Contribution of AMPA Receptor-Mediated LTD in LA/BLA-CeA Pathway to Comorbid Aversive and Depressive Symptoms in Neuropathic Pain.

Comorbid anxiety and depressive symptoms in chronic pain are a common health problem, but the underlying mechanisms remain unclear. Previously, we have demonstrated that sensitization of the CeA neurons via decreased GABAergic inhibition contributes to anxiety-like behaviors in neuropathic pain rats. In this study, by using male Sprague Dawley rats, we reported that the CeA plays a key role in processing both sensory and negative emotional-affective components of neuropathic pain. Bilateral electrolytic lesions of CeA, but not lateral/basolateral nucleus of the amygdala (LA/BLA), abrogated both pain hypersensitivity and aversive and depressive symptoms of neuropathic rats induced by spinal nerve ligation (SNL). Moreover, SNL rats showed structural and functional neuroplasticity manifested as reduced dendritic spines on the CeA neurons and enhanced LTD at the LA/BLA-CeA synapse. Disruption of GluA2-containing AMPAR trafficking and endocytosis from synapses using synthetic peptides, either pep2-EVKI or Tat-GluA2(3Y), restored the enhanced LTD at the LA/BLA-CeA synapse, and alleviated the mechanical allodynia and comorbid aversive and depressive symptoms in neuropathic rats, indicating that the endocytosis of GluA2-containing AMPARs from synapses is probably involved in the LTD at the LA/BLA-CeA synapse and the comorbid aversive and depressive symptoms in neuropathic pain in SNL-operated rats. These data provide a novel mechanism for elucidating comorbid aversive and depressive symptoms in neuropathic pain and highlight that structural and functional neuroplasticity in the amygdala may be important as a promising therapeutic target for comorbid negative emotional-affective disorders in chronic pain.SIGNIFICANCE STATEMENT Several studies have demonstrated the high comorbidity of negative affective disorders in patients with chronic pain. Understanding the affective aspects related to chronic pain may facilitate the development of novel therapies for more effective management. Here, we unravel that the CeA plays a key role in processing both sensory and negative emotional-affective components of neuropathic pain, and LTD at the amygdaloid LA/BLA-CeA synapse mediated by GluA2-containing AMPAR endocytosis underlies the comorbid aversive and depressive symptoms in neuropathic pain. This study provides a novel mechanism for elucidating comorbid aversive and depressive symptoms in neuropathic pain and highlights that structural and functional neuroplasticity in the amygdala may be important as a promising therapeutic target for comorbid negative emotional-affective disorders in chronic pain.

Agent-based modeling of the central amygdala and pain using cell-type specific physiological parameters.

The amygdala is a brain area involved in emotional regulation and pain. Over the course of the last 20 years, multiple researchers have studied sensory and motor connections within the amygdala in trying to understand the ultimate role of this structure in pain perception and descending control of pain. A number of investigators have been using cell-type specific manipulations to probe the underlying circuitry of the amygdala. As data have accumulated in this research space, we recognized a critical need for a single framework to integrate these data and evaluate emergent system-level responses. In this manuscript, we present an agent-based computational model of two distinct inhibitory neuron populations in the amygdala, those that express protein kinase C delta (PKCδ) and those that express somatostatin (SOM). We utilized a network of neural links to simulate connectivity and the transmission of inhibitory signals between neurons. Type-specific parameters describing the response of these neurons to noxious stimuli were estimated from published physiological and immunological data as well as our own wet-lab experiments. The model outputs an abstract measure of pain, which is calculated in terms of the cumulative pro-nociceptive and anti-nociceptive activity across neurons in both hemispheres of the amygdala. Results demonstrate the ability of the model to produce changes in pain that are consistent with published studies and highlight the importance of several model parameters. In particular, we found that the relative proportion of PKCδ and SOM neurons within each hemisphere is a key parameter in predicting pain and we explored model predictions for three possible values of this parameter. We compared model predictions of pain to data from our earlier behavioral studies and found areas of similarity as well as distinctions between the data sets. These differences, in particular, suggest a number of wet-lab experiments that could be done in the future.

