Conditions

FIELD OF INVENTION

[0001] The present invention relates to methods for measuring symptoms in rodent models, particularly in rodent models of Autism Spectrum Disorders and comorbid conditions by measurement of Ultrasonic Vocalizations (USVs).

BACKGROUND OF THE INVENTION

[0002] Autism spectrum disorders (ASDs) represent an early-onset collection of neurodevelopmental deficits that affect as many as 1 in 200 people, with a particularly high prevalence in males. Characterized by repetitive behaviours and impairments in social and communicative skills, ASD diagnosis is currently limited to behavioural symptoms alone. Therefore, accurate symptomatic identification in relevant animal models is imperative, especially since ASDs are frequently comorbid with many other neurological and neuropsychiatric disorders. To date, there are few behavioural test batteries available— with none that provide a comprehensive symptomatic assessment. Animal models can provide a valuable resource for studying neuropathology, as well as for the testing of therapeutic compounds and treatments aimed at slowing or stopping the disorder they mimic. Without the capacity to accurately evaluate symptoms in a relevant ASD animal model, connections between behaviour and neuropathology cannot be made, and our ability to understand this multifaceted disorder and to develop useful ASD treatments is limited.

SUMMARY OF THE INVENTION

[0003] It is an object of the invention to provide a method for measuring symptoms in rodent models, especially in rodent models of Autism Spectrum Disorders and comorbid conditions.

[0004] According to an aspect of the present invention, there is provided a method for measuring symptoms in a rodent model of Autism Spectrum Disorders and comorbid conditions which comprises measuring deficits in one or more developmental, communication, social behaviour, or abnormally repetitive behaviour phenotype, together with at least one measurement of Ultrasonic Vocalizations (USVs).

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SUBSTITUTE SHEET RULE 26 [0005] The method can be used as a screening protocol for evaluating new therapeutants, combinations of therapeutants and/or other relevant medical interventions for treating ASDs. In addition, a variety of symptoms that are frequently found to be comorbid with ASD and characteristic of other diagnosable neuropsychiatric conditions are also readily identifiable and quantifiable using this method.

[0006] Accordingly, there is also provided herein a method to identify a compound having effectiveness or potential effectiveness in treating Autism Spectrum Disorders and comorbid conditions in a rodent model, the method comprising: administering at least one test compound to at least one rodent, said at least one rodent being a model of Autism Spectrum Disorders and comorbid conditions; measuring symptoms in the at least one rodent by measuring deficits in one or more developmental, communication, social behaviour and abnormally repetitive behaviour phenotypes, together with at least one measurement of Ultrasonic Vocalizations (USVs); and identifying said at least one test compound as having effectiveness or potential effectiveness in treating Autism Spectrum Disorders and comorbid conditions if at least one deficit in a developmental, communication, social behaviour and/or abnormally repetitive behaviour phenotype is identified as reduced in the measuring step when compared to controls. [0007] In embodiments of the method, the rodent model is a rat model of Autism Spectrum Disorders and comorbid conditions. As one non-limiting example, the rat model may be induced by prenatal treatment with sodium valproate (NaVPA).

[0008] In further embodiments of the method, the at least one measurement of USVs may be measured during isolation from a litter, with potentiation, without potentiation, or combinations thereof.

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SUBSTITUTE SHEET RULE 26 [0009] In other non-limiting embodiments, the developmental, communication, social behaviour and abnormally repetitive behaviour phenotypes may be measured in combination with the at least one measurement of USVs, the developmental, communication, social behaviour and abnormally repetitive behaviours being measured by one or more of:

Weight;

Auditory startle from about postnatal day (PND) 8 until first startle reflex;

Eye opening from about PND11 until first day eyes open;

Locomotion and repetitive behaviours during isolation at about PND7, PND14, and/or PND21 ;

Surface righting by measurement of latency to turn over from back from about PND8 to about PND 14;

Negative geotaxis by measuring latency to orient body with head higher than rump on 30° inclined plane, from about PND8 to about PND 14;

Home bedding by measuring latency to choose and choice between fresh bedding and home bedding at about PND13;

Tactile stimulation by measurement of aversion to stimulation at about PND16;

Suspension test by measurement of ability to hang on a horizontal wire at about PND24; Tickle test by measurement of response to tickling following group housing and/or isolation at about PND27 and about PND29;

