CeA Neurons Research Articles

Two Ascending Thermosensory Pathways from the Lateral Parabrachial Nucleus That Mediate Behavioral and Autonomous Thermoregulation.

Thermoregulatory behavior in homeothermic animals is an innate behavior to defend body core temperature from environmental thermal challenges in coordination with autonomous thermoregulatory responses. In contrast to the progress in understanding the central mechanisms of autonomous thermoregulation, those of behavioral thermoregulation remain poorly understood. We have previously shown that the lateral parabrachial nucleus (LPB) mediates cutaneous thermosensory afferent signaling for thermoregulation. To understand the thermosensory neural network for behavioral thermoregulation, in the present study, we investigated the roles of ascending thermosensory pathways from the LPB in avoidance behavior from innocuous heat and cold in male rats. Neuronal tracing revealed two segregated groups of LPB neurons projecting to the median preoptic nucleus (MnPO), a thermoregulatory center (LPB→MnPO neurons), and those projecting to the central amygdaloid nucleus (CeA), a limbic emotion center (LPB→CeA neurons). While LPB→MnPO neurons include separate subgroups activated by heat or cold exposure of rats, LPB→CeA neurons were only activated by cold exposure. By selectively inhibiting LPB→MnPO or LPB→CeA neurons using tetanus toxin light chain or chemogenetic or optogenetic techniques, we found that LPB→MnPO transmission mediates heat avoidance, whereas LPB→CeA transmission contributes to cold avoidance. In vivo electrophysiological experiments showed that skin cooling-evoked thermogenesis in brown adipose tissue requires not only LPB→MnPO neurons but also LPB→CeA neurons, providing a novel insight into the central mechanism of autonomous thermoregulation. Our findings reveal an important framework of central thermosensory afferent pathways to coordinate behavioral and autonomous thermoregulation and to generate the emotions of thermal comfort and discomfort that drive thermoregulatory behavior.SIGNIFICANCE STATEMENT Coordination of behavioral and autonomous thermoregulation is important for maintaining thermal homeostasis in homeothermic animals. However, the central mechanism of thermoregulatory behaviors remains poorly understood. We have previously shown that the lateral parabrachial nucleus (LPB) mediates ascending thermosensory signaling that drives thermoregulatory behavior. In this study, we found that one pathway from the LPB to the median preoptic nucleus mediates heat avoidance, whereas the other pathway from the LPB to the central amygdaloid nucleus is required for cold avoidance. Surprisingly, both pathways are required for skin cooling-evoked thermogenesis in brown adipose tissue, an autonomous thermoregulatory response. This study provides a central thermosensory network that coordinates behavioral and autonomous thermoregulation and generates thermal comfort and discomfort that drive thermoregulatory behavior.

Taking a deep breath: How a brainstem pathway integrates pain and breathing

In this issue of Neuron, Liu etal. (2022) shed light on the neural circuits supporting pain- and anxiety-induced elevated breathing rhythms. They reveal PBL core-Oprm1 neurons projecting onto the CeA and shell-Oprm1 neurons projecting onto the preBötC as differential regulators of these behaviors.

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.

Multiple sources of internal calcium stores mediate ethanol-induced presynaptic inhibitory GABA release in the central nucleus of the amygdala in mice.

Ethanol can enhance GABA release in various brain regions via presynaptic mechanisms. However, the presynaptic action of ethanol on inhibitory GABA release is still not well understood. Since calcium is required for neurotransmitter release from presynaptic terminals, the purpose of this study was to investigate the role of both internal and external calcium signaling in ethanol-induced enhancement of GABA release within the central amygdala nucleus (CeA) in acute brain slice preparations. Whole-cell patch clamp electrophysiology was used to record miniature GABAA receptor-mediated inhibitory postsynaptic currents (mIPSCs) from CeA neurons. Ethanol-enhanced mIPSCs were recorded in the presence of antagonists that regulate internal and external calcium-mediated processes. Bath-applied ethanol dose-dependently increased the mean frequency of mIPSCs without altering mIPSC amplitude. Ethanol-induced increases in mIPSC frequency were antagonized by dantrolene, 2-APB, and the endoplasmic reticulum calcium pump (SERCA) antagonists thapsigargin and cyclopiazonic acid (CPA). Blocking calcium release from mitochondria or via exocytosis with ruthenium red also attenuated mIPSCs while frequency was not altered in the presence of a non-selective calcium channel blocker cadmium. The L-type calcium blocker nifedipine, but not its analogue nimodipine, blocked ethanol-induced enhancement in CeA neurons. These results demonstrate ethanol-induced presynaptic release of GABA is mediated by internal calcium stores and by disrupting neurotransmitter exocytosis within the CeA, a critical brain area involved in drugs of abuse and alcohol addiction.

