Vineet C. Chitravanshi

A midline area in the nucleus commissuralis of NTS mediates the phrenic nerve responses to carotid chemoreceptor stimulation

The carotid body chemoreceptor afferents have been reported to project to a discrete area located in the nucleus commissuralis of nucleus tractus solitarius [A. Vardhan et al., Am. J. Physiol., 264 (1993) R41-R50]. The afore-mentioned study was done in spontaneously breathing rats and the afferents and efferents located in the chest wall and the respiratory tract of these animals were intact. In order to exclude the role, if any, of these afferents and efferents, in the present experiments respiratory changes were monitored by recording the phrenic nerve activity instead of tracheal airflow. Experiments were carried out in pentobarbital-anesthetized, bilaterally vagotomized, paralyzed and artificially ventilated rats with a pneumothorax. The carotid body chemoreceptors were stimulated with tracheal administration of nitrogen for 7-10 s. The chemoreceptor stimulation induced an increase in the frequency and amplitude of phrenic nerve bursts. A decrease in the duration of inspiratory (T1), expiratory (TE) and total cycles (TTOT) was observed in the phrenic nerve activity. Inhibition of neuronal cell bodies by microinjections of muscimol (140 pmol/20 nl) into a discrete area in the commissural subnucleus of the nucleus tractus solitarius (coordinates in mm: 0.3 rostral to 0.5 caudal, 0 to 0.5 lateral and 0.3 to 0.5 deep with respect to the calamus scriptorius), attenuated the phrenic nerve responses to the carotid body stimulation. On the other hand, control injections of saline (0.9%) into this site did not alter the phrenic nerve response to the carotid body stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

NMDA as well as non-NMDA receptors mediate the neurotransmission of inspiratory drive to phrenic motoneurons in the adult rat

The neurotransmission of bulbospinal respiratory drive is believed to involve primarily non-NMDA receptors located in the phrenic motonucleus (PMN). This conclusion is based on studies carried out mainly on in vitro brainstem-spinal cord preparations of the neonatal rat. The present study was undertaken to investigate the transmitter/receptor mechanisms in the PMN which are involved in the neurotransmission of inspiratory drive, using an in vivo adult rat model. Microinjections of glutamate, NMDA and AMPA into the PMN elicited an increase in the phrenic nerve (PN) background discharge. These injections did not alter significantly the frequency of spontaneously occurring PN bursts confirming that mechanisms responsible for respiratory rhythm reside in the supraspinal structures. Microinjections of an NMDA receptor blocker (AP-7), in concentrations that did not alter the responses to a non-NMDA receptor agonist (AMPA), reduced the PN amplitude significantly. Similarly, microinjections of a potent non-NMDA receptor blocker (NBQX), in concentrations that did not alter responses to NMDA, reduced the PN amplitude significantly. Sequential microinjections, within an interval of 5 min, of AP-7 and NBQX into the PMN, resulted in a dramatic reduction in the spontaneous PN bursts. The reduction of PN amplitude started immediately after the microinjection of AP-7 and NBQX, either alone or in combination, and reached a maximum within 5-10 min. These results indicate that, unlike in the neonatal rat, both NMDA and non-NMDA receptors located in the PMN play a significant role in the neurotransmission of the inspiratory drive in the adult rat.

Phrenic nerve responses to chemical stimulation of the subregions of ventral medullary respiratory neuronal group in the rat.

