Central Amygdaloid Nucleus Research Articles

The Zebrafish Amygdaloid Complex – Functional Ground Plan, Molecular Delineation, and Everted Topology

In mammals and other tetrapods, a multinuclear forebrain structure, called the amygdala, forms the neuroregulatory core essential for emotion, cognition, and social behavior. Currently, higher circuits of affective behavior in anamniote non-tetrapod vertebrates (“fishes”) are poorly understood, preventing a comprehensive understanding of amygdala evolution. Through molecular characterization and evolutionary-developmental considerations, we delineated the complex amygdala ground plan of zebrafish, whose everted telencephalon has made comparisons to the evaginated forebrains of tetrapods challenging. In this radical paradigm, thirteen telencephalic territories constitute the zebrafish amygdaloid complex and each territory is distinguished by conserved molecular properties and structure-functional relationships with other amygdaloid structures. Central to our paradigm, the study identifies the teleostean amygdaloid nucleus of the lateral olfactory tract (nLOT), an olfactory integrative structure that links dopaminergic telencephalic groups to the amygdala alongside redefining the putative zebrafish olfactory pallium (“Dp”). Molecular characteristics such as the distribution of substance P and the calcium-binding proteins parvalbumin (PV) and calretinin (CR) indicate, that the zebrafish extended centromedial (autonomic and reproductive) amygdala is predominantly located in the GABAergic and isl1-negative territory. Like in tetrapods, medial amygdaloid (MeA) nuclei are defined by the presence of substance P immunoreactive fibers and calretinin-positive neurons, whereas central amygdaloid (CeA) nuclei lack these characteristics. A detailed comparison of lhx5-driven and vGLut2a-driven GFP in transgenic reporter lines revealed ancestral topological relationships between the thalamic eminence (EmT), the medial amygdala (MeA), the nLOT, and the integrative olfactory pallium. Thus, the study explains how the zebrafish amygdala and the complexly everted telencephalon topologically relate to the corresponding structures in mammals indicating that an elaborate amygdala ground plan evolved early in vertebrates, in a common ancestor of teleosts and tetrapods.

Patterns of neural activity in the mouse brain: Wakefulness vs. General anesthesia

In light of the general shift from rats to mice as the leading rodent model in neuroscience research we used c-Fos expression as a tool to survey brain regions in the mouse in which neural activity differs between the states of wakefulness and pentobarbital-induced general anesthesia. The aim was to complement prior surveys carried out in rats. In addition to a broad qualitative review, 28 specific regions of interest (ROIs) were evaluated quantitatively. Nearly all ROIs in the cerebral cortex showed suppressed activity during anesthesia. Subcortically, however, some ROIs showed suppression, some showed little change, and some showed increased activity. The overall picture was similar to the rat. Special attention was devoted to ROIs significantly activated during anesthesia, as such loci might actively drive the transition to anesthetic unconsciousness rather than responding passively to inhbitory agents distributed globally (the “wet blanket” hypothesis). Twelve such “anesthesia-on” ROIs were identified: the paraventricular hypothalamic nucleus, supraoptic nucleus, tuberomamillary nucleus, lateral habenular nucleus, dentate gyrus, nucleus raphe pallidus, central amygdaloid nucleus, perifornical lateral hypothalamus, ventro-lateral preoptic area, lateral septum, paraventricular thalamic nucleus and zona incerta. The same primary anti-FOS antibody was used in all mice, but two alternative reporter systems were employed: ABC-diaminobenzidine and the currently more popular AlexaFluor488. Fluorescence tagging revealed far fewer FOS-immunoreactive neurons, sounding an alert that the reporter system chosen can have major effects on results obtained.

Restraint stress increases the expression of phoenixin immunoreactivity in rat brain nuclei

