Central Amygdala Research Articles

Traumatic Stress-Induced Increases in Anxiety-like Behavior and Alcohol Self-Administration Are Mediated by Central Amygdala CRF1 Neurons That Project to the Lateral Hypothalamus.

Avoidance stress coping, defined as persistent internal and/or external avoidance of stress-related stimuli, is a key feature of anxiety- and stress-related disorders, and contributes to increases in alcohol misuse after stress exposure. Previous work using a rat model of predator odor stress avoidance identified corticotropin-releasing factor (CRF) signaling via CRF Type 1 receptors (CRF1) in the CeA, as well as CeA projections to the lateral hypothalamus (LH) as key mediators of conditioned avoidance of stress-paired contexts and/or increased alcohol drinking after stress. Here, we report that CRF1-expressing CeA cells that project to the LH are preferentially activated in male and female rats that show persistent avoidance of predator odor stress-paired contexts (termed Avoider rats), and that chemogenetic inhibition of these cells rescues stress-induced increases in anxiety-like behavior and alcohol self-administration in male and female Avoider rats. Using slice electrophysiology, we found that prior predator odor stress exposure blunts inhibitory synaptic transmission and increases synaptic drive in CRF1 CeA-LH cells. In addition, we found that CRF bath application reduces synaptic drive in CRF1 CeA-LH cells in Non-Avoiders only. Collectively, these data show that CRF1 CeA-LH cells contribute to stress-induced increases in anxiety-like behavior and alcohol self-administration in male and female Avoider rats.SIGNIFICANCE STATEMENT Stress may lead to a variety of behavioral and physiological negative consequences, and better understanding of the neurobiological mechanisms that contribute to negative stress effects may lead to improved prevention and treatment strategies. This study, performed in laboratory rats, shows that animals that exhibit avoidance stress coping go on to develop heightened anxiety-like behavior and alcohol self-administration, and that these behaviors can be rescued by inhibiting the activity of a specific population of neurons in the central amygdala. This study also describes stress-induced physiological changes in these neurons that may contribute to their role in promoting increased anxiety and alcohol self-administration.

Voluntary alcohol intake alters the motivation to seek intravenous oxycodone and neuronal activation during the reinstatement of oxycodone and sucrose seeking

Opioid-alcohol polysubstance use is prevalent and worsens treatment outcomes. Here we assessed whether co-consumption of oxycodone and alcohol influence the intake of one another, demand for oxycodone, and the neurocircuitry underlying cue-primed reinstatement of oxycodone-seeking. Male and female rats underwent oxycodone intravenous self-administration (IVSA) with homecage access to alcohol (20% v/v) and/or water immediately after the IVSA session. Next, economic demand for intravenous oxycodone was assessed while access to alcohol and/or water continued. Control rats self-administered sucrose followed by access to alcohol and/or water. Rats underwent a cue-primed reinstatement test and brains were processed for c-fos mRNA expression. While both sexes decreased oxycodone intake if they had access to alcohol, and decreased alcohol intake if they had access to oxycodone, only female oxycodone + alcohol rats exhibited decreased demand elasticity and increased cue-primed reinstatement. Alcohol consumption increased the number of basolateral and central amygdala neurons activated during sucrose and oxycodone reinstatement and the number of ventral and dorsal striatum neurons engaged by sucrose reinstatement. Nucleus accumbens shell dopamine 1 receptor expressing neurons displayed activation patterns consistent with oxycodone reinstatement. Thus, alcohol alters the motivation to seek oxycodone in a sex-dependent manner and the neural circuitry engaged by cue-primed reinstatement of sucrose and oxycodone-seeking.

Social behavior and neuronal activation in adolescent female Fos-LacZ transgenic rats: Impact of acute ethanol challenge and baseline levels of social preference

