Central Amygdala Research Articles

Dynorphin/Kappa Opioid Receptor Activity Within the Extended Amygdala Contributes to Stress-Enhanced Alcohol Drinking in Mice

BackgroundWhile there is high comorbidity of stress-related disorders and alcohol use disorder, few effective treatments are available and elucidating underlying neurobiological mechanisms has been hampered by a general lack of reliable animal models. Here, we use a novel mouse model demonstrating robust and reproducible stress-enhanced alcohol drinking to examine the role of dynorphin/kappa opioid receptor (DYN/KOR) activity within the extended amygdala in mediating this stress-alcohol interaction. MethodsMice received repeated weekly cycles of chronic intermittent ethanol exposure alternating with weekly drinking sessions ± forced swim stress exposure. Pdyn messenger RNA expression was measured in the central amygdala (CeA), and DYN-expressing CeA neurons were then targeted for chemogenetic inhibition. Finally, a KOR antagonist was microinjected into the CeA or bed nucleus of the stria terminalis to examine the role of KOR signaling in promoting stress-enhanced drinking. ResultsStress (forced swim stress) selectively increased alcohol drinking in mice with a history of chronic intermittent ethanol exposure, and this was accompanied by elevated Pdyn messenger RNA levels in the CeA. Targeted chemogenetic silencing of DYN-expressing CeA neurons blocked stress-enhanced drinking, and KOR antagonism in the CeA or bed nucleus of the stria terminalis significantly reduced stress-induced elevated alcohol consumption without altering moderate intake in control mice. ConclusionsUsing a novel and robust model of stress-enhanced alcohol drinking, a significant role for DYN/KOR activity within extended amygdala circuitry in mediating this effect was demonstrated, thereby providing further evidence that the DYN/KOR system may be a valuable target in the development of more effective treatments for individuals presenting with comorbidity of stress-related disorders and alcohol use disorder.

A discrete neuronal circuit in central amygdala that modulates social distance and behavior

A discrete neuronal circuit in central amygdala that modulates social distance and behavior

Estrogen Receptor Beta in the Central Amygdala Regulates the Deleterious Behavioral and Neuronal Consequences of Repeated Social Stress in Female Rats

Estrogen Receptor Beta in the Central Amygdala Regulates the Deleterious Behavioral and Neuronal Consequences of Repeated Social Stress in Female Rats

Dopamine Receptor D2, But Not D1, Mediates the Reward Circuit from the Ventral Tegmental Area to the Central Amygdala, Which is Involved in Pain Relief

Dopamine Receptor D2, But Not D1, Mediates the Reward Circuit from the Ventral Tegmental Area to the Central Amygdala, Which is Involved in Pain Relief

Pain-induced potentiation at synapses between pain-activated neurons in the parabrachial nucleus and central amygdala of FosTRAP mice

Pain-induced potentiation at synapses between pain-activated neurons in the parabrachial nucleus and central amygdala of FosTRAP mice

Generation of a CRF1-Cre transgenic rat and the role of central amygdala CRF1 cells in nociception and anxiety-like behavior

Generation of a CRF1-Cre transgenic rat and the role of central amygdala CRF1 cells in nociception and anxiety-like behavior

Interaction between corticosterone and PER2 in regulating emotional behaviors in the rat

Circadian rhythms play a prominent role in psychiatric health with disruption in rhythms associated with poor mental health. Corticosterone (CORT) is an important hormone in entraining the biological rhythms of many cells throughout the body and coordinating peripheral rhythms with the central master clock. Here, we tested the hypothesis that excess CORT during the circadian trough would lead to a flattening of period genes (Per1 and Per2) rhythms in limbic brain areas, and thus impact emotional behaviors. Male rats were injected daily with 2.5 mg/kg CORT or vehicle for 21 days at either ZT0 or ZT12 and sucrose preference, open field, and forced swim behaviors measured during the dark phase of the light cycle. After three weeks of injections, a reduction in sucrose preference was observed in animals injected with CORT at ZT0 and the reduction significantly correlated with reductions in Per2 mRNA expression in the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST). No changes in behavior or period gene expression were observed in animals injected with CORT at ZT12. DsiRNA was used to directly reduce Per2 levels in either the CeA or BNST and behavior was assessed. Despite reductions in Per2 expression in the CeA, no behavioral changes were observed. In contrast, a reduction in Per2 expression in the BNST was sufficient to reduce sucrose preference. The results demonstrate that CORT significantly contributes to the circadian expression of Period genes in certain limbic brain areas and disruption in diurnal CORT or Per2 expression can lead to impaired emotional behavioral responses.

