Bulbospinal serotonin pressor pathways and hypotensive action of methyldopa in the rat.

The administration of methyldopa (200 mg/kg i.p.) induced a green fluorescence typical of catecholamine fluorescence, in regions of the brain stem which coincided with all the major serotonin cell groups, including the B1, B2, and B3 cell groups in the medulla. Prior administration of 5,7-dihydroxytryptamine (5,7-DHT), a neurotoxin relatively specific for serotonin neurons, prevented the appearance of this methyldopa-induced fluorescence. Electrical stimulation of the ventrolateral medulla in areas that coincided with the lateral elements of the B1 and B3 serotonin cell groups evoked pressor responses recorded via cannulae in the abdominal aorta. The pressor responses were frequency-dependent and could be markedly attenuated by prior administration of 5,7-DHT either intracerebroventricularly (i.c.v.) or directly into the cervical cord to ablate descending serotonin nerve fibers. Microinjection of methyldopa (4-16 micrograms) directly into the region of the B1 and B3 cells in the ventrolateral medulla evoked a dose-dependent fall in arterial pressure observed for 4 hours. Here too, prior administration of 5,7-DHT either intracerebroventricularly or directly into the cervical cord largely prevented the hypotensive action of the microinjections of methyldopa. The administration of 5,7-DHT produced a highly selective depletion of serotonin stores without reducing the concentrations of norepinephrine. These experiments suggest that the activity of serotonin nerves descending into the spinal cord from the B1 and B3 cells in the ventrolateral medulla serves to elevate or maintain arterial pressure. They also suggest that these descending serotonin neurons may contribute to the hypotensive action of methyldopa.

Synapses on axons of sympathetic preganglionic neurons in rat and rabbit thoracic spinal cord.

Axosomatic and axodendritic synapses occur on sympathetic preganglionic neurons, but it is not yet known whether their axons receive synaptic input, which could be particularly effective at regulating sympathetic outflow. Here, we examined retrogradely labelled sympathetic preganglionic axons to see if they received synapses. Cholera toxin B subunit (CTB) or CTB conjugated to horseradish peroxidase (CTB-HRP) was used to label neurons projecting to the rat or rabbit superior cervical ganglion, the rat adrenal medulla, or the rabbit stellate ganglion. At the light microscopic level, small groups of CTB-immunoreactive axons travelled through the ventral horn near its lateral boundary, with occasional axons taking a more medial course. The axons passed through the ventrolateral funiculus to exit at the ventral roots. In parasagittal section, a few axons branched within the ventral horn, sending processes rostrally and caudally for short distances before they turned ventrally to exit the spinal cord. At the ultrastructural level, CTB-immunoreactive rat and rabbit sympathetic preganglionic axons were almost exclusively unmyelinated. In contrast, labelling with CTB-HRP revealed both myelinated and unmyelinated axons in the ventral horn, the ventrolateral white matter, and the ventral roots. CTB-HRP also allowed the detection of the initial segment of a sympathetic preganglionic axon. Synapses, with vesicles clustered presynaptically and membrane specializations postsynaptically, were found on some unmyelinated CTB-immunoreactive axons. Occasional axons received several synapses. Synapses were most common on CTB-containing axons just ventral to the intermediolateral cell column. One synapse was found on an axon within 2 microns of its origin from a proximal dendrite. Rare synapses were found several hundred micrometers ventral to the intermediolateral cell column. One branching axon had synapses just below the branch point on both the main axon and the axonal branch. These findings indicate an extensive synaptic input to the axons of at least some sympathetic preganglionic neurons. These axoaxonic synapses could have a profound effect on sympathetic activity.

Serotonin inputs to rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion or adrenal medulla.