Phoenixin is a recently discovered peptide, which has been associated with reproduction, anxiety and food intake. Based on a considerable co-localization it has been linked to nesfatin-1, with a possible antagonistic mode of action. Since nesfatin-1 is known to play a role in anxiety and the response to stress, this study aims to investigate the effects of a well-established psychological stress model, restraint stress, on phoenixin-expressing brain nuclei and phoenixin expression in rats. Male Sprague-Dawley rats were subjected to restraint stress (n = 8) or left undisturbed (control, n = 6) and the brains processed for c-Fos- and phoenixin immunohistochemistry. The number of c-Fos expressing cells was counted and phoenixin expression assessed semiquantitatively. Restraint stress significantly increased c-Fos expression in the dorsal motor nucleus of vagus nerve (DMN, 52-fold, p < 0.001), raphe pallidus (RPa, 15-fold, p < 0.001), medial part of the nucleus of the solitary tract (mNTS, 16-fold, p < 0.001), central amygdaloid nucleus, medial division (CeM, 9-fold, p = 0.01), supraoptic nucleus (SON, 9-fold, p < 0.001) and the arcuate nucleus (Arc, 2.5-fold, p < 0.03) compared to control animals. Also phoenixin expression significantly increased in the DMN (17-fold, p < 0.001), RPa (2-fold, p < 0.001) and mNTS (1.6-fold, p < 0.001) with positive correlations between c-Fos and phoenixin (r = 0.74–0.85; p < 0.01) in these nuclei. This pattern of activation suggests an involvement of phoenixin in response to restraint stress. Whether phoenixin mediates stress effects or is activated in a counterbalancing fashion will have to be further investigated.

Involvement of GABAergic projection from central amygdaloid nucleus to paraventricular nucleus of hypothalamus in electroacupuncture induced regulation of gastric function in GAD2-Cre mice

To explore the effect of γ-aminobutyric acid (GABA)ergic neuronal circuit of the central amygdaloid nucleus (CeA) and the paraventricular nucleus of hypothalamus (PVN) on electroacupuncture (EA)-induced regulation of gastric function by way of CeA-PVN projection. The present study included 3 parts: 1) C57BL/6 mice were randomly divided into control and EA groups (n=6 in each group). EA was applied to right "Weishu"(BL21, Back-shu point) and "Zhongwan"(CV12, Front-mu point) for 20 min, followed by detecting the expression of c-fos in the CeA and PVN by using immunofluorescence staining; 2) Microinjection of anterograde tracer (rAAV-EF1α-DIO-mcherry-WPRE-pA) into the CeA was conducted in GAD2-Cre mice for confirming the projection of GABAergic neurons from CeA to PVN; 3) GAD2-Cre mice were randomly divided into rAAV-DIO-mcherry (intra-CeA injection of rAAV-EF1α-DIO-mcherry-WPRE-pA), rAAV-DIO-hM3D（Gq）-mcherry(intra-CeA injection of rAAV-EF1α-DIO-hM3D(Gq)-mcherry-WPRE-pA) and rAAV-DIO-hM3D（Gq）-mcherry+EA groups(n=6 in each group). The food intake and gastric empty were detected, and the concentration of GABA in the PVN was assayed by using high performance liquid chromatography on the 28th day after intra-CeA injection. 1) The expression of c-fos in the CeA and PVN was significantly increased in the EA group relevant to the control group(P<0.01), suggesting an activation of neurons in both CeA and PVN after EA. 2) Following CeA injection of rAAV-EF1α-DIO-mcherry-WPRE-pA, the densely expressed virus GABAergic neurons were found in CeA and large number of projection fibers found in the PVN, suggesting a direct connection between CeA and PVN. 3) After activating the GABAergic neurons of CeA, the concentration of GABA in the PVN was obviously increased (P<0.01), the food intake and the gastric empty were considerably decreased relevant to the rAAV-DIO-mcherry group(P<0.01). Following EA intervention，the concentration of GABA in the PVN was obviously decreased(P<0.01), the food intake and the gastric empty were significantly increased relevant to the rAAV-DIO-hM3D（Gq）-mcherry group (P<0.01). EA of BL21 and CV12 (Back-shu and Front-mu acupoints) can increase food intake and gastric empty in GAD2-Cre mice, which may be achieved by suppressing the release of GABA in PVN through CeA-PVN GABAergic neural circuit.

Primate Amygdalo-Nigral Pathway for Boosting Oculomotor Action in Motivating Situations.

SummaryA primary function of the primate amygdala is to modulate behavior based on emotional cues. To study the underlying neural mechanism, we first inactivated the amygdala locally and temporarily by injecting a GABA agonist. Then, saccadic eye movements and gaze were suppressed only on the contralateral side. Next, we performed optogenetic activation after injecting a viral vector into the amygdala. Optical stimulation in the amygdala excited amygdala neurons, whereas optical stimulation of axon terminals in the substantia nigra pars reticulata inhibited nigra neurons. Optical stimulation in either structure facilitated saccades to the contralateral side. These data suggest that the amygdala controls saccades and gaze through the basal ganglia output to the superior colliculus. Importantly, this amygdala-derived circuit mediates emotional context information, whereas the internal basal ganglia circuit mediates object value information. This finding demonstrates a basic mechanism whereby basal ganglia output can be modulated by other areas conveying distinct information.

