Role of GABA within the nucleus tractus solitarii in the hypoxic ventilatory decline of awake rats

The purpose of this study was to examine our hypothesis that gamma-aminobutyric acid (GABA) in the nucleus tractus solitarii (NTS) may be related to the hypoxic ventilatory decline (HVD) and that chemoreceptor stimulation was essential to activate this mechanism. We used unanesthetized, freely moving rats in this study. An in vivo microdialysis technique was used to measure the extracellular GABA concentration ([GABA]o), and an in vivo microinjection technique was used to examine the effects of the GABA agonists and antagonists on the ventilation during hypoxia. The GABA agonists injected into the NTS attenuated the ventilation during hypoxia. By hypoxic exposure, [GABA]o was increased during the HVD. However, by carotid body denervation (CBD), this GABA increase was abolished. Although GABA antagonists microinjected into the NTS during the HVD phase significantly increased the depressed ventilation, this effect on the ventilation was abolished by CBD. These results suggest that the GABA in the NTS has a pivotal role in the HVD and that this mechanism is not activated without chemoreceptor stimulation.     

Differential baroreflex control of sympathetic drive by angiotensin II in the nucleus tractus solitarii

Microinjection of angiotensin II into the nucleus tractus solitarii attenuates the baroreceptor reflex-mediated bradycardia by inhibiting both vagal and cardiac sympathetic components. However, it is not known whether the baroreflex modulation of other sympathetic outputs (i.e., noncardiac) also are inhibited by exogenous angiotensin II (ANG II) in nucleus tractus solitarii (NTS). In this study, we determined whether there was a difference in the baroreflex sensitivity of sympathetic outflows at the thoracic and lumbar levels of the sympathetic chain following exogenous delivery of ANG II into the NTS. Experiments were performed in two types of in situ arterially perfused decerebrate rat preparations. Sympathetic nerve activity was recorded from the inferior cardiac nerve, the midthoracic sympathetic chain, or the lower thoracic-lumbar sympathetic chain. Increases in perfusion pressure produced a reflex bradycardia and sympathoinhibition. Microinjection of ANG II (500 fmol) into the NTS attenuated the reflex bradycardia (57% attenuation, P < 0.01) and sympathoinhibition of both the inferior cardiac nerve (26% attenuation, P < 0.05) and midthoracic sympathetic chain (37% attenuation, P < 0.05) but not the lower thoracic-lumbar chain (P = 0.56). We conclude that ANG II in the nucleus tractus solitarii selectively inhibits baroreflex responses in specific sympathetic outflows, possibly dependent on the target organ innervated.     

Successive episodes of synapses production in the developing rat nucleus tractus solitarii

In the rat nucleus tractus solitarii (NTS), synaptogenesis is thought to occur both pre- and postnatally. The present study was performed to precisely define the timetable of synapse formation in the NTS after birth. Changes in synapse morphology and densities were analyzed between postnatal day 3 (P3) and P28 using electron microscopy and ethanol phosphotungstic acid (E-PTA) staining. The proportion of morphologically immature synapses was high at P3 (38%) and P14 (30%) and low (8-14%) at the other ages investigated (P7, P21, and P28). Synaptic density significantly increased between P7 and P14 (60%) and between P21 and P28 (54%), but did not significantly change between P3 and P7 and between P14 and P21. Mean synaptic diameter also increased over the first postnatal month. Significant increases in synaptic size occurred between P3 and P7 (28%) and between P14 and P21 (15%). The present data indicate that, in the NTS, synaptogenesis occurs over a protracted period of time and involves distinct successive episodes of synapse production.     