The input from serotonin-containing nerve fibres to rabbit sympathetic preganglionic neurons projecting to either the superior cervical ganglion or the adrenal medulla was investigated by combining retrograde tracing with the B subunit of cholera toxin and immunocytochemistry for serotonin. There were pronounced rostrocaudal variations in the density of serotonin fibres in the rabbit intermediolateral cell column from T1 to L4; maximum numbers of fibres were found in T3-6 and L3-4 and minimum numbers in T1 and T10-12. By light microscopy, retrogradely labelled sympathetic preganglionic neurons projecting to the superior cervical ganglion or the adrenal medulla received variable densities of close appositions from serotonin-immunoreactive fibres. Some neurons from each population received many close appositions, whereas others received moderate numbers or few appositions. Appositions occurred on the cell bodies, dendrites, and occasionally axons of sympathetic preganglionic neurons. Rare neurons in both groups of retrogradely labelled cells received no appositions from serotonin-containing nerve fibres. At the ultrastructural level, synapses were found between serotonin-positive boutons and sympathetic preganglionic neurons projecting either to the superior cervical ganglion or to the adrenal medulla. These results indicate that, through direct synaptic contacts, serotonin-immunoreactive, presumably bulbospinal, nerve fibres affect the activity of the vast majority of sympathetic preganglionic neurons that send axons either to the superior cervical ganglion or to the adrenal medulla. This serotonin input may be sympathoexcitatory and could mediate increases in sympathetic nerve activity and in the release of catecholamines from the adrenal medulla.

Cholera toxin B-gold, a retrograde tracer that can be used in light and electron microscopic immunocytochemical studies.

The purpose of this study was to test whether a new retrograde tracer, the B subunit of cholera toxin conjugated to colloidal gold particles (CTB-gold), was taken up and transported by neurons in the central nervous system of the rat. Retrograde transport of CTB-gold was assessed from axon terminals, from damaged nerve fibers, and from axons of passage. For light microscopy, CTB-gold was visualized by silver intensification; for electron microscopy, sections were silver-intensified with or without subsequent gold toning. Retrogradely transported CTB-gold was detected in neurons after survival times of 12 hours to 42 days and appeared as black punctate deposits in perikarya and proximal dendrites at the light microscope level. Ultrastructurally, the deposits were usually associated with lysosomes. Injections of CTB-gold into the caudal ventrolateral medulla or into the lateral horn of the spinal cord gave small well-defined injection sites and resulted in retrograde labelling in medullary neurons in the same locations as similarly placed injections of wheat germ agglutinin-horseradish peroxidase. When injected into the superior cervical ganglion, CTB-gold was transported to nerve cell bodies in the spinal cord, but application of CTB-gold to the cut cervical sympathetic trunk did not label neurons in the spinal cord. Injection of CTB-gold into the nodose ganglion retrogradely labelled neurons in the dorsal motor nucleus of the vagus and the nucleus ambiguus. CTB-gold was not transported anterogradely from injections sites within the medulla. Nerve fibers and cell bodies containing neuropeptides, monoamines, or neurotransmitter-synthesizing enzymes were readily immunostained after silver intensification of retrogradely transported CTB-gold. Immunoreactivity for neuropeptides and enzymes was also demonstrated ultrastructurally after silver intensification and gold toning. These results show that CTB-gold is retrogradely transported from nerve terminals and fibers of passage but not from damaged axons. CTB-gold gives well-localized injection sites and persists in neurons for weeks. Transported CTB-gold is easily visualized and its detection is compatible with light and electron microscopic immunocytochemistry. These properties make CTB-gold a valuable tool for studying the connectivity and neurochemistry of pathways in the central nervous system.

Different populations of parvalbumin- and calbindin-D28k-immunoreactive neurons contain GABA and accumulate 3H-D-aspartate in the dorsal horn of the rat spinal cord.

