Jane B Minson

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.

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.

Separate areas of rat medulla oblongata with populations of serotonin- and adrenaline-containing neurons alter blood pressure after L-glutamate stimulation.

Separate populations of serotonin- and adrenaline-containing neurons exist in the ventrolateral medulla oblongata and project to the intermediolateral cell column of the spinal cord. The medullary serotonin nuclei appear to constitute a heterogeneous group with diverse effects on arterial pressure. Microinjections of sodium glutamate (which excites cell bodies but not axons of passage) made in the area of the ventrolateral serotonin cells evokes an increase in arterial pressure which is abolished by prior 5,7-dihydroxytryptamine (5,7-DHT) treatment. In contrast, glutamate microinjection in the area of the serotonin-containing cell bodies in the midline of the medulla evokes falls in arterial pressure and these responses are attenuated by pretreatment with 5,7-DHT. Glutamate microinjection made in the ventrolateral medulla in the area of the adrenaline-containing cells, evokes increases in arterial pressure which are not altered by 5,7-DHT pretreatment. After ablation of the area of the adrenaline-containing cells by electrolytic lesion, the pressor function of the ventrolateral serotonin-containing cells is still observed. These results suggest that although the serotonin-containing neurons of the ventrolateral medulla are closely aligned with the ventrolateral adrenaline area, the serotonin cell groups and the cells of the adrenaline area exert their pressor actions independently.

Spinal cord serotonin release and raised blood pressure after braintem 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.

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.

Glutamate in spinally projecting neurons of the rostral ventral medulla

Phosphate activated glutaminase (PAG), an enzyme of glutamate synthesis, was localized by immunohistochemistry in all PNMT-immunoreactive and all serotonin-immunoreactive neurons in the rostral ventral medulla of the rat. Between 71 and 83% of bulbospinal neurons localised in the rostral ventral medulla projecting to the intermediolateral cell column in the upper thoracic spinal cord contained PAG immunoreactivity. Of these bulbospinal PAG-immunoreactive neurons 17-27% contained PNMT immunoreactivity and 9-16% contained serotonin immunoreactivity. Other bulbospinal PAG-immunoreactive neurons (60-70%) contained neither PNMT- nor serotonin immunoreactivity. The results provide anatomical evidence suggestive of a glutamatergic input to the sympathetic preganglionic neurons of the spinal cord arising from different populations of neurons located in the rostral ventral medulla.

Altered c-fos in Rostral Medulla and Spinal Cord of Spontaneously Hypertensive Rats

Neurons immunoreactive for Fos, the protein product of the immediate early gene c-fos, have been compared in the rostral ventral medulla and spinal cord of conscious normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) after baroreceptor unloading. Hypotension induced by a 60-minute intravenous infusion of sodium nitroprusside reduced baroreceptor activity; controls received intravenous saline. In WKY, 474 +/- 56 (n=6) Fos-positive neurons were identified in the rostral ventral medulla after nitroprusside infusion, a fivefold increase from controls; 50% of the tyrosine hydroxylase-containing neurons in the rostral ventral medulla were activated by this hypotension. Sympathetic preganglionic neurons, mainly sympathoadrenal neurons, were Fos positive after nitroprusside, but Fos-positive sympathetic preganglionic neurons were not observed in control WKY. In SHR, Fos immunoreactivity in the rostral ventral medulla was elevated in the control group compared with the WKY controls (236 +/- 31 and 93 +/- 15, respectively, n=6 for both). Nitroprusside hypotension did not further increase Fos immunoreactivity in the rostral ventral medulla, although the number of Fos-positive spinal sympathetic neurons increased. Our results have identified different neuronal activities between WKY and SHR in sites that are critical to sympathetic outflow. In WKY, nitroprusside effects are consistent with an activation of rostral ventral medulla neurons, including bulbospinal neurons, that are normally inhibited by baroreceptor activity. In SHR, basal nerve activity is increased, so even at rest, rostral ventral medulla neurons and sympathetic preganglionic neurons, mainly sympathoadrenal neurons, are Fos immunoreactive. These activated neurons are likely to contribute to the elevated blood pressure in this rat strain.