Wood block chew by assessment of chewing behaviours at about PND28;

1 on 1 test by measurement of time spent engaged with social partner following isolation housing at about PND31 ;

3 chamber test by measurement of time spent with "stranger" rat versus time spent alone or with novel object at about PND33;

Novel object test by measurement of USVs emitted without and with novel object, latency to approach, interactions with and grooming and rearing with novel object at about PND35; Home cage emergence by measurement of latency to emerge from home cage at about PND38;

Light/dark box testing by measuring time spent and/or or grooming and rearing in light versus dark areas at about PND41; Open field by measuring speed and distance travelled, immobility, thigmotaxic behaviours and/or grooming and rearing in open field at about PND43 and/or about PND142;

Elevated plus maze by measuring number of entries and time spent in open versus closed arms, immobility events, and/or grooming and rearing events in open versus closed arms at about PND45 and/or about PND138;

Prepulse inhibition / startle by measurement of ability to habituate to cued and uncued noise at about PND50;

Morris water maze by measurement of latency to platform location, swim speed and/or path efficiency to platform at about PND58 to about PND61; and

Morris water maze by measurement of latency to platform location, swim speed and/or path efficiency to platform at about PND 144.

[0010] In certain specific embodiments, the at least one measurement of USVs is measured in combination with at least one developmental test, at least one communication test, at least one social behaviour test and at least one abnormally repetitive behaviour test. [001 1] Further embodiments of the invention will become apparent from the following detailed description and related experiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein: FIGURE 1 illustrates a graph showing measurements of weight in control (saline) and

NaVPA-treated male rats from postnatal day 7 to postnatal day 138. Overall, NaVPA rats exhibited less weight gain in adulthood than controls, with males particularly affected. Error bars represent SEM.

FIGURE 2 illustrates a graph showing the mean day of eye opening in control (saline) and NaVPA-treated rats. Eye opening was delayed in NaVPA rats. Error bars represent SEM. FIGURE 3 illustrates a graph showing the percentage of control (saline) and NaVPA-treated rats which respond to touch. NaVPA rats were more sensitive to touch than controls (PND16). Error bars represent SEM.

FIGURE 4 illustrates a graph showing the mean number of grid crosses by control (saline) and NaVPA-treated male rats. During isolation testing on PND7, male NaVPA rats displayed increased locomotor activity compared to controls. No differences were noted in females. Error bars represent SEM.

FIGURE 5 illustrates a graph showing the mean number of grooming episodes in control (saline) and NaVPA-treated male rats. Male NaVPA rats groomed more during isolation testing on PND14, indicative of either heightened anxiety, or increased activity levels. No differences were noted in females. Error bars represent SEM.

FIGURE 6 illustrates a graph showing the latency to first isolation call in control (saline) and NaVPA-treated male and female rats. All rats showed increased USVs during potentiated isolation testing (PND21) (inset; p=0.045 NaVPA more - collapsed for test). In addition,

St

potentiation decreased latency to the 1 USV (but NaVPA overall showed increased latency p=0.033). Error bars represent SEM.

FIGURE 7 illustrates a graph showing the mean duration of USVs in control (saline) and NaVPA-treated rats during tickle testing (PND29; isolation day). NaVPA rats' vocalizations were of shorter duration than controls. In addition, both the number and the frequency range of USVs were altered (data not shown). Error bars represent SEM.

FIGURE 8 illustrates a graph showing the percent of USVs emitted in the 20kHz (aversive) range in control (saline) and NaVPA-treated rats. Mean percent of USVs emitted in the 20kHz were higher for treated rats during Novel Object testing (PND36), even though more traditional behavioural measures were largely unchanged (data not shown). Error bars represent SEM. FIGURE 9 illustrates a graph showing the percent of time spent in the light in control (saline) and NaVPA-treated male rats during Light/Dark box testing. In the Light/Dark box (PND41), male NaVPA rats spent less time in the lighted portion of the box. Error bars represent SEM.

