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. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Alcohol Clin Exp Res. 2019 Aug 14;43(9):1949–1956. doi: 10.1111/acer.14149

Increased maternal care rescues altered reinstatement responding following moderate prenatal alcohol exposure

Sarah L Olguin 1,2, Amber Zimmerman 1, Haikun Zhang 3, Andrea Allan 1,2, Kevin C Caldwell 1,2, Jonathan L Brigman 1,2,3,*
PMCID: PMC6721985  NIHMSID: NIHMS1042504  PMID: 31318985

Abstract

Background:

Fetal Alcohol Spectrum Disorders (FASD) commonly includes deficits in learning, memory and executive control that can have a severe negative impact on quality of life across the lifespan. It is still unclear how prenatal alcohol exposure (PAE) affects executive control processes, such as control over reward seeking, that lead to inappropriate behavior later in life. Learning and reinstatement of a previously learned response after extinction is a simple, well validated, measure of both acquisition of a rewarded instrumental response, and sensitivity to reward and reward-associated cues. We investigated the effects of PAE on learning, extinction and reinstatement of a simple instrumental response for food reward. Next, we assessed the effectiveness of an early intervention, communal nest (CN) housing, on increased reinstatement of an extinguished response seen after PAE.

Methods:

To assess the effects of PAE on control over reward seeking, we tested male and female PAE and saccharine (SAC) controls raised in a standard nest (SN) on the acquisition, extinction, and food reward induced reinstatement of an instrumental response utilizing a touch-screen based paradigm. Next, in order to examine the effects of an early life intervention on these behaviors, we tested PAE and SAC mice raised in a communal nest (CN) early life environment on these behaviors.

Results:

PAE mice readily acquired and extinguished a simple touch response to a white square stimulus. However, PAE mice showed significantly increased and persistent reinstatement compared to controls. Increased maternal care via rearing in CN slowed acquisition and sped extinction learning, and rescued the significantly increased reinstatement responding in PAE mice.

Conclusions:

Together these results demonstrate that even moderate PAE is sufficient to alter control over reward seeking as measured by reinstatement. Importantly, an early life intervention previously shown to improve cognitive outcomes in PAE mice was sufficient to ameliorate this effect.

Keywords: early-life environment, touch-screen, fetal alcohol spectrum disorder (FAD), reward seeking

Introduction

Fetal Alcohol Syndrome (FAS), caused by high dose prenatal alcohol exposure (PAE) can lead to a variety of serious long-term effects in humans including growth restriction, craniofacial dysmorphology, and neurocognitive deficits (Streissguth et al., 1991). Growing evidence suggests that even moderate alcohol exposures can lead to lifelong impairments without the attendant morphological changes seen in FAS. Termed Fetal Alcohol Spectrum Disorders (FASD), these deficits in learning, memory and executive control processes can have a severe negative impact on quality of life across the lifespan.

Deficits in executive control processes can lead to significant decreases in quality of life measures; FASD has been shown to be associated with decreased performance in numerous measures of executive control, beyond what would be predicted by IQ scores alone (Connor et al., 2000). Among other difficulties, young adults with FASD have been shown to have difficulty with control over reward seeking, including significantly increased rates of both inappropriate sexual behaviors and substance use issues (Streissguth et al., 2004). Rodent models are an important tool for studying these effects as studies in human patients and rodent models suggest a congruent effect of blood alcohol content (BAC) on behavioral outcomes (Driscoll et al., 1990). While these models have provided strong insight on the impact of PAE on affective, social and cognitive domains (Marquardt and Brigman, 2016), little is known regarding how PAE alters reward seeking.

Instrumental learning and the reinstatement of a previously learned response after extinction training is a simple behavioral assay to examine numerous aspects of control over reward seeking including: initial learning of a reward-associated cue, persistence of stimulus-response responding when reward is discontinued during extinction, and sensitivity to reward and reward-related cues after a brief re-exposure. While this approach, and reinstatement of responding in particular, has been proposed as a model of relapse in drug addiction (Epstein et al., 2006; Lederle et al., 2011; Sorg, 2012), this paradigm is a valuable tool to understand how motivational systems function optimally, and are altered by developmental insult. Further, this simple approach provides important insight into whether an early life intervention can alter cortico-striatal mediated behaviors in adulthood.