SIRT1 Decreases Emotional Pain Vulnerability with Associated CaMKIIα Deacetylation in Central Amygdala.

Emotional disorders are common comorbid conditions that further exacerbate the severity and chronicity of chronic pain. However, individuals show considerable vulnerability to the development of chronic pain under similar pain conditions. In this study on male rat and mouse models of chronic neuropathic pain, we identify the histone deacetylase Sirtuin 1 (SIRT1) in central amygdala as a key epigenetic regulator that controls the development of comorbid emotional disorders underlying the individual vulnerability to chronic pain. We found that animals that were vulnerable to developing behaviors of anxiety and depression under the pain condition displayed reduced SIRT1 protein levels in central amygdala, but not those animals resistant to the emotional disorders. Viral overexpression of local SIRT1 reversed this vulnerability, but viral knockdown of local SIRT1 mimicked the pain effect, eliciting the pain vulnerability in pain-free animals. The SIRT1 action was associated with CaMKIIα downregulation and deacetylation of histone H3 lysine 9 at the CaMKIIα promoter. These results suggest that, by transcriptional repression of CaMKIIα in central amygdala, SIRT1 functions to guard against the emotional pain vulnerability under chronic pain conditions. This study indicates that SIRT1 may serve as a potential therapeutic molecule for individualized treatment of chronic pain with vulnerable emotional disorders.SIGNIFICANCE STATEMENT Chronic pain is a prevalent neurological disease with no effective treatment at present. Pain patients display considerably variable vulnerability to developing chronic pain, indicating individual-based molecular mechanisms underlying the pain vulnerability, which is hardly addressed in current preclinical research. In this study, we have identified the histone deacetylase Sirtuin 1 (SIRT1) as a key regulator that controls this pain vulnerability. This study reveals that the SIRT1-CaMKIIaα pathway in central amygdala acts as an epigenetic mechanism that guards against the development of comorbid emotional disorders under chronic pain, and that its dysfunction causes increased vulnerability to the development of chronic pain. These findings suggest that SIRT1 activators may be used in a novel therapeutic approach for individual-based treatment of chronic pain.

A balancing act: the role of pro- and anti-stress peptides within the central amygdala in anxiety and alcohol use disorders.

The central nucleus of the amygdala (CeA) is widely implicated as a structure that integrates both appetitive and aversive stimuli. While intrinsic CeA microcircuits primarily consist of GABAergic neurons that regulate amygdala output, a notable feature of the CeA is the heterogeneity of neuropeptides and neuropeptide/neuromodulator receptors that it expresses. There is growing interest in the role of the CeA in mediating psychopathologies, including stress and anxiety states and their interactions with alcohol use disorders. Within the CeA, neuropeptides and neuromodulators often exert pro- or anti- stress actions, which can influence anxiety and alcohol associated behaviours. In turn, alcohol use can cause adaptions within the CeA, which may render an individual more vulnerable to stress which is a major trigger of relapse to alcohol seeking. This review examines the neurocircuitry, neurochemical phenotypes and how pro- and anti-stress peptide systems act within the CeA to regulate anxiety and alcohol seeking, focusing on preclinical observations from animal models. Furthermore, literature exploring the targeting of genetically defined populations or neuronal ensembles and the role of the CeA in mediating sex differences in stress x alcohol interactions are explored.

Dopamine D2 receptor-mediated circuit from the central amygdala to the bed nucleus of the stria terminalis regulates impulsive behavior.

Impulsivity is closely associated with addictive disorders, and changes in the brain dopamine system have been proposed to affect impulse control in reward-related behaviors. However, the central neural pathways through which the dopamine system controls impulsive behavior are still unclear. We found that the absence of the D2 dopamine receptor (D2R) increased impulsive behavior in mice, whereas restoration of D2R expression specifically in the central amygdala (CeA) of D2R knockout mice (Drd2-/-) normalized their enhanced impulsivity. Inhibitory synaptic output from D2R-expressing neurons in the CeA underlies modulation of impulsive behavior because optogenetic activation of D2R-positive inhibitory neurons that project from the CeA to the bed nucleus of the stria terminalis (BNST) attenuate such behavior. Our identification of the key contribution of D2R-expressing neurons in the CeA → BNST circuit to the control of impulsive behavior reveals a pathway that could serve as a target for approaches to the management of neuropsychiatric disorders associated with impulsivity.

