Ghrelin inhibits visceral afferent activation of catecholamine neurons in the solitary tract nucleus.

Brainstem A2/C2 catecholamine (CA) neurons in the solitary tract nucleus (NTS) are thought to play an important role in the control of food intake and other homeostatic functions. We have previously demonstrated that these neurons, which send extensive projections to brain regions involved in the regulation of appetite, are strongly and directly activated by solitary tract (ST) visceral afferents. Ghrelin, a potent orexigenic peptide released from the stomach, is proposed to act in part through modulating NTS CA neurons but the underlying cellular mechanisms are unknown. Here, we identified CA neurons using transgenic mice that express enhanced green fluorescent protein driven by the tyrosine hydroxylase promoter (TH-EGFP). We then determined how ghrelin modulates TH-EGFP neurons using patch-clamp techniques in a horizontal brain slice preparation. Ghrelin inhibited the frequency of spontaneous glutamate inputs (spontaneous EPSCs) onto TH-EGFP neurons, including cholecystokinin-sensitive neurons, an effect blocked by the GHSR1 antagonist, d-Lys-3-GHRP-6. This resulted in a decrease in the basal firing rate of NTS TH-EGFP neurons, an effect blocked by the glutamate antagonist NBQX. Ghrelin also dose-dependently inhibited the amplitude of ST afferent evoked EPSCs (ST-EPSCs) in TH-EGFP NTS neurons, decreasing the success rate for ST-evoked action potentials. In addition, ghrelin decreased the frequency of mini-EPSCs suggesting its actions are presynaptic to reduce glutamate release. Last, inhibition by ghrelin of the ST-EPSCs was significantly increased by an 18 h fast. These results demonstrate a potential mechanism by which ghrelin inhibits NTS TH neurons through a pathway whose responsiveness is increased during fasting.

Lbx1 acts as a selector gene in the fate determination of somatosensory and viscerosensory relay neurons in the hindbrain.

Distinct types of relay neurons in the hindbrain process somatosensory or viscerosensory information. How neurons choose between these two fates is unclear. We show here that the homeobox gene Lbx1 is essential for imposing a somatosensory fate on relay neurons in the hindbrain. In Lbx1 mutant mice, viscerosensory relay neurons are specified at the expense of somatosensory relay neurons. Thus Lbx1 expression distinguishes between the somatosensory or viscerosensory fate of relay neurons.

Modulation of taste sensitivity by GLP-1 signaling.

In many sensory systems, stimulus sensitivity is dynamically modulated through mechanisms of peripheral adaptation, efferent input, or hormonal action. In this way, responses to sensory stimuli can be optimized in the context of both the environment and the physiological state of the animal. Although the gustatory system critically influences food preference, food intake and metabolic homeostasis, the mechanisms for modulating taste sensitivity are poorly understood. In this study, we report that glucagon-like peptide-1 (GLP-1) signaling in taste buds modulates taste sensitivity in behaving mice. We find that GLP-1 is produced in two distinct subsets of mammalian taste cells, while the GLP-1 receptor is expressed on adjacent intragemmal afferent nerve fibers. GLP-1 receptor knockout mice show dramatically reduced taste responses to sweeteners in behavioral assays, indicating that GLP-1 signaling normally acts to maintain or enhance sweet taste sensitivity. A modest increase in citric acid taste sensitivity in these knockout mice suggests GLP-1 signaling may modulate sour taste, as well. Together, these findings suggest a novel paracrine mechanism for the regulation of taste function.

Lateral parabrachial afferent areas and serotonin mechanisms activated by volume expansion.

