Neural Circuit Transitions Supporting Developmentally Specific Social Behavior.

Environmentally appropriate social behavior is critical for survival across the lifespan. To support this flexible behavior, the brain must rapidly perform numerous computations taking into account sensation, memory, motor-control, and many other systems. Further complicating this process, individuals must perform distinct social behaviors adapted to the unique demands of each developmental stage; indeed, the social behaviors of the newborn would not be appropriate in adulthood and vice versa. However, our understanding of the neural circuit transitions supporting these behavioral transitions has been limited. Recent advances in neural circuit dissection tools, as well as adaptation of these tools for use at early time points, has helped uncover several novel mechanisms supporting developmentally appropriate social behavior. This review, and associated Minisymposium, bring together social neuroscience research across numerous model organisms and ages. Together, this work highlights developmentally regulated neural mechanisms and functional transitions in the roles of the sensory cortex, prefrontal cortex, amygdala, habenula, and the thalamus to support social interaction from infancy to adulthood. These studies underscore the need for synthesis across varied model organisms and across ages to advance our understanding of flexible social behavior.

Maturation of a cortical-amygdala circuit limits sociability in male rats.

Prefrontal cortical maturation coincides with adolescent transitions in social engagement, suggesting that it influences social development. The anterior cingulate cortex (ACC) is important for social interaction, including ACC outputs to the basolateral amygdala (BLA). However, little is known about ACC-BLA sensitivity to the social environment and if this changes during maturation. Here, we used brief (2-hour) isolation to test the immediate impact of changing the social environment on the ACC-BLA circuit and subsequent shifts in social behavior of adolescent and adult rats. We found that optogenetic inhibition of the ACC during brief isolation reduced isolation-driven facilitation of social interaction across ages. Isolation increased activity of ACC-BLA neurons across ages, but altered the influence of ACC on BLA activity in an age-dependent manner. Isolation reduced the inhibitory impact of ACC stimulation on BLA neurons in a frequency-dependent manner in adults, but uniformly suppressed ACC-driven BLA activity in adolescents. This work identifies isolation-driven alterations in an ACC-BLA circuit, and the ACC itself as an essential region sensitive to social environment and regulates its impact on social behavior in both adults and adolescents.

Amygdala circuit transitions supporting developmentally-appropriate social behavior.

Social behaviors dynamically change throughout the lifespan alongside the maturation of neural circuits. The basolateral region of the amygdala (BLA), in particular, undergoes substantial maturational changes from birth throughout adolescence that are characterized by changes in excitation, inhibition, and dopaminergic modulation. In this review, we detail the trajectory through which BLA circuits mature and are influenced by dopaminergic systems to guide transitions in social behavior in infancy and adolescence using data from rodents. In early life, social behavior is oriented towards approaching the attachment figure, with minimal BLA involvement. Around weaning age, dopaminergic innervation of the BLA introduces avoidance of novel peers into rat pups' behavioral repertoire. In adolescence, social behavior transitions towards peer-peer interactions with a high incidence of social play-related behaviors. This transition coincides with an increasing role of the BLA in the regulation of social behavior. Adolescent BLA maturation can be characterized by an increasing integration and function of local inhibitory GABAergic circuits and their engagement by the medial prefrontal cortex (mPFC). Manipulation of these transitions using viral circuit dissection techniques and early adversity paradigms reveals the sensitivity of this system and its role in producing age-appropriate social behavior.

Developmental Shifts in Amygdala Activity during a High Social Drive State.

Amygdala abnormalities characterize several psychiatric disorders with prominent social deficits and often emerge during adolescence. The basolateral amygdala (BLA) bidirectionally modulates social behavior and has increased sensitivity during adolescence. We tested how an environmentally-driven social state is regulated by the BLA in adults and adolescent male rats. We found that a high social drive state caused by brief social isolation increases age-specific social behaviors and increased BLA neuronal activity. Chemogenetic inactivation of BLA decreased the effect of high social drive on social engagement. High social drive preferentially enhanced BLA activity during social engagement; however, the effect of social opportunity on BLA activity was greater during adolescence. While this identifies a substrate underlying age differences in social drive, we then determined that high social drive increased BLA NMDA GluN2B expression and sensitivity to antagonism increased with age. Further, the effect of a high social drive state on BLA activity during social engagement was diminished by GluN2B blockade in an age-dependent manner. These results demonstrate the necessity of the BLA for environmentally driven social behavior, its sensitivity to social opportunity, and uncover a maturing role for BLA and its GluN2B receptors in social engagement.SIGNIFICANCE STATEMENT Social engagement during adolescence is a key component of healthy development. Social drive provides the impetus for social engagement and abnormalities underlie social symptoms of depression and anxiety. While adolescence is characterized by transitions in social drive and social environment sensitivity, little is known about the neural basis for these changes. We found that amygdala activity is uniquely sensitive to social environment during adolescence compared with adulthood, and is required for expression of heightened social drive. In addition, the neural substrates shift toward NMDA dependence in adulthood. These results are the first to demonstrate a unique neural signature of higher social drive and begin to uncover the underlying factors that heighten social engagement during adolescence.

