Adrenaline-synthesizing neurons in the medulla of the cat.

In this study, the distribution of neurons containing the adrenaline-synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) was mapped in the medulla of the cat. Data from recent studies in the rat suggest that the anatomical structure responsible for cardiorespiratory changes that occur following application of neurotransmitters and drugs to Schlaefke's area on the ventral medullary surface is the nucleus reticularis rostroventrolateralis (RVL), which is distinguished from adjacent regions of the reticular formation, in part, by the presence of adrenaline-synthesizing neurons. To determine whether an equivalent adrenergic population is present in the RVL of the cat, we used antibodies raised against bovine adrenal PNMT to map the distribution of adrenaline-synthesizing neurons in the reticular formation. In the ventrolateral medulla, we found that labeled cells extended from the level of the retrofacial nucleus to the calamus scriptorius. The majority of labeled cells were seen in a nucleus designated RVL at the level of the rostral one-third of the inferior olive. In the dorsomedial medulla, cells were labeled in the caudal aspect of the nucleus tractus solitarii (NTS) and were especially dense in the subnucleus gelatinosus and commissural nucleus of the vagus. A few lightly labeled cells were also present in the rostral pole of the area postrema (AP). In contrast to the rat, few or no immunoreactive cells were found in the rostral NTS, medial longitudinal fasciculus, nucleus paragigantocellularis dorsalis, or periventricular gray. Our results are consistent with the notion that an area of the RVL containing adrenergic perikarya is the anatomical structure responsible for cardiovascular changes that occur when chemicals are applied to Schlaefke's area.

The effect of intrahypothalamic injection of homodimaprit on blood pressure.

Bilateral injection of the inhibitor of histamine-N-methyltransferase, SKF 91488, which is also known as homodimaprit (5 micrograms), into the preoptic area of the rat produced delayed hypertension, tachycardia and hyperthermia. Some animals exhibited pulmonary edema. These effects were only noted 18-24 hr after an injection and were not an artifact of the injection, since the administration of artificial cerebrospinal fluid produced none of these effects. At the time noted, lesions of the rostral hypothalamus, including the preoptic area, were evident. Injection of a vasopressin antagonist, intravenously, did not lower the blood pressure of the hypertensive animals nor did previous bilateral adrenal demullation prevent or delay the hypertension or tachycardia. Therefore, it does not appear that hypersecretion of either vasopressin or adrenal catecholamines contributed to the cardiovascular effects. Peripheral pretreatment with the sympathetic neurotoxin 6-hydroxydopamine however, did prevent the delayed rise in blood pressure following an injection of homodimaprit. From these studies, it is concluded that the injection of homodimaprit produces lesions in the preoptic area, resulting in hypertension that is maintained by excessive activation of the sympathetic nervous system.

The effect of a vasopressin antagonist on the pressor response to histamine administered centrally.

The mechanism for the pressor response to intracerebroventricularly (i.c.v.) administered histamine was studied. Histamine (HA), when injected intracerebroventricularly in rats, produced a dose-dependent increase in mean arterial pressure (MAP) which was subject to tachyphylaxis. Spinal transection at C-7 in the anesthetized rat did not attenuate the rise in blood pressure. The possibility that the release of 8-arginine vasopressin was responsible for the rise in blood pressure was explored. By pretreating conscious freely-moving rats with a specific antagonist to the vasopressor action of vasopressin viz., [1-beta-mercapto-beta, beta-cyclopentamethyleneproprionic acid), 2-(O-methyl) tyrosine] arginine-vasopressin, there was a statistically-significant attenuation of the pressor response to intracerebroventricularly administered histamine. The antagonist however, did not totally abolish the pressor response, regardless of the dose employed. Concomitant administration of hexamethonium and the vasopressin antagonist did not further attenuate the response. Previous adrenal demedullation did not diminish the response to intracerebroventricularly administered histamine, nor was there any evidence for release of angiotensin II since pretreatment with saralasin did not attenuate the cardiovascular response. These findings suggest that vasopressin along with other as yet undefined substances, are released from the central nervous system to produce the increase in blood pressure after intracerebroventricularly administered histamine.

