Medial Central Nucleus Of The Amygdala Research Articles

Multimodal mapping of cell types and projections in the central nucleus of the amygdala.

The central nucleus of the amygdala (CEA) is a brain region that integrates external and internal sensory information and executes innate and adaptive behaviors through distinct output pathways. Despite its complex functions, the diversity of molecularly defined neuronal types in the CEA and their contributions to major axonal projection targets have not been examined systematically. Here, we performed single-cell RNA-sequencing (scRNA-seq) to classify molecularly defined cell types in the CEA and identified marker genes to map the location of these neuronal types using expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH). We developed new methods to integrate EASI-FISH with 5-plex retrograde axonal labeling to determine the spatial, morphological, and connectivity properties of ~30,000 molecularly defined CEA neurons. Our study revealed spatiomolecular organization of the CEA, with medial and lateral CEA associated with distinct molecularly defined cell families. We also found a long-range axon projection network from the CEA, where target regions receive inputs from multiple molecularly defined cell types. Axon collateralization was found primarily among projections to hindbrain targets, which are distinct from forebrain projections. This resource reports marker gene combinations for molecularly defined cell types and axon-projection types, which will be useful for selective interrogation of these neuronal populations to study their contributions to the diverse functions of the CEA.

Central amygdala corticotropin-releasing factor neurons promote hyponeophagia but do not control alcohol drinking in mice.

Corticotropin-releasing factor (CRF) signaling in the central nucleus of the amygdala (CeA) plays a critical role in rodent models of excessive alcohol drinking. However, the source of CRF acting in the CeA during alcohol withdrawal remains to be identified. In the present study, we hypothesized that CeA CRF interneurons may represent a behaviorally relevant source of CRF to the CeA increasing motivation for alcohol via negative reinforcement. We first observed that Crh mRNA expression in the anterior part of the mouse CeA correlates positively with alcohol intake in C57BL/6J males with a history of chronic binge drinking followed by abstinence and increases upon exposure to chronic intermittent ethanol (CIE) vapor inhalation. We then found that chemogenetic activation of CeA CRF neurons in Crh-IRES-Cre mouse brain slices increases gamma-aminobutyric acid (GABA) release in the medial CeA, in part via CRF1 receptor activation. While chemogenetic stimulation exacerbated novelty-induced feeding suppression (NSF) in alcohol-naïve mice, thereby mimicking the effect of withdrawal from CIE, it had no effect on voluntary alcohol consumption, following either acute or chronic manipulation. Furthermore, chemogenetic inhibition of CeA CRF neurons did not affect alcohol consumption or NSF in chronic alcohol drinkers exposed to air or CIE. Altogether, these findings indicate that CeA CRF neurons produce local release of GABA and CRF and promote hyponeophagia in naïve mice, but do not drive alcohol intake escalation or negative affect in CIE-withdrawn mice. The latter result contrasts with previous findings in rats and demonstrates species specificity of CRF circuit engagement in alcohol dependence.

Central afferents to the nucleus of the solitary tract in rats and mice.

The nucleus of the solitary tract (NTS) regulates life-sustaining functions ranging from appetite and digestion to heart rate and breathing. It is also the brain's primary sensory nucleus for visceral sensations relevant to symptoms in medical and psychiatric disorders. To better understand which neurons may exert top-down control over the NTS, here we provide a brain-wide map of all neurons that project axons directly to the caudal, viscerosensory NTS, focusing on a medial subregion with aldosterone-sensitive HSD2 neurons. Injecting an axonal tracer (cholera toxin b) into the NTS produces a similar pattern of retrograde labeling in rats and mice. The paraventricular hypothalamic nucleus (PVH), lateral hypothalamic area, and central nucleus of the amygdala (CeA) contain the densest concentrations of NTS-projecting neurons. PVH afferents are glutamatergic (express Slc17a6/Vglut2) and are distinct from neuroendocrine PVH neurons. CeA afferents are GABAergic (express Slc32a1/Vgat) and are distributed largely in the medial CeA subdivision. Other retrogradely labeled neurons are located in a variety of brain regions, including the cerebral cortex (insular and infralimbic areas), bed nucleus of the stria terminalis, periaqueductal gray, Barrington's nucleus, Kölliker-Fuse nucleus, hindbrain reticular formation, and rostral NTS. Similar patterns of retrograde labeling result from tracer injections into different NTS subdivisions, with dual retrograde tracing revealing that many afferent neurons project axon collaterals to both the lateral and medial NTS subdivisions. This information provides a roadmap for studying descending axonal projections that may influence visceromotor systems and visceral "mind-body" symptoms.

