered significant.

Results:

Sucrose preference:

Mice bearing the mutation showed the same high preference for sucrose as the wild-type littermates: 95.3% (95% CI=91.1-99.5; n = 10), and 93.0 % (95% CI= 97.4-98.6; n = 8) p=0.46. Sucrose intake:

Mice bearing the mutation consumed 10.5 g sucrose per day per kg bodyweight (S.D. = 3.1; n=10 female mice), compared to 8.5 g in the wt (S.D. = 2.5; n = 8 female mice), p=0.15.

As heterozygote ALCO/22 mutants have a 20% lower bodyweight compared to wt , littermates, the overall consumption of sucrose is comparable.

Interpretation: As sucrose consumption per kg bodyweight (Fig. 6) did not differ between genotypes (factor 1.2; 95% CI = 0.9-1.4) to the same extent as the alcohol consumption (factor 4.6, 95% CI = 3.1-6.2), a caloric motivation for ethanol preference was discounted.

2. SACCHARIN PREFERENCE

Background:

Previous research has shown that inbred mouse strains B6 and 129 not only differ greatly in their preference for ethanol, but also their intake of 2mM saccharin (Sigma) (Bachmanov et al, 1996a+b, 1998).

Any preference of the ALCO/22 mutant line for sweetened solutions (ie saccharin solutions) without the caloric effect offered by a sucrose solution was tested.

Method:

As saccharin is 30Ox sweeter than sucrose, a saccharin solution of 0.4 (0.5) mM

(sodium saccharin, Sigma) should have the same sweetness as the 4% (0.12M) sucrose which was offered. The saccharin preference was tested as the sucrose preference (see 1) and the statistical evaluation was carried out in the same way. N=I 8 animals.

Results: a) Saccharin preference

The preference for 0.5mM saccharin was not different between a group of male wt ALCO/22 mice (n=5) and a group of male ALCO/22 mutant mice (n=7), with a mean of 64.7% and 62.9% respectively, p=0.87. See Figure 7. Including 6 female wt litter mates into the t-test did not change the overall result.

b) Saccharin consumption

Saccharin consumption (in weight of nils of 0.5mM saccharin solution consumed daily, after correction with spillage from control cages) was higher in wt animals (n=5 males of n=5 males and 6 females) compared to mutant ALCO/22 mice (n=7), p=0.018 or p=0.0015 on inclusion of 6 female litter mates into the group of wt. As the body weight between mutant and wt animals differs by 20 % (ALCO/22 mutants have a 20% reduced weight compared to wt littermates), the difference disappeared upon correction for bqdyweight.

Discussion

The results show that ALCO/22 mutants do not have a preference for Saccharin compared to wild type littermates, ruling out the possibility that the ethanol preference of ALCO/22 mutants is caused by a general preference for sweetened solutions or solutions with a different taste to water.

3. FOOD CONSUMPTION

Background: ALCO/22 heterozygote mutants have a lower bodyweight and size compared to their wild-type (+/+) littermates (Fig. 9). This experiment addressed the question if the lower weight and size were a consequence of reduced food intake of the heterozygote mutants. An initial experiment was performed using metabolic cages, but as the mice do not like the environment of the metabolic cage without bedding and their normal standard chow, and the inventor's current home office license only allows the mice to be kept for 24 hours in this environment, all mice lost weight within the 24 hours in the metabolic cage, and the data were not suitable for evaluation. Therefore an alternative method of weighing the food offered for each of the singly caged mice was chosen, and the food weighed and replaced daily over a seven day period.

Method:

Food consumption was measured over seven days by placing a known (weighed) quantity of food pellets (70-80 g) into the cage hopper, which was weighed and replaced each day over a 7 day period, allowing the measurements of daily food consumption.

Mixed model analysis of longitudinal food consumption data was performed using PROC MIXED from SAS Release 8.2 (SAS Institute Inc., Cary, NC, USA) with compound symmetry correlation-structure: The effect estimate in the mixed model analysis used to calculate the food consumption is reported together with its s.e.m.

Result:

ALCO/22 heterozygote mutants have a lower bodyweight and size compared to their wild-type (+/+) littermates (Fig. 9), with a mean food intake of 331.3 g/kg body weight compared to 235.0 g in wild-type littermates, measured daily over 7 days. Mixed linear model analysis for repeated food consumption showed that heterozygotes consumed significantly more food per kg bodyweight than their wild-type littermates (difference: 96.3 ± 12.7 mg/kg, p < 0.01; n = 3 females in both groups), whilst the overall food intake in both groups was comparable (not shown).

Interpretation/Discussion The reduced body weight of the heterozygote ALCO/22 mice is not due to a reduction in food intake. It is more likely due to a change in hormonal levels or other developmental implications of the Gabrbl mutation.

4. EFFECT OF ANTI-RELAPSE DRUG NALTREXONE ON ETHANOL PREFERENCE.

Background:

The effect of anti-relapse drugs which are used in human alcoholic patients to prevent relapse are also reported to reduce the high ethanol preference and intake in the high drinking B6 strain. In the publication by Middaugh and Bandy ⁴ the effect of naltrexone (an opiate receptor antagonist) on the preference of 10% ethanol is best seen if mice are injected with a dose of 6 mg/kg naltrexone before the onset of the dark phase, in which they are most actively drinldng. A dose of 6 mg/kg Naltrexone reduced the ethanol intake of B 6 animals by 50% and reduced increase of extracellular dopamine by 50% ⁵ .

