Excitatory amino acid receptor blockade within the caudal pressor area and rostral ventrolateral medulla alters cardiovascular responses to nucleus raphe obscurus stimulation in rats

Pressor responses elicited by stimulation of the nucleus raphe obscurus (NRO) depend on the integrity of the rostral ventrolateral medulla (RVLM). Therefore, to test the participation of excitatory amino acid (EAA) receptors in the cardiovascular responses evoked by NRO stimulation (1 ms, 100 Hz, 40-70 µA, for 10 s), the EAA antagonist kynurenic acid (Kyn) was microinjected at different sites in the ventrolateral medullar surface (2.7 nmol/200 nl) of male Wistar rats (270-320 g, N = 39) and NRO stimulation was repeated. The effects of NRO stimulation were: hypertension (deltaMAP = +43 ± 1 mmHg, P<0.01), bradycardia (deltaHR = -30 ± 7 bpm, P<0.01) and apnea. Bilateral microinjection of Kyn into the RVLM, which did not change baseline parameters, almost abolished the bradycardia induced by NRO stimulation (deltaHR = -61 ± 3 before vs -2 ± 3 bpm after Kyn, P<0.01, N = 7). Unilateral microinjection of Kyn into the CVLM did not change baseline parameters or reduce the pressor response to NRO stimulation (deltaMAP = +46 ± 5 before vs +48 ± 5 mmHg after Kyn, N = 6). Kyn bilaterally microinjected into the caudal pressor area reduced blood pressure and heart rate and almost abolished the pressor response to NRO stimulation (deltaMAP = +46 ± 4 mmHg before vs +4 ± 2 mmHg after Kyn, P<0.01, N = 7). These results indicate that EAA receptors on the medullary ventrolateral surface play a role in the modulation of the cardiovascular responses induced by NRO stimulation, and also suggest that the RVLM participates in the modulation of heart rate responses and that the caudal pressor area modulates the pressor response following NRO stimulation.

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.

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.

Glutamatergic transmission in the nucleus tractus solitarii: from server to peripherals in the cardiovascular information superhighway

Afferent nerves carrying signals from mechanoreceptors in the aortic arch and carotid sinus terminate predominantly in the nucleus tractus solitarii (NTS). Signal transduction and neurotransmission in the NTS are critical for central cardiovascular reflex control, but little was known about either until the late 1970's. None of the numerous neuroactive chemicals found in the NTS had met strict criteria as a neurotransmitter in the baroreflex arc until data suggested that the excitatory amino acid L-glutamate (GLU) might be released from baroreceptor afferent terminals in the NTS. In anesthetized animals microinjection into the NTS of GLU, which can be demonstrated in terminals in the NTS, produces cardiovascular responses like those seen with activation of the baroreceptor reflex. Similar responses occur in awake animals if the chemoreceptor reflex is eliminated; otherwise, in conscious animals responses mimic those of chemoreceptor reflex activation. GLU is released in the NTS upon selective activation of the baroreceptor, and possibly the chemoreceptor, reflex. Responses to selective agonists as well as baroreflex responses are eliminated by GLU antagonists microinjected into the NTS. Non-NMDA (N-methyl-D-aspartic acid) receptors seem to predominate at primary baroreceptor synapses in the NTS while NMDA receptors may be involved at later synapses. Although inhibition of soluble guanylate cyclase attenuates responses to ionotropic glutamate agonists in the NTS, nitric oxide does not seem to play a role in glutamate transmission in the NTS. GLU may also participate in transmission at cardiovascular neurons beyond the NTS. For example, a role has been suggested for GLU in the ventrolateral medulla and spinal cord. Work continues concerning GLU signal transduction and mechanisms that modulate that transduction both at the NTS and at other cardiovascular nuclei

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

Evidence of interaction between fluoxetine and isosorbide dinitrate on neuroleptic-induced catalepsy in mice

Drugs which influence 5-HTergic mechanisms can modify neuroleptic-induced catalepsy (NC) in rodents, a phenomenon produced by striatal dopamine (DA) receptor blockade. Previous research also suggests a role for endogenous nitric oxide (NO) in the modulation of striatal DAergic neurotransmission; in addition, NO seems to play a role in the 5-HT reuptake mechanism. It is known that clomipramine potentiates NC in mice, but the reported effects of selective 5-HT reuptake inhibitors (SSRIs) in this model are rather contradictory. We then decided to re-address this issue, investigating the effect of fluoxetine (FX), an SSRI, on NC. In view of the ubiquitous role of NO as a central neuromodulator, we also studied the effect of isosorbide dinitrate (ID), a centrally active NO donor, and how both drugs interact to affect the phenomenon of NC. Catalepsy was induced in male albino mice with haloperidol (H; 1 mg/kg, ip) and measured at 30-min interval by means of a bar test. Drugs (FX, ID and FX + ID) or saline (controls) were injected ip 30 min before H, with each animal used only once. FX (5 mg/kg) significantly reduced NC, with maximal attenuation (about 74%) occurring at 150 min after H. ID (5 mg/kg) also inhibited NC (150 min: 62% attenuation). The combined drugs (FX + ID group), however, caused a great potentiation of NC (4.7-fold at its maximum, at 90 min). The effect observed with ID is compatible with the hypothesis that NO increases DA release in the striatum. The attenuation of NC observed with FX may be due to a preferential net effect on the raphe somatodendritic synapse, where inhibitory 5-HT1A autoreceptors are operative. The enhancement of NC caused by combined administration of FX and ID suggests the presence of a pharmacodynamic interaction, whose mechanism, still unclear, may be related to a decrease in striatal DA release

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

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.