Afferents of the mouse linear nucleus

The linear nucleus (Li) was identified in 1978 from its projections to the cerebellum. However, there is no systematic study of its connections with other areas of the central nervous system possibly due to the challenge of injecting retrograde tracers into this nucleus. The present study examines its afferents from some nuclei involved in motor and cardiovascular control with anterograde tracer injections. BDA injections into the central amygdaloid nucleus result in labeled fibers to the ipsilateral Li. Bilateral projections with an ipsilateral dominance were observed after injections in a) jointly the paralemniscal nucleus, the noradrenergic group 7/ Köllike -Fuse nucleus/subcoeruleus nucleus, b) the gigantocellular reticular nucleus, c) and the solitary nucleus/the parvicellular/intermediate reticular nucleus. Retrogradely labeled neurons were observed in Li after BDA injections into all these nuclei except the central amygdaloid and the paralemniscal nuclei. Our results suggest that Li is involved in a variety of physiological functions apart from motor and balance control it may exert via its cerebellar projections.

Whole-Brain Monosynaptic Inputs to Hypoglossal Motor Neurons in Mice

Hypoglossal motor neurons (HMNs) innervate tongue muscles and play key roles in a variety of physiological functions, including swallowing, mastication, suckling, vocalization, and respiration. Dysfunction of HMNs is associated with several diseases, such as obstructive sleep apnea (OSA) and sudden infant death syndrome. OSA is a serious breathing disorder associated with the activity of HMNs during different sleep–wake states. Identifying the neural mechanisms by which the state-dependent activities of HMNs are controlled may be helpful in providing a theoretical basis for effective therapy for OSA. However, the presynaptic partners governing the activity of HMNs remain to be elucidated. In the present study, we used a cell-type-specific retrograde tracing system based on a modified rabies virus along with a Cre/loxP gene-expression strategy to map the whole-brain monosynaptic inputs to HMNs in mice. We identified 53 nuclei targeting HMNs from six brain regions: the amygdala, hypothalamus, midbrain, pons, medulla, and cerebellum. We discovered that GABAergic neurons in the central amygdaloid nucleus, as well as calretinin neurons in the parasubthalamic nucleus, sent monosynaptic projections to HMNs. In addition, HMNs received direct inputs from several regions associated with respiration, such as the pre-Botzinger complex, parabrachial nucleus, nucleus of the solitary tract, and hypothalamus. Some regions engaged in sleep–wake regulation (the parafacial zone, parabrachial nucleus, ventral medulla, sublaterodorsal tegmental nucleus, dorsal raphe nucleus, periaqueductal gray, and hypothalamus) also provided primary inputs to HMNs. These results contribute to further elucidating the neural circuits underlying disorders caused by the dysfunction of HMNs.

Expression Patterns of Inducible Cre Recombinase Driven by Differential Astrocyte-Specific Promoters in Transgenic Mouse Lines.

Astrocytes are the most abundant cell type in the central nervous system (CNS). They provide trophic support for neurons, modulate synaptic transmission and plasticity, and contribute to neuronal dysfunction. Many transgenic mouse lines have been generated to obtain astrocyte-specific expression of inducible Cre recombinase for functional studies; however, the expression patterns of inducible Cre recombinase in these lines have not been systematically characterized. We generated a new astrocyte-specific Aldh1l1-CreERT2 knock-in mouse line and compared the expression pattern of Cre recombinase between this and five widely-used transgenic lines (hGfap-CreERT2 from The Jackson Laboratory and The Mutant Mouse Resource and Research Center, Glast-CreERT2, Cx30-CreERT2, and Fgfr3-iCreERT2) by crossing with Ai14 mice, which express tdTomato fluorescence following Cre-mediated recombination. In adult Aldh1l1-CreERT2:Ai14 transgenic mice, tdTomato was detected throughout the CNS, and five novel morphologically-defined types of astrocyte were described. Among the six evaluated lines, the specificity of Cre-mediated recombination was highest when driven by Aldh1l1 and lowest when driven by hGfap; in the latter mice, co-staining between tdTomato and NeuN was observed in the hippocampus and cortex. Notably, evident leakage was noted in Fgfr3-iCreERT2 mice, and the expression level of tdTomato was low in the thalamus when Cre recombinase expression was driven by Glast and in the capsular part of the central amygdaloid nucleus when driven by Cx30. Furthermore, tdTomato was clearly expressed in peripheral organs in four of the lines. Our results emphasize that the astrocyte-specific CreERT2 transgenic lines used in functional studies should be carefully selected.