Method:

Experiment 1

6 mg/kg dose, applied into ongoing ethanol preference paradigm

18 mice, 8-10 weeks old (7 mutant, 11 wild-type; 12 females, 6 males), which were tested for ethanol preference during 10 days, were allocated to 4 groups (l.High drinking mutants to be injected with Naltrexone; 2.High drinking mutants to be injected with saline; 3.Non drinking wild— type littermates to be injected with Naltrexone; 4.Non drinking wild-type littermates to be injected with Saline) depending on their ethanol intake.

Naltrexone (Sigma) was injected intraperitoneally (i.p.) on day 11 as 6 mg/ml in 0.9% saline into the mice from the mutant and wild-type group (1 and 3), whilst mice from group 2 and 4 were injected with 0.9% saline 30-60 minutes before onset of the dark phase. The following day the weight of the bottles was measured before the next injection was applied.

Experiment 2 Increasing doses of Naltrexone; applied into ongoing ethanol preference paradigm.

In the literature it is unclear which dose of Naltrexone has the best effect. Phillips et al ⁶ report that lower doses of 1-2 mg/kg have the best effect, whereas Middaugh et al. claim that 6 mg/kg have a better effect.

Therefore another cohort of mice, all male, 9 weeks old, were injected with increasing doses of naltrexone in subsequent injections, ranging from 1 mg/kg to 24 mg/kg. From day 17 of the ethanol preference test, 1 mg/kg, 2 mg/kg and 6 mg/kg naltrexone (or saline) were injected on Tuesday, Wednesday, Thursday respectively, and again on Tuesday and Wednesday with 12 and 25 mg/kg Naltrexone respectively. Five male B6 mice, 3 of them injected with Naltrexone, 2 with saline, were monitored in parallel.

Experiment 3 6 mg/kg dose, applied after 5 days break from 10 day ethanol preference test.

After further consideration of published work (Middaugh and Bandy, 2000 ⁴ ), it appears that Naltrexone might need to be applied in a limited access paradigm in order to be effective. Although discussing a 2 bottle choice paradigm, they apply the Naltrexone injection after a 4 day break from previous access to ethanol.

14 mice, 10 weeks old, all female, were used and divided into 4 groups, as described before. After the 10 days ethanol preference test (Mon - Wed), mice were put on distilled water until the following Monday, when the first i.p. injection of 6 mg/kg Naltrexone was applied and the two bottle choice between a bottle containing 10% ethanol and water was introduced. The bottles were weighed each following day, before the next injection was applied. Four injections of Naltrexone were applied, and the weight of the ethanol bottles was measured again on day I ₅ 4 and 6 after the last of the 4 Naltrexone applications.

Result:

Experiment 1

The results of the calculated ethanol preference before, during and after the naltrexone treatment were inserted into tables of graph pad prism, with 5 animals for each of the 4 groups (n=5). The graph showed a slight decrease of ethanol intake in the group of high drinkers (heterozygote mutants) injected with Naltrexone after the first injection Fig 10. The second injection did not obtain the same effect. This observation is consistent with that of Middaugh et al, who describe the first injection as the one which is most effective. However, the effect is statistically not significant when comparing mutant mice injected with Naltexone to the mutant animals injected with saline (t-test between the two groups).

Result: Experiment 2 Increasing doses of Naltrexone

Overall: no effect of repeated Naltrexone injections starting at low doses and increasing to higher doses (Fig. 11). This result is consistent with the observation ofPhillips et al, 1997.

Result: Experiment 3

As in the pilot experiment, the graph (graph pad prism) shows a decrease in ethanol preference after the first Naltrexone injection (Fig. 12), which is consistent with the results of Middaugh and Bandy .

Overall results of Naltrexone experiments:

• Low doses of Naltrexone (1 mg/kg), followed by increasing doses (up to 25 mg/kg) do not obtain reduction of ethanol preference.

• 6 mg/kg dose shows an effect in first application. Subsequent applications don't have the same effect.

• Reduction of absolute ethanol preference from 90% to 60%.

• Reduction of ethanol preference in mutants injected with Naltrexone do not reach statistical significance in comparison to mutants injected with Saline (t- test, non parametric, Mann -Whitney, between two groups), but also not for B 6 animals.

5. EFFECT OF ANTI-RELAPSE DRUG ACAMPROSATE ON a) ETHANOL PREFERENCE b) ABSOLUTE ETHANOL CONSUMPTION

250mg/kg dose, applied after 5 days break from 10 day ethanol preference test, as in Naltrexone experiment 3.

14 mice, 10 weeks old, all female, were used and divided into 4 groups, as described before. After the 10 days ethanol preference test (Mon — Wed), mice were put on distilled water until the following Monday, when the first i.p. injection of 250 mg/kg Acamprosate was applied and the two bottle choice between a bottle containing 10% ethanol and water was introduced. The bottles were weighed each following day, before the next injection was applied. Four injections of Acamprosate were applied, and the weight of the ethanol bottles was measured again on day 1, 4 and 6 after the last of the 4 Acamprosate applications. Five B6 female mice, 3 of them injected with Acamprosate, 2 of them injected with saline, were tested in parallel.