Quantitative analysis of spinally projecting adrenaline-synthesising neurons of C1, C2 and C3 groups in rat medulla oblongata.

Spinal projections of the phenylethanolamine N-methyltransferase (PNMT) immunoreactive neurons of the medulla were investigated using a combination of immunohistochemistry and retrograde transport of colloidal gold particles conjugated to cholera toxin B subunit (CTB-gold). The PNMT-containing adrenergic neurons were localised in three groups, the C1 group in the rostral ventrolateral medulla, the C2 group in the nucleus tractus solitarius/dorsal vagal motor complex in the dorsal medulla and the C3 group in the mediodorsal medulla. The C1 group contained 72% of the medullary PNMT-IR neurons, while C2 comprised 13% and C3 15% of the medullary PNMT-IR neuron population. CTB-gold was injected in the area of the intermediolateral cell column in either upper (T2-T4) or lower (T8-T9) thoracic spinal cord and retrogradely labelled cells were found in the areas of the C1, C2 and C3 groups and in other regions of the medulla which did not contain PNMT-IR neurons. After tracer injections bilaterally at levels T2-T4, retrograde labelling suggested that at least 21% of all medullary PNMT-IR neurons projected to these levels. As a proportion of each group, 26% of C1, 9% of C2 and 33% of C3 neurons projected spinally to T2-T4. After tracer injections bilaterally at levels T8-T9, retrograde labelling suggested that at least 17% of all medullary PNMT-IR neurons projected to these levels. As a proportion of each group, 16% of C1, 9% of C2 and 30% of C3 neurons projected spinally to T8-T9. These figures represent minimum numbers since it is impossible to ensure that every neuron has equal access to the tracer. The results demonstrate that contrary to previous belief, the PNMT-IR innervation of the spinal cord derives from PNMT-IR neurons in the dorsal medulla, as well as from the rostral ventrolateral medulla. Indeed 24% of the PNMT-IR neurons terminating at spinal cord levels T2-T4, and 35% of those terminating at levels T8-T9, derive from the dorsal (C2 and C3) medullary cell groups. Since the PNMT-IR projections are directed towards the intermediolateral cell column, it seems likely that all three groups of medullary adrenaline-containing neurons contribute to the regulation of sympathetic outflow.

Bötzinger neurons project towards bulbospinal neurons in the rostral ventrolateral medulla of the rat.

Sympathetic nerve activity often fluctuates with the respiratory cycle, but the central neurons that impose this respiratory modulation have not been conclusively identified. In the present study, we used intracellular recording and dye‐filling to identify expiratory neurons in the Bötzinger complex. Our aim was to see if Bötzinger neurons project towards putative cardiovascular neurons in the rostral ventrolateral medulla. In the first series of experiments, histochemistry and immunohistochemistry were used to reveal the labelled Bötzinger neurons and neurons immunoreactive for tyrosine hydroxylase. Two out of four Bötzinger neurons had axon varicosities that were closely apposed to tyrosine hydroxylase‐immunoreactive neurons with cell bodies located within 0.6 mm caudal to the facial nucleus (three and five close appositions, respectively). In a second series of studies, rats were injected with cholera toxin B into the intermediolateral cell column of the spinal cord 4–7 days before the electrophysiological recording. Eight of the fourteen labelled Bötzinger neurons had a direct projection towards cholera toxin B‐labelled neurons in the rostral ventrolateral medulla. Close appositions were found on both somata and proximal dendrites (5 ± 2 close appositions/neuron, n=8). The present study supports the idea that a direct projection from Bötzinger neurons to presympathetic neurons in the rostral medulla plays a role in the respiratory modulation of sympathetic nerve activity. J. Comp. Neurol. 388:23–31, 1997.

N‐METHYL‐d‐ASPARTATE RECEPTORS IN THE SPINAL CORD MEDIATE PRESSOR RESPONSES TO STIMULATION OF THE ROSTRAL VENTROLATERAL MEDULLA IN THE RAT

1. Activation of bulbospinal neurons projecting from the C1 area of the rostral ventrolateral medulla evokes a pressor response. The nature of the neurotransmitters involved in mediating this response at spinal cord level has not been established.