Dissecting the Roles of GABA and Neuropeptides from Rat Central Amygdala CRF Neurons in Anxiety and Fear Learning

SUMMARYCentral amygdala (CeA) neurons that produce corticotropin-releasing factor (CRF) regulate anxiety and fear learning. These CeACRF neurons release GABA and several neuropeptides predicted to play important yet opposing roles in these behaviors. We dissected the relative roles of GABA, CRF, dynorphin, and neurotensin in CeACRF neurons in anxiety and fear learning by disrupting their expression using RNAi in male rats. GABA, but not CRF, dynorphin, or neurotensin, regulates baseline anxiety-like behavior. In contrast, chemogenetic stimulation of CeACRF neurons evokes anxiety-like behavior dependent on CRF and dynorphin, but not neurotensin. Finally, knockdown of CRF and dynorphin impairs fear learning, whereas knockdown of neurotensin enhances it. Our results demonstrate distinct behavioral roles for GABA, CRF, dynorphin, and neurotensin in a subpopulation of CeA neurons. These results highlight the importance of considering the repertoire of signaling molecules released from a given neuronal population when studying the circuit basis of behavior.

Orexin Depolarizes Central Amygdala Neurons via Orexin Receptor 1, Phospholipase C and Sodium-Calcium Exchanger and Modulates Conditioned Fear.

Orexins (OX), also known as hypocretins, are excitatory neuropeptides with well-described roles in regulation of wakefulness, arousal, energy homeostasis, and anxiety. An additional and recently recognized role of OX is modulation of fear responses. We studied the OX neurons of the perifornical hypothalamus (PeF) which send projections to the amygdala, a region critical in fear learning and fear expression. Within the amygdala, the highest density of OX-positive fibers was detected in the central nucleus (CeA). The specific mechanisms underlying OX neurotransmission within the CeA were explored utilizing rat brain slice electrophysiology, pharmacology, and chemogenetic stimulation. We show that OX induces postsynaptic depolarization of medial CeA neurons that is mediated by OX receptor 1 (OXR1) but not OX receptor 2 (OXR2). We further characterized the mechanism of CeA depolarization by OX as phospholipase C (PLC)- and sodium-calcium exchanger (NCX)- dependent. Selective chemogenetic stimulation of OX PeF fibers recapitulated OXR1 dependent depolarization of CeA neurons. We also observed that OXR1 activity modified presynaptic release of glutamate within the CeA. Finally, either systemic or intra-CeA perfusion of OXR1 antagonist reduced the expression of conditioned fear. Together, these data suggest the PeF-CeA orexinergic pathway can modulate conditioned fear through a signal transduction mechanism involving PLC and NCX activity and that selective OXR1 antagonism may be a putative treatment for fear-related disorders.

Long-Term Isolation Elicits Depression and Anxiety-Related Behaviors by Reducing Oxytocin-Induced GABAergic Transmission in Central Amygdala.