In human adolescents, females often report drinking for coping reasons to avoid negative affective states. We have shown previously that adolescent female rats with elevated levels of anxiety-like behavior under social test circumstances, indexed via low social preference, are sensitive to anxiolytic effects of ethanol given intraperitoneally (i.p.) in a low-to-moderate dose range. This study was designed to test the hypothesis that patterns of neuronal activation across brain regions implicated in social activity and social preference (used as an index of low versus high anxiety-like social responding) would be affected by acute ethanol differently in adolescent females with high and low social preference, with initial levels of social preference also predicting ethanol-induced changes in social behavior. Adolescent female Fos-LacZ rats were given social interaction tests on postnatal day (P)33 for determination of baseline levels of responding to an unfamiliar social partner and on P35 following administration of 0 or 0.75 g/kg ethanol. Brain tissue was collected, and expression of β-galactoside (β-gal) was used as an index of neuronal activation. Baseline levels of social preference did not predict social responsiveness to an acute ethanol challenge, whereas significant decreases in this social measure that reflects anxiety-like behavioral alterations were evident in adolescent females challenged with ethanol relative to saline-injected controls, suggesting high sensitivity to the anxiogenic effects of ethanol. Ethanol precipitated negative relationships between social preference and prefrontal cortical activation, decreased neuronal activation of the anterior cingulate cortex, but substantially increased β-gal expression in the central amygdala. These results suggest high sensitivity of the prefrontal cortical regions and central amygdala to ethanol-induced alterations in adolescent Fos-LacZ females and provide a background for further phenotyping of neurons activated by ethanol under social test circumstances.

Sex differences in androgen receptor, estrogen receptor alpha, and c-Fos co-expression with corticotropin releasing factor expressing neurons in restrained adult mice

Gonadal hormone actions through androgen receptor (AR) and estrogen receptor alpha (ERα) regulate sex differences in hypothalamic-pituitary-adrenal (HPA) axis responsivity and stress-related behaviors. Here we tested whether corticotropin releasing factor (CRF) expressing neurons, which are widely known to regulate neuroendocrine and behavioral stress responses, co-express AR and ERα as a potential mechanism for gonadal hormone regulation of these responses. Using Crh-IRES-Cre::Ai9 reporter mice we report high co-localization of AR in CRF neurons within the medial preoptic area (MPOA), bed nucleus of the stria terminalis (BST), medial amygdala (MeA), and ventromedial hypothalamus (VMH), moderate levels within the central amygdala (CeA) and low levels in the paraventricular hypothalamus (PVN). Sex differences in CRF/AR co-expression were found in the principal nucleus of the BST (BSTmpl), CeA, MeA, and VMH (males>females). CRF co-localization with ERα was generally lower relative to AR co-localization. However, high co-expression was found within the MPOA, AVPV, and VMH, with moderate co-expression in the arcuate nucleus (ARC), BST, and MeA and low levels in the PVN and CeA. Sex differences in CRF/ERα co-localization were found in the BSTmpl and PVN (males>females). Finally, we assessed neural activation of CRF neurons in restraint-stressed mice and found greater CRF/c-Fos co-expression in females in the BSTmpl and periaqueductal gray, while co-expression was higher in males within the ARC and dorsal CA1. Given the known role of CRF in regulating behavioral stress responses and the HPA axis, AR/ERα co-expression and sex-specific activation of CRF cell groups indicate potential mechanisms for modulating sex differences in these functions.

Disrupting astrocyte-neuron lactate transport prevents cocaine seeking after prolonged withdrawal.

Energy supply, especially the transfer of lactate from astrocytes to neurons, is critical for neuronal plasticity. However, its role in the incubation of cocaine craving remains largely unknown. Using an extended-access self-administration model and in vivo 1H-magnetic resonance spectroscopy, we found that lactate synthesis in the central amygdala (CeA) is required for the intensified cocaine craving after prolonged withdrawal. Furthermore, incubated cocaine seeking was associated with a selective increase in monocarboxylate transporter 2 (MCT2) and MCT4 expression levels. Down-regulation of astrocytic MCT4 or neuronal MCT2 using targeted antisense oligonucleotides or cell type-specific shRNA attenuated cocaine craving and reduced the expression of plasticity-related proteins and excitatory synaptic transmission. Meanwhile, lactate administration rescued MCT4 but not MCT2 disruption-induced behavioral changes due to the inability of lactate to be transported into neurons. Together, our study highlights the critical role of astrocyte-neuron lactate transport in the CeA in the incubation of cocaine craving and suggests a potential therapeutic target for drug addiction.