Cardiorespiratory effects produced by injecting drugs that affect GABA receptors into nuclei associated with the ventral surface of the medulla.

It has recently been shown that L-glutamic acid induced stimulation of cell bodies in a circumscribed area of the rostral ventrolateral medulla (RVLM) in the cat, produced increases in arterial pressure (AP), decreases in heart rate (HR) and transient apnea (Gatti, Norman, DaSilva and Gillis, 1986). The purpose of the present study was to determine if this same area was sensitive to GABA receptor agonists and antagonists. Injection of the GABA agonist muscimol (200 ng), into the rostral ventrolateral medulla of cats anesthetized with chloralose produced a precipitous and immediate fall in arterial pressure (-95 +/- 4.6) and heart rate (-31 +/- 5.9; n = 4, P less than 0.05). Maximal cardiovascular effects could only be achieved if muscimol was injected bilaterally. These effects of muscimol on arterial pressure were dose-dependent. Time-action curves for the effects of muscimol on arterial pressure and respiration were different. Hypotension occurred first and was followed later in time by a decrease in minute ventilation. Within 30 min all animals were apneic after the 200 ng dose. The cardiovascular effects of muscimol were reversed by the injection of the GABA receptor antagonist bicuculline. These data indicate that stimulation of GABA receptors in the rostral ventrolateral medulla produced selective cardiovascular effects and that respiratory neurons sensitive to GABA are apparently not localized with these cardiovascular neurons.

Inhibition of HIV type 1 production by hygromycin B.

HIV infection alters the cellular uptake of ions and other small molecules. This study was designed to determine whether hygromycin B, a low molecular weight (MW 527) aminoglycoside protein synthesis inhibitor that is normally impermeable to mammalian cells at micromolar concentrations, can selectively inhibit HIV expression and cytopathology. CD4+ T lymphoblastoid cells (H9) and peripheral blood mononuclear cells (PBMCs) were infected with HIV-1, then incubated in medium containing various concentrations of hygromycin B. HIV-1-induced formation of multinucleated giant cells and single cell killing were dramatically reduced in the presence of micromolar concentrations of hygromycin B. Hygromycin B also inhibited HIV-1 production in a dose-dependent manner during acute infection. G418, a larger and more hydrophobic aminoglycoside (MW 692), did not display the same selective inhibition of HIV-1 production as hygromycin B. Relative to mock-infected cells, protein synthesis in acutely infected H9 cells was selectively inhibited by hygromycin B. Hygromycin B also reduced HIV production in PBMCs and in H9 cells persistently infected with HIV. PCR analysis demonstrated that hygromycin B did not inhibit HIV-1 reverse transcription. These results demonstrate that HIV-1 infection renders cells more sensitive to hygromycin B than uninfected cells, and provides support for the hypothesis that HIV-1 induces an alteration of plasma membrane permeability. The HIV-modified cell membrane may be a potential target for antiviral intervention and chemotherapy.

A synthetic peptide corresponding to the carboxy terminus of human immunodeficiency virus type 1 transmembrane glycoprotein induces alterations in the ionic permeability of Xenopus laevis oocytes.

The carboxy-terminal 29 amino acids of the human immunodeficiency virus type 1 transmembrane glycoprotein (HIV-1 TM) are referred to as lentivirus lytic peptide 1 (LLP-1). Synthetic peptides corresponding to LLP-1 have been shown to induce cytolysis and to alter the permeability of cultured cells to various small molecules. To address the mechanisms by which LLP-1 induces cytolysis and membrane permeability changes, various concentrations of LLP-1 were incubated with Xenopus laevis oocytes, and two-electrode, voltage-clamp recording measurements were performed. LLP-1 at concentrations of 75 nM and above induced dramatic alterations in the resting membrane potential and ionic permeability of Xenopus oocytes. These concentrations of LLP-1 appeared to induce a major disruption of plasma membrane electrophysiological integrity. In contrast, concentrations of LLP-1 of 20-50 nM induced changes in membrane ionic permeability that mimic changes induced by compounds, such as the bee venom peptide melittin, that are known to form channel-like structures in biological membranes at sublytic concentrations. An analog of LLP-1 with greatly reduced cytolytic activity failed to alter the electrophysiological properties of Xenopus oocytes. Thus, by altering plasma membrane ionic permeability, the carboxy terminus of TM may contribute to cytolysis of HIV-1-infected CD4+ cells.