Recent evidence has shown that the serotonergic mechanism of the lateral parabrachial nucleus (LPBN) participates in the regulation of renal and hormonal responses to isotonic blood volume expansion (BVE). We investigated the BVE-induced Fos activation along forebrain and hindbrain nuclei and particularly within the serotonergic clusters of the raphé system that directly project to the LPBN. We also examined whether there are changes in the concentration of serotonin (5HT) within the raphé nucleus in response to the same stimulus. With this purpose, we analyzed the cells doubly labeled for Fos and Fluorogold (FG) following BVE (NaCl 0.15 M, 2 ml/100 g b.w., 1 min) 7 days after FG injection into the LPBN. Compared with the control group, blood volume-expanded rats showed a significant greater number of Fos-FG double-labeled cells along the nucleus of the solitary tract, locus coeruleus, hypothalamic paraventricular nucleus, central extended amygdala complex, and dorsal raphé nucleus (DRN) cells. Our study also showed an increase in the number of serotonergic DRN neurons activated in response to isotonic BVE. We also observed decreased levels of 5HT and its metabolite 5-hydroxyindoleacetic acid (measured by high-pressure liquid chromatography) within the raphé nucleus 15 min after BVE. Given our previous evidence on the role of the serotonergic system in the LPBN after BVE, the present morphofunctional findings suggest the existence of a key pathway (DRN-LPBN) that may control BVE response through the modulation of 5HT release.

GABAA receptors expression pattern in rat brain following low pressure distension of the stomach.

It is known that gastric mechanoreceptor stimuli are widely integrated into neuronal circuits that involve visceral nuclei of hindbrain as well as several central brain areas. GABAergic neurons are widely represented in hindbrain nuclei controlling gastric motor functions, but limited information is available specifically about GABA(A)-responding neurons in brain visceral areas. The present investigation was designed to determine the central sensory neuronal pathways and their GABA(A)-alpha1 and -alpha3 receptor presenting neurons that respond to gastric mechanoreceptor stimulation within the entire rat brain. Low pressure gastric distension was used to deliver physiological mechanical stimuli in anesthetized rats, and different protocols of gastric distension were performed to mimic different stimulation patterns with and without sectioning vagal and/or splanchnic afferent nerves. Mapping of activated neurons was investigated using double colorimetric immunohistochemistry for GABA(A)-alpha1 or -alpha3 subunits and c-Fos. Following stomach distension, neurons expressing GABA(A) receptors with alpha1 or alpha3 subunits were detected. Low frequency gastric distension induced c-Fos expression in nucleus tractus solitarii (NTS) only, whereas in the high frequency gastric distension c-Fos positive nuclei were found in lateral reticular nucleus and in NTS in addition to some forebrain areas. In contrast, during the tonic-rapid gastric distension the neuronal activation was found in hindbrain, midbrain and forebrain areas. Moreover different protocols of gastric stimulation activated diverse patterns of neurons presenting GABA(A)-alpha1 or -alpha3 receptors within responding brain nuclei, which may indicate a probable functional significance of differential expression of GABA(A)-responding neurons. The same protocol of gastric distension performed in vagotomized rats has confirmed the primary role of the vagus in the response of activation of gastric brain areas, whereas neuronal input of splanchnic origins was shown to play an important role in modulating the mechanogastric response of brain areas.

Acute myocardial ischemia up-regulates nociceptin/orphanin FQ in dorsal root ganglion and spinal cord of rats.

Nociceptin/orphanin FQ (N/OFQ) possesses modulatory effects on somatic noxious signals in spinal cord, while the potential role in visceral nociception remains elusive. We designed this study to investigate the hypothesis that cardiac nociceptive signals from acute ischemic myocardium to the spinal cord are transmitted or modulated by mechanisms including N/OFQ. We examined the changes of N/OFQ and its mRNA in the dorsal root ganglia and spinal cord of upper thoracic segments innervating the heart of rats. Thoracic epidural anesthesia was performed to confirm neural mechanism underlying the changes. We observed that selective coronary artery occlusion significantly up-regulated N/OFQ and ppN/OFQ mRNA in the dorsal root ganglia and spinal cord. Thoracic epidural anesthesia abolished the changes in the expression of N/OFQ and its mRNA. The observations indicate that cardiac noxious neural afferent drive is responsible for the up-regulation of N/OFQ in the primary afferent neurons and intrinsic spinal neurons.

Structure and development of the subesophageal zone of the Drosophila brain. II. Sensory compartments.