Phrenic nerve (PN) responses to unilateral microinjections of L-glutamate (L-Glu, 5 mM) or N-methyl-D-aspartic acid (NMDA, 1 mM) into different subregions of ventral respiratory neuronal group (VRG) were studied in urethane-anesthetized, immobilized, and artificially ventilated, adult male Wistar rats. A 50-nl volume of microinjection was used in all the subregions of VRG except in Pre-Bötzinger complex (Pre-BötC) where a 20-nl volume was used. Unilateral microinjections of L-Glu or NMDA into the Bötzinger complex (BötC) and caudal VRG (cVRG), caused a transient cessation of phrenic nerve (PN) activity. Expiratory neurons, abundant in BötC and cVRG, were excited by stimulation of cardiopulmonary receptors while their responses to carotid chemoreceptor stimulation were variable. Microinjections of L-Glu or NMDA into the Pre-BötC caused an increase in the PN background discharge (this response was unique to Pre-BötC) superimposed on which was an increase in the PN burst frequency. Microinjections of L-Glu or NMDA into the rostral VRG (rVRG) caused an increase in the frequency and amplitude of PN bursts. Inspiratory neurons, abundant in Pre-BötC and rVRG, were excited and inhibited by activation of carotid chemoreceptors and cardiopulmonary receptors, respectively. The coordinates for the location of different subregions of VRG were as follows (reference points are listed in parentheses). BötC: 1.6-2.6 mm rostral (calamus scriptorius), 1.7-2.7 mm lateral (midline), and 2.3-2.8 mm deep (dorsal surface of medulla); Pre-BötC: 1.4-1.6 mm rostral, 1. 8-2.5 mm lateral, and 2.3-2.8 mm deep; rVRG: 0.4-1.4 mm rostral, 1. 6-2.5 mm lateral, and 2.3-2.8 mm deep; and cVRG: 0.5 mm caudal to 0. 5 mm rostral, 1.0-2.2 mm lateral, and 2.1-2.6 mm deep. A detailed map of the subregions of VRG, functionally identified by L-Glu and NMDA-microinjections, has been presented. These data are likely to prove useful in future studies on respiratory reflex mechanisms.

The hypothalamic arcuate nucleus: a new site of cardiovascular action of angiotensin-(1-12) and angiotensin II.

The hypothalamic arcuate nucleus (ARCN) has been reported to play a significant role in cardiovascular regulation. It has been hypothesized that the ARCN may be one of the sites of cardiovascular actions of angiotensins (ANGs). Experiments were carried out in urethane-anesthetized, artificially ventilated, adult male Wistar rats. The ARCN was identified by microinjections of N-methyl-d-aspartic acid (NMDA; 10 mM). Microinjections (50 nl) of ANG-(1-12) (1 mM) into the ARCN elicited increases in mean arterial pressure (MAP), heart rate (HR), and greater splanchnic nerve activity (GSNA). The tachycardic responses to ANG-(1-12) were attenuated by bilateral vagotomy. The cardiovascular responses elicited by ANG-(1-12) were attenuated by microinjections of ANG II type 1 receptor (AT(1)R) antagonists but not ANG type 2 receptor (AT(2)R) antagonist. Combined inhibition of ANG-converting enzyme (ACE) and chymase in the ARCN abolished ANG-(1-12)-induced responses. Microinjections of ANG II (1 mM) into the ARCN also increased MAP and HR. Inhibition of ARCN by microinjections of muscimol (1 mM) attenuated the pressor and tachycardic responses to intravenously administered ANG-(1-12) and ANG II (300 pmol/kg each). These results indicated that 1) microinjections of ANG-(1-12) into the ARCN elicited increases in MAP, HR, and GSNA; 2) HR responses were mediated via both sympathetic and vagus nerves; 3) AT(1)Rs, but not AT(2)Rs, in the ARCN mediated ANG-(1-12)-induced responses; 4) both ACE and chymase were needed to convert ANG-(1-12) to ANG II in the ARCN; and 5) ARCN plays a role in mediating the cardiovascular responses to circulating ANGs.

Cardiovascular function of a glutamatergic projection from the hypothalamic paraventricular nucleus to the nucleus tractus solitarius in the rat.

Experiments were done in urethane-anesthetized, barodenervated, male Wistar rats. Chemical stimulation of the hypothalamic paraventricular nucleus (PVN) by unilateral microinjections of N-methyl-D-aspartic acid (NMDA) elicited increases in mean arterial pressure (MAP) and greater splanchnic nerve activity (GSNA). The increases in the MAP and GSNA induced by chemical stimulation of the PVN were significantly exaggerated by bilateral microinjections of D(-)-2-amino-7-phosphono-heptanoic acid (D-AP7) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydro-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) (ionotropic glutamate receptor antagonists) into the medial subnucleus of the nucleus tractus solitarius (mNTS). These results were confirmed by single unit recordings; i.e. excitation of mNTS barosensitive neurons caused by chemical stimulation of the ipsilateral PVN was blocked by application of D-AP7 and NBQX to these neurons. Bilateral microinjections of D-AP7 and NBQX into the mNTS elicited pressor responses which were significantly attenuated by inhibition of PVN neurons by bilateral microinjections of muscimol. Unilateral microinjections of fluorogold into the mNTS resulted in bilateral retrograde labeling of the PVN neurons. Unilateral microinjections of biotinylated dextran amine into the PVN resulted in anterograde labeling of axons and terminals in the mNTS bilaterally and the labeled terminals exhibited vesicular glutamate transporter-2 immunoreactivity. These results indicated that 1) a tonically active glutamatergic bilateral projection from the PVN to the mNTS exists; 2) bilateral blockade of ionotropic glutamate receptors in the mNTS exaggerates the increases in MAP and GSNA, but not heart rate, to the chemical stimulation of the PVN; and 3) this projection may serve as a restraint mechanism for excitatory cardiovascular effects of PVN stimulation.