Acamprosate (which acts to block the NMDA receptor) was kindly provided by Frederic Landron M.D. Strategic Marketing Leader, Campral, Merck-Sante,

France. The recommendation was to inject twice daily, every 12 hours, with a 200 mg/kg dose. Due to home office regulations and the protocol in the project license only 1 daily injection was possible, 30-60 minutes before onset of dark phase. The single injection may mean that effects are reduced due to quick brealcdown/turnover of Acamprosate in mice. The dose of 250mg/kg was chosen. The recommendations stated that higher doses of 400 mg/kg would be unspecific and lead to adverse effects.

Result a) ETHANOL PREFERENCE (Fig. 13)

Reduction of Ethanol preference in high drinking mutant mice (n=3), and drinking

B6 mice (n=3) with Acamprosate injections, compared to drinking ALCO/22 mutants (n=3) and drinking B6 mice (n==2) treated with saline. Acamprosate treatment reduced ethanol preference in high drinking ALCO/22 mice from 80% to 60%, and inB6 mice from 58% to 40%.

b) ABSOLUTE ETHANOL CONSUMPTION (Fig.14)

A reduction in absolute Ethanol consumption is observed with Saline as well as

Acamprosate injection, both in ALCO/22 drinking as well as in B6 drinking mice. Acamprosate leads to more reduction in ETOH consumption than saline.

A statistical analysis of the effect of Acamprosate (see Figure 15) suggests that

Acamprosate is having an effect in the mutant mice. The effect is around 10% reduction of ethanol preference, but statistically insignificant due to small number of mice. This is consistent with the effect of Acamprosate in man, where it extends the time between relapses by 10 % (Mann et al (2004) alcoholism: Clinical and experimental research 28 (1) 51-63 The efficacy of acamprosate in the maintenance of abstinence in alcohol-dependent individuals: results of a metaanalysis).

6. BLOOD ETHANOL LEVELS

Background:

In order to measure the pharmacological significance of the high ethanol preference and predict the effect of the ethanol intake on the mouse brain, the blood alcohol levels in the mutant mice with high ethanol preference were determined. In the literature a level of ethanol consumption of 6-10 g ethanol /kg/day, as in C57BL/6, is reported to result in more than 100 mg ethanol/dl blood, which is the threshold for pharmacological effects on the mouse brain ⁵ .

As the blood alcohol levels decline very rapidly and become undetectable only a few hours after ethanol exposure/intake, the blood sample needs to be taken towards the end of the active drinking phase of the mice, which is at the end of the dark cycle. In order to make things easier for those experiments to be carried out, mice have been adjusted during 2 weeks in a room with reversed dark:light cycle, with lights off at 10 am. As the absolute ethanol intake of ALCO/22 mutant is around and often higher 1O g ethanol/kg/day, it is expected that blood alcohol levels will be detected reaching 100 mg ethanol/dl.

Figure 16 (taken from Middaugh et al (2003) Conclusion 1: Ingestion of 6 g /kg of ethanol results in BEL near 100 mg/100 ml by 6 hours into active dark phase ( here: eye blood).

Conclusion 2:

Blood alcohol needs to be measured 6 hours after start of dark cycle/drinking.

Method:

Blood from the tail vein of 5 mutant high drinking mice (4 females, one male), 4 non — drinking mice (2 male, 2 female) and 2 ALCO/22 control mice not exposed to alcohol is taken 6 hours after start of the dark phase, during which the maximum of the drinking activity is to be expected according to Middaugh et al, 2003.

Blood ethanol levels are determined immediately using a spectrophotometric Olympus kit.

Results:

No ethanol is detectable in any of the blood samples, even not from the highest drinking mouse, but standards detecting 25, 50 and 100 mg ethanol all work. In order to test the stability of the assay blood from a non drinking mouse is spiked with 20, 50 and 100 mg ethanol from the standards, and those levels are detected. This shows that the assay is able to detect at least levels of 25 mg ethanol, and technical reasons for the lack of detection can be dismissed.

Discussion/Interpretation :

Various explanation for the lack of detectable blood levels are possible: a) Middaugh et al and most researchers in the US who report on detectable levels of blood alcohol in drinking B6 mice use blood samples taken from near the eye (infraorbital sinus), a procedure which is illegal under UK home office regulations. There are publications on the difference between detectable levels of blood alcohol from tail vein and infraorbital sinus (eye). Middaugh et al ⁵ also deprive mice of the access to alcohol 4 hours previously, which might lead to deprivation effects, after which the mice have an over enhanced desire to consume ethanol solutions.

b) The lack of detectable blood ethanol levels (BEL) as observed in the ALCO/22 line is common in mouse models with continuous access to alcohol, where drinking bouts are spread over a 24 hour period and are subject to a high metabolic turnover(Dole and Gentry, 1984). Although they self-administer significant quantities of alcohol, they may achieve significant concentrations only transiently (Dole and Gentry, 1984). It had previously been shown that they will self-administer large amounts of ethanol under conditions where access to fluids is restricted (Dole and Gentry(Dole and Gentry, 1984).

Conclusion:

The lack of detectable BLE is therefore nothing unusual in the ALCO/22 line.

Options of detecting the level of blood alcohol according to the amounts of alcohol consumed might consist in the application of inhibitors for alcohol dehydrogenase (Dole and Gentry ⁷ ). In order to compare and characterize the metabolic rate between animals/lines known amounts of ethanol would need to be inj ected intraperitoneally ² .

