Dental afferents project onto gustatory neurons in the nucleus of the solitary tract.

The aim of this study was to investigate the inferior alveolar nerve (IAN) and chorda tympani (CT) projections onto gustatory neurons of the nucleus of the solitary tract (NST) in the rat by immunochemical and electrophysiological techniques. IAN afferents were retrogradely labeled. NST neurons were labeled either by retrograde tracer injection into the parabrachial nucleus (PBN) or by c-Fos mapping after CT activation. NST neurons responding to tastant stimulation were recorded in vivo before and after electrical stimulation of the IAN. Results from the immunolabeling approach showed IAN boutons "en passant" apposed to retrogradely labeled neurons from PBN and to CT-activated neurons in the NST. Recordings of single NST neurons showed that the electrical stimulation of the IAN significantly decreased CT gustatory responses. Analysis of these data provides an anatomical and physiological basis to support trigeminal dental and gustatory interactions within the brainstem.

The cardiovascular inhibition functions of hydrogen sulfide within the nucleus tractus solitarii are mediated by the activation of KATP channels and glutamate receptors mechanisms.

Hydrogen sulfide (H2S), the colorless gas with the smell of rotten eggs, has been regarded as a novel gaseous signaling molecule. Although H2S has been proved been involved into the cardiovascular functions, the cardiovascular functions of H2S within the nucleus tractus solitarii (NTS) are not clear. Unilateral microinjection of NaHS (2 to 200 pmol), a H2S donor, into the NTS caused transient and dose-dependent hypotension and bradycardia (P<0.01). Microinjection of CBS allosteric activator S-ademetionine (SAM) into the NTS also produced significant decreases in BP (from 101 +/- 8 to 82 +/- 7 mmHg, P < 0.01) and HR (from 469 +/- 16 to 449 +/- 14 bpm, P<0.01), which was very similar to those of NaHS. Pretreatment with hydroxylamine, a CBS inhibitor, failed to affect the cardiovascular functions of intra-NTS NaHS. However, pretreatment with glibenclamide (10 nmol), a KATP channel blocker, eliminated the on BP (from -23 +/- 4 to -5 +/- 1 mmHg, P<0.01) and HR (from -24 +/- 2 to -5 +/- 1 bpm, P<0.01) by 78% and 79%, respectively, of intra-NTS NaHS (20 pmol). Likewise, pretreatment with kynurenic acid (Kyn, 5 nmol) also attenuated the effects of NaHS on BP (from -29 +/- 3 to -12 +/- 3 mmHg, P<0.01) and HR (from -19 +/- 2 to -9 +/- 2 bpm, P<0.01) by 59% and 53%, respectively, of intra-NTS NaHS (20 pmol). These data support the hypothesis that endogenous H2S produces cardiovascular inhibition functions in the NTS, mainly mediated by KATP channels regulation or/and glutamate receptors.

Three-dimensional macronutrient-associated Fos expression patterns in the mouse brainstem.

BACKGROUND: The caudal brainstem plays an important role in short-term satiation and in the control of meal termination. Meal-related stimuli sensed by the gastrointestinal (GI) tract are transmitted to the area postrema (AP) via the bloodstream, or to the nucleus tractus solitarii (NTS) via the vagus nerve. Little is known about the encoding of macronutrient-specific signals in the caudal brainstem. We hypothesized that sucrose and casein peptone activate spatially distinct sub-populations of NTS neurons and thus characterized the latter using statistical three-dimensional modeling. METHODOLOGY/PRINCIPAL FINDINGS: Using immunolabeling of the proto-oncogene Fos as a marker of neuronal activity, in combination with a statistical three-dimensional modeling approach, we have shown that NTS neurons activated by sucrose or peptone gavage occupy distinct, although partially overlapping, positions. Specifically, when compared to their homologues in peptone-treated mice, three-dimensional models calculated from neuronal density maps following sucrose gavage showed that Fos-positive neurons occupy a more lateral position at the rostral end of the NTS, and a more dorsal position at the caudal end. CONCLUSION/SIGNIFICANCE: To our knowledge, this is the first time that subpopulations of NTS neurons have be distinguished according to the spatial organization of their functional response. Such neuronal activity patterns may be of particular relevance to understanding the mechanisms that support the central encoding of signals related to the presence of macronutrients in the GI tract during digestion. Finally, this finding also illustrates the usefulness of statistical three-dimensional modeling to functional neuroanatomical studies.