Hypoxia activates nucleus tractus solitarii neurons projecting to the paraventricular nucleus of the hypothalamus

Peripheral chemoreceptor afferent information is sent to the nucleus tractus solitarii (nTS), integrated, and relayed to other brain regions to alter cardiorespiratory function. The nTS projects to the hypothalamic paraventricular nucleus (PVN), but activation and phenotype of these projections during chemoreflex stimulation is unknown. We hypothesized that activation of PVN-projecting nTS neurons occurs primarily at high intensities of hypoxia. We assessed ventilation and cardiovascular parameters in response to increasing severities of hypoxia. Retrograde tracers were used to label nTS PVN-projecting neurons and, in some rats, rostral ventrolateral medulla (RVLM)-projecting neurons. Immunohistochemistry was performed to identify nTS cells that were activated (Fos-immunoreactive, Fos-IR), catecholaminergic, and GABAergic following hypoxia. Conscious rats underwent 3 h normoxia (n = 4, 21% O(2)) or acute hypoxia (12, 10, or 8% O(2); n = 5 each). Hypoxia increased ventilation and the number of Fos-IR nTS cells (21%, 13 ± 2; 12%, 58 ± 4; 10%, 166 ± 22; 8%, 186 ± 6). Fos expression after 10% O(2) was similar whether arterial pressure was allowed to decrease (-13 ± 1 mmHg) or was held constant. The percentage of PVN-projecting cells activated was intensity dependent, but contrary to our hypothesis, PVN-projecting nTS cells exhibiting Fos-IR were found at all hypoxic intensities. Notably, at all intensities of hypoxia, ～75% of the activated PVN-projecting nTS neurons were catecholaminergic. Compared with RVLM-projecting cells, a greater percentage of PVN-projecting nTS cells was activated by 10% O(2). Data suggest that increasing hypoxic intensity activates nTS PVN-projecting cells, especially catecholaminergic, PVN-projecting neurons. The nTS to PVN catecholaminergic pathway may be critical even at lower levels of chemoreflex activation and more important to cardiorespiratory responses than previously considered.     

Hypoxia-mediated in vivo release of dopamine in nucleus tractus solitarii of rabbits

A wide variety of neuroactive substances have been suggested to be involved in the respiratory depression observed in response to severe hypoxia. By use of the technique of microdialysis, the release of dopamine (DA) was measured in the nucleus tractus solitarii during severe hypoxic provocations (6% O2 in N2) in the adult pentobarbital-anesthetized rabbit. DA release was analyzed by high-performance liquid chromatography with electrochemical detection. Such hypoxic provocations caused pronounced phase of depression in the phrenic nerve activity and enhanced release of DA. After bilateral carotid sinus nerve denervation, acute severe hypoxia did not give rise to enhanced release of DA or to phrenic nerve depression. Mild hypoxic (9% or 12% O2 in N2) or hypercapnic (6% CO2) stimuli resulted in an increased phrenic nerve activity without any concomitant changes in DA release. Decerebration at the midcollicular level in rabbits prevented an enhanced release of DA in the nucleus tractus solitarii during severe hypoxia. The results suggest that 1) DA is involved in the central ventilatory response to severe hypoxia, 2) not only the initial excitatory but also the second depressive phase in response to severe hypoxia is mediated partially by the peripheral chemoreceptors, and 3) the depressive phase is dependent on intact connections from suprapontine structures.     

Angiotensin II increases GABAB receptor expression in nucleus tractus solitarii of rats

Increasing evidence indicates that both the angiotensin II (ANG II) and gamma-aminobutyric acid (GABA) systems play a very important role in the regulation of blood pressure (BP). However, there is little information concerning the interactions between these two systems in the nucleus tractus solitarii (NTS). In the present study, we examined the effects of ANG II on GABAA and GABAB receptor (GAR and GBR) expression in the NTS of Sprague-Dawley rats. The direct effect of ANG II on GBR expression was determined in neurons cultured from NTS. Treatment of neuronal cultures with ANG II (100 nM, 5 h) induced a twofold increase in GBR1 expression, as detected with real-time RT-PCR and Western blots, but had no effect on GBR2 or GAR expression. In electrophysiological experiments, perfusion of neuronal cultures with the GBR agonist baclofen decreased neuronal firing rate by 39% and 63% in neurons treated with either PBS (control) or ANG II, respectively, indicating that chronic ANG II treatment significantly enhanced the neuronal response to GBR activation. In contrast, ANG II had no significant effect on the inhibitory action of the GAR agonist muscimol. In whole animal studies, intracerebroventricular infusion of ANG II induced a sustained increase in mean BP and an elevation of GBR1 mRNA and protein levels in the NTS. These results indicate that ANG II stimulates GBR expression in NTS neurons, and this could contribute to the central nervous system actions of ANG II that result in dampening of baroreflexes and elevated BP in the central actions of ANG II.     