Experimental studies on the neurocardiovascular effects of urapidil.

The major purpose of our study was to determine whether urapidil acts in the central nervous system (CNS) to lower arterial blood pressure. Once demonstrating a CNS antihypertensive action of urapidil we further set out to determine: (1) the relative role of a CNS antihypertensive action to the total antihypertensive effect of urapidil; (2) the brain site of action for the antihypertensive effect of urapidil; and, (3) the receptor mechanism whereby urapidil acts in the CNS to lower arterial blood pressure. Studies were conducted in chloralose-anaesthetised cats, and arterial blood pressure and heart rate were monitored. Drugs were administered intravenously (IV), into the cerebral ventricles (ICV), topically by application to the ventral surface of the medulla and by microinjection into specific nuclei. Receptor binding studies were also conducted using rat cerebral cortex homogenates. We found that injection of urapidil into the fourth ventricle decreased arterial pressure. Local application of urapidil to the ventral medullary surface also decreased arterial blood pressure. Microinjection of urapidil into one of the nuclei associated with the ventral surface of the medulla, the rostral part of the nucleus reticularis lateralis (rLRN), produced a similar degree of antihypertensive effect. The effect of urapidil was not altered by alpha 1-receptor blockade. Instead, the urapidil effect resembled that produced by drugs that stimulate serotonin (5-hydroxytryptamine)-1A receptors (B695-40 and 8-OH-DPAT). Furthermore, urapidil was found to have the highest potency for binding to serotonin-1A receptor sites (as compared to alpha 1- and alpha 2-receptor sites). Urapidil administered IV was shown to lower arterial blood pressure in part by blocking peripheral alpha 1-adrenoceptors but also, in high doses, by acting in the CNS to decrease central sympathetic outflow. These data indicate that urapidil is a unique drug, possessing both peripheral and CNS actions which contribute to its antihypertensive effect. Urapidil may also be unique in that its central action may involve activation of serotonin-1A receptors.

Interaction between pressor and depressor areas in cat ventrolateral medulla.

The purpose of the present study was to investigate whether stimulation of the caudal depressor area (CA) in the medulla lowered blood pressure (BP) by enhancing GABA release at the intermediate pressor area (IA) of the medulla. Application of the excitotoxin kainic acid (KA; 40 mM solution) to the caudal area lowered BP as has been previously described (Gatti, et al., Brain Research, 330 (1985) 21-29). Subsequent IA application of the GABA receptor antagonists picrotoxin (100 micrograms/side) or bicuculline (10 micrograms/side) consistently reversed this hypotensive effect. Picrotoxin raised BP by 92 +/- 10 mmHg (n = 5) after KA while by itself, picrotoxin only increased BP by 23 +/- 7.0 mmHg (n = 3) when applied to the IA. This effect was significantly greater following KA application to the caudal area (P less than 0.05). Likewise, bicuculline raised BP by 74 +/- 8.7 mmHg (n = 7) following KA while by itself, bicuculline only increased BP by 24 +/- 8.2 mmHg when applied to the IA (n = 4). This also was significantly different. This potentiation was not seen with the glycine receptor antagonist, strychnine. These data indicate that there is a GABAergic input from the caudal to the intermediate ventral surface areas of the cat which is involved in the central control of BP.

Increase in coronary vascular resistance produced by stimulating neurons in the region of the area postrema of the cat.