Plastic changes in amygdala subregions by voluntary running contribute to exercise-induced hypoalgesia in neuropathic pain model mice.

Physical exercise has been established as a low-cost, safe, and effective way to manage chronic pain, but exact mechanisms underlying such exercise-induced hypoalgesia (EIH) are not fully understood. Since a growing body of evidence implicated the amygdala (Amyg) as a critical node in emotional affective aspects of chronic pain, we hypothesized that the Amyg may play important roles to produce EIH effects. Here, using partial sciatic nerve ligation (PSL) model mice, we investigated the effects of voluntary running (VR) on the basal amygdala (BA) and the central nuclei of amygdala (CeA). The present study indicated that VR significantly improved heat hyperalgesia which was exacerbated in PSL-Sedentary mice, and that a significant positive correlation was detected between total running distances after PSL-surgery and thermal withdrawal latency. The number of activated glutamate (Glu) neurons in the medal BA (medBA) was significantly increased in PSL-Runner mice, while those were increased in the lateral BA in sedentary mice. Furthermore, in all subdivisions of the CeA, the number of activated gamma-aminobutyric acid (GABA) neurons was dramatically increased in PSL-Sedentary mice, but these numbers were significantly decreased in PSL-Runner mice. In addition, a tracer experiment demonstrated a marked increase in activated Glu neurons in the medBA projecting into the nucleus accumbens lateral shell in runner mice. Thus, our results suggest that VR may not only produce suppression of the negative emotion such as fear and anxiety closely related with pain chronification, but also promote pleasant emotion and hypoalgesia. Therefore, we conclude that EIH effects may be produced, at least in part, via such plastic changes in the Amyg.

Central nucleus of the amygdala as a common substrate of the incubation of drug and natural reinforcer seeking.

Relapse into drug use is a major problem faced by recovering addicts. In humans, an intensification of the desire for the drug induced by environmental cues-incubation of drug craving-has been observed. In rodents, this phenomenon has been modeled by studying drug seeking under extinction after different times of drug withdrawal (or using a natural reinforcer). Although much progress has been made, an integrated approach simultaneously studying different drug classes and natural reward and examining different brain regions is lacking. Lewis rats were used to study the effects of cocaine, heroin, and sucrose seeking incubation on six key brain regions: the nucleus accumbens shell/core, central/basolateral amygdala, and dorsomedial/ventromedial prefrontal cortex. We analyzed PSD95 and gephyrin protein levels, gene expression of glutamatergic, GABAergic and endocannabinoid elements, and amino acid transmitter levels. The relationships between the areas studied were examined by Structural Equation Modelling. Pathways from medial prefrontal cortex and basolateral complex of the amygdala to central nucleus of the amygdala, but not to the nucleus accumbens, were identified as common elements involved in the incubation phenomenon for different substances. These results suggest a key role for the central nucleus of amygdala and its cortical and amygdalar afferences in the incubation phenomenon, and we suggest that by virtue of its regulatory effects on glutamatergic and GABAergic dynamics within amygdalar circuits, the endocannabinoid system might be a potential target to develop medications that are effective in the context of relapse.

A Central Extended Amygdala Circuit That Modulates Anxiety.