Activation of neurons in the hypothalamic dorsomedial nucleus via hypothalamic projections of the nucleus of the solitary tract following refeeding of fasted rats.

We report that satiation evokes neuronal activity in the ventral subdivision of the hypothalamic dorsomedial nucleus (DMH) as indicated by increased c-fos expression in response to refeeding in fasted rats. The absence of significant Fos activation following food presentation without consumption suggests that satiation but not craving for food elicits the activation of ventral DMH neurons. The distribution pattern of the prolactin-releasing peptide (PrRP)-immunoreactive (ir) network showed remarkable correlations with the distribution of activated neurons within the DMH. The PrRP-ir fibers and terminals were immunolabeled with tyrosine hydroxylase, suggesting their origin in lower brainstem instead of local, hypothalamic PrRP cells. PrRP-ir fibers arising from neurons of the nucleus of the solitary tract could be followed to the hypothalamus. Unilateral transections of these fibers at pontine and caudal hypothalamic levels resulted in a disappearance of the dense PrRP-ir network in the ventral DMH while PrRP immunoreactivity was increased in transected fibers caudal to the knife cuts as well as in perikarya of the nucleus of the solitary tract ipsilateral to the transections. In accord with these changes, the number of Fos-expressing neurons following refeeding declined in the ipsilateral but remained high in the contralateral DMH. However, the Fos response in the ventral DMH was not attenuated following chemical lesion (neonatal monosodium glutamate treatment) of the hypothalamic arcuate nucleus, another possible source of DMH inputs. These findings suggest that PrRP projections from the nucleus of the solitary tract contribute to the activation of ventral DMH neurons during refeeding, possibly by transferring information on cholecystokinin-mediated satiation.

Interaction of purinergic and nitrergic mechanisms in the caudal nucleus tractus solitarii of rats.

The interaction of purinergic and nitrergic mechanisms was evaluated in the caudal nucleus tractus solitarii (cNTS) using awake animals and brainstem slices. In awake animals, ATP (1.25 nmol/50 nL) was microinjected into the cNTS before and after the microinjection of a selective neuronal nitric oxide synthase (nNOS) inhibitor N-propyl-l-arginine (NPLA, 3 pmoles/50 nL, n=8) or vehicle (saline, n=4), and cardiovascular and ventilatory parameters were recorded. In brainstem slices from a distinct group of rats, the effects of ATP on the NO concentration in the cNTS using the fluorescent dye DAF-2 DA were evaluated. For this purpose brainstem slices (150 microm) containing the cNTS were pre-incubated with ATP (500 microM; n=8) before and during DAF-2 DA loading. Microinjection of ATP into the cNTS increases the arterial pressure (AP), respiratory frequency (f(R)) and minute ventilation (V(E)), which were significantly reduced by pretreatment with N-PLA, a selective nNOS inhibitor (AP: 39+/-3 vs 16+/-14 mm Hg; f(R): 75+/-14 vs 4+/-3 cpm; V(E): 909+/-159 vs 77+/-39 mL kg(-1) m(-1)). The effects of ATP in the cNTS were not affected by microinjection of saline. ATP significantly increased the NO fluorescence in the cNTS (62+/-7 vs 101+/-10 AU). The data show that in the cNTS: a) the NO production is increased by ATP; b) NO formation by nNOS is involved in the cardiovascular and ventilatory responses to microinjection of ATP. Taken together, these data suggest an interaction of purinergic and nitrergic mechanisms in the cNTS.

Trigeminal projections on gustatory neurons of the nucleus of the solitary tract: a double-label strategy using electrical stimulation of the chorda tympani and tracer injection in the lingual nerve.