NO formation in nucleus tractus solitarii attenuates pressor response evoked by skeletal muscle afferents

We have previously shown that static muscle contraction induces the expression of c-Fos protein in neurons of the nucleus tractus solitarii (NTS) and that some of these cells were codistributed with neuronal NADPH-diaphorase [nitric oxide (NO) synthase]-positive fibers. In the present study, we sought to determine the role of NO in the NTS in mediating the cardiovascular responses elicited by skeletal muscle afferent fibers. Static contraction of the triceps surae muscle was induced by electrical stimulation of the L7 and S1 ventral roots in anesthetized cats. Muscle contraction during microdialysis of artificial extracellular fluid increased mean arterial pressure (MAP) and heart rate (HR) 51 +/- 9 mmHg and 18 +/- 3 beats/min, respectively. Microdialysis of L-arginine (10 mM) into the NTS to locally increase NO formation attenuated the increases in MAP (30 +/- 7 mmHg, P < 0.05) and HR (14 +/- 2 beats/min, P > 0.05) during contraction. Microdialysis of D-arginine (10 mM) did not alter the cardiovascular responses evoked by muscle contraction. Microdialysis of N(G)-nitro-L-arginine methyl ester (2 mM) during contraction attenuated the effects of L-arginine on the reflex cardiovascular responses. These findings demonstrate that an increase in NO formation in the NTS attenuates the pressor response to static muscle contraction, indicating that the NO system plays a role in mediating the cardiovascular responses to static muscle contraction in the NTS.     

AT1 receptors in the nucleus tractus solitarii mediate the interaction between the baroreflex and the cardiac sympathetic afferent reflex in anesthetized rats

The cardiac "sympathetic afferent" reflex (CSAR) has been reported to increase sympathetic outflow and depress baroreflex function via a central angiotensin II (ANG II) mechanism. In the present study, we examined the role of ANG II type 1 (AT(1)) receptors in the nucleus tractus solitarii (NTS) in mediating the interaction between the CSAR and the baroreflex in anesthetized rats. We examined the effects of bilateral microinjection of AT(1) receptor antagonist losartan (100 pmol) into the NTS on baroreflex control of renal sympathetic nerve activity (RSNA) before and after CSAR activation by epicardial application of capsaicin (0.4 microg). Using single-unit extracellular recording, we further examined the effects of CSAR activation on the barosensitivity of barosensitive NTS neurons and the effects of intravenous losartan (2 mg/kg) on CSAR-induced changes in activity of NTS barosensitive neurons. Bilateral NTS microinjection of losartan significantly attenuated the increases in arterial pressure, heart rate, and RSNA evoked by capsaicin but also markedly (P < 0.01) reversed the CSAR-induced blunted baroreflex control of RSNA (Gain(max) from 1.65 +/- 0.10 to 2.22 +/- 0.11%/mmHg). In 17 of 24 (70.8%) NTS barosensitive neurons, CSAR activation significantly (P < 0.01) inhibited the baseline neuronal activity and attenuated the neuronal barosensitivity. In 11 NTS barosensitive neurons, intravenous losartan effectively (P < 0.01) normalized the decreased neuronal barosensitivity induced by CSAR activation. In conclusion, blockade of NTS AT(1) receptors improved the blunted baroreflex during CSAR activation, suggesting that the NTS plays an important role in processing the interaction between the baroreflex and the CSAR via an AT(1) receptor-dependent mechanism.     