Divergent brainstem opioidergic pathways that coordinate breathing with pain and emotions

Breathing can be heavily influenced by pain or internal emotional states, but the neural circuitry underlying this tight coordination is unknown. Here we report that Oprm1 (μ-opioid receptor)-expressing neurons in the lateral parabrachial nucleus (PBL) are crucial for coordinating breathing with affective pain in mice. Individual PBLOprm1 neuronal activity synchronizes with breathing rhythm and responds to noxious stimuli. Manipulating PBLOprm1 activity directly changes breathing rate, affective pain perception, and anxiety. Furthermore, PBLOprm1 neurons constitute two distinct subpopulations in a "core-shell" configuration that divergently projects to the forebrain and hindbrain. Through non-overlapping projections to the central amygdala and pre-Bötzinger complex, these two subpopulations differentially regulate breathing, affective pain, and negative emotions. Moreover, these subsets form recurrent excitatory networks through reciprocal glutamatergic projections. Together, our data define the divergent parabrachial opioidergic circuits as a common neural substrate that coordinates breathing with various sensations and behaviors such as pain and emotional processing.

Oxytocin Attenuates the Stress-Induced Reinstatement of Alcohol-Seeking in Male Rats: Role of the Central Amygdala.

Factors such as stress and anxiety often contribute to alcohol-dependent behavior and can trigger a relapse of alcohol addiction and use. Therefore, it is important to investigate potential pharmacological interventions that may alleviate the influence of stress on addiction-related behaviors. Previous studies have demonstrated that the neuropeptide oxytocin has promising anxiolytic potential in mammals and may offer a pharmacological target to diminish the emotional impact on reinstatement of alcohol-seeking. The purpose of the present study was to investigate the effect of oxytocin on stress-induced alcohol relapse and identify a neural structure mediating this effect through the use of an ethanol self-administration and yohimbine-induced reinstatement paradigm. While yohimbine administration resulted in the reinstatement of ethanol-seeking behavior, the concurrent administration of yohimbine and oxytocin attenuated this effect, suggesting that oxytocin may disrupt stress-induced ethanol-seeking behavior. The central amygdala (CeA) is a structure that drives emotional responses and robustly expresses oxytocin receptors. Intra-CeA oxytocin similarly attenuated the yohimbine-induced reinstatement of ethanol-seeking behavior. These results demonstrate that oxytocin has the potential to attenuate stress-induced relapse into ethanol-seeking behavior, and that this mechanism occurs specifically within the central amygdala.

Astrocytes play a critical role in mediating the effect of acute ethanol on central amygdala glutamatergic transmission

The Central Amygdala (CeA) has been heavily implicated in many aspects of alcohol use disorder. Ethanol (EtOH) has been shown to modulate glutamatergic transmission in the lateral subdivision of the CeA, however, the exact mechanism of this modulation is still unclear. EtOH exposure is associated with increased pro-inflammatory cytokines in the CeA, and inhibition of neuroimmune cells (microglia and astrocytes) has previously been shown to reduce EtOH drinking in animal models. Since neuroimmune activation seems to be involved in many of the effects of EtOH, we hypothesized that acute EtOH exposure will increase excitatory glutamatergic transmission in the CeA via modulation of neuroimmune cells. Using ex vivo brain slice whole-cell patch clamp electrophysiology, it was found that a physiologically relevant concentration of EtOH (20 mM) significantly increased presynaptic glutamatergic transmission in the CeA. Pharmacologic and chemogenetic inhibition of astrocyte function significantly reduced the ability of EtOH to modulate CeA glutamatergic transmission with minimal impact of microglia inhibition. This finding prompted additional studies examining whether direct neuroimmune activation through lipopolysaccharide (LPS) might lead to an increase in the glutamatergic transmission in the CeA. It was found that LPS modulation of glutamatergic transmission was limited by microglia activation and required astrocyte signaling. Taken together these results support the hypothesis that acute EtOH enhances lateral CeA glutamatergic transmission through an astrocyte mediated mechanism.This article is part of the special Issue on ‘Neurocircuitry Modulating Drug and Alcohol Abuse'.