Early life environment can exert long-lasting effects on an organism (Gluckman and Hanson, 2004; Monk et al., 2012; Monk et al., 2013) and animal models can provide an important translational tool to test how early-life interventions may provide an important treatment avenue, as the vastly shorter time scales and high level of experimental control allow for approaches not available in the human population. Specifically, rearing in a communal nest (CN) in rodents is an ethologically relevant intervention that has previously been shown effective in increasing time on nest by dams, improving measures of pup care, and increased pup-pup interactions prior to weaning compared to the standard nest (SN) (Branchi and Alleva, 2006; Branchi et al., 2013b; Branchi et al., 2013a). This early life intervention has also been shown to significantly reduce social anxiety and response to social, but not physical, stress in adulthood (Branchi et al., 2011). Importantly, previous studies combining CN with PAE have found that increased maternal and pup interaction measures seen in CN are not altered by alcohol consumption in dams, and neither is maternal alcohol drinking reduced by CN, making this intervention well suited for PAE studies (Caldwell et al., 2015b). Previously, we have shown that the communal nest intervention can mitigate the effects of moderate PAE on hippocampal dependent learning and memory as measured by context discrimination (Caldwell et al., 2015b). While these findings suggest that CN is a simple, effective, intervention to ameliorate the effects of PAE on hippocampal learning and memory, the impact of this approach on cortical mediated executive control deficits has not been explored.

Here, we assessed the effect of PAE on the acquisition, extinction, and food reward induced reinstatement of a touch-screen based instrumental response paradigm previously shown to distinguish alterations in reward seeking between strains and genetic manipulations (Lederle et al., 2011; Oliver et al., 2018). Next, given behavioral alterations were seen after PAE, we investigated whether CN was sufficient to rescue increases in reinstatement seen in PAE mice.

Materials and Methods

Prenatal Alcohol Exposure and Housing.

All experimental procedures were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the University of New Mexico Health Sciences Center Institutional Animal Care and Use Committee. Male and female C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Females consumed either 0.066% (w/v) saccharin or an ethanol solution (5% w/v for four days, then 10% w/v until parturition) sweetened with 0.066% (w/v) saccharin for four hours (from 1000 to 1400 hrs), as previously described (Brady et al., 2012a; Brady et al., 2013). We have shown this protocol reliably produces blood ethanol concentrations of 80–90 mg/dL at the end of the 4-hour drinking period in both standard nest (SN) and communal nest (CN) dams (Caldwell et al. 2015). Blood ethanol concentrations were not directly measured during the present study, however, alcohol consumption was determined by weighing drinking tubes and the amount of ethanol consumed was divided by the number of dams in the cage. CN and SN mice drank to levels consistent with our previous studies (CN=3.75±0.50 SN=4.46±0.48 g/kg/4 hrs) and no significant difference were seen between consumption level by housing (t(14)=1.46, p=0.17). On post-natal day 0 both the ethanol and control saccharin solutions were diluted 1:2 with tap water every 2 days for a 6 day step-down procedure.

After approximately one week of drinking, the Whitten effect (Heiderstadt et al., 2014; Caldwell et al., 2015b) was used to synchronize the estrous cycle and facilitate the efficiency of a limited (two-day) mating paradigm. The mating protocol involved placing either two solo-housed females (SN) or all females from a communal cage (CN; 3–4 dams) into the cage of a solo-housed male from 1400 to 0800 h for two successive days. Females were weighed to check for pregnancy 9 days following mating and again 13–14 days following mating. Non-pregnant females were removed from CN cages, if fewer than two females were pregnant in a CN cage, females were transferred between communal cages. Communal cages had dams that delivered litters within two days of each other. Following parturition, the SN and CN cages were left undisturbed until weaning at approximately 23 days of age, excluding a single cage bedding change approximately 10 days following delivery of the litter(s) and placement of drinking tubes. Standard Nest (SN) females/dams were housed alone prior to and throughout pregnancy. For communal nest (CN) rearing, three or four females were housed together prior to positive identification of pregnancy. Offspring remained in CN or SN housing until weaning and then were maintained in groupings of 2–4 per cage in a temperature and humidity- controlled vivarium under a reverse 12 h light/dark cycle (lights off 0800 h) and tested during the dark phase. For behavioral testing, n=7–8 offspring per sex/treatment/maternal care condition was used. To maximize breeding output, a maximum of 3 pups per litter were utilized and analyzed as an independent variable to examine litter effects. In the case offspring were removed from group cages due to fighting or diet issues, solo housing was analyzed across variable.