NMDA receptors in nucleus tractus solitarii are linked to soluble guanylate cyclase

We sought to test the hypothesis that cardiovascular responses to activation of ionotropic, but not metabotropic, glutamate receptors in the nucleus tractus solitarii (NTS) depend on soluble guanylate cyclase (sGC) and that inhibition of sGC would attenuate baroreflex responses to changes in arterial pressure. In adult male Sprague-Dawley rats anesthetized with chloralose, the ionotropic receptor agonists N-methyl-d-aspartate (NMDA) and dl-alpha-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) and the metabotropic receptor agonist trans-dl-amino-1,3-cyclopentane-dicarboxylic acid (ACPD) were microinjected into the NTS before and after microinjection of sGC inhibitors at the same site. Inhibition of sGC produced significant dose-dependent attenuation of cardiovascular responses to NMDA but did not alter responses produced by injection of AMPA or ACPD. Bilateral inhibition of sGC did not alter arterial pressure, nor did it attenuate baroreflex responses to pharmacologically induced changes in arterial pressure. This study links sGC with NMDA, but not AMPA or metabotropic, receptors in cardiovascular signal transduction through NTS.     

Electrically elicited cardiovascular and respiratory responses from the nucleus tractus solitarii in unrestrained cats

Heart rate, arterial blood pressure and respiratory rate responses to electrical stimulation of the nucleus tractus solitarii (NTS) were studied in unanaesthetized freely moving cats. Complex cardiovascular response patterns, mainly pressor responses, were obtained from stimulation of the portion of the NTS rostral to the obex. No significant difference was observed between the effects produced by stimulation of the NTS on the right and on the left side. These results indicate that the rostral portion of the NTS also plays a role in the cardiovascular control, and a functional asymmetry between the two sides does not exist at the level of the NTS.     

Ventilatory effects of substance P-saporin lesions in the nucleus tractus solitarii of chronically hypoxic rats

During ventilatory acclimatization to hypoxia (VAH), time-dependent increases in ventilation lower Pco(2) levels, and this persists on return to normoxia. We hypothesized that plasticity in the caudal nucleus tractus solitarii (NTS) contributes to VAH, as the NTS receives the first synapse from the carotid body chemoreceptor afferents and also contains CO(2)-sensitive neurons. We lesioned cells in the caudal NTS containing the neurokinin-1 receptor by microinjecting the neurotoxin saporin conjugated to substance P and measured ventilatory responses in awake, unrestrained rats 18 days later. Lesions did not affect hypoxic or hypercapnic ventilatory responses in normoxic control rats, in contrast to published reports for similar lesions in other central chemosensitive areas. Also, lesions did not affect the hypercapnic ventilatory response in chronically hypoxic rats (inspired Po(2) = 90 Torr for 7 days). These results suggest functional differences between central chemoreceptor sites. However, lesions significantly increased ventilation in normoxia or acute hypoxia in chronically hypoxic rats. Hence, chronic hypoxia increases an inhibitory effect of neurokinin-1 receptor neurons in the NTS on ventilatory drive, indicating that these neurons contribute to plasticity during chronic hypoxia, although such plasticity does not explain VAH.     

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.     

Changes in the alpha-adrenoceptors in the medulla oblongata including nucleus tractus solitarii of spontaneously hypertensive rats

The nucleus tractus solitarii (NTS) is a brain stem center mediating depression of blood pressure. In order to elucidate a possible mechanism for the central regulation of blood pressure, we studied noradrenergic indices in the medulla oblongata, a region including the NTS, in spontaneously hypertensive rats (SHR) as compared with normotensive controls of the Wistar Kyoto strain (WKY) at 12 weeks of age. The medulla oblongata was the only brain region showing a significantly low noradrenaline level in the SHR as compared with WKY rats; the level is also significantly decreased at 8 weeks of age. The alpha 1-adrenergic binding sites, as measured with 2-(2, 6-dimethoxy) phenoxyethylamine-methylbenzodioxan [³H]WB4101 showed significant increases inK _D andB _max values in medulla oblongata homogenates from rats of both strains from 4–12 weeks after birth, with no significant interstrain difference. On the other hand, theK _D andB _max of the alpha 2-sites, measured by [³H]yohimbine binding, were reduced in SHR as compared to WKY animals, even at 4 weeks after birth when hypertension was not yet apparent. As expected, the relatively selective alpha 2-antagonist, clonidine, was a potent inhibitor of [³H]yohimbine binding but not of [³H]WB4101 binding in these homogenates. The results suggest that some genetic disorder in the alpha 2-adrenergic transmission system in the NTS region may be involved in the development of hypertension in the SHR rats.Dedicated to Professor Yasuzo Tsukada.     

