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.

Developmental differences in amygdala projection neuron activation associated with isolation-driven changes in social preference.

Adolescence is a developmental period characterized by brain maturation and changes in social engagement. Changes in the social environment influence social behaviors. Memories of social events, including remembering familiar individuals, require social engagement during encoding. Therefore, existing differences in adult and adolescent social repertoires and environmentally-driven changes in social behavior may impact novel partner preference, associated with social recognition. Several amygdala subregions are sensitive to the social environment and can influence social behavior, which is crucial for novelty preference. Amygdala neurons project to the septum and nucleus accumbens (NAc), which are linked to social engagement. Here, we investigated how the social environment impacts age-specific social behaviors during social encoding and its subsequent impact on partner preference. We then examined changes in amygdala-septal and -NAc circuits that accompany these changes. Brief isolation can drive social behavior in both adults and adolescents and was used to increase social engagement during encoding. We found that brief isolation facilitates social interaction in adolescents and adults, and analysis across time revealed that partner discrimination was intact in all groups, but there was a shift in preference within isolated and non-isolated groups. We found that this same isolation preferentially increases basal amygdala (BA) activity relative to other amygdala subregions in adults, but activity among amygdala subregions was similar in adolescents, even when considering conditions (no isolation, isolation). Further, we identify isolation-driven increases in BA-NAc and BA-septal circuits in both adults and adolescents. Together, these results provide evidence for changes in neuronal populations within amygdala subregions and their projections that are sensitive to the social environment that may influence the pattern of social interaction within briefly isolated groups during development.

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.

Sex differences in the Activity of Basolateral Amygdalar Neurons that Project to the Bed Nucleus of the Stria Terminalis and their Role in Anticipatory Anxiety.

Abnormal fear and anxiety can manifest as psychiatric disorders. The bed nucleus of the stria terminalis (BNST) is implicated in sustained responding to, or anticipation of, an aversive event which can be expressed as anticipatory anxiety. The basolateral amygdala (BLA) is also active during anticipatory anxiety and sends projections to the BNST. However, little is known about the role for BLA neurons that project to BNST (BLA-BNST) in anticipatory anxiety in rodents. To address this, we tested if chemogenetic inactivation of the BLA-BNST pathway attenuates sustained conditioned responses produced by anticipation of an aversive stimulus. For comparison, we also assessed BLA-BNST inactivation during social interaction, which is sensitive to unlearned anxiety. We found that BLA-BNST inactivation reduced conditioned sustained freezing and increased social behaviors, but surprisingly, only in males. To determine if sex differences in BLA-BNST neuronal activity contribute to the differences in behavior, we used in vivo and ex vivo electrophysiological approaches. In males, BLA-BNST projection neurons were more active and excitable which coincided with a smaller after-hyperpolarization current (I AHP) compared to other BLA neurons; whereas in females, BLA-BNST neurons were less excitable and had larger I AHP compared to other BLA neurons. These findings demonstrate that activity of BLA-BNST neurons mediates conditioned anticipatory anxiety-like behavior in males. The lack of a role of BLA-BNST in females in this behavior, possibly due to low excitability of these neurons, also highlights the need for caution when generalizing the role of specific neurocircuits in fear and anxiety. Significance Statement :Anxiety disorders disproportionately affect women. This hints towards sex differences within anxiety neurocircuitry, yet most of our understanding is derived from male rodents. Furthermore, debilitating anticipation of adverse events is among the most severe anxiety symptoms, but little is known about anticipatory anxiety neurocircuitry. Here we demonstrated that BLA-BNST activity is required for anticipatory anxiety to a prolonged aversive cue, but only in males. Moreover, BLA-BNST neurons are hypoactive and less excitable in females. These results uncover BLA-BNST as a key component of anticipatory anxiety circuitry, and cellular differences may explain the sex-dependent role of this circuit. Uncovering this disparity provides evidence that the assumed basic circuitry of an anxiety behavior might not readily transpose from males to females.

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.

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.

Repeated stress induces a pro-inflammatory state, increases amygdala neuronal and microglial activation, and causes anxiety in adult male rats.

