The invention relates to treatments for depressive disorders based on reducing the extracellular matrix in the hippocampus. The invention also relates to methods of screening for new compounds to treat cognitive impairment in depressive disorders. BACKGROUND OF THE INVENTION

Depression is a common, debilitating disorder. The symptoms of depression are often treated with medication or psychotherapy. Pharmacological agents include serotonin- selective reuptake inhibitors (SSRIs), such as fluoxetine; norepinephrine reuptake inhibitors (NERIs)' combined serotonin-norepinephrine reuptake inhibitors (SNRIs); monoamine oxidase inhibitors (MAOIs); and phosphodiesterase-4 (PDE4) inhibitors. However, even with these options available, many patients fail to respond, or respond only partially to the treatment. Traditional therapies can also have significant side effects, including sexual dysfunction, gastrointestinal disturbances, agitation, insomnia, and weight gain. In addition, the present treatment options do not often address the cognitive impairments associated with depression.

Therefore, there remains a need for the development of improved therapies for the treatment of depression. SUMMARY OF THE INVENTION

An object of the present disclosure is to provide methods of treating an individual suffering from a depressive disorder, preferably major depressive disorder (MDD), comprising administering to an individual in need thereof one or more compounds that alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus of said individual. Accordingly, compounds that alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus of said individual for preparing a medicament for treating an individual suffering from a depressive disorder are also provided. Preferably, cognitive impairment in depressive disorders is treated.

Another object of the disclosure provides methods of treating an individual suffering from a depressive disorder, comprising determining the amount of one or more extracellular matrix (ECM) components in said individual, and administering one or more compounds to an individual having an increased amount of ECM components as compared to a reference, wherein said one or more compounds alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus of said individual. Preferably, the amount of one or more ECM components is determined by detecting said one or more ECM components in the hippocampus, preferably using non-invasive detection. Preferably, the method also comprises administering to said individual a positron emission tomography (PET) compatible tracer, wherein said tracer binds to said one or more ECM components; carrying out a PET scan on said individual; and determining whether the hippocampus of said individual has an increased level of said ECM component as compared to a reference value. Preferably, the level of one or more ECM components is determined by detecting one or more ECM components in a biological sample from said individual, preferably from blood, serum, or cerebrospinal fluid.

Preferably, said one or more administered compounds increase the degradation of the ECM in the hippocampus or decrease the production of the ECM in the hippocampus. Preferably, said one or more compounds is Chondroitinase ABC. Preferably, said one or more administered compounds decreases the amount of at least one ECM

component the ECM of the hippocampus.

Preferably, the alteration of the amount of or the composition of the ECM in the hippocampus increases synaptic plasticity in the hippocampus.

Preferably, wherein at least one of the one or more administered compounds comprises a nucleic acid encoding a membrane-anchored protein that alters the amount of or the composition of the extracellular matrix. An object of the present disclosure is to provide methods of identifying a compound for treating an individual suffering from depressive disorder, comprising administering to a rodent model of social defeat-induced persistent stress (SDPS) one or more compounds and determining the effect of said one or more compounds on said rodent. Preferably, said effect is measured by determining the effect on the extracellular matrix (ECM) in the hippocampus of said rodent, the effect on hippocampal plasticity, and/or the effect on memory, cognitive or affective dysfunction.

An object of the present disclosure is to provide methods of identifying a compound for treating an individual suffering from depressive disorder, comprising contacting hippocampal tissue from a rodent model of social defeat- induced persistent stress (SDPS) with one or more compounds and determining the effect on the extracellular matrix (ECM) and determining the effect of said one or more compounds on said tissue. Preferably, said effect is measured by determining the effect on the

extracellular matrix (ECM) or the effect on plasticity in said tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. SDPS impacts on cognitive and affective behavioral tasks.

a) Rats (>10-week old) were subjected to the social defeat-induced persistent stress (SDPS) paradigm. SDPS rats received a daily social defeat episode (5 min) for 5 days and were subsequently individually housed for ~3 months (Van Bokhoven et al., 2011). Control animals were handled daily for 5 days and were housed in pairs.

Pharmacotherapy (Imi) or behavioral therapy (BT) were applied only during the last three weeks of the individual housing period, until behavioral assessment (object place recognition (OPR; b), announced anticipation to a sucrose reward (AASR; c) or decapitation (†;see Figs 2 and 3) for physiological and biochemical analyses. A control- treatment group (H2O) was included. Independent cohorts of animals were used for behavioral, physiological and biochemical analyses, b) Exploration rate during the test phase of an OPR task. SDPS suppressed spatial memory performance, whereas BT and Imi reversed this effect (treatment: F(2,52)=5.481; treatment x SDPS:

F(2,49)=3.397; n=9-10 for al experimental groups), c) Anticipation towards 5% sucrose expressed as the difference in activity in the CS-US interval post-training compared with pre-training. SDPS significantly suppressed reward anticipation, whereas BT and Imi reversed this effect (SDPS: F(l,35)=3.594, P=0.068; treatment: F(2,34)=3.547; SDPS x treatment: F(2,30)=5.486; n=6 for al experimental groups). Post hoc: *P<0.05, **P<0.001.

Figure 2. SDPS impairs LTP maintenance in the CA1 of the dorsal

hippocampus.

a) Coronal section of the dorsal hippocampus placed on a MED64 electrode array. An electrode in the Schaffer collateral pathway (white) was used as stimulating electrode. Field potentials (fEPSPs) were recorded and averaged for the electrodes in the dendritic field of CA1 (marked x). b) Representative traces from one experiment recorded before (gray) and after (black) LTP induction, c) Time course of percentage change in fEPSP measured before and after high frequency stimulation (2 trains of 100 Hz stimulus), d) Change in fEPSP 40-50 min after LTP induction (gray indicated in c). SDPS significantly suppressed CA1 LTP, whereas Imi and BT reversed this effect (Treatment: F(2,58)=4.563; treatment x SDPS: F(2,54)=2.727; n=7-15 slices from 3-7 rats). Post hoc *P<0.05, **P<0.001.

Figure 3. SDPS increases peri-synaptic expression of ECM proteins in the dorsal hippocampus.

a) Dorsal hippocampus synaptic membrane proteomics analysis using iTRAQ (n=5 per group). Quantification of iTRAQ labeling revealed 37 proteins regulated by SDPS. Among these, the group of extracellular matrix (ECM) proteins were overrepresented in GO analysis. From the 37 proteins, 26 were rescued by behavioral therapy (BT; P<0.1 SDPS_BT vs. SDPS) and of these, 18 were rescued by imipramine (Imi; P<0.1 SDPS_Imi vs. SDPS). From these 18 proteins, the ECM group was again

overrepresented. b) Examples (upper panel) of immunoblot analysis of several ECM proteins for SDPS vs. control (H2O) in an independent set of animals (n=4-6 per group) revealed a trend for general upregulation (lower panel) by SDPS in the synaptic membrane fraction (Fold change: 1.35), a trend for upregulation of Neurocan, and a significant regulation of Phosphacan and Brevican. Blots show the specific protein band (apparent molecular weight), as well as total protein input used for normalization, c) Samples were run adjacent to Con H2O samples. Examples (upper panel) of immunoblot analysis revealed that the SDPS-induced increased expression of Brevican and Phosphacan in the synaptic membrane fraction was rescued by Imi and BT (lower panel, ANOVA for treatment: Brevican, F(2,20)=4.207, P=0.030; Phosphacan, F(2, 19)=4.098, P=0.042; n=6-8 per group). Samples were run adjacent to Con H2O samples. The dotted line indicates the expression level of water-treated controls. Gray bars indicate the SDPS-induced regulation of Brevican and

Phosphacan, as seen in panel b, and bars above indicate P-values for the expression difference vs. these SDPS water-treated animals. Brevican and Phosphacan

upregulation was specific for the synaptic membrane fraction, as the corresponding total homogenate revealed no difference in expression (Brevican, F(l, 12)=0.288, n.s. and Phosphacan, F(l, 12)=1.882, n.s. (n=6-7)). Expression differences between samples are indicated: P<0.1, *P< 0.05, or n.s., non-significant expression differences from water-treated controls (dotted line) are indicated in each bar.

Figure 4. ECM breakdown rescues SDPS-induced impairments of LTP maintenance and memory.

a) Experimental schedule. Following the SDPS paradigm animals were subjected to an OPR test (at -10 weeks) followed by local application of Chondroitinase ABC (ChABC) or Penicillinase (Peni; control) at the dorsal hippocampus. Treatment effect was then estimated by electrophysiology, measuring maintenance of LTP (LTP; 2-3.5 weeks after injection, c) and behaviorally, by examining OPR performance (10-13 days after injection, d) that 4 weeks, albeit that less intense staining is still visible within the CAl and dentate gyrus. Scale bar indicates 500 μπι. b) Paraformaldehyde- fixed coronal sections from the brain of a naive rat were probed with fluorescein labeled WFL after Penicillinase or at several time points after ChABC treatment. WFL staining in control animals (Con Peni acute) is visible in distinct layers of the hippocampus, e.g., high expression in CA2 (open arrowhead), mostly in the dentate gyrus (molecular and granular layer), and low expression in CAl (stratum lacunosum- moleculare, radiatum, and oriens), whereas the pyramidal layer was devoid of WFL staining. ChABC injection (0.03 U per side) aimed at the CAl area of the

hippocampus led to ECM breakdown that spread mostly medial and to some extent lateral, for which the boundaries are indicated (white arrowhead). Note that at 4 weeks after ChABC treatment there is some recovery of the WFL-staining. c).

Example of coronal section of the dorsal hippocampus with external stimulator (black rod), and recording electrode (arrowhead). Change in fEPSP 40-50 min after LTP induction showed that the suppression of CAl LTP by SDPS could be completely rescued by ChABC treatment (ANOVA: SDPS: F(l,46)=10.028; treatment: F(l,46)=3.956; SDPS x Treatment: F(l,43)=5.969; n=12-13 slices from 6-8 animals), d) Exploration rates during the test phase of an OPR task. SDPS suppressed spatial memory performance at 12 weeks after the start of the SDPS paradigm, and was rescued by ChABC treatment in SDPS animals, and was slightly impaired in control animals (ANOVA: treatment: F(l,35)=3.121; treatment x Group: F(l,32)=7.035).

Presence of a spatial memory (P<0.05 vs. fictional control (Akkerman et al., 2012)) memory is indicated ($). Post hoc *P<0.05, **P<0.01 (vs. all other groups, c).

Figure 5. Individual housing has no effect on hippocampus-dependent memory Interaction ratio during the test phase of an allocentric object place task. Animals that were individually housed for 3 months (n=16) showed similar memory performance compared with control paired housed animals (n=9; *P=0.021), and showed a trend ( P=0.080) for difference compared with the social defeat group (n=10). The individually housed group showed significant discrimination of the novel vs.

familiar place of the object (P=0.048) similar as the control group (P=0.013), whereas the social defeat group showed no discrimination (P=0.8811).

Figure 6. SDPS does not affect novel object place recognition, sucrose preference and intake, a) Discrimination index during the test phase of a novel object recognition task. The SDPS paradigm did not affect performance on this task. b,c) Social defeat did not affect sucrose preference (b), neither did it affect sucrose intake. Preference = (sucrose intake - water intake) / total fluid intake (%).