Fos expression induced by olanzapine and risperidone in the central extended amygdala

The extended amygdala has been proposed to play an essential role in cognitive and affective processes and in neuropsychiatric disorders. In the present study, we examined the induction of Fos-like nuclei in the central amygdaloid nucleus (CeA), sublenticular extended amygdala (SLEA), interstitial nucleus of the posterior limb of the anterior commissure (IPAC), and bed nucleus of the stria terminalis (BSTL) of rodents to improve the knowledge regarding the pharmacological profile, therapeutic efficacy, and side-effects of olanzapine, an atypical antipsychotic drug and risperidone, a mixed atypical/typical antipsychotic drug in the rat brain. In addition, we evaluated the induction of Fos-like-nuclei in areas connected with these structures such as prefrontal cortex (PFCx), and nucleus accumbens shell, and in other important areas including the lateral septum and caudate-putamen that are involved in the therapeutic efficacy or side-effects of antipsychotic drugs. Fos-like-immunoreactivity induced by olanzapine and risperidone was compared with that by the atypical antipsychotic clozapine and typical antipsychotic haloperidol.Regarding the extended amygdala, and similarly to clozapine, olanzapine (5–10 mg/kg) and, with a lower efficacy, risperidone (1–3 mg/kg), induced Fos-like-nuclei in CeA, IPAC, SLEA, and BSTL. Both these drugs increased the induction of Fos-like-nuclei in PFCx, nucleus accumbens shell, lateral septum, and caudate-putamen. On the contrary, the increase of Fos-like-nuclei in the extended amygdala by haloperidol was restricted to IPAC only. These findings, consistent with the important role of extended amygdala in neuropsychiatric disorders characterized by affective disturbances, showed that olanzapine and risperidone, contrary to haloperidol, preferentially activated Fos-expression in these brain areas.

Brain-wide Mapping of Mono-synaptic Afferents to Different Cell Types in the Laterodorsal Tegmentum

The laterodorsal tegmentum (LDT) is a brain structure involved in distinct behaviors including arousal, reward, and innate fear. How environmental stimuli and top-down control from high-order sensory and limbic cortical areas converge and coordinate in this region to modulate diverse behavioral outputs remains unclear. Using a modified rabies virus, we applied monosynaptic retrograde tracing to the whole brain to examine the LDT cell type specific upstream nuclei. The LDT received very strong midbrain and hindbrain afferents and moderate cortical and hypothalamic innervation but weak connections to the thalamus. The main projection neurons from cortical areas were restricted to the limbic lobe, including the ventral orbital cortex (VO), prelimbic, and cingulate cortices. Although different cell populations received qualitatively similar inputs, primarily via afferents from the periaqueductal gray area, superior colliculus, and the LDT itself, parvalbumin-positive (PV+) GABAergic cells received preferential projections from local LDT neurons. With regard to the different subtypes of GABAergic cells, a considerable number of nuclei, including those of the ventral tegmental area, central amygdaloid nucleus, and VO, made significantly greater inputs to somatostatin-positive cells than to PV+ cells. Diverse inputs to the LDT on a system-wide level were revealed.

Increased protein and mRNA expression of corticotropin-releasing factor (CRF), decreased CRF receptors and CRF binding protein in specific postmortem brain areas of teenage suicide subjects