FIGURE 10 illustrates a graph showing the time to first USV during open field testing in control (saline) and NaVPA-treated male and female rats. Both number of USVs (p=0.035;

NaVPA more - not shown) and latency to 1 USV were altered in NaVPA rats during Open Field testing(PND43). However, while most behavioural data showed no differences between groups, grooming and rearing were decreased in the NaVPA groups, particularly in males (see Figure 21). Error bars represent SEM. FIGURE 11 illustrates a graph showing the mean frequency (Hz) of USVs in control (saline) and NaVPA-treated rats during Open Field testing. In the Open Field in adulthood (PND142), NaVPA rats were more likely to emit a USV than were controls (inset; p=0.044). The mean frequency of these USVs were less commonly in the 20kHz "aversive" range. As during early life, grooming and rearing were also decreased in the NaVPA groups (data not shown). Error bars represent SEM, while the asterisk denotes statistical significance (p=0.044).

FIGURE 12 illustrates a graph showing the percent startle attenuation in control (saline) and NaVPA-treated rats. Male NaVPA rats showed no attenuation of startle response to a 120Hz noise. However, PPI results were not different between the groups (data not shown). Error bars represent SEM. FIGURE 13 illustrates a graph showing the mean distance travelled by control (saline) and NaVPA-treated rats during Morris Water Maze (MWM) testing. NaVPA rats displayed difficulty in finding the platform during watermaze testing (PND58-60). However, there were no differences between groups during reversal testing (data not shown). Error bars represent SEM. FIGURE 14 illustrates a graph showing the distance travelled in non-platform quadrant regions by control (saline) and NaVPA-treated male rats during probe trial testing. In adulthood (PND145) during probe trial testing, male NaVPA rats displayed an altered search strategy from controls. Error bars represent SEM.

FIGURE 15 illustrates a graph showing the mean % of aversive isolation calls in control (saline) and NaVPA-treated rats. Error bars indicate SEM. The asterisk denotes a significant difference from controls (p=0.014).

FIGURE 16 illustrates a graph showing the mean % of aversive USVs in control (saline) and NaVPA-treated rats during tickle test at day 2. Error bars indicate SEM, while the asterisk denotes a significant difference from controls (p=0.014).

FIGURE 17 illustrates a graph showing the mean % of aversive vocalizations in control (saline) and NaVPA-treated male and female rats during novel object testing. Treated rats emitted more aversive USVs (B; p=0.005) than controls. This increase was primarily a result of aversive vocalizations from treated males (A; p=0.021). Error bars indicate SEM, while asterisks denote a significant difference from the corresponding control group.

FIGURE 18 illustrates a graph showing the mean number and duration (sec) of USVs in control (saline) and NaVPA-treated male and female rats during isolation testing in PND7 pups. Error bars indicate SEM. The pound sign indicates a trend toward a statistically significant difference from the appropriate control group (p=0.062), while the asterisk indicates a significant difference (p=0.045).

FIGURE 19 illustrates a graph showing the mean latency (sec) to first USV with and without object, in control (saline) and NaVPA-treated female rats during Novel object testing. Error bars indicate SEM, while the asterisk denotes a significant difference from controls (p=0.024).

FIGURE 20 illustrates a graph showing the mean latency (sec) to first USV in male and female rats during light/dark box testing. Error bars indicate SEM. FIGURE 21 illustrates a graph showing the mean number of grooming and rearing events in control (saline) and NaVPA-treated male rats (postnatal day 43). Error bars indicate SEM.

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SUBSTITUTE SHEET RULE 26 FIGURE 22 illustrates graphs showing the mean time spent interacting (A) and mean number of USV calls (B) in 1 on 1 social testing for treated and control rat pairs. Error bars indicate SEM.

DETAILED DESCRIPTION

[0013] A comprehensive rodent ASD behavioural test battery has been developed and validated using the most commonly utilized environmental ASD model - the valproic acid (NaVPA) rat. This new test battery, which combines traditional behavioural testing paradigms with recent technologies that allow evaluation of rat "speech" in the ultrasonic spectrum, provides the capability to behaviourally assess current and new ASD rat models for alterations in social communication and social interactions in a variety of situations, and restricted/repetitive patterns of behaviour as defined in the Diagnostic and Statistical Manual V (DSM-V).

[0014] The new test battery also provides a comprehensive behavioural screening protocol for evaluating new therapeutants and/or combinations of therapeutants and/or other relevant medical interventions for treating ASDs. In addition, a variety of symptoms that are frequently found to be comorbid with ASD and are characteristic of other diagnosable neuropsychiatric conditions are also readily identifiable and quantifiable using this highly sensitive paradigm.