Reference List

1. Bachmanov, A. A. et al. Sucrose consumption in mice: major influence of two genetic loci affecting peripheral sensory responses.

Mamm.Genome 8, 545-548 (1997).

2. Spanagel, R. et al. The clock gene Per2 influences the glutamatergic system and modulates alcohol consumption. Nat.Med. 11, 35-42 (2005). 3. Pirker, S., Schwarzer, C, Wieselthaler, A., Sieghart, W., & Sperk,

G. GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101, 815-850 (2000).

4. Middaugh, L. D. & Bandy, A. L. Naltrexone effects on ethanol consumption and response to ethanol conditioned cues in C57BL/6 mice. Psychopharmacology (Berl) 151, 321 -327 (2000).

5. Middaugh, L. D., Szumlinski, K. K., Van Patten, Y., Marlowe, A. L., & Kalivas, P. W. Chronic ethanol consumption by C57BL/6 mice promotes tolerance to its interoceptive cues and increases extracellular dopamine, an effect blocked by naltrexone. Alcohol Clin.Exp.Res. 27, 1892-1900 (2003).

6. Phillips, T. J., Wenger, C. D., & Dorow, J. D. Naltrexone effects on ethanol drinking acquisition and on established ethanol consumption in C57BL/6J mice. Alcohol Clin.Exp.Res. 2I ₅ 691-702 (1997).

7. Dole, V. P. & Gentry, R. T. Toward an analogue of alcoholism in mice: scale factors in the model. Proc.Natl.Acad.Sci. U.S.A 81, 3543-

3546 (1984).

Example 4: A mutation in the GABAA receptor βl gene promotes alcohol preference in mice

Alcohol misuse/dependence is a familial disorder with a complex behavioural phenotype ¹ . A number of quantitative trait loci within GABA _A receptor gene clusters associate strongly with alcohol dependence ² and genetic manipulations of several GABA _A receptor subunits influence alcohol intake in mice ³ . Using ENU mutagenesis, we have generated a mouse line with a strong heritable preference for alcohol consumption. This phenotype is associated with a single mutation (L285R) in the third transmembrane domain of the GABA _A receptor βl subunit, which in vitro results in tonically open receptors. In humans we found weak linkage and allelic associations between the GABA _A receptor beta 1 gene [GABRBl) and alcohol dependence. Our findings indicate that GAB A _A receptors containing the βl subunit play a role in promoting increased alcohol intake in mice and possibly man.

Recently several studies have focused attention on the GABRBl gene, Parsian and colleagues ¹³ found significant association (p= 0.004) between the alleles of a tetranucleotide marker ¹⁴ in GABRBl and alcoholism in 133 alcohol misusers and

89 control subjects. In a second study Song et al ⁵ used the transmission disequilibrium test (TDT) in the COGA Study data set and showed linkage disequilibrium between GABRBl and 'alcoholism' (p< 0.03).

Individuals who misuse alcohol also exhibit a number of electrophysiological abnormalities ¹⁶ . The cognitive evoked potential (P300) is reduced in size and the EEG beta activity is increased in alcohol misusers, consistent with alterations in cerebral neurotransmission. These phenotypes are genetically determined with linkage to a region on chromosome 4 ⁶ ' ¹⁷ . Porjesz et al ¹⁸ report linkage between the increased EEG beta activity in alcoholics and a tetranucleotide marker ¹⁴ in intron 8 of GABRBl.

The in vitro experiments described here show how a single base mutation in GABRBl results in tonically open channels and how in vivo this mutation substantially increases voluntary alcohol consumption in a line of mice. Human data shows weak linkage between a region near GABRBl and alcohol dependence

in families and a statistically significant allelic association between two microsatellite markers within GABRBl and alcohol dependence in a case control study. Taken together, these findings indicate that GABRBl has a role in modulating alcohol consumption and in the development of alcohol dependence.

In this study a non-alcohol drinldng mouse strain (BALB/c) was exposed to N- ethyl-N-nitrosourea (ENU) mutagenesis, establishing a new mouse line (denoted ALCO/22) which exhibited a strong preference for alcohol. ALCO/22 heterozygotes animals imbibe significantly greater quantities of alcohol than their wild type counterparts (factor 4.6, 95% CI - 3.1-6.2); 75 - 80 % of their total fluid intake is imbibed as 10% v/v alcohol (Fig. 17a, Table 1), equating to an average daily intake of 11 g/kg bodyweight (Fig. 17b). The total fluid intake was not affected by the mutation (data not shown). The ALCO/22 heterozygotes animals were of lower bodyweight and size than their wild-type counterparts (Fig. 17c) but nevertheless consumed significantly more food per kg bodyweight. Sucrose consumption did not differ significantly between the genotypes (factor 1.2; 95% CI = 0.9-1.4) (Fig. 17d), making it unlikely that their consumption of alcohol was motivated by energy needs. The alcohol drinking preference phenotype was found in 123 out of the 130 animals (93%) carrying the mutation and has been followed for seven generations.

The site of the ENU-induced mutation was mapped to a region of mouse chromosome 5, between markers D5MUSK2 and D5MU356 (Fig. 19a) which contains the Gabra4 and Gabrbl subunit genes. Sequencing the coding regions of both genes revealed only a single T to G base mutation at position 94 of exon 8 in the βl gene, resulting in a leucine to arginine exchange at residue 285 (L285R; numbering according to the mature polypeptide, i.e. lacking the signal sequence) (Fig. 19b). L285, which is located near the external end of the subunit's third transmembrane domain, is highly conserved across species, suggesting that it plays an important role in GABAA receptor function.

