Varicose Fibers Research Articles

Orexin mechanisms in the prelimbic cortex modulate the expression of contextual conditioned fear.

Despite the existing anatomical and physiological evidence pointing to the involvement of orexinergic projections from the lateral hypothalamus (LH) in regulating fear-related responses, little is known regarding the contribution of the orexin system in the prelimbic cortex (PL) on contextual fear. We investigated the role of orexin-A (OrxA) and orexin type 1 receptors (Orx1R) in the PL during the expression of contextual conditioned fear in mice. Neural tract tracing of the LH-PL pathway and Orx1R immunoreactivity in the PL of C57BL/6 male mice were performed. In a pharmacological approach, the animals were treated with either the Orx1R selective antagonist SB 334,867 (3, 30, and 300 nM/0.1 µL) or OrxA (28, 70, and 140 pmol/0.1 µL) in the PL before the test session of contextual fear conditioning. Neural tract tracing deposits in the LH showed some perikarya, mainly axons and terminal buttons in the PL, suggesting LH-PL reciprocate pathways. Furthermore, we showed a profuse network comprised of Orx1R-labeled thin varicose fibers widely distributed in the same field of LH-PL pathways projection. The selective blockade of Orx1R with SB 334,867 at 30 and 300 nM in the PL caused a decrease in freezing response, whereas the treatment with OrxA at 140 pmol promoted an increase in freezing response. In summary, these data confirmed an anatomical link between LH and PL, established the presence of Orx1R in the PL, and a modulatory role of the orexin system in such structure, possibly mainly via Orx1R, during contextual fear conditioning.

Distribution and anti-nociceptive function of endomorphin-1 in the rat cranial sensory ganglia.

Distribution of endomorphin-1 (EM-1) was immunohistochemically investigated in the rat cranial sensory ganglia. Small to medium-sized neurons in the trigeminal (TG), petrosal (PG), and jugular ganglia (JG) expressed EM-1-immunoreactivity. However, EM-1-immunoreactive (-ir) neurons were infrequent in the nodose ganglion. In the brainstem, EM-1-ir varicose fibers were detected in the superficial layer of the medullary dorsal horn and the caudal part of the nucleus tractus solitarius. By trichrome immunofluorescence analysis, approximately 70% of EM-1-ir neurons were also immunoreactive for transient receptor potential vanilloid 1 (TRPV1) in all the examined ganglia. Additionally, 56.8% of EM1-ir TG neurons and approximately 30% of EM-1-ir PG and JG neurons showed calcitonin gene-related peptide (CGRP)-immunoreactivity. By a retrograde tracing method, several TG, PG, and JG neurons innervating the facial and external ear canal skin expressed EM-1-immunoreactivity. However, EM-1-ir neurons innervating the tooth pulp, circumvallate papilla, and pharynx were relatively rare. Thus, EM-1 expression and its coexistence with TRPV1 and CGRP in the cranial sensory neurons may depend on their various peripheral targets. EM1-ir neurons probably project to the superficial layer of the medullary dorsal horn and caudal part of the nucleus tractus solitarius. EM-1 may be involved in nociceptive transmission from the skin.

Distribution, quantification, and characterization of substance P enteric neurons in the submucosal and myenteric plexuses of the porcine colon

The pig is an important translational model for studying intestinal physiology and disorders for its many homologies with humans, including the organization of the enteric nervous system (ENS), the major regulator of gastrointestinal functions. This study focused on the quantification and neurochemical characterization of substance P (SP) neurons in the pig ascending (AC) and descending colon (DC) in wholemount preparations of the inner submucosal plexus (ISP), outer submucosal plexus (OSP), and myenteric plexus (MP). We used antibodies for the pan-neuronal marker HuCD, and choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS), markers for excitatory and inhibitory transmitters, for multiple labeling immunofluorescence and high-resolution confocal microscopy. The highest density of SP immunoreactive (IR) neurons was in the ISP (222/mm2 in the AC, 166/mm2 in the DC), where they make up about a third of HuCD-IR neurons, compared to the OSP and MP (19–22% and 13–17%, respectively, P < 0.001–0.0001). HuCD/SP/ChAT-IR neurons (up to 23%) were overall more abundant than HuCD/SP/nNOS-IR neurons (< 10%). Most SP-IR neurons contained ChAT-IR (62–85%), whereas 18–38% contained nNOS-IR with the highest peak in the OSP. A subpopulation of SP-IR neurons contains both ChAT- and nNOS-IR with the highest peak in the OSP and ISP of DC (33–36%) and the lowest in the ISP of AC (< 10%, P < 0.001). SP-IR varicose fibers were abundant in the ganglia. This study shows that SP-IR neurons are functionally distinct with variable proportions in different plexuses in the AC and DC reflecting diverse functions of specific colonic regions.

