Chemogenetic Manipulation of Amygdala Kappa Opioid Receptor Neurons Modulates Amygdala Neuronal Activity and Neuropathic Pain Behaviors.

Neuroplasticity in the central nucleus of the amygdala (CeA) plays a key role in the modulation of pain and its aversive component. The dynorphin/kappa opioid receptor (KOR) system in the amygdala is critical for averse-affective behaviors in pain conditions, but its mechanisms are not well understood. Here, we used chemogenetic manipulations of amygdala KOR-expressing neurons to analyze the behavioral consequences in a chronic neuropathic pain model. For the chemogenetic inhibition or activation of KOR neurons in the CeA, a Cre-inducible viral vector encoding Gi-DREADD (hM4Di) or Gq-DREADD (hM3Dq) was injected stereotaxically into the right CeA of transgenic KOR-Cre mice. The chemogenetic inhibition of KOR neurons expressing hM4Di with a selective DREADD actuator (deschloroclozapine, DCZ) in sham control mice significantly decreased inhibitory transmission, resulting in a shift of inhibition/excitation balance to promote excitation and induced pain behaviors. The chemogenetic activation of KOR neurons expressing hM3Dq with DCZ in neuropathic mice significantly increased inhibitory transmission, decreased excitability, and decreased neuropathic pain behaviors. These data suggest that amygdala KOR neurons modulate pain behaviors by exerting an inhibitory tone on downstream CeA neurons. Therefore, activation of these interneurons or blockade of inhibitory KOR signaling in these neurons could restore control of amygdala output and mitigate pain.

Morphological heterogeneity of neurons in the human central amygdaloid nucleus.

The central amygdaloid nucleus (CeA) has an ancient phylogenetic development and functions relevant for animal survival. Local cells receive intrinsic amygdaloidal information that codes emotional stimuli of fear, integrate them, and send cortical and subcortical output projections that prompt rapid visceral and social behavior responses. We aimed to describe the morphology of the neurons that compose the human CeA (N = 8 adult men). Cells within CeA coronal borders were identified using the thionine staining and were further analyzed using the "single-section" Golgi method followed by open-source software procedures for two-dimensional and three-dimensional image reconstructions. Our results evidenced varied neuronal cell body features, number and thickness of primary shafts, dendritic branching patterns, and density and shape of dendritic spines. Based on these criteria, we propose the existence of 12 morphologically different spiny neurons in the human CeA and discuss the variability in the dendritic architecture within cellular types, including likely interneurons. Some dendritic shafts were long and straight, displayed few collaterals, and had planar radiation within the coronal neuropil volume. Most of the sampled neurons showed a few to moderate density of small stubby/wide spines. Long spines (thin and mushroom) were observed occasionally. These novel data address the synaptic processing and plasticity in the human CeA. Our morphological description can be combined with further transcriptomic, immunohistochemical, and electrophysiological/connectional approaches. It serves also to investigate how neurons are altered in neurological and psychiatric disorders with hindered emotional perception, in anxiety, following atrophy in schizophrenia, and along different stages of Alzheimer's disease.

Social buffering in rats reduces fear by oxytocin triggering sustained changes in central amygdala neuronal activity.

The presence of a companion can reduce fear, but the neural mechanisms underlying this social buffering of fear are incompletely known. We studied social buffering of fear in male and female, and its encoding in the amygdala of male, auditory fear-conditioned rats. Pharmacological, opto,- and/or chemogenetic interventions showed that oxytocin signaling from hypothalamus-to-central amygdala projections underlied fear reduction acutely with a companion and social buffering retention 24 h later without a companion. Single-unit recordings with optetrodes in the central amygdala revealed fear-encoding neurons (showing increased conditioned stimulus-responses after fear conditioning) inhibited by social buffering and blue light-stimulated oxytocinergic hypothalamic projections. Other central amygdala neurons showed baseline activity enhanced by blue light and companion exposure, with increased conditioned stimulus responses that persisted without the companion. Social buffering of fear thus switches the conditioned stimulus from encoding "fear" to "safety" by oxytocin-mediated recruitment of a distinct group of central amygdala "buffer neurons".

Neuronal nicotinic acetylcholine receptor of the central amygdala modulates the ethanol-induced tolerance to anxiolysis and withdrawal-induced anxiety in male rats.

