key: cord-0257749-xi1fdmif
authors: Noback, Michael; Barrow, James C.; Carr, Gregory V.
title: Post-weaning social isolation increases reward-seeking behavior in a sex-specific manner in mice
date: 2021-04-20
journal: bioRxiv
DOI: 10.1101/2021.04.20.440665
sha: 75ace8f27190404657142428ebefb881f88cac3a
doc_id: 257749
cord_uid: xi1fdmif

Social isolation is a growing concern in public health. Although isolation at any age is harmful, previous studies have shown that isolation during adolescence, correlating with critical periods of brain development, can impair cognitive function and increase the risk for psychiatric illness later in life. In this study, we utilized a mouse model of adolescent social isolation (SI) and compared performance of isolated and group-housed mice on touchscreen-based continuous performance test (CPT) and fixed ratio/progressive ratio (FR/PR) tasks in adulthood. SI increased sensitivity in the CPT in male mice and had no effect in female mice. The increase in sensitivity was consistent across time bins within the 45-minute testing session and there were no SI effects on reaction times or reward retrieval latencies. A possible confound for performance in the CPT would be SI-induced changes in reward-seeking or motivation for the strawberry milk reward. We next compared the SI mice to their group-housed littermate controls on both FR and PR schedules of reinforcement and found that male SI mice earned significantly more reinforcers on FR schedules of reinforcement and had higher breakpoints on PR schedules compared to their group-housed littermates. SI had no effect on FR or PR performance in female mice. These data indicate that SI during adolescence has striking, sex-specific effects on reward-seeking behavior in adult mice and may provide a useful behavioral model for studying the link between SI and risk for neuropsychiatric disorders.

Social isolation (SI) is a stressor with significant acute and chronic effects, including increased risk for 45 developing psychiatric disorders. Specifically, there is a clear link between SI and anxiety, depression, 46 substance use disorder, and schizophrenia [1] . Despite the confirmation of SI as a risk factor, the 47 biological link between SI and psychiatric disorders is unknown. 48 49

The relevance of SI as a source of environmental stress has increased dramatically over the past year 50 due to the COVID-19 pandemic. It is too early to definitively assess the societal and economic impacts of 51 the increase in SI due to social distancing measures, but its role in public health is already being closely 52 monitored [2] [3] [4] . A deeper understanding of the biological effects of SI will be critical to address the 53 long-term effects of COVID-19-related SI. 54 55

There is an extensive literature on the effects of SI on older people (Reviewed in [5] ), but recent studies 56 have identified increases in the prevalence of SI and loneliness among children and adolescents [6] . SI in 57 younger people is particularly important for disorders with significant neurodevelopmental components, 58

including schizophrenia and substance use disorders [7] . Given the increased plasticity of the brain 59 during childhood and adolescence [8] , SI may have different or greater effects compared to SI in adults. 60 SI has also been studied in animal models, and both chronic and acute isolation have been associated 61

with anxiety-and depression-like behavior in rodents [9] . SI during postnatal days 21-35, roughly 62 correlating developmentally to ages 11 to 18 in humans, [8] produced deficits in myelination in the 63 prelimbic and infralimbic cortices through an IL-6-dependent mechanism [10, 11] . Additionally, SI during 64 this period also enhances the activity of interneurons in the prelimbic and infralimbic cortices [12] . 65 66

It is not clear why isolation during postnatal days 21-35 produces such drastic effects on the structure 67 and function of the brain. Interestingly, postnatal days 21-35 is a period of significant amounts of social 68 play behavior in mice and isolation during this period can lead to deficits in social interactions in 69 adulthood [13, 14] . Moreover, the social behavior deficits are directly related to abnormal interneuron 70 function in the prefrontal cortex [ Breeding pairs of C57BL/6J mice were purchased at 6-7 weeks old from The Jackson Laboratory (Bar 94

Harbor, ME, USA). Only one litter from each breeding pair was used in these experiments. Pups were 95 raised according to an isolation protocol adapted from one used by Makinodan and colleagues [10] . 96

Pups were weaned at postnatal day 21 (P21) and housed either in isolation or in a group of three same 97 sex littermates. Isolated mice were rehoused with another same sex isolated littermate at P35 for the 98 duration of the experiment (Figure 1a ). 99 100

2.2 Handling, food restriction, and habituation 101

At P63, mice were placed on food restriction and briefly handled for three days. Food amounts were set 102 to keep the animals at no less than 85% of their free feeding weight. During the initial handling days, 103 mice were also introduced to a Bussey-Saksida testing chamber (Lafayette Instrument, Lafayette, IN, 104 USA) for habituation. Mice were placed in the chamber, and 1 mL of reward (strawberry Nesquik, 105