Taste and sensory information are closely associated and our electrophysiological studies showed a trigeminal modulation of gustatory neurons in the nucleus of the solitary tract (NST). Chorda tympani (CT) and lingual nerves (LN) converge centrally in the rostral subdivision of the NST in hamsters and rats. However, no study has yet revealed the details of this overlap on a same section. We therefore used a double-label strategy to visualize neurons in the NST that receive both trigeminal and gustatory inputs. An anterograde tracer (BDA, Biotinylated Dextran Amine) was applied unilaterally to the cut central end of the LN in male Sprague-Dawley rats. One week later, the ipsilateral CT was electrically stimulated, after which animals were perfused and brainstem sections double-labelled for Fos immunoreactivity of activated NST neurons and BDA labelling of LN afferents. Our results permitted to circumscribe the regional overlap of the trigeminal and CT afferents mainly in the rostral central (RC) subdivision of the gustatory NST. Fos-immunoreactive neurons were observed to be closely apposed by BDA-labelled fibres and terminal boutons. Such varicosities mainly "en passant" were especially present in the RC zone of the nucleus. These observations provide an anatomical substrate for trigemino-gustatory interactions.

Oestrogen receptors in the central nervous system and evidence for their role in the control of cardiovascular function.

Oestrogen is considered beneficial to cardiovascular health through protective effects not only on the heart and vasculature, but also on the autonomic nervous system via actions on oestrogen receptors. A plethora of evidence supports a role for the hormone within the central nervous system in modulating the pathways regulating cardiovascular function. A complex interaction of several brainstem, spinal and forebrain nuclei is required to receive, integrate and co-ordinate inputs that contribute appropriate autonomic reflex responses to changes in blood pressure and other cardiovascular parameters. Central effects of oestrogen and oestrogen receptors have already been demonstrated in many of these areas. In addition to the classical nuclear oestrogen receptors (ERalpha and ERbeta) a recently discovered G-protein coupled receptor, GPR30, has been shown to be a novel mediator of oestrogenic action. Many anatomical and molecular studies have described a considerable overlap in the regional expression of these receptors; however, the receptors do exhibit specific characteristics and subtype specific expression is found in many autonomic brain areas, for example ERbeta appears to predominate in the hypothalamic paraventricular nucleus, whilst ERalpha is important in the nucleus of the solitary tract. This review provides an overview of the available information on the localisation of oestrogen receptor subtypes and their multitude of possible modulatory actions in different groups of neurochemically and functionally defined neurones in autonomic-related areas of the brain.

Role of reactive oxygen species in brainstem in neural mechanisms of hypertension.

The involvement of reactive oxygen species such as superoxide is implicated in the pathogenesis of hypertension. The brain contains a high concentration of polyunsaturated fatty acids in its cell membranes. These fatty acids are targets of oxygen-derived free radicals. Thiobarbituric acid-reactive substances (TBARS), an indirect marker of oxidative stress, are increased in the brainstem of stroke-prone spontaneously hypertensive rats (SHRSP) compared with those of Wistar-Kyoto rats (WKY). In addition, the intensity of electron spin resonance signals taken from the rostral ventrolateral medulla (RVLM), a cardiovascular center, decreases more rapidly in SHRSP than in WKY. To confirm the role of reactive oxygen species in the RVLM or the nucleus tractus solitarius (NTS) in SHRSP, we transfected adenovirus vectors encoding the manganese superoxide dismutase (MnSOD) gene (AdMnSOD) or Cu/Zn-SOD gene (AdCu/ZnSOD) bilaterally into the RVLM or the NTS. After the gene transfer, blood pressure and heart rate of SHRSP, monitored by radio-telemetry system, were significantly decreased compared with non-treated SHRSP, but not WKY. Urinary norepinephrine excretion was significantly decreased in AdMnSOD- or AdCu/ZnSOD-transfected SHRSP, but not in WKY. Furthermore, we found that activation of NAD(P)H oxidase via Rac1 is a source of reactive oxygen species generation in the brain of hypertensive rats. Taken together, these results suggest that the increased oxidative stress in the RVLM and the NTS contribute to the central nervous system mechanisms underlying hypertension in SHRSP. We also found that atorvastatin has actions of reducing oxidative stress in the brain associated with sympatho-inhibitory effects.

Similarities of the neuronal circuit for the induction of fictive vomiting between ferrets and dogs.