Figure 7. Individual housing has no effect on hippocampus-dependent LTP. Average change (± SEM) from baseline (dotted gray line) in fEPSP 40-50 min after LTP induction in the CA1 region of the dorsal hippocampus. Individual housing had no effect on the maintenance phase of LTP and shows similar increased fEPSPs as controls that were analyzed at the same time (right). For comparison (left), the fEPSP change is shown for the controls and social defeat group (P=8.09* 10 ⁵ ; c.f. Fig. 2). Thus, although individual housing after social defeat is a necessary component to induce a depressive -like state in animals that underwent social defeat stress on the long-term, it does not by itself result in a severe stress experience affecting basal physiology (see Fig. 10) or hippocampus-dependent memory and neuronal plasticity. Figure 8. Synaptic NMDA and AMPA receptor expression are not affected long-term after social defeat. Quantitative immunoblot analysis of glutamate receptor subunits revealed that from the NMDA (left) and the AMPA (right) receptor subunits no difference was measured in the synaptic fraction of the dorsal

hippocampus long-term after social defeat stress. Data presented are mean+SEM; n=6 for all groups (typical examples shown in inserts above each sample).

Figure 9. ChABC treatment results in decreased WFL staining 12 days after injection. Rats from the SDPS group were treated with either Penicillinase (left) or ChABC (right) and were perfused after 12 days. Coronal sections were probed with fluorescein labeled WFL. WFL staining is visible in the CA2 (open arrowhead) and in the stratum lacunosum-moleculare of CAl and granular layer of the dentate gyrus (}). After ChABC injection (0.03 U per side) aimed at the CAl area of the hippocampus, WFL-positive levels in the CA2 and strata of the CAl/dentate gyrus were less intense. Staining in the corpus callosum, as well as the perineuronal nets in the retrosplenial cortex (arrow) are visible in both pictures and are not affected by ChABC treatment. Scale bar indicates 250 μηι.

Figure 10. Acute stress effects are only observed in the first three weeks of the SDPS paradigm, a) Mean levels of plasma corticosterone measured acutely (2.5 month-old animals) after a social defeat episode, or long-term (6 month-old animals), i.e., at the end of treatment with water (H2O), imipramine (Imi) or behavioral therapy (BT), in animals that underwent social defeat or served as controls, as well as in animals that were individually housed (and their respective paired-housed controls). Although the acute stress of social defeat altered corticosterone levels, long after social defeat no difference between any of the groups was observed. Individual housing for 3 months did not affect corticosterone levels, b-d) Various physiology parameters were acquired over the first five weeks after the start of the SDPS paradigm. The acute stress of social defeat trials reduced body weight (b), food intake (c), and water intake (d). These parameters were normalized after 4, 3, and 2 weeks, respectively, indicative of the absence of an acute stressor in the SDPS paradigm, and hence the validity of this model to capture behavioral and physiological changes related to a depressive-like state rather than the acute effects of a stressor. *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS

The disclosure is based, in part, on the finding that the extracellular matrix is upregulated in a rodent model of depression. The SDPS (social defeat-induced persistent stress) paradigm described in the examples elicits hippocampus-dependent memory impairments, as well as reduced maintenance of induced long-term potentiation (LTP) in the hippocampus. The SDPS paradigm induces an enduring depressive-like state, including deficits in short-term spatial memory, that can be reversed by pharmacotherapy and behavioral therapy (examples 1 and 2). This rodent model thus mimics the affective and the cognitive part of depressive disorders in humans.

Surprisingly, the SDPS paradigm also alters the expression of proteins of the extracellular matrix (example 3). ECM protein expression could be normalized after behavioral therapy or by treatment with the tricyclic antidepressant imipramine. Intervention at the level of the ECM in vivo also mediates the restoration of SDPS- induced impairments of hippocampus-dependent plasticity and memory.

ECM proteins are highly expressed in the brain and are produced by both neurons and glial cells. During postnatal maturation of neuronal circuits these ECM pack into netlike structures, termed perineuronal nets (PNN), localized around a subset of neurons. The main component of ECM in the brain is polysaccharide hyaluronic acid. It acts as a backbone to engage proteoglycans and other glycoproteins into ECM structures. These protein components of the hyaluronic-based ECM are chondroitin sulfate proteoglycans (CSPGs; e.g., aggrecan, brevican, neurocan, NG2, versican and phosphacan), tenascins (e.g., Tnc and Tnr), and so-called hyaluronan link proteins (e.g., Haplnl) (Bandtlow and Zimmermann 2000; Yamaguchi 2000; Rauch 2004). Agrin is a heparan sulfate proteoglycan component of the extracellular matrix. CD44 is a cell surface receptor with its principle ligand being hyaluronic acid (HA) (Ponta, et al., Nature Rev. Mol. Cell. Biol. 2003, 4, 33-45; herein incorporated by reference). Additional components of the ECM include perlecan, laminin, fibronectin, collagen, pentraxins, pleiotrophin/HB-GAM, reelin, thrombospondin. Regulation of the ECM includes not only the production of new ECM but also controlling the rate at which the ECM is degraded. Hyaluronidases are a family of enzymes that degrade hyaluronic acid. Matrix metallopro teases (MMP) are enzymes involved in the proteolysis of the ECM, which themselves can be inhibited by the tissue inhibitor of metalloproteinase family (TIMP1, -2, -3, and -4). Neurotrypsin is a brain-specific serine protease responsible for agrin cleavage. Tissue-type plaminogen activator (tPA) is a serine protease. It has been demonstrated that application of tPA to the brain has effects on synaptic plasticity. The inhibition of CSPGs is also an important means to affect the ECM.

Chondroitinase ABC (ChABC) is an enzyme that cleaves glycosaminoglycan side chains from a protein core. CSPGs are involved in inhibiting synaptic plasticity, and treatment with ChABC after spinal core injury reduced such inhibition (reviewed in Busch and Silver, Current Opinion in Neurobiology 2007, 17: 120-127). CSPG function can also be affected by disrupting the glycosylation of CSPG. Such glycosylation is carried out by Xylosytransferase-1 (XT- 1). Inhibition of XT- 1, for example via a DNA enzyme, reduces GAG chains (Hurtado et al. Brain 2008 131: 2596-2605). Such a strategy promoted spinal cord repair. Antibodies that bind sulphation motifs of CSPGs have also been demonstrated to inhibit CSPG function (Gama et al. Nat. Chem. Biol. 2006 2:467-473; the disclosure and in particular the antibodies described therein are hereby incorporated by reference). The synthesis of CSPGs can also be affected by inhibiting enzymes such as chondroitin 4 sulphotransferase.

Lysyl oxidase (LOX or protein-lysine 6-oxidase) is an enzyme that cross-links collagens or elastins in the ECM. Beta-aminopropionitrile (BAPN) is an irreversible inhibitor of LOX that has been used to reduce breast cancer metastasis.

In one aspect, the disclosure provides a method of treating an individual suffering from a depressive disorder comprising administering to an individual in need thereof one or more compounds that alter the amount of or the composition of the

extracellular matrix (ECM) in the hippocampus of said individual. Depressive disorders describe conditions characterized by a long-lasting depressed mood and/or marked loss of interest or pleasure (anhedonia). Such disorders include mild depression (dysthymia), major depressive disorder (MMD), seasonal affective disorder, bipolar disorder, cyclothymic disorder, neurotic depression, atypical

(reactive) depression, and post- traumatic stress disorder (PTSD). Preferably, said disorder is MMD.

Major depressive disorder may also be referred to as clinical depression, major depression, unipolar depression, unipolar disorder or recurrent depression. Major depressive disorder (MDD) is a complex neuropsychiatric disorder that is

characterized by persistent negative mood, a multifaceted anhedonic state, and impaired cognitive function(Bradley and Power, 1988; Der-Avakian and Markou, 2012; Gould et al., 2007; Treadway et al., 2012; Zakzanis et al.,

I !)!)8) K X Ii K 1 K R ^' i . It may also be referred to as clinical depression, major depression, unipolar depression, unipolar disorder or recurrent depression. MDD is considered to be the second leading cause of disability of all somatic and psychiatric disorder world-wide. According to the World Health Organization it globally accounts for more lost productivity than any other psychiatric disorder(WHO-depression, 2012). A substantial part of this is attributed to the cognitive deficits that accompanies depression, as specified in the diagnostic criteria of both DSM-IV and ICD- 10, including impairments related to attention, working and episodic memory and executive functions(Gould et al., 2007; McClintock et al., 2010; Murrough et al., 2011; Zakzanis et al., 1998), that could persist beyond recovery of the depression(Baune et al., 2010). The debilitating properties of MDD in the affective and cognitive domains pose questions concerning the underlying neurobiological mechanisms and efficacy of current therapies. Various subtypes of depression are described in, e.g., DSM IV.

The term "bipolar disorder" is used to describe a mood disorder that is characterized by extreme variations in mood, from mania and/or irritability to depression. Diagnosis of bipolar disorder is described in, e.g., DSM IV. Bipolar disorders include bipolar disorder I (mania with or without major depression) and bipolar disorder II

(hypomania with major depression), see, e.g., DSM IV. The term "dysthymic disorder" is used to describe a mood disorder characterized by a variety of depressive symptoms in a patient are not numerous or severe enough to qualify for major depressive disorder. In both disorders, individuals may have changes in their sleep patterns or appetite, low energy or fatigue, low self-esteem, poor concentration or difficulty making decisions, or hopelessness during periods of depressed mood. However, individuals with dysthymic disorder may have more prominent cognitive or interpersonal symptoms, such as pessimism, feelings of inadequacy, and social withdrawal. Posttraumatic stress disorder (PTSD) is a chronic psychiatric disorder that is triggered by extreme psychological trauma, including rape, exposure to warfare, and even cancer. It was described in veterans of the American Civil War, and has been called "shell shock," "combat neurosis," and "operational fatigue." Symptoms of the disorder may include nightmares, flashbacks, emotional detachment or numbing of feelings (emotional self-mortification or dissociation), insomnia, avoidance of reminders and extreme distress when exposed to the reminders ("triggers"), loss of appetite, irritability, hypervigilance, memory loss (may appear as difficulty paying attention), excessive startle response, clinical depression, and anxiety. Seasonal affective disorder (SAD) is a form of depression that occurs in relation to the seasons, most commonly beginning in winter. This disorder is marked by symptoms of depression profound enough to seriously affect work and relationships.

In preferred embodiments, the individual suffering from a depressive disorder is not suffering from dementia or Alzheimer's disease, i.e., the depressive disorder is not due to dementia or Alzheimer's disease.

The disclosure provides methods using one or more compounds that alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus of said individual. Preferably, these methods induce an alteration of the amount of or the composition of the ECM in the hippocampus and thereby increase synaptic plasticity in the hippocampus. Preferably, the alteration of the amount of or the composition of the ECM in the hippocampus slows or prevents the progression of cognitive and/or memory impairment associated with the depressive disorder. It is within the purview of a skilled person to recognize whether a treatment slows or prevents the progression of cognitive and/or memory impairment in a particular individual as compared to the normal progression of such disorder in an untreated population of depressed subjects. In preferred embodiments, said or more compounds decrease the ECM in the hippocampus by at least 10, 20, 30, 40, or 60%. It is clear to a skilled person that the ECM should not be completely ablated, but rather reduced. For example, a

combination of high dose of both chondroitinase and hyaluronidase would be expected to completely ablate the ECM functionally. Such treatment would be expected to negatively affect memory, whereas low to moderate dose would be beneficial.