The subesophageal zone (SEZ) of the Drosophila brain processes mechanosensory and gustatory sensory input from sensilla located on the head, mouth cavity and trunk. Motor output from the SEZ directly controls the movements involved in feeding behavior. In an accompanying paper (Hartenstein et al., ), we analyzed the systems of fiber tracts and secondary lineages to establish reliable criteria for defining boundaries between the four neuromeres of the SEZ, as well as discrete longitudinal neuropil domains within each SEZ neuromere. Here we use this anatomical framework to systematically map the sensory projections entering the SEZ throughout development. Our findings show continuity between larval and adult sensory neuropils. Gustatory axons from internal and external taste sensilla of the larva and adult form two closely related sensory projections, (a) the anterior central sensory center located deep in the ventromedial neuropil of the tritocerebrum and mandibular neuromere, and (b) the anterior ventral sensory center (AVSC), occupying a superficial layer within the ventromedial tritocerebrum. Additional, presumed mechanosensory terminal axons entering via the labial nerve define the ventromedial sensory center (VMSC) in the maxilla and labium. Mechanosensory afferents of the massive array of chordotonal organs (Johnston's organ) of the adult antenna project into the centrolateral neuropil column of the anterior SEZ, creating the antenno-mechanosensory and motor center (AMMC). Dendritic projections of dye back-filled motor neurons extend throughout a ventral layer of the SEZ, overlapping widely with the AVSC and VMSC. Our findings elucidate fundamental structural aspects of the developing sensory systems in Drosophila.

Activity-dependent reorganization of local circuitry in the developing visceral sensory system.

Neural activity during critical periods could fine-tune functional synaptic connections. N-methyl-d-aspartate (NMDA) receptor activation is critically implicated in this process and blockade leads to disruption of normal circuit formation. This phenomenon has been well investigated in several neural systems including the somatosensory system, but not yet evidenced in the visceral sensory system. Ultrastructural analysis of GABAergic synapses and electrophysiological analysis of inhibitory and excitatory postsynaptic currents of the rat caudal nucleus tractus solitarii (NTS) cells revealed that developmental changes in the synaptic organizations were blocked by MK-801, an NMDA receptor antagonist, when administered at postnatal days 5-8, a presumed critical period for the visceral sensory system. Normal synapse reorganization during postnatal development dictates undifferentiated neonatal caudal NTS neurons in terms of synaptic input patterns measured by electron microscopy and electrophysiology into two cell groups: small and large cells under far stronger excitatory and inhibitory influence, respectively. Blockade by MK-801 during the critical period might leave adult neurons wired in the undifferentiated synaptic networks, possibly preventing synapse elimination and subsequent stabilization of the proper wiring.

Organization of immune-responsive medullary projections to the bed nucleus of the stria terminalis, central amygdala, and paraventricular nucleus of the hypothalamus: evidence for parallel viscerosensory pathways in the rat brain.

Immune-responsive neurons in the brainstem, primarily in the nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM), contribute to a significant drive on forebrain nuclei responsible for brain-mediated host defense responses. The current study investigated the relative contribution of brainstem-derived ascending pathways to forebrain immune-responsive nuclei in the rat by means of retrograde tract tracing and c-Fos immunohistochemistry. Fluorogold was iontophoresed into the bed nucleus of stria terminalis (BST), central nucleus of the amygdala (CEA), paraventricular nucleus of the hypothalamus (PVN), and the pontine lateral parabrachial nucleus (PBL; an important component of ascending viscerosensensory pathways) followed 2 weeks later by intraperitoneal injection of lipopolysaccharide (LPS, 0.1 mg/kg) or saline. The NTS and VLM provide immune-responsive input to all four regions, via direct, predominantly catecholaminergic, projections to the PVN, the lateral BST, and the CEA, and mostly non-catecholaminergic projections to the PBL. The PBL provides a major LPS-activated input to the BST and CEA. The pattern of LPS-activated catecholaminergic projections from the VLM and NTS to the forebrain is characterized by a strong predominance of VLM input to the PVN, whereas the NTS provides a greater contribution to the BST. These findings indicate that direct and indirect pathways originate in the caudal brainstem that propagate immune-related information from the periphery with multiple levels of processing en route to the forebrain nuclei, which may allow for integration of brain responses to infection.

