Disentangling the effects of Corticotrophin Releasing Factor and GABA release from the ventral bed nucleus of the stria terminalis on ethanol self-administration in mice

Excessive alcohol use causes a great deal of harm and negative health outcomes. Corticotrophin releasing factor (CRF), a stress-related neuropeptide, has been implicated in binge ethanol intake and ethanol dependence. CRF containing neurons in the bed nucleus of the stria terminalis (BNSTCRF) can control ethanol consumption. These BNSTCRF neurons also release GABA, raising the question, is it CRF or GABA release or both that is controlling alcohol consumption. Here, we used viral vectors to separate the effects of CRF and GABA release from BNSTCRF neurons on the escalation of ethanol intake in an operant self-administration paradigm in male and female mice. We found that CRF deletion in BNST neurons reduces ethanol intake in both sexes, with a stronger effect in males. For sucrose self-administration there was no effect of CRF deletion. Suppression of GABA release, via knockdown of vGAT, from BNSTCRF produced a transient increase in ethanol operant self-administration following in male mice, and reduced in motivation to work for sucrose on a progressive ratio schedule of reinforcement in a sex-dependent manner. Together, these results highlight how different signaling molecules from the same populations of neurons can bidirectionally control behavior. Moreover, they suggest that BNST CRF release is important for high intensity ethanol drinking that precedes dependence, whereas GABA release from these neurons may play a role in regulating motivation.


Background
In 2014 approximately 137.9 million people in the United States were current alcohol users, of whom 43.6% were binge alcohol drinkers and 6.4% met criteria for Alcohol Use Disorder (AUD) (1). Binge drinking, which the NIAAA defines as a pattern of drinking over a short period of time that brings blood alcohol concentration levels to 0.08 g/dl, may represent an important vulnerability to the development of AUD (2,3). Escalation of drinking is a key process in the transition from casual to excessive drinking in AUD; however, the discrete mechanisms that drive this process are not fully understood. Corticotrophin releasing factor (CRF) is a stressresponsive neuropeptide that is released from the paraventricular nucleus of the hypothalamus (PVN) and from additional regions like the bed nucleus of the stria terminalis (BNST). CRF is engaged in escalated alcohol intake, theorized to be compensating for excessive reward as a major component of the pathological progression to alcohol use disorder (4, for review: 5).
Although early clinical studies of corticotrophin releasing factor receptor type 1 (CRF-R1) antagonists were unsuccessful (6), multiple independent genetic association studies have revealed a link between excessive alcohol consumption and this gene (7,8). Basic mechanistic research into how corticotrophin releasing factor (CRF) signaling is engaged over the course of escalated alcohol consumption is thus warranted, and may elucidate up or downstream regulators of CRF that may serve as successful therapeutic targets. Repeated binge drinking is one form of escalated intake that engages CRF neurons in the BNST (9)(10)(11). The majority of CRF-expressing neurons within the BNST are GABAergic (12-15). Inhibition of CRF-BNST neurons reduces binge drinking (10).
Over the past ten years, rates of AUD have increased in women by 84%, relative to a 35% increase in men (16). More research is needed into sex differences in the neurobiological response to alcohol (17). The BNST is a sexually dimorphic brain region, and differences in structure are due to organizational influences of sex hormones during early development (18,19). Additionally, sex-specific differences in CRF and GAD67 (one enzyme that synthesizes GABA) were observed within the BNST of mice (20), highlighting the importance of studying males and females. Here, we use viral vectors to selectively reduce expression of CRF or the vesicular GABA transporter (vGAT) in the ventral BNST to determine the role of CRF release or GABA release on binge ethanol intake in an operant model of self-administration in male and female mice. counterbalanced across mice and maintained throughout the duration of the experiment. Active lever responses were initially reinforced using a fixed ratio 1 (FR1) schedule, where each press resulted in delivery of a single reinforcer. Once individual mice earned a criterion of 10 reinforcers in a single FR1 session (1)(2)(3)(4)(5) days, mean 1.5 days), they were then trained on a fixed ratio 2 (FR2) schedule for three days. Mice that took longer than 2 days to achieve the performance criterion (n=6 total in all experiments) underwent remedial FR1 sessions that were extended to a duration of 2 hours. After three days of FR2 training, they were then trained on a fixed ratio 4 (FR4) schedule for 9 days.
After 9 days of FR4 training, the mice underwent progressive ratio testing to examine the motivation to work for liquid reinforcers. These progressive ratio tests were modified for use with this ethanol and sucrose solution by changing the session timeout to 90 seconds from the last earned reward, which was three times the average C57Bl6/J self-administration inter-reinforcer interval (23). First mice were tested with an arithmetic progression, where the ratios progressed 1, 1, 2, 2, 3, 3, 4, 4, 5, 5, 7, 7, 9, 9, 11, 11, 13, 13, 15, 15, 18, 18, 21, 21, 24, 24,… (24). Mice received FR4 self-administration days between progressive ratio sessions to re-establish stable responding. The second progressive ratio test followed an exponential progression where the ratios were determined using (5*e 0.2*reward )-5, rounded up to the nearest integer (25). Breakpoint is not a continuous measure, so we report rewards earned, which is proportional to the log transformed breakpoint (23,25).