Dopamine D2 receptors in WFS1-neurons regulate food-seeking and avoidance behaviors

The selection and optimization of appropriate adaptive responses depends on interoceptive and exteroceptive stimuli as well as on the animal's ability to switch from one behavioral strategy to another. Although growing evidence indicate that dopamine D2R-mediated signaling events ensure the selection of the appropriate strategy for each specific situation, the underlying neural circuits through which they mediate these effects are poorly characterized. Here, we investigated the role of D2R signaling in a mesolimbic neuronal subpopulation expressing the Wolfram syndrome 1 (Wfs1) gene. This subpopulation is located within the nucleus accumbens, the central amygdala, the bed nucleus of the stria terminalis, and the tail of the striatum, all brain regions critical for the regulation of emotions and motivated behaviors. Using a mouse model carrying a temporally controlled deletion of D2R in WFS1-neurons, we demonstrate that intact D2R signaling in this neuronal population is necessary to regulate homeostasis-dependent food-seeking behaviors in both male and female mice. In addition, we found that reduced D2R signaling in WFS1-neurons impaired active avoidance learning and innate escape responses. Collectively, these findings identify a yet undocumented role for D2R signaling in WFS1-neurons as a novel effector through which dopamine optimizes appetitive behaviors and regulates defensive behaviors.

Evidence for the existence of facilitatory interactions between the dopamine D2 receptor and the oxytocin receptor in the amygdala of the rat. Relevance for anxiolytic actions.

Introduction: The amygdala is a limbic region of high value for understanding anxiety and its treatment. Dopamine D2 receptors (D2Rs) and oxytocin receptors (OXTRs) have both been shown to participate in modulating anxiety involving effects in the amygdala. The goal is to understand if D2R-OXTR heterocomplexes exist in the central amygdala and if, through enhancing allosteric receptor-receptor interactions, may enhance anxiolytic actions. Methods: The methods used involve the shock-probe burying test, the in situ proximity ligation assay (PLA), image acquisition and analysis, and the BRET2 assay. Bilateral cannulas were introduced into the amygdala, and the effects of the coadministration of oxytocin and the D2R-like agonist quinpirole into the amygdala were studied. Results: The combination treatment enhanced the anxiolytic effects compared to the single treatment. The D2R/D3R antagonist raclopride blocked the effects of the combination treatment of oxytocin and the D2R agonist, although oxytocin is regarded as a distinct modulator of fear-mediating anxiolytic effects. In situ PLA results indicate the existence of D2R-OXTR heteroreceptor complexes and/or the co-location of OXTR and D2R within the same cell membrane nanodomains in the central amygdala. With BRET2, evidence is given for the existence of D2R-OXTR heteromers in HEK293 cells upon co-transfection. Discussion: The enhanced behavioral effects observed upon co-treatment with OXTR and D2R agonists may reflect the existence of improved positive receptor-receptor interactions in the putative D2R-OXTR heterocomplexes in certain neuronal populations of the basolateral and central amygdala. The D2R-OXTR heterocomplex, especially upon agonist co-activation in the central amygdala, may open a new pharmacological venue for the treatment of anxiety.

Possible involvement of GABAergic system on central amygdala Mediated anxiolytic effect of agmatine in rats

Objectives To study the pharmacological interactions between agmatine and gamma aminobutyric acid (GABA) modulatory agents in the regulation of anxiety-like behavior in rats. Materials and methods Male Wistar rats were treated drugs per se or in combination and 15 min after last injection were subjected to elevated plus-maze (EPM) test. Anxiety-like behavior was evaluated by measuring behavioral conventional readout, open arm activity (duration and/or entries) for 5-minute duration. Results Acute intra-central amygdala (CeA) injection of agmatine (0.1-0.6 μmol/site/rat), muscimol (0.25-1 nmol/site/rat), diazepam (5-20 μg/site/rat) and allopregnanolone (2-8 μg/site/rat) increased open arm entries of the rats in EPM suggesting anxiolytic effect in dose dependent manner. Moreover, the anxiolytic effect at subeffective dose of agmatine (0.1 μmol/site/rat) was potentiated by subeffective dose of muscimol (0.25 nmol/site/rat), diazepam (5 μg/site/rat) and allopregnanolone (4 μg/site/rat). Whereas, pretreatment with GABAA receptor antagonist, bicuculline (10 ng/site/rat) blocked the anxiolytic effect of agmatine and its synergistic effect of agmatine plus muscimol. Similarly, benzodiazepine (BZD) receptor antagonist, flumazenil (15 μg/site/rat) and GABA allosteric modulator antagonist, RO 15-45 13 (10 μg/site/rat) reduced the anxiolytic effect of agmatine, given alone and with diazepam and allopregnanolone, respectively. Conclusion These results indicated that anxiolytic effect of agmatine is medicated via GABAergic mechanisms, probably conciliated by the GABAA receptor subtypes. Modulation of interplay between agmatine and GABAA receptor activity might be a pertinent solution for the regulation of anxiety.