Microglia Control Escalation of Drinking in Alcohol-Dependent Mice: Genomic and Synaptic Drivers

BackgroundMicroglia, the primary immune cells of the brain, are implicated in alcohol use disorder. However, it is not known if microglial activation contributes to the transition from alcohol use to alcohol use disorder or is a consequence of alcohol intake. MethodsWe investigated the role of microglia in a mouse model of alcohol dependence using a colony stimulating factor 1 receptor inhibitor (PLX5622) to deplete microglia and a chronic intermittent ethanol vapor two-bottle choice drinking procedure. Additionally, we examined anxiety-like behavior during withdrawal. We then analyzed synaptic neuroadaptations in the central nucleus of the amygdala (CeA) and gene expression changes in the medial prefrontal cortex and CeA from the same animals used for behavioral studies. ResultsPLX5622 prevented escalations in voluntary alcohol intake and decreased anxiety-like behavior associated with alcohol dependence. PLX5622 also reversed expression changes in inflammatory-related genes and glutamatergic and GABAergic (gamma-aminobutyric acidergic) genes in the medial prefrontal cortex and CeA. At the cellular level in these animals, microglia depletion reduced inhibitory GABAA and excitatory glutamate receptor-mediated synaptic transmission in the CeA, supporting the hypothesis that microglia regulate dependence-induced changes in neuronal function. ConclusionsOur multifaceted approach is the first to link microglia to the molecular, cellular, and behavioral changes associated with the development of alcohol dependence, suggesting that microglia may also be critical for the development and progression of alcohol use disorder.

Molecular, Morphological, and Functional Characterization of Corticotropin-Releasing Factor Receptor 1-Expressing Neurons in the Central Nucleus of the Amygdala.

The central nucleus of the amygdala (CeA) is a brain region implicated in anxiety, stress-related disorders and the reinforcing effects of drugs of abuse. Corticotropin-releasing factor (CRF, Crh) acting at cognate type 1 receptors (CRF1, Crhr1) modulates inhibitory and excitatory synaptic transmission in the CeA. Here, we used CRF1:GFP reporter mice to characterize the morphological, neurochemical and electrophysiological properties of CRF1-expressing (CRF1+) and CRF1-non-expressing (CRF1–) neurons in the CeA. We assessed these two neuronal populations for distinctions in the expression of GABAergic subpopulation markers and neuropeptides, dendritic spine density and morphology, and excitatory transmission. We observed that CeA CRF1+ neurons are GABAergic but do not segregate with calbindin (CB), calretinin (CR), parvalbumin (PV), or protein kinase C-δ (PKCδ). Among the neuropeptides analyzed, Penk and Sst had the highest percentage of co-expression with Crhr1 in both the medial and lateral CeA subdivisions. Additionally, CeA CRF1+ neurons had a lower density of dendritic spines, which was offset by a higher proportion of mature spines compared to neighboring CRF1– neurons. Accordingly, there was no difference in basal spontaneous glutamatergic transmission between the two populations. Application of CRF increased overall vesicular glutamate release onto both CRF1+ and CRF1– neurons and does not affect amplitude or kinetics of EPSCs in either population. These novel data highlight important differences in the neurochemical make-up and morphology of CRF1+ compared to CRF1– neurons, which may have important implications for the transduction of CRF signaling in the CeA.