Previous studies suggested that the following neuronal circuit participates in the induction of vomiting by afferent vagal stimulation in decerebrated paralyzed dogs: (1) afferent fibers of the vagus nerve, (2) neurons of the solitary nucleus (NTS), (3) neurons of the prodromal sign center near the semicompact part of the nucleus ambiguus (scAMB), (4) neurons of the central pattern generator in the reticular area adjacent to the compact part of nucleus ambiguus (cAMB), (5) respiratory premotor neurons in the caudal medulla, (6) motor neurons of the diaphragm and abdominal muscles. However, the commonality of this neuronal circuit in different species has not yet been clarified. Thus, this study was conducted to clarify this point. This study clarified for the first time that fictive vomiting in decerebrated paralyzed ferrets could be induced by vagal stimulation, and could be identified by centrifugal activity patterns of the phrenic and abdominal muscle nerves. The distributions of c-Fos immunoreactive neurons in the NTS, scAMB and cAMB areas in ferrets that exhibited fictive vomiting were denser than those in ferrets that did not. Application of the nonNMDA receptor antagonist into the 4th ventricle produced the reversible suppression of fictive vomiting. The NK1 receptor immunoreactive puncta were found in the reticular area adjacent to the scAMB. Microinjections of NK1 receptor antagonist into the reticular areas on both sides abolished fictive vomiting. All these results in the ferrets are identical with results previously obtained in dogs and cats. Therefore, this suggests that the above neuronal circuit commonly participates in the induction of emesis in these animal species.

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.

Chemical stimulation of visceral afferents activates medullary neurones projecting to the central amygdala and periaqueductal grey.

Cholecystokinin (CCK) stimulates gastrointestinal vagal afferent neurones that signal visceral sensations. We wished to determine whether neurones of the nucleus of the solitary tract (NTS) or ventrolateral medulla (VLM) convey visceral afferent information to the central nucleus of the amygdala (CeA) or periaqueductal grey region (PAG), structures that play a key role in adaptive autonomic responses triggered by stress or fear. Male Sprague-Dawley rats received a unilateral microinjection of the tracer cholera toxin subunit B (CTB, 1%) into the CeA or PAG followed, 7 days later, by an injection of CCK (100 microg/kg, i.p.) or saline. Brains were processed for detection of Fos protein (Fos-IR) and CTB. CCK induced increased expression of Fos-IR in the NTS and the VLM, relative to control. When CTB was injected into the CeA, CTB-immunoreactive (CTB-IR) neurones were more numerous in the rostral NTS ipsilateral to the injection site, whereas they were homogeneously distributed throughout the VLM. Double-labelled neurones (Fos-IR+CTB-IR) were most numerous in the ipsilateral NTS and caudal VLM. The NTS contained the higher percentage of CTB-IR neurones activated by CCK. When CTB was injected into the PAG, CTB-IR neurones were more numerous in the ipsilateral NTS whereas they were distributed relatively evenly bilaterally in the rostral VLM. Double-labelled neurones were not differentially distributed along the rostrocaudal axis of the NTS but were more numerous in this structure when compared with the VLM. NTS and VLM neurones may convey visceral afferent information to the CeA and the PAG.

Hemodynamic and respiratory responses to microinjection of ATP into the intermediate and caudal NTS of awake rats.

The nucleus tractus solitarii (NTS) is the site of integration of the peripheral chemoreceptor afferents in the brainstem. Previous studies from our laboratory have shown that microinjection of ATP into the intermediate NTS produced increases in arterial pressure and bradycardia. In the present study, we evaluated the hemodynamic and respiratory responses to microinjection of ATP into the intermediate and caudal commissural NTS. In the same group of rats the responses were compared with cardiorespiratory responses to chemoreflex activation (KCN, i.v.). The data show that microinjection of ATP into the intermediate NTS produced pressor and bradycardic responses similar to those observed in response to chemoreflex activation but apnoea instead of tachypnoea. Microinjection of ATP into caudal commissural NTS produced increase in arterial pressure and tachypnoea similar to the chemoreflex but a minor bradycardia. The data show that microinjection of ATP into different sub-regions of the NTS produces a diverse pattern of hemodynamic and respiratory responses and suggest the involvement of this purine in the neurotransmission of the cardiovascular reflex in the NTS.

Medial prefrontal cortical integration of psychological stress in rats.