The functional consequences of the L285R mutation were determined by expressing wild-type (α2βlγ2S/L) and mutant (α2βl ^L285R γ2S/L) GABA _A R in HEK293 cells. The amplitude of the maximal GABA response was reduced in the mutant receptor to approximately 10% of that in the wild-type, whilst GABA potency was marginally increased (~2-fold, Table 1; Fig. 18a). In the absence of GABA, increased holding currents were required to voltage clamp cells expressing the mutant subunit, suggesting these receptors opened spontaneously. This was confirmed with cyanotriphenylborate (CTB, 100 μM), a blocker of open GABR _A channels ²⁰ , which reduced the holding currents near to those of wild-type receptors (Fig. 18a, inset). The ability of the mutant channels to gate spontaneously was also demonstrated in outside-out patches taken from cells expressing α2βl ^L2S5R γ2L receptors, but not in those expressing α2βlγ2L receptors (Fig. 18b). Individual single channel currents for the mutants (γ = 26.2 ± 0.8 pS, n = 4) were similar to those of the wild-type α2βlγ2L receptors (γ = 27.6 ± 0.8 pS, n = 4, P > 0.05) following lOμM GABA exposure (Fig. 18b), but the βl ^L285R - expressing GABA _A receptors were frequently open in the absence of GABA and showed a reduced response to GABA application.

No significant differences were evident between the patterns of surface expression of mutant and wild-type forms using the 9E10 antibody to myc epitope-labeled βl subunits and confocal microscopy (Fig. 18c). This further suggests that the reduction in GABA current amplitude is due to a defect in receptor function rather than a reduction in the number of cell surface GABA _A receptors.

Although GABA _A receptors may mediate several behavioral effects of alcohol, studies of the direct effects of ethanol on GABAA receptors are inconclusive ⁹ ' ²¹ ' ²² In the present study, 200 mM alcohol had little effect on the amplitude of EC ₁₀ GABA responses on either wild-type or mutant receptors expressed as either α2βl (data not shown), α2βlγ2S or α2βlγ2L (Table 1; Fig. 18d). Alcohol intoxication is also associated with increased production of the endogenous neurosteroid, 5α- pregnan-3α-ol-20-one (allopregnanolone), a potent potentiator at GABA _A receptors . However, no substantial change in the sensitivity of wild-type and βl

mutant receptors to potentiation by 1 μM allopregnanolone was observed (Fig. 18d).

The family based genetic linkage analysis found evidence supportive for linkage between the genetic marker D4S3248 which is 13 Megabases from GABRBJ and alcoholism with a maximum lod score of 1.4, assuming a recessive model. Multipoint linkage analysis with three adjacent markers on chromosome 4 confirmed this positive lod score. In the case control sample of 360 individuals with alcohol dependence syndrome and 450 healthy controls a tetranucleotide marker marker in the βl receptor subunit gene showed association with alcohol dependence (CLUMP T3, p = 0.028, empirical p = 0.155).

This monogenic animal model supports a role for the GABA _A receptor βl subunit in modulating alcohol consumption. A single mutation in a critical functional region of Gabrbl, previously not identified as playing a key role in determining alcohol preference in animal models, results in altered GABA _A receptor function in HEK293 cells. The electrophysiological data suggest that the observed L285R mutation in the βl subunit results in some degree of tonic activation of their ion channels. Previous mutagenesis studies in Xenopus oocytes have shown that a different mutation (M286W) in the transmembrane domain (TM3) of the βl subunit also results in a tonically open receptor: however detailed pharmacology of this mutant is not available and its relationship to alcohol consumption in vivo was not shown . The mechanism by which our mutation in GABAA receptor βl influences alcohol consumption is not apparent at present. These data do, however, exclude the possibility that alcohol simply modulates the sensitivity of the mutant receptor to GABA. The probability that this phenotype develops as a consequence of changes in other receptor subtypes or as a result of imbalance in excitatory/inhibitory signaling in neuronal circuits mediating ethanol reward, needs to be considered. Only limited inferences can be made in regard to the functional consequences of the mutant GABA subunit in vivo , because the animals are heterozygous for the mutant gene and there are more than 15 different

GABA subunits available in the brain for assembly into a fully functional receptor.

The involvement of the Gabrbl gene in determining alcohol preference in our mice, is complemented by the previously demonstrated significant allelic association between alcohol dependence and the GABRBl polymorphisms in man ^13ib ' ¹⁸ ' ²⁴ . The present study demonstrates only a weak linkage and association between drinking behavior and GABRBl polymorphism, possibly because of genetic heterogeneity of the patient group tested.

The recent finding that a single nucleotide polymorphism in the intronic regions of GABRA2 is associated with 'alcoholism', has led to the suggestion that altered transcription of this gene is involved in the pathogenesis of the disorder ²⁵ . However, our mouse and human data point towards involvement of GABRBL It is unlikely that the microsatellite marker alleles at the GABRBl locus are in linkage disequilibrium with GABRA2 susceptibility alleles as they are 700 kb apart. 'Alcoholism' is a heterogeneous disorder and genetic variations of several GABA _A receptor subunit genes as well as other genes ²⁶ may contribute to the development of "alcoholism" in subsets of patients. These data indicate that GABA _A receptors containing the βl subunit, modulate alcohol consumption in mice and probably also in man. The electrophysiological data show at least one way in which this receptor function may be altered to produce increased alcohol consumption. Whether the same altered receptor function is present in a subset of human subjects with alcoholism remains to be investigated by more detailed phenotyping of the patients of this cohort.