Altered Neuronal Activity in the Central Nucleus of the Amygdala Induced by Restraint Water-Immersion Stress in Rats

Restraint water-immersion stress (RWIS), a compound stress model, has been widely used to induce acute gastric ulceration in rats. A wealth of evidence suggests that the central nucleus of the amygdala (CEA) is a focal region for mediating the biological response to stress. Different stressors induce distinct alterations of neuronal activity in the CEA; however, few studies have reported the characteristics of CEA neuronal activity induced by RWIS. Therefore, we explored this issue using immunohistochemistry and in vivo extracellular single-unit recording. Our results showed that RWIS and restraint stress (RS) differentially changed the c-Fos expression and firing properties of neurons in the medial CEA. In addition, RWIS, but not RS, induced the activation of corticotropin-releasing hormone neurons in the CEA. These findings suggested that specific neuronal activation in the CEA is involved in the formation of RWIS-induced gastric ulcers. This study also provides a possible theoretical explanation for the different gastric dysfunctions induced by different stressors.

The amygdalo-nigrostriatal network is critical for an optimal temporal performance.

The amygdalo-nigrostriatal (ANS) network plays an essential role in enhanced attention to significant events. Interval timing requires attention to temporal cues. We assessed rats having a disconnected ANS network, due to contralateral lesions of the medial central nucleus of the amygdala (CEm) and dopaminergic afferents to the lateral striatum, as compared to controls (sham and ipsilateral lesions of CEm and dopaminergic afferents to LS) in a temporal bisection task. ANS disconnection induced poorer temporal precision and increased response latencies to a short duration. The present results reveal a role of the ANS network in temporal processing.

A Transgenic Rat for Investigating the Anatomy and Function of Corticotrophin Releasing Factor Circuits.

Corticotrophin-releasing factor (CRF) is a 41 amino acid neuropeptide that coordinates adaptive responses to stress. CRF projections from neurons in the central nucleus of the amygdala (CeA) to the brainstem are of particular interest for their role in motivated behavior. To directly examine the anatomy and function of CRF neurons, we generated a BAC transgenic Crh-Cre rat in which bacterial Cre recombinase is expressed from the Crh promoter. Using Cre-dependent reporters, we found that Cre expressing neurons in these rats are immunoreactive for CRF and are clustered in the lateral CeA (CeL) and the oval nucleus of the BNST. We detected major projections from CeA CRF neurons to parabrachial nuclei and the locus coeruleus, dorsal and ventral BNST, and more minor projections to lateral portions of the substantia nigra, ventral tegmental area, and lateral hypothalamus. Optogenetic stimulation of CeA CRF neurons evoked GABA-ergic responses in 11% of non-CRF neurons in the medial CeA (CeM) and 44% of non-CRF neurons in the CeL. Chemogenetic stimulation of CeA CRF neurons induced Fos in a similar proportion of non-CRF CeM neurons but a smaller proportion of non-CRF CeL neurons. The CRF1 receptor antagonist R121919 reduced this Fos induction by two-thirds in these regions. These results indicate that CeL CRF neurons provide both local inhibitory GABA and excitatory CRF signals to other CeA neurons, and demonstrate the value of the Crh-Cre rat as a tool for studying circuit function and physiology of CRF neurons.

Effects of chronic ethanol exposure on neuronal function in the prefrontal cortex and extended amygdala

Chronic alcohol consumption and withdrawal leads to anxiety, escalated alcohol drinking behavior, and alcohol dependence. Alterations in the function of key structures within the cortico-limbic neural circuit have been implicated in underlying the negative behavioral consequences of chronic alcohol exposure in both humans and rodents. Here, we used chronic intermittent ethanol vapor exposure (CIE) in male C57BL/6J mice to evaluate the effects of chronic alcohol exposure and withdrawal on anxiety-like behavior and basal synaptic function and neuronal excitability in prefrontal cortical and extended amygdala brain regions. Forty-eight hours after four cycles of CIE, mice were either assayed in the marble burying test (MBT) or their brains were harvested and whole-cell electrophysiological recordings were performed in the prelimbic and infralimbic medial prefrontal cortex (PLC and ILC), the lateral and medial central nucleus of the amygdala (lCeA and mCeA), and the dorsal and ventral bed nucleus of the stria terminalis (dBNST and vBNST). Ethanol-exposed mice displayed increased anxiety in the MBT compared to air-exposed controls, and alterations in neuronal function were observed in all brain structures examined, including several distinct differences between subregions within each structure. Chronic ethanol exposure induced hyperexcitability of the ILC, as well as a shift toward excitation in synaptic drive and hyperexcitability of vBNST neurons; in contrast, there was a net inhibition of the CeA. This study reveals extensive effects of chronic ethanol exposure on the basal function of cortico-limbic brain regions, suggests that there may be complex interactions between these regions in the regulation of ethanol-dependent alterations in anxiety state, and highlights the need for future examination of projection-specific effects of ethanol in cortico-limbic circuitry.