The colocalization of parvalbumin (PV), calbindin-D28k (CaBP), GABA immunoreactivities, and the ability to accumulate 3H-D-aspartate selectively were investigated in neurons of laminae I-IV of the dorsal horn of the rat spinal cord. Following injection of 3H-D-aspartate into the basal dorsal horn (laminae IV-VI), perikarya selectively accumulating 3H-D-aspartate were detected in araldite embedded semithin sections by autoradiography, and consecutive semithin sections were treated to reveal PV, CaBP and GABA by postembedding immunocytochemistry. Perikarya accumulating 3H-D-aspartate were found exclusively in laminae I-III, and no labelled somata were found in deeper layers or in the intermediolateral column although the labelled amino acid clearly spread to these regions. More than half of the labelled cells were localized in lamina II. In this layer, 16.4% of 3H-D-aspartate-labelled perikarya were also stained for CaBP. In contrast to CaBP, PV or GABA was never detected in neurons accumulating 3H-D-aspartate. A high proportion of PV-immunoreactive perikarya were also stained for GABA in laminae II and III (70.0% and 61.2% respectively). However, the majority of CaBP-immunoreactive perikarya were GABA-negative. GABA-immunoreactivity was found in less than 2% of the total population of cells stained for CaBP in laminae I-IV. A significant proportion of the GABA-negative but PV-immunoreactive neurons also showed CaBP-immunoreactivity in laminae II and IV. These results show that out of the two calcium-binding proteins, CaBP is a characteristic protein of a small subpopulation of neurons using excitatory amino acids and PV is a characteristic protein of a subpopulation of neurons utilizing GABA as a transmitter. However, both proteins are present in additional subgroups of neurons, and neuronal populations using inhibitory or excitatory amino acid transmitters are heterogeneous with regard to their content of calcium-binding proteins in the dorsal horn of the rat spinal cord.

Effects of sinoaortic baroreceptor denervation on blood pressure and PNMT activity in medulla oblongata and spinal cord of normotensive and genetically hypertensive rats.

The effects of sinoaortic denervation on arterial blood pressure and central activity of phenylethanolamine-N-methyl transferase (PNMT, the last enzyme in adrenaline biosynthesis), were compared in normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHR-SP). Denervation of the arterial baroreceptors caused immediate increases in mean arterial blood pressure (MAP) in all three strains which were maximal at 90 min (32 mmHg in WKY, 51 mmHg in SHR and 80 mmHg in SHR-SP). Spinal cord PNMT activity increased above sham-operated levels in WKY at 90 min, but PNMT levels in SHR and SHR-SP, already significantly higher than in WKY, were not altered acutely after sinoaortic denervation. Over a seven day period after baroreceptor denervation, MAP rose by 15 mmHg in WKY and PNMT activity was about 100% greater in spinal cord and ventral medulla. In the two genetically hypertensive strains sinoaortic denervation failed to produce a further sustained rise in pressure or and PNMT activity in the ventral medulla or spinal cord. We suggest that increased activity of bulbospinal adrenaline neurons contribute to the sustained elevation in pressure seen in intact SHR and SHR-SP, as well as in WKY after denervation of arterial baroreceptors.

Influence of dietary sodium on blood pressure in baroreceptor-denervated rats.

One possible explanation for the salt sensitivity of blood pressure (BP) in certain hypertensive individuals is that neural mechanisms which normally counteract the pressor effect of a high dietary sodium intake are defective. We have tested this possibility in normotensive Wistar-Kyoto rats (WKY) by surgically ablating the arterial baroreflex mechanism. This manoeuvre, by itself, conferred substantial salt-sensitivity on the WKY rats whose BP is normally relatively insensitive to dietary sodium intake. The treated rats responded to a high sodium diet with a significant rise in systolic BP which was reversed by substituting a low sodium diet. Thus, impaired baroreflex function which has been observed in essential hypertension and in hypertensive animals, may be responsible for the hypertensive effect of sodium.

Bulbospinal sympatho-excitatory neurons in the rat caudal raphe.

OBJECTIVES: To explore the rat caudal raphe nuclei for neurons that respond to activation of baroreceptor nerves and that have a spinal axon, and to compare the behavioural properties of barosensitive bulbospinal neurons in the rat caudal raphe with the properties of barosensitive bulbospinal neurons in the rostral ventrolateral medulla. DESIGN: Extracellular unit recordings were obtained from an area extending up to 1.0 mm caudally from the caudal edge of the facial nucleus. Two sites were explored: the rostral ventrolateral medulla and the midline. MATERIALS AND METHODS: Single-unit recordings were made in anaesthetized (75 mg/kg chloral hydrate and 30 mg/kg sodium pentobarbitone then 3-6 mg intravenously as required), immobilized (2 mg pancuronium as required) Sprague-Dawley rats. Central respiratory drive was recorded from phrenic nerve discharge. The barosensitivity of single units was assessed by R-wave triggered histograms and by histograms of their responses to aortic nerve stimulation or to intravenous injection of phenylephrine. Nociceptors were activated by a brief pinch of the tail. RESULTS: Eleven spontaneously active units in the midline that were inhibited by baroreceptor stimulation and had a spinal axon were studied. Respiratory modulation was present and was predominantly inspiratory. Barosensitive neurons in the rostral ventrolateral medulla were activated by nociceptive inputs; midline barosensitive neurons were not. CONCLUSIONS: The behavioural characteristics of midline neurons differ from those of the bulbospinal barosensitive neurons in the rostral ventrolateral medulla, indicating that raphe spinal neurons have different sets of afferent inputs and may subserve to a distinct physiological role. The present paper is the first report of bulbospinal neurons in the rat caudal raphe that are inhibited by activation of arterial baroreceptors.