Compulsive Eating in a Rat Model of Binge Eating Disorder Under Conditioned Fear and Exploration of Neural Mechanisms With c-fos mRNA Expression.

Compulsive eating is the most obstinate feature of binge eating disorder. In this study, we observed the compulsive eating in our stress-induced binge-like eating rat model using a conflicting test, where sucrose and an aversively conditioned stimulus were presented at the same time. In this conflicting situation, the binge-like eating prone rats (BEPs), compared to the binge-like eating resistant rats (BERs), showed persistent high sucrose intake and inhibited fear response, respectively, indicating a deficit in palatability devaluation and stronger anxiolytic response to sucrose in the BEPs. We further analyzed the neuronal activation with c-fos mRNA in situ hybridization. Surprisingly, the sucrose access under conditioned fear did not inhibit the activity of amygdala; instead, it activated the central amygdala. In the BEPs, sucrose reduced the response of the paraventricular hypothalamic nucleus (PVN), while enhancing activities in the lateral hypothalamic area (LHA) to the CS. The resistance to devaluating the palatable food in the BEPs could be a result of persistent Acb response to sucrose intake and attenuated recruitment of the medial prefrontal cortex (mPFC). We interpret this finding as that the reward system of the BEPs overcame the homeostasis system and the stress-responding system.

Synaptic effects of IL-1β and CRF in the central amygdala after protracted alcohol abstinence in male rhesus macaques.

A major barrier to remission from an alcohol use disorder (AUD) is the continued risk of relapse during abstinence. Assessing the neuroadaptations after chronic alcohol and repeated abstinence is important to identify mechanisms that may contribute to relapse. In this study, we used a rhesus macaque model of long-term alcohol use and repeated abstinence, providing a platform to extend mechanistic findings from rodents to primates. The central amygdala (CeA) displays elevated GABA release following chronic alcohol in rodents and in abstinent male macaques, highlighting this neuroadaptation as a conserved mechanism that may underlie excessive alcohol consumption. Here, we determined circulating interleukin-1β (IL-1β) levels, CeA transcriptomic changes, and the effects of IL-1β and corticotropin releasing factor (CRF) signaling on CeA GABA transmission in male controls and abstinent drinkers. While no significant differences in peripheral IL-1β or the CeA transcriptome were observed, pathway analysis identified several canonical immune-related pathways. We addressed this potential dysregulation of CeA immune signaling in abstient drinkers with an electrophysiological approach. We found that IL-1β decreased CeA GABA release in controls while abstinent drinkers were less sensitive to IL-1β's effects, suggesting adaptations in the neuromodulatory role of IL-1β. In contrast, CRF enhanced CeA GABA release similarly in controls and abstinent drinkers, consistent with rodent studies. Notably, CeA CRF expression was inversely correlated with intoxication, suggesting that CRF levels during abstinence may predict future intoxication. Together, our findings highlight conserved and divergent actions of chronic alcohol on neuroimmune and stress signaling on CeA GABA transmission across rodents and macaques.

Basolateral and central amygdala orchestrate how we learn whom to trust

Cooperation and mutual trust are essential in our society, yet not everybody is trustworthy. In this fMRI study, 62 healthy volunteers performed a repeated trust game, placing trust in a trustworthy or an untrustworthy player. We found that the central amygdala was active during trust behavior planning while the basolateral amygdala was active during outcome evaluation. When planning the trust behavior, central and basolateral amygdala activation was stronger for the untrustworthy player compared to the trustworthy player but only in participants who actually learned to differentiate the trustworthiness of the players. Independent of learning success, nucleus accumbens encoded whether trust was reciprocated. This suggests that learning whom to trust is not related to reward processing in the nucleus accumbens, but rather to engagement of the amygdala. Our study overcomes major empirical gaps between animal models and human neuroimaging and shows how different subnuclei of the amygdala and connected areas orchestrate learning to form different subjective trustworthiness beliefs about others and guide trust choice behavior.