Presence of a remote fear memory engram in the central amygdala.

Fear memory formation and recall are highly regulated processes, with the central amygdala (CeA) contributing to fear memory-related behaviors. We recently reported that a remote fear memory engram is resident in the anterior basolateral amygdala (aBLA). However, the extent to which downstream neurons in the CeA participate in this engram is unknown. We tested the hypothesis that CeA neurons activated during fear memory formation are reactivated during remote memory retrieval such that a CeA engram participates in remote fear memory recall and its associated behavior. Using contextual fear conditioning in TRAP2;Ai14 mice, we identified, by persistent Cre-dependent tdTomato expression (i.e., "TRAPing"), CeA neurons that were c-fos-activated during memory formation. Twenty-one days later, we quantified neurons activated during remote memory recall using Fos immunohistochemistry. Dual labeling was used to identify the subpopulation of CeA neurons that was both activated during memory formation and reactivated during recall. Compared with their context-conditioned (no shock) controls, fear-conditioned (electric shock) mice (n = 5/group) exhibited more robust fear memory-related behavior (freezing) as well as larger populations of activated (tdTomato+) and reactivated (dual-labeled) CeA neurons. Most neurons in both groups were mainly located in the capsular CeA subdivision (CeAC). Notably, however, only the size of the TRAPed population distributed throughout the CeA was significantly correlated with time spent freezing during remote fear memory recall. Our findings indicate that fear memory formation robustly activates CeA neurons and that a subset located mainly in the CeAC may contribute to both remote fear memory storage/retrieval and the resulting fear-like behavior.

Memory impairment was ameliorated by corticolimbic microinjections of arachidonylcyclopropylamide (ACPA) and miRNA-regulated lentiviral particles in a streptozotocin-induced Alzheimer's rat model

The present study aimed to investigate the effect of corticolimbic cannabinoid CB1 receptors activity on memory impairment in the intracerebroventricular (ICV)-streptozotocin (STZ) animal model of Alzheimer's like-disease. This study also assessed whether the corticolimbic overexpression of miRNA-137 or -let-7a could increase the endocannabinoids by inhibiting the monoglyceride lipase (MAGL) to ameliorate STZ response. The results showed that ICV microinjection of STZ (3 mg/kg/10 μl) impaired passive avoidance memory retrieval. The chronic microinjection of arachidonylcyclopropylamide (ACPA; 10 ng/0.5 μl), a selective cannabinoid CB1 receptor agonist, into the hippocampal CA1 region, the central amygdala (CeA) or the medial prefrontal cortex (mPFC) ameliorated the amnesic effect of ICV-STZ. Intra-CA1 or -CeA microinjection of ACPA alone did not affect memory retrieval, while its microinjection into the mPFC impaired memory formation. Based on bioinformatics analysis and verification of the MAGL gene, miRNA-137 and -let-7a were chosen to target the expression levels of MAGL in the corticolimbic regions. The chronic corticolimbic microinjection of lentiviral particles containing miRNA-137 or -let-7a ameliorated ICV-STZ-induced memory impairment. The high transfection efficiency was determined for each virus using comparing fluorescent and conventional vision. Corticolimbic overexpression of miRNA-137 or -let-7a decreased the MAGL gene expression that encodes the MAGL enzyme to increase the endocannabinoids. Thus, among the molecular mechanisms and signaling pathways involved in the pathophysiology of Alzheimer's disease (AD), it is worth mentioning the role of endocannabinoids in the corticolimbic regions. CB1 receptor agonists, miRNA-137 or -let-7a, may be potential therapeutic targets against cognitive decline in AD.

Acute Ethanol Modulates Synaptic Inhibition in the Basolateral Amygdala via Rapid NLRP3 Inflammasome Activation and Regulates Anxiety-Like Behavior in Rats.