New insight on the enteric cholinergic innervation of the pig colon by central and peripheral nervous systems: reduction by repeated loperamide administration.

The central and peripheral nervous systems provide cholinergic innervation in the colon. The ability to assess their neuroanatomical distinctions is still a challenge. The pig is regarded as a relevant translational model due to the close similarity of its enteric nervous system (ENS) with that of human. Opioid-induced constipation is one of the most common side effects of opioid therapy. We developed an approach to differentiate the central and peripheral cholinergic innervation of the pig colon using double immunolabeling with a novel mouse anti-human peripheral type of choline acetyltransferase (hpChAT) antibody combined with a rabbit anti-common type of ChAT (cChAT) antibody, a reliable marker of cholinergic neurons in the central nervous system. We examined their spatial configurations in 3D images of the ENS generated from CLARITY-cleared colonic segments. The density was quantitated computationally using Imaris 9.7. We assessed changes in the distal colon induced by daily oral treatment for 4 weeks with the μ opioid receptor agonist, loperamide (0.4 or 3 mg/kg). The double labeling showed strong cChAT immunoreactive (ir) fibers in the cervical vagus nerve and neuronal somata and fibers in the ventral horn of the sacral (S2) cord while hpChAT immunoreactivity was visualized only in the ENS but not in the vagus or sacral neural structures indicating the selectivity of these two antibodies. In the colonic myenteric plexus, dense hpChAT-ir neurons and fibers and varicose cChAT-ir fibers surrounding hpChAT-ir neurons were simultaneously visualized in 3D. The density of cChAT-ir varicose fibers in the outer submucosal plexus of both males and females were higher in the transverse and distal colon than in the proximal colon and in the myenteric plexus compared to the outer submucosal plexus and there was no cChAT innervation in the inner submucosal plexus. The density of hpChAT in the ENS showed no segmental or plexus differences in both sexes. Loperamide at the highest dose significantly decreased the density hpChAT-ir fibers + somata in the myenteric plexus of the distal colon. These data showed the distinct density of central cholinergic innervation between myenteric and submucosal plexuses among colonic segments and the localization of cChAT-ir fibers around peripheral hpChAT neurons in 3D. The reduction of cholinergic myenteric innervation by chronic opiate treatment points to target altered prokinetic cholinergic pathway to counteract opiate constipation.

Localization of Substance P-Immunoreactive Nerve Structures in the Rat Colon

The tachykinins, including their major representative substance P (SP), are an essential component of its non-adrenergic, non-cholinergic transmission of the myenteric plexus of the enteric nervous system. Hence, the understanding of the role that SP plays in the conduction of a wide variety of motility and secretory reflexes lies in the evidence-based understanding of the sites of its expression. In the present study, the presence, distribution and staining intensity of the SP-positive nerve structures in the myenteric plexus of the proximal and distal colon of the adult rat have been investigated by means of immunohistochemistry at the light microscopic level. We observed an abundance of intensely stained beaded SP-immunopositive fibres, surrounding the SP-immunonegative myenteric neuronal cell bodies in a basket-like manner. Fine bundles of SP-immunostained varicose fibres were also registered in the adjacent smooth muscle layers. The statistical analysis revealed discernible prevalence of the staining intensity in the proximal colon, compared to the distal one. In summary, our findings provide immunohistochemical evidence for the occurrence of abundant SP-immunopositive nerve structures in the colonic region of the rat. Taken together with previous morphological and pharmacological research the results of this study suggest that the SP-mediated neurotransmission is of physiological and likely of pathological significance for the examined region.