Preferably, said or more compounds do not affect the ECM outside of the

hippocampus. Such localized effect may be the result of, for example, localized administration to the hippocampus and/or targeting the one or more compounds to the hippocampus (for example expressing said compounds under the control of a hippocampal specific promoter).

Preferably, said individual is a mammal, more preferably a human. Compounds which alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus of said individual include those that decrease the amount or composition of ECM proteins and/or hyaluronic acid. Preferably, said compounds decrease the amount or composition of chondroitin sulphate proteoglycans (preferably selected from aggrecan, brevican, neurocan, NG2, versican or phosphacan); hyaluronic acid; TIMP1; TIMP2; TIMP3; TIMP4; CD44, lysyl oxidase; perlecan; laminin;

fibronectin; collagen; tenascins (such as Tenascin-C and Tenascin-R); pentraxins; pleiotrophin/HB-GAM; hyaluronan link proteins (e.g., Haplnl); reelin;

thrombospondin; heparin-sulfate proteoglycan agarin; Xylosytransferase- 1; and chondroitin 4 sulphotransferase. More preferably said compounds decrease the amount or composition of chondroitin sulphate proteoglycans (preferably selected from aggrecan, brevican, neurocan, NG2, versican or phosphacan); hyaluronic acid; CD44, and hyaluronan link proteins (e.g., Haplnl). More preferably said compounds decrease the amount or composition of chondroitin sulphate proteoglycans, as can be experimentally verified by WFA- or WFL staining in post-mortem brains.

Such decrease in amount or composition can include a reduction in the expression or secretion of an ECM protein or hyaluronic acid or the increase in (partial)

degradation. It also includes a disruption in the post-translational modifications of an ECM protein. Preferably, said compounds decrease the amount or composition of one or more chondroitin sulphate proteoglycans. Preferably, said amount or composition is altered using a ChABC or an enzyme capable of cleaving glycosaminoglycan side chains from a protein core.

The disclosure further provides pharmaceutical compositions useful in the treatment of depressive disorder comprising the one or more compounds for altering the amount of ECM or the composition of the ECM in the hippocampus and a pharmaceutically acceptable excipient.

Preferably, the compound is an inhibitor of one or more of the following, chondroitin sulphate proteoglycans (preferably selected from aggrecan, brevican, neurocan, NG2, versican or phosphacan); hyaluronic acid; TIMP1; TIMP2; TIMP3; TIMP4; CD44 (e.g., an inhibitory anti-CD44 antibody), lysyl oxidase; perlecan; laminin; fibronectin;

collagen; tenascins (such as Tenascin-C and Tenascin-R); pentraxins;

pleiotrophin/HB-GAM; hyaluronan link proteins (e.g., Haplnl); reelin;

thrombospondin; heparin-sulfate proteoglycan agarin; Xylosytransferase- 1; and chondroitin 4 sulphotransferase; and/or a polypeptide or a functional fragment thereof or a nucleic acid encoding said polypeptide or functional fragment thereof selected from chondroitin ase ABC, hyaluronidase, a matrix metalloprotease, neurotrypsin, Tissue-type plaminogen activator (tPA), an antibody that bind sulphation motifs of CSPGs. Preferably, the compound decreases the function or expression of one or more chondroitin sulphate proteoglycans (CSPGs). Preferably the compound is a

polypeptide or a functional fragment thereof or a nucleic acid encoding said

polypeptide or functional fragment thereof selected from chondoitinase ABC, an antibody that bind sulphation motifs of CSPGs, an inhibitor of Xylosytransferase- 1 (XT-1), and an inhibitor of chondroitin 4 sulp ho transferase. Preferably, said ChABC is a mammalian ChABC, more preferably a human ChABC. The ChABC can be any enzyme having chondroitin-sulfate-ABC endolyase activity.

In a preferred embodiment, the compound promotes the degradation or destabilzation of the ECM. Such compounds are known in the art and in preferred embodiments said compound is a polypeptide or a functional fragment thereof or a nucleic acid encoding said polypeptide or functional fragment thereof selected from chondroitinase ABC, hyaluronidase, a matrix metallopro tease, neurotrypsin, Tissue-type plaminogen activator (tPA), an antibody that bind sulphation motifs of CSPGs; or an inhibitor of hyaluronic acid, or an inhibitor of lysyl oxidase, TIMP1, TIMP2, TIMP3, or TIMP4.

In a preferred embodiment, the compound decreases the production of the ECM, in particular decreases the production of ECM proteins and/or decreases post- translational modification thereof. Such compounds are known in the art and in preferred embodiments said compound is an inhibitor of perlecan, laminin, fibronectin, collagen, xylosytransferase- 1, chondroitin 4 sulphotransferase, tenascins, such as Tenascin-C (Tnc) and Tenascin-R (Tnr); pentraxins, pleiotrophin/HB-GAM, reelin, hyaluronan link protein (e.g., Haplnl); thrombospondin, heparin-sulfate proteoglycan agarin, and a chondroitin sulphate proteoglycans (CSPGs), preferably a CSPG selected from aggrecan, brevican (Bean), neurocan (Ncan), NG2, versican (Vcan) and phosphacan. Preferred hyaluronidases include HYAL1, HYAL2, HYAL3, and PH-20/SPAM1.

Preferred metalloproteinases include MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMPll, MMP12, MMP13, MMP14, MMP15, MMP16, MMP23, and MMP24, more preferably MMP9 Preferably the compound is ChABC or an inhibitor of V-can or Tenascin-C. Preferably the compound is ChABC or an inhibitor of V-can. Preferably the compound is not Tenascin-C and/or a lysyl oxidase inhibitor.

Preferably, the compound is not ChABC or a CD44 inhibitor. Preferably, the compound is not Tenascin-C, a lysyl oxidase inhibitor, ChABC, or a CD44 inhibitor. Preferably, the compound is not Tenascin-C, a lysyl oxidase inhibitor, or ChABC.

Inhibitors include polypeptides, small molecules (e.g., β-aniinopropionitrile), and nucleic acid based inhibitors. Preferably, the inhibitor of a protein is a nucleic acid molecule such as an antisense oligonucleotide, an RNA interference molecule or a binding molecule (e.g., an antibody or antibody fragment) that binds to the protein interferes with its function. A preferred inhibitor of hyaluronic acid is hyaluronidase. A preferred inhibitor of CD44 is an anti-CD44 antibody or antigen binding fragment thereof.

Preferably, the compounds are (polypeptides. In order to facilitate uptake into the cells, the (polypeptides may be modified. For example, the peptides may be fused to cell-penetrating peptides such as Tat and Penatratin (see, e.g., Richard et al. J Biol Chem. 2003 Jan 3;278(l):585-90).

Preferably the compounds are nucleic acids encoding polypeptides in which the polypeptide is membrane anchored. Preferably, said protein is selected from chondroitinase ABC, hyaluronidase, a matrix metalloprotease, neurotrypsin, Tissue- type plaminogen activator (tPA), and an antibody, preferably an antigen binding fragment, that bind sulphation motifs of CSPGs. The addition of a membrane anchor is thought to limit the spread of the expressed protein so that the effects are localized to the hippocampus. Preferably, said nucleic acids also comprise a hippocampal specific promoter.

Accordingly, the disclosure further provides the use of an isolated nucleic acid encoding a recombinant protein, which is membrane anchored upon expression in a cell, wherein said recombinant protein alters the amount or the composition of the extracellular matrix (ECM) in the hippocampus. Preferably the protein and the membrane anchor are from different proteins, i.e., a chimeric protein is expressed. Said nucleic acids are useful in the treatment of depressive disorder. In some embodiments the compound contains an endogenous membrane anchor (e.g., GPI-linked MMP17, type I transmembrane MMP14, and type II transmembrane MMP23). In other embodiments, the compound is a chimeric protein comprising an exogenous membrane anchor, e.g., the type I transmembrane MMP14 fused to neurotrypsin.

Suitable means to anchor a polypeptide to the membrane are known in the art and include the transmembrane domain from a membrane protein (e.g., the

transmembrane region of the HLA class I or CD4 proteins) as well as GPI-anchors. In some embodiments, the polypeptide comprises a GPI-signal peptide. Such a signal peptide is a C-terminal amino acid sequence of a polypeptide which consists of one amino acid to which the GPl-anchor will be attached, an optional spacer peptide, and a hydrophobic peptide. Almost all of this signal peptide, i.e. the optional spacer peptide and the hydrophobic peptide, is removed posttranslationally by the enzyme GPI- transaminase and a bond between the amino group of the core ethanolamine phosphate of the GPl-anchor and the amino acid to which the GPl-anchor is attached is formed. Methods for preparing such recombinant proteins are known in the art and additional details may be found in WO2007131774, which is hereby incorporated by reference.

Preferably, the nucleic acids encoding polypeptides as disclosed herein also contain a signal sequence directing the polypeptide to the cell membrane, usually a N-terminal stretch of around 20 amino acids. The nucleic acids may also contain a myristoylation signal (see, e.g., Vilas et al. PNAS 2006 103;6542-6547). Myristoylation localizes the protein to the cell membrane.

The compounds may be provided as isolated nucleic acids. As used herein, "isolated" means that the polypeptides are separated from other components of either (a) a natural source, such as a plant or cell, preferably bacterial culture, or (b) a synthetic organic chemical reaction mixture. Said nucleic acids may be operably linked to additional sequences such as promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention. In a preferred embodiment, the nucleic acid is operably linked to a hippocampal promoter. Such promoters are described, e.g., in Valen et al. Genome Res. 2009 Feb; 19(2):255-65 and the Dlx5/6 promoter described in Delzor et al. Human Gene Therapy Methods 2012 23:242-254. In some embodiments, the compound is an inhibitor of a protein. Preferably the inhibitor is a peptide that interferes with the catalytic sites or function of its target protein. Preferably the inhibitor is a nucleic acid molecule whose presence in a cell causes the degradation of or inhibits the function, transcription, or translation of its target gene, e.g., Bean, in a sequence-specific manner. Exemplary nucleic acid molecules include aptamers, siRNA, artificial microRNA, interfering RNA or RNAi, dsRNA, ribozymes, antisense oligonucleotides, and DNA expression cassettes encoding said nucleic acid molecules.

Preferably, the nucleic acid molecule is an antisense oligonucleotide. Antisense oligonucleotides (ASO) generally inhibit their target by binding target mRNA and sterically blocking expression by obstructing the ribosome. ASOs can also be used for "exon-skipping". Exon-skipping oligonucleotides bind to pre-mRNA and modulate splicing such that one or more exons are skipped in the resulting mRNA. Exon- skipping may lead to an in frame deletion resulting in a truncated protein or protein lacking internal amino acids or skipping may lead to a premature stop codon resulting in nonsense-mediated decay. The design of such oligonucleotides is well-known in the art (see, e.g., Aartsma-Rus et al Mol Ther 17(3):548 (2009)).