The nicotine acetylcholinergic receptor (nAchR) in the central nucleus of the amygdala (CeA) is known to modulate anxiety traits as well as ethanol-induced behavioral effects. Therefore, the present study investigated the role of CeA nAChR in the tolerance to ethanol anxiolysis and withdrawal-induced anxiety-related effects in rats on elevated plus maze (EPM). To develop ethanol dependence, rats were given free access to an ethanol-containing liquid diet for 10 days. To assess the development of tolerance, separate groups of rats were challenged with ethanol (2 g/kg, i.p.) on days 1, 3, 5, 7 and 10 during the period of ethanol exposure, followed by an EPM assessment. Moreover, expression of ethanol withdrawal was induced after switching ethanol-dependent rats to a liquid diet on day 11, and withdrawal-induced anxiety-like behavior was noted at different post-withdrawal time points using the EPM test. The ethanol-dependent rats were pretreated with intra-CeA (i.CeA) (bilateral) injections of nicotine (0.25 µg/rat) or mecamylamine (MEC) (5 ng/rat) before the challenge dose of ethanol on subthreshold tolerance on the 5th day or on peak tolerance day, that is, 7th or 10th, and before assessment of postwithdrawal anxiety on the 11th day on EPM. Bilateral i.CeA preadministration of nicotine before the challenge dose of ethanol on days 5, 7 and 10 exhibited enhanced tolerance, while injection of MEC, completely mitigated the tolerance to the ethanol-induced antianxiety effect. On the other hand, ethanol-withdrawn rats pretreated i.CeA with nicotine exacerbated while pretreatment with MEC, alleviated the ethanol withdrawal-induced anxiety on all time points. Thus, the present investigation indicates that stimulation of nAChR in CeA negatively modulates the ethanol-induced chronic behavioral effects on anxiety in rats. It is proposed that nAChR antagonists might be useful in the treatment of alcohol use disorder and ethanol withdrawal-related anxiety-like behavior.

Alteration of serotonin release response in the central nucleus of the amygdala to noxious and non-noxious mechanical stimulation in a neuropathic pain model rat.

Previously, we found that serotonin (5-HT) release in the central nucleus of the amygdala (CeA) of anesthetized rats decreases in response to innocuous stroking of the skin, irrespective of stimulus laterality, but increases in response to noxious pinching applied to a hindlimb contralateral to the 5-HT measurement site. The aim of the present study was to determine whether intra-CeA 5-HT release responses to cutaneous stimulation were altered in an animal model of neuropathic pain induced by ligation of the left L5 spinal nerve. In anesthetized neuropathic pain model rats, stroking of the left hindlimb increased 5-HT release in the CeA, whereas stroking of the right hindlimb decreased it. Meanwhile, pinching of the left hindlimb increased intra-CeA 5-HT release irrespective of stimulus laterality. In conclusion, the present study demonstrated that intra-CeA 5-HT release responses to cutaneous stimulation are altered in an animal model of neuropathic pain.

Nausea-induced suppression of feeding is mediated by central amygdala Dlk1-expressing neurons.

The motivation to eat is suppressed by satiety and aversive stimuli such as nausea. The neural circuit mechanisms of appetite suppression by nausea are not well understood. Pkcδ neurons in the lateral subdivision of the central amygdala (CeA) suppress feeding in response to satiety signals and nausea. Here, we characterized neurons enriched in the medial subdivision (CeM) of the CeA marked by expression of Dlk1. CeADlk1 neurons are activated by nausea, but not satiety, and specifically suppress feeding induced by nausea. Artificial activation of CeADlk1 neurons suppresses drinking and social interactions, suggesting a broader function in attenuating motivational behavior. CeADlk1 neurons form projections to many brain regions and exert their anorexigenic activity by inhibition of neurons of the parabrachial nucleus. CeADlk1 neurons are inhibited by appetitive CeA neurons, but also receive long-range monosynaptic inputs from multiple brain regions. Our results illustrate a CeA circuit that regulates nausea-induced feeding suppression.

Development of activity-based anorexia requires PKC-δ neurons in two central extended amygdala nuclei.

Anorexia nervosa (AN) is a serious psychiatric disease, but the neural mechanisms underlying its development are unclear. A subpopulation of amygdala neurons, marked by expression of protein kinase C-delta (PKC-δ), has previously been shown to regulate diverse anorexigenic signals. Here, we demonstrate that these neurons regulate development of activity-based anorexia (ABA), a common animal model for AN. PKC-δ neurons are located in two nuclei of the central extended amygdala (EAc): the central nucleus (CeA) and oval region of the bed nucleus of the stria terminalis (ovBNST). Simultaneous ablation of CeAPKC-δ and ovBNSTPKC-δ neurons prevents ABA, but ablating PKC-δ neurons in the CeA or ovBNST alone is not sufficient. Correspondingly, PKC-δ neurons in both nuclei show increased activity with ABA development. Our study shows how neurons in the amygdala regulate ABA by impacting both feeding and wheel activity behaviors and support a complex heterogeneous etiology of AN.