Substance P expression and neurochemical characterization in the enteric nervous system of the porcine colon

The pig is a good model for studying intestinal functions and disorders for its homologies with humans such as microbiome composition, size, nutrition being both omnivores and colon fermenters. The enteric nervous system (ENS) of pigs and humans has a multilayered submucosal plexus with an inner submucous plexus (ISP) near the mucosa and an outer plexus (OSP) near the circular muscle in addition to the myenteric plexus (MP) between the muscle layers. We have shown differences in the density and distribution of functionally distinct neurons in different regions and plexuses of the porcine colon. Aim: This study focused on Substance P, a peptide that modulates many functions in the gastrointestinal (GI) tract and plays an important role in neurogenic inflammation. We tested whether there were differences in the density and neurochemical profile of SP neurons in the ISP, OSP and MP of the ascending (AC) and descending (DC) colon of 15 Yucatan minipigs (12M, 3F, 7-months-old, body weight 25-30 kg). We processed colonic wholemounts for multiple labeling immunofluorescence using the HuCD, choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) as neuronal markers with high resolution confocal microscopy and Imaris software to quantify the number of neurons/mm2 and the % of total neurons identified by the pan-neuronal marker HuCD. Results: HuCD/SP-immunoreactive (IR) neurons were most abundant in the ISP vs. OSP and MP in both AC and DC (p&lt;0.01-p&lt;0.0001) with highest density in ISP of AC (p&lt;0.0001 vs. DC). SP-IR neurons represent 27-28% of HuCD-IR neurons in ISP followed by OSP (19-22%) and MP (13-17%, p&lt;0.05- p&lt;0.0001) in AC and DC. The highest proportion of SP-IR neurons contains ChAT-IR (62-67% in the MP, 74-76% in the ISP, 70-85% in OSP) with no significant differences in the AC and DC. By contrast, SP-IR neurons containing nNOS-IR were much less abundant in both AC and DC with the highest peak in the OSP 36% vs. 16% in the ISP, p&lt;0.01, and 20% in the MP in AC, whereas in the DC the highest peak was in the ISP (31%) followed by OSP and MP (25% and 18%) without significant differences. There was a small population of SP-/ChAT-/nNOS-IR enteric neurons in all plexuses in AC and DC. Varicose SP-IR fibers were abundant in the ganglia and distributed to the muscle layers and mucosa. Conclusions: SP-IR neurons include excitatory, inhibitory and interneurons based on the co-expression of ChAT, nNOS or both. The different density and neurochemical profile likely reflect different functions of SP-IR neurons such as immune modulation, regulation of mucosal function and control of vascular, myogenic and neurogenic activities. Mapping neuromodulators expression in a pre-clinical model provides the basis for elucidating neuronal circuits underlying GI functions and advance our understanding of human diseases. Supported by NIH SPARC OT2OD24899 &amp; NIH-P30DK41301. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Regional differences of extrinsic and intrinsic cholinergic innervation and alterations by chronic opioid-induced constipation in the porcine colonic enteric nerve system