ASOs can also inhibit their target by binding target mRNA thus forming a DNA-RNA hybrid that can be a substance for RNase H. ASOs may also be produced as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, oligonucleotide mimetics, or regions or portions thereof. Such compounds have also been referred to in the art as hybrids or gapmers. Methods for designing and modifying such gapmers are described in, for example, U.S. Patent Publication Nos. 20110092572 and 20100234451. Preferred ASOs include Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), and morpholinos. Preferably, the nucleic acid molecule is an RNAi molecule, i.e., RNA interference molecule. Preferred RNAi molecules include siRNA, shRNA, and artificial miRNA. siRNA comprises a double stranded structure typically containing 15 to 50 base pairs and preferably 19 to 25 base pairs and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. An siRNA may be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure. As used herein "shRNA" or "small hairpin RNA" (also called stem loop) is a type of siRNA. In one embodiment, these shRNAs are composed of a short, e.g. about 10 to about 25 nucleotide, antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand. Alternatively, the sense strand can precede the nucleotide loop structure and the antisense strand can follow.

The design and production of siRNA molecules is well known to one of skill in the art (Hajeri PB, Singh SK. Drug Discov Today. 2009 14(17- 18):851-8). Methods of administration of therapeutic siRNA is also well-known to one of skill in the art (Manjunath N, and Dykxhoorn DM. Discov Med. 2010 May;9(48):418-30; Quo J et al, Mol Biosyst. 2010 Jul 15;6(7): 1143-61). siRNA molecule comprises an antisense strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense strand is complementary to a RNA sequence or a portion thereof encoding.

Artificial miRNA molecules are pre-miRNA or pri-miRNA comprising a stem-loop structure(s) derived from a specific endogenous miRNA in which the stem(s) of the stem-loop structure(s) incorporates a mature strand-star strand duplex where the mature strand sequence is distinct from the endogenous mature strand sequence of the specific referenced endogenous miRNA. (See, e.g., U.S. Patent Publication Nos. 20050075492 and 20100292310 for the design and production of artificial miRNA molecules).

RNA interference refers to a decrease in the mRNA level in a cell for a heterologous target gene by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in the cell without the presence of the, e.g., miRNA or siRNA interference molecule

RNAi molecules may also include chemical analogues such as, e.g., 2'-0-Methyl ribose analogues of RNA, DNA, LNA and RNA chimeric oligonucleotides, and other chemical analogues of nucleic acid oligonucleotides.

The nucleic acid molecule inhibitors may be chemically synthesized and provided directly to cells of interest. The nucleic acid compound may be provided to a cell as part of a gene delivery vehicle. Such a vehicle is preferably a liposome or a viral gene delivery vehicle. Liposomes are well known in the art and many variants are available for gene transfer purposes. Vectors comprising said nucleic acids are also provided. A "vector" is a recombinant nucleic acid construct, such as plasmid, phase genome, virus genome, cosmid, or artificial chromosome, to which another DNA segment may be attached. The term "vector" includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers. Viral vectors include retrovirus, adeno-associated virus (AAV), pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr and adenovirus vectors. Vector sequences may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.). Lentiviruses have been previously described for transgene delivery to the hippocampus (van Hooijdonk BMC Neuroscience 2009, 10:2) There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11:205- 210 (1993)).

Cells comprising said nucleic acids or vectors comprising nucleic acids are also provided. The method of introduction is largely dictated by the targeted cell type include, e.g., CaP04 precipitation, liposome fusion, lipofectin, electroporation, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, , viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. The nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction, outlined below), or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.). Such cells are useful for producing isolated polypeptides which may be used in the methods described herein.

The compounds as described herein may be provided as isolated (polypeptides.

Preferably, via conventional techniques, the (polypeptides are synthesized

(chemically or in vitro) and/or purified.

Polypeptides as described herein may be produced by culturing a host cell

transformed with an expression vector containing nucleic acid encoding a dominant negative polypeptide. Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, Pichia pastoris, etc. Preferably, said polypeptides are expressed in mammalian cells. Mammalian expression systems are also known in the art, and include retroviral systems.

Suitable cell types include tumor cells, Jurkat T cells, NIH3T3 cells, CHO, and Cos, cells.

In some embodiments, the inhibitors described herein are antibodies, e.g., antibodies that block the sulfation motifs of CSPGs. As used herein, the term "antibody" includes, for example, both naturally occurring and non-naturally occurring antibodies, polyclonal and monoclonal antibodies, chimeric antibodies and wholly synthetic antibodies and fragments thereof, such as, for example, the Fab', F(ab')2, Fv or Fab fragments, or other antigen recognizing immunoglobulin fragments. Methods of making antibodies are well known in the art and many suitable antibodies are commercially available. Preferably, the antibodies disclosed herein include antigen binding fragments (e.g., Fab', F(ab')2, Fv or Fab fragments).

The disclosure further provides compositions comprising a compound that alters the amount of or the composition of the extracellular matrix in the hippocampus and a pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient is also provided. Such pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000.

When administering the pharmaceutical preparations thereof to an individual, it is preferred that the compound is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Preferred are excipients capable of forming complexes, vesicles and/or liposomes that deliver such a compound as defined herein in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients comprise polyethylenimine (PEI) or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, ExGen 500, synthetic amphiphils (SAINT- 18), lipofectin™, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver such compounds, to a cell.

In a preferred embodiment of the methods, a compound is provided directly to the hippocampus. The compound may be delivered by way of a catheter or other delivery device having one end implanted in a tissue, e.g., the brain by, for example, intracranial infusion. Such methods are known in the art and are further described in U.S. Publications 20120116360 and 20120209110, which are hereby incorporated by reference.

A compound as described herein may also be administered into the cerebral spinal fluid. Such compounds are preferably linked to molecules that preferentially bind hippocampal cells (e.g., molecules that bind hippocampal specific cell surface molecules).

Methods that use a catheter to deliver a therapeutic agent to the brain generally involve inserting the catheter into the brain and delivering the composition to the desired location. To accurately place the catheter and avoid unintended injury to the brain, surgeons typically use stereotactic apparatus/procedures, (see, e.g., U.S. Pat. No. 4,350, 159) During a typical implantation procedure, an incision may be made in the scalp to expose the patient's skull. After forming a burr hole through the skull, the catheter may be inserted into the brain.

Other delivery devices useful with methods disclosed herein include a device providing an access port, which can be implanted subcutaneously on the cranium through which therapeutic agents may be delivered to the brain, such as the model 8506 ICV Access Port and the 8507 Intraspinal Port, developed by Medtronic, Inc. of Minneapolis, Minn. Actual dosage levels of the pharmaceutical preparations described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start with doses of the compounds described herein at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. The disclosure also contemplates the treatment of individuals having a depressive disorder, comprising conjointly treating an individual in need thereof with 1) one or more compounds that alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus of said individual and 2) a treatment of depressive disorder. Said treatments for depressive disorder include psychotherapy,

pharmacological agents, and electroconvulsive therapy. Pharmacological agents include serotonin-selective reuptake inhibitors (SSRIs), such as fluoxetine;

norepinephrine reuptake inhibitors (NERIs)' combined serotonin-norepinephrine reuptake inhibitors (SNRIs); monoamine oxidase inhibitors (MAOIs); and

phosphodiesterase-4 (PDE4) inhibitors. Compounds that alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus may be

administered simultaneously or sequentially with other pharmacological agents for treating depressive disorders.

In a further aspect, the disclosure provides a method of treating an individual suffering from a depressive disorder, comprising determining the amount of one or more extracellular matrix (ECM) components in said individual, and administering one or more compounds to an individual having an increased amount of ECM components as compared to a reference, wherein said one or more compounds alter the amount of or the composition of the extracellular matrix (ECM) ) in the

hippocampus of said individual. Preferably, an increased amount of ECM components provides an indication of the probability that said individual will respond well to treatment with said compound, in particular that cognitive and/or memory

impairments are likely to improve.

Preferably, said ECM component is selected from perlecan, laminin, fibronectin, collagen, tenascins, such as Tenascin-C (Tnc) and Tenascin-R (Tnr); pentraxins, pleiotrophin/HB-GAM, reelin, hyaluronan link protein (e.g., Haplnl);

thrombospondin, heparin-sulfate proteoglycan agarin, chondroitin sulphate

proteoglycans (preferably a CSPG selected from aggrecan, brevican (Bean), neurocan (Ncan), NG2, versican (Vcan) and phosphacan); chondroitin sulfate; heparin;

glycosaminoglycan, and sulfated glycosarninoglycan. More preferably, said ECM component is selected from brevican, phosphacan, and neurocan.

In a preferred embodiment of the disclosure, the amount of one or more ECM components is determined by detecting one or more ECM components in the hippocampus, preferably using non-invasive detection. In preferred embodiments, the components in the hippocampus may be detected using an extracellular matrix binding compound (ECM-binding compound). ECM-binding compounds suitable for use include compounds that bind, e.g., to perlecan, laminin, fibronectin, collagen, xylosytransferase- 1, tenascins, such as Tenascin-C (Tnc) and Tenascin-R (Tnr); pentraxins, pleiotrophin/HB-GAM, reelin, hyaluronan link protein (e.g., Haplnl); thrombospondin, heparin-sulfate proteoglycan agarin, chondroitin sulphate proteoglycans (preferably a CSPG selected from aggrecan, brevican (Bean), neurocan (Ncan), NG2, versican (Vcan) and phosphacan); chondroitin sulfate; heparin; glycosaminoglycan, and sulfated glycosarninoglycan. In some embodiments, the ECM-binding compound is a small molecule, a peptide, a protein, or an antibody. The term "antibody" includes, for example, both naturally occurring and non-naturally occurring antibodies, polyclonal and monoclonal antibodies, chimeric antibodies and wholly synthetic antibodies and fragments thereof, such as, for example, the Fab', F(ab')2, Fv or Fab fragments, or other antigen recognizing immunoglobulin fragments.

In some embodiments, the ECM-binding compound is a hyaluronan binding protein. Suitable proteins or binding fragments thereof include a CD44 polypeptide, a TSG6 polypeptide, an HABP4 polypeptide, an HAPLN1 polypeptide, an RHAMM

polypeptide, a STAB- 1 polypeptide, a STAB-2 polypeptide,; an XLKDl polypeptide, a brevican polypeptide, an LYVE-1 polypeptide, an aggrecan polypeptide a versican polypeptide, a neurocan polypeptide.

In some embodiments, the ECM-binding compound is Wisteria floribunda agglutin (WFA), Wisteria floribunda lectin (WFL), which labels chondroitin sulfates.

Preferably, said ECM-binding compound further comprises a label allowing for its detection.

In preferred embodiments, positron emission tomography is used to determine the level of one or more ECM components. Accordingly, a method is provided comprising providing an individual with a PET compatible tracer, wherein said tracer binds one or more ECM components, and scanning said individual with a PET scanner.