Recently, Somberg, in a preliminary report (1983), noted that electrical stimulation of the area postrema causes an increase in coronary vascular resistance. The increase in resistance was mediate by an increase in sympathetic activity as it was counteracted by alpha-adrenergic receptor blockade. The purpose of our study was to confirm the findings of Somberg by using the method of chemical-induced activation of cell bodies in the area postrema. This was done by bilateral topical application of kainic acid to the area postrema of cats while monitoring coronary blood flow, heart rate, arterial blood pressure and the ECG. Topical application of kainic acid produced an increase in coronary vascular resistance (43 +/- 13%, P less than 0.05), S-T segment changes, increases in heart rate (60 +/- 10, P less than 0.05) and arterial blood pressure (88 +/- 20, P less than 0.05) and ventricular tachyarrhythmias. All of these effects were prevented by pretreating animals with the alpha-adrenoceptor blocking agent, phentolamine. These results suggest that chemical excitation of area postrema neurons produces coronary constriction that is mediated by the sympathetic nervous system and alpha-adrenoceptors on coronary vessels.

Cardiotopic organization of the nucleus ambiguus? An anatomical and physiological analysis of neurons regulating atrioventricular conduction.

Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic postganglionic vagal motoneurons on the surface of the heart which are capable of selective control of sinoatrial rate, atrioventricular conduction and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates atrioventricular conduction (the AV ganglion). Medullary tissues were processed for the histochemical detection of retrogradely labeled neurons by light and electron microscopic methods. Negative dromotropic retrogradely labeled cells were found in a long column in the ventrolateral nucleus ambiguus (NA-VL), which enlarged somewhat at the level of the area postrema, but reached its largest size rostral to the area postrema in an area termed the rostral ventrolateral nucleus ambiguus (rNA-VL). Three times as many cells were observed in the left rNA-VL as compared to the right (P < 0.025). Retrogradely labeled cells were also consistantly observed in the dorsal motor nucleus of the vagus (DMV). The DMV contained one third as many cells as the NA-VL. The right DMV contained twice as many cells as the left (P < 0.05). These data are consistent with physiological evidence that suggests that the left vagus nerve is dominant in the regulation of AV conduction, but that the right vagus nerve is also influential. While recording the electrocardiogram in paced and non-paced hearts, L-glutamate (GLU) was microinjected into the rNA-VL. Microinjections of GLU caused a 76% decrease in the rate of atrioventricular (AV) conduction (P < 0.05) and occasional second degree heart block, without changing heart rate. The effects of GLU were abolished by ipsilateral cervical vagotomy. These physiological data therefore support the anatomical inference that CNS neurons that are retrogradely labeled from the AV ganglion selectively exhibit negative dromotropic properties. Retrogradely labeled negative dromotropic neurons displayed a round nucleus with ample cytoplasm, abundant rough endoplasmic reticulum and the presence of distinctive somatic and dendritic spines. These neurons received synapses from afferent terminals containing small pleomorphic vesicles and large dense core vesicles. These terminals made both asymmetric and symmetric contacts with negative dromotropic dendrites and perikarya, respectively. In conclusion, the data presented indicate that there is a cardiotopic organization of ultrastructurally distinctive negative dromotropic neurons in the NA-VL. This central organization of parasympathetic preganglionic vagal motoneurons mirrors the functional organization of cardioinhibitory postganglionic neurons of the peripheral vagus nerve. These data are further discussed in comparison to a recent report on the light microscopic distribution and ultrastructural characteristics of negative chronotropic neurons in the NA-VL42.(ABSTRACT TRUNCATED AT 400 WORDS)

Synaptic interactions of substance P immunoreactive nerve terminals in the baro- and chemoreceptor reflexes of the cat.