A link exists between immune function and psychiatric conditions, particularly depressive and anxiety disorders. Psychological stress is a powerful trigger for these disorders and stress influences immune state. However, the nature of peripheral immune changes after stress conflicts across studies, perhaps due to the focus on few measures of pro-inflammatory or anti-inflammatory processes. The basolateral amygdala (BLA) is critical for emotion, and plays an important role in the effects of stress on anxiety. As such, it may be a primary central nervous system (CNS) mediator for the effects of peripheral immune changes on anxiety after stress. Therefore, this study aimed to delineate the influence of stress on peripheral pro-inflammatory and anti-inflammatory aspects, BLA immune activation, and its impact on BLA neuronal activity. To produce a more encompassing view of peripheral immune changes, this study used a less restrictive approach to categorize and group peripheral immune changes. We found that repeated social defeat stress in adult male Sprague-Dawley rats increased the frequencies of mature T-cells positive for intracellular type 2-like cytokine and serum pro-inflammatory cytokines. Principal component analysis and hierarchical clustering was used to guide grouping of T-cells and cytokines, producing unique profiles. Stress shifted the balance towards a specific set that included mostly type 2-like T-cells and pro-inflammatory cytokines. Within the CNS component, repeated stress caused an increase of activated microglia in the BLA, increased anxiety-like behaviors across several assays, and increased BLA neuronal firing in vivo that was prevented by blockade of microglia activation. Because repeated stress can trigger anxiety states by actions in the BLA, and altered immune function can trigger anxiety, these results suggest that repeated stress may trigger anxiety-like behaviors by inducing a pro-inflammatory state in the periphery and the BLA. These results begin to uncover how stress may recruit the immune system to alter the function of brain regions critical to emotion.

Disruptive effects of repeated stress on basolateral amygdala neurons and fear behavior across the estrous cycle in rats.

Stress is a precipitating factor in depression and anxiety disorders. Patients with these disorders often show amygdala abnormalities. The basolateral amygdala (BLA) is integral in mood and emotion, and is sensitive to stress. While much is known about effects of stress on BLA neuron activity and morphology in males, less is known in females. We tested whether repeated stress exerts distinct effects on BLA in vivo neuronal activity and morphology of Golgi-stained BLA neurons [lateral (LAT) and basal (BA) nuclei] in adult female rats. Repeated restraint stress increased BLA neuronal firing and caused hypertrophy of BLA neurons in males, while it decreased LAT and BA neuronal firing and caused hypotrophy of neurons in the LAT of females. BLA neuronal activity and function, such as fear conditioning, shifts across the estrous cycle. Repeated stress disrupted this pattern of BLA activity and fear expression over the estrous cycle. The disruptive effects of stress on the pattern of BLA function across estrous may produce behavior that is non-optimal for a specific phase of the estrous cycle. The contrasting effects of stress may contribute to sex differences in the effects of stress on mood and psychiatric disorders.

Limited prefrontal cortical regulation over the basolateral amygdala in adolescent rats.

Cognitive regulation of emotion develops from childhood into adulthood. This occurs in parallel with maturation of prefrontal cortical (PFC) regulation over the amygdala. The cellular substrates for this regulation may include PFC activation of inhibitory GABAergic elements in the amygdala. The purpose of this study was to determine whether PFC regulation over basolateral amygdala area (BLA) in vivo is immature in adolescence, and if this is due to immaturity of GABAergic elements or PFC excitatory inputs. Using in vivo extracellular electrophysiological recordings from anesthetized male rats we found that in vivo summation of PFC inputs to the BLA was less regulated by GABAergic inhibition in adolescents (postnatal day 39) than adults (postnatal day 72-75). In addition, stimulation of either prelimbic or infralimbic PFC evokes weaker inhibition over basal (BA) and lateral (LAT) nuclei of the BLA in adolescents. This was dictated by both weak recruitment of inhibition in LAT and weak excitatory effects of PFC in BA. The current results may contribute to differences in adolescent cognitive regulation of emotion. These findings identify specific elements that undergo adolescent maturation and may therefore be sensitive to environmental disruptions that increase risk for psychiatric disorders.

Sex- and Estrus-Dependent Differences in Rat Basolateral Amygdala.

Depression and anxiety are diagnosed almost twice as often in women, and the symptomology differs in men and women and is sensitive to sex hormones. The basolateral amygdala (BLA) contributes to emotion-related behaviors that differ between males and females and across the reproductive cycle. This hints at sex- or estrus-dependent features of BLA function, about which very little is known. The purpose of this study was to test whether there are sex differences or estrous cyclicity in rat BLA physiology and to determine their mechanistic correlates. We found substantial sex differences in the activity of neurons in lateral nuclei (LAT) and basal nuclei (BA) of the BLA that were associated with greater excitatory synaptic input in females. We also found strong differences in the activity of LAT and BA neurons across the estrous cycle. These differences were associated with a shift in the inhibition-excitation balance such that LAT had relatively greater inhibition during proestrus which paralleled more rapid cued fear extinction. In contrast, BA had relatively greater inhibition during diestrus that paralleled more rapid contextual fear extinction. These results are the first to demonstrate sex differences in BLA neuronal activity and the impact of estrous cyclicity on these measures. The shift between LAT and BA predominance across the estrous cycle provides a simple construct for understanding the effects of the estrous cycle on BLA-dependent behaviors. These results provide a novel framework to understand the cyclicity of emotional memory and highlight the importance of considering ovarian cycle when studying the BLA of females.SIGNIFICANCE STATEMENT There are differences in emotional responses and many psychiatric symptoms between males and females. This may point to sex differences in limbic brain regions. Here we demonstrate sex differences in neuronal activity in one key limbic region, the basolateral amygdala (BLA), whose activity fluctuates across the estrous cycle due to a shift in the balance of inhibition and excitation across two BLA regions, the lateral and basal nuclei. By uncovering this push-pull shift between lateral and basal nuclei, these results help to explain disparate findings about the effects of biological sex and estrous cyclicity on emotion and provide a framework for understanding fluctuations in emotional memory and psychiatric symptoms.