Common and distinct neural inputs to the medial central nucleus of the amygdala and anterior ventrolateral bed nucleus of stria terminalis in rats

The central nucleus of the amygdala (CEA) and lateral bed nucleus of stria terminalis (BST) are highly interconnected limbic forebrain regions that share similar connectivity with other brain regions that coordinate behavioral and physiological responses to internal and environmental stressors. Their similar connectivity is frequently referred to when describing the CEA and lateral BST together as a unified "central extended amygdala". However, the CEA and BST reportedly play distinct roles in behavioral and physiological responses associated with fear, anxiety, and social defeat, presumably due to differences in connectivity. To identify common and unique sources of input to the CEA and lateral BST, we performed dual retrograde tracing. Fluorogold and cholera toxin β were iontophoresed into the medial CEA (CEAm) and the anterior ventrolateral BST (BSTvl) of adult male rats. The anatomical distribution of tracer-labeled neurons was mapped throughout the brain. Regions with overlapping populations of CEAm- and BSTvl-projecting neurons were further examined for the presence of double-labeled neurons. Although most regions with input to the mCEA also projected to the BSTvl, and vice versa, cortical and sensory system-related regions projected more robustly to the CEAm, while motor system-related regions primarily innervated the BSTvl. The incidence of double-labeled neurons with collateralized axonal inputs to the CEAm and BSTvl was relatively small (~2 to 13%) and varied across regions, suggesting regional differences in the degree of coordinated CEAm and BSTvl input. The demonstrated similarities and differences in inputs to CEAm and BSTvl provide new anatomical insights into the functional organization of these limbic forebrain regions.

Aldosterone-sensitive neurons in the nucleus of the solitary tract: bidirectional connections with the central nucleus of the amygdala.

The HSD2 (11-beta-hydroxysteroid dehydrogenase type 2-expressing) neurons in the nucleus of the solitary tract (NTS) of the rat are aldosterone-sensitive and have been implicated in sodium appetite. The central nucleus of the amygdala (CeA) has been shown to modulate salt intake in response to aldosterone, so we investigated the connections between these two sites. A prior retrograde tracing study revealed only a minor projection from the HSD2 neurons directly to the CeA, but these experiments suggested that a more substantial projection may be relayed through the parabrachial nucleus. Small injections of cholera toxin beta subunit (CTb) into the external lateral parabrachial subnucleus (PBel) produced both retrograde cell body labeling in the HSD2 neurons and anterograde axonal labeling in the lateral subdivision of the CeA. Also, injections of either CTb or Phaseolus vulgaris leucoagglutinin into the medial subdivision of the CeA labeled a descending projection from the amygdala to the medial NTS. Axons from the medial CeA formed numerous varicosities and terminals enveloping the HSD2 neurons. Complementary CTb injections, centered in the HSD2 subregion of the NTS, retrogradely labeled neurons in the medial CeA. These bidirectional projections could form a functional circuit between the HSD2 neurons and the CeA. The HSD2 neurons may represent one of the functional inputs to the lateral CeA, and their activity may be modulated by a return projection from the medial CeA. This circuit could provide a neuroanatomical basis for the modulation of salt intake by the CeA.