METHODS

MURINE STUDIES.

Mice. BALB/c mice were exposed twice weeldy to 100mg/kg ENU and backcrossed to female C3H mice (http://www.irιut.har.mrc.ac.ιil<). The wild-type and ALCO/22 mutant mice in this study were littermates derived from intercrosses

between heterozygous ALCO/22 males and C3H females. Heterozygote ALCO/22 mutant females are infertile and no homozygote animals could be produced. Mice were given free access to food and water and kept under a 12 hr light/dark cycle with artificial light. Animals were housed and treated under standard conditions using procedures approved by the UK Home Office.

Phenotyping. Mice aged 7-10 weeks from first generation backcrosses (B ALB /c mut x C3H), and subsequent backcrosses, were housed in single cages and offered a choice of water and 10 % v/v ethanol in water (diluted from 96% Ethanol, AnalaR, BDH) from two bottles randomly positioned in the cage. The consumption of water and ethanol was measured by weighing bottles filled with 100-150 ml, on days 1, 3, 5, 8 and 10, on a 10 day cycle. Ethanol preference was calculated as the ratio of ethanol over the total amount of liquid imbibed. The weights were corrected for spillage determined by loss of fluids in the water and ethanol bottle in two empty cages measured in parallel. For the determination of absolute ethanol consumption the consumed amount from each bottle was similarly corrected for spillage. The weights of the animals were recorded at the start of the 10 day period. Food consumption was measured over 7 days by placing a known quantity of food pellets (70-80 g) in the cage hopper, which was weighed and replaced each day. Sucrose preference was assessed by offering the animals a 4% (w/v) sucrose solution and water in a two bottle choice paradigm over a 10 day period. Sucrose preference was calculated as the ratio of sucrose solution over the total amount of liquid imbibed and calculated as absolute daily amount of sucrose consumed per kg bodyweight.

Statistical analysis. Two group comparisons for the characterization of the phenotype of the mice were performed with two-sided exact Wilcoxon rank sum tests. The mean values are represented in the graphs of Figure 17 with significance set at p < 0.05. Mixed model analysis of longitudinal food consumption was performed using PROC MIXED from SAS Release 8.2 (SAS Institute Inc., Gary, NC, USA) with compound symmetry correlation-structure. The effect estimate in the mixed model analysis used to calculate the food consumption is reported

together with its SEM. Approximate 95% confidence intervals of the factors comparing alcohol as well as sucrose consumption between heterozygote mutant and wt were calculated using the delta-method.

Mapping. DNA was extracted, using a DNA purification kit (Nucleon), from the tail tips of 13 mice displaying high ethanol preference (2 G2, 11 G3), originating from the same founder with the desired phenotype. An initial Genome Scan Mapping procedure using 86 fluorescently labeled microsatellite markers, which are polymorphic between BALB/c and C3H, revealed a region of common BALB/c DNA between DSMU15 (64.1Mb) and DSMitlO (101.6 Mb) on mouse chromosome 5.

For fine mapping and identification of informative recombinants, DNA was extracted from ear biopsies by incubation in a lysis buffer containing: 50 mM TrisHCl, 1 mM EDTA, 0.5 % Tween 20, and 150-300 μg/ml proteinase K at pH 8.5 for 4-12 hr at 55 ⁰ C, and then denatured for 12 min at 95 ⁰ C. To reduce the region of BALB/c DNA that carried the point mutation, a further 169 animals from backcross experiments were tested and phenotyped for alcohol preference and investigated whether recombination had occurred in the region of the GABAA receptor genes by detecting the presence of BALB/c DNA, using eleven further polymorphic markers between D5MU15 and DSMitlO. In 82 mice with the high ethanol preference phenotype, eight recombination events were found in the interval between D5MU15 and DSMitlO, two of which were located in a region of common BALB/c DNA between the markers D5MU304 and D5MU356 (interval of 68.78 Mb and 71.84 Mb). Additional polymorphic markers were placed between DSMU304 and D5MU355 and between D5MU134 and D5MH356 to facilitate identification of candidate genes on mouse chromosome 5. Marker D5MUSK2 (69.66 Mb) was used to exclude Gabrgl and Gabτa2 as candidate genes because of the absence of BALB/c DNA in one of the recombinants. Size differences for the fragments of the additional polymorphic markers were between 4-20 bp and separated on 4% agarose gels (AGTC Bio Products Ltd). Oligonucleotide sequences are available on request.

Sequencing. The two main candidate genes Gabra4 and Gabrbl in the mutant mice were sequenced from both directions and compared with the sequence obtained from the two parental strains. The nine coding exons were amplified using intronic primers (Sigma Genosys), including intron/exon borders, from genomic DNA for the GABAA R βl subunit and also the nine coding exons for the α4 gene, from heterozygote ALCO/22 mutant (+/Mut) and parental mouse strains BALB/c and C3H. PCR products were subjected to gel purification using the Wizard kit (Promega). Products were sequenced on a 3700 ABI Sequencer using "Big Dye" chemistry (Applied Biosystems) and analyzed with BioEdit software. Sequences were derived from the information of ENSMUST00000031122 in "Ensembl", version 33.

