Autonomic processing of the cardiovascular reflexes in the nucleus tractus solitarii

The nucleus tractus solitarii (NTS) receives afferent projections from the arterial baroreceptors, carotid chemoreceptors and cardiopulmonary receptors and as a function of this information produces autonomic adjustments in order to maintain arterial blood pressure within a narrow range of variation. The activation of each of these cardiovascular afferents produces a specific autonomic response by the excitation of neuronal projections from the NTS to the ventrolateral areas of the medulla (nucleus ambiguus, caudal and rostral ventrolateral medulla). The neurotransmitters at the NTS level as well as the excitatory amino acid (EAA) receptors involved in the processing of the autonomic responses in the NTS, although extensively studied, remain to be completely elucidated. In the present review we discuss the role of the EAA L-glutamate and its different receptor subtypes in the processing of the cardiovascular reflexes in the NTS. The data presented in this review related to the neurotransmission in the NTS are based on experimental evidence obtained in our laboratory in unanesthetized rats. The two major conclusions of the present review are that a) the excitation of the cardiovagal component by cardiovascular reflex activation (chemo- and Bezold-Jarisch reflexes) or by L-glutamate microinjection into the NTS is mediated by N-methyl-D-aspartate (NMDA) receptors, and b) the sympatho-excitatory component of the chemoreflex and the pressor response to L-glutamate microinjected into the NTS are not affected by an NMDA receptor antagonist, suggesting that the sympatho-excitatory component of these responses is mediated by non-NMDA receptors.

Localization of NMDA receptors in the cerebral cortex: a schematic overview

The fundamental role of N-methyl-D-aspartate (NMDA) receptors in many cortical functions has been firmly defined, as has its involvement in a number of neurological and psychiatric diseases. However, until recently very little was known about the anatomical localization of NMDA receptors in the cerebral cortex of mammals. The recent application of molecular biological techniques to the study of NMDA receptors has provided specific tools which have greatly expanded our understanding of the localization of NMDA receptors in the cerebral cortex. In particular, immunocytochemical studies on the distribution of cortical NMDA receptors have shown that NMDA receptors are preferentially localized on dendritic spines, have disclosed an unknown fraction of presynaptic NMDA receptors on both excitatory and inhibitory axon terminals, and demonstrated that cortical astrocytes do express NMDA receptors. These studies suggest that the effects induced by the activation of NMDA receptors are not due solely to the opening of NMDA channels on neuronal postsynaptic membranes, as previously assumed, but that the activation of presynaptic and glial NMDA receptors may mediate part of these effects

Cardiovascular responses to microinjection of trans-(±)-ACPD into the NTS were similar in conscious and chloralose-anesthetized rats

The changes in mean arterial pressure (MAP) and heart rate (HR) in response to the activation of metabotropic receptors in the nucleus tractus solitarii (NTS) with trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-(±)-ACPD) were evaluated in conscious and anesthetized Wistar, male rats weighing 240-260 g (N = 8). The responses obtained with trans-(±)-ACPD were compared with the responses to L-glutamate (1 nmol/100 nl), since in a previous study we showed that anesthesia converted a pressor response to L-glutamate microinjected into the NTS of conscious rats to a depressor response in the same rats under urethane or chloralose anesthesia. Microinjection of 3 doses of trans-(±)-ACPD (100, 500 and 1000 pmol/100 nl) produced a dose-dependent fall in MAP (range, -20 to -50 mmHg) and HR (range, -30 to -170 bpm) under both conscious and chloralose anesthesia conditions. These data indicate that the cardiovascular responses to the activation of metabotropic receptors by trans-(±)-ACPD are not affected by chloralose anesthesia while the cardiovascular responses to the activation of excitatory amino acid (EAA) receptors by L-glutamate are significantly altered

Inhibition of carbachol-induced inositol phosphate accumulation in the embryonic retina promoted by kainate and veratridine

In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 µM carbachol was used, the estimated IC50 value for kainate was 0.2 µM and the maximal inhibition of ~50% was obtained with 1 µM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 µM veratridine, but not 50 mM KCl, inhibited ~65% of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 ± 38.0 to 2044.5 ± 299.9 cpm/mg protein, retinal response decreased to 861.6 ± 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.