Characterization of neurons in the rat central nucleus of the amygdala: Cellular physiology, morphology, and opioid sensitivity

The central nucleus of the amygdala (CeA) orchestrates autonomic and other behavioral and physiological responses to conditioned stimuli that are aversive or elicit fear. As a related CeA function is the expression of hypoalgesia induced by conditioned stimuli or systemic morphine administration, we examined postsynaptic opioid modulation of neurons in each major CeA subdivision. Following electrophysiological recording, biocytin-filled neurons were precisely located in CeA regions identified by chemoarchitecture (enkephalin-immunoreactivity) and cytoarchitecture (DAPI nuclear staining) in fixed adult rat brain slices. This revealed a striking distribution of physiological types, as 92% of neurons in capsular CeA were classified as late-firing, whereas no neurons in the medial CeA were of this class. In contrast, 60% or more of neurons in the lateral and medial CeA were low-threshold bursting neurons. Mu-opioid receptor (MOPR) agonists induced postsynaptic inhibitory potassium currents in 61% of CeA cells, and this ratio was maintained in each subdivision and for each physiological class of neuron. However, MOPR agonists more frequently inhibited bipolar/fusiform cells than triangular or multipolar neurons. A subpopulation of MOPR-expressing neurons were also inhibited by delta opioid receptor agonists, whereas a separate population were inhibited kappa opioid receptors (KOPR). The MOPR agonist DAMGO inhibited 9/9 CeM neurons with projections to the parabrachial nucleus identified by retrograde tracer injection. These data support models of striatopallidal organization that have identified striatal-like and pallidal-like CeA regions. Opioids can directly inhibit output from each subdivision by activating postsynaptic MOPRs or KOPRs on distinct subpopulations of opioid-sensitive neurons.

Opposing Influence of Basolateral Amygdala and Footshock Stimulation on Neurons of the Central Amygdala

The basolateral complex (BLA) and the central nucleus of the amygdala (CeA) are believed to mediate the expression of affective responses. After affective learning, conditioned stimulus-related information is thought to be conveyed from the BLA to the CeA; the medial CeA (Cem), in turn, projects to hypothalamic and brainstem structures involved with induction of affective responses. Although the conditioned stimulus and unconditioned stimulus both evoke affective responses, the precise response often differs. It is unknown whether this difference is represented by distinct activity patterns of single Cem neurons. Furthermore, the nature of the interaction between the BLA and Cem is unknown. Using in vivo extracellular and intracellular recordings, we examined how the BLA affects the Cem and compared this with effects induced by footshock (unconditioned stimulus) in the same neurons. Our results demonstrate that, contrary to conventional views, BLA stimulation primarily inhibits Cem neurons by a polysynaptic circuit, and show that single Cem neurons respond to both BLA input and footshock in an opposite manner. These results demonstrate the predominantly inhibitory nature of the BLA-Cem interaction. These data further demonstrate the distinct cellular events that might lead to differential modulation of conditioned and unconditioned affective responses.

Inhibition of the central extended amygdala by loud noise and restraint stress

It is well established that the central nucleus of the amygdala (CEA) is involved in responses to stress, fear and anxiety. Many studies have used c-fos expression to map the brain's response to processive stress, but curiously the CEA generally is not highly activated. We have previously shown that exposure to a novel vs. home environment reduces amphetamine-induced activation of the lateral CEA (CEAl) and the oval nucleus of the bed nucleus of the stria terminalis (BSTov). This is consistent with the idea that processive stress inhibits neurons in these nuclei. We have tested this hypothesis by exposing rats to noise, at a range of intensities from non-stressful to stressful, or to restraint conditions, immediately after a remote injection of amphetamine, 2 mg/kg i.p., or interleukin-1beta (IL-1beta) 0.5 microg/kg i.p. (used to obtain a level of c-fos mRNA against which to measure inhibition). In keeping with our hypothesis, amphetamine- or IL-1beta-induced c-fos and zif-268 mRNA were significantly decreased in the CEAl and BSTov under conditions of loud noise or restraint stress compared with control conditions. This inhibition does not require a stress-induced rise in corticosterone because data were similar in animals that had been adrenalectomized with a low-dose corticosterone replacement. As both the CEAl and BSTov are highly gamma-aminobutyric acid (GABA) -ergic and project to the medial CEA (CEAm), their inhibition potentially causes an increased input to the CEAm. As the CEAm is a major output nucleus of the amygdala, this could have important consequences within the neural circuitry controlling responses to processive stress.