Cardiovascular responses to microinjections of nicotine into the caudal ventrolateral medulla of the rat

This study focuses on the role of nicotinic receptors located in the caudal ventrolateral medullary depressor area (CVLM) in regulating/modulating cardiovascular function. Blood pressure and heart rate were monitored by standard techniques in urethane-anesthetized, artificially ventilated, adult male Wistar rats. Multi-barreled glass-micropipettes (tip size 20-40 microm) were used to make microinjections (100 nl) into the CVLM. Concentrations of nicotine ranging from 250 micromto 10 mM were microinjected unilaterally into the CVLM. The maximum depressor and bradycardic responses were elicited by a 1 mM concentration of nicotine. Sequential microinjections of mecamylamine (1 mM), an antagonist for nicotinic receptors containing alpha3beta4 subunits, then alpha-bungarotoxin (1 microm), an antagonist for nicotinic receptors containing alpha-7 subunits, were made into the CVLM. Microinjecting a combination of a nicotinic receptor blocker and toxin resulted in the complete blockade of the cardiovascular responses induced by nicotine (1 mM, 100 nl). These results indicate that: (1) nicotinic receptors are present in the CVLM; (2) activation of these receptors results in depressor and bradycardic responses; (3) for a complete blockade of nicotine-induced cardiovascular responses, it is necessary to use a combination of mecamylamine and alpha-bungarotoxin; (4) since mecamylamine and alpha-bungarotoxin are known to block nicotinic receptors containing alpha3beta4 and alpha-7 subunits, respectively, two different subtypes of nicotinic receptors (one which contains a combination of alpha3beta4 subunits, and one which contains alpha-7 subunits) must be present in the CVLM; and (5) it is not clear whether these two subtypes of nicotinic receptor are located on the same or different populations of CVLM-neurons.

Cardiovascular effects of adrenocorticotropin microinjections into the rostral ventrolateral medullary pressor area of the rat.

The presence of adrenocorticotropic hormone (ACTH)-immunoreactive cells and melanocortin (MC) receptors (MC4 and to a lesser extent MC3) has been demonstrated in the medullary reticular formation in the general area where rostral ventrolateral medullary pressor area (RVLM) is located. The importance of RVLM in the regulation of cardiovascular function is well established. Based on these reports, it was hypothesized that ACTH may play a role in the regulation of cardiovascular function. To test this hypothesis, experiments were carried out on artificially ventilated, adult male, urethane-anesthetized and unanesthetized mid-collicular decerebrate rats. The RVLM was identified by microinjections (100 nl) of L-glutamate (L-Glu). Microinjections (100 nl) of ACTH (0.5, 1 and 2 mmol/l) into the RVLM elicited increases in MAP and HR; tachycardic responses were relatively inconsistent. The effects of ACTH were blocked by SHU9119 and agouti-related protein (AGRP). SHU9119 (a synthetic compound) and AGRP (an endogenous peptide) are antagonists for MC4, and to a lesser extent MC3, receptors. The specificity of these antagonists for MC receptors was indicated by their lack of effect on l-Glu responses. Microinjection of ACTH into the RVLM increased the efferent discharge in the greater splanchnic nerve. It was concluded that (1) ACTH exerts excitatory effects on RVLM neurons resulting in pressor and tachycardic responses, (2) these responses were mediated via MC4 and to a lesser extent MC3 receptors in the RVLM, and (3) the pressor effects of ACTH were mediated via sympathetic activation. This is the first report showing central cardiovascular actions of ACTH.