Sphingosine-1-phosphate receptor 1 agonist SEW2871 alters membrane properties of late-firing somatostatin expressing neurons in the central lateral amygdala

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates a wide spectrum of biological processes including apoptosis, immune response and inflammation. Here, we sought to understand how S1P signaling affects neuronal excitability in the central amygdala (CeA), which is a brain region associated with fear learning, aversive memory, and the affective dimension of pain. Because the G-protein coupled S1P receptor 1 (S1PR1) has been shown to be the primary mediator of S1P signaling, we utilized S1PR1 agonist SEW2871 and S1PR1 antagonist NIBR to determine a potential role of S1PR1 in altering the cellular physiology of neurons in the lateral division of the CeA (CeL) that share the neuronal lineage marker somatostatin (Sst). CeL-Sst neurons play a critical role in expression of conditioned fear and pain modulation. Here we used transgenic breeding strategies to identify fluorescently labeled CeL-Sst neurons for electrophysiological recordings. Using principal component analysis, we identified two primary subtypes of Sst neurons within the CeL in both male and female mice. We denoted the two types regular-firing (type A) and late-firing (type B) CeL-Sst neurons. In response to SEW2871 application, Type A neurons exhibited increased input resistance, while type B neurons displayed a depolarized resting membrane potential and voltage threshold, increased current threshold, and decreased voltage height. NIBR application had no effect on CeL Sst neurons, indicating the absence of tonic S1P-induced S1PR1. Our findings reveal subtypes of Sst neurons within the CeL that are uniquely affected by S1PR1 activation, which may have implications for how S1P alters supraspinal circuits.

Periaqueductal gray neurons encode the sequential motor program in hunting behavior of mice

Sequential encoding of motor programs is essential for behavior generation. However, whether it is critical for instinctive behavior is still largely unknown. Mouse hunting behavior typically contains a sequential motor program, including the prey search, chase, attack, and consumption. Here, we reveal that the neuronal activity in the lateral periaqueductal gray (LPAG) follows a sequential pattern and is time-locked to different hunting actions. Optrode recordings and photoinhibition demonstrate that LPAGVgat neurons are required for the prey detection, chase and attack, while LPAGVglut2 neurons are selectively required for the attack. Ablation of inputs that could trigger hunting, including the central amygdala, the lateral hypothalamus, and the zona incerta, interrupts the activity sequence pattern and substantially impairs hunting actions. Therefore, our findings reveal that periaqueductal gray neuronal ensembles encode the sequential hunting motor program, which might provide a framework for decoding complex instinctive behaviors.

Central nucleus of the amygdala projections onto the nucleus accumbens core regulate binge-like alcohol drinking in a CRF-dependent manner

RationaleThe nucleus accumbens (NAc) is important for regulating a number of behaviors, including alcohol and substance use. We previously found that chemogenetically manipulating neuronal activity in the NAc core regulates binge-like drinking in mice. The central amygdala (CeA) is also an important regulator of alcohol drinking, and projects to the NAc core. We tested whether neuronal projections from the CeA to the NAc core, or neuropeptides released by the CeA in the NAc core, could regulate binge drinking. MethodsFor experiment 1, mice were administered AAV2 Cre-GFP into the NAc core and a Cre-inducible DREADD [AAV2 DIO- hM3Dq, -hM4Di, or -mCherry control] into the CeA. We tested the effects of altering CeA to NAc core activity on binge-like ethanol intake (via “Drinking in the Dark”, DID). For experiment 2, we bilaterally microinfused corticotropin releasing factor (CRF), neuropeptide Y (NPY), or somatostatin (SST) into the NAc core prior to DID. For experiment 3, we tested whether intra-NAc CRF antagonism prevented reductions in drinking induced by CNO/hM3Dq stimulation of CeA->NAc projections. ResultsChemogenetically increasing activity in neurons projecting from the CeA to NAc core decreased binge-like ethanol drinking (p < 0.01). Intra-NAc core CRF mimicked chemogenetic stimulation of this pathway (p < 0.05). Binge-like drinking was unaffected by the doses of NPY and SST tested. Lastly, we found that intra-NAc CRF antagonism prevented reductions in drinking induced by chemogenetic stimulation of CeA->NAc projections. These findings demonstrate that neurons projecting from the CeA to NAc core that release CRF are capable of regulating binge-like drinking in mice.This article is part of the special Issue on ‘Neurocircuitry Modulating Drug and Alcohol Abuse'.