Isolation driven changes in Iba1-positive microglial morphology are associated with social recognition memory in adults and adolescents.

Microglia are critical for regulation of neuronal circuits that mature from adolescence to adulthood. The morphological complexity and process length of microglia can indicate different activation states. These states are sensitive to a variety of environmental and stress conditions. Microglia are sensitive to many factors that also regulate social behavior, and in turn, microglial manipulations can impact social function. Brief social isolation is one factor that can lead to robust social changes. Here, we explored the role of microglia in the effects of brief social isolation on social recognition memory. Using morphological measures of Iba1 to index microglial intensity, complexity, and process length, we identified different effects of brief isolation on microglial complexity in the basal region of the amygdala between adults and adolescents alongside overall increases in intensity of Iba1 in several cortical brain regions. Short-term social recognition memory is sensitive to the amount of social engagement, and provides an opportunity to test if social engagement produced by brief isolation enhances social learning in a manner that relies on microglia. We found that brief isolation facilitated social interaction across ages but had opposing effects on short-term social recognition. Isolation increased novel partner investigation in adolescents, which is consistent with better social recognition, but increased familiar partner investigation in adults. Depletion of microglia with PLX3397 prevented these effects of brief isolation in adolescents, and reduced them in adults. These results suggest that distinct changes in microglial function driven by the social environment may differentially contribute to subsequent social recognition memory during development.

Optogenetic inhibition of either the anterior or posterior retrosplenial cortex disrupts retrieval of a trace, but not delay, fear memory.

Previous work investigating the role of the retrosplenial cortex (RSC) in memory formation has demonstrated that its contributions are not uniform throughout the rostro-caudal axis. While the anterior region was necessary for encoding CS information in a trace conditioning procedure, the posterior retrosplenial cortex was needed to encode contextual information. Using the same behavioral procedure, we tested if there was a similar dissociation during memory retrieval. First, we found that memory retrieval following trace conditioning results in increased neural activity in both the anterior and posterior retrosplenial cortex, measured using the immediate early gene zif268. Similar increases were not found in either RSC subregion using a delay conditioning task. We then found that optogenetic inhibition of neural activity in either subregion impairs retrieval of a trace, but not delay, memory. Together these results add to a growing literature showing a role for the retrosplenial cortex in memory formation and retention. Further, they suggest that following formation, memory storage becomes distributed to a wider network than is needed for its initial consolidation.

Regulation of learned fear expression through the MgN-amygdala pathway.

Heightened fear responding is characteristic of fear- and anxiety-related disorders, including post-traumatic stress disorder. Neural plasticity in the amygdala is essential for both initial fear learning and fear expression, and strengthening of synaptic connections between the medial geniculate nucleus (MgN) and amygdala is critical for auditory fear learning. However, very little is known about what happens in the MgN-amygdala pathway during fear recall and extinction, in which conditional fear decreases with repeated presentations of the auditory stimulus alone. In the present study, we found that optogenetic inhibition of activity in the MgN-amygdala pathway during fear retrieval and extinction reduced expression of conditional fear. While this effect persisted for at least two weeks following pathway inhibition, it was specific to the context in which optogenetic inhibition occurred, linking MgN-BLA inhibition to facilitation of extinction-like processes. Reduced fear expression through inhibition of the MgN-amygdala pathway was further characterized by similar synaptic expression of GluA1 and GluA2 AMPA receptor subunits compared to what was seen in controls. Inhibition also decreased CREB phosphorylation in the amygdala, similar to what has been reported following auditory fear extinction. We then demonstrated that this effect was reduced by inhibition of GluN2B-containing NMDA receptors. These results demonstrate a new and important role for the MgN-amygdala pathway in extinction-like processes, and show that suppressing activity in this pathway results in a persistent decrease in fear behavior.

Decreased cued fear discrimination learning in female rats as a function of estrous phase.

Relative to males, female rats can show enhanced contextual fear generalization (demonstrating a fear response in a safe or neutral context) dependent on estrogen receptor activation. The current experiment aimed to extend this finding to cued fear conditioning. Females in low-estrogen phases of the estrous cycle showed good discrimination, similar to males, between a conditional stimulus that predicted shock (CS+) and an equally familiar one that did not (CS-), while females in the proestrus (high estrogen) phase demonstrated similar levels of fear between the CS+ and CS-. These results demonstrate that cued fear generalization is similarly influenced by endogenous estrogens.