Operant apparatus.

Behavior was conducted in a chamber measuring 21.6 × 17.8 × 12.7 cm (model # ENV-307W, Med Associates, St. Albans, VT) housed within a sound- and light-attenuating box (Med Associates, St. Albans, VT). The standard grid floor of the chamber was covered with a solid acrylic plate to facilitate ambulation. A magazine that could deliver either 14 mg dustless pellets (#F05684, BioServ, Frenchtown, NJ) from a pellet dispenser, a house-light, tone generator and an ultra-sensitive lever was located at one end of the chamber. At the opposite end of the chamber there was a touch-sensitive screen (Conclusive Solutions, Sawbridgeworth, U.K.) covered by a black acrylic aperture plate allowing two 7.5 × 7.5 cm touch areas separated by 1 cm and located at a height of 0.8 cm from the floor of the chamber. Stimulus presentation in the response windows and touches were controlled and recorded by the K Limbic Software Package v1.20.2 (Conclusive Solutions, Sawbridgeworth, U.K.).

Pretraining.

Beginning at 8 weeks of age, all mice were handled daily and were food-restricted to 85% of their free-feeding body weight. Operant training began once mice reached food-restricted weight at ~10 weeks of age. Mice were first acclimated to the reward pellets by provision of ~10 pellets/mouse in the home cage for 3–5 days and then habituated to retrieving reward in the operant chamber. Mice were allowed 30 min to freely retrieve pellets available in the magazine. Mice retrieving at least 10 rewards within 30 min were moved to lever press training. Here, mice could only obtain reward by responding on an ultrasensitive lever within the chamber. Reward delivery was accompanied by the presentation of secondary reinforcers: a 2-sec, 65 dB auditory tone and illumination of a magazine light. For each trial, mice were required to collect the delivered reward (measured by magazine beam-break) before another reward was available via an active lever response. Mice were required to lever-press and collect 30 rewards in under 30 minutes before moving to acquisition testing.

Acquisition of a touch-screen instrumental response.

During acquisition, lever press now led to presentation of two stimuli (1 × 2.8 cm2 white square per window) in both response apertures on the touch-screen. Stimuli remained until a touch response was made (Figure 1A). A nose-poke of either stimulus led to the delivery of a single primary (food reward) and both secondary reinforcers described above. Mice were given 30 trials per 60 minute session daily (5 sec inter-trial-interval). Acquisition criterion was 30 responses within 15 minutes for 5 consecutive testing sessions.

Figure 1. Touch-screen Acquisition, Extinction and Reinstatement.

Figure 1.

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A. Acquisition training required mice to initiate the onset of 2 white square stimuli via lever press and touch either stimulus for food reward. Reward in the magazine was concomitant with 1s tone and illumination of the magazine light as secondary reinforcers. B. Extinction training presented the same visual stimuli automatically for 10sec and touch at either stimulus produced neither primary nor secondary reinforcement. C. Reinstatement of instrumental responding consistent of 6 primed trials where the stimuli automatically appeared in both response windows for 10 sec followed by reward delivery and secondary reinforcers. On the following 54 trials, stimuli were still automatically presented in the absence of reinforcement. Reinstatement was determined by calculating the difference in responding during reinstatement minus the response rate during the criterion extinction session. Extinction retention was tested 7 days following reinstatement training.

Extinction of instrumental response training.

Extinction training began on the first session following an individual mouse attaining acquisition criterion. During extinction testing, the visual stimuli were now automatically presented (no initiation required) in each aperture for 10 sec or until a touch was made at either response window (10 sec ITI; Figure 1B). A nose-poke during stimulus presentation only caused the stimuli to disappear, with no primary or secondary reinforcement. 30 stimulus presentation trials were presented in each daily extinction session. Criterion extinction learning was set at omission of response on 77% of trials for 2 consecutive daily sessions.