Sex and context differences in the effects of trauma on comorbid alcohol use and post-traumatic stress phenotypes in actively drinking rats.

Alcohol use disorder (AUD) and affective disorders are frequently comorbid and share underlying mechanisms that could be targets for comprehensive treatment. Post-traumatic stress disorder (PTSD) has high comorbidity with AUD, but comprehensive models of this overlap are nascent. We recently characterized a model of comorbid AUD and PTSD-like symptoms, wherein stressed rats receive an inhibitory avoidance (IA)-related footshock on two occasions followed by two-bottle choice (2BC) voluntary alcohol drinking. Stressed rats received the second footshock in a familiar (FAM, same IA box as the first footshock) or novel context (NOV, single-chambered apparatus); the FAM paradigm more effectively increased alcohol drinking in males and the NOV paradigm in females. During abstinence, stressed males displayed avoidance-like PTSD symptoms, and females showed hyperarousal-like PTSD symptoms. Rats in the model had altered spontaneous action potential-independent GABAergic transmission in the central amygdala (CeA), a brain region key in alcohol dependence and stress-related signaling. However, PTSD sufferers may have alcohol experience prior to their trauma. Here, we therefore modified our AUD/PTSD comorbidity model to provide 3weeks of intermittent extended alcohol access before footshock and then studied the effects of NOV and FAM stress on drinking and PTSD phenotypes. NOV stress suppressed the escalation of alcohol intake and preference seen in male controls, but no stress effects were seen on drinking in females. Additionally, NOV males had decreased action potential-independent presynaptic GABA release and delayed postsynaptic GABAA receptor kinetics in the CeA compared to control and FAM males. Despite these changes to alcohol intake and CeA GABA signaling, stressed rats showed broadly similar anxiogenic-like behaviors to our previous comorbid model, suggesting decoupling of the PTSD symptoms from the AUD vulnerability for some of these animals. The collective results show the importance of alcohol history and trauma context in vulnerability to comorbid AUD/PTSD-like symptoms.

Methamphetamine and Modulation Functionality of the Prelimbic Cortex for Developing a Possible Treatment of Alzheimer's Disease in an Animal Model.

Alzheimer’s disease (AD) is a progressive neurodegenerative condition that causes cognitive impairment and other neuropsychiatric symptoms. Previously, little research has thus far investigated whether methamphetamine (MAMPH) can enhance cognitive function or ameliorate AD symptoms. This study examined whether a low dose of MAMPH can induce conditioned taste aversion (CTA) learning, or can increase plasma corticosterone levels, neural activity, and neural plasticity in the medial prefrontal cortex (mPFC) (responsible for cognitive function), the nucleus accumbens (NAc) and the amygdala (related to rewarding and aversive emotion), and the hippocampus (responsible for spatial learning). Furthermore, the excitations or lesions of the prelimbic cortex (PrL) can affect MAMPH-induced CTA learning, plasma corticosterone levels, and neural activity or plasticity in the mPFC [i.e., PrL, infralimbic cortex (IL), cingulate cortex 1 (Cg1)], the NAc, the amygdala [i.e., basolateral amygdala (BLA) and central amygdala (CeA)], and the hippocampus [i.e., CA1, CA2, CA3, and dentate gyrus (DG)]. In the experimental procedure, the rats were administered either saline or NMDA solutions, which were injected into the PrL to excite or destroy PrL neurons. Additionally, rats received 0.1% saccharin solution for 15 min, followed by intraperitoneal injections of either normal saline or 1 mg/kg MAMPH to induce CTA. A one-way ANOVA was performed to analyze the effects of saccharin intake on CTA, plasma corticosterone levels, and the expression of c-Fos and p-ERK. The results showed that the MAMPH induced CTA learning and increased plasma corticosterone levels. The mPFC, and particularly the PrL and IL and the DG of the hippocampus, appeared to show increased neural activity in c-Fos expression or neural plasticity in p-ERK expression. The excitation of the PrL neurons upregulated neural activity in c-Fos expression and neural plasticity in p-ERK expression in the PrL and IL. In summary, MAMPH may be able to improve cognitive and executive function in the brain and reduce AD symptoms. Moreover, the excitatory modulation of the PrL with MAMPH administration can facilitate MAMPH-induced neural activity and plasticity in the PrL and IL of the mPFC. The present data provide clinical implications for developing a possible treatment for AD in an animal model.