Patch clamp electrophysiology and data analysis. Human α2 and murine βl, γ2S and γ2L cDNAs were used for expression studies. Site-directed mutagenesis of L285R was performed using the Quickchange method (Stratagene); oligonucleotide sequences are available on request. Sequences of the full coding region of mutated cDNAs were determined by automated fluorescent sequencing at the Wolfson Institute for Biomedical Research, UCL. Plasmid DNAs for transfection were purified using the Hi- Speed Plasmid Midi Kit (Qiagen).

Human embryonic kidney (HEK) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum, 2 mM glutamine, 100 units/ml penicillin G and 100 mg/ml streptomycin at 37 ⁰ C in 95% air/5% CO ₂ . HEK cells were transiently transfected by calcium phosphate co-precipitation of 1 μg of each plasmid DNA encoding αl, β2 and γ2 and 0.5 μg pEGFP (Clontech). Cells were used for electrophysiology after 20 hr.

Whole-cell currents were recorded at room temperature (20-22 ⁰ C) from single HEK cells voltage clamped at -4OmV, using an Axopatch 200B amplifier (Axon

Instruments, California, USA). Patch electrodes (3-5 Mω) contained (mM): 144

KCl, 2 MgCl ₂ , 1 CaCl ₂ , 10 HEPES, 11 EGTA and 2 adenosine triphosphate, pH

7.2. Cells were continuously superfused with Krebs solution containing (mM): 140 NaCl, 4.7 KCl, 1.2 MgCl ₂ , 2.5 CaCl ₂ , 10 HEPES and 11 glucose, pH 7.4. Recordings were filtered at 5 kHz (6-pole Bessel, 36dB/octave). Drugs and Krebs solution were applied (exchange rate 100 ms) to HEK cells using a modified U- tube. GABA, CTB and ethanol were dissolved directly into Krebs solution and the pH adjusted to 7.4. Allopregnanolone was diluted in Krebs from a 10 mM stock in DMSO. For studies of ethanol potentiation, the GABA current evoked by a low GABA concentration (EC ₁₀ ) was initially determined then 200 mM ethanol was applied , for 30-60 s prior to the co-application of EC ₁₀ GABA and ethanol. Allopregnanolone potentiation was determined by co-application Of EC ₁₀ GABA and the steroid. Dose-response relationships were fitted with a non-linear least squares fitting routine using Origin 6.1 (Microcal). Data points represent the mean ± SEM. of at least three experiments. The fractional degree of spontaneous receptor activation was ascertained by dividing the amplitude of currents induced by saturating concentrations of CTB ( _ICTB ) by the sum of those induced by saturating concentrations of CTB and GABA (ICTB + I _G AB _A )- Single GABA- activated channels were recorded from excised outside-out patches maintained at a potential of -7OmV as previously described .

Confocal microscopy. Human α2 and murine βl ^myc or βl ^myc ' ^L285R , and γ2L cDNAs were expressed to form αβγ receptors in concert with enhanced Green Fluorescent Protein (EGFP). The βl ^myc subunit containing the myc epitope in its N-terminal, extracellular domain is an addition which is proven to be electrophysio logically silent ² . Transfected HEK293 cells were fixed in phosphate- buffered saline (PBS) containing 4% paraformaldehyde for 15 min before being quenched with 5OmM NH ₄ Cl in PBS for 10 min. After washing in PBS, cells were incubated for 45 min at room temperature with 9E10 antibody (to the extracellular myc epitope, 1:100 dilution of rabbit polyclonal; Santa Cruz Biotechnology). Cells were washed in PBS containing 10% foetal calf serum (FCS) and 0.4% bovine serum albumin (BSA) before incubation for 45 min with a TPJTC-conjugated secondary antibody. Cells were then thoroughly washed and mounted in glycerol. Mounted slides were viewed using an upright Zeiss

Axiophot microscope and confocal images were obtained from a Zeiss LSM510 Meta laser-scanning confocal microscope. The confocal detector gain, amplifier offset and amplifier gain were set at identical levels for all FITC and TRITC images of wild-type and mutant receptors in order that expression intensities could be compared. The scanning slice depth was set to 1.2 μm providing maximal EGFP fluorescence in the cell; this was occasionally associated with the appearance of an apparent halo of TRITC fluorescence.

HUMAN STUDIES.

Family and Case Control Samples

Ethical permission was obtained for these studies from the UK NHS Metropolitan Multicentre Research Ethics Committee. All subjects provided written consent. The family sample consisted of 18 large Caucasian families of 300 individuals multiply affected by 'alcoholism' selected as likely to be informative for genetic linkage analysis ²⁷ . Subject were classified as having the alcohol dependence syndrome (ADS), Research Diagnosis Criteria Alcoholism (RDCA) or as Heavy Drinking (HD) based on the Clinical Alcoholism Interview Schedule, the Schedule for Affective Disorders and Schizophrenia - Lifetime version (SADS-L) and the Royal College of Psychiatrists thresholds for heavy drinking of 14 units (112 g) per week for women or 21 units (168) per week for men over at least one month (Royal College of Psychiatrists, 1986). All subjects were interviewed by two independent psychiatrists and a consensus categorization agreed. One hundred and seventy-six family members were genotyped for this study classified as normal (50), heavy drinking (HD) (22), RDCA (50) and alcohol dependence syndrome (ADS) (54).