Reinstatement via brief-reward delivery.

On the next session following extinction criterion, mice were tested for reinstatement of instrumental responding. During the reinstatement sessions, stimuli automatically appeared in both response windows for 10 sec as during extinction (10 sec ITI). For the first 6 reinstatement trials, primary (food pellet) and secondary (tone and magazine light) reinforcers were delivered to a touch or after 10 second presentation to induce reinstatement of responding. The first 6 rewarded trials were followed by 54 non-rewarded trials during which stimuli were again automatically presented but no reinforcers were delivered regardless of response. The reinstatement effect was calculated via the difference in responding during reinstatement versus the response rate during criterion extinction session performance.

Extinction Retention.

1 week following reinstatement mice were given an additional session of extinction training as described above. Total number of unreinforced responses made to stimuli on 30 automatically presented trials were recorded.

Results

As no main effect of sex was seen on any measure, males and females were collapsed across groups. No litter or solo housing effect was seen across behavioral tasks, and was therefore removed from further analysis. All mice across treatments underwent habituation to consuming food reward in the touch-screen chamber in the first session. Similarly, the total sessions to acquire lever-press behavior for a non-sucrose food reward did not significantly differ across treatment or housing (main effect of treatment: F1,30=0.91, p=0.35; main effect of housing: F1,30=0.86, p=0.48). Mice of both treatments and housing conditions rapidly acquired the simple instrumental response to a visual stimulus. Analysis of performance across acquisition trials (Figure 2A) found that treatment did not significantly affect speed of acquisition (main effect of treatment F1,30=0.91, p=0.34) but housing condition did, with SN mice requiring significantly fewer sessions to reach acquisition criterion than CN mice (main effect of housing F1.30=14.64, p<0.01) with no significant interaction (F1,30=0.27, p=0.60). Similarly, latency from initiation to response (Figure 2B) did not differ significantly between PAE and SAC groups (main effect of treatment F1,30=0.12, p=0.73) but SN mice had significantly shorter latencies than CN (F1,30=50.23, p<0.01) with no significant interaction (F1,30=0.27, p=0.60). Early life environment also significantly affected latency to retrieve food reward (Figure 2C) after a touch (main effect of housing: F1,30=21.39, p<0.01) while PAE and SAC did not significantly differ and no interaction was present (main effect of treatment: F1,30=2.36, p=0.14; interaction: F1,30=2.14, p=0.154).

Figure 2. PAE did not alter acquisition of an instrumental response.

Figure 2.

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A. All mice readily learned the touch-screen response and no significant difference was seen on session to acquire the response, although animals in a communal nest required significantly more sessions regardless of treatment. CN mice required significantly more sessions to criterion regardless of treatment. B. PAE did not significantly alter latency to respond to rewarded stimuli, but communal nest mice responded at significantly slower rates. C. Communal nest, but not prenatal treatment, significantly increased latency to retrieve reward. Data are Means± SEM. *=main effect of housing p<.05.

During extinction training where primary and secondary reinforcers were removed, all mice rapidly decreased response to the visual stimuli. Analysis of extinction sessions found that PAE and SAC did not significantly differ on trials to criterion (main effect of treatment: F1,30=0.40, p=0.53). As in acquisition, early life environment groups differed significantly with SN mice being significantly slower to extinguish (Figure 3A; main effect of housing: F1,30=4.75, p=0.04; interaction: F1,30=0.07, p=0.79). Average latency to touch on non-reinforced response trials increased across sessions and was significantly shorter in SN mice versus communal (Figure 3B) but did not differ across treatment with no interaction (main effect of housing: F1,30=5.52, p=0.03; main effect of treatment: F1,30=0.18, p=0.68; interaction: F1,30=0.79, p=0.38). Analysis of responding on the session where criterion was reached (Figure 3C) showed that although CN mice were faster to extinguish than standard reared mice, all mice responded to non-reinforced stimuli on the final session of extinction at similar levels with no significant differences or interactions between groups (main effect of housing: F1,30=1.03, p=0.32; main effect of treatment: F1,30=1.50, p=0.23; interaction: F1,30=0.34, p=0.56).

Figure 3. PAE did not alter extinction of the previously learned response.