Chronic alcohol exposure leads to a neuroinflammatory response involving activation of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome and proinflammatory cytokine production. Acute ethanol (EtOH) exposure activates GABAergic synapses in the central and basolateral amygdala (BLA) ex vivo, but whether this rapid modulation of synaptic inhibition is because of an acute inflammatory response and alters anxiety-like behavior in male and female animals is not known. Here, we tested the hypotheses that acute EtOH facilitates inhibitory synaptic transmission in the BLA by activating the NLRP3 inflammasome-dependent acute inflammatory response, that the alcohol-induced increase in inhibition is cell type and sex dependent, and that acute EtOH in the BLA reduces anxiety-like behavior. Acute EtOH application at a binge-like concentration (22-44 mm) stimulated synaptic GABA release from putative parvalbumin (PV) interneurons onto BLA principal neurons in ex vivo brain slices from male, but not female, rats. The EtOH facilitation of synaptic inhibition was blocked by antagonists of the Toll-like receptor 4 (TLR4), the NLRP3 inflammasome, and interleukin-1 receptors, suggesting it was mediated by a rapid local neuroinflammatory response in the BLA. In vivo, bilateral injection of EtOH directly into the BLA produced an acute concentration-dependent reduction in anxiety-like behavior in male but not female rats. These findings demonstrate that acute EtOH in the BLA regulates anxiety-like behavior in a sex-dependent manner and suggest that this effect is associated with presynaptic facilitation of parvalbumin-expressing interneuron inputs to BLA principal neurons via a local NLRP3 inflammasome-dependent neuroimmune response.SIGNIFICANCE STATEMENT Chronic alcohol exposure produces a neuroinflammatory response, which contributes to alcohol-associated pathologies. Acute alcohol administration increases inhibitory synaptic signaling in the brain, but the mechanism for the rapid alcohol facilitation of inhibitory circuits is unknown. We found that acute ethanol at binge-like concentrations in the basolateral amygdala (BLA) facilitates GABA release from parvalbumin-expressing (PV) interneuron synapses onto principal neurons in ex vivo brain slices from male rats and that intra-BLA ethanol reduces anxiety-like behavior in vivo in male rats, but not female rats. The ethanol (EtOH) facilitation of inhibition in the BLA is mediated by Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation and proinflammatory IL-1β signaling, which suggests a rapid NLRP3 inflammasome-dependent neuroimmune cascade that plays a critical role in acute alcohol intoxication.

Activation of the basolateral or the central amygdala dampened the incentive motivation for food reward on high fixed-ratio schedules

The amygdala plays crucial roles in emotional processing, motivated behaviors, and stress responses. It receives sensory information and modulates fear- and anxiety-related behaviors. Neuronal activations are induced in the basolateral complex of the amygdala (BLA) and the central nucleus of the amygdala (CeA) when exposing to acute stress, leading to increased alertness and proper behavioral adaptation. Previous studies have shown that animals displayed a decrease in appetitive motivated behaviors under stress conditions. However, whether the hyperactive amygdala is responsible for the decrease in appetitive motivated behaviors remains unknown. In this study, we aimed to examine the role of BLA or CeA activation in effort-based motivated behavior. We pharmacologically activated the BLA or the CeA with N-methyl-D-aspartate (NMDA) before the lever-pressing for food reward test on different fixed-ratio (FR) schedules (FR1, FR16, or FR32) in male Long-Evans rats. Our data showed that activation of either the BLA or the CeA with NMDA (0.05 μg in 0.5 μl per site) decreased the lever-pressing behavior on higher FR schedules of FR16 and FR32, but not on the FR1 test. Importantly, locomotor activity and free-feeding food intake were intact under amygdala activation, suggesting that the decrease in lever-pressing behavior was not due to motor disablement or decreased appetite. These results suggested that activation of the BLA or the CeA negatively impaired the effort-based motivated behavior that the animals were less willing to work for food reward.