Overactivity of hypothalamic-pituitary-adrenal (HPA) axis function has been implicated in depression and suicidal behavior. This is based on the observation of an abnormal dexamethasone (DEX) and DEX-adrenocorticotropic hormone (ACTH) test in patients with depression and suicidal behavior. Recently, some studies have also found abnormalities of glucocorticoid receptors (GR), mineralocorticoid receptors (MR), corticotropin releasing factor (CRF), CRF receptors (CRF-R) and CRF binding protein (CRF-BP) in depressed and suicidal patients. Some investigators have also observed increased levels of CRF in the cerebrospinal fluid (CSF) and altered levels of MR, GR and CRF in the postmortem brain of depressed and suicidal subjects. We have earlier reported decreased protein and mRNA expression of GR and GILZ, a chaperone protein, in the postmortem brain of teenage suicide subjects. We have further studied CRF and its receptors in different areas of the postmortem brain of suicide subjects, i.e., the prefrontal cortex (PFC), hippocampus (HIPPO), subiculum and amygdala (AMY) from teenage suicide subjects. The CRF and its receptors were determined in the PFC (Brodmann area 9), HIPPO, subiculum and different amygdaloid nuclei from 24 normal control subjects and 24 teenage suicide subjects. Protein expression of CRF, its receptors and CRF-BP was determined by immunolabeling using the Western blot technique and mRNA expression was determined by real-time PCR (qPCR) technique. We found that the mRNA levels of CRF were significantly increased in the PFC, in the central amygdaloid nucleus (CeAMY) and in the subiculum. mRNA levels of CRF-R1 and CRF-BP were significantly decreased in the PFC. We did not find any changes in the HIPPO of any of the CRF components we studied. When we compared the protein expression of CRF components we found that CRF was significantly increased and CRF-R1, CRF-R2 and CRF-BP significantly decreased in the PFC. On the other hand, there were no changes in the protein expression of CRF components in the HIPPO. Our results in the postmortem brain suggest that, as found by clinical studies in the CSF, there are significant alterations of CRF and its receptors in the postmortem brain of teenage suicide subjects. These alterations of CRF and its components were region-specific, as changes were not generally observed in the HIPPO.

Collateral Projections from the Lateral Parabrachial Nucleus to the Central Amygdaloid Nucleus and the Ventral Tegmental Area in the Rat.

ABSTRACTLateral parabrachial nucleus (LPB) is a critical region in the integration and transmission of peripheral nociceptive information. The parabrachio‐amygdaloid (P‐Amy) pathway and parabrachio‐ventral tegmental area (P‐VTA) pathway is thought to be significant in regulation of pain‐related negative emotions. In present study, retrograde tract tracers Fluoro‐gold (FG) and tetramethylrhodramine‐dextran (TMR) were stereotaxically injected into the right central amygdaloid nucleus (CeA) and right VTA, respectively. Then, part of these rats were performed with the spare nerve injury (SNI) in the controlateral side of FG and TMR injection. Afterwards, double‐ or triple‐immunofluorescent histochemistry was used to examine FG/TMR double‐ and FG/TMR/FOS or FG/TMR/CGRP triple‐labeled neurons in the LPB. The results showed that all of FG, TMR single‐ and FG/TMR double‐labeled neurons were distributed in the LPB bilaterally with an ipsilateral predominance. The proportion of FG/TMR double‐labeled neurons to the total number of FG‐ and TMR‐labeled neurons was 10.78% and 13.07%, respectively. Nearly all of the FG/TMR double‐labeled neurons (92.67%) showed calcitonin gene‐related peptide (CGRP) immunopositive. On the other hand, in the SNI rats, about 89.49% and 77.87% of FG‐ and TMR‐labeled neurons were FG/FOS‐ and TMR/FOS‐positive neurons; about 93.33% of the FG/TMR double‐labeled neurons were FOS‐LI. Our results suggest that the part of CGRP immunopositive neurons in the LPB send projection fibers to both the CeA and VTA by the way of axon collaterals, which are activated by the nociceptive stimulation in the SNI condition, and may play an important role in the transmission of peripheral nociceptive information. Anat Rec, 302:1178–1186, 2019. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

The offspring of rats selected for high or low ethanol intake at adolescence exhibit differential ethanol-induced Fos immunoreactivity in the central amygdala and in nucleus accumbens core