The colon is richly innervated by extrinsic (ext) and intrinsic (int) cholinergic nerves from central and enteric nervous system (CNS and ENS) modulating colonic motility, secretion and inflammation. However, a challenge lies in our inability to anatomically distinguish such innervation in the colon. The porcine colon is regarded as a useful translational research model due to many similarities with human. The present study developed an approach to differentiate ext- and int-cholinergic innervation of the porcine colon using double immunolabeling with a novel mouse antibody against human peripheral choline acetyltransferase (hpChAT) combined with a rabbit antibody anti-common ChAT (cChAT) respectively. The spatial configuration of ext- and int- cholinergic innervation in the ENS was assessed on 3D images generated from CLARITY-cleared porcine colonic samples. Their densities were computationally quantitated using Imaris 9 within the inner and outer submucosal and myenteric plexuses (ISP, OSP, MP) along the proximal, transverse and distal colonic regions (pC, tC, dC) collected from 18 adult Yucatan minipigs including 6 naïve (3 of each sex), 4 vehicle controls (2 of each sex), 8 treated with loperamide, a μ opioid receptor agonist (LOP, 0.4 or 3 mg/kg/day, po, 4 weeks, 4/group, 2 of each sex). The specificities of cChAT and hpChAT antibodies were confirmed with porcine cervical vagal trunk (CVT), sacral spinal cord (SSC, S2-S4) and colonic ENS. The double labeling showed the strong cChAT immunoreactive (+) fibers in the CVT and neuronal somata and fibers in the SSC, but no hpChAT+ labeling was visualized in these structures. In the colonic MP, the dense hpChAT+ neurons and fibers and varicose cChAT+ fibers were simultaneously visualized. 3D images demonstrated that int- cholinergic neurons (hpChAT+) were closely surrounded by ext- cholinergic varicose fibers and dot like structures (cChAT+), presumably nerve terminals. The density of cChAT+ but not hpChAT+ fibers+somata showed significant differences among three segments (dC&gt;tC&gt;pC) within three enteric plexuses (MP&gt;OSP&gt;ISP) in naïve pigs. Compared to control groups, the high dose LOP induced a 1.3-fold decrease of cChAT+ fiber density in males (p&lt;0.05) and 1.5-fold in females (p&lt;0.05), and 2.2-fold decrease of hpChAT+ fibers+somata density in males (p&lt;0.05) and 3.6-fold in female (p&lt;0.01) with a 1.5-fold greater decrease in females than males (p&lt;0.05) in the MP of the dC. This study demonstrates for the first time the ext- and int- cholinergic innervation, the spatial configuration in 3D structure, regional variations in the porcine colonic ENS and the reduction by chronic opiate use with greater in females than males. These findings provide neuroanatomical evidence of CNS-gut cross-talk via cholinergic innervation of the colon and point to the neuromodulation of this pathway to alleviate functional colonic disorders like chronic opioid-induced constipation. Supported by NIH/SPARC OT2OD024899. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Anatomical Development of the Cerebellothalamic Tract in Embryonic Mice

The main connection from cerebellum to cerebrum is formed by cerebellar nuclei axons that synapse in the thalamus. Apart from its role in coordinating sensorimotor integration in the adult brain, the cerebello-thalamic tract (CbT) has also been implicated in developmental disorders, such as autism spectrum disorders. Although the development of the cerebellum, thalamus and cerebral cortex have been studied, there is no detailed description of the ontogeny of the mammalian CbT. Here we investigated the development of the CbT at embryonic stages using transgenic Ntsr1-Cre/Ai14 mice and in utero electroporation of wild type mice. Wide-field, confocal and 3D light-sheet microscopy of immunohistochemical stainings showed that CbT fibers arrive in the prethalamus between E14.5 and E15.5, but only invade the thalamus after E16.5. We quantified the spread of CbT fibers throughout the various thalamic nuclei and found that at E17.5 and E18.5 the ventrolateral, ventromedial and parafascicular nuclei, but also the mediodorsal and posterior complex, become increasingly innervated. Several CbT fiber varicosities express vesicular glutamate transporter type 2 at E18.5, indicating cerebello-thalamic synapses. Our results provide the first quantitative data on the developing murine CbT, which provides guidance for future investigations of the impact that cerebellum has on thalamo-cortical networks during development.

The pigment-dispersing factor neuronal network systematically grows in developing honey bees.

The neuropeptide pigment-dispersing factor (PDF) plays a prominent role in the circadian clock of many insects including honey bees. In the honey bee brain, PDF is expressed in about 15 clock neurons per hemisphere that lie between the central brain and the optic lobes. As in other insects, the bee PDF neurons form wide arborizations in the brain, but certain differences are evident. For example, they arborize only sparsely in the accessory medulla (AME), which serves as important communication center of the circadian clock in cockroaches and flies. Furthermore, all bee PDF neurons cluster together, which makes it impossible to distinguish individual projections. Here, we investigated the developing bee PDF network and found that the first three PDF neurons arise in the third larval instar and form a dense network of varicose fibers at the base of the developing medulla that strongly resembles the AME of hemimetabolous insects. In addition, they send faint fibers toward the lateral superior protocerebrum. In last larval instar, PDF cells with larger somata appear and send fibers toward the distal medulla and the medial protocerebrum. In the dorsal part of the medulla serpentine layer, a small PDF knot evolves from which PDF fibers extend ventrally. This knot disappears during metamorphosis and the varicose arborizations in the putative AME become fainter. Instead, a new strongly stained PDF fiber hub appears in front of the lobula. Simultaneously, the number of PDF neurons increases and the PDF neuronal network in the brain gets continuously more complex.

