Neurons In Central Nucleus Of Amygdala Research Articles

Ethanol Metabolite, Acetate, Increases Excitability of the Central Nucleus of Amygdala Neurons through Activation of NMDA Receptors.

The central nucleus of the amygdala (CeA) is a key brain region involved in emotional and stressor responses due to its many projections to autonomic regulatory centers. It is also a primary site of action from ethanol consumption. However, the influence of active metabolites of ethanol such as acetate on the CeA neural circuitry has yet to be elucidated. Here, we investigated the effect of acetate on CeA neurons with the axon projecting to the rostral ventrolateral medulla (CeA-RVLM), as well as quantified cytosolic calcium responses in primary neuronal cultures. Whole-cell patch-clamp recordings in brain slices containing autonomic CeA-RVLM neurons revealed a dose-dependent increase in neuronal excitability in response to acetate. N-Methyl-d-aspartate receptor (NMDAR) antagonists suppressed the acetate-induced increase in CeA-RVLM neuronal excitability and memantine suppressed the direct activation of NMDAR-dependent inward currents by acetate in brain slices. We observed that acetate increased cytosolic Ca2+ in a time-dependent manner in primary neuronal cell cultures. The acetate enhancement of calcium signaling was abolished by memantine. Computational modeling of acetic acid at NMDAR/NR1 glutamatergic and glycinergic sites suggests potential active site interactions. These findings suggest that within the CeA, acetate is excitatory at least partially through activation of NMDAR, which may underlie the impact of ethanol consumption on autonomic circuitry.

Kappa Opioid Receptor Blockade in the Amygdala Mitigates Pain Like-Behaviors by Inhibiting Corticotropin Releasing Factor Neurons in a Rat Model of Functional Pain.

Functional pain syndromes (FPS) occur in the absence of identifiable tissue injury or noxious events and include conditions such as migraine, fibromyalgia, and others. Stressors are very common triggers of pain attacks in various FPS conditions. It has been recently demonstrated that kappa opioid receptors (KOR) in the central nucleus of amygdala (CeA) contribute to FPS conditions, but underlying mechanisms remain unclear. The CeA is rich in KOR and encompasses major output pathways involving extra-amygdalar projections of corticotropin releasing factor (CRF) expressing neurons. Here we tested the hypothesis that KOR blockade in the CeA in a rat model of FPS reduces pain-like and nocifensive behaviors by restoring inhibition of CeA-CRF neurons. Intra-CeA administration of a KOR antagonist (nor-BNI) decreased mechanical hypersensitivity and affective and anxiety-like behaviors in a stress-induced FPS model. In systems electrophysiology experiments in anesthetized rats, intra-CeA application of nor-BNI reduced spontaneous firing and responsiveness of CeA neurons to peripheral stimulation. In brain slice whole-cell patch-clamp recordings, nor-BNI increased feedforward inhibitory transmission evoked by optogenetic and electrical stimulation of parabrachial afferents, but had no effect on monosynaptic excitatory transmission. Nor-BNI decreased frequency, but not amplitude, of spontaneous inhibitory synaptic currents, suggesting a presynaptic action. Blocking KOR receptors in stress-induced FPS conditions may therefore represent a novel therapeutic strategy.

Locus coeruleus anchors a trisynaptic circuit controlling fear-induced suppression of feeding

The circuit mechanisms underlying fear-induced suppression of feeding are poorly understood. To help fill this gap, mice were fear conditioned, and the resulting changes in synaptic connectivity among the locus coeruleus (LC), the parabrachial nucleus (PBN), and the central nucleus of amygdala (CeA)-all of which are implicated in fear and feeding-were studied. LC neurons co-released noradrenaline and glutamate to excite PBN neurons and suppress feeding. LC neurons also suppressed inhibitory input to PBN neurons by inducing heterosynaptic, endocannabinoid-dependent, long-term depression of CeA synapses. Blocking or knocking down endocannabinoid receptors in CeA neurons prevented fear-induced depression of CeA synaptic transmission and fear-induced suppression of feeding. Altogether, these studies demonstrate that LC neurons play a pivotal role in modulating the circuitry that underlies fear-induced suppression of feeding, pointing to new ways of alleviating stress-induced eating disorders.

