William T. Talman

Evidence that glutamic acid is the neurotransmitter of baroreceptor afferents terminating in the nucleus tractus solitarius (NTS)

The possibility that L-glutamic acid (L-Glu) might be the neurotransmitter of baroreceptor afferents terminating in the nucleus tractus solitarius (NTS) and affecting activity of the autonomic nervous system was tested using a variety of techniques. Effects on blood pressure and heart rate of microinjections of L-Glu and antagonists into the NTS supported this contention. Use of blocking agents was confirmatory. Biochemical studies provided evidence that the agent (glutamic acid) is synthesized, stored and reaccumulated in these nerve endings. Furthermore, it was shown that L-Glu is released from the NTS in vivo. Degeneration of afferent terminals due to removal of the nodose ganglia reduced L-Glu levels in the NTS. After discussion of this and additional evidence that L-Glu is the transmitter and a consideration of reasons for eliminating other possibilities (substance P, etc.) it was concluded that L-glutamic acid is the neurotransmitter of baroreceptor afferents.

Acute hypertension after the local injection of kainic acid into the nucleus tractus solitarii of rats.

Kainic acid (KA), an analogue of L-glutamate, was microinjected in 0.1 pi of saline into the nucleus tractus solitarii (NTS) of adult rats. In rats anesthetized with halothane or a-chloralose, KA injected unilaterally elicited hypotension, bradycardia, and apnea. The threshold dose was 0.1-0.2 ng (10~13 mol). Doses >0.2 ng blocked responses to subsequent injections for at least 30 minutes. Doses of KA >15 ng reduced the reflex bradycardia elicited by raising the arterial pressure with phenylephrine and produced arterial hypertension in rats anesthetized with a-chloralose or in other rats within 15 minutes of terminating halothane anesthesia. Bilateral injection of KA in doses >15 ng completely blocked baroreflexes and resulted in a dose-dependent elevation of arterial pressure (167 ± 9.4; P < 0.001) both in a-chloralose-anesthetized rats and in awake rats after the termination of halothane anesthesia. The hypertension rapidly led to pulmonary edema and death. Procaine microinjected also elicited fulminating hypertension; vehicle did not. Doses of KA producing hypertension caused no histological or biochemical evidence of neuronal death. The cardiovascular responses to KA were restricted to sites in the intermediate one-third of NTS and could not be elicited by injection into adjacent sites in brainstem. The results indicate that, in low doses, KA injected into NTS stimulates neurons which mediate the baroreflex, whereas, in higher doses, it produces baroreflex blockade and neurogenic hypertension. The results suggest that fulminating hypertension can be produced by nondestructive perturbations of neurochemical transmission in brain. Since the cadiovascular responses of KA are similar to those produced by microinjection into NTS of the amino acid neurotransmitter glutamic acid, the study adds further support to the hypothesis that L-glutamate is the neurotransmitter released by baroreceptor afferent nerves. Circ Res 48: 292-298, 1981

Content and in vitro release of endogenous amino acids in the area of the nucleus of the solitary tract of the rat

Abstract: We sought to identify amino acid neurotransmitter candidates within the nucleus of the solitary tract in rats. Twenty endogenous amino acids were quantified by reverse‐phase HPLC with fluorescence detection (30‐fmol limit). Mi‐cropunches (1 mm) of the intermediate area of the solitary nucleus were prepared, and the amino acid content determined. Of all the components measured, the putative transmitters Glu, Gly, γ‐aminobutyric acid, taurine, Asp, and Ala appeared in greatest concentrations. Bilateral micropunches superfused in vitro with buffered medium containing 56 mM potassium released Glu, γ‐aminobutyric acid, and Gly in a significant manner (p < 0.05) compared with basal levels. With Glu, 78% was calcium‐dependent and, therefore, presumably from nerve endings; 99% of γ‐aminobutyric acid and 42% of Gly were dependent on calcium. After removal of the nodose ganglion, a bilateral decrease in the calcium‐dependent release of Glu and γ‐aminobutyric acid, but not Gly, was observed; decreases were significant ipsilateral to the site of ablation. We conclude that (a) Glu is a transmitter of primary afferents in the nucleus of the solitary tract; (b) glutamatergic afferents may interact with γ‐aminobutyric acid system(s) in this region; (c) Gly also may participate in the mediation and/or modulation of cardiovascular or other visceral reflexes; and (d) amino acid neurotransmission may play an integral role in the neurogenic control of arterial pressure

Kynurenic acid microinjected into the nucleus tractus solitarius of rat blocks the arterial baroreflex but not responses to glutamate

The selective excitatory amino acid antagonist kynurenic acid was bilaterally microinjected into the nucleus tractus solitarius of rats to determine its effects on the arterial baroreflex and on the cardiovascular responses to glutamate and nonglutamate agonists injected at the same site. Kynurenic acid blocked the responses to N-methyl-D-aspartate and kainate as well as the baroreflex, but did not block the response to glutamate, quisqualate, or acetylcholine. The data may suggest that glutamate is not a neurotransmitter in the baroreflex arc or that only certain glutamate receptors are integral to the baroreflex in the nucleus tractus solitarius.