Both the amygdala and the bed nucleus of the stria terminalis (BNST) have been implicated in maladaptive anxiety characteristics of anxiety disorders. However, the underlying circuit and cellular mechanisms have remained elusive. Here we show that mice with Erbb4 gene deficiency in somatostatin-expressing (SOM+) neurons exhibit heightened anxiety as measured in the elevated plus maze test and the open field test, two assays commonly used to assess anxiety-related behaviors in rodents. Using a combination of electrophysiological, molecular, genetic, and pharmacological techniques, we demonstrate that the abnormal anxiety in the mutant mice is caused by enhanced excitatory synaptic inputs onto SOM+ neurons in the central amygdala (CeA), and the resulting reduction in inhibition onto downstream SOM+ neurons in the BNST. Notably, our results indicate that an increase in dynorphin signaling in SOM+ CeA neurons mediates the paradoxical reduction in inhibition onto SOM+ BNST neurons, and that the consequent enhanced activity of SOM+ BNST neurons is both necessary for and sufficient to drive the elevated anxiety. Finally, we show that the elevated anxiety and the associated synaptic dysfunctions and increased dynorphin signaling in the CeA-BNST circuit of the Erbb4 mutant mice can be recapitulated by stress in wild-type mice. Together, our results unravel previously unknown circuit and cellular processes in the central extended amygdala that can cause maladaptive anxiety.SIGNIFICANCE STATEMENT The central extended amygdala has been implicated in anxiety-related behaviors, but the underlying mechanisms are unclear. Here we found that somatostatin-expressing neurons in the central amygdala (CeA) controls anxiety through modulation of the stria terminalis, a process that is mediated by an increase in dynorphin signaling in the CeA. Our results reveal circuit and cellular dysfunctions that may account for maladaptive anxiety.

Resting-State Functional Connectivity Between Centromedial Amygdala and Insula as Related to Somatic Symptoms in Depressed Patients: A Preliminary Study.

OBJECTIVE: Somatic symptoms are prevalent in patients with depression. The centromedial amygdala (CMA) is a key brain region that mediates autonomic and somatic responses. Abnormal function in the CMA may contribute to the development of somatic symptoms in depressed patients. METHODS: We compared the resting-state functional connectivity (RSFC) based on the seed of the left and right CMA between 37 patients with depression and 30 healthy controls. The severity of depressive and somatic symptoms was assessed using the Hamilton Depression Rating Scale (HDRS) and the 15-item somatic symptom severity scale of the Patient Health Questionnaire (PHQ-15). Correlation analysis was performed to investigate the relationship between the RSFC and clinical variables (HDRS and PHQ-15) in depressed patients. RESULTS: Compared with healthy controls, patients with depression exhibited decreased RSFC between the CMA and insula, and superior temporal gyrus. In addition, functional connectivity between the left CMA and left insula was negatively correlated with PHQ-15 (r = -0.348, p = .037) in depressed patients. No significant relation was found between the RSFC and HDRS in depressed patients. CONCLUSIONS: Functional connectivity between the CMA and insula is reduced in depressive patients, which is associated with the severity of somatic symptoms. Our findings may provide a potential neural substrate to interpret the co-occurrence of depression with somatic symptoms.

Resting-state connectivity of the bed nucleus of the stria terminalis and the central nucleus of the amygdala in clinical anxiety

Background: The central nucleus of the amygdala and bed nucleus of the stria terminalis are involved primarily in phasic and sustained aversive states. Although both structures have been implicated in pathological anxiety, few studies with a clinical population have specifically focused on them, partly because of their small size. Previous work in our group used high-resolution imaging to map the restingstate functional connectivity of the bed nucleus of the stria terminalis and the central nucleus of the amygdala in healthy subjects at 7 T, confirming and extending structural findings in humans and animals, while providing additional insight into cortical connectivity that is potentially unique to humans. Methods: In the current follow-up study, we contrasted resting-state functional connectivity in the bed nucleus of the stria terminalis and central nucleus of the amygdala at 7 T between healthy volunteers (n = 30) and patients with generalized and/or social anxiety disorder (n = 30). Results: Results revealed significant voxel-level group differences. Compared with healthy volunteers, patients showed stronger resting-state functional connectivity between the central nucleus of the amygdala and the lateral orbitofrontal cortex and superior temporal sulcus. They also showed weaker resting-state functional connectivity between the bed nucleus of the stria terminalis and the dorsolateral prefrontal cortex and occipital cortex. Limitations: These findings depart from a previous report of resting-state functional connectivity in the central nucleus of the amygdala and bed nucleus of the stria terminalis under sustained threat of shock in healthy volunteers. Conclusion: This study provides functional MRI proxies of the functional dissociation of the bed nucleus of the stria terminalis and central nucleus of the amygdala, and suggests that resting-state functional connectivity of key structures in the processing of defensive responses do not recapitulate changes related to induced state anxiety. Future work needs to replicate and further probe the clinical significance of these findings.