Β-endorphin-immunoreactive perikarya appear to receive innervation from NPY-immunoreactive fiber varicosities in the human hypothalamus.

Morphological and pharmacological studies indicate that hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons communicate with each other in rats and regulate a variety of hypothalamic and extrahypothalamic functions. Indeed, electron microscopic studies revealed NPY-immunoreactive (NPI-IR) synapses on β-endorphin-IR neurons in the hypothalamus. However, no such connections have been reported in humans. Here, we studied the putative NPY-β-endorphin associations with high-resolution light microscopic double-label immunocytochemistry in the human hypothalamus. The majority of β-endorphin-IR perikarya appear to be innervated by abutting NPY-IR fibers in the infundibulum/median eminence, receiving more than 6 contacts (38% of the counted neurons) or three to six contacts (42% of the counted neurons). The rest of the β-endorphin-IR neurons are lightly innervated by NPY fibers (14%, one-three contacts) or do not receive any detectable NPY-IR axon varicosities (6% of the counted neurons). Since β-endorphin is cleaved from the proopiomelanocortin (POMC) precursor, the NPY-β-endorphin connections also provide the foundation for NPY-α-MSH and NPY-ACTH connections and their subsequent physiology. The close anatomical connections between NPY-IR nerve terminals and β-endorphin-IR neurons reported herein may represent functional synapses and provide the foundation for NPY-stimulated β-endorphin release. By interacting with β-endorphin, NPY may have a more widespread regulatory capacity than acting alone on different neurotransmitter systems.

Immunocytochemical localization of tyrosine hydroxylase in the visual cortex of the microbat, Rhinolophus ferrumequinum.

In order to enhance our understanding of bat vision, we investigated tyrosine hydroxylase (TH)-immunoreactive (IR) fibers in the visual cortex of the microbat. The study was conducted on 12 freshly-caught adult bats (Rhinolophus ferrumequinum, both sexes, weighing 15-20 g). We used standard immunocytochemistry and confocal microscopy. TH-IR fibers were distributed throughout all layers of the visual cortex, with the highest density in layer I. Two types of TH-IR fibers were observed: small and large varicose fibers. TH-IR cells were not found in the microbat visual cortex. The microbat substantia nigra and ventral tegmental areas, previously identified sources of TH-IR fibers in the mammalian visual cortex, all contained strongly labeled TH-IR cells. The average diameters of TH-IR cells in the substantia nigra and the ventral tegmental areas were 14.39 ± 0.13 μm (mean ± SEM) and 11.85 ± 0.13 μm, respectively. Our results suggest that the microbat has a well-constructed neurochemical organization of THIR fibers. This observation should provide fundamental insights into a better understanding of the nocturnal, echolocating bat visual system.

Differential synapse density between Purkinje cell dendritic spine and parallel fiber varicosity in the rat cerebellum among the phylogenic lobules

The cerebellum is a region of the brain that plays an important role in motor control. It is classified phylogenetically into archicerebellum, paleocerebellum and neocerebellum. The Purkinje cells are lined in a row called Purkinje cell layer and it has a unique dendritic branches with many spines.The previous study reported that there is a difference of synapse density according to the lobules based on large two-dimensional data. However, recent study with high voltage electron microscopy showed there was no differences in dendritic spine density of the Purkinje cell according to its phylogenetic lobule. We analyzed Purkinje cell density in the II, VI and X lobules by stereological modules and synaptic density was estimated by double disector based on Purkinje cell density in the molecular layer of each lobule.The results showed that there was significant difference in the Purkinje cell density and synapse number according to their phylogenetic lobules. The number of Purkinje cell in a given volume was larger in the archicerebellum, but synapse density was higher in the neocerebellum.These data suggest that cellular and synaptic organization of the Purkinje cell is different according to their phylogenetic background.