Fear Learning Enhances Prefrontal Cortical Suppression of Auditory Thalamic Inputs to the Amygdala in Adults, but Not Adolescents.

Adolescence is characterized by increased susceptibility to the development of fear- and anxiety-related disorders. Adolescents also show elevated fear responding and aversive learning that is resistant to behavioral interventions, which may be related to alterations in the circuitry supporting fear learning. These features are linked to ongoing adolescent development of medial prefrontal cortical (PFC) inputs to the basolateral amygdala (BLA) that regulate neural activity and contribute to the refinement of fear responses. Here, we tested the hypothesis that the extent of PFC inhibition of the BLA following fear learning is greater in adults than in adolescents, using anesthetized in vivo recordings to measure local field potentials (LFPs) evoked by stimulation of PFC or auditory thalamic (MgN) inputs to BLA. We found that BLA LFPs evoked by stimulation of MgN inputs were enhanced in adults following fear conditioning. Fear conditioning also led to reduced summation of BLA LFPs evoked in response to PFC train stimulation, and increased the capacity of PFC inhibition of MgN inputs in adults. These data suggest that fear conditioning recruits additional inhibitory capacity by PFC inputs to BLA in adults, but that this capacity is weaker in adolescents. These results provide insight into how the development of PFC inputs may relate to age differences in memory retention and persistence following aversive learning.

The dorsal hippocampus mediates synaptic destabilization and memory lability in the amygdala in the absence of contextual novelty.

The recall of a previously formed fear memory triggers a process through which synapses in the amygdala become "destabilized". This labile state at retrieval may be critical for the plasticity required to modify, update, or disrupt long-term memories. One component of this process involves the rapid internalization of calcium impermeable AMPA receptors (CI-AMPAR). While some recent work has focused on the details of modifying amygdala synapses, much less is known about the environmental factors that control memory updating and the important circuit level processes. Synchrony between the hippocampus and amygdala increases during memory retrieval and stable memories can sometimes be made labile with hippocampal manipulations. Recent work shows that memory lability at retrieval is influenced by the novelty of the retrieval environment, and detection of this novelty likely relies on the dorsal hippocampus (DH). Our goal was to determine how local activity in the DH contributes to memory lability and synaptic destabilization in the amygdala during retrieval when contextual novelty is introduced. We found that contextual novelty during retrieval is necessary for alterations in amygdala activity and CI-AMPAR internalization. In the absence of novelty, suppression of local activity in the DH prior to learning allowed for retrieval-dependent CI-AMPAR internalization in the amygdala. We next tested whether the changes in AMPAR internalization were accompanied by differences in memory lability. We found that a memory was made labile when activity within the DH was disrupted in the absence of contextual novelty. These results suggest that the DH is important for encoding contextual information during learning that regulates retrieval-dependent memory modification in the amygdala.

Context memory formation requires activity-dependent protein degradation in the hippocampus.

Numerous studies have indicated that the consolidation of contextual fear memories supported by an aversive outcome like footshock requires de novo protein synthesis as well as protein degradation mediated by the ubiquitin-proteasome system (UPS). Context memory formed in the absence of an aversive stimulus by simple exposure to a novel environment requires de novo protein synthesis in both the dorsal (dHPC) and ventral (vHPC) hippocampus. However, the role of UPS-mediated protein degradation in the consolidation of context memory in the absence of a strong aversive stimulus has not been investigated. In the present study, we used the context preexposure facilitation effect (CPFE) procedure, which allows for the dissociation of context learning from context-shock learning, to investigate the role of activity-dependent protein degradation in the dHPC and vHPC during the formation of a context memory. We report that blocking protein degradation with the proteasome inhibitor clasto-lactacystin ß-lactone (ßLac) or blocking protein synthesis with anisomycin (ANI) immediately after context preexposure significantly impaired context memory formation. Additionally, we examined 20S proteasome activity at different time points following context exposure and saw that the activity of proteasomes in the dHPC increases immediately after stimulus exposure while the vHPC exhibits a biphasic pattern of proteolytic activity. Taken together, these data suggest that the requirement of increased proteolysis during memory consolidation is not driven by processes triggered by the strong aversive outcome (i.e., shock) normally used to support fear conditioning.

Input from the medial geniculate nucleus modulates amygdala encoding of fear memory discrimination.