Combining USVs with Communication, Social, and Other Behavioural Studies [0015] In humans, autism spectrum disorder (ASD) is diagnosed if the following criteria in the Diagnostic and Statistical Manual V (DSM-V; American Psychiatric Association (2013), Diagnostic and Statistical Manual of Mental Disorders (5 ^th Ed.), Arlington, VA, incorporated herein by reference) are met:

A. Persistent deficits in social communication and social interaction across contexts, not accounted for by general developmental delays B. Restricted, repetitive patterns of behavior, interests, or activities (e.g. stereotyped or repetitive speech, motor movement, resistance and/or distress at small changes, hyper- or hypo-reactivity to the environment, etc).

C. Symptoms must be present in early childhood (but may not become fully manifest until social demands exceed limited capacities).

D. Symptoms together limit and impair everyday functioning.

[0016] In addition, ASD in humans is often associated with a high rate of comorbidity (Tuchman & Rapin, 2002), with mood (e.g. depression, anxiety) and conduct (e.g. aggression, impulsivity) disorders particularly prevalent. [0017] The technology that has been developed is focused on providing a comprehensive rat test battery aimed at targeting these criteria, thus offering a highly relevant and predictive measure to evaluate potential ASD therapeutics and/or treatment regimes, as well as to assess the validity of currently established and newly developed rat models for ASD testing.

[0018] At present, evaluation of ASD rat models utilize traditional behavioural tests; if ultrasonic vocalization (USV) measures are included at all, they are run as a separate, standalone component, almost exclusively during an early (postnatal day 7 or 8) isolation period from the mother. While this approach allows for basic assessment, it does not even begin to tap into the complexities of the human disorder it attempts to evaluate.

[0019] As highly social creatures, rats possess a broad behavioural repertoire, making them the preferred species for modeling cognition, affective, and neurological disorders (http://www.sageresearchmodels .com/research-models). Their vocalizations, particularly in the ultrasonic range, are equally sophisticated. For example, neonatal rats emit at least 4 distinct, multicomponent ultrasonic waveforms while other species (mice, hamsters, etc), utilize only one call pattern (Portfors, 2007; Hashimoto et al., 2004). In addition, USV frequency in adulthood is considered indicative of a rat's affective state, with emissions in the 22kHz range highly associated with aversive situations (e.g. confronting intruders, predator scent, losing fights), while 50kHz emissions are utilized for more positive circumstances (e.g.

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SUBSTITUTE SHEET RULE 26 during, or in anticipation of play, receipt of food reward, etc). While adult mice also ultilize USVs, their communication is limited primarily to mating encounters. Unlike rats, mice do not indicate negative or positive affect through USVs, and do not produce USVs at all during aversive situations (Portfors, 2007).

[0020] Combining behavioural tests with corresponding USV emissions as outlined in the present test battery allows for multifaceted evaluation of ASD rat models from infancy to adulthood, including potential comorbid activity. All tests were carefully chosen, and run in very specific combinations to ensure optimal behavioural and USV results. The technology has also been effective for identifying sex differences that would be undetectable using more traditional assessments. This sensitivity is especially important given that ASD is 4x more prevalent in males.

[0021] The following examples provide results that highlight the importance of assessing USVs in combination with other test methods in models of ASD.

EXAMPLES

Methods

[0022] Animals: Sprague-Dawley rats (Charles River, PQ) were bred in-house with pregnancies confirmed by vaginal swab (gestational day 0) and ultrasound (G10). On G12, pregnant rats were injected with an acute dose (600mg/kg i.p.) of sodium valproate (NaVPA). Litters were assessed on day of birth for number of pups, as well as for pup weight and length. Behavioural testing began on postnatal day (PND) 7 according to the test battery described in the table below.