Neural Circuit Mechanism Underlying the Feeding Controlled by Insula-Central Amygdala Pathway.

SummaryThe Central nucleus of amygdala (CeA) contains distinct populations of neurons that play opposing roles in feeding. The circuit mechanism of how CeA neurons process information sent from their upstream inputs to regulate feeding is still unclear. Here we show that activation of the neural pathway projecting from insular cortex neurons to the CeA suppresses food intake. Surprisingly, we find that the inputs from insular cortex form excitatory connections with similar strength to all types of CeA neurons. To reconcile this puzzling result, and previous findings, we developed a conductance-based dynamical systems model for the CeA neuronal network. Computer simulations showed that both the intrinsic electrophysiological properties of individual CeA neurons and the overall synaptic organization of the CeA circuit play a functionally significant role in shaping CeA neural dynamics. We successfully identified a specific CeA circuit structure that reproduces the desired circuit output consistent with existing experimentally observed feeding behaviors.

Sleep Regulation by Neurotensinergic Neurons in a Thalamo-Amygdala Circuit

A crucial step in understanding the sleep-control mechanism is to identify sleep neurons. Through systematic anatomical screening followed by functional testing, we identified two sleep-promoting neuronal populations along a thalamo-amygdala pathway, both expressing neurotensin (NTS). Rabies-mediated monosynaptic retrograde tracing identified the central nucleus of amygdala (CeA) as a major source of GABAergic inputs to multiple wake-promoting populations; gene profiling revealed NTS as a prominent marker for these CeA neurons. Optogenetic activation and inactivation of NTS-expressing CeA neurons promoted and suppressed non-REM (NREM) sleep, respectively, and optrode recording showed they are sleep active. Further tracing showed that CeA GABAergic NTS neurons are innervated by glutamatergic NTS neurons in a posterior thalamic region, which also promote NREM sleep. CRISPR/Cas9-mediated NTS knockdown in either the thalamic or CeA neurons greatly reduced their sleep-promoting effect. These results reveal a novel thalamo-amygdala circuit for sleep generation in which NTS signaling is essential for both the upstream glutamatergic and downstream GABAergic neurons.

Optogenetic Long-Term Depression Induction in the PVT-CeL Circuitry Mediates Decreased Fear Memory.

The dysregulation of fear learning and abnormal activities of cerebral networks may contribute to the etiologies of anxiety disorders. Although it has been proposed that decreased activity in the paraventricular nucleus of the thalamus (PVT) to the lateral central nucleus of amygdala (CeL) pathway could induce an attenuation of learned fear, no study has shown the effect of the direct optogenetic activation of PVT projecting CeL neurons in vivo on unconditioned fear-related behaviors or learned fear expression. The mechanisms that control the neuronal activity of the PVT-CeL pathway involved in anxiety are rare. Here, we found that CeL neurons have varied responses to optogenetic excitation of PVT terminals in the CeL: neurons with relative high excitability(~ 30%), neurons with relative low excitability(~60%), and neurons with no excitability (~ 10%). We next explored the role of the PVT-CeL pathway in unconditioned and conditioned fear-related behaviors by using optogenetics and anxiety assays in freely moving mice. We observed that temporally precise optogenetic activation of the CeL-projecting PVT neurons had no effect on unconditioned fear-related behaviors on the elevated plus maze test and the open field test. But optogenetic activation of the CeL-projecting PVT neurons increased conditioned fear expression. We then found that optogenetic long-term depression (LTD) induction in the CeL receiving PVT afferents effectively exerted a persistent attenuation of learned fear. The percentage of neurons with relative high excitability was decreased by the LTD induction, and the percentage of neurons with relative low excitability was increased by the LTD induction. Taking these results together, we identify that increased activity of the PVT-CeL pathway could lead to as excessive learned fear. The CeL neurons with relative high responses to the photo-stimulation of PVT afferents in the CeL may be the key neurons that regulate the output of learned fear expression. Our optogenetic LTD protocol may inspire the development of novel treatments for anxiety disorders involving deep brain stimulation to induce plasticity at relevant brain areas.