Isolation stress is a major risk factor for neuropsychiatric disorders such as depressive and anxiety disorders. However, the molecular mechanisms underlying isolation-induced neuropsychiatric disorders remain elusive. In the present study, we investigated the subcellular mechanisms by which long-term isolation elicits depression and anxiety-related behaviors in mice. First, we found that long-term isolation induced depression-related behaviors in the forced swimming test (FST) and the sucrose preference test, as well as anxiety-related behaviors in the elevated zero maze test (EZMT) and the open field test. Next, we showed that intracentral amygdala (CeA) injection of oxytocin (OXT), but not intracerebroventricular injection, attenuated isolation-induced depression and anxiety-related behaviors via oxytocin receptor (OXTR), not vasopressin-1a receptor (V1aR), in the FST and EZMT, respectively. Quantitative real-time polymerase chain reaction analysis revealed that after 5 weeks of isolation, mRNA transcription of OXTR in the CeA, but not that of V1aR, significantly decreased, whereas OXT and vasopressin mRNA transcription in the paraventricular nucleus of hypothalamus did not change significantly. Whole-cell patch clamping of acute brain slices demonstrated that the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in CeA neurons, but not their amplitude, was lower in isolated mice than in group-housed mice. Notably, OXT treatment increased the mIPSC frequency in the CeA neurons, but to a lesser extent in the case of isolated mice than in that of group-housed mice via OXTR. Taken together, our findings suggest that long-term isolation down-regulates OXTR mRNA transcription and diminishes OXT-induced inhibitory synaptic transmission in the CeA and may contribute to the development of depression and anxiety-related behaviors in isolated mice through the enhancement of CeA activity.

Small conductance calcium activated potassium (SK) channel dependent and independent effects of riluzole on neuropathic pain-related amygdala activity and behaviors in rats

Background and purposeChronic neuropathic pain is an important healthcare issue with significant emotional components. The amygdala is a brain region involved in pain and emotional-affective states and disorders. The central amygdala output nucleus (CeA) contains small-conductance calcium-activated potassium (SK) channels that can control neuronal activity. A clinically available therapeutic, riluzole can activate SK channels and may have antinociceptive effects through a supraspinal action. We tested the hypothesis that riluzole inhibits neuropathic pain behaviors by inhibiting pain-related changes in CeA neurons, in part at least through SK channel activation. Experimental approachBrain slice physiology and behavioral assays were done in adult Sprague Dawley rats. Audible and ultrasonic vocalizations and von Frey thresholds were measured in sham and neuropathic rats 4 weeks after left L5 spinal nerve ligation (SNL model). Whole cell patch-clamp recordings of regular firing CeA neurons in brain slices were used to measure synaptic transmission and neuronal excitability. Key resultsIn brain slices, riluzole increased the SK channel-mediated afterhyperpolarization and synaptic inhibition, but inhibited neuronal excitability through an SK channel independent action. SNL rats had increased vocalizations and decreased withdrawal thresholds compared to sham rats, and intra-CeA administration of riluzole inhibited vocalizations and depression-like behaviors but did not affect withdrawal thresholds. Systemic riluzole administration also inhibited these changes, demonstrating the clinical utility of this strategy. SK channel blockade in the CeA attenuated the inhibitory effects of systemic riluzole on vocalizations, confirming SK channel involvement in these effects. Conclusions and implicationsThe results suggest that riluzole has beneficial effects on neuropathic pain behaviors through SK channel dependent and independent mechanisms in the amygdala.

Monomethyl fumarate inhibits pain behaviors and amygdala activity in a rat arthritis model.

Neuroplasticity in the amygdala, a brain center for emotions, leads to increased neuronal activity and output that can generate emotional-affective behaviors and modulate nocifensive responses. Mechanisms of increased activity in the amygdala output region (central nucleus, CeA) include increased reactive oxygen species, and so we explored beneficial effects of monomethyl fumarate (MMF), which can have neuroprotective effects through the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) antioxidant response pathway. Systemic (intraperitoneal) MMF dose-dependently inhibited vocalizations and mechanosensitivity (hindlimb withdrawal reflexes) of rats in an arthritis pain model (kaolin-carrageenan-induced monoarthritis in the knee). Stereotaxic administration of MMF into the CeA by microdialysis also inhibited vocalizations but had a limited effect on mechanosensitivity, suggesting a differential contribution to emotional-affective vs sensory pain aspects. Extracellular single-unit recordings of CeA neurons in anesthetized rats showed that stereotaxic administration of MMF into the CeA by microdialysis inhibited background activity and responses of CeA neurons to knee joint stimulation in the arthritis pain model. Monomethyl fumarate had no effect on behaviors and neuronal activity under normal conditions. The results suggest that MMF can inhibit emotional-affective responses in an arthritis pain model through an action that involves the amygdala (CeA).