PET is a well-known technique to determine the distribution of a tracer in vivo. A radioactive tracer is administered to an individual. The individual is then subjected to a scanning procedure using a PET or PET/CT scanner. Quantification of

radiopharmaceutical (radio-tracer) uptake by the target tissue can be performed using methods known in the art (see, e.g., Boellaard R. et al. Journal of Nuclear Medicine, Vol. 45, No. 9, pp 1519-1527, 2004 and U.S. Publications 20100196274 and

20110148861, which are hereby incorporated by reference). The distribution of ECM binding tracer can be determined in "normal" individuals to determine a baseline which can be compared to the level in subject suspected of cognitive/memory impairment. A suitable tracer binds to said one or more ECM components and is preferably a peptide sequence. The tracer is labelled with a short-lived radioactive tracer isotope, such as carbon- 11, nitrogen- 13, oxygen- 15, or fluorine- 18. In other preferred embodiments of the disclosure, the amount of one or more ECM components is determined by detecting one or more ECM components in a biological sample from said individual, preferably from blood, serum, or cerebrospinal fluid, more preferably from blood or serum. As is understood by a skilled person, the detection of ECM components includes and is preferably the detection of peptide fragments of ECM proteins. Peptide fragments are understood as being from 10 to 100 amino acids in length, preferably from 10 to 50 amino acids in length. An ECM component can be detected using a number of assays known to a skilled person. Preferred assays are based on antibody binding to ECM component, in particular peptide fragments of ECM proteins. Commercially available antibodies exist for the detection of ECM proteins and additional antibodies can easily be prepared by methods known in the art. Suitable immunoassays include, e.g., western blots, radio-immunoassay, ELISA (enzyme -linked immunosorbant assay), "sandwich" immunoassay, immunoradiometric assay, gel diffusion precipitation reaction, immunodiffusion assay, precipitation reaction, agglutination assay (e.g., gel agglutination assay, hemagglutination assay, etc.), complement fixation assay, immunofluorescence assay, protein A assay, and immunoelectrophoresis assay. In addition to the use of antibody based assays, assays using other ECM component binders may also be used. For example, an ECM binding peptide can be immobilized on a solid support such as a chip. A biological sample is passed over the solid support. Bound ECM components are then detected using any suitable method, such as surface plasmon resonance (SPR) (See e.g., WO 90/05305, herein incorporated by reference).

Preferably, the method comprises the detection of brevican or a brevican peptide with a brevican specific antibody. Preferably, the method comprises the detection of phosphacan or a phosphacan peptide with a phosphacan specific antibody. Preferably, the method comprises the detection of neurocan or a neurocan peptide with a neurocan specific antibody.

Preferably, an individual is treated with one or more compounds alter the amount of or the composition of the extracellular matrix (ECM) when there is a significant increase in one or more ECM components in said individual as compared to a reference value. The significant increase relates preferably to an increase in the hippocampus, more preferably at the synapses of the hippocampus. The in vivo and in vitro methods provide herein provide an indication as to the changes in the ECM in the hippocampus.

A "significant" increase in a value, as used herein, can refer to a difference which is reproducible or statistically significant, as determined using statistical methods that are appropriate and well-known in the art, generally with a probability value of less than five percent chance of the change being due to random variation. Preferably, a significant increase is at least 20, at least 40, or at least 50% higher than the reference value.

A reference value refers to the level (amount) of a protein in a comparable sample (e.g., from the same type of tissue as the tested tissue, such as blood or serum), from a "normal" healthy subject that does not exhibit a depressive disorder. If desired, a pool or population of the same tissues from normal subjects can be used, and the reference value can be an average or mean of the measurements. A further aspect of the disclosure provides a method of identifying a compound for treating an individual suffering from depressive disorder, comprising administering to a rodent model of social defeat-induced persistent stress (SDPS) one or more compounds and determining the effect of said one or more compounds on said rodent (in vivo screening model). The test compound can be administered to the rodent in the "in vivo screening model" by any route, e.g., orally (such as in the food or drinking water), intranasally, intracranialy, etc. In addition, further methods are provided comprising contacting hippocampal tissue from a rodent model of social defeat-induced persistent stress (SDPS) with one or more compounds and determining the effect on hippocampal tissue (in vitro screening model). Brain slices can be used as models for disease (see for review Cho et al.

Current Neuropharmacology 2007 5: 19-33). Brain slices retain the tissue architecture of specific brain regions and share similar processes as the in vivo brain region. Test compounds can be administered continuously, e.g., by being present in the media, or can be administered over particular times, e.g., by washing out the compound with media.

While any rodent may be used in the SDPS, preferred are social animals, e.g., rats, guinea pigs, and gerbils. A preferred rodent is a rat, in which the SDPS paradigm has been shown. More acute, or short-term changes that affect both the ECM and cognitive function can be modeled in a social defeat paradigm using mice.

Methods to induce the SDPS paradigm in rodents is described in the examples as well as, e.g., Ruis et al., 1999. Briefly, animals are first exposed to social defeat (e.g., by being placed together with a more dominant animal). This normally comprises at least two consecutive days of a single exposure to social defeat (daily exposure), preferably the animals encounter social defeat for at least 5 days. At least after the first encounter with social defeat the animal is housed individually.

The effects of acute social defeat stress in rodents can be observed at least as early as several hours after exposure to the last defeat. These effects include an increase in plasma corticosterone levels, a reduction in synaptic expression of several

glutamatergic receptor subunits in the hippocampus, and a reduction in hippocampus- dependent special memory.

More specifically, in rats, social defeat-induced persistent stress results in a reduction in hippocampus-dependent special memory in the absence of changes in plasma corticosterone levels and glutamatergic receptor subunits in the hippocampus.

Induction of the SDPS paradigm requires that the animals are individually housed for an extended period of time. Preferably, this is for at least 3 weeks, at least 6 weeks, at least 8 weeks, or at least 12 weeks.

Preferably, the effect of the administered compound effect is measured by determining the effect on the extracellular matrix (ECM) in the hippocampus of said rodent, the effect on hippocampal plasticity, and/or the effect on memory, cognitive or affective dysfunction.

The effect on the extracellular matrix can measured for methods using the in vivo screening model or in vitro screening model. The effect can be determined by, e.g., the methods already discussed herein. For example, blood or CSF samples may be taken and the amount of various ECM components measured. Preferably, the amount of various ECM components is directly measured in the hippocampal tissue. For example, total homogenates or, preferably, synaptic membranes of the hippocampus can be isolated. The amount of ECM components, preferably aggregan, phosphacan, or neurocan, can be determined using, for example, an immunoassay. Suitable immunoassays have already been described herein and include, e.g., immunoblotting for the level of protein in a synaptic membrane fraction (see Figure 3). The amount of ECM components can also be visualized using microscopy on tissue sections. The alteration of the ECM can also be measured using Wisteria floribunda agglutin (WFA) or Wisteria floribunda lecithin (WFL), or using antibodies against said ECM

components.

Compounds that increase the ECM or amount of ECM protein expression are not expected to treat depressive disorders and may in fact worsen the conditions.

Conversely, compounds that decrease the ECM or amount of ECM protein expression are expected to treat depressive disorders.

The effect on hippocampal plasticity can measured for the methods that use either the in vivo screening model or in vitro screening model. As used herein, the term

"plasticity" refers to the capacity of the nervous system, or a portion thereof, to change (e.g., to reorganize) its structure and/or function, generally in response to an environmental condition, injury, experience, or ongoing nervous system activity. Plasticity may involve the proliferation of neurons or glia, the growth or movement of neuronal processes and/or alterations in their shape. Plasticity may involve formation of new synaptic connections between neurons and/or strengthening or weakening of existing synaptic connections. Formation of new synaptic connections may involve growth or movement of neuronal processes.

Long-term potentiation (LTP) is a form of synaptic plasticity that results in a lasting increase in synaptic efficacy. It can be characterized by a sustained increase in amplitude of a postsynaptic potential due to a facilitating stimulation. Under LTP, the increase in postsynaptic potential persists well after the facilitating stimulus has subsided. In preferred methods, the effect of the administered compound effect is measured by determining the effect on hippocampal LTP.

In general, for cultured neurons or neural slices, short term potentiation (STP) is characterized by an increased potential that can be measured within 1-10 minutes post induction and substantially decays thereafter. LTP, in contrast, is characterized by an increased potential that can be measured within about 20-30 minutes post induction and is able to be maintained hours thereafter, depending on whether an early form (E-LTP) or late form (L-LTP) is measured. For example, in the case of LTP for hippocampal CA1 neurons following tetanic stimulation as described in the examples (Fig. 2 and 4), LTP is measured 40-50 minutes post induction.

Hippocampal LTP can be measured in a number of ways known to one of skill in the art. The examples include a preferred method of using a microelectrode array (see example in Figure 2) localized at the Schafer collateral pathway, connecting CA3 to CA1 subareas of the dorsal hippocampus, or via an external electrode for providing the stimulus and in the presence of an extracellular electrode for measuring the change in field excitatory postsynaptic potentials (fEPSPs) (see example in Figure 4). A tetanus of extracellular stimulation is applied and fEPSPs are measured extracellularly.

SDPS results in reduced hippocampal LTP. Compounds that restore (partially or completely) the reduction in LTP, such as imipramine and ChABC (see examples), are expected to treat depressive disorders. Compounds that have little or no effect on LTP, or reduce LTP even further, are not expected to treat depressive disorders and may in fact worsen the conditions. The effect on the SDPS-induced effect on memory, cognitive or affective dysfunction can be measured for the methods that use the in vivo screening model. These entail, amongst others: spatial and/or contextual memory, social memory, anhedonia, as measured by, e.g., altered reward intake, anticipation to announced reward, or motivation to obtain a reward.

Depressed patients often suffer from cognitive impairments. SDPS results in hippocampus-dependent cognitive impairments that persist up to three months following social defeat. The effects on memory or cognitive or affective dysfunction can be measured by a number of assays known in the art. Examples include a location recognition task- object place recognition task (OPR) (Dere et al., 2007) ENREF 17 , which measures impairments in short-term spatial memory. Both imipramine and ChABC treatment improved OPR task performance. Deficits in the affective domain, i.e., an anhedonic phenotype (Von Frijtag et al., 2001; Von Frijtag et al., 2000; Von Frijtag et al., 2002), can be measured in "anticipatory reward" assays. SDPS results in the reduced anticipation towards a 5% sucrose solution compared with control rats. This decrease in anhedonia is reversed by both imipramine and ChABC treatment.

Compounds that restore (partially or completely) the memory, cognitive or affective dysfunctions induced by SDPS, such as imipramine and ChABC (see examples), are expected to treat depressive disorders. Compounds with little or no effect on said behaviours, or negatively affect memory, cognitive or affective functions, are not expected to treat depressive disorders and may in fact worsen the conditions.

As used herein, "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The word "approximately" or "about" when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value.