Presynaptic inhibition of excitatory synaptic transmission from the calcitonin gene-related peptide-containing parabrachial neurons to the central amygdala in mice - unexpected influence of systemic inflammation thereon.

The monosynaptic connection from the lateral parabrachial nucleus (LPB) to the central amygdala (CeA) serves as a fundamental pathway for transmitting nociceptive signals to the brain. The LPB receives nociceptive information from the dorsal horn and spinal trigeminal nucleus and sends it to the "nociceptive" CeA, which modulates pain-associated emotions and nociceptive sensitivity. To elucidate the role of densely expressed mu-opioid receptors (MORs) within this pathway, we investigated the effects of exogenously applied opioids on LPB-CeA synaptic transmission, employing optogenetics in mice expressing channelrhodopsin-2 in LPB neurons with calcitonin gene-related peptide (CGRP). A MOR agonist ([D-Ala2,N-Me-Phe4,Glycinol5]-enkephalin, DAMGO) significantly reduced the amplitude of light-evoked excitatory postsynaptic currents (leEPSCs), in a manner negatively correlated with an increase in the paired-pulse ratio. An antagonist of MORs significantly attenuated these effects. Notably, this antagonist significantly increased leEPSC amplitude when applied alone, an effect further amplified in mice subjected to lipopolysaccharide injection 2 h before brain isolation, yet not observed at the 24-h mark. We conclude that opioids could shut off the ascending nociceptive signal at the LPB-CeA synapse through presynaptic mechanisms. Moreover, this gating process might be modulated by endogenous opioids, and the innate immune system influences this modulation.

Rhesus infant nervous temperament predicts peri-adolescent central amygdala metabolism & behavioral inhibition measured by a machine-learning approach.

Anxiety disorders affect millions of people worldwide and impair health, happiness, and productivity on a massive scale. Developmental research points to a connection between early-life behavioral inhibition and the eventual development of these disorders. Our group has previously shown that measures of behavioral inhibition in young rhesus monkeys (Macaca mulatta) predict anxiety-like behavior later in life. In recent years, clinical and basic researchers have implicated the central extended amygdala (EAc)-a neuroanatomical concept that includes the central nucleus of the amygdala (Ce) and the bed nucleus of the stria terminalis (BST)-as a key neural substrate for the expression of anxious and inhibited behavior. An improved understanding of how early-life behavioral inhibition relates to an increased lifetime risk of anxiety disorders-and how this relationship is mediated by alterations in the EAc-could lead to improved treatments and preventive strategies. In this study, we explored the relationships between infant behavioral inhibition and peri-adolescent defensive behavior and brain metabolism in 18 female rhesus monkeys. We coupled a mildly threatening behavioral assay with concurrent multimodal neuroimaging, and related those findings to various measures of infant temperament. To score the behavioral assay, we developed and validated UC-Freeze, a semi-automated machine-learning (ML) tool that uses unsupervised clustering to quantify freezing. Consistent with previous work, we found that heightened Ce metabolism predicted elevated defensive behavior (i.e., more freezing) in the presence of an unfamiliar human intruder. Although we found no link between infant-inhibited temperament and peri-adolescent EAc metabolism or defensive behavior, we did identify infant nervous temperament as a significant predictor of peri-adolescent defensive behavior. Our findings suggest a connection between infant nervous temperament and the eventual development of anxiety and depressive disorders. Moreover, our approach highlights the potential for ML tools to augment existing behavioral neuroscience methods.

Activation of the CXCR4 Receptor by Chemokine CXCL12 Increases the Excitability of Neurons in the Rat Central Amygdala.