The case control sample consisted of 360 consecutive attenders at an alcohol problems service (267 men 93 women: median [range] age 53 [23-78] years) who fulfilled the International Classification of Diseases criteria (ICDlO) and DSMIV for alcohol dependence syndrome. The majority of these individuals (81%) had

been misusing alcohol for > 20 years; with median alcohol intakes of 125 (48-400) g/day in women and 200 (64-680) g/day in men.

The 450 healthy controls subjects were interviewed with the SADS-L to exclude psychiatric disorder including alcohol misuse/dependence. All the case and control subjects had white Irish, Welsh, Scottish or English ancestry.

Genotyping and laboratory procedures

Fifty nanograms of total genomic DNA was amplified by Polymerase Chain Reaction (PCR) with oligonucleotide primers using standard techniques. For the family linkage study, chromosome 4 markers showing positive lods in the COGA study were genotyped as well as D4S2632, D4S1627, D4S3242, D4S3248 and

D4S3243. In order to test for association between alcohol dependence and

GABRBl a tetranucleotide marker ¹⁴ within intron 8 and a dinucleotide marker ²⁸ within intron 2 were genotyped. For all markers an Ml 3 tail was added to one of the oligonucleotide primers which was used to hybridise with a complementary oligonucleotide pre-labelled with an infra red dye. Gel electrophoresis and laser scanning enabled genotyping of the polymorphic fragment lengths using LI-COR

Model 4200 automated fluorescent DNA sequencers.

Statistical Genetics

The linkage analysis was performed using the classical lod score method and using likelihood-based model-free analysis carried out with the MFLINK program as previously described by us ⁹ . For lod score analyses, the programs MLINK and LINKMAP were used from the FASTLINK package. Three nested affection models were used. These were ADS, RDCA and HD. Each affection model was analysed assuming dominant transmission, recessive transmission and using the model-free method. For dominant models the ^' frequency of the abnormal allele was set to 0.02 and for recessive models to 0.2. The penetrances for normal and abnormal genotypes respectively were set to 0.02 and 0.5 for ADS, to 0.04 and 0.7 for RDCA and to 0.2 and 0.9 for HD. These penetrance values were chosen to produce models approximately consistent with prevalence data from previous

epidemiological research of 0.04, 0.06 and 0.2 for ADS, RDCA and HD respectively. Two-point and three-point analysis was carried out for all affection definitions and transmission models. Classical linkage analysis was carried out under the assumption that locus heterogeneity might be present, yielding an HLOD statistic. The allelic association analysis was carried out with CLUMP which employs an empirical Monte Carlo test of significance and which does not require correction for multiple alleles

GenBank accession numbers. Mouse GABA _A receptor βl proteinNP_032095; GL6679907.

References for Methods

1. Mortensen, M. et al. Activation of single heteromeric GABAA receptor ion channels by full and partial agonists. J. Physiol 557, 389-413 (2004).

2. Connolly, C. N., Krishek, B. J., McDonald, B. J., Smart, T. G. & Moss, S. J. Assembly and cell surface expression of heteromeric and homomeric γ- aminobutyric acid type A receptors. J. Biol. Chem. 271, 89-96 (1996).

Table 1. Properties of wild-type and βl i L ^L 2 ² 8 ^!i S ⁵ R ^K mutant GABA _A receptors expressed in HEK293 cells. Data represent the mean ± s.e. of 3-15 experiments (number of

experiments in parentheses). I _max is the maximal GABA current and I _h0Id signifies the holding current. EC ₅₀ and nπ have their usual meanings (see Supplementary

Subjects Allele frequencies CLUMP tests of association

Allele (bp) 108 110 112 114 116 118 120 122 Tl T2 T3 T4

Healthy controls 1 4 64 319 377 111 7 1 x ² 9.373 5.664 3.490 4.695

(n =442 ) 0% 0% 7% 36% 43% 13% 1% 0% f 0.191 0.225 0.248 0.2 ₅ 9

Alcohol dependents 0 1 65 267 291 67 9 0 P ^b 0.227 0.226 0.062

(n=3 ₅ 0) 0% 0% 9% 38% 42% 10% 1% 0%

Dean 17

Allele (bp) 160 164 168 172 176 180 184

Healthy controls 36 8 146 167 382 135 2 x ² 9.283 9.028 4.850 2.459

(n=438) 4% 1% 17% 19% 44% 15% 0% P ^a 0.158 0.108 0.155 0.647

Alcohol dependents I ₅ 14 117 140 293 121 2 P ^b 0.159 0.155 0.028

(n=351) 2% 2% 17% 20% 42% 17% 0% methods).

Table 2. Allele frequencies and CLUMP analysis of the microsatellite markers

Poly ..25 and Dean .17 in alcohol dependent individuals and healthy controls. Subtests of the CLUMP programme are Tl: Pearson's χ2 statistic of the 'raw' contingency

10 table. T2: The χ2 statistic of a table with rare alleles grouped together to prevent small expected cell counts. T3: The largest of the χ2 statistics of 2x2 tables each of which compares one allele against the rest grouped together. T4: The largest of the χ2 statistics of all possible 2x2 tables comparing any combination of alleles against the rest. ^a p value after Monte Carlo simulation of the data to correct for

15 multiple alleles; ^b uncorrected p value.

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