The term "treating" includes prophylactic and/or therapeutic treatments. The term "prophylactic or therapeutic" treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

Therapeutic efficacy can alternatively be demonstrated by a decrease in the frequency or severity of symptoms associated with the treated condition or disorder, or by altering the nature, recurrence, or duration of symptoms associated with the treated condition or disorder. Therapeutic efficacy with the treated condition or disorder, or by altering the nature, recurrence, or duration of symptoms associated with the treated condition or disorder. In this context, "effective amounts," "therapeutic amounts," "therapeutically effective amounts," and "effective doses" of CNS drugs and lithium agents within the invention can be readily determined by ordinarily skilled artisans following the teachings of this disclosure and employing tools and methods generally known in the art, often based on routine clinical or patient-specific factors.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention. EXAMPLES

Example 1. Restoration of SDPS-induced cognitive and affective dysfunction by imipramine and behavioral therapy

We used a rat model for the chronic phase of a depressive-like state, the social defeat- induced persistent stress paradigm (SDPS; Fig. la). We examined hippocampus- dependent cognitive decline, and in particular, impairments in short-term spatial memory, based on a location recognition task (Dere et al., 2007) K R ^' 1 7. the object place recognition task (OPR; Fig. lb). The SDPS paradigm decreased performance in the OPR task (_P=0.034 vs. control; Fig. lb) and SDPS animals displayed no memory for the object location (_P=0.477; vs. fictive control (Akkerman et al., 2012)), whereas controls did (P=0.044). This effect was specific to the SDPS paradigm, as individual housing alone did not affect OPR performance (Fig. 5). Thus, individual housing is, by virtue of interaction with social defeat, likely necessary component to induce the depressive-like state in the SDPS paradigm (Ruis et al., 1999), however, by itself it does not result in a severe depressive-like state affecting hippocampus-dependent memory. SDPS had no effect on object-recognition (Fig. 6), a task that is hippocampus-independent. Thus, novelty preference and discriminative ability were similar among groups and did not confound spatial memory of the OPR task.

Different forms of therapy, namely oral administration of imipramine or behavioral therapy (1 h daily enriched housing) during the last three weeks of the SDPS paradigm (Fig. lb), were able to reverse the lasting SDPS-induced cognitive deficits (ANOVA, treatment: i^0.007; treatment x SDPS: i^0.042). The tricyclic

antidepressant imipramine normalized OPR task performance (P=0.002 vs. water- treated SDPS; -P=0.527 vs. water-treated control) (Fig. lb), illustrating the predictive value of the SDPS model(Vythilingam et al., 2004). Behavioral therapy was able to rescue the SDPS-evoked impairment in spatial memory (P=0.002 vs. water-treated SDPS; -P=0.166 vs. water-treated control; Fig. lb). This is of interest because in humans physical exercise and positive psychosocial activities can reduce depressive symptoms, including deficits in cognitive function, and increase stress resiliency (Southwick et al., 2005). Pharmacological and behavioral therapies had no effect in control animals.

Furthermore, we confirmed that SDPS induces a deficit in the affective domain, i.e., an anhedonic phenotype (Von Frijtag et al., 2001; Von Frijtag et al., 2000; Von Frijtag et al., 2002), as shown by a reduced anticipation towards a 5% sucrose solution compared with control rats (P=0.012; Fig. lc). This increase in anhedonia was reversed to control levels by both imipramine and behavioral therapy (ANOVA, SDPS x treatment: P=0.0093; treatment: P=0.041; PO.001 SDPS BT or SDPS Imi vs. SDPS H ₂ 0; P>0.5 SDPS BT or SDPS Imi vs. Con H2O) (Fig. lc; Supplemental Fig. 2 (Kamal et al., 2010; Von Frijtag et al., 2002)).

Example 2. Restoration of SDPS-induced LTP impairment by

antidepressants and behavioral therapy

As imipramine treatment and the behavioral therapy were both capable of rescuing cognitive performance in a hippocampus-dependent memory task, we questioned whether these forms of treatment were also able to recover reduced hippocampal LTP as observed following SDPS (Artola et al., 2006; Van Bokhoven et al., 2011; Von Frijtag et al., 2001). Using a microelectrode array localized at the Schafer collateral pathway, connecting CA3 to CA1 subareas of the dorsal hippocampus (Fig. 2a), a tetanus of extracellular stimulation was applied and excitatory postsynaptic potentials (fEPSPs) were measured extracellularly. Following LTP induction, control animals showed an increase in average fEPSP amplitude, which at 40-50 min post- tetanus reached 111% (Fig. 2b). Importantly, socially defeated rats displayed a highly significant reduction in LTP when compared with controls (97% SDPS H2O vs. 111% Con H2O; PO.0001), confirming previous reports on SDPS-induced impairments in hippocampal physiology (Von Frijtag et al., 2001). This impairment was not observed after individual housing only (Fig. 7).

Similar to restoring cognitive performance, the reduced potential to maintain LTP following SDPS was reversed by both types of therapy (ANOVA, treatment: P=0.015; SDPS x treatment: P=0.074; Fig. 2c). Both imipramine (P=0.0301 vs. water-treated SDPS) and behavioral therapy (P=0.002 vs. water-treated SDPS (Kamal et al., 2010)) rescued plasticity, further supporting the beneficial influence of both treatments in counteracting depressive-like symptomatology. Imipramine treatment in controls slightly enhanced LTP (121%; P=0.057 vs. water-treated control), whereas behavioral therapy alone had no effect on LTP maintenance (112%; P=0.838 vs. water-treated control).

Example 3. SDPS-induced increased synaptic expression of extracellular matrix proteins is restored by antidepressants and behavioral therapy

We subsequently aimed to identify molecular substrates underlying SDPS-mediated deficits in hippocampus- dependent memory and reduced hippocampal plasticity that could be rescued by pharmacological and behavioral treatment. For this we used an unbiased proteomics analysis of hippocampal synaptic membrane fractions of SDPS and control animals, with and without treatment, in 5 biologically independent samples. In total, 519 proteins were identified with at least 2 distinct peptides with a confidence of > 95%. From these, 37 proteins were significantly regulated by the SDPS paradigm (P<0.05; adjusted for multiple testing (Pavelka et al., 2004)).

Overrepresentation analysis using GO annotation revealed a large contribution of extracellular matrix (ECM) proteins (Fig. 3a; adjusted P-value=0.039), of which all showed upregulation by SDPS (Table 1). Subsequent analysis revealed that of these 37 proteins, a set of 18 proteins were rescued by behavioral therapy or imipramine compared with the SDPS group that was treated with water as control (P<0.1).

Among this group of proteins that might potentially counteract the SDPS effect, again ECM proteins prevailed (Fig. 3a; adjusted P-value=0.025).

In an independent set of animals, the enhanced expression level after SDPS of six core components of adult brain ECM, namely Aggrecan, Phosphacan, Neurocan, Tenascin- R, Brevican and Hyaluronan and proteoglycan link protein 1 (Haplnl), was examined by means of quantitative immunoblotting in the synaptic membrane fraction (Fig. 3b). We validated significant upregulation of Brevican (1.7-fold, P=0.023) and

Phosphacan (1.5-fold, P=0.032), and a trend for upregulation of Neurocan (1.4-fold, P= 0.068) in the SDPS group compared to controls. Together, these data indicate that depressive-like state persistently alters the ECM composition surrounding synapses of the dorsal hippocampus.

To substantiate that dysregulation of ECM components and the subsequent rescue by imipramine and behavioral therapy is at the basis of SDPS-induced changes, we measured the effect of both treatments on Brevican and Phosphacan levels. There was an overall treatment effect (imipramine administration and behavioral therapy) for Brevican and Phosphacan levels in the SDPS group (ANOVA: P=0.030 and P=0.042, respectively). Indeed, in SDPS animals Brevican and Phosphacan levels were not increased compared with their own treatment controls (BT: _P=0.315, _P=0.630; Imi: -P=0.835, -P=0.810, respectively, Fig. 3c). Imipramine normalized both ECM proteins after SDPS to that of water-treated controls (Brevican Imi: P=0.016 vs. water-treated SDPS; Phosphacan Imi: P=0.020 vs. water-treated SDPS). Although with a trend, behavioral therapy was able to rescue the SDPS-induced increase in Brevican

(P=0.090 vs. water-treated SDPS). In contrast, behavioral therapy was not able to normalize Phosphacan after SDPS to that of water-treated controls (P=0.789 vs.

water-treated SDPS). This was mainly due to a non-significant increase of

Phosphacan by behavioral therapy in control animals (P=0.300 vs. water-treated controls).

Importantly, the increased level of these ECM proteins is apparently specific to the synapse, as no increase in Brevican or Phosphacan expression was detected in the corresponding total cell lysates (P=0.755, _P=0.195; Fig. 3d). These data indicated that both therapeutic approaches act at the synaptic expression of the extracellular matrix and counteract the upregulation introduced by the persistent depressive-like state in hippocampal synapses. Example 4. Extracellular matrix degradation by ChABC reverses SDPS- induced deficits in memory and hippocampal plasticity

To assess causality between synaptic ECM protein expression, memory deficits and reduced hippocampal plasticity as displayed by the SDPS paradigm, we locally administered chondroitinase ABC (ChABC), an enzyme performing chondroitin sulphate proteoglycan digestion (Pizzorusso et aL 2(302), in the dorsal hippocampus of an independent group of SDPS animals (Fig. 4a). The magnitude, spread and duration of ECM degradation was measured by immunohistochemical stainings with Wisteria Floribunda Lectin (WFL), a well-established marker for perineuronal nets showing a long-term decrease in WFL-positive staining that, using this low dose, lasts up to a month after ChABC application (Fig. 4b).

Controlling for the stereotactic injection of ChABC, an independent group of animals was injected with Penicillinase (Peni (Pizzonisso et al.. 2002)). an enzyme with no endogenous substrate. At the physiological level, we observed that ChABC administration exerted a significant group-specific effect on the impaired LTP following SDPS (ANOVA, SDPS x ChABC interaction: P=0.019; treatment: P=0.053; SDPS: P=0.003; Fig. 4c). Post-hoc comparisons further demonstrated that whereas SDPS induced a robust reduction in LTP maintenance as compared to Peni-treated controls (control Peni, 123%; SDPS Peni, 106%; P<0.001), ChABC administration reversed this effect (SDPS ChABC 119%; P=0.413 vs. control Peni; P=0.004 vs. SDPS Peni rats). ChABC application had no effect on LTP response as recorded from control animals (control ChABC, 121%; -P=0.755 vs. control Peni).

At the behavioral level following ChABC administration, OPR task performance showed a significant interaction of SDPS x ChABC treatment (ANOVA, P=0.012), indicating that defeated animals and controls showed differential OPR performance due to proteoglycan ECM degradation. In particular, ChABC treatment significantly improved cognitive performance in SDPS rats (P=0.015; 73% SDPS ChABC vs. 51% SDPS Peni), and no group differences between Peni-treated controls and SDPS ChABC-treated rats were observed (P=0.111; 73% SDPS ChABC vs. 63% control Peni). Both ChABC-treated SDPS and Peni-treated control rats showed a clear object location memory (_P=0.001 and 0.012 vs. fictive control (Akkerman et al., 2012), respectively), whereas this was absent in the SDPS Peni group (_P=0.477). Whereas ChABC-treated controls did not differ from the Peni-treated animals (P=0.465; 58% control ChABC vs. control Peni), they displayed slightly impaired discrimination memory P=0.114). Thus, ECM degradation following local hippocampal intervention restores both the physiological and the behavioral depression-reminiscing cognitive impairments.

Using the SDPS paradigm, modeling a perpetuated depression, we show that we can mimic sustained impairments in the affective and cognitive domains in the absence of acute stress (Fig. 10). The data suggests that persistent upregulation of the ECM contributes to a lasting state of depression-associated cognitive decline, and affected maintenance of LTP at CA1 synapses and hippocampus- dependent memory.