The neurochemical anatomy and synaptic interactions of morphologically identified chemoreceptor or baroreceptor afferents in the nucleus of the solitary tract (NTS) are poorly understood. A substantial body of physiological and light microscopic evidence suggests that substance P (SP) may be a neurotransmitter contained in first order sensory chemo- or baroreceptor afferents, however ultrastructural support of this hypothesis is lacking. In the present report we have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase. Medullary tissues including the commissural NTS (cNTS) were processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical detection of SP by dual labeling light and electron microscopic methods. At the light microscopic level, dense bilateral labeling with TMB was found in the tractus solitarius (TS) and cNTS, caudal to the obex. Rostral to the obex, significant ipsilateral TMB labeling was detected in the dorsal, dorso-lateral, and medial subnuclei of the NTS, as well as in the TS. Significant staining of SP immunoreactive processes was detected in most subnuclei of the NTS. The cNTS was examined by electron microscopy. Either HRP or SP were readily identified in single labeled unmyelinated axons, myelinated axons, and nerve terminals in the cNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the cNTS which were simultaneously identified as CSN primary afferents. These ultrastructural data support the hypothesis that SP immunoreactive first order neurons are involved in the origination of the chemo- and baroreceptor reflexes. Axo-axonic synapses were observed between CSN primary afferent terminals and: (a) unlabeled nerve terminals; (b) other CSN primary afferent terminals; and (c) terminals containing SP. Axo-axonic synapses were also observed between CSN primary afferents which contained SP, and other SP terminals. These observations may mediate the morphological bases for multiple forms of presynaptic inhibition in the cNTS, including those involved in cardiorespiratory integration. In conclusion, our results indicate that SP immunoreactive nerve terminals may be important in both the origination and the modulation of the chemo- and/or baroreceptor reflexes.

Cardiorespiratory effects produced by application of L-glutamic and kainic acid to the ventral surface of the cat hindbrain.

The purpose of our study was to determine the cardiorespiratory effects of exciting cell bodies at the rostral (area M), intermediate (area S), and caudal (area L) chemosensitive sites on the ventral surface of the medulla. To do this, L-glutamic and kainic acid were applied bilaterally to each chemosensitive site while monitoring tidal volume (VT), respiratory rate (f), mean arterial pressure (BP), and heart rate (HR) in chloralose-anesthetized cats. Application of solutions (5 microliter) of L-glutamic acid ranging from 62.5 to 2000 mM to the intermediate area produced concentration-dependent increases in VT (from 2.0 +/- 0.6 ml to 14 +/- 1.1 ml) and BP (from 2.0 +/- 1.7 mm Hg to 39 +/- 5.3 mm Hg). No significant changes in f and HR were noted. Similar effects were observed with application of kainic acid. Application of L-glutamic acid to the caudal area produced hypotension (-24 +/- 5.4 mm Hg) with no accompanying changes in VT and f. No responses were observed after application of L-glutamic acid to the rostral area. These data suggest that activation of cell bodies on the intermediate area produces simultaneous stimulatory effects on BP and VT, whereas activation of cell bodies at the caudal area produces selective depressant effects on BP.

Substance P-immunoreactive boutons closely appose inspiratory protruder hypoglossal motoneurons in the cat.

In anesthetized cats, we recorded intracellularly from 26 hypoglossal motoneurons which were antidromically activated following electrical stimulation of either the medial or lateral branches of the hypoglossal nerve. Twenty-one of these neurons were protruder motoneurons 6 of which had inspiratory activity. Three of the protruder motoneurons with inspiratory activity were filled with Neurobiotin and found to be closely apposed to substance P-like immunoreactive nerve terminals.

Cardiorespiratory effects produced by microinjecting L-glutamic acid into medullary nuclei associated with the ventral surface of the feline medulla.