Neurokinin-1 receptor-immunoreactive sympathetic preganglionic neurons: target specificity and ultrastructure.

Substance P is involved in cardiovascular control at the spinal cord level, where it acts through neurokinin-1 receptors. In this study we used immunocytochemistry and retrograde tracing to investigate the presence of the neurokinin-1 receptor and its ultrastructural localization in rat sympathetic preganglionic neurons that project to the superior cervical ganglion or the adrenal medulla. Immunofluorescence for the neurokinin-1 receptor outlined the somatic and dendritic surfaces of neurons in autonomic subnuclei of spinal cord segments T1-T12, whereas immunofluorescence for the tracer, cholera toxin B subunit, filled retrogradely labelled cells. There was a significant difference in the proportion of neurokinin-1 receptor-immunoreactive sympathetic preganglionic neurons supplying the superior cervical ganglion and the adrenal medulla. Thirty-eight percent of the neurons that projected to the superior cervical ganglion were immunoreactive for the neurokinin-1 receptor compared to 70% of neurons innervating the adrenal medulla. Of neurons projecting to the superior cervical ganglion, significantly different proportions showed neurokinin-1 receptor immunoreactivity in spinal cord segment T1 (15%) versus segments T2 T6 (45%). At the ultrastructural level, neurokinin-1 receptor staining occurred predominantly on the inner leaflets of the plasma membranes of retrogradely labelled sympathetic preganglionic neurons. Deposits of intracellular label were often observed in dendrites and in the rough endoplasmic reticulum and Golgi apparatus of cell bodies. Neurokinin-1 receptor immunoreactivity was present at many, but not all, synapses as well as at non-synaptic sites, and occurred at synapses with substance P-positive as well as substance P-negative nerve fibres. Only 37% of the substance P synapses occurred on neurokinin-1-immunoreactive neurons in the intermediolateral cell column. These results show that presence of the neurokinin-1 receptor in sympathetic preganglionic neurons is related to their target. The ultrastructural localization of the receptor suggests that sympathetic preganglionic neurons may be affected (i) by substance P released at neurokinin-1 receptor-immunoreactive synapses, (ii) by other tachykinins (e.g., neurokinin A), which co-localize in substance P fibres in the intermediolateral cell column, acting through other neurokinin receptors, and (iii) by substance P that diffuses to neurokinin-1 receptors from distant sites.

Complete penetration of antibodies into vibratome sections after glutaraldehyde fixation and ethanol treatment: light and electron microscopy for neuropeptides.