Adolescents exhibit, when compared to adults, altered responsivity to the unconditional effects of ethanol. It is unclear if this has a role in the excessive ethanol intake of adolescents. Wistar rats from the third filial generation (F3) of a short-term breeding program which were selected for high (STDRHI) vs. low (STDRLO) ethanol intake during adolescence, were assessed for ethanol-induced (0.0, 1.25 or 2.5 g/kg) Fos immunoreactivity (Fos-ir) in the central (Ce), basolateral (BLA) and medial (Me) amygdaloid nuclei; nucleus accumbens core and shell (AcbC, AcbSh), ventral tegmental area (VTA), as well as prelimbic and infralimbic (PrL, IL) prefrontal cortices. Following i.p. administration of saline, and across the structures measured, Fos-ir was significantly greater in STDRHI than in STDRLO rats. Across both lines, baseline Fos-ir was significantly lower in BLA than in any other structure, whereas PrL, IL and Shell did not differ between each other and exhibited significantly greater level of baseline neural activation than Ce, Me, AcbC and VTA. STDRLO, but not STDRHI, rats exhibited ethanol-induced Fos-ir in Ce. STRDHI, but not STDRLO, rats exhibited an ethanol-induced Fos-ir depression in AcbC. Key maternal care behaviors (i.e., grooming of the pups, latency to retrieve the pups, time spent in the nest and time adopting a kiphotic posture) were fairly similar across lines. There were significant intergenerational variations in the amount self-licking behaviors in STDRHI dams as well as an increased amount of exploration of the cage in these animals, when compared to STDRLO counterparts. These results indicate that short term selection for differential alcohol intake during adolescence yields heightened neural activity at baseline (i.e., after vehicle) in STRDHI vs. STDRLO adolescent rats, and differential sensitivity to ethanol-induced Fos immunoreactivity in Ce and in AcbC. It is unlikely that rearing patterns explained the neural differences reported, between STDRHI and STDRLO rats.

Neurochemical fingerprinting of amygdalostriatal and intra-amygdaloid projections: a tracing–immunofluorescence study in the rat

Amygdalostriatal and intra-amygdaloid fiber connectivity was studied in rats via injections of one of the tracers Phaseolus vulgaris leucoagglutinin (PHA-L) or biotinylated dextran amine (BDA) into various amygdaloid nuclei. To determine the neurotransmitter identity of labeled fibers we combined tracer detection with immunofluorescence staining, using antibodies against vesicular transporters (VTs) associated with glutamatergic (VGluT1, VGluT2) or GABAergic (VGAT) neurotransmission. High-magnification confocal laser scanning images were screened for overlap: occurrence inside tracer labeled fibers or axon terminals of immunofluorescence signal associated with one of the VTs.Labeled amygdalostriatal fibers were seen when tracer had been injected into the magnocellular and parvicellular portions of the basal amygdaloid nucleus and the lateral amygdaloid nucleus (nuclei belonging to 'cortical type' amygdaloid nuclei). Intra-amygdaloidal projection fibers were mostly found after tracer injections in the central and medial amygdaloid nuclei ('striatal type' amygdaloid nuclei).Terminals of tracer-labeled amygdalostriatal fibers contained immunofluorescence signal associated mostly with VGluT1 and to a lesser degree with VGluT2 or VGAT. Intra-amygdaloid labeled fibers showed colocalization mostly of VGluT1, followed by VGAT. VGluT2 co-occurred in a minority of intra-amygdaloid tracer-containing fiber terminals.We conclude from our observations that both amygdalostriatal and intra-amygdaloid projections, arising from, respectively, 'cortical type' and 'striatal type' amygdaloid nuclei contain strong glutamatergic and modest GABAergic components. The glutamatergic fibers express either VGluT1 or VGluT2. The absence in large numbers of tracer labeled fibers of expression of one of the selected VTs leads us to suspect that amygdalostriatal projection fibers may contain hitherto neglected neurotransmitters in these connections, e.g., aspartate.

Cortical and Subcortical Projections from Granular Insular Cortex Receiving Orofacial Proprioception

We have recently revealed that the proprioceptive signal from jaw-closing muscle spindles (JCMSs) is conveyed to the dorsal part of granular insular cortex rostroventrally adjacent to the rostralmost part of secondary somatosensory cortex (dGIrvs2) via the caudo-ventromedial edge (VPMcvm) of ventral posteromedial thalamic nucleus (VPM) in rats. However, it remains unclear to which cortical or subcortical structures the JCMS proprioceptive information is subsequently conveyed from the dGIrvs2. To test this issue, we injected an anterograde tracer, biotinylated dextranamine, into the electophysiologically identified dGIrvs2, and analyzed the resultant distribution profiles of labeled axon terminals in rats. Labeled terminals were distributed with an ipsilateral predominance. In the cerebral cortex, they were seen in the primary and secondary somatosensory cortices, lateral and medial agranular cortices and dorsolateral orbital cortex. In the basal ganglia, they were found in the caudate putamen, core part of accumbens nucleus, lateral globus pallidus, subthalamic nucleus, and substantia nigra pars compacta and pars reticulata. They were also observed in the central amygdaloid nucleus and extended amygdala (the interstitial nucleus of posterior limb of anterior commissure and the juxtacapsular part of lateral division of bed nucleus of stria terminalis). In the thalamus, they were seen in the reticular nucleus, ventromedial nucleus, core VPM, parvicellular part of ventral posterior nucleus, oval paracentral nucleus, medial and triangular parts of posterior nucleus, and zona incerta as well as the VPMcvm. These data suggest that the JCMS proprioceptive information through the dGIrvs2 is transmitted to the emotional ‘limbic’ regions as well as sensorimotor regions.