Nitric oxide signaling pathway mediates the L-arginine-induced cardiovascular effects in the nucleus tractus solitarii of rats

We have previously demonstrated that L-arginine produces profound cardiovascular effects when microinjected into the nucleus tractus solitarii (NTS) of the rat. The present study extended our earlier work and examined further the underlying mechanisms of action of L-arginine in the NTS. Our results showed that intra-NTS microinjection of L-arginine (0.1-10 nmol) elicited dose-dependent depressor and bradycardic effects that were not significantly evoked by equivalent doses of D-arginine. The effects of L-arginine were blocked by pre-injection of 7-nitroindazole (0.02-1 nmol), a neuronal nitric oxide synthase inhibitor. Additionally, application of the calmodulin inhibitor W-7 (0.01-0.33 nmol) reduced cardiovascular responses to L-arginine (10 nmol) in a dose-dependent manner. Pre-injections of soluble guanylyl cyclase inhibitors, LY83583 (0.01-0.33 nmol) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 0.03-1 pmol) both suppressed the L-arginine-induced depressor and bradycardic effects. Finally, the cardiovascular effects of L-arginine in the NTS were attenuated by HA1004 (0.1-1 nmol), a cGMP-dependent protein kinase inhibitor, but not by the protein kinase C inhibitor H-7 (1 nmol). Taken together, the results indicate that the cardiovascular effects produced by L-arginine in the NTS are inhibited by pharmacological interventions that block nitric oxide production and cGMP-PKG signaling pathway within the nucleus.     

Aortic coarctation hypertension induces fibroblast growth factor-2 immunoreactivity in the stimulated nucleus tractus solitarii

The actions of neurotrophic factors i.e. basic fibroblast growth factor (bFGF, FGF-2) to neurons are related not only to neuronal development and maintenance but also to synaptic plasticity regarding neurotransmission. We analyzed here the levels of FGF-2 immunoreactivity in the nucleus tractus solitarii (NTS) of Wistar Kyoto rats in response to alterations of neuronal activity promoted by the stimulation of the baroreceptor reflex following an aortic coarctation-induced-hypertension. The FGF-2 immunoreactivity (IR) was found in the cytoplasm of the neurons and in the nuclei of the glial cells in the NTS. A large number of NTS neurons expressed FOS immunoreactivity 4 h after coarctation, as an indication of neuronal activity. Stereological methods showed an increased number of FGF-2 immunoreactive (ir) neuronal profiles (90%) and glial profiles (149%) in the NTS of the 72 h aortic coarctated rats. 1-week later, FGF-2 ir neurons were still increased (54%) but no change was found in the number of FGF-2 ir glial profiles. The double immunoperoxidase method revealed that the majority of the FGF-2 ir glial cells was glial fibrillary acidic protein (GFAP) positive astrocytes. GFAP immunohistochemistry showed an astroglial reaction at 72 h time-interval (55%) but not 1 week after stimulation. The number of the cresyl violet positive neurons and OX42 ir profiles (marker of activated microglia) in the NTS of coarctated rats were not different from control by 1 week and 1 month after the surgery, indicating a lack of NTS injury in this period following coarctation hypertension. FGF-2 may be an important neurotrophic factor in areas involved in the control of blood pressure. The increased FGF-2 IR in the NTS cells following neuronal stimulation may represent trophic and plastic adaptive responses in this nucleus in an autocrine/paracrine fashion.