To develop a method for quantitative electron microscopic immunocytochemistry on neural tissue of CNS, we tested the extent to which ethanol treatment would improve the penetration of immunoreagents through vibratome sections fixed in high concentrations of glutaraldehyde without compromising ultrastructure. Transverse or sagittal vibratome sections (60-80 microns) of spinal cord perfused with 1% formaldehyde plus 1% or 2.5% glutaraldehyde were washed in 50% ethanol for 0-70 min and stained to reveal immunoreactivity for neuropeptide Y (NPY). Semi-thin (1 micron) or ultra-thin sections were used to assess the depth to which NPY nerve fibers in the dorsal horn were stained. Without ethanol washing, immunoreactive nerve fibers were visualized only in the surface 5-10 microns of transverse or sagittal vibratome sections. In transverse vibratome sections, NPY nerve fibers, which ran perpendicular to the cut surfaces of the sections, were entirely stained after a 30-min wash in 50% ethanol. The numbers of NPY-immunoreactive varicosities and synapses were comparable at the surfaces and in the centers of the vibratome sections. In sagittal sections, where NPY nerve fibers ran parallel to the cut surfaces, fibers in the centers of vibratome sections could not be labeled even after 70 min in 50% ethanol. Substance P- and enkephalin (Enk)-immunoreactive nerve fibers could also be completely stained in transverse sections of spinal cord or medulla oblongata after 30-min exposure to ethanol. Ethanol washing had no significant deleterious effects on ultrastructure, although the amount of cytoplasmic matrix in neurons decreased with increasing exposure. These results indicate that washing with 50% ethanol for at least 30 min allows immunoreagents to penetrate completely through nerve fibers fixed with high concentrations of glutaraldehyde, as long as the fibers have cut ends at both surfaces of a vibratome section. This technique makes possible quantitative electron microscopic immunocytochemical studies and is proving a useful tool for defining synaptic connections in the CNS.

Central serotonergic mechanisms in hypertension.

Serotonin-containing neurons in the central nervous system are grouped into a number of discrete and distinctive collections with cell bodies in the brainstem and projections passing to many regions of the brain and spinal cord. Evidence is presented that activation of one projection of serotonin-containing neurons from the midbrain to the hypothalamus elevates arterial pressure. Evidence is also presented that activation of a projection descending from the lateral B3 serotonin cell group to the spinal cord elicits a pressor response that is accompanied by increased release of serotonin in the spinal cord and is independent of the C1 adrenaline-containing neurons that lie close by. In contradistinction, experiments are described demonstrating that activation of the midline group of B3 serotonin cells in the raphe nucleus causes a fall in arterial pressure, consistent with the view that different groups of serotonin neurons in the brain and spinal cord participate in the control of blood pressure in diverse ways and can have different effects on blood pressure. Finally, experiments are described showing that the hypotensive action of methyldopa is mediated in part through central serotonin nerves.

c-fos identifies GABA-synthesizing barosensitive neurons in caudal ventrolateral medulla.

Hypertension in the conscious rat, elicited by i.v. infusion of phenylephrine, evoked expression of the immediate early gene c-fos in discrete groups of brain stem neurons. Fos-immunoreactive neurons were located in the caudal ventrolateral medulla (CVLM); others were located in the nucleus of the tractus solitarius (NTS). Because of their sensitivity to alterations in arterial pressure, these neurons are likely to subserve the arterial baroreceptor reflex. The aim of this study was to identify the brain stem projections and the neurotransmitter content of the barosensitive CVLM neurons using neuronal tracing and immunohistochemistry. Some of the barosensitive CVLM neurons projected directly to the rostral ventrolateral medulla (RVLM), and many contained the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD). Other CVLM neurons, containing markers of glutamate or catecholamine synthesis, were insensitive to baroreceptor stimulation. This study delineates neuronal pathways acting in the arterial baroreceptor reflex and identifies precisely GABA-synthesizing CVLM neurons as the source of inhibitory input to the RVLM.

Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons.

Aiming to ablate sympathetic preganglionic neurons (SPN) innervating a defined target, we injected saporin conjugated to cholera toxin B subunit (CTB) unilaterally into the superior cervical ganglion of rats. In spinal cord segments T1-T3, the numbers of cholinergic neurons in the intermediolateral cell column ipsilateral and contralateral to the injected ganglion were significantly different by 3 days post-injection. By day 14, 77% of ipsilateral cholinergic neurons had disappeared. A higher percentage of neurons were killed in T1-T2 than in T3. Comparing SPN counts from CTB-saporin injected rats and counts from rats receiving unconjugated CTB into the superior cervical ganglion indicated that 84% of SPN supplying the ganglion had died by 14 days. Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons and may also eliminate other types of neurons that transport CTB.

The tungstate-stabilized tetramethylbenzidine reaction for light and electron microscopic immunocytochemistry and for revealing biocytin-filled neurons.