Figure 3.

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A. Mice rapidly extinguished the response in the absence of reward. No significant difference was seen on sessions to reach extinction criterion (≥80% omitted trials). Communal nest animals required significantly fewer sessions to extinguish regardless of treatment. B. PAE did not significantly alter latency to respond during unrewarded extinction and Communal nest mice responded at significantly slower rates. C. There were no significant differences between PAE or SAC or Communal or Standard nest animals on the criterion session of extinction prior to reinstatement. Data are Means± SEM. *=main effect of housing p<.05.

When mice were tested for reinstatement via a 6 trial re-exposure to primary and secondary reinforcement, PAE mice differed significantly from SAC controls. During the first 6 primed trials of reinstatement, PAE and SAC mice responded to the stimuli at similar rates as controls regardless of housing condition (Figure 4A; main effect of housing: F1,30=0.29, p=0.59; main effect of treatment: F1,30<0.01 p=0.99; interaction: F1,30=1.45, p=0.24). In contrast, there was a significant interaction whereby PAE mice responded to the 54 unrewarded trials at a rate that was significantly higher than SAC control, but this effect was significantly ameliorated in CN housing (Figure 4B; main effect of housing: F1,30=1.8. p=0.19; main effect of treatment: F1,30=1.24 p=0.28; interaction: F1,30=8.68, p<0.01). The effects of PAE and CN were consistent for reinstatement uncorrected for last extinction session. Analysis of the latency to respond to stimuli during reinstatement found no significant differences between groups (Figure 4C; main effect of housing: F1,30=1.49, p=0.23; main effect of treatment: F1,30=0.06 p=0.82; interaction: F1,30=0.40, p=0.54). When tested for extinction retention following one week of no testing, a significant interaction was seen in which PAE SN mice still responded significantly more to unreinforced stimuli while this effect was decreased in PAE CN mice (Figure 4D; main effect of housing: F1,30=2.46, p=0.12; main effect of treatment: F1,30=0.87 p=0.36; interaction: F1,30=5.746, p=0.02).

Figure 4. PAE significantly increases unrewarded reinstatement responding and is ameliorated by communal nest rearing.

Figure 4.

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A. Treatment and rearing groups did not differ in responding on the first 6 rewarded trials. PAE standard nest mice showed significantly increased SN PAE mice did not respond at higher rates than the other groups during the 6 trial reinforced trials, but had significantly higher responding more during the 54 unrewarded trials that followed B. Standard housed PAE mice had a significantly larger reinstatement effect (reinstatement touches – touches on last extinction session) versus all other groups. Communal PAE did not differ from SAC control. C. No differences between rearing condition or treatment were seen for latency to touch on unrewarded trials. D. Standard rearing PAE mice had significantly higher responses to unrewarded trials even 1 week following reinstatement. Data are Means± SEM. *=main effect of treatment p<.05; *=interaction term p<.05.

Discussion

In the current study we found that PAE did not adversely affect acquisition or extinction of a simple instrumental response in a touch-screen paradigm, but led to significantly increased reinstatement of the extinguished response after a brief burst of reward. This effect persisted when reinstatement retention was tested one week later, with no further priming. When examining the effect of CN on PAE and SAC performance, we found that CN intervention led to significantly slower acquisition of the instrumental responding, although all mice attained acquisition criterion. CN also significantly accelerated extinction as measured by sessions to extinction criterion, regardless of exposure group. In addition to alteration in acquisition and extinction that were consistent across prenatal treatment, CN rescued the significant increase in reinstatement seen in PAE animals raised in standard conditions.

While it has been well-established that the moderate PAE model used here leads to deficits in both hippocampal function and memory (Brady et al., 2012) the effects on cortical and striatal learning, such as reinstatement and acquisition respectively, are not as well described. However, the lack of a PAE effect on acquisition seen in the current study is consistent with previous findings in other tasks. Specifically, we have shown that this PAE model does not alter the ability to acquire instrumental touch responding, or discrimination of pairwise visual stimuli, however reversal of the learned association was significantly impaired (Marquardt et al., 2014). Moderate PAE during the first and second trimester similarly impairs reversal, but did not alter acquisition of a spatial response in the Y maze (Allan et al., 2014). Results from studies in the rat utilizing similar exposures, have consistently found that PAE spares learning while significantly increasing perseveration during spatial reversals (Riley et al., 1979; Wainwright et al., 1990; Hamilton et al., 2014). Together with the current results, these findings suggest that moderate PAE during the first and second trimester does not lead to global deficits in learning, but rather more clinically relevant impairments when executive function is taxed.

