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

Bone and dental tissues are richly innervated by sensory and sympathetic neurons. However, the characterization of the morphology, molecular phenotype, and distribution of nerves that innervate hard tissue has so far mostly been limited to thin histological sections. This approach does not adequately capture dispersed neuronal projections due to the loss of important structural information during three‐dimensional (3D) reconstruction. In this study, we modified the immunolabeling‐enabled imaging of solvent‐cleared organs (iDISCO/iDISCO+) clearing protocol to image high‐resolution neuronal structures in whole femurs and mandibles collected from perfused C57Bl/6 mice. Axons and their nerve terminal endings were immunolabeled with antibodies directed against protein gene product 9.5 (pan‐neuronal marker), calcitonin gene–related peptide (peptidergic nociceptor marker), or tyrosine hydroxylase (sympathetic neuron marker). Volume imaging was performed using light sheet fluorescence microscopy. We report high‐quality immunolabeling of the axons and nerve terminal endings for both sensory and sympathetic neurons that innervate the mouse femur and mandible. Importantly, we are able to follow their projections through full 3D volumes, highlight how extensive their distribution is, and show regional differences in innervation patterns for different parts of each bone (and surrounding tissues). Mapping the distribution of sensory and sympathetic axons, and their nerve terminal endings, in different bony compartments may be important in further elucidating their roles in health and disease. [ABSTRACT FROM AUTHOR]

The epithalamus, an area of the dorsal diencephalon found in all vertebrates, consists of the habenula, the subhabenular nuclei, and associated tracts. The habenula is itself divisible into two parts—a medial and a lateral nucleus differing in their inputs, outputs, and cellular morphology. The medial component is related to the limbic system and serotonergic raphe, while the lateral nucleus is more interconnected with the basal ganglia and midbrain dopamine systems. These findings, which come from experiments mainly done on mammals, serve as a basis for comparison with other vertebrates. However, similar studies in other amniotes, such as reptiles, are few. To fill this gap in knowledge, two species of crocodiles were examined utilizing a variety of histological methods in various planes of section. The following results were obtained. First, the habenula was divided into medial and lateral parts based on its cytoarchitecture. Neurons in the medial habenula were small, were closely packed, and had a limited dendritic arbor characterized by unusual distal dendritic appendages, whereas neurons in the lateral habenula were larger, were more loosely packed, and had longer dendritic processes that were commonly beaded. Second, the stria medullaris, the major input to the habenula, was identified by its immunoreactivity to parvalbumin. Third, the fasciculus retroflexus (habenulointerpeduncular tract), the primary output of the habenula, was visualized by staining with acetylcholinesterase. Fourth, nuclei associated with the habenula, the subhabenular nuclei, have been identified and characterized. These features provide a means to recognize the major nuclei and tracts in the epithalamus in crocodiles and are likely applicable to other reptiles. [ABSTRACT FROM AUTHOR]

The avian homologue of oxytocin (OT), formerly called mesotocin, influences social behaviors in songbirds and potentially song production. We sought to characterize the distribution of OT peptide in the brain of two songbird species: canaries (Serinus canaria) and zebra finches (Taeniopygia guttata). To visualize OT, we performed immunocytochemistry using an antibody previously shown to identify OT in avian species. In both canaries and zebra finches, dense OT‐ir perikarya were located in the paraventricular nucleus (PVN), preoptic area (POA), supraoptic nucleus (SON), and medial bed nucleus of the stria terminalis (BNSTm). We also observed morphologically distinct OT‐ir cells scattered throughout the mesopallium. OT‐ir fibers were observed in the PVN, ventral medial hypothalamus (VMH), periaqueductal gray (PAG), intercollicular nucleus (ICo), and ventral tegmental area (VTA). We also observed punctate OT‐ir fibers in the song control nucleus HVC. In both male and female canaries, OT‐ir fibers were present in the lateral septum (LS), but innervation was greater in males. We did not observe this sex difference in zebra finches. Much of the OT staining observed is consistent with general distributions within the vertebrate hypothalamus, indicating a possible conserved function. However, some extra‐hypothalamic distributions, such as perikarya in the mesopallium, may be specific to songbirds and play a role in song perception and production. The presence of OT‐ir fibers in HVC and song control nuclei projecting dopaminergic regions provides anatomical evidence in support of the idea that OT can influence singing behavior—either directly via HVC or indirectly via the PAG, VTA, or POA. [ABSTRACT FROM AUTHOR]