Endocannabinoid Signaling in the Central Amygdala and Bed Nucleus of the Stria Terminalis: Implications for the Pathophysiology and Treatment of Alcohol Use Disorder.

High rates of relapse are a chronic and debilitating obstacle to effective treatment of alcohol use disorder (AUD); however, no effective treatments are available to treat symptoms induced by protracted abstinence. In the first part of this 2-part review series, we examine the literature supporting the effects of alcohol exposure within the extended amygdala (EA) neural circuitry. In Part 2, we focus on a potential way to combat negative affect associated with AUD, by exploring the therapeutic potential of the endogenous cannabinoid (eCB) system. The eCB system is a potent modulator of neural activity in the brain, and its ability to mitigate stress and negative affect has long been an area of interest for developing novel therapeutics. This review details the recent advances in our understanding of eCB signaling in 2 key regions of the EA, the central nucleus of the amygdala and the bed nucleus of the stria terminalis (BNST), and their role in regulating negative affect. Despite an established role for EA eCB signaling in reducing negative affect, few studies have examined the potential for eCB-based therapies to treat AUD-associated negative affect. In this review, we present an overview of studies focusing on eCB signaling in EA and cannabinoid modulation on EA synaptic activity. We further discuss studies suggesting dysregulation of eCB signaling in models of AUD and propose that pharmacological augmentation of eCB could be a novel approach to treat aspects of AUD. Lastly, future directions are proposed to advance our understanding of the relationship between AUD-associated negative affect and the EA eCB system that could yield new pharmacotherapies targeting negative affective symptoms associated with AUD.

GABA Cells in the Central Nucleus of the Amygdala Promote Cataplexy.

Cataplexy is a hallmark of narcolepsy characterized by the sudden uncontrollable onset of muscle weakness or paralysis during wakefulness. It can occur spontaneously, but is typically triggered by positive emotions such as laughter. Although cataplexy was identified >130 years ago, its neural mechanism remains unclear. Here, we show that a newly identified GABA circuit within the central nucleus of the amygdala (CeA) promotes cataplexy. We used behavioral, electrophysiological, immunohistochemical, and chemogenetic strategies to target and manipulate CeA activity selectively in narcoleptic (orexin-/-) mice to determine its functional role in controlling cataplexy. First, we show that chemogenetic activation of the entire CeA produces a marked increase in cataplexy attacks. Then, we show that GABA cells within the CeA are responsible for mediating this effect. To manipulate GABA cells specifically, we developed a new mouse line that enables genetic targeting of GABA cells in orexin-/- mice. We found that chemogenetic activation of GABA CeA cells triggered a 253% increase in the number of cataplexy attacks without affecting their duration, suggesting that GABA cells play a functional role in initiating but not maintaining cataplexy. We show that GABA cell activation only promotes cataplexy attacks associated with emotionally rewarding stimuli, not those occurring spontaneously. However, we found that chemogenetic inhibition of GABA CeA cells does not prevent cataplexy, suggesting these cells are not required for initiating cataplexy attacks. Our results indicate that the CeA promotes cataplexy onset and that emotionally rewarding stimuli may trigger cataplexy by activating GABA cells in the CeA.SIGNIFICANCE STATEMENT Although cataplexy has been closely linked to positive emotions for >130 years, the neural circuitry that underlies this relationship is poorly understood. Recent work suggests that the amygdala, a brain area important for processing emotion, may be part of this circuit. This study provides the first functional evidence to implicate GABA cells in the amygdala as regulators of cataplexy triggered by positive emotions and identifies the amygdala as the brain region important more for gating the entrance into rather than the exit from cataplexy. We also generated a new mouse model for studying GABA neurons in narcoleptic mice, which could serve as a useful tool for studying the neurobiological underpinnings of narcolepsy.