Generalization of fear can involve abnormal responding to cues that signal safety and is common in people diagnosed with post-traumatic stress disorder. Differential auditory fear conditioning can be used as a tool to measure changes in fear discrimination and generalization. Most prior work in this area has focused on elevated amygdala activity as a critical component underlying generalization. The amygdala receives input from auditory cortex as well as the medial geniculate nucleus (MgN) of the thalamus, and these synapses undergo plastic changes in response to fear conditioning and are major contributors to the formation of memory related to both safe and threatening cues. The requirement for MgN protein synthesis during auditory discrimination and generalization, as well as the role of MgN plasticity in amygdala encoding of discrimination or generalization, have not been directly tested. GluR1 and GluR2 containing AMPA receptors are found at synapses throughout the amygdala and their expression is persistently up-regulated after learning. Some of these receptors are postsynaptic to terminals from MgN neurons. We found that protein synthesis-dependent plasticity in MgN is necessary for elevated freezing to both aversive and safe auditory cues, and that this is accompanied by changes in the expressions of AMPA receptor and synaptic scaffolding proteins (e.g., SHANK) at amygdala synapses. This work contributes to understanding the neural mechanisms underlying increased fear to safety signals after stress.

CaMKII regulates proteasome phosphorylation and activity and promotes memory destabilization following retrieval.

Numerous studies have suggested that memories "destabilize" and require de novo protein synthesis in order to reconsolidate following retrieval, but very little is known about how this destabilization process is regulated. Recently, ubiquitin-proteasome mediated protein degradation has been identified as a critical regulator of memory trace destabilization following retrieval, though the specific mechanisms controlling retrieval-induced changes in ubiquitin-proteasome activity remain equivocal. Here, we found that proteasome activity is increased in the amygdala in a CaMKII-dependent manner following the retrieval of a contextual fear memory. We show that in vitro inhibition of CaMKII reversed retrieval-induced increases in proteasome activity. Additionally, in vivo pharmacological blockade of CaMKII abolished increases in proteolytic activity and activity related regulatory phosphorylation in the amygdala following retrieval, suggesting that CaMKII was "upstream" of protein degradation during the memory reconsolidation process. Consistent with this, while inhibiting CaMKII in the amygdala did not impair memory following retrieval, it completely attenuated the memory impairments that resulted from post-retrieval protein synthesis blockade. Collectively, these results suggest that CaMKII controls the initiation of the memory reconsolidation process through regulation of the proteasome.

Enhancing fear extinction.

Gradually reducing a source of fear during extinction treatments may weaken negative memories in the long term.

Long-lasting effects of disturbing the circadian rhythm or sleep in adolescence.

Circadian rhythms are endogenous, near 24-hour rhythms that regulate a multitude of biological and behavioral processes across the diurnal cycle in most organisms. Over the lifespan, a bell curve pattern emerges in circadian phase preference (i.e. chronotype), with children and adults generally preferring to wake earlier and fall asleep earlier, and adolescents and young adults preferring to wake later and fall asleep later than their adult counterparts. This well-defined shift speaks to the variability of circadian rhythmicity over the lifespan and the changing needs and demands of the brain as an organism develops, particularly in the adolescent period. Indeed, adolescence is known to be a critical period of development during which dramatic neuroanatomical changes are occurring to allow for improved decision-making. Due to the large amount of re-structuring occurring in the adolescent brain, circadian disruptions during this period could have adverse consequences that persist across the lifespan. While the detrimental effects of circadian disruptions in adults have been characterized in depth, few studies have longitudinally assessed the potential long-term impacts of circadian disruptions during adolescence. Here, we will review the evidence that disruptions in circadian rhythmicity during adolescence have effects that persist into adulthood. As biological and social time often conflict in modern society, with school start times misaligned with adolescents' endogenous rhythms, it is critical to understand the long-term impacts of disrupted circadian rhythmicity in adolescence.

Elevated fear states facilitate ventral hippocampal engagement of basolateral amygdala neuronal activity.

Fear memory formation and retention rely on the activation of distributed neural circuits. The basolateral amygdala (BLA) and ventral hippocampus (VH) in particular are two regions that support contextual fear memory processes and share reciprocal connections. The VH → BLA pathway is critical for increases in fear after initial learning, in both fear renewal following extinction learning and during fear generalization. This raises the possibility that functional changes in VH projections to the BLA support increases in learned fear. In line with this, fear can also be increased with alterations to the original content of the memory via reconsolidation, as in fear elevation procedures. However, very little is known about the functional changes in the VH → BLA pathway supporting reconsolidation-related increases in fear. In this study, we used in vivo extracellular electrophysiology to examine the functional neuronal changes within the BLA and in the VH → BLA pathway as a result of fear elevation and standard fear retrieval procedures. Elevated fear expression was accompanied by higher BLA spontaneous firing compared to a standard fear retrieval condition. Across a range of stimulation frequencies, we also found that VH stimulation evoked higher BLA firing following fear elevation compared to standard retrieval. These results suggest that fear elevation is associated with an increased capacity of the VH to drive neuronal activity in the BLA, highlighting a potential circuit involved in strengthening existing fear memories.