Histochemical and immunohistochemical localization of nitrergic structures in the carotid body of spontaneously hypertensive rats

The carotid body (CB) is a multipurpose metabolic sensor that acts to initiate cardiorespiratory reflex adjustments to maintain homeostasis of blood-borne chemicals. Emerging evidence suggests that nitric oxide increases the CB chemosensory activity and this enhanced peripheral chemoreflex sensitivity contributes to sympathoexcitation and consequent pathology. The aim of this study was to examine by means of NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry the presence and distribution of nitrergic structures in the CB of spontaneously hypertensive rats (SHRs) and to compare their expression patterns to that of age-matched normotensive Wistar rats (NWRs). Histochemistry revealed that the chemosensory glomus cells were NADPH-d-negative but were encircled by fine positive varicosities, which were also dispersed in the stroma around the glomeruli. The NADPH-d-reactive fibers showed the same distributional pattern in the CB of SHRs, however their staining activity was weaker when compared with NWRs. Thin periglomerular, intraglomerular and perivascular varicose fibers, but not glomus or sustentacular cells in the hypertensive CB, constitutively expressed two isoforms of NOS, nNOS and eNOS. In addition, clusters of glomus cells and blood vessels in the CB of SHRs exhibited moderate immunoreactivity for the third known NOS isoenzyme, iNOS. The present study demonstrates that in the hypertensive CB nNOS and eNOS protein expression shows statistically significant down-regulation whereas iNOS expression is up-regulated in the glomic tissue compared to normotensive controls. Our results suggest that impaired NO synthesis could contribute to elevated blood pressure in rats via an increase in chemoexcitation and sympathetic nerve activity in the CB.

Presence of substance P positive terminals on hypothalamic somatostatinergic neurons in humans: the possible morphological substrate of the substance P-modulated growth hormone secretion.

Substance P is an undecapeptide affecting the gastrointestinal, cardiovascular, and urinary systems. In the central nervous system, substance P participates in the regulation of pain, learning, memory, and sexual homeostasis. In addition to these effects, previous papers provided solid evidence that substance P exhibits regulatory effects on growth. Indeed, our previous study revealed that growth hormone-releasing hormone (GHRH) neurons appear to be densely innervated by substance P fibers in humans. Since growth hormone secretion is regulated by the antagonistic actions of both GHRH and somatostatin, in the present paper we have examined the possibility that SP may also affect growth via the somatostatinergic system. Therefore, we have studied the putative presence of juxtapositions between the substance P-immunoreactive (IR) and somatostatinergic systems utilizing double label immunohistochemistry combined with high magnification light microscopy with oil immersion objective. In the present study, we have revealed a dense network of substance P-IR axonal varicosities contacting the majority of somatostatin-IR neurons in the human hypothalamus. Somatostatinergic perikarya are often covered by these fiber varicosities that frequently form basket-like encasements with multiple en passant type contacts, particularly in the infundibular nucleus/median eminence and in the basal periventricular area of the tuberal region. In addition, numerous substance-P-somatostatinergic juxtapositions can be found in the basal perifornical zone of the tuberal area. If these contacts are indeed functional synapses, they may represent the morphological substrate of the control of substance P on growth. Indeed, the frequency and density of these juxtapositions indicate that in addition to the regulatory action of substance P on GHRH secretion, substance P also influences growth by regulating hypothalamic somatostatinergic system via direct synaptic contacts.

Altered serotonin innervation in the rat epileptic brain

Studies in animal models of epilepsy revealed compromised serotonin (5-HT) transmission between the raphe nuclei and the brain limbic system. The goal of the present study was to evaluate the effects of epilepsy on the structural integrity of the dorsal (DR) and median (MnR) raphe nuclei and on the morphology of serotonergic fiber terminals in the dentate gyrus (DG), infralimbic cortex (IL) and medial septum (MS). The study was performed in adult Wistar rats using the kainate (9.5 mg/kg) status epilepticus (SE) model. Four months post-SE, the brainstem sections of the animals were immunostained for 5-HT, whereas the forebrain sections were immunostained for serotonin transporter (SERT). Stereological analysis revealed that epileptic rats, as compared to controls, had approximately 30% less 5-HT-stained cells in the interfascicular part of the DR, but twice as many 5-HT-stained cells in the MnR. Another finding was the reorganization of the 5-HT fiber network in all target areas analyzed, as indicated by the rightward shift of the density-size distribution histograms of SERT-stained fiber varicosities. Nonlinear regression analysis of these histograms revealed that SERT-stained varicosities were represented by two subpopulations characterized by distinct cross-sectional areas. The areal density of the small-sized varicosities was decreased in the DG (hilus and molecular layer), IL cortex (layers II/III) and MS, while that of the larger-sized varicosities was increased. The present results support the hypothesis that chronic epilepsy can trigger profound structural reorganization of the ascending serotonergic pathways in the rat brain.