Glutamatergic vestibular neurons express Fos after vestibular stimulation and project to the NTS and the PBN in rats.

In this study, retrograde tracing method combined with phosphate-activated glutaminase (PAG) and Fos immunofluorescence histochemistry was used to identify glutamatergic vestibular nucleus (VN) neurons receiving vestibular inputs and projecting to the nucleus of the solitary tract (NTS) and the parabrachial nucleus (PBN). Conscious animals were subjected to 120 min Ferris-wheel like rotation stimulation. Neuronal activation was assessed by Fos expression in the nucleus of VN neurons. After Fluoro-gold (FG) injection into the caudal NTS, approximately 48% FG-labeled VN neurons were immunoreactive for PAG, and about 14% PAG/FG double-labeled neurons co-existed with Fos. Following FG injection into the PBN, approximately 56% FG-labeled VN neurons were double-labeled with PAG, and about 12% of the PAG/FG double-labeled neurons also expressed Fos. Careful examination of the typology and distribution pattern of these PAG-immunoreactive neurons indicated that the vast majority of these neurons were glutamatergic rather than GABAergic. These results suggest that PAG-immunoreactive VN neurons might constitute excitatory glutamatergic VN-NTS and VN-PBN transmission pathways and these pathways might be involved in vestibulo-autonomic reflexes during vestibular stimulation.

Two closely located areas in the suboesophageal ganglion and the tritocerebrum receive projections of gustatory receptor neurons located on the antennae and the proboscis in the moth Heliothis virescens.

Sucrose stimulation of gustatory receptor neurons on the antennae, the tarsi, and the mouthparts elicits the proboscis extension reflex in many insect species, including lepidopterans. The sensory pathways involved in this reflex have only partly been investigated, and in hymenopterans only. The present paper concerns the pathways of the gustatory receptor neurons on the antennae and on the proboscis involved in the proboscis extension reflex in the moth Heliothis virescens (Lepidoptera; Noctuidae). Fluorescent dyes were applied to the contact chemosensilla, sensilla chaetica on the antennae, and sensilla styloconica on the proboscis, permitting tracing of the axons of the gustatory receptor neurons in the central nervous system. The stained axons showed projections from the two appendages in two closely located but distinct areas in the suboesophageal ganglion (SOG)/tritocerebrum. The projections of the antennal gustatory receptor neurons were located posterior-laterally to those from the proboscis. Electrophysiological recordings from the receptor neurons in s. chaetica during mechanical and chemical stimulation were performed, showing responses of one mechanosensory and of several gustatory receptor neurons. Separate neurons showed excitatory responses to sucrose and sinigrin. The effect of these two tastants on the proboscis extension reflex was tested by repeated stimulations with solutions of the two compounds. Whereas sucrose elicited extension in 100% of the individuals in all repetitions, sinigrin elicited extension in fewer individuals, a number that decreased with repeated stimulation.

Gustatory terminal field organization and developmental plasticity in the nucleus of the solitary tract revealed through triple-fluorescence labeling.

Early dietary sodium restriction has profound influences on the organization of the gustatory brainstem. However, the anatomical relationships among multiple gustatory nerve inputs have not been examined. Through the use of triple-fluorescence labeling and confocal laser microscopy, terminal fields of the greater superficial petrosal (GSP), chorda tympani (CT), and glossopharyngeal (IX) nerves were visualized concurrently in the nucleus of the solitary tract (NTS) of developmentally sodium-restricted and control rats. Dietary sodium restriction during pre- and postnatal development resulted in a twofold increase in the volume of both the CT and the IX nerve terminal fields but did not affect the volume of the GSP terminal field. In controls, these nerve terminal fields overlapped considerably. The dietary manipulation significantly increased the overlapping zones among terminal fields, resulting in an extension of CT and IX fields past their normal boundaries. The differences in terminal field volumes were exaggerated when expressed relative to the respective NTS volumes. Furthermore, increased terminal field volumes could not be attributed to an increase in the number of afferents because ganglion cell counts did not differ between groups. Taken together, selective increases in terminal field volume and ensuing overlap among terminal fields suggest an increased convergence of these gustatory nerve terminals onto neurons in the NTS. The genesis of such convergence is likely related to disruption of cellular and molecular mechanisms during the development of individual terminal fields, the consequences of which have implications for corresponding functional and behavioral alterations.