5-HT2C Receptor Knockdown in the Amygdala Inhibits Neuropathic-Pain-Related Plasticity and Behaviors.

Neuroplasticity in the amygdala drives pain-related behaviors. The central nucleus (CeA) serves major amygdala output functions and can generate emotional-affective behaviors and modulate nocifensive responses. The CeA receives excitatory and inhibitory inputs from the basolateral nucleus (BLA) and serotonin receptor subtype 5-HT2CR in the BLA, but not CeA, has been implicated anxiogenic behaviors and anxiety disorders. Here, we tested the hypothesis that 5-HT2CR in the BLA plays a critical role in CeA plasticity and neuropathic pain behaviors in the rat spinal nerve ligation (SNL) model. Local 5-HT2CR knockdown in the BLA with stereotaxic injection of 5-HT2CR shRNA AAV vector decreased vocalizations and anxiety- and depression-like behaviors and increased sensory thresholds of SNL rats, but had no effect in sham controls. Extracellular single-unit recordings of CeA neurons in anesthetized rats showed that 5-HT2CR knockdown blocked the increase in neuronal activity (increased responsiveness, irregular spike firing, and increased burst activity) in SNL rats. At the synaptic level, 5-HT2CR knockdown blocked the increase in excitatory transmission from BLA to CeA recorded in brain slices from SNL rats using whole-cell patch-clamp conditions. Inhibitory transmission was decreased by 5-HT2CR knockdown in control and SNL conditions to a similar degree. The findings can be explained by immunohistochemical data showing increased expression of 5-HT2CR in non-GABAergic BLA cells in SNL rats. The results suggest that increased 5-HT2CR in the BLA contributes to neuropathic-pain-related amygdala plasticity by driving synaptic excitation of CeA neurons. As a rescue strategy, 5-HT2CR knockdown in the BLA inhibits neuropathic-pain-related behaviors.SIGNIFICANCE STATEMENT Neuroplasticity in the amygdala has emerged as an important pain mechanism. This study identifies a novel target and rescue strategy to control abnormally enhanced amygdala activity in an animal model of neuropathic pain. Specifically, an integrative approach of gene transfer, systems and brain slice electrophysiology, behavior, and immunohistochemistry was used to advance the novel concept that serotonin receptor subtype 5-HT2C contributes critically to the imbalance between excitatory and inhibitory drive of amygdala output neurons. Local viral vector-mediated 5-HT2CR knockdown in the amygdala normalizes the imbalance, decreases neuronal activity, and inhibits neuropathic-pain-related behaviors. The study provides valuable insight into serotonin receptor (dys)function in a limbic brain area.

Age-related differences in anxiety-like behavior and amygdalar CCL2 responsiveness to stress following alcohol withdrawal in male Wistar rats.

Behavioral and neuroimmune vulnerability to withdrawal from chronic alcohol varies with age. The relation of anxiety-like behavior to amygdalar CCL2 responses following stress after withdrawal from chronic intermittent alcohol (CIA) was investigated in adolescent and adult rats. Adolescent and adult Wistar rats were exposed to CIA (three 5-day blocks of dietary alcohol separated by 2days of withdrawal) at concentrations that created similar blood alcohol levels across age. Twenty-four hours into the final withdrawal, half of the rats were exposed to 1h of restraint stress. Four hours post-stress, rats were used for behavior or tissue assays. Anxiety-like behavior was increased versus controls by CIA in adolescents and by CIA+stress in adults. CCL2 mRNA was increased versus controls by CIA in adolescents and by CIA and CIA+stress in adults. CCL2 co-localization with neuronal marker NeuN was decreased versus controls by CIA in adolescents and by CIA+stress in adults. CCL2 co-localization with astrocytic marker GFAP was decreased versus controls by CIA and CIA+stress in adolescents, but experimental groups did not differ from controls in adults. CCL2 co-localization with microglial marker Iba1 was decreased versus controls by stress alone in adolescents and by CIA+stress in adults. Changes in CCL2 protein might control behavior at either age but are particularly associated with CIA alone in adolescents and with CIA+stress in adults. That the number of CeA neurons expressing CCL2 was altered after CIA and stress is consistent with CCL2 involvement in neural function.