Role of Endogenous Carbon Monoxide in Central Regulation of Arterial Pressure

We investigated the contribution of neural mechanisms to the arterial pressure increase produced by zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG), an inhibitor of endogenous carbon monoxide synthesis. The arterial baroreceptor reflex control of heart rate was examined in rats with and without ZnDPBG pretreatment (45 micromol/kg IP) by analysis of the arterial pressure-heart rate relationship during infusions of phenylephrine or sodium nitroprusside to vary arterial pressure. ZnDPBG increased arterial pressure from 110 +/- 3 to 126 +/- 2 mm Hg without eliciting bradycardia. The maximum gain of the heart rate response to changes in arterial pressure was attenuated by ZnDPBG treatment (-1.9 +/- 0.3 versus -4.8 +/- 1.0 bpm/mm Hg). The possibility that ZnDPBG elevates arterial pressure by attenuating baroreceptor reflex function was addressed by comparing the pressor response to ZnDPBG (45 micromol/kg IP) in rats with and without sinoaortic denervation. The pressor effect of ZnDPBG was similar in rats with and without arterial baroreceptor deafferentation, implying that the increase in pressure is not simply the consequence of attenuated baroreceptor reflex function per se. The possibility that ZnDPBG increases arterial pressure via an effect on the nucleus tractus solitarii (NTS) also was investigated. ZnDPBG (1 nmol in 100 nL) injected into the NTS of rats increased arterial pressure from 111 +/- 4 to 126 +/- 5 mm Hg, and this effect was reversed by an ipsilateral microinjection of carbon monoxide into the NTS. Accordingly, the pressor effect of ZnDPBG may rely on inhibition of carbon monoxide production in the NTS. This implies that carbon monoxide formed by brain heme oxygenase plays a role in the central regulation of arterial pressure.

Chronic lability of arterial pressure produced by destruction of A2 catecholaminergic neurons in rat brainstem.

SUMMARY The cardiovascular effects of electrolytic lesions of the A2 group of catecholaminergic neurons of the dorsal medulla were analyzed in chronically instrumented rats. A2 lesions resulted after 24 hours, in an enduring increase in lability (variability) and augmented reactivity of the arterial pressure during spontaneous or elicited behaviors, without a change in the average arterial pressure. Heart rate and its variability were unchanged. A2 lesions almost abolished the bradycardia elicited by acutely elevating arterial pressure with phenylephrine, but not the hypotension elicited under anesthesia by carotid sinus stretch. Lability of arterial pressure could not be attributed to damage to cardiovagal neurons. Vagal blockade with atropine or methylatropine did not alter the mean or variability of the arterial pressure. Lability of arterial pressure was produced only by damage to A2 neurons and not by lesions in the area postrema or by transection of commissural fibers of the nucleus tractus solitarii (NTS). Moreover, the magnitude of lability was correlated directly with the amount of damage to A2. A2 lesions resulted in a reduction of dopamine-/3-hydroxylase activity to 60% of control in the NTS. We conclude that destruction of A2 neurons produces persistent lability and exaggerated reactivity of arterial pressure as a consequence of partial removal of the noradrenergic innervation of the NTS. The results suggest that noradrenergic neurons of A2 serve to modulate baroreceptor reflexes in NTS. The observation that lability of arterial pressure can occur in the absence of changes of average arterial pressure suggests distinctive anatomical networks subserving phasic and tonic control of the arterial pressure in the brainstem. Circ Res 46: 842-853, 1980

Cholinergic mechanisms in the nucleus tractus solitarii and cardiovascular regulation in the rat

To determine the role played by cholinergic neurons in cardiovascular regulation by the nucleus tractus solitarii (NTS), microinjections (0.1 microliter) of acetylcholine (ACh) or carbachol (C) were made into the NTS of rats, and the changes in arterial pressure and heart rate were recorded. The dose-dependent hypotension and bradycardia which resulted were mediated by the activation of muscarinic, but not nicotinic, receptors confined to the NTS. Blockade of muscarinic receptors bilaterally in the NTS resulted in mild hypertension and attenuated, but did not abolish, the baroreceptor reflex. Eserine, injected into the NTS, augmented and prolonged the action of acetylcholine and slightly increased the maximal baroreceptor reflex response. Thus, cholinergic mechanisms in the NTS tend tonically to lower arterial pressure and may modulate the baroreceptor reflex, without being an integral part of the reflex arc.