[0023] Ultrasonic Vocalizations: To record USVs, a specialized microphone is required, which provides access to vocalizations in all frequencies used by rats. A large body of literature is available regarding interpretation of these USV frequencies, and there are 3 main types: 22kHz, 40kHz, and 50kHz. 40kHz calls are emitted solely by rat pups (isolation calls when separated from their litter and mother). These types of calls are believed to be important for communication with the mother, eliciting increased maternal care. At PND 14+, a potentiated USV effect can be produced when pups are returned to the mother following an initial isolation period (with USVs recorded), allowed individualized attention for several minutes, and then re-isolated. Interestingly, 40kHz calls have been related to emotional development, with studies showing that pups who emit more of these calls during infancy (thus receiving more maternal care) display less anxiety-related behaviour in adulthood. Further, adult call characteristics (e.g. amplitude, frequency) show a relationship to early-life maternal care as well. In adulthood, 22kHz calls are emitted primarily in response to aversive stimuli, while 50kHz calls are associated with positive affect. For the test battery outlined, USV calls were recorded in conjunction with more traditional behavioural testing, thus giving an extra 'social/communication' and 'repetitive behaviour/communication dimension to the measures under study.

[0024] Ultrasonic Vocalization (USV) Acquisition Protocol (A visoft-UltraSoundGate 116Hb converter; condenser microphone; Dell Latitude D630 laptop):

1. Attach microphone to converter unit, plug converter into USB port on laptop. Turn power on.

2. Choose "workstation only" on computer start up.

3. Start Avisoft Acquisition program "Recorder USGH".

4. Carefully remove protective cover from microphone, position it as required, and perform function test:

-select "pause" in software unless a recording of the test is needed;

-to begin test, select "record". Check microphone pickup by rubbing fingertips together;

- USV noise will be visible on the screen in real time as a diffused grey/black 'cloud'; -check all areas of testing arena; when finished, remember to stop recording; and deselect "pause"; failure to turn pause off will result in recordings not saving.

5. Check that time of test run in program parameters corresponds to the actual time of the test (seconds). Note: the USV recording will automatically stop when the test is complete, and a prompt screen will appear for labeling the saved file.

6. To begin testing, select "record". As during the function test, a real time monitor screen will appear. Any USVs will be visibly apparent here, as well as a count of the USVs, and the time left until the test is complete.

4 1- [0025] Ultrasonic Vocalization (USV) Data Acquisition Protocol (Avisoft SASLab Pro USB key)

1. Open SASLab Pro (make sure USB key is plugged in).

2. Open file to be analyzed. Once it is loaded, click on the 1st icon in the top row

(computation of spectrogram according to the selected spectrogram parameters). Both a spectrogram and a measurements window will appear when the analysis is complete.

3. Check the spectrogram for any "noise" (a constant line running throughout).

4. In the measurements window, click on "export" and select "copy parameter measurement values for entire file" from the drop-down menu. Paste this information into the appropriate Excel spreadsheet.

5. Close measurements window (spectrogram will close simultaneously), and then close the USV file.

6. Continue from #2 as required. Table 1. Comprehensive behavioural test battery.

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SUBSTITUTE SHEET RULE 26

and away Results:

[0026] Weight measurements in control (saline) and NaVPA-treated rats were recorded from postnatal day 7 to postnatal day 138. Overall, NaVPA rats exhibited less weight gain in adulthood than controls (p=0.010), with males particularly affected (Figure 1). [0027] The mean day of eye opening in control (saline) and NaVPA-treated rats was found to be delayed in NaVPA rats (p=0.022; Figure 2).

[0028] The percentage of control (saline) and NaVPA-treated rats which respond to touch is illustrated in Figure 3. As shown, NaVPA rats were more sensitive to touch than controls (p=0.045; PND16). [0029] The mean number of grid crosses by control (saline) and NaVPA-treated male rats is illustrated in Figure 4. During isolation testing on PND7, male NaVPA rats displayed increased locomotor activity compared to controls (p=0.030). No differences were noted in females.

[0030] The mean number of grooming episodes in control (saline) and NaVPA-treated male rats is illustrated in Figure 5. Male NaVPA rats groomed more during isolation testing on

PND14 (p=0.001), indicative of either heightened anxiety, or increased activity levels. No differences were noted in females.

[0031] The measurements of latency to first isolation call in control (saline) and NaVPA- treated male and female rats are illustrated in Figure 6. All rats showed increased USVs during potentiated isolation testing (PND21) (inset; p=0.045 NaVPA more when collapsed for

St

test). In addition, potentiation decreased latency to the 1 USV, although NaVPA overall showed increased latency, p=0.033).