Adolescent Alcohol Exposure Produces Sex-Specific Long-term Hyperalgesia via Changes in Central Amygdala Circuit Function

BackgroundExposure to alcohol during adolescence produces many effects that last well into adulthood. Acute alcohol use is analgesic, and people living with pain report drinking alcohol to reduce pain, but chronic alcohol use produces increases in pain sensitivity. MethodsWe tested the acute and lasting effects of chronic adolescent intermittent ethanol (AIE) exposure on pain-related behavioral and brain changes in male and female rats. We also tested the long-term effects of AIE on synaptic transmission in midbrain (ventrolateral periaqueductal gray [vlPAG])-projecting central amygdala (CeA) neurons using whole-cell electrophysiology. Finally, we used circuit-based approaches (DREADDs [designer receptors exclusively activated by designer drugs]) to test the role of vlPAG-projecting CeA neurons in mediating AIE effects on pain-related outcomes. ResultsAIE produced long-lasting hyperalgesia in male, but not female, rats. Similarly, AIE led to a reduction in synaptic strength of medial CeA cells that project to the vlPAG in male, but not female, rats. Challenge with an acute painful stimulus (i.e., formalin) in adulthood produced expected increases in pain reactivity, and this effect was exaggerated in male rats with a history of AIE. Finally, CeA-vlPAG circuit activation rescued AIE-induced hypersensitivity in male rats. ConclusionsOur findings are the first, to our knowledge, to show long-lasting sex-dependent effects of adolescent alcohol exposure on pain-related behaviors and brain circuits in adult animals. This work has implications for understanding the long-term effects of underage alcohol drinking on pain-related behaviors in humans.

Distinct Circuits From the Central Lateral Amygdala to the Ventral Part of the Bed Nucleus of Stria Terminalis Regulate Different Fear Memory

BackgroundThe ability to differentiate stimuli that predict fear is critical for survival; however, the underlying molecular and circuit mechanisms remain poorly understood. MethodsWe combined transgenic mice, in vivo transsynaptic circuit-dissecting anatomical approaches, optogenetics, pharmacological methods, and electrophysiological recording to investigate the involvement of specific extended amygdala circuits in different fear memory. ResultsWe identified the projections from central lateral amygdala (CeL) protein kinase C δ (PKCδ)–positive neurons and somatostatin (SST)-positive neurons to GABAergic (gamma-aminobutyric acidergic) and glutamatergic neurons in the ventral part of the bed nucleus of stria terminalis (vBNST). Prolonged optogenetic activation or inhibition of the PKCδCeL-vBNST pathway specifically reduced context fear memory, whereas the SSTCeL-vBNST pathway mainly reduced tone fear memory. Intriguingly, optogenetic manipulation of vBNST neurons that received the projection from PKCδCeL neurons exerted bidirectional regulation of context fear, whereas manipulation of vBNST neurons that received the projection from SSTCeL neurons could bidirectionally regulate both context and tone fear memory. We subsequently demonstrated the presence of δ and κ opioid receptor protein expression within the CeL-vBNST circuits, potentially accounting for the discrepancy between prolonged activation of GABAergic circuits and inhibition of downstream vBNST neurons. Finally, administration of an opioid receptor antagonist cocktail on the PKCδCeL-vBNST or SSTCeL-vBNST pathway successfully restored context or tone fear memory reduction induced by prolonged activation of the circuits. ConclusionsTogether, these findings establish a functional role for distinct CeL-vBNST circuits in the differential regulation and appropriate maintenance of fear.

Divergent Population-Specific Effects of Chronic Ethanol Exposure on Excitability and Inhibitory Transmission in Male and Female Rat Central Amygdala.

The central nucleus of the amygdala (CeA) is implicated in alcohol use disorder (AUD) and AUD-associated plasticity. The CeA is a primarily GABAergic nucleus that is subdivided into lateral and medial compartments with genetically diverse subpopulations. GABAA receptors are heteromeric pentamers with subunits conferring distinct physiological characteristics. GABAA receptor signaling in the CeA has been implicated in ethanol-associated plasticity; however, population-specific changes in inhibitory signaling and subunit expression remain unclear. Here, we combined electrophysiology with single-cell gene expression analysis of population markers and GABAA receptor subunits to examine population-specific changes in inhibitory control in male and female rats following chronic ethanol exposure. We found that chronic ethanol exposure and withdrawal produced global changes in GABAA receptor subunit expression at the transcript and protein levels, increased excitability in female CeA neurons, and increased inhibitory synaptic transmission in male CeA neurons. When we examined CeA neurons at the single-cell level we found heterogenous populations, as previously reported. We observed ethanol-induced increases in excitability only in somatostatin neurons in the CeA of females, decreases in excitability only in the protein kinase C delta (PKCd) population in males, and ethanol-induced increases in inhibitory transmission in male PKCd and calbindin 2-expressing CeA neurons. There were no population-specific differences in GABAA receptor (Gabr) subunits in males but reduced GabrA5 expression in female somatostatin neurons. Collectively, these findings suggest that defined CeA populations display differential ethanol sensitivity in males and females, which may play a role in sex differences in vulnerability to AUD or expression of AUD pathology.SIGNIFICANCE STATEMENT The CeA is involved in the effects of ethanol in the brain; however, the population-specific changes in CeA activity remain unclear. We used recordings of CeA neuronal activity and single-cell gene expression to examine population-specific changes in inhibitory control in male and female rats following chronic ethanol exposure and found sex- and population-specific effects of chronic ethanol exposure and withdrawal. Specifically, female CeA neurons displayed increased excitability in the somatostatin CeA population, whereas male CeA neurons displayed increased inhibitory control in both PKCd and calbindin populations and decreased excitability in the PKCd population. These findings identify CeA populations that display differential sensitivity to ethanol exposure, which may contribute to sex differences in vulnerability to alcohol use disorder.