Respiratory effects of kynurenic acid microinjected into the ventromedullary surface of the rat

Several studies demonstrate that, within the ventral medullary surface (VMS), excitatory amino acids are necessary components of the neural circuits involved in the tonic and reflex control of respiration and circulation. In the present study we investigated the cardiorespiratory effects of unilateral microinjections of the broad spectrum glutamate antagonist kynurenic acid (2 nmol/200 nl) along the VMS of urethane-anesthetized rats. Within the VMS only one region was responsive to this drug. This area includes most of the intermediate respiratory area, partially overlapping the rostral ventrolateral medulla (IA/RVL). When microinjected into the IA/RVL, kynurenic acid produced a respiratory depression, without changes in mean arterial pressure or heart rate. The respiratory depression observed was characterized by a decrease in ventilation, tidal volume and mean inspiratory flow and an increase in respiratory frequency. Therefore, the observed respiratory depression was entirely due to a reduction in the inspiratory drive. Microinjections of vehicle (200 nl of saline) into this area produced no significant changes in breathing pattern, blood pressure or heart rate. Respiratory depression in response to the blockade of glutamatergic receptors inside the rostral VMS suggests that neurons at this site have an endogenous glutamatergic input controlling the respiratory cycle duration and the inspiratory drive transmission.

Participation of nitric oxide in the nucleus isthmi in CO2-drive to breathing in toads

The nucleus isthmi (NI) is a mesencephalic structure of the amphibian brain. It has been reported that NI plays an important role in integration of CO2 chemoreceptor information and glutamate is probably involved in this function. However, very little is known about the mechanisms involved. Recently, it has been shown that nitric oxide synthase (NOS) is expressed in the brain of the frog. Thus the gas nitric oxide (NO) may be involved in different functions in the brain of amphibians and may act as a neurotransmitter or neuromodulator. We tested the hypothesis that NO plays a role in CO2-drive to breathing, specifically in the NI comparing pulmonary ventilation, breathing frequency and tidal volume, after microinjecting 100 nmol/0.5 µl of L-NAME (a nonselective NO synthase inhibitor) into the NI of toads (Bufo paracnemis) exposed to normocapnia and hypercapnia. Control animals received microinjections of vehicle of the same volume. Under normocapnia no significant changes were observed between control and L-NAME-treated toads. Hypercapnia caused a significant (P<0.01) increase in ventilation only after intracerebral microinjection of L-NAME. Exposure to hypercapnia caused a significant increase in breathing frequency both in control and L-NAME-treated toads (P<0.01 for the control group and P<0.001 for the L-NAME group). The tidal volume of the L-NAME group tended to be higher than in the control group under hypercapnia, but the increase was not statistically significant. The data indicate that NO in the NI has an inhibitory effect only when the respiratory drive is high (hypercapnia), probably acting on tidal volume. The observations reported in the present investigation, together with other studies on the presence of NOS in amphibians, indicate a considerable degree of phylogenetic conservation of the NO pathway amongst vertebrates.

Anxiolytic effect of methylene blue microinjected into the dorsal periaqueductal gray matter

The dorsal periaqueductal gray (DPAG) has been implicated in the behavioral and autonomic expression of defensive reactions. Several results suggest that, along with GABA, glutamate and serotonin, nitric oxide (NO) may play a role in defense reactions mediated by this region. To further investigate this possibility we microinjected methylene blue (MB; 10, 30 or 100 nmol/0.5 µl) into the DPAG of rats submitted to the elevated plus-maze test, an animal model of anxiety. MB has been used as an inhibitor of soluble guanylate cyclase (sGC) to demonstrate cGMP-mediated processes, and there is evidence that NO may exert its biological effects by binding to the heme part of guanylate cyclase, causing an increase in cGMP levels. The results showed that MB (30 nmol) significantly increased the percent of time spent in the open arms (saline = 11.57 ± 1.54, MB = 18.5 ± 2.45, P<0.05) and tended to do the same with the percentage of open arm entries (saline = 25.8 ± 1.97, MB = 33.77 ± 3.07, P<0.10), but did not change the number of enclosed arm entries. The dose-response curve, however, had an inverted U shape. These results indicate that MB, within a limited dose range, has anxiolytic properties when microinjected into the DPAG.