The oral somatosensory system relays essential information about mechanical stimuli to enable oral functions such as feeding and speech. The neurochemical and anatomical diversity of sensory neurons across oral cavity sites have not been systematically compared. To address this gap, we analyzed healthy human tongue and hard‐palate innervation. Biopsies were collected from 12 volunteers and underwent fluorescent immunohistochemistry (≥2 specimens per marker/structure). Afferents were analyzed for markers of neurons (βIII tubulin), myelinated afferents (neurofilament heavy, NFH), and Merkel cells and taste cells (keratin 20, K20). Hard‐palate innervation included Meissner corpuscles, glomerular endings, Merkel cell‐neurite complexes, and free nerve endings. The organization of these somatosensory endings is reminiscent of fingertips, suggesting that the hard palate is equipped with a rich repertoire of sensory neurons for pressure sensing and spatial localization of mechanical inputs, which are essential for speech production and feeding. Likewise, the tongue is innervated by afferents that impart it with exquisite acuity and detection of moving stimuli that support flavor construction and speech. Filiform papillae contained end bulbs of Krause, as well as endings that have not been previously reported, including subepithelial neuronal densities, and NFH+ neurons innervating basal epithelia. Fungiform papillae had Meissner corpuscles and densities of NFH+ intraepithelial neurons surrounding taste buds. The differing compositions of sensory endings within filiform and fungiform papillae suggest that these structures have distinct roles in mechanosensation. Collectively, this study has identified previously undescribed neuronal endings in human oral tissues and provides an anatomical framework for understanding oral mechanosensory functions. [ABSTRACT FROM AUTHOR]

Gonadotropin‐inhibitory hormone (GnIH) is a neuropeptide first discovered in the quail brain that is involved in the control of reproductive physiology and behaviors, and stress response. GnIH gene encodes a second peptide, GnIH‐related peptide‐2 (RP2), the distribution and function of which remain unknown. We therefore studied GnIH‐RP2 distribution by immunohistochemistry using a novel antibody capable of discriminating between GnIH and GnIH‐RP2. The overall distribution of GnIH‐RP2 is similar to that of GnIH. The vast majority of labeled neurons is located in the paraventricular nucleus (PVN) of the hypothalamus. Labeling of fibers is conspicuous in the diencephalon, but present also in the mesencephalon and telencephalon. Several regions involved in the control of reproduction and stress response (the PVN, septum, bed nucleus of the stria terminalis and nucleus commissura pallii) showed a dense network of immunolabeled fibers. To investigate the potential function of GnIH‐RP2 we compared its expression in two quail lines genetically selected for divergence in their emotional reactivity. A quantitative analysis in the above‐mentioned brain regions showed that the density of fibers was similar in the two lines. However, the number of GnIH‐RP2 labeled neurons was higher in the median portion of the PVN in birds with higher emotional reactivity. These results point to a possible involvement of GnRH‐RP2 in modulating stress response and/or emotional reactivity. [ABSTRACT FROM AUTHOR]