The present study aimed to determine whether the medial prefrontal cortex (mPFC) (prelimbic and infralimbic regions) is implicated in the integration of a stress response. Sprague-Dawely rats were implanted with telemetry probes and guide cannulae so that either muscimol or vehicle could be administered locally within the mPFC or dorsomedial hypothalamus (DMH). The heart rate and blood pressure of rats was continuously recorded as either muscimol or vehicle was administered centrally and rats were either exposed to restraint stress or left alone in their home cages. After the stress challenge, or equivalent period, rats that had received intra-mPFC injections were processed for immunohistochemical detection of Fos throughout the neuraxis. Bilateral microinjection of muscimol into the mPFC had no effect upon either baseline cardiovascular parameters or restraint stress-induced tachycardia or pressor responses whereas, in the DMH, pretreatment with muscimol attenuated the cardiovascular stress response. Analysis of Fos expression throughout the CNS of nonstressed rats showed no effect of muscimol injections into the mPFC on baseline expression in the nuclei examined. In contrast, rats that had received muscimol injections into their mPFC and were subsequently restrained exhibited an increase in the number of Fos-positive cells in the DMH, medial amygdala, and medial nucleus tractus solitarius as compared to vehicle-injected rats that experienced restraint stress. These results indicate that, during acute psychological stress, the mPFC does not modulate the cardiovascular system in rats but does inhibit specific subcortical nuclei to exert control over aspects of an integrated response to a stressor.

Cardiovascular responses to microinjection of ATP into the nucleus tractus solitarii of awake rats.

Microinjection of increasing doses of ATP (0.31, 0.62, 1.25, and 2.5 nmol/50 nl) into the nucleus tractus solitarii (NTS) produced a dose-dependent pressor response. Prazosin abolished the pressor response and produced no change in the bradycardic response to ATP. Microinjection of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (0.25 nmol/50 nl), a nonselective P2 receptor antagonist into the NTS, reduced the bradycardic response but had no effect on the pressor response to microinjection of ATP (1.25 nmol/50 nl) into the NTS. Microinjection of suramin (2 nmol/50 nl), another nonselective P2 receptor antagonist, had no effect on the pressor and bradycardic responses to microinjection of ATP (1.25 nmol/50 nl) into the NTS. Antagonism of A1 receptors of adenosine with 1,3-dipropyl-8-cyclopentylxanthine also produced no changes in the cardiovascular responses to microinjection of ATP into the NTS. The involvement of excitatory amino acid (EAA) receptors in the pressor and bradycardic responses to microinjection of ATP into the NTS was also evaluated. Microinjection of kynurenic acid, a nonselective EAA receptor antagonist (10 nmol/50 nl), into the NTS reduced the bradycardic response and had no effect on the pressor response to microinjection of ATP into the NTS. The data show that 1) microinjection of ATP into the NTS of awake rats produced pressor and bradycardic responses by independent mechanisms, 2) the activation of parasympathetic component may involve an interaction of P2 and EAA receptors in the NTS, and 3) the sympathoexcitatory response to microinjection of ATP into the NTS was not affected by the blockade of P2, A1, or EAA receptors.

Fos expression by glutamatergic neurons of the solitary tract nucleus after phenylephrine-induced hypertension in rats.

The baroreflex pathway might include a glutamatergic connection between the nucleus of the solitary tract (NTS) and a segment of the ventrolateral medulla (VLM) called the caudal ventrolateral medulla. The main goal of this study was to seek direct evidence for such a connection. Awake rats were subjected to phenylephrine- (PE-) induced hypertension (N=5) or received saline (N=5). Neuronal activation was gauged by the presence of Fos-immunoreactive (Fos-ir) nuclei. Fos-ir neurons that contained vesicular glutamate transporter 2 mRNA (glutamatergic neurons) or glutamic acid decarboxylase mRNA (GABAergic neurons) were mapped throughout the medulla oblongata. Saline-treated rats had very few Fos-ir neurons. In PE-treated rats, Fos-ir neurons were detected in both NTS and VLM. In NTS, 72% of Fos-ir neurons were glutamatergic and 26% were GABAergic. In the VLM, 41% of Fos-ir neurons were glutamatergic and 56% were GABAergic. In VLM, Fos-ir glutamatergic neurons were evenly distributed and were often catecholaminergic, whereas Fos-ir GABAergic cells were clustered around Bregma -13.0 mm. This region of the VLM was injected with Fluoro-Gold (FG) in eight rats, four of which received PE and the rest saline. Fos-ir NTS neurons retrogradely labeled with FG were detected only in PE-treated rats. These cells were exclusively glutamatergic and were concentrated within the NTS subnuclei that receive the densest inputs from arterial baroreceptors. In conclusion, PE, presumably via baroreceptor stimulation, induces Fos in glutamatergic and GABAergic neurons in both NTS and VLM. At least 29% of the Fos-ir glutamatergic neurons of NTS project to the vicinity of the VLM GABAergic interneurons that are presumed to mediate the sympathetic baroreflex.