Blood ethanol concentration
In an experiment with male and female C57Bl6/J mice, we measured blood ethanol concentration to compare this to our calculated grams per kilogram measure. Within 5 minutes of the end of the 9 th FR4 session mice were deeply anesthetized with isofluorane, and trunk blood was taken. Blood was centrifuged and plasma was collected. Blood ethanol concentration was measured using an Analox-AM1 alcohol analyzer (Analox Technologies, Atlanta, GA, USA). using a drill to burr small holes in the skull directly above the injection targets. Microinjections were performed with a 1 µL Neuros Hamilton syringe (Hamilton, Reno, NV, USA) and a micro-infusion pump (Nanoject III, Drummond Scientific; Broomall, PA, USA) that infused virus at 100 nL/min. Viruses were administered bilaterally with 200 nL per side (relative to bregma: ML ±0.9 mm, AP, 0.23 mm, DV -4.75 mm), and the needle was left in place for 5 min to allow for diffusion of the virus before the needle was slowly withdrawn. Most mice were administered a single injection of buprenorphine (0.1mg/kg, s.c.) for surgical analgesia, with supplemental pain management from Tylenol water. For a total of 9 mice in behavioral studies and 4 mice in the electrophysiology experiment, the approved surgical analgesia protocol changed to ketoprofen injections (5 mg/kg, s.c.) on the day of surgery and at least two days post-surgery. Mice were allowed to recover for at least 3 weeks prior to being used for behavioral studies, and 8 weeks prior to being used for slice electrophysiology.

Viral placement validation for behavioral studies
Mice were anesthetized with an overdose of the anesthetic Tribromoethanol (Avertin, 1 mL, i.p.), and transcardially perfused with chilled 0.01 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were extracted and post-fixed in 4% PFA for 24 hours and then stored in PBS at 4°C. 45 µm coronal sections were collected using a Leica VT1000S vibratome (Leica Biosystems; Deer Park, IL, USA) and then stored in 0.02% sodium azide (Sigma Aldrich) in PBS. The tissue was then mounted on slides and allowed to dry before cover slipping with Vecta-Shield Hardset Mounting Medium with DAPI (Vector Laboratories; Newark, CA, USA). Viral placements were imaged at 4x magnification using a Keyence BZ-X800 All-in-one Fluorescence microscope (Keyence; Itasca, IL, USA).
Neurons were identified using infrared differential interference contrast on a Scientifica Slicescope II and low pass filtered at 3 kHz. Access resistance was continuously monitored and changes greater than 20% from the initial value were excluded from data analyses.
Data are presented in figures as the mean ± standard error of the mean. Data are presented in sex-separated graphs for clarity, sex effects were analyzed and reported in the text. Response rates and rewards earned across virus, sex, and behavioral sessions were analyzed using repeated measures GLM with a Poisson distribution, because this distribution is the most appropriate for count data. Regression coefficients were tested with Wald χ 2 to determine if they were significantly different from zero. Significant interaction effects were analyzed pairwise among relevant conditions (sex or virus) across days with a least significant difference adjustment for multiple comparisons. Significance level was set at α= 0.05. For ethanol self-administration studies, grams per kilogram were calculated based on the volume consumed using the following formula ((0.014 L * number of rewards earned)volume leftover in drinking trough after session) *0.09*0.789) / (body weight converted to kg). Grams per kilogram of ethanol consumed, and blood ethanol concentration were analyzed using the normal distribution since these are continuous measures. Blood ethanol concentration and calculated grams per kilogram were correlated with a simple linear regression. Percentage of responsive neurons in viral conditions was compared using Fisher's exact test. Amplitude and latency of oIPSCs were analyzed with unpaired t tests between viral conditions, with Welch's correction applied for unequal variances when appropriate.