Direct evidence for nitric oxide synthase in vagal afferents to the nucleus tractus solitarii

The anatomical relationship between vagal afferents and brain nitric oxide synthase containing terminals in the nucleus tractus solitarii was studied by means of anterograde tracing combined with immunocytochemistry and immuno-electron microscopy. Biotinylated dextran amine was injected into the nodose ganglion with a glass micropipette. Four to eight days following the injection, regions of the nucleus tractus solitarii containing biotinylated dextran amine-labelled vagal afferents and those containing nitric oxide synthase-immunopositive terminals were congruent. Many neurons exhibiting nitric oxide synthase immunoreactivity were found within the biotinylated dextran amine-containing terminal field. However dense labeling of terminals with biotinylated dextran amine precluded determination if the terminals were nitric oxide synthase-immunoreactive. Therefore, we combined degeneration of vagal afferents after removal of one nodose ganglion with nitric oxide synthase immuno-electron microscopy. Axon terminals that possessed characteristic vesicle clusters and were partially or completely engulfed by glial processes were identified as degenerating vagal afferents. Degenerating axon terminals comprised 38% of the total axon terminals in the nucleus tractus solitarii in a sample of sections; and of the degenerating axon terminals, 67% were nitric oxide synthase-immunoreactive. Nitric oxide synthase immunoreactivity was present in 41% of the non-degenerating axon terminals. Prominent staining of dendrites for nitric oxide synthase immunoreactivity indicated that much of the nitric oxide synthase in the nucleus tractus solitarii is not derived from peripheral afferents. Of the total number of dendritic profiles sampled, half were nitric oxide synthase-immunoreactive. Our data support the hypothesis that nitric oxide or nitric oxide donors may be present in primary vagal afferents that terminate in the nucleus tractus solitarii. While this study confirms that vagal afferents contain brain nitric oxide synthase, it demonstrates for the first time that the majority of nitric oxide synthase immunoreactivity in the nucleus tractus solitarii is found in intrinsic structures in the nucleus. In addition, our data show that second or higher order neurons in the nucleus tractus solitarii may be nitroxidergic and receive both nitroxidergic and non-nitroxidergic vagal input.

Baroreflex actions of substance P microinjected into the nucleus tractus solitarii in rat: a consequence of local distortion

Biologically active substance P (SP) (1000 ng in 0.1 microliter saline) microinjected into the nucleus tractus solitarii (NTS) of 25 rats did not affect arterial pressure, heart rate, or the baroreceptor reflex. However, microinjection of saline alone in volumes greater than 0.3 microliter consistently elicited hypotension and bradycardia followed occasionally by transient hypertension. These data suggest that previously reported cardiovascular effects of SP microinjected into the NTS resulted from local distortion.

Modulation of cardiovascular and electrocortical activity through serotonergic mechanisms in the nucleus tractus solitarius of the rat

The nucleus tractus solitarius (NTS) is an integral part of the baroreceptor reflex arc. Thus, stimulation of the NTS elicits changes in arterial pressure and heart rate as well as in numerous other physiologic parameters including electrocortical activity. Serotonin (5-HT), which has been implicated in cardiovascular and electrocortical control, is present in nerve terminals within the NTS. Therefore, this study was designed to determine whether 5-HT may effect that control within the NTS. Serotonin injected into the NTS of anesthetized rats produced marked changes in the EEG, arterial pressure, and heart rate. EEG activity changed from irregular 1-5 Hz, 350-500 microV waves with an overlying 13-15 Hz, low voltage rhythm to a regular, 5 Hz, 250-300 microV rhythm. The dose-dependent cardiovascular changes were maximal at a dose of 400 pmol which produced a fall of mean arterial pressure of 48 +/- 2 mm Hg from a baseline of 96 +/- 4 mm Hg and of heart rate of 90 +/- 9 bpm from a baseline of 400 +/- 18 bpm (n = 6; P less than 0.001). Both the cardiovascular and EEG effects of 5-HT injected into the NTS were blocked by the prior injection of the 5-HT antagonist metergoline at the same site. However, the bilateral microinjection of metergoline into the NTS did not affect the baroreceptor reflex. Thus, although serotonergic mechanisms in the NTS may be involved in the modulation of electrocortical and cardiovascular activity, they are not integral to the baroreceptor reflex arc.