Metabolite and light regulation of metabolism in plants: lessons from the study of a single biochemical pathway

We are using molecular, biochemical, and genetic approaches to study the structural and regulatory genes controlling the assimilation of inorganic nitrogen into the amino acids glutamine, glutamate, aspartate and asparagine. These amino acids serve as the principal nitrogen-transport amino acids in most crop and higher plants including Arabidopsis thaliana. We have begun to investigate the regulatory mechanisms controlling nitrogen assimilation into these amino acids in plants using molecular and genetic approaches in Arabidopsis. The synthesis of the amide amino acids glutamine and asparagine is subject to tight regulation in response to environmental factors such as light and to metabolic factors such as sucrose and amino acids. For instance, light induces the expression of glutamine synthetase (GLN2) and represses expression of asparagine synthetase (ASN1) genes. This reciprocal regulation of GLN2 and ASN1 genes by light is reflected at the level of transcription and at the level of glutamine and asparagine biosynthesis. Moreover, we have shown that the regulation of these genes is also reciprocally controlled by both organic nitrogen and carbon metabolites. We have recently used a reverse genetic approach to study putative components of such metabolic sensing mechanisms in plants that may be conserved in evolution. These components include an Arabidopsis homolog for a glutamate receptor gene originally found in animal systems and a plant PII gene, which is a homolog of a component of the bacterial Ntr system. Based on our observations on the biology of both structural and regulatory genes of the nitrogen assimilatory pathway, we have developed a model for metabolic control of the genes involved in the nitrogen assimilatory pathway in plants.

Protective effects of centrally acting sympathomodulatory drugs on myocardial ischemia induced by sympathetic overactivity in rabbits

It is recognized that an imbalance of the autonomic nervous system is involved in the genesis of ventricular arrhythmia and sudden death during myocardial ischemia. In the present study we investigated the effects of clonidine and rilmenidine, two centrally acting sympathomodulatory drugs, on an experimental model of centrally induced sympathetic hyperactivity in pentobarbital-anesthetized New Zealand albino rabbits of either sex (2-3 kg, N = 89). We also compared the effects of clonidine and rilmenidine with those of propranolol, a ß-blocker, known to induce protective cardiovascular effects in patients with ischemic heart disease. Central sympathetic stimulation was achieved by intracerebroventricular injection of the excitatory amino acid L-glutamate (10 µmol), associated with inhibition of nitric oxide synthesis with L-NAME (40 mg/kg, iv). Glutamate triggered ventricular arrhythmia and persistent ST-segment shifts in the ECG, indicating myocardial ischemia. The intracisternal administration of clonidine (1 µg/kg) and rilmenidine (30 µg/kg) or of a nonhypotensive dose of rilmenidine (3 µg/kg) decreased the incidence of myocardial ischemia (25, 14 and 25%, respectively, versus 60% in controls) and reduced the mortality rate from 40% to 0.0, 0.0 and 12%, respectively. The total number of ventricular premature beats per minute fell from 30 ± 9 in the control group to 7 ± 3, 6 ± 3 and 2 ± 2, respectively. Intravenous administration of clonidine (10 µg/kg), rilmenidine (300 µg/kg) or propranolol (500 µg/kg) elicited similar protective effects. We conclude that clonidine and rilmenidine present cardioprotective effects of central origin, which can be reproduced by propranolol, a lipophilic ß-blocking agent.