Steps in the formation of neurites and synapses studied in cultured leech neurons

Leech neurons in culture have provided novel insights into the steps in the formation of neurite outgrowth patterns, target recognition and synapse formation. Identified adult neurons from the central nervous system of the leech can be removed individually and plated in culture under well-controlled conditions, where they retain their characteristic physiological properties, grow neurites and form specific chemical or electrical synapses. Different identified neurons develop distinctive outgrowth patterns that depend on their identities and on the molecular composition of the substrate. On native substrates, the patterns displayed by these neurons reproduce characteristics from the adult or the developing neurons. In addition, the substrate may induce selective directed growth between pairs of neurons that normally make contact in the ganglion. Upon contact, pairs of cultured leech neurons form chemical or electrical synapses, or both types depending on the neuronal identities. Anterograde and retrograde signals during membrane contact and synapse formation modify the distribution of synaptic terminals, calcium currents, and responses to 5-hydroxytryptamine.

Blockade of NK-1 receptors in the lateral commissural nucleus tractus solitarii of awake rats had no effect on the cardiovascular responses to chemoreflex activation

The neurotransmission of the chemoreflex in the nucleus tractus solitarii (NTS), particularly of the sympatho-excitatory component, is not completely understood. There is evidence that substance P may play a role in the neurotransmission of the chemoreflex in the NTS. Microinjection of substance P (50 pmol/50 nl, N = 12, and 5 nmol/50 nl, N = 8) into the commissural NTS of unanesthetized rats produced a significant increase in mean arterial pressure (101 ± 1 vs 108 ± 2 and 107 ± 3 vs 115 ± 4 mmHg, respectively) and no significant changes in heart rate (328 ± 11 vs 347 ± 15 and 332 ± 7 vs 349 ± 13 bpm, respectively) 2 min after microinjection. Previous treatment with WIN, an NK-1 receptor antagonist (2.5 nmol/50 nl), microinjected into the NTS of a specific group of rats, blocked the pressor (11 ± 5 vs 1 ± 2 mmHg) and tachycardic (31 ± 6 vs 4 ± 3 bpm) responses to substance P (50 pmol/50 nl, N = 5) observed 10 min after microinjection. Bilateral microinjection of WIN into the lateral commissural NTS (N = 8) had no significant effect on the pressor (50 ± 4 vs 42 ± 6 mmHg) or bradycardic (-230 ± 16 vs -220 ± 36 bpm) responses to chemoreflex activation with potassium cyanide (iv). These data indicate that the activation of NK-1 receptors by substance P in the NTS produces an increase in baseline mean arterial pressure and heart rate. However, the data obtained with WIN suggest that substance P and NK-1 receptors do not play a major role in the neurotransmission of the chemoreflex in the lateral commissural NTS.

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.

Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons

A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 ± 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 µM and a maximum increase of 51.2 ± 4.4%. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons.

Neo-Timm staining in the thalamus of chronically epileptic rats

The thalamus is an important modulator of seizures and is severely affected in cholinergic models of epilepsy. In the present study, chronically epileptic rats had their brains processed for neo-Timm and acetylcholinesterase two months after the induction of status epilepticus with pilocarpine. Both controls and pilocarpine-treated animals presented neo-Timm staining in the anterodorsal nucleus, laterodorsal nucleus, reticular nucleus, most intralaminar nuclei, nucleus reuniens, and rhomboid nucleus of the thalamus, as well as in the zona incerta. The intensity of neo-Timm staining was similar in control and pilocarpine-treated rats, except for the nucleus reuniens and the rhomboid nucleus, which had a lower intensity of staining in the epileptic group. In animal models of temporal lobe epilepsy, zinc seems to modulate glutamate release and to decrease seizure activity. In this context, a reduction of neo-Timm-stained terminals in the midline thalamus could ultimately result in an increased excitatory activity, not only within its related nuclei, but also in anatomical structures that receive their efferent connections. This might contribute to the pathological substrate observed in chronic pilocarpine-treated epileptic animals.

Glutamate receptors as seen by light: spectroscopic studies of structure-function relationships

Ionotropic glutamate receptors are major excitatory receptors in the central nervous system and also have a far reaching influence in other areas of the body. Their modular nature has allowed for the isolation of the ligand-binding domain and for subsequent structural studies using a variety of spectroscopic techniques. This review will discuss the role of specific ligand:protein interactions in mediating activation in the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptors as established by various spectroscopic investigations of the GluR2 and GluR4 subunits of this receptor. Specifically, this review will provide an introduction to the insight gained from X-ray crystallography and nuclear magnetic resonance investigations and then go on to focus on studies utilizing vibrational spectroscopy and fluorescence resonance energy transfer to study the behavior of the isolated ligand-binding domain in solution and discuss the importance of specific ligand:protein interactions in the mechanism of receptor activation.