Primarily regarded as immune proteins, chemokines are emerging as a family of molecules serving neuromodulatory functions in the developing and adult brain. Among them, CXCL12 is constitutively and widely expressed in the CNS, where it was shown to act on cellular, synaptic, network, and behavioral levels. Its receptor, CXCR4, is abundant in the amygdala, a brain structure involved in pathophysiology of anxiety disorders. Dysregulation of CXCL12/CXCR4 signaling has been implicated in anxiety-related behaviors. Here we demonstrate that exogenous CXCL12 at 2 nM but not at 5 nM increased neuronal excitability in the lateral division of the rat central amygdala (CeL) which was evident in the Late-Firing but not Regular-Spiking neurons. These effects were blocked by AMD3100, a CXCR4 antagonist. Moreover, CXCL12 increased the excitability of the neurons of the basolateral amygdala (BLA) that is known to project to the CeL. However, CXCL12 increased neither the spontaneous excitatory nor spontaneous inhibitory synaptic transmission in the CeL. In summary, the data reveal specific activation of Late-Firing CeL cells along with BLA neurons by CXCL12 and suggest that this chemokine may alter information processing by the amygdala that likely contributes to anxiety and fear conditioning.

Somatosensory cortex and central amygdala regulate neuropathic pain-mediated peripheral immune response via vagal projections to the spleen.

Pain involves neuroimmune crosstalk, but the mechanisms of this remain unclear. Here we showed that the splenic T helper 2 (TH2) immune cell response is differentially regulated in male mice with acute versus chronic neuropathic pain and that acetylcholinergic neurons in the dorsal motor nucleus of the vagus (AChDMV) directly innervate the spleen. Combined in vivo recording and immune cell profiling revealed the following two distinct circuits involved in pain-mediated peripheral TH2 immune response: glutamatergic neurons in the primary somatosensory cortex (GluS1HL)→AChDMV→spleen circuit and GABAergic neurons in the central nucleus of the amygdala (GABACeA)→AChDMV→spleen circuit. The acute pain condition elicits increased excitation from GluS1HL neurons to spleen-projecting AChDMV neurons and increased the proportion of splenic TH2 immune cells. The chronic pain condition increased inhibition from GABACeA neurons to spleen-projecting AChDMV neurons and decreased splenic TH2 immune cells. Our study thus demonstrates how the brain encodes pain-state-specific immune responses in the spleen.

Top-down control of flight by a non-canonical cortico-amygdala pathway.

Survival requires the selection of appropriate behaviour in response to threats, and dysregulated defensive reactions are associated with psychiatric illnesses such as post-traumatic stress and panic disorder1. Threat-induced behaviours, including freezing and flight, are controlled by neuronal circuits in the central amygdala (CeA)2; however, the source of neuronal excitation of the CeA that contributes to high-intensity defensive responses is unknown. Here we used a combination of neuroanatomical mapping, in vivo calcium imaging, functional manipulations and electrophysiology to characterize a previously unknown projection from the dorsal peduncular (DP) prefrontal cortex to the CeA. DP-to-CeA neurons are glutamatergic and specifically target the medial CeA, the main amygdalar output nucleus mediating conditioned responses to threat. Using a behavioural paradigm that elicits both conditioned freezing and flight, we found that CeA-projecting DP neurons are activated by high-intensity threats in a context-dependent manner. Functional manipulations revealed that the DP-to-CeA pathway is necessary and sufficient for both avoidance behaviour and flight. Furthermore, we found that DP neurons synapse onto neurons within the medial CeA that project to midbrain flight centres. These results elucidate a non-canonical top-down pathway regulating defensive responses.

Suppressing fear in the presence of a safety cue requires infralimbic cortical signaling to central amygdala.

Stressful events can have lasting and impactful effects on behavior, especially by disrupting normal regulation of fear and reward processing. Accurate discrimination among environmental cues predicting threat, safety or reward adaptively guides behavior. Post-traumatic stress disorder (PTSD) represents a condition in which maladaptive fear persists in response to explicit safety-predictive cues that coincide with previously learned threat cues, but without threat being present. Since both the infralimbic cortex (IL) and amygdala have each been shown to be important for fear regulation to safety cues, we tested the necessity of specific IL projections to the basolateral amygdala (BLA) or central amygdala (CeA) during safety recall. Male Long Evans rats were used since prior work showed female Long Evans rats did not acquire the safety discrimination task used in this study. Here, we show the infralimbic projection to the central amygdala was necessary for suppressing fear cue-induced freezing in the presence of a learned safety cue, and the projection to the basolateral amygdala was not. The loss of discriminative fear regulation seen specifically during IL->CeA inhibition is similar to the behavioral disruption seen in PTSD individuals that fail to regulate fear in the presence of a safety cue.

Analysis of Monosynaptic Inputs to Thalamic Paraventricular Nucleus Neurons Innervating the Shell of the Nucleus Accumbens and Central Extended Amygdala.