While acquisition and extinction performance was not affected by PAE, a brief (6 trial) re-exposure to a food reward was sufficient to induce significantly higher reinstatement of responding in alcohol exposed animals versus controls. As previously discussed, reinstatement is a direct measure of control over reward seeking (Lederle et al., 2011), and the robust reinstatement over control levels suggests that PAE drives the recovery of previously extinguished learned responses after re-exposure to reward. Given the highly elevated rates of substance use and treatment rates in those with FASD, the fact that moderate PAE is sufficient to reinstate a previously extinguished response for up to a week, is a potential highly significant finding (Streissguth et al., 2004). While reinstatement of a learned response after extinction activates specific neural circuits depending on how it is elicited, dopaminergic signaling from the ventral tegmental area (VTA) to the prelimbic cortex (prL), nucleus accumbens core (NAcC), and ventral pallidum (VP) play a central role in this process (Kalivas and Volkow, 2005; Farrell et al., 2018). Additionally, the basolateral amygdala (BLA) is thought to coordinate inputs from cortical regions including the orbitofrontal area and its output to the NAc and PrL during reinstatement (See, 2005). While much is known regarding the effects of a range of PAE models on specific brain regions (Marquardt and Brigman, 2016), the effect of moderate developmental exposure on NAc, and PrL is still not fully understood. Studies utilizing PAE models in the rat have shown that moderate exposure is sufficient to significantly increase the immediate early gene (IEG) c-Fos immuno reactivity in multiple regions associated with reinstatement including the VTA, NAc, and PrL cortex (Fabio et al., 2015). In a rat PAE model analogous to the one used in the current study, NMDA receptor subunit levels within the PrL showed a moderate decrease while receptor levels were significantly elevated in the agranular insular cortex, suggesting long-lasting alterations in excitatory tone which may mediate learning and memory (Bird et al., 2015). BLA function and morphology is also altered by PAE, with moderate and high dose exposure in the rat significantly decreasing dopaminergic modulation of inhibitory GABAergic transmission leading to hyper-excitability (Zhou et al., 2010; Diaz et al., 2014) while more moderate doses increases BLA spine number (Cullen et al., 2013). While alterations in both cortical and amygdala activity after PAE suggest that the significantly increased reinstatement seen here are due to altered circuit function, future studies are needed to examine both indirect (IEG) and direct (in vivo recording) measures of PrL and BLA activity elicited by reinstatement.

Our most important finding was that a simple early life intervention, CN housing, was sufficient to reduce the significantly increased reinstatement effect in PAE animals. CN conditions have been shown to have numerous effects on the development and behavior of offspring, with the most well described being more complex social behaviors. CN mice have been shown to have higher levels of social investigation and social adaptation to exposure to novel conspecifics or novel home cage environments (Branchi et al., 2006a; Branchi et al., 2006b; D’Andrea et al., 2007; Branchi et al., 2013b). Being raised in a CN environment also alters anxiety-and depression- like behavior in later life. CN significantly reduces anxiety induced by social interactions, while increasing anxiety-like behaviors when faced with an anxiety provoking environment such as an elevated plus maze (Branchi and Alleva, 2006). Similarly, CN mice show significantly reduced depression-like symptoms, including anhedonia, after social defeat or isolation stress and alters immobility in the forced swim task (Palanza et al., 2001; Branchi et al., 2010; D’Andrea et al., 2010). Overall, behavioral studies suggest that increased maternal care and opportunity for pup-pup interaction in the CN leads to increased social resilience in adulthood. Behavioral alterations seen after CN are accompanied by persistent neurobiological alterations including increased levels of nerve growth factor NGF and brain-derived neurotrophic factor (BDNF) in the hippocampus and hypothalamus (Branchi et al., 2006a; Branchi et al., 2006b). Not surprisingly, CN has also been shown to increase hippocampal neurogenesis, both increasing the rate and survival of newborn neurons in the dentate gyrus (Branchi et al., 2006b).