Antihypertensive and neuroprotective effects of catestatin in spontaneously hypertensive rats: Interaction with GABAergic transmission in amygdala and brainstem

The chromogranin A-derived peptide catestatin (CST) exerts sympathoexcitatory and hypertensive effects when microinjected into the rostral ventrolateral medulla (RVLM: excitatory output); it exhibits sympathoinhibitory and antihypertensive effects when microinjected into the caudal ventrolateral medulla (CVLM: inhibitory output) of vagotomized normotensive rats. Here, continuous infusion of CST into the central amygdalar nucleus (CeA) of spontaneously hypertensive rats (SHRs) for 15days resulted in a marked decrease of blood pressure (BP) in 6-month- (by 37mmHg) and 9-month- (by 65mmHg)old rats. Whole-cell patch-clamp recordings on pyramidal CeA neurons revealed that CST increased both spontaneous inhibitory postsynaptic current (sIPSC) amplitude plus frequency, along with reductions of sIPSC rise time and decay time. Inhibition of GABAA receptors (GABAARs) by bicuculline completely abolished CST-induced sIPSC, corroborating that CST signals occur through this major neuroreceptor complex. Hypertension is a major risk factor for cerebrovascular diseases, leading to vascular dementia and neurodegeneration. We found a marked neurodegeneration in the amygdala and brainstem of 9-month-old SHRs, while CST and the GABAAR agonist Muscimol provided significant neuroprotection. Enhanced phosphorylation of Akt and ERK accounted for these neuroprotective effects through anti-inflammatory and anti-apoptotic activities. Overall our results point to CST exerting potent antihypertensive and neuroprotective effects plausibly via a GABAergic output, which constitute a novel therapeutic measure to correct defects in blood flow control in disorders such as stroke and Alzheimer’s disease.

Ethanol metabolite increases activity of CeA neurons and requires activation of local NMDA receptors (1125.5)

The central nucleus of amygdala (CeA) is involved in the sympathoexcitatory and pressor responses to ethanol intake. However, the underlying neural mechanisms have not been determined. Here we investigated the role of acetate, the ethanol metabolite, 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 18 ± 1 Hz (n=7). 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 53 ± 3 Hz (n=4), which was significantly (p<0.01) higher than vehicle control. Pre‐treatment with AP5 (60 µM, n=7), the NMDA receptor blocker, significantly prevented increase in excitability (37 ± 5 Hz, p<0.05) elicited by acetate. AP5 alone was without effect on baseline excitability. The contribution of acetate in regulating sympathetic nerve activity (SNA) was assessed by microinjecting acetate into the CeA of anesthetized rat. Acetate (0.2 µmol/100 nl) significantly (P<0.05) increased lumber SNA by 90 ± 1 %, splanchnic SNA by 76 ± 18% and MAP by 4 ± 1 mmHg (n=3). 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. The CeA acetate elicited sympathoexcitatory responses involve the activation of CeA‐RVLM neurons, which may underlie the mechanisms of increased SNA during the development of alcohol associated hypertension.Grant Funding Source: Supported by AHA 10SDG2640130

Role of Amygdala Central Nucleus in Aversive Learning Produced by Shock or by Unexpected Omission of Food

Many psychological learning theories have noted commonalities between aversive states produced by presentation of negative reinforcers, such as electric shock, and the omission of expected positive reinforcers, such as food. Here, three groups of rats received training with one auditory cue paired with shock and another with the omission of expected food, a shock-paired cue and a food-omission control cue, or a food-omission cue and a shock control cue. Food-omission cues were established by contrast with food delivery; after extensive light-food pairings, the light was followed by the food-omission cue instead of food. Aversiveness of the food-omission cue was assessed with a conditioned punishment procedure, in which presentation of that cue was made contingent on performance of one previously trained instrumental response, whereas a second response had no consequences. We found that rats with lesions of amygdala central nucleus (CeA) showed impaired acquisition of freezing to the cue paired with shock and no evidence for acquisition of aversive properties by the cue that accompanied the omission of expected food. Furthermore, analyses of Arc and Homer1a mRNAs after rats were exposed to a two-epoch test procedure that allowed assessment of gene expression produced by two different test stimuli showed that both food-omission and shock-paired cues generated more neuronal activity in CeA than appropriate control cues. However, the number of neurons that were activated by both shock and food-omission cues was not significantly greater than expected by chance. Thus, under these test conditions, different subsets of CeA neurons represented these two aversive states.