The paraventricular nucleus of the thalamus (PVT) sends dense projections to the shell of the nucleus accumbens (NAcSh), dorsolateral region of the bed nucleus of the stria terminalis (BSTDL) and the lateral region of central nucleus of the amygdala (CeL). Projection specific modulation of these pathways has been shown to regulate appetitive and aversive behavioral responses. The present investigation applied an intersectional monosynaptic rabies tracing approach to quantify the brain-wide sources of afferent input to PVT neurons that primarily project to the NAcSh, BSTDL and CeL. The results demonstrate that these projection neurons receive monosynaptic input from similar brain regions. The prefrontal cortex and the ventral subiculum of the hippocampus were major sources of input to the PVT projection neurons. In addition, the lateral septal nucleus, thalamic reticular nucleus and the hypothalamic medial preoptic area, dorsomedial, ventromedial, and arcuate nuclei were sources of input. The subfornical organ, parasubthalamic nucleus, periaqueductal gray matter, lateral parabrachial nucleus, and nucleus of the solitary tract were consistent but lesser sources of input. This input-output relationship is consistent with recent observations that PVT neurons have axons that bifurcate extensively to divergently innervate the NAcSh, BSTDL and CeL.

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.

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

BACKGROUND: Exposure 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. METHODS: We 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. RESULTS: AIE 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. CONCLUSIONS: Our 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.

Subpopulations of corticotropin-releasing factor containing neurons and internal circuits in the chicken central extended amygdala.

In mammals, the central extended amygdala is critical for the regulation of the stress response. This regulation is extremely complex, involving multiple subpopulations of GABAergic neurons and complex networks of internal and external connections. Two neuron subpopulations expressing corticotropin-releasing factor (CRF), located in the central amygdala and the lateral bed nucleus of the stria terminalis (BSTL), play a key role in the long-term component of fear learning and in sustained fear responses akin to anxiety. Very little is known about the regulation of stress by the amygdala in nonmammals, hindering efforts for trying to improve animal welfare. In birds, one of the major problems relates to the high evolutionary divergence of the telencephalon, where the amygdala is located. In the present study, we aimed to investigate the presence of CRF neurons of the central extended amygdala in chicken and the local connections within this region. We found two major subpopulations of CRF cells in BSTL and the medial capsular central amygdala of chicken. Based on multiple labeling of CRF mRNA with different developmental transcription factors, all CRF neurons seem to originate within the telencephalon since they express Foxg1, and there are two subtypes with different embryonic origins that express Islet1 or Pax6. In addition, we demonstrated direct projections from Pax6 cells of the capsular central amygdala to BSTL and the oval central amygdala. We also found projections from Islet1 cells of the oval central amygdala to BSTL, which may constitute an indirect pathway for the regulation of BSTL output cells. Part of these projections may be mediated by CRF cells, in agreement with the expression of CRF receptors in both Ceov and BSTL. Our results show a complex organization of the central extended amygdala in chicken and open new venues for studying how different cells and circuits regulate stress in these animals.

Maternal separation early in life induces excessive activity of the central amygdala related to abnormal aggression.

Epidemiological studies have indicated that child maltreatment, such as neglect, is a risk factor of escalated aggression, potentially leading to delinquency and violent crime in the future. However, little is known about the mechanisms by which an early adverse environment may later cause violent behavior. In this study, we aimed to thoroughly examine the association between aggression against conspecific animals and the activity of amygdala subnuclei using the maternal separation (MS) model, which is a common model of early life stress. In the MS group, pups of Sprague-Dawley rats were separated from their dam during postnatal days 2-20 (twice a day, 3 h each). We only included 9-week-old male offspring for each analysis and compared the MS group with the mother-reared control group; both groups were raised by the same dam during postnatal days 2-20. The results revealed that the MS group exhibited higher aggression and excessive activity of only the central amygdala (CeA) among the amygdala subnuclei during the aggressive behavior test. Moreover, a significant positive correlation was observed between higher aggression and CeA activation. While CeA activity is known to be involved in hunting behavior for prey, some previous studies have also indicated a relationship between CeA and intraspecific aggression. It remains unclear, however, whether excessive CeA activity directly induces intraspecific aggression. Therefore, we stimulated the CeA using optogenetics with 8-week-old rats to clarify the relationship between intraspecific aggression and CeA activity. Notably, CeA activation resulted in higher aggression, even when the opponent was a conspecific animal. In particular, bilateral CeA activation resulted in more severe displays of aggressive behavior than necessary, such as biting a surrendered opponent. These findings suggest that an adverse environment during early development intensifies aggression through excessive CeA activation, which can increase the risk of escalating to violent behavior in the future.