We have previously shown that maternal care in early life reverses the effect of PAE on hippocampal formation-dependent learning and memory. Specifically, whereas SN PAE mice displayed a deficit in context discrimination on day 5 and 6 of testing, CN housing mitigated performance in PAE to control levels (Caldwell et al., 2015a). Importantly, the reversal of the PAE deficit was associated with a normalization of PAE-dependent alterations in nuclear glucocorticoid receptor (GR) levels and factors controlling glucocorticoid receptor signaling in the hippocampal formation to control levels. PAE also significantly increased corticotrophin releasing hormone (CRH), although this increase was not altered by housing condition. However, CN was sufficient to increase basal anticorticotropin-releasing hormone receptor type 1 levels in PAE animals back to control levels (Caldwell et al., 2015b).

Together with our current rescue effect in CN, this raises the intriguing possibility that dam-pup and pup-pup interactions that occur during CN may alter stress responsivity after PAE. The question of whether the SN commonly used in research is a “impoverished”, stressful environment, or if the increased interaction in CN is “enriched” is still open to debate (Branchi et al., 2011). While multi-dam nests are more common in the wild, encompassing as much as 80% of nests, single dam nests are also present, and nest type is highly dependent on population density (Manning et al., 1995). What is clear is that CN significantly increases both maternal and peer interactions that are critical for the structural and functional brain development (Wurbel, 2001). Together with previous findings that CN during early life may prepare offspring to adapt to more complex dynamic social and environmental situations in later life, our current findings further support CN as a simple, effective early life intervention to lessen the impact of developmental insults such as PAE.

While the motivation to examine a CN intervention was focused on reinstatement, we also report that CN housing significantly increased the number of sessions required to attain initial acquisition criterion, regardless of exposure condition. While all animals in both housing conditions were able to reach the acquisition criterion, these results suggest that CN may alter learning of the instrumental response, or alter motivation to perform for food reward. Interestingly, CN mice took significantly longer to respond to stimuli after initiation, and also took significantly longer to retrieve a delivered reward after a response versus SN mice, regardless of treatment. Alterations in performance in CN mice was not limited to acquisition. Regardless of treatment, CN mice more readily extinguish responding when reinforces were removed, and were slower to respond to stimuli when they did touch versus SN animals, regardless of PAE treatment. While it is possible that CN led to a general impairment in learning our previous work found no alteration in training rates by housing (Caldwell et al., 2015b). Further, all animals were trained to the same level of acquisition regardless of number of sessions, and CN improved extinction performance, a process that is not simply the unlearning of the previous association, but rather the formation of a new association (Bernal-Gamboa et al., 2017b; Bernal-Gamboa et al., 2017a), Taken together, slowed acquisition and accelerated extinction coupled with consistently slower reward retrieval rates suggests that differences in CN performance may not be driven by changes in learning rate, but rather an alteration in the motivation for reward. Moderate PAE has previously been shown to alter motivation, increasing responding for both ethanol and other non-ethanol (milk) rewards (March et al., 2009). While further studies are needed to examine how PAE and early life environment interact to alter reward sensitivity, the current results suggest that mice raised with increased maternal care may be less focused on appetitive food rewards than mice reared in a standard environment.

In conclusion, we found that moderate PAE did not alter acquisition or extinction of a simple instrumental response, but significantly increased reinstatement after a brief re-exposure to reward. This effect was sufficient to persist up to a week later. Importantly, a simple early life intervention, CN housing ameliorated aberrant reinstatement behavior seen after PAE, even 1 week post reinstatement. Additionally, CN mice were slower to acquire the initial response, but extinguished the response when reward was discontinued at faster rates than control. Taken together, this data demonstrates that PAE can have a long lasting effect on control over reward seeking in a simple paradigm. Further, increased maternal and conspecific interaction during the early post-natal period via CN is an effective and ethological relevant approach to ameliorate the effects of PAE on cortical function.

Acknowledgements

This work was supported by the NIAAA Intramural Research Program and grants 1R01AA025652-01, 1P50AA022534-01 and 5T32AA014127e13.

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