Nestle) was placed in the feeding tray. The habituation schedule consisted of a 30-minute session where 106 the screen was responsive to touch, but touches were not rewarded. Mice were considered habituated 107 when they had undergone at least three habituation session and had fully consumed the reward during 108 at least one session. In stage 1, mice were trained to touch a visual stimulus (white square). The square was displayed 113 (stimulus duration) for 10 seconds. The stimulus duration was followed by a 0.5 second limited hold (LH) 114

period during which the screen was blank, but a touch would still yield a reward. Upon interacting with 115 the stimulus, a one-second 3 kHz tone would sound, a small amount of reward would be dispensed, and 116 the reward tray would be illuminated. Head entry into the reward tray was detected by an infrared (IR)  117 beam, and upon head entry the intertrial interval (ITI) of 2 seconds would begin. If the mouse did not 118 interact with the stimulus, the ITI would begin immediately following the LH period. The next trial would 119 begin immediately following the ITI. The criterion for advancement to stage 2 was obtaining 60 rewards 120 within a single 45-minute session. 121 122

In stage 2, the white square pattern was replaced with either horizontal or vertical bars -the mouse's 123 assigned positive stimulus, or S+. The S+ was counterbalanced within groups. The SD was reduced from 124 10 seconds to 2 seconds, and the LH was increased from 0.5 seconds to 2.5 seconds. The criterion for 125 advancement was the same as stage 1. 126 127

In stage 3, a negative stimulus (S-; snowflake pattern) was added. Each trial had a 50% chance of being 128 either an S+ or S-trial. SD and LH were identical to stage 2, but ITI was increased to 5 seconds. 129

Interacting with S-during the SD or LH would not yield a reward and would start the ITI. The criteria for 130 advancement to stage 4 were a minimum of seven sessions, during which at least two consecutive 131 sessions had a discrimination index (d') score of 0.6 or higher. A discrimination index is a measurement 132 derived from signal detection theory [24]that is used to distinguish meaningful response to stimulus 133 from noise. The discrimination index was calculated as follows: 134 135 (Figure 1c ). Fixed ratio 1 (FR1) required a 143 mouse to touch a white square, similar to CPT stage 1 without the stimulus duration timer. The session 144 ended after 45 minutes, or when a mouse obtained 30 rewards within one session. After a mouse 145 obtained 30 rewards within a single session, they progressed onto the next stage. FR2 and FR3 followed 146 the same rules as FR1, but with the respective number of touches per reward. FR5 consisted of multiple 147 sessions in which the mouse had to touch the square five times to obtain a reward. The same timing and 148 cutoff rules from the previous stages applied, but mice were required to obtain 30 rewards during each 149 of at least three sessions before advancing to progressive ratio testing. Progressive ratio 4 (PR4) 150

followed the same parameters as the previous FR stages, but the number of touches required for a 151 reward increased by four each time a reward was obtained. The initial PR4 phase consisted of seven 152 sessions, and during each session the break point of each mouse was recorded. The break point was 153 defined as the highest number of touches committed within one session to obtain a reward. 154 155

Following the initial PR4 phase, mice were again tested on FR1, but the 30-reward cap was removed. 156

Three similar sessions of FR5 followed. The amount of rewards obtained per session was recorded. 157

After the free-feeding sessions, mice underwent another seven-session PR4 phase. 158 159 After observing that SI males scored higher in the CPT, a test sensitive to changes in attention, we tested 202 males in a progressive ratio regimen, a battery sensitive to changes in motivation and reward-seeking 203

behavior. This test allowed us to determine whether the observed CPT performance in SI males was 204 being driven by an increase in attentional reserves or changes in reward-seeking behavior. We found 205 that male mice that had undergone post-weaning SI expended more effort to obtain rewards during PR4 206 trials compared to group-housed littermates. (Figure 5a , F13,11 = 3.430, p = 0.0113) 207 208 4.6 Male SI mice consume more reward in an uncapped FR1 session compared to group-housed controls 209