A peroxidase reaction product that can be easily distinguished from standard diaminobenzidine (DAB) reaction products is needed for pre-embedding electron microscopic double-antibody labelling studies. Benzidine dihydrochloride (BDHC) and gold-substituted silver peroxidase reactions are unsatisfactory for double labelling because they lack sensitivity and reliability and/or compromise ultrastructure. We show here that light and electron microscopic immunocytochemistry can be done with a modification of the tungstate-stabilized tetramethylbenzidine (TMB) reaction (Weinberg and Van Eyck 1991) which yields a crystalline reaction product. With this method, we have obtained excellent immunolabelling for a variety of antigens, including tyrosine hydroxylase, enkephalin, serotonin, Fos protein and retrogradely transported cholera toxin B subunit (CTB). The TMB-tungstate reaction is useful for ultrastructural double labelling because the crystals contrast well with the amorphous product of diaminobenzidine reactions. The TMB-tungstate reaction is more sensitive and reliable for immunocytochemistry than the benzidine dihydrochloride reaction and gives better ultrastructure than the gold-substituted silver peroxidase reaction. We also show that neurons filled with biocytin by intracellular injection can be visualized with TMB-tungstate for either light (LM) or electron (EM) microscopy.

Tracer-toxins: cholera toxin B-saporin as a model.

We have shown previously that retrogradely-transported cholera toxin B (CTB)-saporin has eliminated sympathetic preganglionic neurons by 7 days after injection (Llewellyn-Smith, I.J., Martin, C.L., Arnolda, L.F., Minson, J.B., 1999. NeuroReport 10, 307). To ascertain whether this tracer-toxin can kill other types of neurons that transport CTB retrogradely with a similar time course, we injected CTB-saporin into the facial nerves of rats and allowed them to survive for 7 days. Facial motoneurons were counted ipsilateral and contralateral to the injected nerves in sections of perfused medulla processed to reveal immunoreactivity for choline acetyltransferase (ChAT). There was a statistically significant decrease in the number of ChAT-immunoreactive neurons ipsilateral to the injected nerve in three out of nine rats. Inadequate injections were probably the reason that most rats showed no decrease in motoneurons numbers after treatment with CTB-saporin, since the staining intensity and numbers of facial motoneurons that showed CTB-immunoreactivity varied markedly between rats after retrograde tracing with unconjugated CTB. These results show that CTB-saporin can eliminate motoneurons as well as sympathetic preganglionic neurons, indicate that protocols for the injection of tracer-toxins should be optimized to ensure maximum neuronal death and support our contention that CTB-saporin should kill any central neuron that expresses GM1 ganglioside, the membrane component to which CTB binds.

Evidence for an excitatory amino acid pathway in the brainstem and for its involvement in cardiovascular control.

The source and possible role of excitatory amino acid projections to areas of the ventrolateral medulla (VLM) involved in cardiovascular control were studied. Following the injection of [3H]D-aspartate ([3H]D-Asp), a selective tracer for excitatory amino acid pathways, into vasopressor or vasodepressor areas of the VLM in rats, more than 90% of retrogradely labelled neurones were found in the nucleus of the solitary tract (NTS). Very few of the [3H]D-Asp-labelled cells were immunoreactive for tyrosine hydroxylase, none for phenylethanolamine-N-methyltransferase or gamma-aminobutyric acid. The density of labelled cells in the NTS was similar to that obtained with the non-selective tracers wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and WGA-colloidal gold, but these tracers also labelled other cell groups in the medulla. Furthermore, the decrease in blood pressure, caused by pharmacological activation of neurones in the NTS of rats, or by electrical stimulation of the aortic depressor nerve in rabbits could be blocked by the selective N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate injected into the caudal vasodepressor area of the VLM. This area corresponds to the termination of [3H]D-Asp transporting NTS neurones. These results provide evidence that a population of NTS neurones projecting to the VLM use excitatory amino acids as transmitters. Among other possible functions, this pathway may mediate tonic and reflex control of blood pressure via NMDA receptors in the VLM.

Spinal cord serotonin release and raised blood pressure after brainstem kainic acid injection.

The recently developed technique of in vivo dialysis has permitted us to make direct measurements of serotonin release in a specific region of the spinal cord and to relate this to changes in blood pressure elicited by chemical stimulation of the brainstem. In the present experiments we have shown that chemical stimulation of bulbospinal neurons in the region of the B3 cell group in the ventromedial medulla, causes an increase in the release of serotonin in the thoracic spinal cord and that this release is associated with an increase in blood pressure.