The purpose of our study was to use microinjections of L-glutamic acid to better localize the cell bodies in the intermediate area of the ventral medullary surface that exert control over cardiorespiratory activity. L-glutamic acid (200 nl of a 1-M solution) was microinjected into the nucleus paragigantocellularis lateralis, lateral reticular nucleus and into an area which is part of the 'glycine-sensitive area', which lies in the center of the intermediate area. Normally, when L-glutamic acid is applied to the surface of the intermediate area, increases in arterial pressure and tidal volume are observed. Increases in tidal volume were never observed upon microinjection into the 3 sites associated with the intermediate area, suggesting that the tidal volume change elicited from surface application occurs because of L-glutamic acid interacting with cell bodies either on the surface or extremely close to the surface. Pressor responses were elicited with microinjection of L-glutamic acid into the lateral reticular nucleus and the 'glycine-sensitive area', but not the nucleus paragigantocellularis lateralis; indeed, microinjection of L-glutamic acid into the nucleus paragigantocellularis lateralis caused hypotension. Hence, cell bodies responsible for raising arterial pressure may reside in either the lateral reticular nucleus or the 'glycine-sensitive area'.

What are the roles of substance P and neurokinin-1 receptors in the control of negative chronotropic or negative dromotropic vagal motoneurons? A physiological and ultrastructural analysis.

Recent data indicate that there is a cardiotopic organization of negative chronotropic and negative dromotropic neurons in the nucleus ambiguus (NA). Negative dromotropic neurons are found in the rostral ventrolateral NA (rNA-VL), negative chronotropic neurons are found in the caudal ventrolateral NA (cNA-VL), and both types of neurons are found in an intermediate level of the ventrolateral NA (iNA-VL). Substance P (SP) immunoreactive nerve terminals synapse upon negative chronotropic vagal motoneurons in the iNA-VL, and SP microinjections in the NA cause bradycardia. In the present report we have attempted to: (1) define the type of tachykinin receptor which mediates the negative chronotropic effect of SP microinjections into the iNA-VL; (2) define the physiological effect of microinjections of a selective SP agonist into the rNA-VL on atrioventricular (AV) conduction: and (3) find ultrastructural evidence for synaptic interactions of SP-immunoreactive nerve terminals with negative dromotropic vagal motoneurons in the rNA-VL. Microinjections of the excitatory amino acid glutamate (Glu) into the iNA-VL to activate all local vagal preganglionic neurons caused both bradycardia and a decrease in the rate of AV conduction. Injections of the selective neurokinin-1 (NK-1) receptor agonist drug GR-73632 also caused bradycardia, however the rapid onset of agonist induced desensitization prevented an evaluation of potential effects on AV conduction in the iNA-VL. These data suggest that the SP-induced bradycardia which can be elicited from the NA is mediated, at least in part, by NK-1 receptors. Microinjections of Glu into the rNA-VL caused a decrease in AV conduction without an effect on cardiac rate. On the other hand, GR-73632 microinjections into rNA-VL did not affect AV conduction. Following injections of the beta subunit of cholera toxin conjugated to horseradish peroxidase (CTB-HRP) into the left atrial fat pad ganglion which selectively mediates changes in AV conduction, retrogradely labeled neurons were histochemically visualized in the rNA-VL. These tissues were subsequently processed for the simultaneous immunocytochemical visualization of SP, and examined by electron microscopy. Histochemically labeled neurons were large, multipolar, with abundant cytoplasm containing large masses of rough endoplasmic reticulum, and exhibited distinctive dendritic and somatic spines. Unlabeled nerve terminals were noted to form either asymmetric or symmetric synapses with dendrites, dendritic spines, and perikarya of histochemically labeled neurons. SP-immunoreactive nerve terminals were also detected in the rNA-VL. SP terminals typically contained numerous small pleomorphic vesicles, multiple large dense core vesicles, and several mitochondria, and they synapsed upon unlabeled dendritic profiles. A total of 154 SP-immunoreactive nerve terminals were observed on photomicrographs of tissues which also contained histochemically labeled profiles. None made an identifiable synapse with a retrogradely labeled profile on the sections examined. In summary, both physiological and ultrastructural data indicate that SP terminals in the iNA-VL do modify the output of negative chronotropic vagal motoneurons. This effect is mediated by NK-1 receptors. On the other hand both physiological and ultrastructural data indicate that SP terminals in the rNA-VL do not modify the output of negative dromotropic vagal motoneurons. Therefore different mechanisms (neurotransmitters or receptors) mediate the central vagal control of cardiac rate and AV conduction.