[0032] The mean duration of USVs in control (saline) and NaVPA-treated rats during tickle testing (PND29; isolation day) is illustrated in Figure 7. NaVPA rats' vocalizations were of shorter duration than controls (p=0.000). In addition, both the number and the frequency range of USVs were altered (as discussed in further detail below and in Figure 17).

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SUBSTITUTE SHEET RULE 26 [0033] The percent of USVs emitted in the 20kHz (aversive) range in control (saline) and NaVPA-treated rats is shown in Figure 8. Mean percent of USVs emitted in the 20kHz range were higher for treated rats during Novel Object testing (p=0.005; PND36), even though more traditional behavioural measures were largely unchanged (data not shown). [0034] The percent of time spent in the light in control (saline) and NaVPA-treated male rats during Light/Dark box testing is shown in Figure 9. In the Light/Dark box (PND41), male NaVPA rats spent less time in the lighted portion of the box (p=0.035).

[0035] The time to first USV during open field testing in control (saline) and NaVPA-treated male and female rats is shown in Figure 10. Both number of USVs (p=0.035; NaVPA more -

St

not shown) and latency to 1 USV were altered in NaVPA rats during juvenile Open Field testing (PND43). However, while most behavioural data showed no differences between groups, grooming and rearing were decreased in the NaVPA groups, particularly in males (see Figure 21).

[0036] The mean frequency of USVs in control (saline) and NaVPA-treated rats during adult Open Field testing are shown in Figure 11. In the Open Field in adulthood (PND 142), NaVPA rats were more likely to emit a USV than were controls (inset; p=0.044). The mean frequency of these USVs were less commonly in the 20kHz "aversive" range (p=0.044). As during early life, grooming and rearing were also decreased in the NaVPA groups (data not shown).

[0037] The percent startle attenuation in control (saline) and NaVPA-treated rats is shown in Figure 12. Male NaVPA rats showed no attenuation of startle response to a 120Hz noise (p=0.018). However, PPI results were not different between the groups (data not shown).

[0038] The mean distance travelled by control (saline) and NaVPA-treated rats during Morris Water Maze (MWM) testing is shown in Figure 13. NaVPA rats displayed difficulty in finding the platform during watermaze testing (PND58-60). However, there were no differences between groups during reversal testing (data not shown).

[0039] The distance travelled in non-platform quadrant regions by control (saline) and NaVPA-treated male rats during probe trial testing is shown in Figure 14. In adulthood

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SUBSTITUTE SHEET RULE 26 (PND 145) during probe trial testing, male NaVPA rats displayed an altered search strategy from controls (p=0.040).

[0040] Referring to Figure 15, although the number and duration of potentiated isolation USVs at postnatal day (PND) 14 were equivalent, the figure shows that NaVPA rat pups emitted more USVs in the 22kHz (aversive) range (p=0.014). These data highlight the importance of assessing USV frequency range in models of ASD. In addition, these data provide an example of the importance of appropriate test combinations. Rat pups at this age do not emit many calls (sonic or ultrasonic) when isolated from their dam, and in this study, there was no difference between groups during this first isolation period. However, if the pup is placed back with the dam for several minutes following the initial isolation, then re- isolated, USV call rates will subsequently increase. This phenomenon is referred to as potentiation. In controls, potentiation increased USV call rates by 20%. In treated rats, the call rate was more than double at 42%. Therefore, Figure 15 presents alterations in aversive USV emissions between treated and control rats during the potentiated trial only. [0041] During interactions with the investigator (2nd day of tickle testing) on PND29, treated rats emitted a greater percentage of 22kHz aversive USVs (p=0.014; Figure 16). This test depends upon rats' enjoyment of social contact. When tickled by a familiar experimenter, rats will emit 50kHz "laughter".