An intra-amygdala circuit specifically regulates social fear learning.

Adaptive social behavior requires transmission and reception of salient social information. Impairment of this reciprocity is a cardinal symptom of autism. The amygdala is a critical mediator of social behavior and is implicated in social symptoms of autism. Here we found that a specific amygdala circuit, from the lateral nucleus to the medial nucleus (LA-MeA), is required for using social cues to learn about environmental cues that signal imminent threats. Disruption of the LA-MeA circuit impaired valuation of these environmental cues and subsequent ability to use a cue to guide behavior. Rats with impaired social guidance of behavior due to knockout of Nrxn1, an analog of autism-associated gene NRXN, exhibited marked LA-MeA deficits. Chemogenetic activation of this circuit reversed these impaired social behaviors. These findings identify an amygdala circuit required to guide emotional responses to socially significant cues and identify an exploratory target for disorders associated with social impairments.

Greater physiological and behavioral effects of interrupted stress pattern compared to daily restraint stress in rats.

Repeated stress can trigger a range of psychiatric disorders, including anxiety. The propensity to develop abnormal behaviors after repeated stress is related to the severity, frequency and number of stressors. However, the pattern of stress exposure may contribute to the impact of stress. In addition, the anxiogenic nature of repeated stress exposure can be moderated by the degree of coping that occurs, and can be reflected in homotypic habituation to the repeated stress. However, expectations are not clear when a pattern of stress presentation is utilized that diminishes habituation. The purpose of these experiments is to test whether interrupted stress exposure decreases homotypic habituation and leads to greater effects on anxiety-like behavior in adult male rats. We found that repeated interrupted restraint stress resulted in less overall homotypic habituation compared to repeated daily restraint stress. This was demonstrated by greater production of fecal boli and greater corticosterone response to restraint. Furthermore, interrupted restraint stress resulted in a lower body weight and greater adrenal gland weight than daily restraint stress, and greater anxiety-like behavior in the elevated plus maze. Control experiments demonstrated that these effects of the interrupted pattern could not be explained by differences in the total number of stress exposures, differences in the total number of days that the stress periods encompased, nor could it be explained as a result of only the stress exposures after an interruption from stress. These experiments demonstrate that the pattern of stress exposure is a significant determinant of the effects of repeated stress, and that interrupted stress exposure that decreases habituation can have larger effects than a greater number of daily stress exposures. Differences in the pattern of stress exposure are therefore an important factor to consider when predicting the severity of the effects of repeated stress on psychiatric disorders.

Effects of repeated stress on excitatory drive of basal amygdala neurons in vivo.

Chronic stress leads to heightened affective behaviors, and can precipitate the emergence of depression and anxiety. These disorders are associated with increased amygdala activity. In animal models, chronic stress leads to increased amygdala-dependent behaviors, as well as hyperactivity of amygdala neurons. However, it is not known whether increased excitatory synaptic drive after chronic stress contributes to hyperactivity of basolateral amygdala (BLA; comprised of basal, lateral, and accessory basal nuclei) neurons. This study tested whether repeated stress causes an increase in excitatory drive of basal amygdala (BA) neurons in vivo, and whether this is correlated with an increase in the number of dendritic spines and a shift in dendritic distribution. Using in vivo intracellular recordings, this study found that repeated restraint stress caused an increase in the frequency of spontaneous excitatory synaptic events in vivo, which correlated with the number of dendritic spines in reconstructed neurons. Furthermore, parallel changes in the kinetics of the synaptic events and the distribution of spines indicated a more prominent functional contribution of synaptic inputs from across the dendritic tree. The shift in spine distribution across the dendritic tree was further confirmed with the examination of Golgi-stained tissue. This abnormal physiological drive of BA neurons after repeated stress may contribute to heightened affective responses after chronic stress. A reduction in the impact of excitatory drive in the BA may therefore be a potential treatment for the harmful effects of chronic stress in psychiatric disorders.