The lifetime impact of stress on fear regulation and cortical function.

A variety of stressful experiences can influence the ability to form and subsequently inhibit fear memory. While nonsocial stress can impact fear learning and memory throughout the lifespan, psychosocial stressors that involve negative social experiences or changes to the social environment have a disproportionately high impact during adolescence. Here, we review converging lines of evidence that suggest that development of prefrontal cortical circuitry necessary for both social experiences and fear learning is altered by stress exposure in a way that impacts both social and fear behaviors throughout the lifespan. Further, we suggest that psychosocial stress, through its impact on the prefrontal cortex, may be especially detrimental during early developmental periods characterized by higher sociability. This article is part of the Special Issue on 'Fear, Anxiety and PTSD'.

Memory retrieval, reconsolidation, and extinction: Exploring the boundary conditions of post-conditioning cue exposure.

Following fear conditioning, behavior can be reduced by giving many CS-alone presentations in a process known as extinction or by presenting a few CS-alone presentations and interfering with subsequent memory reconsolidation. While the two share procedural similarities, both the behavioral outcomes and the neurobiological underpinnings are distinct. Here we review the neural and behavioral mechanisms that produce these separate behavioral reductions, as well as some factors that determine whether or not a retrieval-dependent reconsolidation process or an extinction process will be in effect.

The impact of social defeat on basomedial amygdala neuronal activity in adult male rats.

Social stressors negatively impact social function, and this is mediated by the amygdala across species. Social defeat stress is an ethologically relevant social stressor in adult male rats that increases social avoidance, anhedonia, and anxiety-like behaviors. While amygdala manipulations can mitigate the negative effects of social stressors, the impact of social defeat on the basomedial subregion of the amygdala is relatively unclear. Understanding the role of the basomedial amygdala may be especially important, as prior work has demonstrated that it drives physiological responses to stress, including heart-rate related responses to social novelty. In the present study, we quantified the impact of social defeat on social behavior and basomedial amygdala neuronal responses using anesthetized in vivo extracellular electrophysiology in adult male Sprague Dawley rats. Socially defeated rats displayed increased social avoidance behavior towards novel Sprague Dawley conspecifics and reduced time initiating social interactions relative to controls. This effect was most pronounced in rats that displayed defensive, boxing behavior during social defeat sessions. We next found that socially defeated rats showed lower overall basomedial amygdala firing and altered the distribution of neuronal responses relative to the control condition. We separated neurons into low and high Hz firing groups, and neuronal firing was reduced in both low and high Hz groups but in a slightly different manner. This work demonstrates that basomedial amygdala activity is sensitive to social stress, displaying a distinct pattern of social stress-driven activity relative to other amygdala subregions.

Fear Reduced Through Unconditional Stimulus Deflation Is Behaviorally Distinct From Extinction and Differentially Engages the Amygdala.

Background: Context fear memory can be reliably reduced by subsequent pairings of that context with a weaker shock. This procedure shares similarities with extinction learning: both involve extended time in the conditioning chamber following training and reduce context-elicited fear. Unlike extinction, this weak-shock exposure has been hypothesized to engage reconsolidation-like processes that weaken the original memory. Methods: We directly compared the weak-shock procedure with extinction using male and female Long Evans rats. Results: Both repeated weak-shock exposure and extinction resulted in decreased context freezing relative to animals that received context fear conditioning but no subsequent context exposure. Conditioning with the weak shock was not enough to form a persistent context-shock association on its own, suggesting that the weak-shock procedure does not create a new memory. Weak-shock exposure in a new context can still reduce freezing elicited by the training context, suggesting that it reduces responding through a different process than extinction, which does not transcend context. Finally, reduced fear behavior produced through both extinction and weak-shock exposure was mirrored by reduced zif268 expression in the basolateral amygdala. However, only the weak-shock procedure resulted in changes in lysine-48 polyubiquitin tagging in the synapse of the basolateral amygdala, suggesting that this procedure produced long-lasting changes in synaptic function within the basolateral amygdala. Conclusions: These results suggest that the weak-shock procedure does not rely on the creation of a new inhibitory memory, as in extinction, and instead may alter the original representation of the shock to reduce fear responding.