Evidence for peptide co-transmission in retrograde- and anterograde-labelled central nucleus of amygdala neurones projecting to NTS

Synaptic terminals in the nucleus of the solitary tract (NTS) from axons originating in the central nucleus of the amygdala (CeA) are known to contain γ-aminobutyric acid (GABA) immunoreactivity. Here, we have investigated whether such projections contain neuropeptides as putative co-transmitters. Somata in the medial and lateral CeA that were retrogradely labelled with cholera toxin B (CTb) injected into the commissural NTS were found to be immunoreactive for GABA, somatostatin (SOM), neurotensin (NT), vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS). Subpopulations of fibres in the NTS that were anterogradely labelled with biotin dextran amine (BDA) injected into the CeA and examined using both fluorescence and electron microscopy appeared to colocalise somatostatin, but not other neuropeptides. Their varicosities were observed in proximity to NTS neurones that were immunoreactive for the somatostatin receptor sst 2A subtype, substance P (SP) NK1 receptor, and the GABA A receptor α 3, β 1 and γ 2 subunits. This morphological evidence is consistent with the possibility of GABA-somatostatin co-transmission at synapses of some of the CeA projection neurones to NTS that might inhibit cardiovascular reflex responses in response to fear or emotion-related stimuli.

Evidence for corticotropin‐releasing factor, neurotensin, and somatostatin in the neural pathway from the central nucleus of the amygdala to the parabrachial nucleus

The central nucleus of the amygdala (CNA) and the parabrachial nucleus of the pons (PBN) are included within a group of brain nuclei involved in autonomic responses. Previous studies have shown that the CNA sends a considerable projection to the PBN and that both nuclei contain neurons immunoreactive to many different peptides. In the present study, we used the combined retrograde fluorescence-immunofluorescence method to determine whether the CNA projection to the PBN contains any of the following neuropeptides: corticotropin-releasing factor (CRF), neurotensin (NT), somatostatin (SS), and enkephalin (ENK). Following injections of fluorescent dye into the PBN, neurons within both lateral and medial subdivisions of the CNA were retrogradely labeled. A significant percentage of CRF (54-66%)-, NT (40-53%)-, and SS (31-50%)-immunoreactive neurons were retrogradely labeled, predominantly within the lateral CNA. Enkephalin-immunoreactive neurons were never retrogradely labeled, although they were often found adjacent to retrogradely labeled neurons. Our results show that the lateral CNA is a major source of CRF, NT, and SS terminals within the PBN. Neurons in the medial CNA also provide a significant contribution to the CNA-PBN pathway, but their chemical nature remains to be determined. We conclude that CRF, NT, and SS are important putative neurotransmitters in the CNA's regulation of PBN function. This CNA-PBN peptidergic pathway may participate in stress-related cardiovascular and respiratory responses.

Afferent connections to cardiovascular sites in the amygdala: a horseradish peroxidase study in the cat

To investigate afferent connections to nuclei of the amygdala that have been shown electrophysiologically to receive inputs from baro- and chemoreceptors, small discrete deposits of horseradish peroxidase (HRP) were placed in the region of the central and lateral nuclei of the amygdala in cats. HRP deposits localized to the medial central nucleus of the amygdala labeled neurons in the ipsilateral hypothalamus, primarily in the paraventricular and ventromedial nuclei. In addition, the parabrachial nuclei and the locus coeruleus were observed to project to the region of the central nucleus. After HRP deposits in the medial portion of the lateral nucleus of the amygdala only a few labeled neurons were found scattered throughout the ipsilateral hypothalamus. In addition thalamic and cortical projections shown by previous investigators were confirmed. This study has demonstrated that in the cat several areas in the hypothalamus and brain stem project to the medial portions of the central and lateral nuclei of the amygdala suggesting that these areas may be relay stations for baro- and chemoreceptor information projecting to the amygdala.