Substance P nerve terminals synapse upon negative chronotropic vagal motoneurons.

Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic ganglia on the surface of the heart which are capable of selective control of sino-atrial rate, atrio-ventricular conduction, and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates heart rate (the SA ganglion). Medullary tissues were processed for the histochemical visualization of retrogradely labeled neurons and for the immunohistochemical detection of the neurotransmitter substance P (SP) by dual labeling light and electron microscopic methods. Negative chronotropic retrogradely labeled cells were found in a long slender column in the ventrolateral nucleus ambiguous (NA-VL) which enlarged somewhat at the level of the area postrema. These cells were found bilaterally, but they were asymmetrically distributed. Half the animals showed a pronounced right side predominance in retrograde labeling, while the other half of the animals showed a lesser left side predominance. These observations may help to explain some of the controversy in the literature concerning the relative influence of the right and left vagus nerves on sinus rate. Ultrastructural examination demonstrated axo-somatic and axo-dendritic contacts between SP nerve terminals and retrogradely labeled negative chronotropic NA-VL neurons. SP immunoreactivity was often associated with large dense-core vesicles in terminals forming either symmetric or asymmetric synapses. These observations provide a potential anatomical substrate for the centrally mediated bradycardia elicited by microinjections of SP into the NA. SP immunoreactive terminals were also observed to make axo-somatic, axo-dendritic, and axo-axonic synapses with unlabeled neurons in NA-VL.(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P immunoreactive nerve terminals in the dorsolateral nucleus of the tractus solitarius: roles in the baroreceptor reflex.

Physiological and light microscopic evidence suggest that substance P (SP) may be a neurotransmitter contained in first-order sensory baroreceptor afferents; however, ultrastructural support for this hypothesis is lacking. We have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase (HRP). The dorsolateral subnucleus of the nucleus tractus solitarius (dlNTS) was processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical visualization of SP by dual labeling light and electron microscopic methods. Either HRP or SP was readily identified in single-labeled unmyelinated axons, myelinated axons, and nerve terminals in the dlNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the dlNTS, which were simultaneously identified as CSN primary afferents. However, only 15% of CSN terminals in the dlNTS were immunoreactive for SP. Therefore, while the ultrastructural data support the hypothesis that SP immunoreactive first-order neurons are involved in the origination of the baroreceptor reflex, they suggest that only a modest part of the total sensory input conveyed from the carotid sinus baroreceptors to the dlNTS is mediated by SP immunoreactive CSN terminals. Five types of axo-axonic synapses were observed in the dlNTS. SP immunoreactive CSN afferents were very rarely involved in these synapses. Furthermore, SP terminals were never observed to form the presynaptic element in an axo-axonic synapse with a CSN afferent. Therefore, SP does not appear to be involved in the modulation of the baroreceptor reflex in the dlNTS.

Chemical stimulation of the area postrema induces cardiorespiratory changes in the cat.

The purpose of our study was to determine the cardiorespiratory effects of exciting cell bodies of the area postrema of the cat. This was accomplished by local application of L-glutamic acid (bilateral application of 5 microliter of a 250-1000 mM solution) and kainic acid (bilateral application of 5 microliter of a 40 mM solution) to the area postrema of chloralose-anesthetized cats while monitoring arterial pressure, heart rate, tidal volume and respiratory rate. These excitatory amino acids activate neuronal cell bodies but not axons of passage. L-Glutamic acid produced a dose-dependent increase in arterial pressure, decreases in respiratory rate and minute volume and, occasionally, ventricular tachyarrhythmias. Kainic acid produced effects similar to those seen with L-glutamic acid except the changes in respiratory activity were more pronounced with each animal exhibiting respiratory arrest. In artificially respired animals, kainic acid produced similar cardiovascular changes as those occurring in spontaneously breathing animals (i.e. increases in arterial pressure of 61 +/- 5.7 mm Hg, and in heart rate of 32 +/- 8.3 beats/min). Finally, application of kainic acid to the area postrema abolished the pressor and tachycardic responses to bilateral occlusion of the carotid arteries. These results suggest that activation of cell bodies in the area postrema can result in pronounced cardiorespiratory changes.