The motivation testing battery included an unlimited FR1 session between blocks of PR4 sessions to 210 eliminate potential reward satiety as a confounding factor. We found that while both GH and SI mice 211 consumed more rewards during the FR1 session than during the PR4 sessions, thus confirming that they 212

were not reaching reward satiety, SI mice consumed significantly more rewards than their group-housed 213 littermates. (Figure 5b , F13,11 = 1.349, p = 0.0037) The number of rewards received during this session 214 also exceeded the amount of rewards received during any one session of the CPT, indicating that satiety 215

was not a confounding factor in that test either. 216 217

Adolescent social isolation causes measurable behavioral changes that persist through adulthood. Using 219 a CPT, we found that SI increased sensitivity measures, usually interpreted as improved sustained 220 attention, in male mice. SI also caused increases in reward-seeking behavior in males as measured by a 221 FR and PR tasks, indicating the effects in the CPT may not have been specific to the domain of sustained 222

attention. The effects of SI appeared to be sex-specific, as the performance of female mice in the CPT 223 was unaffected by SI. These results together form a consistent, robust profile of the effects of 224 adolescent SI on reward-seeking behavior. 225 226

This study investigated the effects of adolescent SI on sustained attention via CPT, which has established 228 usage in rodent studies and human diagnostics [25] . Adolescent SI male mice performed significantly 229 better on stage 4 of the CPT than group-housed males and both group-housed and SI females. Group-230 housed and SI females did not score significantly differently on the CPT. 231 232 To further characterize how mice engaged with the task, we analyzed the CPT session data using 15-233 minute time bins. Performance throughout each CPT session was consistent among the housing 234 conditions, with male SI mice scoring significantly higher than the other housing conditions in all three 235 bins. Female SI and group-housed mice again were not significantly different from each other. 236

Higher scores in CPT are usually associated with improvements in sustained attention [23] , but many 237 studies of SI indicate impaired attention as a key feature of the phenotype [26] . In response to this 238 finding, we considered performance in the CPT as an interaction between the mouse's willingness to 239 engage in the task and their interest in the food reward. If SI impaired attention but also increased 240 reward-seeking behavior, this may read as an increase in CPT score depending on the weight of each 241 change. 242 243

To further investigate this interaction between attention and motivation, we utilized a fixed-245 ratio/progressive-ratio regimen. Only males were tested due to the fact that females did not show a 246 significant effect of isolation in the CPT. Progressive ratio tasks measure effort committed to obtain a 247 reward without the cognitively demanding rules of the CPT, so the PR results would possibly allow us to 248 further refine the potential implications of the CPT results. 249 250 SI males showed increased effort in the PR, which is interpreted as increased reward-seeking behavior. 251

This validates our hypothesis that changes in CPT performance caused by SI are due at least in part to 252 significant differences in reward-seeking and motivation in SI males compared to the other groups 253

tested. This finding is in agreement with the known role of isolation as a risk factor for substance use 254 disorders in humans. 

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Short-Term Analysis

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The Epidemiology of 308 Social Isolation: National Health and Aging Trends Study

Perceived social isolation in a community sample: its prevalence and correlates 311 with aspects of peoples' lives

Impact of adolescent social experiences on 314 behavior and neural circuits implicated in mental illnesses

Stress and the developing adolescent brain

Brief periods of socialization and later behavior in the rat

A Critical Period for Social Experience-Dependent 321

Oligodendrocyte Maturation and Myelination

Social 325 isolation impairs remyelination in mice through modulation of IL-6

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Scoring of social interactions and play in mice during adolescence

Prefrontal parvalbumin interneurons require juvenile social experience to establish 336 adult social behavior

Post-weaning social isolation increases 338

ΔFosB/FosB protein expression in sex-specific patterns in the prelimbic/infralimbic cortex and 339 hippocampus in mice

Cortical Circuit for Depression-and Anxiety-Related Behaviors Mediated by Cholecystokinin: Role 344 of FosB

Molecular neurobiology of addiction: What's all the (Δ)FosB about?

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New frontiers in translational 355 research: Touchscreens, open science, and the mouse translational research accelerator 356 platform

Attention/vigilance in 360 schizophrenia: Performance results from a large multi-site study of the Consortium on the 361 Genetics of Schizophrenia (COGS)

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Functional Dissociations Between Subregions 374 of the Medial Prefrontal Cortex on the Rodent Touchscreen Continuous Performance Test (rCPT)

of Attention

Behavioural and neurochemical effects of post-weaning social isolation 377 in rodents-Relevance to developmental neuropsychiatric disorders

Early life adversity promotes 382 resilience to opioid addiction-related phenotypes in male rats and sex-specific transcriptional 383 changes

Early life stress 386 causes sex-specific changes in adult fronto-limbic connectivity that differentially drive learning

ΔfosB in brain reward 391 circuits mediates resilience to stress and antidepressant responses