Chronic stress causes amygdala hyperexcitability in rodents.

BACKGROUND: Chronic stress is a major health concern, often leading to depression, anxiety, or when severe enough, posttraumatic stress disorder. While many studies demonstrate that the amygdala is hyperresponsive in patients with these disorders, the cellular neurophysiological effects of chronic stress on the systems that underlie psychiatric disorders, such as the amygdala, are relatively unknown. METHODS: In this study, we examined the effects of chronic stress on the activity and excitability of amygdala neurons in vivo in rats. We used in vivo intracellular recordings from single neurons of the lateral amygdala (LAT) to measure neuronal properties and determine the cellular mechanism for the effects of chronic stress on LAT neurons. RESULTS: We found a mechanism for the effects of chronic stress on amygdala activity, specifically that chronic stress increased excitability of LAT pyramidal neurons recorded in vivo. This hyperexcitability was caused by a reduction of a regulatory influence during action potential firing, facilitating LAT neuronal activity. The effects of stress on excitability were occluded by agents that block calcium-activated potassium channels and reversed by pharmacological enhancement of calcium-activated potassium channels. CONCLUSIONS: These data demonstrate a specific channelopathy that occurs in the amygdala after chronic stress. This enhanced excitability of amygdala neurons after chronic stress may explain the observed hyperresponsiveness of the amygdala in patients with posttraumatic stress disorder and may facilitate the emergence of depression or anxiety in other patients.

The Notch ligands Jagged2, Delta1, and Delta4 induce differentiation and expansion of functional human NK cells from CD34+ cord blood hematopoietic progenitor cells.

Notch receptor signaling is required for T cell development, but its role in natural killer (NK) cell development is poorly understood. We compared the ability of the 5 mammalian Notch ligands (Jagged1, Jagged2, Delta1, Delta3, or Delta4) to induce NK cell development from human hematopoietic progenitor cells (HPCs). CD34(+) HPCs were cultured with OP9 stromal cell lines transduced with 1 of the Notch ligands or with OP9 stromal cells alone, in the presence of IL-7, Flt3L, and IL-15. Differentiation and expansion of CD56(+)CD3(-) cells were greatly accelerated in the presence of Jagged2, Delta-1, or Delta-4, versus culture in the absence of ligand or in the presence of Jagged1 or Delta3. At 4 weeks, cultures containing Jagged2, Delta1, or Delta4 contained 80% to 90% NK cells, with the remaining cells being CD33(+) myelogenous cells. Notch-induced NK (N-NK) cells resembled CD56(bright) NK cells in that they were CD16(-), CD94(-), CD117(+), and killer immunoglobulin-like receptors (KIR(-)). They also expressed NKp30, NKp44, NKp46, 2B4, and DNAM-1, with partial expression of NKG2D. The N-NK cells displayed cytotoxic activity against the K562 and RPMI-8226 cell lines, at levels similar to activated peripheral blood (PB) NK cells, although killing of Daudi cells was not present. N-NK cells were also capable of interferon (IFN)-gamma secretion. Thus, Notch ligands have differential ability to induce and expand immature, but functional, NK cells from CD34(+) HPCs. The use of Notch ligands to generate functional NK cells in vitro may be significant for cellular therapy purposes.

Association of PSD-95 with ErbB4 facilitates neuregulin signaling in cerebellar granule neurons in culture.

The growth factor neuregulin 1 (NRG) selectively induces an increase in the gamma-aminobutyric acid (GABA)(A) receptor beta2 subunit protein in rat cerebellar granule neurons in culture. We previously demonstrated that NRG acts by triggering ErbB4 receptor phosphorylation and subsequent signaling through the mitogen-activated kinase (MAPK), phosphatidyl inositol-3 kinase (PI-3K) and cyclin-dependent kinase 5 (cdk5) pathways. In this report we show that the scaffolding protein, PSD-95, plays a key role in mediating the effects of NRG and that reducing its level attenuates the NRG-induced increase in beta2 subunit expression. PSD-95 appears to facilitate the effects of NRG through its association with ErbB4, an interaction that is augmented by NRG-activated cdk signaling. Inhibition of cdk activity with roscovitine attenuates the association of PSD-95 with ErbB4. The effects of cdk5 are not blocked by U0126, an inhibitor of MAPK signaling, indicating that cdk5 functions independently of cross-talk with this pathway. These findings raise the possibility that NRG-induced activation of cdk5 works in part by recruiting PSD-95, a protein involved in regulating synaptic plasticity, to associate with ErbB4. This interaction may be a positive feedback loop that augments NRG signaling and its downstream effects on GABA(A) receptor beta2 subunit expression.