Analgesic Effects of Duloxetine on Formalin-Induced Hyperalgesia and Its Underlying Mechanisms in the CeA.

In rodents, the amygdala has been proposed to serve as a key center for the nociceptive perception. Previous studies have shown that extracellular signal-regulated kinase (ERK) signaling cascade in the central nucleus of amygdala (CeA) played a functional role in inflammation-induced peripheral hypersensitivity. Duloxetine (DUL), a serotonin and noradrenaline reuptake inhibitor, produced analgesia on formalin-induced spontaneous pain behaviors. However, it is still unclear whether single DUL pretreatment influences formalin-induced hypersensitivity and what is the underlying mechanism. In the current study, we revealed that systemic pretreatment with DUL not only dose-dependently suppressed the spontaneous pain behaviors, but also relieved mechanical and thermal hypersensitivity induced by formalin hindpaw injection. Consistent with the analgesic effects of DUL on the pain behaviors, the expressions of Fos and pERK that were used to check the neuronal activities in the spinal cord and CeA were also dose-dependently reduced following DUL pretreatment. Meanwhile, no emotional aversive behaviors were observed at 24 h after formalin injection. The concentration of 5-HT in the CeA was correlated with the dose of DUL in a positive manner at 24 h after formalin injection. Direct injecting 5-HT into the CeA suppressed both the spontaneous pain behaviors and hyperalgesia induced by formalin injection. However, DUL did not have protective effects on the formalin-induced edema of hindpaw. In sum, the activation of CeA neurons may account for the transition from acute pain to long-term hyperalgesia after formalin injection. DUL may produce potent analgesic effects on the hyperalgesia and decrease the expressions of p-ERK through increasing the concentration of serotonin in the CeA.

Long-Term Plasticity in Amygdala Circuits: Implication of CB1-Dependent LTD in Stress.

The amygdala mediates many forms of emotional learning, during which the central nucleus of amygdala (CeA) functions as a major output of the amygdala by converging inputs from the basolateral nucleus (BLA) and other amygdalar subregions. However, the contribution of BLA-CeA synaptic transmission and plasticity of this transmission after exposure to emotional stimuli remains to be completely understood. Using paired recording, we simultaneously recorded BLA and CeA neurons, and observed that BLA-CeA transmission was glutamatergic. In this transmission, high-frequency stimulation induced NMDA receptor (NMDAR)-dependent LTP, low-frequency stimulation induced NMDAR-dependent LTD, whereas modest-frequency stimulation induced cannabinoid receptor1 (CB1)-dependent LTD. After acute stress, CB1-dependent LTD of this transmission was selectively abolished. This effect of stress was mimicked by intra-CeA administration of CB1-selective agonists and prevented by CB1-selective antagonists. Furthermore, intra-CeA administration of CB1 antagonists prevented stress-induced reduction of explorative behaviors. These results indicate that CB1 signaling-mediated plasticity in local circuits of the amygdala plays a critical role in emotional responses.

Intraneuronal Acidification with Acetic Acid, an Ethanol Metabolite, Increases Excitability of Central Nucleus of Amygdala Neurons with Axon Projecting to Rostral Ventrolateral Medulla (CeA‐RVLM)