Domoic acid lesions in nucleus of the solitary tract: time-dependent recovery of hypoxic ventilatory response and peripheral afferent axonal plasticity.

The nucleus of the solitary tract (NTS) plays a pivotal role in the ventilatory response to hypoxia (HVR). However, the effects of excitotoxic lesions and the potential for functional recovery and plasticity remain unknown. Domoic acid (DA) or vehicle were bilaterally injected within the NTS of adult male Sprague Dawley rats. HVR (10% O(2)) and anatomical changes were assessed at 5-90 d after surgery. DA induced dose-dependent HVR attenuations ( approximately 70% at peak effect) that exhibited saturation at concentrations of 0.75-1.0 mm. However, although sodium cyanide-induced ventilatory responses were virtually abolished, DA did not modify baroreceptor gain. Consistent with ventilatory reductions, NTS neurons showed a significant degeneration 3 d after DA injection. In addition, the projection fields and the density of vagal afferent terminals to the NTS, and the motor neurons in the dorsal motor nucleus of the vagus were substantially reduced at 15 d. At 30 d, no functional or neural recovery were apparent. However, at day 60, the reduction in HVR was only approximately 40% of control, and at 90 d, HVR returned to control levels, paralleling regeneration of vagal afferent terminals within the NTS. The regeneration was particularly prominent in the commissural and dorsomedial subnuclei in the absence of cellular recovery. Thus, the integrity of the NTS is critical for HVR, spontaneous HVR recovery occurs after excitotoxic lesions in the NTS, and vagal-glossopharyngeal terminal sprouting in the NTS may underlie the anatomical substrate for such spontaneous functional recovery. The adult brainstem/NTS has self-repairing capabilities and will compensate for functional losses after structural damage by rewiring of its neural circuitry.

Cardiac afferents to the nucleus of the tractus solitarius: A WGA-HRP study in the rat.

Central distribution of the sensory fibers of the heart was investigated in the rat by the use of transganglionic transport of horseradish peroxidase (HRP). After the left intercostal thoracotomy was done under deep anesthesia and artificial respiration, wheat germ agglutinin-conjugated HRP (WGA-HRP) was injected into the left and right ventricular walls and the apex of the heart. HRP-labeled fibers were observed to be distributed to the dorsomedial portion of the medulla oblongata through the vagal nerve. The labeled fibers were present in various subnuclei of the nucleus of the tractus solitarius (NTS) bilaterally at the level of +0.36 to -1.74 mm to the obex. However, the most conspicuous feature in the present study was that the labeled fibers were exclusively confined to the medial, ventrolateral and commissural NTS with some distribution to the dorsolateral NTS. Although the labeling in the medial and ventrolateral NTS was observed to extend rostrocaudally, it was of interest that the labeling in the medial NTS was divided into the ventral and dorsal parts at the level around the obex. Accumulation of the labeled fibers in the commissural NTS was found at the level caudal to the obex and these fibers were traced to the caudal portion of its subnucleus with a gradual decrease in number. This pattern of distribution of cardiac afferents in the NTS was considered to be peculiar to the rat, because it was quite different from that reported previously in the cat.

Mapping of carotid baroreceptor subtype projections to the nucleus tractus solitarius using c-fos immunohistochemistry.