Moreover, our data demonstrate that the ECM-mediated, SDPS-induced, memory and plasticity deficits can be completely reversed by antidepressants, behavioral therapy, and, importantly also by local enzymatic degradation of the ECM. Together this implies a causal link between SPDS-increased ECM levels, reduced plasticity potential, and dysfunctional memory processes.

Animals, Materials and methods

Animals in the SDPS paradigm

Wistar rats (age > 10 weeks at the start of the experiment) were habituated (2 weeks), and subsequently exposed to the social defeat-induced persistent stress paradigm (SDPS)(Van Bokhoven et al., 2011), starting with 5 single daily exposures to social defeat stress. From the first defeat episode onwards, SDPS rats were single-housed in macrolon class III cages (lights on at 7:00 p.m. and off at 7:00 a.m.). Food and water were available ad libitum. The individual housing group was housed individually for three months, and control rats were housed in pairs in macrolon class IV cages, and were daily handled during the defeat exposure of the SDPS group. During the last three weeks of this social isolation, rats were treated by oral (gavage) administration of the antidepressant imipramine (20 mg/kg per 0.5 mL water; Sigma- Aldrich, Germany), behavioral therapy (BT), consisting of housing in an enriched environment for one hour every day, or water as control, resulting in six experimental groups. All behavioral, electrophysiological and molecular analyses were performed at the end of treatment in independent groups, unless stated otherwise.

Cognitive and affective behavior

Object place recognition task - Hippocampal-dependent short-term memory was determined with an object place recognition test (Dere et al., 2007; Howland and

Cazakoff, 2010), using a 15-minute retention interval. Discrimination between spatial locations of objects was used as measurement for spatial memory (exploration rate = time spent in active zone (novel location)/ total exploration time (novel + familiar location) in a 4-minute test.

Reward anticipatory behavior - A classical Pavlovian conditioning setup was used to investigate anticipatory behavior, as described earlier(Von Frijtag et al., 2001).

Differences in activity (reflected by frequency or transitions of behavioral elements) displayed before training compared with those after training were used as parameter for reward anticipation.

Reward anticipatory behavior - To investigate the behavioral response to the conditioning stimulus (repetitive sound (keyboard) and light flashes (three times)), animals were observed before training (trial 0) to determine baseline activity, and again after 35 training trials of pairing with a 5% sucrose-reward, using the computer program 'The Observer' (Noldus Information Technology, Wageningen, The

Netherlands). Differences in activity (reflected by frequency or transitions of behavioral elements) displayed before training compared with those after training were used as parameter for reward anticipation.

Sucrose preference - The preference for sucrose (5%) was measured in a two-bottle (sucrose and water) consumption test. Consumption was assessed after 24 h by reweighing the pre-weighted bottles. After 2 days, the consumption test was repeated. In case of social housing, consumption for each subject was set to half of the total consumption. Sucrose preference was expressed as the increase in consumption (gram) relative to water (gram), and this difference was represented as percentage of the total consumption (gram) [100% x (Δ sucrose-water)/total volume sucrose and water consumed].

Corticosterone assay

Trunk blood samples were collected via decapitation between 9:00 am and 11:00 am. Samples were collected into a 7 mL heparin-coated tube (Greiner Bio-One, Monroe, North Carolina) and kept on ice. The samples were spun at 1000 x g for 10 min. Plasma was decanted and stored at -80 °C until the assay was used. Levels of serum corticosterone were assessed using a rat Glucocorticoid (GC) ELISA kit (Cusabio Biotech Co., LTD), according to the manufacturers instructions.

Long-term potentiation (LTP) measurements

Rats were decapitated and subsequently, brains were rapidly removed, and placed in ice-cold artificial cerebrospinal fluid (ACSF; in mM: NaCl 124, KC1 3.3, KH2PO4 1.2, MgS0 ₄ 1.3, CaC12 2.5, NaHC03 20 and Glucose 10.0, constantly gassed with 95% 02/5% CO2). Horizontal hippocampal slices were cut on a vibrating microtome at 400 pm thickness and then placed in a submerged-style holding chamber in ACSF, bubbled with carbogen (95% O2, 5% CO2). Slices were allowed to recover for 1 hour following slicing. A planar multi-electrode recording setup (MED64 system, Alpha Med Sciences Co., Ltd, Tokyo, Japan) was employed to record field excitatory post- synaptic potential (fEPSP), and to study LTP as described in Shimono et al.(Shimono et al., 2002) (see supplemental materials and methods).

iTRAQ-based Proteomics To analyze differential expression of hippocampal synaptic membrane proteins between experimental groups, quantitative iTRAQ proteomics was performed. To this end, tissue preparation, iTRAQ labeling, two-dimensional liquid chromatography, MS ^/ MS and protein identification and quantification were performed as described previously(Counotte et al., 2011; Van den Oever et al., 2008) EN KEF 28. For overrepresentation or enrichment analysis, the list of regulated proteins

(genesymbols), or parts thereof, was uploaded on the WebGestalt (WEB-based GEne SeT AnaLysis Toolkit; and compared with the total list of 519 proteins used, with the hypergeometric test analysis and adjustment for multiple testing(Benjamini and Hochberg, 1995) using P<0.05 and a minimum of 2 genes per group.

Immunoblotting

Total homogenate and synaptic membranes of the dorsal hippocampus were isolated from an independent group of animals (n=6). Samples (10 μg) were lysed in Laemli lysis buffer, separated by electrophoresis on a Criterion 7.5% Tris-HCl sodium dodecyl sulfate-polyacrylamide precast gel (Bio Rad Laboratories), and blotted to PVDF membrane. The following antibodies were used: rabbit anti-Aggrecan (Millipore;

1: 1000), mouse anti-Phosphacan (Developmental Studies Hybridoma Bank; 1: 1000), mouse anti-Neurocan (Alpha Diagnostics; 1: 1000), mouse anti-TenascinR (Acris Antibodies; 1:2000), guinea-pig anti-Brevican (generously provided by Magdeburg; 1:2000) and rabbit anti-HLPNl (Abeam; 1: 1000). To correct for input differences, we compared the total protein amount from each sample, as this is a reliable

method(Counotte et al., 2011; Van den Oever et al., 2008) not dependent on a single protein for normalization.

Immunohistochemistry

Naive (Fig. 4b) or SDPS (Fig. 9) rats were perfused (4% paraformaldehyde), and free- floating coronal hippocampal sections (30 μηι) were labeled overnight with

Fluorescein labeled Wisteria Floribunda Lectin (1:400, FL-1351, Vector Laboratories). Dorsal hippocampus injections

SDPS and control rats received a single infusion of 0.03 U per side of ChABC (Sigma Aldrich; C3667) or Penicillinase (Sigma Aldrich; P0389) as control in 0.5 μL in the dorsal hippocampus (Bregma: -3.8 AP, ± 2.1 ML, and -2.9 DV) 3 months after the last social defeat trial. After a 2-week recovery period, an OPR task was performed, and subsequently animals were used on a daily basis for LTP measurements, with all animals ranging between 16 - 24 days after operation (see Fig. 4).

Statistical analysis

Memory for the novel place of the object or the novel object itself was statistically tested by comparing the data (Student's t-test) to a fictive control (Akkerman et al., 2012). This fictive control had an average value of 50%, but had the same distribution as the original data, thereby giving a more realistic comparison with higher statistical power than performing a single-sample t-test. The iTRAQ-based proteomics was performed in five biological independent experiments. Proper correction for multiple measurements was carried out using the PLGEM (Pavelka et al., 2004), or using the WebGestalt Go enrichment analysis. For all other data, statistical analysis was performed using SPSS20.0. SDPS and treatment effects were assessed with one-way or two-way analysis of variance (ANOVA), followed by Student's t-test post-hoc analyses. Testing was two-sided, unless the initial experiment (proteomics to immunoblot, or LTP to behavior after ChABC treatment) directed the experiment. All results are expressed as group means ± SEM.

References

Akkerman, S., Prickaerts, J., Steinbusch, H.W., and Blokland, A. (2012). Object recognition testing: statistical considerations. Behavioural brain research 232, 317- 322.

Artola, A., von Frijtag, J.C., Fermont, P.C.J., Gispen, W.H., Schrama, L.H., Kamal, A., and Spruijt, B.M. (2006). Long-lasting modulation of the induction of LTD and LTP in rat hippocampal CA1 by behavioural stress and environmental enrichment. The European journal of neuroscience 23, 261-272.

Baune, B.T., Miller, R., McAfoose, J., Johnson, M., Quirk, F., and Mitchell, D. (2010). The role of cognitive impairment in general functioning in major depression.

Psychiatry research 176, 183- 189.

Benjamini, Y., and Hochberg, Y. (1995). Controlling the False Discovery Rate - a Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B-Methodological 57, 289-300.

Berardi, N., Pizzorusso, T., and Maffei, L. (2004). Extracellular matrix and visual cortical plasticity: freeing the synapse. Neuron 44, 905-908. Bradley, V.A., and Power, R. (1988). Aspects of the relationship between cognitive theories and therapies of depression. The British journal of medical psychology 61 ( Pt 4), 329-338.

Brakebusch, C, Seidenbecher, C.I., Asztely, F., Rauch, U., Matthies, H., Meyer, H., Krug, M., Bockers, T.M., Zhou, X., Kreutz, M.R., et al. (2002). Brevican- deficient mice display impaired hippocampal CA1 long-term potentiation but show no obvious deficits in learning and memory. Molecular and cellular biology 22, 7417-7427.

Bruckner, G., Grosche, J., Schmidt, S., Hartig, W., Margolis, R.U., Delpech, B., Seidenbecher, C.I., Czaniera, R., and Schachner, M. (2000). Postnatal development of perineuronal nets in wild-type mice and in a mutant deficient in tenascin-R. The Journal of comparative neurology 428, 616-629.

Bukalo, O., Schachner, M., and Dityatev, A. (2001). Modification of extracellular matrix by enzymatic removal of chondroitin sulfate and by lack of tenascin-R differentially affects several forms of synaptic plasticity in the hippocampus.

Neuroscience 104, 359-369.

Buschler, A., and Manahan-Vaughan, D. (2012). Brief environmental enrichment elicits metaplasticity of hippocampal synaptic potentiation in vivo. Frontiers in behavioral neuroscience 6, 85.

Counotte, D.S., Goriounova, N.A., Li, K.W., Loos, M., van der Schors, R.C., Schetters, D., Schoffelmeer, A.N., Smit, A.B., Mansvelder, H.D., Pattij, T., et al. (2011). Lasting synaptic changes underlie attention deficits caused by nicotine exposure during adolescence. Nature neuroscience 14, 417-419.

Der-Avakian, A., and Markou, A. (2012). The neurobiology of anhedonia and other reward-related deficits. Trends in neurosciences 35, 68-77.

Dere, E., Huston, J.P., and De Souza Silva, M.A. (2007). The pharmacology, neuroanatomy and neurogenetics of one-trial object recognition in rodents. Neurosci Biobehav Rev 31, 673-704.

Dityatev, A., and Schachner, M. (2003). Extracellular matrix molecules and synaptic plasticity. Nat Rev Neurosci 4, 456-468.