We have reported that activation of central nucleus of amygdala (CeA) is involved in the in vivo sympathoexcitatory and pressor responses to acute local challenge of acetate, the ethanol metabolite. However, the underlying neural mechanisms have not been determined. Evidence indicates weak organic acids are capable of decreasing intracellular pH due to intracellular transport of the organic acid and alternating neuronal activity. Recently, we have found that bath application of acetate caused a dose dependent increase in the in vitro excitability of CeA neurons with axon projecting to rostral ventrolateral medulla (CeA‐RVLM), which may underlie the mechanisms of increased sympathetic outflow in response to acetate challenge. We hypothesized that intraneuronal acidification due to intracellular transport of acetic acid may be a contributing factor. The role of intraneuronal acidification with the ethanol metabolite acetic acid was investigated under brain slice preparation. Specifically, we compared the action potential firing rate, depolarizing input resistance (Rinput), and voltage threshold (Vt) for firing action potentials between control intracellular recording solution (pH = 7.35) and 7.5 mM acetic acid loaded intracellular recording solution (pH = 5.20). In current‐clamp recordings, graded current injections evoked graded increases in discharge frequency among neurons from both groups. Maximum discharge evoked by +250 pA current injection in the 7.5 mM acetic acid loaded (pH = 5.20) group was 31 ± 1 Hz (n=6), which was significantly greater (P < 0.05) than that of controls group (pH = 7.35, 19 ± 1 Hz, n=7). Intraneuronal acidification with 7.5 mM acetic acid significantly (p < 0.05) increased Rinput of CeA‐RVLM neurons (731 ± 64 MΩ) compared to control (531 ± 48 MΩ). Likewise, 7.5 mM acetic acid caused a significant (p < 0.05) hyperpolarizing shift in Vt (−49.28 ± 1.37 mV) compared to control neurons (−43.83 ± 1.76 mV). In cultured neurons incubated with a pH sensitive probe, increases in bath application of acetate (7.5 mM) significantly (p < 0.05) increased the corrected fluorescence intensity of the neurons compared to control, indicative of a decrease in intraneuronal pH. This data suggests that extracellular acetate leads to intraneuronal acidification via transport of acetic acid which has profound effects on CeA‐RVLM membrane properties that enhance neuronal excitability and may contribute to the mechanism underlying alcohol associated sympathetic activation and hypertension.Support or Funding Information11SDG7420029 (ZYS) and R15‐HL122952 (QHC)

Acetate is an Active Metabolite of Ethanol: Increases Firing and Evokes Inward Currents through Activation of NMDA Receptors in RVLM Projecting CeA Neurons

We have recently reported that activation of central nucleus of amygdala (CeA) is involved in the in vivo sympathoexcitatory and pressor responses to acute challenge of acetate, the ethanol metabolite. However, the underlying neural mechanisms have not been determined. Here we investigated the role of acetate in regulating the in vitro excitability of CeA neurons with axon projecting to rostral ventrolateral medulla (CeA‐RVLM) under brain slice preparation. In current‐clamp recordings, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +250 pA injections and averaged 19 ± 1 Hz (n=8). Bath application of acetate (0, 7.5, 37.5 and 75 mM) increased the excitability of CeA‐RVLM neurons in a dose‐dependent manner. Maximum spike discharge in the presence of acetate (75 mM) was 44 ± 5 Hz (n=7). Pre‐treatment with AP5 (60 µM, n=7), the NMDA receptor competitive blocker or memantine (30 µM, n=9) a NMDA receptor channel pore blocker, significantly attenuated the increased excitability elicited by acetate (34 ± 4 Hz for AP5 and 20 ± 5 Hz for memantine respectively, p<0.05 vs 75 mM acetate alone). AP5 or memantine alone did not alter baseline excitability. In voltage‐clamp recordings, acetate application produced inward currents (22.4 ± 2 pA, n=6) which were completely abolished by pre‐treatment with AP5 (n=4) or memantine (n=6). Immunohistochemistry study demonstrates expression of NMDA NR1 in CeA‐RVLM neurons. Our data indicate that activation of NMDA receptors by acetate contributes to the increased excitability of CeA‐RVLM neurons, which may underlie the mechanism of sympathoexcitation and pressor response in vivo. AHA2640130 (QHC)

Somatostatin and nociceptin inhibit neurons in the central nucleus of amygdala that project to the periaqueductal grey