Vagal afferent neurons projecting to the stomach and small intestine exhibit multiple N-methyl-D-aspartate receptor subunit phenotypes.

Previous reports suggest that NMDA receptors participate in control of food intake via vagal afferent neurons that innervate the upper gastrointestinal (GI) tract. While messenger RNA coding for the NR1 NMDA receptor subunit is present in a majority of vagal afferent neurons of nodose ganglia (NG), immunoreactivity for other NMDA receptor subunits (NR2B, NR2C and NR2D) are expressed in more limited subpopulations of vagal afferents. To determine whether vagal afferent neurons that project to the stomach or duodenum exhibit distinct NMDA receptor subunit phenotypes, we examined immunoreactivity (IR) for NMDA receptor NR1, NR2B, NR2C and NR2D subunits in NG neurons that were labeled by injections of the retrograde tracer Fast Blue (FB) into the wall of the stomach or duodenum. FB injections into the fundus or corpus of the stomach labeled comparable numbers of neurons in both the left and right NG, while proximal duodenal injections labeled only neurons of left NG. NR1-IR expression was observed in most neurons innervating the upper GI tract (fundus, 97%; corpus, 95%; duodenum, 98%). Likewise, most neurons that innervated the upper GI tract expressed NR2B-IR (fundus, 98%; corpus, 85%; duodenum, 81%). NR2C-IR was observed in only 52%, 46% and 32% of FB-positive neurons projecting to the fundus, corpus or duodenum respectively, while NR2D-IR occurred in an even more restricted FB-labeled subpopulation (fundus, 13%; corpus, 26%; and duodenum, 18%). Our observations indicate that different subpopulations of vagal afferents express distinct NMDA receptor subunit phenotypes. However, the neuronal distribution of NMDA receptor subunits is not correlated with innervation of either the stomach or duodenum.

Local axonal arborization patterns of distinct neuronal types in the caudal nucleus of the tractus solitarius.

Neurons in the caudal nucleus of the tractus solitarius (cNTS) are quite heterogeneous in cell size (50 to 450 microm(2) in somal area) and other morphologic characteristics. For a more objective classification of cNTS neurons, their morphologic features were analyzed quantitatively based on reconstructed biocytin-filled cells after whole-cell patch-clamp recordings. According to the patterns of axonal branching behaviors, cNTS cells could be classified into two groups: smaller cells (94.1 microm(2) in mean somal area, range 62-120 microm(2), n = 22) and larger cells (245 microm(2) in mean somal area, range 142-411 microm(2), n = 23). Extensive axonal arborization with numerous possible synaptic boutons was specifically associated with smaller neurons, while larger cells possessed no or few axon collaterals, suggesting their distinct roles as local circuit neurons (or interneurons) and projection neurons, respectively. With regard to somatodendritic characteristics, the following correlations with cell size were found: smaller cells had larger form factors than larger cells (P < 0.05). Larger neurons had more extensive dendritic arborization, expressed by total dendritic length (P < 0.01) and number of dendritic branching points (P < 0.01), than smaller cells. It was suggested that small cNTS neurons contribute specifically to an integration of input information generated in the local circuits, while large neurons convey the integrated information to other autonomic brain regions.

Expression of hyperpolarization-activated cyclic nucleotide-gated cation channels in rat dorsal root ganglion neurons innervating urinary bladder.