Overexpression of the dopamine D3 receptor in the rat dorsal striatum induces dyskinetic behaviors

L-DOPA-induced dyskinesias (LID) are motor side effects associated with treatment of Parkinsons disease (PD). The etiology of LID is not clear; however, studies have shown that the dopamine D3 receptor is upregulated in the basal ganglia of mice, rats and non-human primate models of LID. It is not known if the upregulation of D3 receptor is a cause or result of LID. In this paper we tested the hypothesis that overexpression of the dopamine D3 receptor in dorsal striatum, in the absence of dopamine depletion, will elicit LID. Replication-deficient recombinant adeno-associated virus-2 expressing the D3 receptor or enhanced green fluorescent protein (EGFP) were stereotaxically injected, unilaterally, into the dorsal striatum of adult rats. Post-hoc immunohistochemical analysis revealed that ectopic expression of the D3 receptor was limited to neurons near the injection sites in the dorsal striatum. Following a 3-week recovery period, rats were administered saline, 6 mg/kg L-DOPA, 0.1 mg/kg PD128907 or 10 mg/kg ES609, i.p., and motor behaviors scored. Rats overexpressing the D3 receptor specifically exhibited contralateral axial abnormal involuntary movements (AIMs) following administration of L-DOPA and PD128907 but not saline or the novel agonist ES609. Daily injection of 6 mg/kg L-DOPA to the rats overexpressing the D3 receptor also caused increased vacuous chewing behavior. These results suggest that overexpression of the D3 receptor in the dorsal striatum results in the acute expression of agonist-induced axial AIMs and chronic L-DOPA-induced vacuous chewing behavior. Agonists such as ES609 might provide a novel therapeutic approach to treat dyskinesia.

Cardiovascular responses elicited by a new endogenous angiotensin in the nucleus tractus solitarius of the rat

Cardiovascular effects of angiotensin-(1-12) [ANG-(1-12)] were studied in the medial nucleus of the tractus solitarius (mNTS) in anesthetized, artificially ventilated, adult male Wistar rats. Microinjections (100 nl) of ANG-(1-12) (0.06 mM) into the mNTS elicited maximum decreases in mean arterial pressure (MAP; 34 ± 5.8 mmHg) and heart rate (HR; 39 ± 3.7 beats/min). Bilateral vagotomy abolished ANG-(1-12)-induced bradycardia. Efferent greater splanchnic nerve activity was decreased by microinjections of ANG-(1-12) into the mNTS. Blockade of ANG type 1 receptors (AT(1)Rs; using ZD-7155 or L-158,809), but not ANG type 2 receptors (AT(2)Rs; using PD-123319), significantly attenuated ANG-(1-12)-induced cardiovascular responses. Simultaneous inhibition of both angiotensin-converting enzyme (ACE; using captopril) and chymase (using chymostatin) completely blocked the effects of ANG-(1-12). Microinjections of A-779 [ANG-(1-7) antagonist] did not attenuate ANG-(1-12)-induced responses. Pressure ejection of ANG-(1-12) (0.06 mM, 2 nl) caused excitation of barosensitive mNTS neurons, which was blocked by prior application of the AT(1)R antagonist. ANG-(1-12)-induced excitation of mNTS neurons was also blocked by prior sequential applications of captopril and chymostatin. These results indicate that 1) microinjections of ANG-(1-12) into the mNTS elicited depressor and bradycardic responses by exciting barosensitive mNTS neurons; 2) the decreases in MAP and HR were mediated via sympathetic and vagus nerves, respectively; 3) AT(1)Rs, but not AT(2)Rs, mediated these actions of ANG-(1-12); 4) the responses were mediated via the conversion of ANG-(1-12) to ANG II and both ACE and chymase were involved in this conversion; and 5) ANG-(1-7) was not one of the metabolites of ANG-(1-12) in the mNTS.

Bradycardic effects mediated by activation of G protein-coupled estrogen receptor in rat nucleus ambiguus

•  What is the central question of this study? Clinical and experimental studies indicate that estrogen increases cardiac vagal tone. The role of the G protein‐coupled estrogen receptor in autonomic cardiac control is not known. •  What is the main finding and its importance? Using calcium imaging, electrophysiological techniques and in vivo studies of microinjection into the nucleus ambiguus and measurement of cardiovascular responses, we show that activation of the G protein‐coupled estrogen receptor in cardiac vagal neurons of the nucleus ambiguus increases cytosolic Ca2+ and depolarizes these neurons, leading to a decrease in heart rate. Our findings suggest a novel role for the G protein‐coupled estrogen receptor in modulation of cardiac vagal tone.