This study has combined physiological pressure stimulation of carotid baroreceptors via a vascularly isolated carotid sinus and anodal block of baroreceptor afferent fibers in the carotid sinus nerve to examine the medullary projections of type I vs. type II (large A- vs. small A- and C-fiber afferent) baroreceptors. The control distribution of cells in the nucleus tractus solitarius expressing c-fos in response to physiological activation of carotid baroreceptors in the isolated sinus was compared to that during anodal block of large A-fibers in the carotid sinus nerve. Carotid baroreceptor stimulation primarily activated neurons in the ipsilateral commissural and medial subnuclei of the caudal nucleus tractus solitarius and the dorsolateral, dorsomedial and medial subnuclei in the intermediate and rostral levels of the nucleus tractus solitarius. Elimination of large A-fiber carotid baroreceptor afferents, during similar carotid baroreceptor stimulation resulted in a decrease in the number of cells expressing c-fos in the dorsomedial subnucleus of the rostral nucleus tractus solitarius. These data indicate that projections of larger A-fiber (type I) carotid baroreceptors are localized primarily to the rostral dorsomedial subnucleus, while those of smaller A- and C-fiber baroreceptors are more widely distributed to the commissural, medial and dorsal subnuclei of the nucleus tractus solitarius.

Immunohistochemical localization of neuropeptides and neurotransmitters in the nucleus solitarius.

The nucleus tractus solitarii (NTS), which receives visceral afferent information from the cardiovascular, respiratory, gastrointestinal and taste systems, contains multiple neurotransmitters and neuropeptides throughout its rostral to caudal extent. The neurotransmitters and neuropeptides immunoreactivity is located predominately in varicose fibers and small puncta throughout the neuropil. In addition, immunoreactive NTS neurons for a variety of neurotransmitters and neuropeptides are present in subnuclear regions. The neuroactive substances localized immunohistochemically in the NTS include acetylcholine, the neuropeptides, substance P, methionine- and leucine-enkephalin, beta-endorphin, cholecystokinin, neurotensin, galanin, calcitonin gene-related peptide, somatostatin, FMRMamide, neuropeptide Y, angiotensin II, vasoactive intestinal polypeptide, vasopressin, oxytocin, thyrotropin-releasing hormone, luteinizing hormone-releasing hormone, atrial natriuretic peptide, the catecholamines, dopamine, norepinephrine, epinephrine, serotonin, histamine and the amino acids, GABA and glutamate. The pattern of innervation for each neurotransmitter and neuropeptide is not homogeneously distributed throughout the NTS. Each substance has a unique pattern within the NTS as each subnuclear region contains different immunohistochemical staining patterns and densities of fibers. At the ultrastructural level both neurotransmitters and neuropeptides are present in synaptic terminals that are in contact with different parts of the neuronal membranes. Typically, the labeled terminals contain both small, clear vesicles and large, dense core vesicles with the exception of synaptic terminals containing acetylcholine, GABA and glutamate which do not typically have the large, dense core vesicles. The most frequent post-synaptic target are dendrites and spinous processes. Less frequently, synaptic contacts are present on the cell soma.

Effect of sinus denervation and vagotomy on c-fos expression in the nucleus tractus solitarius after exposure to CO2.

Exposure to hypercapnia and electrical stimulation of the carotid sinus nerve (CSN) has been shown to induce c-fos expression in several brain stem regions including the nucleus tractus solitarius (NTS). To test whether the labeled neurons were activated directly by hypercapnia or secondarily via the carotid bodies (sinus nerve), adult rats were exposed to either air or 14-16% CO2 for 1 h. Experiments were done on eight groups: (1) exposure to air, (2) exposure to CO2, (3) chronic CSN denervation/CO2, (4) chronic unilateral CSN denervation/CO2, (5) chronic sham CSN denervation/CO2, (6) anesthetized/CO2, (7) anesthetized and acute vagotomy/CO2, and (8) premedicated with morphine, 10 mg s.c., 20 min before exposure to CO2. After exposure to CO2 or air the rats were anesthetized, perfused with 4% paraformaldehyde and the brains processed for immunohistochemical staining for c-fos protein using the PAP (i.e. peroxidase anti-peroxidase) technique. Labeled neurons in the area of the NTS in every second 50- "mu"m section were counted and their position plotted using a microscope and camera lucida attachment. Rats exposed to CO2 had a significantly greater number of labeled neurons in the NTS than those exposed to air. Other interventions, such as CSN denervation, surgery, anesthesia, vagotomy or injection of morphine did not significantly affect the level of c-fos expression in rats exposed to hypercapnia, indicative of central stimulation rather than secondary peripheral input. These responsive neurons may be part of a widespread central chemoreceptive complex.