Operant Binge Ethanol Self-Administration
High levels of ethanol consumption are achieved in our operant self-administration model for 9% (v/v) ethanol with 2% sucrose in food restricted, post-prandial mice during the dark cycle. The level of ethanol and sucrose included in the ethanol solution is about equivalent to a Moscato wine. Male and female C57BL/6J mice responded on the active lever to self-administer ethanol ( Figure 1A). Females tended to respond at higher rates than males did across days, with a significantly higher response rate on day 9 of FR4 (Main effect of sex:

Ethanol self-administration ventral BNST CRF Knockdown
Knocking down expression of CRF peptide within the ventral bed nucleus of the stria terminalis led to reductions in ethanol self-administration. This effect was more pronounced in males: when active lever responses across days are tested for sex and virus effects there is a significant sex-by-virus interaction (Wald χ 2 (1)=9.0, p=0.003) and a sex-by-virus-by-day interaction (Wald χ 2 (8)=15.3, p=0.05). When examining the . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.03.02.530838 doi: bioRxiv preprint GFP-expressing control mice, was no significant main effect of sex, or sex-by-day interaction, which indicates that the sex effects are driven by the CRF knockdown groups. When examining the CRF knockdown mice, there was a significant main effect of sex (Wald χ 2 (1)=11.6, p< 0.0001) and a significant sex-by-day interaction (Wald χ 2 (8)=43.0, p<0.0001). Male CRF knockdown mice had significantly lower active lever responding than female CRF knockdown mice on all days except day 4. Male CRF knockdown mice self-administered less ethanol than male GFP controls (Figure 2A. Main effect of virus Wald χ 2 (1)=18.9, p<0.0001). There was a trend for a reduction in active lever responding in female CRF Knockdown mice ( Figure 2B. Main effect of virus: Wald χ 2 (1)=3.3, p=0.07).
Knocking down expression of the CRF peptide also reduced the consumption of ethanol as measured by g/kg, with a similar pattern to the active lever responding. When assessing g/kg across days of FR4 by sex and virus, there is a significant main effect of sex (Wald χ 2 (1)=13.1, p<0.0001), and sex-by-virus interaction (Wald χ 2 (1)=4.1, p=0.04). Examining the role for sex in the GFP-expressing control mice revealed a significant sexby-day interaction (Wald χ 2 (8)=18.6, p=0.017), where female GFP mice had significantly higher g/kg on day 6 (p<0.05) compared to male GFP mice, reflecting the tendency for female mice to consume higher levels of ethanol than males. Examining the role for sex in the Cre-expressing control mice revealed a significant main effect of sex (Wald χ 2 (1)=28.5, p<0.0001) and sex-by-day interaction (Wald χ 2 (8)=28.5, p<0.0001), where male CRF knockdown mice consumed less ethanol than female CRF knockdown mice did (all days' p<0.01). Male CRF knockdown mice consumed less ethanol than male GFP controls ( Figure 2C. Main effect of virus: Wald χ 2 (1)=12.8, p<0.0001, virus-by-day interaction: Wald χ 2 (8)=72.5, p<0.0001, all days' p≤ 0.01). There was a tendency for female CRF knockdown mice to consume less ethanol than female GFP controls, reflected in a trend for a day-by-virus interaction ( Figure 2D. Wald χ 2 (8)=14.7, p=0.066), and significantly lower g/kg values on day 6 (p=0.001) and day 8 (p=0.02).
Knocking down CRF peptide expression in the ventral BNST reduced motivation to work for ethanol reinforcers in the progressive ratio tests. Rewards earned on the arithmetic progressive ratio test ( Figure 2E . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.03.02.530838 doi: bioRxiv preprint

Sucrose self-administration ventral BNST CRF Knockdown
Sucrose self-administration was not affected by knockdown of CRF peptide in the BNST. When examining active lever responses across days by sex and virus, there was no significant main effect of virus (Wald χ 2 (1)=0.012, p=0.9) or sex-by-virus interaction (Wald χ 2 (1)=1.7, p=0.2). There was a significant main effect of sex (Wald χ 2 (1)=20.2, p<0.0001), and sex-by-virus-by-day interaction (Wald χ 2 (8)=15.7, p<0.05), due to higher levels of active lever responses in females compared to males. When examining GFP-expressing control mice, females responded more for sucrose than GFP-expressing males did (main effect of sex: Wald χ 2 (1)=14.5, Knocking down CRF peptide expression in the ventral BNST did not alter motivation to work for sucrose reinforcers in the progressive ratio tests. In the arithmetic progressive ratio test, there was a significant main effect of sex ( Figure 3C. Main effect of sex: Wald χ 2 (1)=12.9, p<0.0001) reflecting the higher level of rewards earned in females. In the exponential progressive ratio test, there was a significant main effect of sex ( Figure   3D. Main effect of sex: Wald χ 2 (1)=9.6, p=0.002, sex-by-virus interaction: Wald χ 2 (1)=4.5, p=0.03). There were no significant effects of virus for males (p=0.1) or for females (p=0.1) on rewards earned in the exponential progressive ratio test.