Afferent pathways innervating the urinary bladder consist of myelinated Adelta- and unmyelinated C-fibers, the neuronal cell bodies of which correspond to medium and small-sized cell populations of dorsal root ganglion (DRG) neurons, respectively. Since hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel currents have been identified in various peripheral sensory neurons, we examined the expression of isoforms of HCN channels in the L6-S1 spinal cord and bladder afferent neurons from L6-S1 DRG in rats. Among HCN-1, HCN-2 and HCN-4 channel subtypes, positive staining with HCN-2 antibodies was found in the superficial dorsal horn of the spinal cord and small- and medium-sized unidentified DRG neurons. In dye-labeled bladder afferent neurons, HCN-2-positive cells were found in approximately 60% of neurons, and HCN-2 was expressed in both small- and medium-sized neurons with a higher ratio (expression ratio: 61% and 50% of neurons, respectively) compared with unidentified DRG neurons, in which the HCN expression ratio was 47% and 21% of small- and medium-sized cells, respectively. These results suggest that HCN-2 is the predominant subtype of HCN channels, which can control neuronal excitability, in small-sized C-fiber and medium-sized Adelta fiber DRG neurons including bladder afferent neurons, and might modulate activity of bladder afferent pathways controlling the micturition reflex.

Ablation of NK1 receptor bearing neurons in the nucleus of the solitary tract blunts cardiovascular reflexes in awake rats.

The nucleus of the solitary tract (NTS) receives primary afferents involved in cardiovascular regulation. We investigated the role of NK(1)-receptor bearing neurons in the NTS on cardiovascular reflexes in awake rats fitted with chronic venous and arterial cannulae. These neurons were lesioned selectively with saporin conjugated with substance P (SP-SAP, 2 microM, bilateral injections of 20 nL in the subpostremal NTS, or 200 nL in both the subpostremal and the commissural NTS). Before, and 7 and 14 days after injection of SP-SAP, we measured changes in blood pressure and heart rate induced by i.v. injection of phenylephrine and nitroprusside (baroreceptor reflex), cyanide (arterial chemoreceptor reflex), and phenylbiguanide (Bezold-Jarisch reflex). The smaller injections with SP-SAP completely abolished NK1 receptor staining in the subpostremal NTS. The larger injections abolished NK1 receptor immunoreactivity in an area that extended from the commissural NTS to the rostral end of the subpostremal NTS. The lesions seemed to affect only a limited number of neurons, since neutral red stained sections did not show any obvious reduction in cell number. The smaller lesions reduced the gain of baroreflex bradycardia and the hypotension induced by phenylbiguanide. The larger lesions completely abolished the response to phenylbiguanide, blocked the baroreflex bradycardia induced by phenylephrine, severely blunted the baroreflex tachycardia, and blocked the bradycardia and reduced the hypertension induced by cyanide. Thus, these responses depend critically on NK(1)-receptor bearing neurons in the NTS.

Novel gut afferents: Intrinsic afferent neurons and intestinofugal neurons.

Information about the conditions of all tissues in the body is conveyed to the central nervous system through afferent neurons. Uniquely amongst peripheral organs, the intestine has numerous additional afferent neurons, intrinsic primary afferent neurons that have their cell bodies and processes in the enteric plexuses and do not project to the central nervous system. They detect conditions within the gut and convey that information to intrinsic reflex pathways that are also entirely contained inside the gut wall. Intrinsic primary afferent neurons respond both to the presence of material in the gut lumen and to distension of the gut wall and initiate reflex changes in contractile activity, fluid transport across the mucosa and local blood flow. They also function as nociceptors that initiate tissue-protective propulsive and secretory reflexes to rid the gut of pathogens. The regulation of excitability of intrinsic primary afferent neurons is through multiple ion channels and ion channel regulators, and their excitability is critical to setting the strength of enteric reflexes. The intestine also provides afferent signals to sympathetic pre-vertebral ganglia. The signals are conveyed from the gut by intestinofugal neurons that have their cell bodies within enteric ganglia and form synapses in the sympathetic ganglia. Intestinofugal neurons form parts of the afferent limbs of entero-enteric inhibitory reflexes. Because the unusual afferent neurons of the small intestine and colon make their synaptic connections outside the central nervous system, the neurons and the reflex centres that they affect are potential targets for non-central penetrant therapeutic compounds.

Signalling the state of the digestive tract.