Cerebral oxidative metabolism mapping in four genetic mouse models of anxiety and mood disorders

The psychopathology of depression is highly complex and the outcome of studies on animal models is divergent. In order to find brain regions that could be metabolically distinctively active across a variety of mouse depression models and to compare the interconnectivity of brain regions of wild-type and such genetically modified mice, histochemical mapping of oxidative metabolism was performed by the measurement of cytochrome oxidase activity. We included mice with the heterozygous knockout of the vesicular glutamate transporter (VGLUT1−/+), full knockout of the cannabinoid 1 receptor (CB1−/−), an anti-sense knockdown of the glucocorticoid receptor (GRi) and overexpression of the human 5-hydroxytryptamine transporter (h5-HTT). Altogether 76 mouse brains were studied to measure oxidative metabolism in one hundred brain regions, and the obtained dataset was submitted to a variety of machine learning algorithms and multidimensional scaling. Overall, the top brain regions having the largest contribution to classification into depression model were the lateroanterior hypothalamic nucleus, the anterior part of the basomedial amygdaloid nucleus, claustrum, the suprachiasmatic nucleus, the ventromedial hypothalamic nucleus, and the anterior hypothalamic area. In terms of the patterns of inter-regional relationship between wild-type and genetically modified mice there was little overall difference, while the most deviating brain regions were cortical amygdala and ventrolateral and ventral posteromedial thalamic nuclei. The GRi mice that most clearly differed from their controls exhibited deviation of connectivity for a number of brain regions, such as ventrolateral thalamic nucleus, the intermediate part of the lateral septal nucleus, the anteriodorsal part of the medial amygdaloid nucleus, the medial division of the central amygdaloid nucleus, ventral pallidum, nucleus of the vertical limb of the diagonal band, anteroventral parts of the thalamic nucleus and parts of the bed nucleus of the stria terminalis. Conclusively, the GRi mouse model was characterized by changes in the functional connectivity of the extended amygdala and stress response circuits.

Distribution of vesicular glutamate transporters in the brain of the turtle (Pseudemys scripta elegans).

The distribution of glutamatergic neurons has been extensively studied in mammalian and avian brains, but its distribution in a reptilian brain remains unknown. In the present study, the distribution of subpopulations of glutamatergic neurons in the turtle brain was examined by in situ hybridization using probes for vesicular glutamate transporter (VGLUT) 1-3. Strong VGLUT1 expression was observed in the telencephalic pallium; the mitral cells of the olfactory bulb, the medial, dorsomedial, dorsal, and lateral parts of the cerebral cortex, pallial thickening, and dorsal ventricular ridge; and also, in granule cells of the cerebellar cortex. Moderate to weak expression was found in the lateral and medial amygdaloid nuclei, the periventricular cellular layer of the optic tectum, and in some brainstem nuclei. VGLUT2 was weakly expressed in the telencephalon but was intensely expressed in the dorsal thalamic nuclei, magnocellular part of the isthmic nucleus, brainstem nuclei, and the rostral cervical segment of the spinal cord. The cerebellar cortex was devoid of VGLUT2 expression. The central amygdaloid nucleus did not express VGLUT1 or VGLUT2. VGLUT3 was localized in the parvocellular part of the isthmic nucleus, superior and inferior raphe nuclei, and cochlear nucleus. Our results indicate that the distribution of VGLUTs in the turtle brain is similar to that in the mammalian brain rather than that in the avian brain.