GABA knockdown functional validation
Using short hairpin RNA interference directed at the vesicular GABA transporter (referred to as shvGAT), we were able to selectively reduce GABA release from CRF neurons in the BNST (26,27). We injected a mixture of channelrhodopsin and either the shvGAT virus or the scrambled sequence control virus into the BNST of CRF-Cre mice to measure optically evoked inhibitory currents. Neurons in the BNST are densely inter-. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; connected (29,30), so whole-cell patch clamp recordings from non-fluorescent were able to detect reductions in GABA release from CRF neurons with GABA knockdown. There were no significant differences in the percentage of responsive neurons recorded from scramble controls ( Figure 4A) or shvGAT-expressing mice ( Figure 4B). There was a significant reduction in oIPSC amplitude in shvGAT-expressing mice compared to scramble controls ( Figure 4C. t(7.6)=3.6, p=0.007), indicating that GABA release was reduced by the viral manipulation. There was no difference in oIPSC latency between viral conditions ( Figure 4D. t(13)=1.5, p=0.15). was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Sucrose self-administration following BNST CRF neuron vGAT knockdown
Reducing GABA release from BNST CRF neurons had various impacts on sucrose self-administration. For active lever responding for sucrose, when analyzing the effect of virus and sex across days, there was a significant main effect of sex (Wald χ 2 (1)=5.8, p=0.02), and a significant sex-by-virus-by-day interaction (Wald χ 2 (8)=16.2, p=0.04). Analyzing active lever responding for scramble control mice, there was a significant sexby-day interaction (Wald χ 2 (8)=24.1, p=0.002), where females tended to respond more on the active lever than males did, reaching significance on day 8 (p=0.02) and day 9 (p=0.001). When examining active lever responding for shvGAT-expressing animals, there was a significant main effect of sex (Wald χ 2 (1)=4.8, p=0.03), and a significant sex-by-day interaction (Wald χ 2 (8)=30.0, p<0.0001), with females responding more on the active lever than males (day 2 p=0.03, day 3 p=0.04, day 7 p=0.02, day 9 p=0.005). Within male mice ( Figure 6A  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Knocking down GABA release from BNST CRF neurons had a larger effect on males than females on motivation to work for sucrose reinforcement. In the arithmetic progressive ratio test ( Figure 6C) there was a significant main effect of sex (Wald χ 2 (1)=19.1, p<0.0001), a trend for a main effect of virus (Wald χ 2 (1)=3.6, p=0.06), and a significant sex-by-virus interaction (Wald χ 2 (1)=6.7, p=0.009). GABA knockdown in BNST CRF neurons in males reduced motivation to work for sucrose on an arithmetic progressive ratio test (p=0.015), but did not alter motivation to work for sucrose in females on this test (p=0.3). In the exponential progressive ratio test, there was a significant main effect of virus (Wald χ 2 (1)=4.5, p=0.04), indicating that GABA knockdown reduced motivation to work for sucrose for both males and females.