Alcohol Dependence Disrupts Amygdalar L-Type Voltage-Gated Calcium Channel Mechanisms.

L-type voltage-gated calcium channels (LTCCs) are implicated in several psychiatric disorders that are comorbid with alcoholism and involve amygdala dysfunction. Within the amygdala, the central nucleus (CeA) is critical in acute alcohol's reinforcing actions, and its dysregulation in human alcoholics drives their negative emotional state and motivation to drink. Here we investigated the specific role of CeA LTCCs in the effects of acute alcohol at the molecular, cellular physiology, and behavioral levels, and their potential neuroadaptation in alcohol-dependent rats. Alcohol increases CeA activity (neuronal firing rates and GABA release) in naive rats by engaging LTCCs, and intra-CeA LTCC blockade reduces alcohol intake in nondependent rats. Alcohol dependence reduces CeA LTCC membrane abundance and disrupts this LTCC-based mechanism; instead, corticotropin-releasing factor type 1 receptors (CRF1s) mediate alcohol's effects on CeA activity and drive the escalated alcohol intake of alcohol-dependent rats. Collectively, our data indicate that alcohol dependence functionally alters the molecular mechanisms underlying the CeA's response to alcohol (from LTCC- to CRF1-driven). This mechanistic switch contributes to and reflects the prominent role of the CeA in the negative emotional state that drives excessive drinking.SIGNIFICANCE STATEMENT The central amygdala (CeA) plays a critical role in the development of alcohol dependence. As a result, much preclinical alcohol research aims to identify relevant CeA neuroadaptions that promote the transition to dependence. Here we report that acute alcohol increases CeA neuronal activity in naive rats by engaging L-type calcium channels (LTCCs) and that intra-CeA LTCC blockade reduces alcohol intake in nondependent rats. Alcohol dependence disrupts this LTCC-based mechanism; instead, corticotropin-releasing factor type 1 receptors (CRF1s) mediate alcohol's effects on CeA activity and drive the escalated alcohol intake of alcohol-dependent rats. This switch reflects the important role of the CeA in the pathophysiology of alcohol dependence and represents a new potential avenue for therapeutic intervention during the transition period.

Pain-Associated Neural Plasticity in the Parabrachial to Central Amygdala Circuit : Pain Changes the Brain, and the Brain Changes the Pain.

In addition to the canonical spino-thalamo-cortical pathway, lines of recently accumulated anatomical and physiological evidence suggest that projections originating in nociception-specific neurons in lamina I of the dorsal horn or the spinal nucleus of the trigeminal nerve to the lateral parabrachial nucleus (LPB) and then to the central amygdala (CeA) play essential roles in the nociception-emotion link and its tightening in chronic pain. With recent advances in the artificial manipulation of central neuronal activity, such as those with optogenetics, it is now possible to address many unanswered questions regarding the molecular and cellular mechanisms underlying the plastic changes in this pathway and their role in the pain chronification process.

Contributions of the Central Extended Amygdala to Fear and Anxiety.

It is widely thought that phasic and sustained responses to threat reflect dissociable circuits centered on the central nucleus of the amygdala (Ce) and the bed nucleus of the stria terminalis (BST), the two major subdivisions of the central extended amygdala. Early versions of this hypothesis remain highly influential and have been incorporated into the National Institute of Mental Health Research Research Domain Criteria framework. However, new observations encourage a different perspective. Anatomical studies show that the Ce and BST form a tightly interconnected unit, where different kinds of threat-relevant information can be integrated and used to assemble states of fear and anxiety. Imaging studies in humans and monkeys show that the Ce and BST exhibit similar functional profiles. Both regions are sensitive to a range of aversive challenges, including uncertain or temporally remote threat; both covary with concurrent signs and symptoms of fear and anxiety; both show phasic responses to short-lived threat; and both show heightened activity during sustained exposure to diffusely threatening contexts. Mechanistic studies demonstrate that both regions can control the expression of fear and anxiety during sustained exposure to diffuse threat. These observations compel a reconsideration of the central extended amygdala's contributions to fear and anxiety and its role in neuropsychiatric disease.