Cardiovascular neurons in cat caudal ventrolateral medulla: location and characterization of GABAergic input.

The purposes of the present study were to: (1) characterize the GABAergic input to vasodepressor neurons in the caudal ventrolateral medulla of the cat, and (2) define more precisely the anatomical localization of these neurons in this species. This was done by microinjecting GABA receptor antagonists and agonists, and a negative allosteric modulator of the GABA receptor, namely, ethyl-beta-carboline-3-carboxylate, into the caudal ventrolateral medulla of alpha-chloralose-anesthetized animals while monitoring arterial blood pressure and heart rate. Localization studies where performed relating injection sites in the caudal ventrolateral medulla where cardiovascular responses were elicited, to neurons exhibiting immunoreactivity to tyrosine hydroxylase (TH) and phenethyl-N-methyl-transferase (PNMT). Microinjection of 1 and 10 ng of bicuculline into the caudal ventrolateral medulla produced decreases in mean blood pressure and heart rate of -34 +/- 6.4 and -49 +/- 9.2 mmHg, and -22 +/- 4.3 and -35 +/- 8.2 beats/min, respectively. Hypotension and bradycardia were also observed with picrotoxin microinjection (120 ng). Microinjection of muscimol (100-200 ng) and GABA (12 microgram) had no effect on mean blood pressure and heart rate. Microinjection of ethyl-beta-carboline-3-carboxylate also decreased mean blood pressure (-39 +/- 7.0 mmHg). The location of the micropipette tip after bicuculline microinjection in relation to TH and PNMT immunoreactive cells was as follows: (1) TH-immunoreactive cells of the A1 cell group were visible in the same relative location as the micropipette tip, and (2) no PNMT-positive cells were noted at the sites where bicuculline elicited hypotension. These results indicate that there is a tonic GABAergic input to neurons in the caudal ventrolateral medulla. The location of these neurons overlaps with the A1 cells.

Ultrastructural circuitry of cardiorespiratory reflexes: there is a monosynaptic path between the nucleus of the solitary tract and vagal preganglionic motoneurons controlling atrioventricular conduction in the cat.

We have tested the hypothesis: (1) that presumptive negative dromotropic vagal preganglionic neurons in the ventrolateral nucleus ambiguus (NA-VL) can be selectively labelled from the heart, by injecting one of two fluorescent tracers into the two intracardiac ganglia which independently control sino-atrial (SA) rate or atrioventricular (AV) conduction; i.e., the SA and AV ganglia, respectively. The NA-VL was examined for the presence of single and/or double labelled cells. Over 91% of vagal preganglionic neurons in the NA-VL projecting to either intracardiac ganglion did not project to the second ganglion. Consequently, we also tested the hypothesis: (2) that there is a monosynaptic connection between neurons of the medial, and/or dorsolateral nucleus of the solitary tract (NTS), rostral to obex, and negative dromotropic neurons in the NA-VL. An anterograde tracer was injected into the NTS, and a retrograde tracer into the AV ganglion. The anterograde marker was found in both myelinated and unmyelinated axons in the NA-VL, as well as in nerve terminals. Axo-somatic and axo-dendritic synapses were detected between terminals labelled from the NTS, and retrogradely labelled negative dromotropic neurons in the NA-VL. This is the first ultrastructural demonstration of a monosynaptic pathway between neurons in the NTS and functionally associated (negative dromotropic) cardioinhibitory neurons. The data are consistent with the hypothesis that the neuroanatomical circuitry mediating the vagal baroreflex control of AV conduction may be composed of as few as four neurons in series, although interneurons may also be interposed within the NTS.