The gastrointestinal tract has a rich sensory innervation. Extrinsic afferents in vagal, splanchnic and pelvic nerves project to the CNS where gut reflex function is coordinated and integrated with behavioural responses (e.g. regulation of food intake) and mediate sensations. The afferent information conveyed by vagal and spinal mechanosensitive afferents can be very different. Vagal afferents have low thresholds of activation and reach maximal responses within physiological levels of distension. In contrast, spinal afferents, although many have corresponding thresholds for activation, are able to respond beyond the physiological range and encode both physiological and noxious levels of stimulation. However, mechanosensitivity is not fixed but can be influenced by a wide range of chemical mediators released as a consequence of ischemia, injury and inflammation. Indeed, previously mechanical insensitive afferents can develop mechanosensitivity during inflammation and a variety of chemical mediators are implicated in this sensitisation process. Chemosensitivity is also a property of vagal mucosal afferents that detect the chemical milieu for chemicals absorbed across the epithelium or released from enteroendocrine cells that are strategically positioned to "taste" luminal contents. Thus, there exists a complex interplay between immunomodulators, neurotransmitters and neuroendocrine factors that underlie gastrointestinal sensing mechanisms and enable orchestration of appropriate host responses.

Development of the human nucleus of the solitary tract: a cyto- and chemoarchitectural study.

The present study investigated the prenatal development of the cyto- and chemoarchitecture of the human nucleus of the solitary tract from 9 to 35 weeks, by using Nissl staining and immunoreactivity to calbindin, calretinin, tyrosine hydroxylase and GAP-43. The nucleus began to gain heterogeneity and show different subnuclei as early as 13 weeks, and approached cytoarchitectural maturation from 21 to 25 weeks. The subnuclear division pattern observed in the fetal nucleus of the solitary tract at 25 weeks was very similar to that of the adult. Neurons immunoreactive to calbindin first appeared in the medial gastrointestinal area of the nucleus at 13 weeks, particularly within a putative gelatinosus subnucleus, while calretinin immunoreactivity during fetal life suggested the possible presence of a central subnucleus. Tyrosine hydroxylase immunoreactive neurons were seen in the medial subdivisions of the nucleus of the solitary tract as early as 13 weeks, but the population continued to increase until 25 weeks. Strong GAP-43 immunoreactivity was also present in the nucleus of the solitary tract at 13 weeks, especially in the dorsolateral and commissural subnuclei, while at 21 weeks there was a significant decline of GAP-43 expression. Results from the chemoarchitectural study showed that the human nucleus of the solitary tract expressed various neurochemical substances at an early developmental age (13 weeks), even before cellular and neuropil maturation was fully attained. Expression of these factors may play an important role in establishment and integration of viscerosensory function in the nucleus.

Soluble guanylate cyclase and neuronal nitric oxide synthase colocalize in rat nucleus tractus solitarii.

Nitric oxide has been implicated in transmission of cardiovascular signals in the nucleus tractus solitarii (NTS). Pharmacological studies suggest that activation of neurons by nitric oxide in the NTS may involve soluble guanylate cyclase (sGC). However, anatomical data supporting this suggestion have not been available. In this study, we tested the hypothesis that neurons and fibers containing neuronal nitric oxide synthase (nNOS) lie in close proximity to those containing sGC and the two enzymes colocalize in some neurons and fibers in the NTS. We perfused six rats and obtained brain stem sections for double immunofluorescent staining utilizing antibodies selective for sGC and for nNOS combined with confocal microscopy. The distribution and staining intensity of nNOS-immunoreactivity (IR) was similar to our earlier reports. IR of sGC was present in cell bodies, proximal dendrites and fibers of many brain stem regions. Strong sGC-IR was noted in the hypoglossal, dorsal motor nucleus of vagus and gracilis nuclei. The NTS exhibited moderate sGC-IR. Superimposed images showed that many NTS neurons contained both nNOS-IR and sGC-IR. The percentage of sGC-IR positive cells that were also nNOS-IR positive differed among NTS subnuclei. Similarly, the percentage of nNOS-IR positive cells that were also sGC positive differed among NTS subnuclei. Fibers stained for both nNOS-IR and sGC-IR were also present in NTS subnuclei. In addition, we identified fibers that were stained for nNOS-IR or sGC-IR alone and often found such singly labeled fibers apposed to each other. These data support our hypothesis and provide anatomical support for the suggestion that nitroxidergic activation of the NTS involves sGC.