The central amygdala projection to the substantia nigra reflects prediction error information in appetitive conditioning

The central amygdala nucleus (CeA) plays a critical role in cognitive processes beyond fear conditioning. For example, intact CeA function is essential for enhancing attention to conditioned stimuli (CSs). Furthermore, this enhanced attention depends on the CeA's connections to the nigrostriatal system. In the current study, we examined the role of the CeA's connections to two midbrain dopamine regions, the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA), in processing CS information when predictions of reward or nonreward were confirmed or disconfirmed. Initially, two different retrograde tracers were injected into the SNc and the VTA of rats, to label CeA cells. Different groups of rats then received a visual CS either paired or unpaired with food. Finally, Fos induction was assessed after a test session in which rats were exposed to the visual CS alone or paired with food. Colabeling of Fos and the retrograde tracer(s) showed that CeA neurons projecting to the SNc, but not to the VTA, were engaged in processing CS information when the training and testing conditions differed. These results suggest that the CeA-nigral pathway represents prediction error information during appetitive conditioning.

Central amygdaloid axon terminals are in contact with retrorubral field neurons that project to the parvicellular reticular formation of the medulla oblongata in the rat

The retrorubral field (RRF) contains numerous dopaminergic neurons and projects to the parvicellular reticular formation (RFp) of the medullary and pontomedullary brainstem, where many premotor neurons project to the orofacial motor nuclei. To know how the amygdala affects the RRF–RFp pathway in the rat, we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the RFp-projecting RRF neurons by using combined anterograde and retrograde tracing techniques. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and Fluoro-gold (FG) into the RFp, the prominent overlapping distribution of BDA-labeled axon terminals and FG-labeled neurons was found in the lateral part of the RRF ipsilateral to the injection sites, where the BDA-labeled axon terminals made symmetrical synapses with somata and dendrites of the FG-labeled neurons. Using a combination of retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH), we secondly demonstrated that the RFp-projecting RRF neurons were immunonegative for TH. Using a combination of anterograde tracing and immunohistochemistry for glutamic acid decarboxylase (GAD), we finally revealed that the CeA axon terminals in the RRF were immunoreactive for GAD. The present results suggest that GABAergic CeA neurons may exert inhibitory influences on non-dopaminergic RRF neurons that project to the RFp in the control of orofacial movements closely related to emotional behavior.

Neuroanatomical and neurochemical organization of projections from the central amygdaloid nucleus to the nucleus retroambiguus via the periaqueductal gray in the rat

The periaqueductal gray (PAG)-nucleus retroambiguus (NRA) pathway has been known to be involved in the control of vocalization and sexual behavior. To know how the amygdaloid complex influences the PAG-NRA pathway, here we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the PAG neurons that project to the NRA by using anterograde and retrograde tract-tracing techniques in the rat. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and cholera toxin B subunit (CTb) into the NRA, the prominent overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found ipsilaterally in the lateral/ventrolateral PAG, where some of the BDA-labeled terminals made symmetrical synaptic contacts with somata and dendrites of the CTb-labeled neurons. After CTb injection into the lateral/ventrolateral PAG, CTb-labeled neurons were distributed mainly in the medial division of the CeA. After BDA injection into the lateral/ventrolateral PAG, BDA-labeled fibers were distributed mainly in and around the NRA within the medulla oblongata. Using a combined retrograde tracing and in situ hybridization technique, we further demonstrated that more than half of the CeA neurons labeled with Fluoro-Gold (FG) injected into the lateral/ventrolateral PAG were positive for glutamic acid decarboxylase 67 mRNA and that the vast majority of PAG neurons labeled with FG injected into the NRA expressed vesicular glutamate transporter 2 mRNA. The present results suggest that the glutamatergic PAG-NRA pathway is under the inhibitory influence of the GABAergic CeA neurons.