Dityatev, A., Schachner, M., and Sonderegger, P. (2010). The dual role of the extracellular matrix in synaptic plasticity and homeostasis. Nat Rev Neurosci 11, 735- 746. Driessen, E., and Hollon, S.D. (2010). Cognitive behavioral therapy for mood disorders: efficacy, moderators and mediators. Psychiatr Clin North Am 33, 537-555. Fairhall, S.L., Sharma, S., Magnusson, J., and Murphy, B. (2010). Memory related dysregulation of hippocampal function in major depressive disorder. Biol Psychol 85, 499-503.

Frischknecht, R., and Gundelfinger, E.D. (2012). The brain's extracellular matrix and its role in synaptic plasticity. Advances in experimental medicine and biology 970, 153-171.

Frischknecht, R., Heine, M., Perrais, D., Seidenbecher, C.I., Choquet, D., and

Gundelfinger, E.D. (2009). Brain extracellular matrix affects AMPA receptor lateral mobility and short-term synaptic plasticity. Nature neuroscience 12, 897-904.

Gogolla, N., Caroni, P., Luthi, A., and Herry, C. (2009). Perineuronal nets protect fear memories from erasure. Science 325, 1258-1261.

Gould, N.F., Holmes, M.K., Fantie, B.D., Luckenbaugh, D.A., Pine, D.S., Gould, T.D., Burgess, N., Manji, H.K., and Zarate, C.A., Jr. (2007). Performance on a virtual reality spatial memory navigation task in depressed patients. Am J Psychiatry 164, 516-519.

Hammen, C. (2005). Stress and depression. Annu Rev Clin Psychol 1, 293-319.

Hartig, W., Brauer, K., and Bruckner, G. (1992). Wisteria floribunda agglutinin- labelled nets surround parvalbumin-containing neurons. Neuroreport 3, 869-872.

Hasler, G., Drevets, W.C., Manji, H.K., and Charney, D.S. (2004). Discovering endophenotypes for major depression. Neuropsychopharmacology 29, 1765-1781.

Heinrich, L.M., and Gullone, E. (2006). The clinical significance of loneliness: a literature review. Clin Psychol Rev 26, 695-718.

Howland, J.G., and Cazakoff, B.N. (2010). Effects of acute stress and GluN2B- containing NMDA receptor antagonism on object and object-place recognition memory. Neurobiol Learn Mem 93, 261-267.

Kamal, A., Van der Harst, J.E., Kapteijn, CM., Baars, A.J.M., Spruijt, B.M., and Ramakers, G.M.J. (2010). Announced reward counteracts the effects of chronic social stress on anticipatory behavior and hippocampal synaptic plasticity in rats.

Experimental brain research Experimentelle Hirnforschung Experimentation cerebrale 201, 641-651. Kapfhammer, J.P., and Schwab, M.E. (1992). Modulators of neuronal migration and neurite growth. Current opinion in cell biology 4, 863-868.

Krishnan, V., and Nestler, E.J. (2008). The molecular neurobiology of depression. Nature 455, 894-902.

McClintock, S.M., Husain, M.M., Greer, T.L., and CuUum, C.M. (2010). Association between depression severity and neurocognitive function in major depressive disorder: a review and synthesis. Neuropsychology 24, 9-34.

McKinnon, M.C., Yucel, K., Nazarov, A., and MacQueen, G.M. (2009). A meta-analysis examining clinical predictors of hippocampal volume in patients with major depressive disorder. J Psychiatry Neurosci 34, 41-54.

Meighan, S.E., Meighan, P.C., Choudhury, P., Davis, C.J., Olson, M.L., Zornes, P.A., Wright, J.W., and Harding, J.W. (2006). Effects of extracellular matrix-degrading proteases matrix metalloproteinases 3 and 9 on spatial learning and synaptic plasticity. J Neurochem 96, 1227-1241.

Murrough, J.W., Iacoviello, B., Neumeister, A., Charney, D.S., and Iosifescu, D.V. (2011). Cognitive dysfunction in depression: neurocircuitry and new therapeutic strategies. Neurobiol Learn Mem 96, 553-563.

Nagy, V., Bozdagi, O., and Huntley, G.W. (2007). The extracellular protease matrix metalloproteinase-9 is activated by inhibitory avoidance learning and required for long-term memory. Learn Mem 14, 655-664.

Nagy, V., Bozdagi, O., Matynia, A., Balcerzyk, M., Okulski, P., Dzwonek, J., Costa, R.M., Silva, A.J., Kaczmarek, L., and Huntley, G.W. (2006). Matrix metalloproteinase- 9 is required for hippocampal late-phase long-term potentiation and memory. The Journal of neuroscience : the official journal of the Society for Neuroscience 26, 1923- 1934.

Nestler, E.J., and Hyman, S.E. (2010). Animal models of neuropsychiatric disorders. Nature neuroscience 13, 1161-1169.

Pavelka, N., Pelizzola, M., Vizzardelli, C, Capozzoli, M., Splendiani, A., Granucci, F., and Ricciardi-Castagnoli, P. (2004). A power law global error model for the

identification of differentially expressed genes in microarray data. BMC

Bioinformatics 5, 203.

Pittenger, C, and Duman, R.S. (2008). Stress, depression, and neuroplasticity: a convergence of mechanisms. Neuropsychopharmacology 33, 88- 109. Pizzorusso, T., Medini, P., Berardi, N., Chierzi, S., Fawcett, J.W., and Maffei, L.

(2002). Reactivation of ocular dominance plasticity in the adult visual cortex. Science 298, 1248- 1251.

Ruis, M.A., te Brake, J.H., Buwalda, B., De Boer, S.F., Meerlo, P., Korte, S.M., Blokhuis, H.J., and Koolhaas, J.M. (1999). Housing familiar male wildtype rats together reduces the long-term adverse behavioural and physiological effects of social defeat. Psychoneuroendocrinology 24, 285-300.

Saghatelyan, A.K., Dityatev, A., Schmidt, S., Schuster, T., Bartsch, U., and

Schachner, M. (2001). Reduced perisomatic inhibition, increased excitatory

transmission, and impaired long-term potentiation in mice deficient for the

extracellular matrix glycoprotein tenascin-R. Molecular and cellular neurosciences 17, 226-240.

Sapolsky, R.M. (2000). Glucocorticoids and hippocampal atrophy in neuropsychiatric disorders. Arch Gen Psychiatry 57, 925-935.

Sheline, Y.I., Sanghavi, M., Mintun, M.A., and Gado, M.H. (1999). Depression duration but not age predicts hippocampal volume loss in medically healthy women with recurrent major depression. J Neurosci 19, 5034-5043.

Shimono, K., Baudry, M., Ho, L., Taketani, M., and Lynch, G. (2002). Long-term recording of LTP in cultured hippocampal slices. Neural Plast 9, 249-254.

Southwick, S.M., Vythilingam, M., and Charney, D.S. (2005). The psychobiology of depression and resilience to stress: implications for prevention and treatment. Annu

Rev Clin Psychol 1, 255-291.

Treadway, M.T., Bossaller, N.A., Shelton, R.C., and Zald, D.H. (2012). Effort-based decision-making in major depressive disorder: a translational model of motivational anhedonia. Journal of abnormal psychology 121, 553-558.

Van Bokhoven, P., Oomen, C.A., Hoogendijk, W.J., Smit, A.B., Lucassen, P. J., and Spijker, S. (2011). Reduction in hippocampal neurogenesis after social defeat is long- lasting and responsive to late antidepressant treatment. The European journal of neuroscience 33, 1833- 1840.

Van den Oever, M.C., Goriounova, N.A., Li, K.W., Van der Schors, R.C., Binnekade, R., Schoffelmeer, A.N., Mansvelder, H.D., Smit, A.B., Spijker, S., and De Vries, T.J. (2008). Prefrontal cortex AMPA receptor plasticity is crucial for cue-induced relapse to heroin-seeking. Nature neuroscience 11, 1053-1058. Van den Oever, M.C., Lubbers, B.R., Goriounova, N.A., Li, K.W., Van der Schors, R.C., Loos, M., Riga, D., Wiskerke, J., Binnekade, R., Stegeman, M., et al. (2010). Extracellular matrix plasticity and GABAergic inhibition of prefrontal cortex pyramidal cells facilitates relapse to heroin seeking. Neuropsychopharmacology 35, 2120-2133.

Von Frijtag, J.C., Kamal, A., Reijmers, L.G., Schrama, L.H., van den Bos, R., and Spruijt, B.M. (2001). Chronic imipramine treatment partially reverses the long-term changes of hippocampal synaptic plasticity in socially stressed rats. Neuroscience letters 309, 153- 156.

Von Frijtag, J.C., Reijmers, L.G., Van der Harst, J.E., Leus, I.E., Van den Bos, R., and Spruijt, B.M. (2000). Defeat followed by individual housing results in long-term impaired reward- and cognition-related behaviours in rats. Behavioural brain research 117, 137- 146.

Von Frijtag, J.C., Van den Bos, R., and Spruijt, B.M. (2002). Imipramine restores the long-term impairment of appetitive behavior in socially stressed rats.

Psychopharmacology 162, 232-238.

Vythilingam, M., Vermetten, E., Anderson, G.M., Luckenbaugh, D., Anderson, E.R., Snow, J., Staib, L.H., Charney, D.S., and Bremner, J.D. (2004). Hippocampal volume, memory, and Cortisol status in major depressive disorder: effects of treatment. Biol Psychiatry 56, 101-112.

Wang, X.B., Bozdagi, O., Nikitczuk, J.S., Zhai, Z.W., Zhou, Q., and Huntley, G.W. (2008). Extracellular proteolysis by matrix metalloproteinase-9 drives dendritic spine enlargement and long-term potentiation coordinately. Proceedings of the National Academy of Sciences of the United States of America 105, 19520-19525.

Weber, P., Bartsch, U., Rasband, M.N., Czaniera, R., Lang, Y., Bluethmann, H.,

Margolis, R.U., Levinson, S.R., Shrager, P., Montag, D., et al. (1999). Mice deficient for tenascin-R display alterations of the extracellular matrix and decreased axonal conduction velocities in the CNS. The Journal of neuroscience : the official journal of the Society for Neuroscience 19,

WHO-depression (2012).

Young, K.D., Erickson, K., Nugent, A.C., Fromm, S.J., Mallinger, A.G., Furey, M.L., and Drevets, W.C. (2012). Functional anatomy of autobiographical memory recall deficits in depression. Psychol Med 42, 345-357. Zakzanis, K.K., Leach, L., and Kaplan, E. (1998). On the nature and pattern of neurocognitive function in major depressive disorder. Neuropsychiatry Neuropsychol Behav Neurol 11, 111- 119.

Table 1. Synaptic protein changes after SDPS. From the total of 519 proteins identified, 37 proteins were significantly regulated 3 months after social defeat in the SDPS paradigm (log2; P-value<0.05 after adjustment for multiple hypothesis-testing using PLGEM; indicated in bold). The rescue by Imipramine (Imi; 18 proteins) or behavioral therapy (BT; 26 proteins) is indicated (P-value<0.1 vs. SDPS H2O after adjustment for multiple hypothesis-testing using PLGEM; bold) as the expression in SDPS Imi or SDPS BT animals vs. the expression by SDPS H2O. Indicated is Protein symbol, Gene symbol, and protein description.