The central nucleus of amygdala (CeA) plays an important role in modulation of the descending antinociceptive pathways. Using whole-cell patch clamp recordings from brain slices, we found that CeA neurons responded to the endogenous ligands somatostatin (SST) and nociceptin/orphanin FQ (OFQ) via an increased K-conductance. Co-application with selective antagonists suggested that SST and OFQ act on SSTR2 and ORL1 receptors, respectively. Taking account of anatomical localisation of recorded neurons, the present study showed that many responsive neurons were located within the medial subdivision of CeA and all CeA projection neurons to the midbrain periaqueductal grey invariably responded to these peptides. Randomly selected agonist-responsive neurons in CeA predominantly classified physiologically as low-threshold spiking neurons. The similarity of SST, OFQ and, as previously reported, opioid responsiveness in a sub-population of CeA neurons suggests converging roles of these peptides to inhibit the activity of projections from CeA to vlPAG, and potentially similar antinociceptive actions in this pathway.

Chronic morphine treatment induces functional delta-opioid receptors in amygdala neurons that project to periaqueductal grey

Chronic morphine treatment and persistent pain stimuli trigger translocation of delta-opioid receptors (DORs) from cytosolic pools to the surface membrane. Previously, we reported that chronic treatment with morphine induces functional DORs on GABAergic nerve terminals impinging on some neurons in the midbrain periaqueductal grey. In the present investigation, we used chronic administration of morphine in adult rats to study delta and mu-opioid receptors in the central nucleus of amygdala (CeA), a brain region with a substantial (presumed) GABAergic projection to the periaqueductal grey. Chronic morphine treatment increased the proportion of neurons displaying an increased potassium conductance in response to a selective DOR-agonist. There was a corresponding reduction in responsiveness of CeA neurons to a selective mu-opioid agonist. By combining retrograde labelling and live cell recording of CeA-periaqueductal grey projection neurons, we found nearly all (6/7 or 86%) projection neurons responded to delta agonist after chronic treatment with morphine versus only 2/7 neurons (29%) from vehicle-treated animals. Other physiological properties of amygdala neurons did not differ between neurons from vehicle and morphine-treated animals. Taken together, these results indicate that chronic treatment with morphine upregulates functional DORs in neurons projecting from the CeA to periaqueductal grey. CeA-periaqueductal grey projections form part of the descending antinociceptive and autonomic control systems suggesting an upregulation of functional DOR in antinociception, emotion and anxiety following chronic morphine treatment.

Serotonergic System in the Central Nucleus of Amygdala Mediates Cannabidiol- Induced Sleep Alteration

Cannabidiol (CBD) is one of the psycho-inactive constituents of marijuana, the Cannabis sativa. The pharma- cological property of CBD, especially the anxiolytic effect, is significant in the therapeutic purposes. The central nucleus of amygdala (CeA) plays a key role in the anxiety and its related behavioral responses (e.g. sleep-wake activity), and sero- tonin is one of the major mediators. However, the sleep-wake effect of CBD remains unclear. This study was designed to elucidate the effects of CBD on sleep-wake alteration and the involvement of serotonin in the CeA. Administrations of 5- hydroxytryptamine (5-HT), 5-HT1A receptor partial agonist (buspirone), 5-HT2 antagonist (ritanserin), cannabinoid CB1 receptor agonist (ACEA), or CB1 antagonist (AM-251) were employed to elucidate the action of CBD on CeA presynaptic CB1 receptors, serotonergic activity and the subsequent sleep alteration. We found that microinjection of CBD into the CeA prior to the beginning of the light period dose-dependently decreased slow wave sleep (SWS) with limited effect on rapid eye movement sleep (REMS). CBD-induced SWS suppression during the light period could be mimicked by admin- istering serotonin into the CeA. Buspirone and ritanserin dose-dependently blocked CBD-induced SWS decrease. Fur- thermore, administration of AM-251 exhibits similar effect as that of CBD on sleep-wake activity, and the CBD-induced SWS decrease was partially blocked by ACEA. These observations suggest that CBD acting on the CeA neurons de- creases SWS during the light phase, which is at least partially mediated by the consequence of antagonizing presynaptic CB1 receptors, enhancing serotonin release from the presynaptic terminals and subsequently acting on the postsynaptic 5- HT2 receptors.