Nocistatin and nociceptin exert opposite effects on the excitability of central amygdala nucleus-periaqueductal gray projection neurons

Central amygdala nucleus (CeA)-periaqueductal gray (PAG) pathway is the component of descending antinociceptive circuitry. Nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) produce supraspinal pronociceptive and antinociceptive effects, respectively. We hypothesized that opposite effects of N/OFQ and NST on supraspinal pain modulation result from their opposing effects on the excitability of CeA-PAG projection neurons. This hypothesis was tested by investigating electrophysiological effects of N/OFQ and NST on medial CeA neurons that project to PAG (CeA M-PAG). N/OFQ hyperpolarized CeA M-PAG projection neurons by enhancing inwardly rectifying potassium conductance. In contrast, NST depolarized CeA M-PAG neurons by causing the opening of TRPC cation channels via G αq/11-PLC-PKC pathway. CeA M-PAG neurons hyperpolarized by N/OFQ express CRF or neurotensin mRNA. NST-responsive CeA M-PAG neurons contain CRF or substance P mRNA. Our study provides the evidence that the molecular and cellular basis for opposite effects of N/OFQ and NST on supraspinal pain regulation is their opposing effects on the excitability of peptidergic CeA M-PAG neurons.

Role of Amygdalo-Nigral Circuitry in Conditioning of a Visual Stimulus Paired with Food

The amygdala central nucleus (CeA) plays an important part in associative learning. Although most research has focused on functions of its descending projections to brainstem areas involved in autonomic and somatomotor responses, the ascending projections of CeA also play critical roles in learning. For example, a CeA-nigrostriatal pathway is important for acquiring orienting responses (ORs) to conditioned stimuli (CSs) that signal food delivery. In this study, the function of this CeA-nigrostriatal pathway in appetitive conditioning of rats was considered in more detail. In experiment 1, we combined anatomical tracing and methods for detecting neuronal activation to examine whether CeA neurons that project to the substantia nigra pars compacta (SNc) are activated by a visual CS for food. After injection of the retrograde tracer Fluoro-Gold (FG) into SNc, the rats received pairings of a visual CS with food. After a test with the CS alone, the brains were prepared to assess FG labeling and CS-induced Fos expression in CeA with immunohistochemical procedures. Colocalization of Fos and FG in CeA neurons was visualized with confocal-fluorescence microscopy. The CS induced Fos expression in CeA, and a majority of these Fos-positive neurons were also FG positive, indicating activation of the CeA-SNc pathway by the CS. In experiment 2, lesions that disconnected CeA and SNc prevented the acquisition of conditioned ORs but did not affect the acquisition of conditioned food-related responses or the display of unconditioned ORs. These experiments demonstrate a role for amygdalo-nigral circuitry in learned modulation of attention to signals for biologically significant events.

Inhibitory avoidance learning altered ensemble activity of amygdaloid neurons in rats

In this study, we examined single-unit activity in the amygdala before and after a rat had acquired an inhibitory avoidance task. Long-Evans rats with microwires chronically implanted into the central nucleus (CeA) or basolateral complex (BLC) of the amygdala were acclimatized to the apparatus of a step-through inhibitory avoidance task for three sessions. On the fourth session, rats in the experimental group received an inescapable footshock (3 mA, 1 s) as they stepped from the lit side into the dark side of the task apparatus, whereas rats in the control group received the same amount of shock on a different apparatus. All rats were tested for retention in the task apparatus 1 day after shock training. The experimental rats showed better retention than the controls as they stayed longer in the lit side. Ensemble unit activities were recorded in the amygdala nuclei from the indwelling wire bundles during the acclimation and test sessions. The data collected from well-isolated amygdala units showed that neuronal discharge habituated from the first to the third acclimation session. In the test session, the experimental group, but not the control group, showed elevated firing rates in the CeA or BLC neurons located on either side of the brain. These findings provide the first piece of evidence showing that learning of an inhibitory avoidance task leads to an increase in amygdala neuronal discharges during a retention test.