Cardiovascular responses to microinjections of GABA or anesthetics into the rostral ventrolateral medulla of conscious and anesthetized rats

The rostral ventrolateral medulla (RVLM) contains neurons involved in tonic and reflex control of arterial pressure. We describe the effects of gamma-aminobutyric acid (GABA) and anesthetics injected into the RVLM of conscious and urethane (1.2 g/kg, iv) anesthetized Wistar rats (300-350 g). In conscious rats, bilateral microinjection of GABA (50 nmol/200 nl) induced a small but significant decrease in blood pressure (from 130 ± 3.6 to 110 ± 5.6 mmHg, N = 7). A similar response was observed with sodium pentobarbital microinjection (24 nmol/200 nl). However, in the same animals, the fall in blood pressure induced by GABA (from 121 ± 8.9 to 76 ± 8.8 mmHg, N = 7) or pentobarbital (from 118 ± 4.5 to 57 ± 11.3 mmHg, N = 6) was significantly increased after urethane anesthesia. In contrast, there was no difference between conscious (from 117 ± 4.1 to 92 ± 5.9 mmHg, N = 7) and anesthetized rats (from 123 ± 6.9 to 87 ± 8.7 mmHg, N = 7) when lidocaine (34 nmol/200 nl) was microinjected into the RVLM. The heart rate variations were not consistent and only eventually reached significance in conscious or anesthetized rats. The right position of pipettes was confirmed by histology and glutamate microinjection into the RVLM. These findings suggest that in conscious animals the RVLM, in association with the other sympathetic premotor neurons, is responsible for the maintenance of sympathetic vasomotor tone during bilateral RVLM inhibition. Activity of one or more of these premotor neurons outside the RVLM can compensate for the effects of RVLM inhibition. In addition, the effects of lidocaine suggest that fibers passing through the RVLM are involved in the maintenance of blood pressure in conscious animals during RVLM inhibition.

Possible etiologies for tropical spastic paraparesis and human T lymphotropic virus I-associated myelopathy

The epidemiology of tropical spastic paraparesis/human T lymphotropic virus I (HTLV-I)-associated myelopathy (TSP/HAM) is frequently inconsistent and suggests environmental factors in the etiology of these syndromes. The neuropathology corresponds to a toxometabolic or autoimmune process and possibly not to a viral disease. Some logical hypotheses about the etiology and physiopathology of TSP and HAM are proposed. Glutamate-mediated excitotoxicity, central distal axonopathies, cassava, lathyrism and cycad toxicity may explain most cases of TSP. The damage caused to astrocytes and to the blood-brain barrier by HTLV-I plus xenobiotics may explain most cases of HAM. Analysis of the HTLV-I/xenobiotic ratio clarifies most of the paradoxical epidemiology of TSP and HAM. Modern neurotoxicology, neuroimmunology and molecular biology may explain the neuropathology of TSP and HAM. It is quite possible that there are other xenobiotics implicated in the etiology of some TSP/HAMs. The prevention of these syndromes appears to be possible today.

Differentiation of autonomic reflex control begins with cellular mechanisms at the first synapse within the nucleus tractus solitarius

Visceral afferents send information via cranial nerves to the nucleus tractus solitarius (NTS). The NTS is the initial step of information processing that culminates in homeostatic reflex responses. Recent evidence suggests that strong afferent synaptic responses in the NTS are most often modulated by depression and this forms a basic principle of central integration of these autonomic pathways. The visceral afferent synapse is uncommonly powerful at the NTS with large unitary response amplitudes and depression rather than facilitation at moderate to high frequencies of activation. Substantial signal depression occurs through multiple mechanisms at this very first brainstem synapse onto second order NTS neurons. This review highlights new approaches to the study of these basic processes featuring patch clamp recordings in NTS brain slices and optical techniques with fluorescent tracers. The vanilloid receptor agonist, capsaicin, distinguishes two classes of second order neurons (capsaicin sensitive or capsaicin resistant) that appear to reflect unmyelinated and myelinated afferent pathways. The differences in cellular properties of these two classes of NTS neurons indicate clear functional differentiation at both the pre- and postsynaptic portions of these first synapses. By virtue of their position at the earliest stage of these pathways, such mechanistic differences probably impart important differentiation in the performance over the entire reflex pathways.