The Australian lungfish Neoceratodus forsteri is the only extant species of the order Ceratodontiformes, which retained most of the primitive features of ancient lobe finned‐fishes. Lungfishes are the closest living relatives of land vertebrates and their study is important for deducing the neural traits that were conserved, modified, or lost with the transition from fishes to land vertebrates. We have investigated the nitrergic system with neural nitric oxide synthase (NOS) immunohistochemistry and NADPH‐diaphorase (NADPH‐d) histochemistry, which yielded almost identical results except for the primary olfactory projections and the terminal and preoptic nerve fibers labeled only for NADPH‐d. Combined immunohistochemistry was used for simultaneous detection of NOS with catecholaminergic, cholinergic, and serotonergic structures, aiming to establish accurately the localization of the nitrergic elements and to assess possible interactions between these neurotransmitter systems. The results demonstrated abundant nitrergic cells in the basal ganglia, amygdaloid complex, preoptic area, basal hypothalamus, mesencephalic tectum and tegmentum, laterodorsal tegmental nucleus, reticular formation, spinal cord, and retina. In addition, low numbers of nitrergic cells were observed in the olfactory bulb, all pallial divisions, lateral septum, suprachiasmatic nucleus, prethalamic and thalamic areas, posterior tubercle, pretectum, torus semicircularis, cerebellar nucleus, interpeduncular nucleus, the medial octavolateral nucleus, nucleus of the solitary tract, and the dorsal column nucleus. Colocalization of NOS and tyrosine hydroxylase was observed in numerous cells of the ventral tegmental area/substantia nigra complex. Comparison with other vertebrates, using a neuromeric analysis, reveals that the nitrergic system of Neoceratodus shares many neuroanatomical features with tetrapods and particularly with amphibians. [ABSTRACT FROM AUTHOR]

The transition between the breeding and nonbreeding states is often marked by a shift in energy balance. Despite this well‐known shift in energy balance, little work has explored seasonal differences in the orexigenic neuropeptides that regulate food intake in wild animals. Here we tested the hypothesis that free‐living male song sparrows (Melospiza melodia) show seasonal changes in energetic state, circulating steroids, and both neuropeptide Y (NPY) and orexin (OX) immunoreactivity. Nonbreeding song sparrows had more fat and muscle, as well as a ketone and triglyceride profile suggesting a greater reliance on lipid reserves. Breeding birds had higher plasma androgens; however, nonbreeding birds did maintain androgen precursors in circulation. Nonbreeding birds had more NPY immunoreactivity, specifically in three brain regions: lateral septum, bed nucleus of the stria terminalis, and ventral tegmental area. Furthermore, nonbreeding birds had more OX immunoreactivity in multiple brain regions. Taken together, the data indicate that a natural shift in energy balance is associated with changes in NPY and OX in a region‐specific manner. Seasonal variation in immunoreactivity of neuropeptide Y or NPY (above) and orexin (below) were investigated in sparrows. Nonbreeding birds had higher NPY and orexin immunoreactivity than breeding birds throughout the brain, including areas that regulate social behavior. This coincided with nonbreeding shifts in fat reserve usage and circulating steroid profiles. [ABSTRACT FROM AUTHOR]

The neuropeptides vasopressin (AVP) and oxytocin (OT) have been implicated in the regulation of numerous social behaviors in adult and juvenile animals. AVP and OT signaling predominantly occur within a circuit of interconnected brain regions known collectively as the 'social behavior neural network' (SBNN). Importantly, AVP and OT signaling within the SBNN has been shown to differentially regulate diverse social behaviors, depending on the age and/or sex of the animal. We hypothesized that variation in the display of these behaviors is due in part to age and sex differences in AVP and OT synthesis within the SBNN. However, a thorough characterization of AVP and OT-immunoreactive (ir) fibers and cell bodies across age and sex within the SBNN has been lacking in rats. We therefore quantified AVP- and OT-ir fibers and cell bodies in 22 subregions of the forebrain SBNN in juvenile and adult, male and female rats. We found numerous age (16 subregions) and sex (10 subregions) differences in AVP-ir fiber fractional areas, and AVP-ir cell body numbers, which were mainly observed in the medial amygdala/bed nucleus of the stria terminalis to lateral septum circuit. In contrast to AVP, we observed no age or sex differences in OT-ir fiber fractional areas or cell bodies in any of the 22 subregions of the forebrain SBNN. Thus, unlike the static pattern observed for OT, AVP innervation of the forebrain SBNN appears to undergo developmental changes, and is highly sexually dimorphic, which likely has significant functional consequences for the regulation of social behavior. [ABSTRACT FROM AUTHOR]