5-HT1A receptor in the central amygdala and 5-HT2A receptor in the basolateral amygdala are involved in social hierarchy in male mice

Most social animals self-organize into dominance hierarchies that strongly influence their behavior and health. The serotonin (5-HT) system is believed to play an important role in the formation of social hierarchy. 5-HT receptors are abundantly expressed in the amygdala, which is considered as the central node for the perception and learning of social hierarchy. In this study, we assessed the functions of various 5-HT receptor subtypes related to social rank determination in different subregions of the amygdala using the confrontation tube test in mice. We revealed that most adult C57BL/6 J male mice exhibited a linear social rank after a few days of cohousing. The tube test ranks were slightly related to anxiety-like behavioral performance. After the tube test, the amygdala and 5-HT neurons in the dorsal raphe nucleus were activated in lower-rank individuals. Quantitative real-time polymerase chain reaction analysis revealed that despite the high expression of 5-HT1A receptor mRNA in the central amygdala (CeA), 5-HT2A receptor mRNA expression was downregulated in the basolateral amygdala (BLA) in higher-rank individuals. The dominant–subordinate relationship between mouse pairs could be switched via pharmacological modulation of these receptors in CeA and BLA, suggesting that these expression changes are essential for establishing social ranks. Our findings provide novel insights into the divergent functions of 5-HT receptors in the amygdala related to social hierarchy, which is closely related to our health and welfare.

The Role of Genetically Distinct Central Amygdala Neurons in Appetitive and Aversive Responding Assayed with a Novel Dual Valence Operant Conditioning Paradigm.

To survive, animals must meet their biological needs while simultaneously avoiding danger. However, the neurobiological basis of appetitive and aversive survival behaviors has historically been studied using separate behavioral tasks. While recent studies in mice have quantified appetitive and aversive conditioned responses simultaneously (Jikomes et al., 2016; Heinz et al., 2017), these tasks required different behavioral responses to each stimulus. As many brain regions involved in survival behavior process stimuli of opposite valence, we developed a paradigm in which mice perform the same response (nose poke) to distinct auditory cues to obtain a rewarding outcome (palatable food) or avoid an aversive outcome (mild footshoock). This design allows for both within-subject and between-subject comparisons as animals respond to appetitive and aversive cues. The central nucleus of the amygdala (CeA) is implicated in the regulation of responses to stimuli of either valence. Considering its role in threat processing (Wilensky et al., 2006; Haubensak et al., 2010) and regulation of incentive salience (Warlow and Berridge, 2021), it is important to examine the contribution of the CeA to mechanisms potentially underlying comorbid dysregulation of avoidance and reward (Sinha, 2008; Bolton et al., 2009). Using this paradigm, we tested the role of two molecularly defined CeA subtypes previously linked to consummatory and defensive behaviors. Significant strain differences in the acquisition and performance of the task were observed. Bidirectional chemogenetic manipulation of CeA somatostatin (SOM) neurons altered motivation for reward and perseveration of reward-seeking responses on avoidance trials. Manipulation of corticotropin-releasing factor neurons (CRF) had no significant effect on food reward consumption, motivation, or task performance. This paradigm will facilitate investigations into the neuronal mechanisms controlling motivated behavior across valences.