Substance P appears to affect growth via growth hormone-releasing hormone (GHRH) neurons in the human hypothalamus.

Substance P is an eleven-amino acid neuropeptide (undecapeptide) with multiple effects on the gastrointestinal, cardiovascular, and urinary systems as well as complex central nervous system functions such as pain, learning, memory, and sexual homeostasis. Previous studies also revealed that substance P exhibits regulatory effects on growth possibly via influencing hypothalamic GHRH release in human. However, the morphological substrate of this phenomenon has not been elucidated yet. In the present study, we examined the putative presence of juxtapositions between the substance P- and GHRH-immunoreactive (IR) systems using double-label immunocytochemistry. High-magnification light microscopy with oil immersion was used to identify putative juxtapositions between these systems. Our studies revealed substance P-IR fiber network abutting on the surface of the majority of GHRH-immunoreactive neurons in the human hypothalamus. These fiber varicosities often cover a significant surface area on the GHRH-IR neurons, forming basket-like encasements with multiple en passant type contacts. The majority of these densely innervated GHRH-IR neurons were found in the infundibular nucleus/median eminence, while substance P-IR fibers often abut on the GHRH-IR neurons in the periventricular zone and basal perifornical area of the tuberal region and in the dorsomedial subdivision of the ventromedial nucleus. The posterior hypothalamus did not contain observable substance P-GHRH associations. The density and the morphology of these intimate associations suggest that substance P influences growth by regulating hypothalamic GHRH release by direct synaptic contacts.

Roles of cocaine- and amphetamine-regulated transcript peptide in the rostral ventrolateral medulla in cardiovascular regulation in rats

Cocaine- and amphetamine-regulated transcript peptide (CARTp) is present in neurons and varicose fibers in the rostral ventrolateral medulla (RVLM) that is crucial in the control of cardiovascular function. Prior research indicated that intracisternal administration of CARTp evokes hypertension and accumulation of Fos in the RVLM. Despite the interaction among CARTp, cardiovascular effect, and the RVLM, no studies have directly examined whether CARTp participates in cardiovascular regulation in the RVLM. The current study directly examined the modulation of blood pressure and baroreflex sensitivity by CARTp in the RVLM in the different strain of rats. Immunohistochemical study showed that CARTp immunoreactive (CART-IR) cell bodies and varicose CART-IR fibers were observed throughout the RVLM in the SD, WKY, and SHRs. Varicose CART-IR nerve fibers were particularly abundant in the WKY and SHRs. Bilateral microinjection of CARTp (30 pmol) into the RVLM caused a significant increase in mean arterial pressure (MAP) in WKY and SHRs. Bilateral microinjection of CARTp antibody (1:5000) into the RVLM displayed a fall in the basal level of the MAP in SHRs but had no effects in WKY rats. In SD rats, bilateral microinjection of CARTp (6, 30 or 60 pmol) into the RVLM did not change the MAP but attenuated phenylephrine-induced bradycardia in a dose-dependent manner. We propose that CARTp acting in the RVLM may involvement in the cardiovascular regulation either by increases in the blood pressure or by decreases in the baroreflex sensitivity in rats. Moreover, endogenous CARTp in the RVLM is associated with the maintenance of basal blood pressure of SHRs.

GnRH Neurons Provide Direct Input to Hypothalamic Tyrosine Hydroxylase Immunoreactive Neurons Which Is Maintained During Lactation.