Presynaptic δ Opioid Receptors Regulate Ethanol Actions in Central Amygdala

Endogenous opioid systems are implicated in the reinforcing effects of ethanol consumption. For example, delta opioid receptor (DOR) knockout (KO) mice show greater ethanol consumption than wild-type (WT) mice (Roberts et al., 2001). To explore the neurobiological correlates underlying these behaviors, we examined effects of acute ethanol application in brain slices from DOR KO mice using whole-cell patch recording techniques. We examined the central nucleus of amygdala (CeA) because the CeA is implicated in alcohol reinforcement (Koob et al., 1998). We found that the acute ethanol effects on GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) were greater in DOR KO mice than in WT mice. Ethanol increased the frequency of miniature IPSCs (mIPSCs) significantly more in DOR KO mice than in WT mice. In CeA of WT mice, application of ICI 174864 [[allyl]2-Tyr-alpha-amino-isobutyric acid (Aib)-Aib-Phe-Leu-OH], a DOR inverse agonist, augmented ethanol actions on mIPSC frequency comparable with ethanol effects seen in DOR KO mice. Superfusion of the selective DOR agonist D-Pen(2),D-Pen(5)-enkephalin decreased the mean frequency of mIPSCs; this effect was reversed by the DOR antagonist naltrindole. These findings suggest that endogenous opioids may reduce ethanol actions on IPSCs of CeA neurons in WT mice through DOR-mediated inhibition of GABA release and that the increased ethanol effect on IPSCs in CeA of DOR KO mice could be, at least in part, due to absence of DOR-mediated inhibition of GABA release. This result supports the hypothesis that endogenous opioid peptides modulate the ethanol-induced augmentation of GABA(A) receptor-dependent circuitry in CeA (Roberto et al., 2003).

Acute and chronic ethanol alter glutamatergic transmission in rat central amygdala: an in vitro and in vivo analysis.

The modulation of glutamatergic transmission by ethanol may contribute to ethanol intoxication, reinforcement, tolerance, and dependence. Therefore, we used in vitro electrophysiological and in vivo microdialysis techniques to investigate the effects of acute and chronic ethanol on glutamatergic transmission in the central nucleus of amygdala (CeA). Superfusion of 5-66 mM ethanol decreased compound glutamatergic EPSPs and EPSCs in CeA neurons, with half-maximal inhibition elicited by 14 mM ethanol. Ethanol (44 mM) decreased both non-NMDAR- and NMDAR-mediated EPSPs and EPSCs by 21%. Both the ethanol- and ifenprodil-induced depression of NMDAR-mediated EPSPs and EPSCs was enhanced in rats that received chronic ethanol treatment (CET). Ifenprodil also occluded the ethanol effect, suggesting that NR2B subunit-containing receptors may be involved. With local applications of NMDA, acute ethanol elicited a greater inhibition of NMDA currents in slices taken from CET (47%) compared with naive (30%) animals, suggesting that CET sensitizes NMDA receptors to ethanol. Acute ethanol also reduced paired pulse facilitation of EPSPs and EPSCs only in CET animals, suggesting acute ethanol-induced increase of glutamate release. This finding was supported by in vivo experiments showing that infusion of ethanol (0.1-1 M) via reverse microdialysis significantly increased glutamate release into the CeA dialysate but only after CET. Moreover, baseline CeA glutamate content was significantly higher in CET compared with naive animals. These combined findings suggest that CET and withdrawal lead to neuroadaptations of glutamatergic transmission at both presynaptic and postsynaptic sites in CeA, and glutamatergic synapses in CeA may play an important role in ethanol dependence.