Evidence for a bulbospinal serotonergic pressor pathway in the rat brain.

The cardiovascular role of spinal serotonin (5-HT) neurones descending from 5-HT cells near the ventrolateral surface of the medulla oblongata was investigated by stimulating these cells in normal animals and in animals with selective chemical ablation of 5-HT nerves. These laterally placed 5-HT nerves fall within the B1 and B3 groups in the medulla and were identified using immunohistochemistry. 5,7-Dihydroxytryptamine (5,7-DHT) was injected into the lateral cerebral ventricle (i.c.v.) to produce a generalized destruction of central 5-HT pathways, with preliminary intraperitoneal administration of desipramine to prevent depletion of noradrenaline stores. In other experiments, 5,7-DHT was injected directly into the cervical spinal cord, after preliminary treatment with desipramine, to produce selective destruction of spinal 5-HT nerves, confirmed both biochemically and immunohistochemically. Electrical stimulation near the lateral 5-HT cells in the B1 and B3 cell groups elicited pressor responses in control (vehicle-injected) rats; the increase in mean arterial pressure was proportional to the intensity and to the frequency of stimulation. Microinjections of kainic acid or L-glutamate at the same sites also produced an increase in mean arterial pressure. Selective destruction of 5-HT nerves, whether produced by i.c.v. or intra-spinal administration of 5,7-DHT, reduced the magnitude of the pressor response to electrical stimulation by over 50%. These experiments suggest the activity of 5-HT nerve cells adjacent to the ventrolateral surface of the medulla oblongata and projecting to the intermediolateral cell column serves to elevate arterial pressure and maintain vasomotor tone.

Glutamate-immunoreactive synapses on retrogradely-labelled sympathetic preganglionic neurons in rat thoracic spinal cord.

Retrograde tracing with cholera toxin B subunit (CTB) combined with post-embedding immunogold labelling was used to demonstrate the presence of glutamate-immunoreactive synapses on sympathetic preganglionic neurons that project to the adrenal medulla or to the superior cervical ganglion in rat thoracic spinal cord. At the electron microscope level, glutamate-immunoreactive synapses were found on retrogradely labelled nerve cell bodies and on dendrites of all sizes. Two-thirds of the vesicle-containing axon profiles that were directly apposed to, or synapsed on, CTB-immunoreactive sympathoadrenal neurons were glutamate positive. The proportion of glutamate-immunoreactive contacts and synapses on sympathoadrenal neurons decreased to zero when the anti-glutamate antiserum was absorbed with increasing concentrations of glutamate from 0.1 mM to 10 mM. Double immunogold labelling for glutamate and gamma-aminobutyric acid (GABA) showed that glutamate-immunoreactive profiles did not contain GABA and that GABA-immunoreactive profiles did not contain glutamate. These results suggest that glutamate is the major excitatory neurotransmitter to sympathoadrenal neurons and possibly to other sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord.

Inhibition of vasodepressor neurons in the caudal ventrolateral medulla of the rabbit increases both arterial pressure and the release of neuropeptide Y-like immunoreactivity from the spinal cord.

The role of bulbospinal neuropeptide Y (NPY)-containing neurons of the rostral ventrolateral medulla in the rabbit in mediating the increase in blood pressure that occurs during inhibition of cells in the caudal ventrolateral medulla was investigated in urethane-anaesthetized rabbits. In the present experiments bilateral injections of the GABA agonist, muscimol, into the caudal ventrolateral medulla elicited a slowly-developing rise in arterial pressure that was maximal 15 min after the injection. Accompanying this increase in arterial pressure was an increase in the release of NPY-like immunoreactivity (NPY-LI) into the spinal subarachnoid space. This pattern of response is similar to that seen after direct chemical stimulation of the NPY-containing cells of the rostral ventrolateral medulla. Taken together, these findings suggest that tonically active neurons in the caudal ventrolateral medulla exert their effects by inhibiting sympathoexcitatory NPY-containing neurons whose cell bodies are situated in the rostral ventrolateral medulla.