Effects of unilatral- and bilateral inhibition of rostral ventral tegmental area and central nucleus of amygdala on morphine-induced place conditioning in male Wistar rat.

The rostral ventral tegmental area (VTAR) and central nucleus of amygdala (CeA) are considered the main regions for induction of psychological dependence on abused drugs, such as morphine. The main aim of this study was to investigate the transient inhibition of each right and left side as well as both sides of the VTAR and the CeA by lidocaine (2%) on morphine reward properties using the conditioned place preference (CPP) method. Male Wistar rats (250±20 g) 7 days after recovery from surgery and cannulation were conditioned to morphine (7.5 mg/kg) in CPP apparatus. Five minutes before morphine injection in conditioning phase, lidocaine was administered either uni- or bilaterally into the VTAR (0.25 µL/site) or CeA (0.5 µL/site). The results revealed that lidocaine administration into the left side, but not the right side of the VTAR and the CeA reduced morphine CPP significantly. The reduction was potentiated when lidocaine was injected into both sides of the VTAR and the CeA. The number of compartment crossings was reduced when lidocaine was injected into both sides of the VTAR and the CeA as well as the left side. Rearing was reduced when lidocaine was injected into the right, but not the left side of the VTAR. Sniffing and rearing increased when animals received lidocaine in the right side and reduced in the group that received lidocaine in the left side of the CeA. It was concluded that the right and the left side of VTAR and the CeA play different roles in morphine-induced activity and reward.

Synaptic and network consequences of monosynaptic nociceptive inputs of parabrachial nucleus origin in the central amygdala.

A large majority of neurons in the superficial layer of the dorsal horn projects to the lateral parabrachial nucleus (LPB). LPB neurons then project to the capsular part of the central amygdala (CeA; CeC), a key structure underlying the nociception-emotion link. LPB-CeC synaptic transmission is enhanced in various pain models by using electrical stimulation of putative fibers of LPB origin in brain slices. However, this approach has limitations for examining direct monosynaptic connections devoid of directly stimulating fibers from other structures and local GABAergic neurons. To overcome these limitations, we infected the LPB of rats with an adeno-associated virus vector expressing channelrhodopsin-2 and prepared coronal and horizontal brain slices containing the amygdala. We found that blue light stimulation resulted in monosynaptic excitatory postsynaptic currents (EPSCs), with very small latency fluctuations, followed by a large polysynaptic inhibitory postsynaptic current in CeC neurons, regardless of the firing pattern type. Intraplantar formalin injection at 24 h before slice preparation significantly increased EPSC amplitude in late firing-type CeC neurons. These results indicate that direct monosynaptic glutamatergic inputs from the LPB not only excite CeC neurons but also regulate CeA network signaling through robust feed-forward inhibition, which is under plastic modulation in response to persistent inflammatory pain.

Parvalbumin Interneurons of Central Amygdala Regulate the Negative Affective States and the Expression of Corticotrophin-Releasing Hormone During Morphine Withdrawal.

BACKGROUND: The central nucleus of the amygdala (CeA) is a crucial component of the neuronal circuitry mediating aversive emotion. Its role in the negative affective states during drug withdrawal includes changes in opioidergic, GABAergic, and corticotropin-releasing factor neurotransmission. However, the modulation of the neurobiological interconnectivity in the CeA and its effects in the negative reinforcement of drug dependents are poorly understood. METHOD: We performed electrophysiological recordings to assess the membrane excitability of parvalbumin (PV)+ interneurons in the CeA during chronic morphine withdrawal. We tested the morphine withdrawal-induced negative affective states, such as the aversive (assessed by conditioned place aversion), anxiety (assessed by elevated plus maze), and anhedonic-like (assessed by saccharin preference test) behaviors, as well as the mRNA level of corticotropin-releasing hormone (CRH) via optogenetic inhibition or activation of PV+ interneurons in the CeA. RESULT: Chronic morphine withdrawal increased the firing rate of CeA PV+ interneurons. Optogenetic inhibition of the activity of CeA PV+ interneurons attenuated the morphine withdrawal-induced negative affective states, such as the aversive, anxiety, and anhedonic-like behaviors, while direct activation of CeA PV+ interneurons could trigger those negative affective-like behaviors. Optogenetic inhibition of the CeA PV+ interneurons during the morphine withdrawal significantly attenuated the elevated CRH mRNA level in the CeA. CONCLUSION: The activity of PV+ interneurons in the CeA was up-regulated during chronic morphine withdrawal. The activation of PV+ interneurons during morphine withdrawal was crucial for the induction of the negative emotion and the up-regulation of CRH mRNA levels in the CeA.