Neurokinin B-Expressing Neurons of the Central Extended Amygdala Mediate Inhibitory Synaptic Input onto Melanin-Concentrating Hormone Neuron Subpopulations.

The lateral hypothalamic area (LHA) is a highly conserved brain region critical for maintaining physiological homeostasis and goal-directed behavior. LHA neurons that express melanin-concentrating hormone (MCH) are key regulators of arousal, energy balance, and motivated behavior. However, cellular and functional diversity among LHAMCH neurons is not well understood. Previous anatomic and molecular data suggest that LHAMCH neurons may be parsed into at least two distinct subpopulations, one of which is enriched in neurokinin-3 receptor (NK3R), the receptor for neurokinin B (NKB), encoded by the Tac2 gene. This tachykininergic ligand-receptor system has been implicated in reproduction, fear memory, and stress in other brain regions, but NKB interactions with LHAMCH neurons are poorly understood. We first identified how LHAMCH subpopulations may be distinguished anatomically and electrophysiologically. To dissect functional connectivity between NKB-expressing neurons and LHAMCH neurons, we used Cre-dependent retrograde and anterograde viral tracing in male Tac2-Cre mice and identified Tac2/EYFP+ neurons in the bed nucleus of the stria terminalis and central nucleus of the amygdala, the central extended amygdala, as major sources of NKB input onto LHAMCH neurons. In addition to innervating the LHA, these limbic forebrain NKB neurons also project to midbrain and brainstem targets. Finally, using a dual-virus approach, we found that optogenetic activation of these inputs in slices evokes GABA release onto a subset of LHAMCH neurons but lacked specificity for the NK3R+ subpopulation. Overall, these data define parallel tachykininergic/GABAergic limbic forebrain projections that are positioned to modulate multiple nodes of homeostatic and behavioral control.SIGNIFICANCE STATEMENT The LHA orchestrates fundamental behavioral states in the mammalian hypothalamus, including arousal, energy balance, memory, stress, and motivated behavior. The neuropeptide MCH defines one prominent population of LHA neurons, with multiple roles in the regulation of homeostatic behavior. Outstanding questions remain concerning the upstream inputs that control MCH neurons. We sought to define neurochemically distinct pathways in the mouse brain that may communicate with specific MCH neuron subpopulations using viral-based retrograde and anterograde neural pathway tracing and optogenetics in brain slices. Here, we identify a specific neuropeptide-defined forebrain circuit that makes functional synaptic connections with MCH neuron subpopulations. This work lays the foundation for further manipulating molecularly distinct neural circuits that modulate innate behavioral states.

CRHCeA\u2192VTA inputs inhibit the positive ensembles to induce negative effect of opiate withdrawal

Plasticity of neurons in the ventral tegmental area (VTA) is critical for establishment of drug dependence. However, the remodeling of the circuits mediating the transition between positive and negative effect remains unclear. Here, we used neuronal activity-dependent labeling technique to characterize and temporarily control the VTA neuronal ensembles recruited by the initial morphine exposure (morphine-positive ensembles, Mor-Ens). Mor-Ens preferentially projected to NAc, and induced dopamine-dependent positive reinforcement. Electrophysiology and rabies viral tracing revealed the preferential connections between the VTA-projective corticotrophin-releasing hormone (CRH) neurons of central amygdala (CRHCeA→VTA) and Mor-Ens, which was enhanced after escalating morphine exposure and mediated the negative effect during opiate withdrawal. Pharmacologic intervention or CRISPR-mediated repression of CRHR1 in Mor-Ens weakened the inhibitory CRHCeA→VTA inputs, and alleviated the negative effect during opiate withdrawal. These data suggest that neurons encoding opioid reward experience are inhibited by enhanced CRHCeA→VTA inputs induced by chronic morphine exposure, leading to negative effect during opiate withdrawal, and provide new insight into the pathological changes in VTA plasticity after drug abuse and mechanism of opiate dependence.