[0042] Again, the presentation of this test was carefully designed to maximize call rate and provide a comparison of situational measures. To this end, USVs were recorded during an initial test period when rats had been group housed (Day 1 testing PND27) and were therefore much less interested in social interactions with the experimenter. Rats were then re-tested on a subsequent day (Day 2 testing PND29) following individual housing, which increased interest in experimenter-associated social activity and thus USV calls. Between day measures showed that, as expected, both control (p=0.002) and treated (p=0.044) rats vocalized more on Day 2 compared to Day 1. There were no differences between conditions on Day 1 in any USV measures. However, there were differences on Day 2 for number of USVs (treated rats tended to vocalize less than controls, p=0.08) and USV duration (treated rats' vocalizations were shorter than controls, p=0.000; see Figure 7). In addition, treated rats also tended to

A T- emit shorter vocalizations on the 2nd test day compared to their vocalizations on the 1st day (p=0.059). In line with the results presented in Figure 16, the overall mean frequency range of treated rats' USVs also tended to be lower (p=0.075).

[0043] During novel object testing, treated rats emitted more aversive USVs (B; p=0.005) than controls (Figure 17). This increase was primarily a result of aversive vocalizations from treated males (A; p=0.021). Novel object testing assesses response to a novel object - in this case an unfamiliar object inserted into the rat's home cage. The latency to approach the object and the time spent with the object are the usual measures recorded, and are considered to provide information about curiosity and anxiety. Note that this figure is also representative of sex differences that may be revealed with USV measurements (also see Figure 19).

[0044] Figure 18 demonstrates an example of sex differences in testing responses. On PND7 during isolation testing, treated females tended to emit less USVs than control females (p=0.062) (A). The USVs that treated females did emit, however, were of a longer duration (p=0.045) (B). There were no differences between groups for either measure in males. Figure 19 demonstrates another example of sex differences in testing responses in the novel object test. Prior to novel object presentation, there were no differences noted between groups in latency to emit USVs (w/o object). Once the novel object was placed in the cage however, treated females did not vocalize again for almost one minute (P=0.024; with object).

[0045] As mentioned above, novel object testing assesses curiosity and anxiety. In this test, purely behavioural measures such as latency to approach the object and the time spent with the object were not different between groups for either male or female rats. However, this doesn't mean the rats were not affected. In fact, USV measures show a very intriguing difference in how male and female responses differ. As shown in figure 17, there were differences found in aversive USV calls, with treated males particularly affected by the presence of the object. And as shown here in figure 19, treated females also responded to the object's presence through USV - in this case, by withholding vocalizations. Note that female USV vocalizations did not differ between groups prior to object presentation, and behavioural responses (latency to approach object, time spent, etc) were also not different. Without the combination of USV and behaviour, these important social / communication differences would have remained undetected.

[0046] In another test of anxiety (light/dark box; Figure 20), females overall showed a tendency toward longer latency to USV emission than males (p=0.086), suggesting that withholding vocalizations may be a normal female response to situations they find aversive.

[0047] During open field testing at PND43, treated males groomed and reared less than controls (Figure 21). Another type of anxiety test, the open field provides information about a rat's normal fear of bright open spaces. In this test, behavioural measures showed differences between treated and control males for grooming and rearing (treated males did less of both; heightened grooming and rearing are believed to be indicative of increased anxiety or increased activity or increased compulsivity / repetitive behaviour). As well, treated males moved around a lot, entering both the inner and the outer maze areas more often, although the distance they travelled overall, and their movement speed was not different from controls (data not shown). Entries to the inner maze area is often considered a sign of decreased fear/anxiety. With the information from the addition of USV measures from figure 10, it appears that the exhibited behaviour is indeed an indication of an altered response to dangerous situations, as treated males vocalized almost as soon as they were placed in the maze, while the other groups withheld USVs until 3-4 minutes had passed. In addition, treated rats (both male and female) emitted more USVs overall than controls during this test (p=0.035, data not shown), with call frequency within the positive affect (50kHz) range.

Interestingly, when this test was re-run in adulthood (PND140), a similar behavioural response for treated males was seen in grooming, rearing, and inner/outer maze entries - and this time, there was also a difference in mean frequency of USVs as well, with treated rats' vocalizations in the positive (50kHz) range, while control rats' mean vocalization range was closer to aversive frequencies (p=0.044; Figure 11).

[0048] In 1 on 1 social testing, treated rat pairs spent less time than control rat pairs interacting (p=0.023; Figure 22A). In addition, they also emitted fewer USV calls during this time (p=0.000; Figure 22B), and the calls that they did make were of a shorter duration (p=0.000).

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SUBSTITUTE SHEET RULE 26 [0049] One or more currently preferred embodiments have been described by way of example. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.