Chronic ethanol exposure decreases CB1 receptor function at GABAergic synapses in the rat central amygdala.

The endogenous cannabinoids (eCBs) influence the acute response to ethanol and the development of tolerance, dependence and relapse. Chronic alcohol exposure alters eCB levels and Type 1 cannabinoid receptor (CB1 ) expression and function in brain regions associated with addiction. CB1 inhibits GABA release, and GABAergic dysregulation in the central nucleus of the amygdala (CeA) is critical in the transition to alcohol dependence. We investigated possible disruptions in CB1 signaling of rat CeA GABAergic transmission following intermittent ethanol exposure. In the CeA of alcohol-naive rats, CB1 agonist WIN 55,212-2 (WIN) decreased the frequency of spontaneous and miniature GABAA receptor-mediated inhibitory postsynaptic currents (s/mIPSCs). This effect was prevented by CB1 antagonism, but not Type 2 cannabinoid receptor (CB2 ) antagonism. After 2-3 weeks of intermittent ethanol exposure, these WIN inhibitory effects were attenuated, suggesting ethanol-induced impairments in CB1 function. The CB1 antagonist AM251 revealed a tonic eCB/CB1 control of GABAergic transmission in the alcohol-naive CeA that was occluded by calcium chelation in the postsynaptic cell. Chronic ethanol exposure abolished this tonic CB1 influence on mIPSC, but not sIPSC, frequency. Finally, acute ethanol increased CeA GABA release in both naive and ethanol-exposed rats. Although CB1 activation prevented this effect, the AM251- and ethanol-induced GABA release were additive, ruling out a direct participation of CB1 signaling in the ethanol effect. Collectively, these observations demonstrate an important CB1 influence on CeA GABAergic transmission and indicate that the CeA is particularly sensitive to alcohol-induced disruptions of CB1 signaling.

Evolutionarily conserved prefrontal-amygdalar dysfunction in early-life anxiety.

Some individuals are endowed with a biology that renders them more reactive to novelty and potential threat. When extreme, this anxious temperament (AT) confers elevated risk for the development of anxiety, depression and substance abuse. These disorders are highly prevalent, debilitating and can be challenging to treat. The high-risk AT phenotype is expressed similarly in children and young monkeys and mechanistic work demonstrates that the central (Ce) nucleus of the amygdala is an important substrate. Although it is widely believed that the flow of information across the structural network connecting the Ce nucleus to other brain regions underlies primates' capacity for flexibly regulating anxiety, the functional architecture of this network has remained poorly understood. Here we used functional magnetic resonance imaging (fMRI) in anesthetized young monkeys and quietly resting children with anxiety disorders to identify an evolutionarily conserved pattern of functional connectivity relevant to early-life anxiety. Across primate species and levels of awareness, reduced functional connectivity between the dorsolateral prefrontal cortex, a region thought to play a central role in the control of cognition and emotion, and the Ce nucleus was associated with increased anxiety assessed outside the scanner. Importantly, high-resolution 18-fluorodeoxyglucose positron emission tomography imaging provided evidence that elevated Ce nucleus metabolism statistically mediates the association between prefrontal-amygdalar connectivity and elevated anxiety. These results provide new clues about the brain network underlying extreme early-life anxiety and set the stage for mechanistic work aimed at developing improved interventions for pediatric anxiety.