Central and peripheral expression sites of phoenixin-14 immunoreactivity in rats

Phoenixin is a pleiotropic peptide involved in reproduction, anxiety and recently also implicated in the control of food intake. Besides the 20-amino acid phoenixin, the 14-amino acid phoenixin-14 also shows bioactive properties. However, the expression sites of phoenixin-14 in the brain and peripheral tissues are not yet described in detail. Therefore, a mapping of the brain and peripheral tissues from male and female Sprague-Dawley rats with a specific phoenixin-14 antibody was performed using western blot and immunohistochemistry. High density of phoenixin-14 immunoreactivity was detected in the medial division of the brain central amygdaloid nucleus, in the spinal trigeminal tract and in the spinocerebellar tract as well as in cells between the crypts of duodenum, jejunum and ileum. Medium density immunoreactivity was observed in the bed nucleus of the stria terminalis, in the area postrema, the nucleus of the solitary tract and the dorsal motor nucleus of the vagus nerve as well as in the peripheral parts of the islets of Langerhans in the pancreas. A low density of phoenixin-14 immunoreactivity was detected in the arcuate nucleus, the supraoptic nucleus and the raphe pallidus. After pre-absorption of the antibody with phoenixin-14 peptide, no immunosignals were observed indicating specificity of the antibody. Taken together, the widespread distribution of phoenixin-14 immunoreactivity gives additional rise to the pleiotropic functions of the peptide such as possible effects in gastrointestinal motility, immune functions and glucose homeostasis.

α-Synucleinopathy in the Human Amygdala in Parkinson Disease: Differential Vulnerability of Somatostatin- and Parvalbumin-Expressing Neurons.

Olfactory dysfunction and emotional impairment are nonmotor symptoms in Parkinson disease (PD). These symptoms might be correlated with the appearance of Lewy bodies and neurites (ubiquitin and α-synuclein aggregates) in the amygdala (Braak stage 3). α-Synucleinopathy in the amygdala has been studied only occasionally, and no data on cell types involved are available. This work aimed to analyze α-synuclein expression in the basolateral, central, and cortical amygdaloid nuclei in 5 PD patients (Braak stages 3-5) and 5 controls. Expression of somatostatin and parvalbumin as well as its colocalization with α-synuclein was quantified under confocal microscopy. α-synuclein expression did not differ significantly between the central and other nuclei. The density of somatostatin was significantly decreased in the basolateral and central complex. The density of parvalbumin was significantly diminished in the basolateral complex. Parvalbumin-positive cells colocalized frequently with α-synuclein (68.44%), whereas, somatostatin-positive cells colocalized only occasionally (6.98%). These data revealed the differential vulnerability among interneuron populations in the human amygdala and could help to explain nonmotor symptoms such as anhedonia in PD.

Acute Impact of Selected Pyridoindole Derivatives on Fos Expression in Different Structures of the Rat Brain.

The impacts of three pyridoindole derivatives (PDs), designated as PD144, PD143, and PD104, which have previously been shown to have antidepressant (PD144) and anxiolytic (PD143, PD104) properties, were investigated on the Fos expressions in 11 different rat brain areas, including the medial prefrontal cortex, striatum, septum, accumbens nucleus (shell, core), bed nucleus of the stria terminalis, hypothalamic paraventricular nucleus, central amygdala, locus coeruleus, dorsal raphe nucleus, and the solitary tract nucleus. Control rats received vehicle, while the other three groups the PDs in a dose of 25mg/kg/b.w. The animals were transcardially perfused with a fixative 90min after the treatments. Coronal sections of 40-µm thickness were processed for Fos-immunostaining by avidin-biotin-peroxidase complex and visualized by nickel-intensified diaminobenzidine complex. Fos-labeled sections were counterstained with neuropeptides including corticoliberine (CRH), oxytocin (OXY), vasopressin (AVP), and vasoactive intestinal polypeptide (VIP) and processed for immunofluorescence staining using Alexa Fluor 555 dye. In all the three groups of animals, the upregulation of PDs-induced Fos expression only in 2 of 11 brain areas was investigated, namely, in the hypothalamic paraventricular nucleus (PVN) and the central amygdaloid nucleus (CeA). The other brain structures studied were devoid of Fos expression. Counterstaining of the Fos-labeled CeA-containing sections with VIP antibody revealed that the Fos expression stimulated by the PDs was upregulated in all the CeA subdivisions (lateral, ventral, capsular), except the medial one. Dual immunoprocessings showed Fos/CRH-labeling in both the PVN and the amygdala and Fos/OXY in the PVN. No Fos/AVP colocalizations were seen in the PVN. The obtained data provide the first view on the intracerebral effects of three new PDs derivatives, which effects were restricted only to the PVN and CeA areas. The present data may help to improve our understanding of the impact of the selected PDs on the brain and to anticipate possible behavioral and neuroendocrine consequences.