Gonadotropin releasing hormone (GnRH) neurons provide neuronal input to the preoptic area (POA) and the arcuate nucleus (Arc), two regions involved critically in the regulation of neuroendocrine functions and associated behaviors. These areas contain tyrosine hydroxylase immunoreactive (TH-IR) neurons, which play location-specific roles in the neuroendocrine control of both the luteinizing hormone and prolactin secretion, as well as, sexually motivated behaviors. Concerning changes in the activity of GnRH neurons and the secretion pattern of GnRH seen under the influence of rising serum estrogen levels and during lactation, we tested the hypothesis that the functional state of GnRH neurons is mediated via direct synaptic connections to TH-IR neurons in the POA and Arc. In addition, we examined putative changes of these inputs in lactating mice and in mothers separated from their pups. Confocal microscopic and pre-embedding immunohistochemical studies on ovariectomized mice treated with 17β-estradiol (OVX+E2) provided evidence for direct appositions and asymmetric synapses between GnRH-IR fiber varicosities and TH-IR neurons in the POA and the Arc. As TH co-localizes with kisspeptin (KP) in the POA, confocal microscopic analysis was continued on sections additionally labeled for KP. The TH-IR neurons showed a lower level of co-labeling for KP in lactating mice compared to OVX+E2 mice (16.1 ± 5% vs. 57.8 ± 4.3%). Removing the pups for 24 h did not alter significantly the KP production in TH-IR neurons (17.3 ± 4.6%). The mean number of GnRH-IR varicosities on preoptic and arcuate TH cells did not differ in the three animal models investigated. This study shows evidence that GnRH neurons provide direct synaptic inputs to POA and Arc dopaminergic neurons. The scale of anatomical connectivity with these target cells was unaltered during lactation indicating a maintained GnRH input, inspite of the altered hormonal condition.

Immunocytochemical Localization of Choline Acetyltransferase in the Microbat Visual Cortex.

The purpose of the present study was to investigate the organization of choline acetyltransferase (ChAT)-immunoreactive (IR) fibers in the visual cortex of the microbat, using standard immunocytochemistry and confocal microscopy. ChAT-IR fibers were distributed throughout all layers of the visual cortex, with the highest density in layer III and the lowest density in layer I. However, no ChAT-IR cells were found in the microbat visual cortex. ChAT-IR fibers were classified into two types: small and large varicose fibers. Previously identified sources of cholinergic fibers in the mammalian visual cortex, the nucleus of the diagonal band, the substantia innominata, and the nucleus basalis magnocellularis, all contained strongly labeled ChAT-IR cells in the microbat. The average diameter of ChAT-IR cells in the nucleus of the diagonal band, the substantia innominata, and the nucleus basalis magnocellularis was 16.12 μm, 13.37 μm, and 13.90 μm, respectively. Our double-labeling study with ChAT and gamma-aminobutyric acid (GABA), and triple labeling with ChAT, GABA, and post synaptic density 95 (PSD-95), suggest that some ChAT-IR fibers make contact with GABAergic cells in the microbat visual cortex. Our results should provide a better understanding of the nocturnal bat visual system.

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the brain of the teleost Cyprinus carpio.

Cholinergic systems play a role in basic cerebral functions and its dysfunction is associated with deficit in neurodegenerative disease. Mechanisms involved in human brain diseases are often approached by using fish models, especially cyprinids, given basic similarities of the fish brain to that of mammals. In the present paper, the organization of central cholinergic systems have been described in the cyprinid Cyprinus carpio, the common carp, by using specific polyclonal antibodies against ChAT, the synthetic enzyme of acetylcholine, that is currently used as a specific marker for cholinergic neurons in all vertebrates. In this work, serial transverse sections of the brain and the spinal cord were immunostained for ChAT. Results showed that positive neurons are present in several nuclei of the forebrain, the midbrain, the hindbrain and the spinal cord. Moreover, ChAT-positive neurons were detected in the synencephalon and in the cerebellum. In addition to neuronal bodies, afferent varicose fibers were stained for ChAT in the ventral telencephalon, the preoptic area, the hypothalamus and the posterior tuberculum. No neuronal cell bodies were present in the telencephalon. The comparison of cholinergic distribution pattern in the Cyprinus carpio central nervous system has revealed similarities but also some interesting differences with other cyprinids. Our results provide additional information on the cholinergic system from a phylogenetic point of view and may add new perspectives to physiological roles of cholinergic system during evolution and the neuroanatomical basis of neurological diseases.