Discussion
In the present study, we found that deletion of CRF from the BNST led to a reduction of operant ethanol selfadministration in male mice and reduced motivation to consume alcohol in both male and female mice. This appeared selective for ethanol, as we did not see similar effects of CRF knockdown when examining operant sucrose self-administration in parallel behavior-matched controls. Suppression of vGAT from BNST CRF neurons transiently increased ethanol responses in male, but not female mice. Additionally, suppression of vGAT from BNST CRF neurons reduced motivation to work for sucrose reinforcers, with females showing a reduction in motivation only on the exponential progressive ratio schedule. Together, these findings suggest that CRF release from BNST neurons supports high intensity drinking that precedes ethanol dependence. Suppressing GABA release from BNST CRF neurons may produce a state of anhedonia, which may underlie the transient increase in ethanol self-administration in males due to stronger negative reinforcement from the ethanol, as well as the reduction in motivation for sucrose reinforcers. This study supports a model in which different compounds released from the same population of neurons can bidirectionally influence behavior. In support of this model, a recent report demonstrated opposing functions in anxiety-like behavior for CRF and GABA released from central amygdala CRF-expressing neurons in rats (27). This is an important consideration to take into account when designing and interpreting experiments that drive activation and inhibition of neuronal populations.
Previous work in our lab found that CRF-containing neurons in the BNST appeared to be important for bingelike ethanol consumption. Specifically, we found that activating a Gi-coupled DREADD led to reduced alcohol consumption, in both male and female mice (9,10). These BNST CRF neurons are GABAergic as well, raising . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; the question was it the CRF or the GABA, or both that was driving increased alcohol intake. In male mice, there was a clear model that emerged, with CRF driving alcohol responding and motivation, and GABA playing an opposing role on alcohol responding. In females, it was less clear. Deletion of CRF led to a reduction in motivation, but minimal impact on responding. This is interesting, as in a recent study we found that there were sex differences in the physiological properties of BNST CRF neurons, with the neurons from female mice being more excitable with greater synaptic excitation (31).
Reducing GABA release from BNST CRF neurons in male mice produced a transient increase in selfadministration of ethanol, but did not alter motivation to work for ethanol on our progressive ratio tests. It is possible that if we had tested for motivation differences earlier in FR4 training, we may have seen an increase.
It is possible that there is some form of compensatory mechanism at play to adjust ethanol self-administration behavior following GABA release reductions, which seems more likely to happen with fast neurotransmitters like GABA compared to neuromodulators like CRF. Taken together, these data sets support a model in which there may be different mechanisms and circuits engaged to support high level alcohol consumption. These sex-differences are important to consider translationally, as there have been negative results reported for CRFR1 antagonists clinical trials for AUD that have focused on female patient populations (32).  (34). Previous work using fiber photometry recordings has shown that food consumption increases (35), and cocaine administration decreases (36), bulk neural activity in the BNST. Combined with our results, this might indicate that GABA release from BNST-CRF neurons may lessen the motivational value of sucrose reinforcers. For cocaine reinforcers, the role for decreased BNST-CRF neural activity is not related to motivation, so optogenetic stimulation does not alter cocaine-motivated behavior.

Potential caveats of our model
Food restriction impacts the mouse behavior in this study, as does the inclusion of sweetener. Notably, in humans this is the case also. Food insecurity risk is associated with moderate and severe alcohol use disorder . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.03.02.530838 doi: bioRxiv preprint diagnosis in young adults (37). According to the CDC, about half of American adults drink sugar-sweetened beverages on a given day (38), and about 1 in 5 American adults is dieting, which often involves some form of food restriction (39). Indeed, the term "drunkorexia" was coined 15 years ago to describe patterns of behavior involving food restriction and binge drinking of alcohol in humans that is associated with higher risk for alcoholrelated problems (40). The model used herein, rather than studying the impact of these factors in isolation to determine their individual effect, is using them as a model for common human behaviors and social determinants of health that represent important risk factors for adverse outcomes. Other operant studies for ethanol (25,41), food (42-45), and other rewards including cocaine (34,46,47) utilize food restriction as a means to enhance motivation. It is possible that enhanced motivation through food restriction may produce a ceiling effect for measuring increases in operant responding.

Conclusion
These data demonstrate that the same genetically-defined neural population within a brain region can influence motivated behavior in opposing ways depending on which neurotransmitter is released. Additionally, neural circuits underlying motivated behavior are sexually dimorphic.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023.  C. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.03.02.530838 doi: bioRxiv preprint Figure 1: Operant Binge Ethanol Self-Administration model a. Male (n=8) and female (n=8) C57Bl6/J mice learn to respond on the active lever for ethanol reinforcers.
Females tended to respond on the active lever more than males did across days, with the effect being significant on day 9. Main effect of sex: Wald χ2 (1)   was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.03.02.530838 doi: bioRxiv preprint was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.03.02.530838 doi: bioRxiv preprint mice to female scramble mice across days: day 1 p=0.7, day 2 p=0.6, day 3 p=0.96, day 4 p=0.7, day 5 p=0.4, day 6 p=0.3, day 7 p=0.4, day 8 p=0.5, day 9 p=0.6 c. BNST-CRF GABA knockdown reduced motivation to work for sucrose reinforcement on an arithmetic progressive ratio schedule of reinforcement in male mice. Main effect of sex Wald χ 2 (1) = 19.1, p<0.0001, main effect of virus Wald χ 2 (1) = 3.6, p=0.06, sex-by-virus interaction Wald χ 2 (1) = 6.7, p=0.009. Effect of virus in males p=0.015, effect of virus in females p=0.3. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.03.02.530838 doi: bioRxiv preprint g.

Figure 7: Summary of behavioral results
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 1, 2023. ; https://doi.org/10.1101/2023.03.02.530838 doi: bioRxiv preprint