Effects of centrally acting antihypertensive drugs on the microcirculation of spontaneously hypertensive rats

We investigated the acute effects of centrally acting antihypertensive drugs on the microcirculation of pentobarbital-anesthetized spontaneously hypertensive rats (SHR). The effects of the sympatho-inhibitory agents clonidine and rilmenidine, known to activate both alpha2-adrenoceptors and nonadrenergic I1-imidazoline binding sites (I1BS) in the central nervous system, were compared to those of dicyclopropylmethyl-(4,5-dimethyl-4,5-dihydro-3H -pyrrol-2-yl)-amine hydrochloride (LNP 509), which selectively binds to the I1BS. Terminal mesenteric arterioles were observed by intravital microscopy. Activation of the central sympathetic system with L-glutamate (125 µg, ic) induced marked vasoconstriction of the mesenteric microcirculation (27 ± 3%; N = 6, P < 0.05). In contrast, the marked hypotensive and bradycardic effects elicited by intracisternal injection of clonidine (1 µg), rilmenidine (7 µg) and LNP 509 (60 µg) were accompanied by significant increases in arteriolar diameter (12 ± 1, 25 ± 10 and 21 ± 4%, respectively; N = 6, P < 0.05). The vasodilating effects of rilmenidine and LNP 509 were two-fold higher than those of clonidine, although they induced an identical hypotensive effect. Central sympathetic inhibition elicited by baclofen (1 µg, ic), a GABA B receptor agonist, also resulted in vasodilation of the SHR microvessels. The acute administration of clonidine, rilmenidine and LNP 509 also induced a significant decrease of cardiac output, whereas a decrease in systemic vascular resistance was observed only after rilmenidine and LNP 509. We conclude that the normalization of blood pressure in SHR induced by centrally acting antihypertensive agents is paralleled by important vasodilation of the mesenteric microcirculation. This effect is more pronounced with substances acting preferentially (rilmenidine) or exclusively (LNP 509) upon I1BS than with those presenting important alpha2-adrenergic activity (clonidine).

Glutamatergic neurotransmission modulates hypoxia-induced hyperventilation but not anapyrexia

The interaction between pulmonary ventilation (V E) and body temperature (Tb) is essential for O2 delivery to match metabolic rate under varying states of metabolic demand. Hypoxia causes hyperventilation and anapyrexia (a regulated drop in Tb), but the neurotransmitters responsible for this interaction are not well known. Since L-glutamate is released centrally in response to peripheral chemoreceptor stimulation and glutamatergic receptors are spread in the central nervous system we tested the hypothesis that central L-glutamate mediates the ventilatory and thermal responses to hypoxia. We measured V E and Tb in 40 adult male Wistar rats (270 to 300 g) before and after intracerebroventricular injection of kynurenic acid (KYN, an ionotropic glutamatergic receptor antagonist), alpha-methyl-4-carboxyphenylglycine (MCPG, a metabotropic glutamatergic receptor antagonist) or vehicle (saline), followed by a 1-h period of hypoxia (7% inspired O2) or normoxia (humidified room air). Under normoxia, KYN (N = 5) or MCPG (N = 8) treatment did not affect V E or Tb compared to saline (N = 6). KYN and MCPG injection caused a decrease in hypoxia-induced hyperventilation (595 ± 49 for KYN, N = 7 and 525 ± 84 ml kg-1 min-1 for MCPG, N = 6; P < 0.05) but did not affect anapyrexia (35.3 ± 0.2 for KYN and 34.7 ± 0.4ºC for MCPG) compared to saline (912 ± 110 ml kg-1 min-1 and 34.8 ± 0.2ºC, N = 8). We conclude that glutamatergic receptors are involved in hypoxic hyperventilation but do not affect anapyrexia, indicating that L-glutamate is not a common mediator of this interaction.

Electrophysiological evidence for the presence of NR2C subunits of N-methyl-D-aspartate receptors in rat neurons of the nucleus tractus solitarius

The nucleus tractus solitarius (NTS) plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs) in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 µM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (± SEM) amplitude of -41.2 ± 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 ± 0.17 for parameter delta obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 ± 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 ± 51 and 418 ± 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV). The biophysical properties of the NMDA receptors observed in the present study suggest that these receptors in the NTS contain NR2C subunits and may contribute to the synaptic signal integration.