Diverse processing of pharmacological and natural rewards by the central amygdala

The central amygdala (CeA) with its medial (CeM) and lateral (CeL) nuclei is the brain hub for processing stimuli with emotional context. CeL nucleus gives a strong inhibitory input to the CeM, and this local circuitry assigns values (positive or negative) to incoming stimuli, guiding appropriate behavior (approach or avoid). However, the particular involvement of CeA in processing such emotionally relevant information and adaptations of the CeA circuitry are not yet well understood. In this study, we examined synaptic plasticity in the CeA after exposure to two types of rewards, pharmacological (cocaine) and natural (sugar). We found that both rewards engage CeM, where they generate silent synapses resulting in the strengthening of the network. However, only cocaine triggers plasticity in the CeL, which leads to the weakening of its excitatory inputs. Finally, chemogenetic inhibition of CeM attenuates animal preference for sugar, while activation delays cocaine-induced increase in locomotor activity.

Sexually dimorphic effects of monoacylglycerol lipase inhibitor MJN110 on stress-related behaviour and drinking in Marchigian Sardinian alcohol-preferring rats.

The endocannabinoid (eCB) system plays an important homeostatic role in the regulation of stress circuits and has emerged as a therapeutic target to treat stress disorders and alcohol use disorder (AUD). Extensive research has elucidated a role for the eCB anandamide (AEA), but less is known about 2-arachidonoylglycerol (2-AG) mediated signalling. We pharmacologically enhanced eCB signalling by inhibiting the 2-AG metabolizing enzyme, monoacylglycerol lipase (MAGL), in male and female Marchigian Sardinian alcohol-preferring (msP) rats, a model of innate alcohol preference and stress hypersensitivity, and in control Wistar rats. We tested the acute effect of the selective MAGL inhibitor MJN110 in alleviating symptoms of alcohol drinking, anxiety, irritability and fear. A single systemic administration of MJN110 increased 2-AG levels in the central amygdala, prelimbic and infralimbic cortex but did not acutely alter alcohol drinking. MAGL inhibition reduced aggressive behaviours in female msPs, and increased defensive behaviours in male msPs, during the irritability test. Moreover, in the novelty-induced hypophagia test, MJN110 selectively enhanced palatable food consumption in females, mitigating stress-induced food suppression. Lastly, msP rats showed increased conditioned fear behaviour compared with Wistar rats, and MJN110 reduced context-associated conditioned fear responses, but not cue-probed fear expression, in male msPs. Acute inhibition of MAGL attenuated some stress-related responses in msP rats but not voluntary alcohol drinking. Our results provide new insights into the sex dimorphism documented in stress-induced responses. Sex-specific eCB-based approaches should be considered in the clinical development of therapeutics.

The parabrachial to central amygdala pathway is critical to injury-induced pain sensitization in mice

The spino-ponto-amygdaloid pathway is a major ascending circuit relaying nociceptive information from the spinal cord to the brain. Potentiation of excitatory synaptic transmission in the parabrachial nucleus (PBN) to central amygdala (CeA) pathway has been reported in rodent models of persistent pain. However, the functional significance of this pathway in the modulation of the somatosensory component of pain was recently challenged by studies showing that spinal nociceptive neurons do not target CeA-projecting PBN cells and that manipulations of this pathway have no effect on reflexive-defensive somatosensory responses to peripheral noxious stimulation. Here, we showed that activation of CeA-projecting PBN neurons is critical to increase both stimulus-evoked and spontaneous nociceptive responses following an injury in male and female mice. Using optogenetic-assisted circuit mapping, we confirmed a functional excitatory projection from PBN→CeA that is independent of the genetic or firing identity of CeA cells. We then showed that peripheral noxious stimulation increased the expression of the neuronal activity marker Fos in CeA-projecting PBN neurons and that chemogenetic inactivation of these cells decreased behavioral hypersensitivity in models of neuropathic and inflammatory pain without affecting baseline nociception. Lastly, we showed that chemogenetic activation of CeA-projecting PBN neurons is sufficient to induced bilateral hypersensitivity without injury. Together, our results indicate that the PBN→CeA pathway is a key modulator of pain-related behaviors that can increase reflexive-defensive and affective-motivational responses to somatosensory stimulation in injured states without affecting nociception under normal physiological conditions.