Brain Research, 555 ( 1991) 326-33 I © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.5(/ A DONIS 000689939124676V
Glutamate in spinally projecting neurons of the rostral ventral medulla Jane Minson 1, Paul Pilowsky 1, Ida Llewellyn-Smith 1, Takeshi Kaneko e, Vimal Kapoor 1 and John Chalmers 1 t Department of Medicine and Centre for Neuroscience, The Flinders University of South Australia, Adelaide, South Australia (Australia) and 2Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Kyoto (Japan)
(Accepted 26 February 1991) Key words: Phosphate activated glutaminase; PNMT; Serotonin; Bulbospinal; Rostral ventrolateral medulla; Sympathetic preganglionic
neuron; Cholera toxin B, Rat
Phosphate activated glutaminase (PAG), an enzyme of glutamate synthesis, was localized by immunohistochemistry in all PNMTimmunoreactive and all serotonin-immunoreaetive 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. Bulbospinal neurons in the rostral ventral medulla are involved in regulating the activity of the sympathetic preganglionic neurons located in the intermediolateral cell column of the thoracic spinal cord 8'27'36. A variety of putative neurotransmitters or their biosynthetic enzymes have been localised within these bulbospinal neurons, including phenylethanolamine-N-methyltransferase (PNMT) 28,35,36, serotonin 7,14'21, neuropeptide y6 and substance p14. Furthermore, these substances have all been identified within nerve terminals that form synapses within the intermediolateral cell column 2'3A9'26 and functional studies have provided some evidence suggesting possible excitatory transmitter roles for serotonin 13'22"34, NPY 3°, substance e41 and adrenaline 18'29 in the intermediolateral cell column. There is, however, no definitive evidence that these transmitters mediate the excitation of sympathetic preganglionic neurons that follows stimulation of the rostral ventral medulla. Recently, we have demonstrated that the sympathoexcitatory responses after rostral ventral medulla stimulation are attenuated by the intrathecal administration of excitatory amino acid antagonists 23-25. Since neurons in the intermediolateral cell column are richly innervated by axon terminals containing glutamate immunoreactivity32, it seems appropriate to consider glutamate as another sympathoexcitatory transmitter candidate in the intermediolateral cell
column. The glutamate synthesising enzyme, phosphate activated glutaminase (PAG), has now been observed to be extensively colocalised with PNMT and serotonin in neurons of the rostral ventral medulla 16, and it was the aim of the present study to determine whether or not P A G immunoreactivity could be localised in bulbospinal neurons in this brainstem region. Male Wistar Kyoto rats weighing 250--350 g were anaesthetised by intraperitoneal administration of sodium pentobarbitone (30 mg/kg) and chloral hydrate (100 mg/kg). The rats were intubated and ventilated. A superficial branch of the femoral artery was cannulated and the rats were placed in a stereotaxic holder. Suxamethonium chloride (4 mg/kg i.a.) induced short lasting neuromuscular blockade. The upper thoracic spinal cord (T2-T4) was exposed by a dorsal incision and partial laminectomy. A retrograde neuronal tracer, cholera toxin B subunit conjugated to 7 nm colloidal gold (CTB-gold2°; Gilt Products, Department of Medicine, Hinders Medical Centre), was injected at 4 sites bilaterally in the spinal cord in the area of the intermediolateral cell column. A total of 1 ~1 of tracer was injected in each rat over 2 mm of exposed spinal cord. After a recovery period of at least 7 days, the rats were deeply anaesthetised with sodium pentobarbitone (60 mg/kg i.p.) and perfused with 200 ml of tissue culture medium (Dulbec-
Correspondence: J. Minson, Department of Medicine, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia. Fax: (61) (8)
327 co's modified Eagle's medium/F-12 H a m ; Sigma) that had been equilibrated with 95% 0 2 and 5% CO2, followed by 1 1 of 4% formaldehyde in 0.1 M sodium phosphate, p H 7.4. The brain and thoracic spinal cord were post-fixed in the same fixative solution overnight at r o o m temperature. Tissues were processed using a Silver Enhancement Kit (Sigma) to intensify the retrogradely transported gold particles prior to revealing P A G - , and PNMT- or scrotonin immunoreactivities. In order to prevent high background staining during the silver intensification process, due to contamination with phosphate ions, serial 30 /zm transverse sections of the medulla oblongata and 50 ~ m sections of spinal cord were cut into distilled water. Sections were washed in two changes of distilled water and incubated for 15 min (3 x 5 min) in a 1:1 mix of silver enhancer solutions A and B, rinsed in water and fixed in 2.5% sodium thiosulphate in distilled water for 5 rain. After several washes in water the sections were processed for immunohistochemistry. Sections were washed in 10 m M Tris, 0.05% merthiolate in 0.1 M phosphate-buffered saline, p H 7.4 (TPBS), containing 0.3% Triton X-100, then exposed to 10% normal horse serum (NHS) in TPBS-Triton for 30 min. Sections were incubated in a mouse monoclonal IgM antibody to P A G (Kaneko Mab-120~5'16; 5 ~g/ml) in 10% N H S TPBS-Triton at 4 °C for 3 days, and subsequently in 1:200 biotinylated goat anti-mouse IgM ~ chain specific, Sigma) in 1% N H S TPBS-Triton overnight at room temperature, and in 1:1000 Avidin-HRP (Sigma) in TPBS-Triton for 4 h. Sections were washed in 3 changes of TPBS-Triton after each incubation. Immunoreactivity was visualised by a peroxidase reaction. The reaction mixture contained 0.05% 3,3"-diaminobenzidine tetrahydrochloride ( D A B ) , 0.04% nickel ammonium sulphate, 0.004% ammonium chloride and 0.2% D-glucose ~ in 0.1 M phosphate buffer, p H 7.4. Sections were soaked in this reaction mix for 10 rain and then the reaction was started by adding 1/zl of glucose oxidase (Type V-S in 0.1 M sodium acetate buffer; Sigma) per ml of reaction mixture. After processing to visualise P A G immunoreactivity, alternate sections were further incubated in rabbit antiP N M T serum diluted 1:8000 or in rat monoclonal anti-serotonin (Seralab) 1:1000 in 10% NHS TPBS for 2 days at r o o m temperature, in 1:200 biotinylated sheep anti-rabbit or anti-mouse IgG (Sigma), respectively, in 1% N H S TPBS overnight and in 1:1000 Avidin-HRP in TPBS for 4 h at r o o m temperature, washes between incubations being in TPBS. PNMT- or serotonin immunoreactivity was visualised by a peroxidase reaction, where the D A B reaction mixture, in 50 m M Tris-HCl, pH 7.6, contained no nickel ammonium sulphate. Control sections that had either of the monoclonal antibodies omitted, or that had normal rabbit serum substituted for
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Fig. 1. A: distribution of PAG-immunoreactive neurons (open circle) and bulbospinal neurons containing PAG immunoreactivity (filled circle) in the rostral ventral medulla of one rat at a level approximately 250 #m caudal to the facial nucleus. B: distribution of bulbospinai neurons at the same level as A. Bulbospinal neurons are immunoreactive for PAG only (open circle with filled circle inside), immunoreactive for PAG and PNMT (star), immunoreactive for PAG and serotonin (filled triangle) or non-immunoreactive (asterisk). The inset shows the area used for the quantitative analysis of retrogradely labelled and immunoreactive neurons. It is delineated laterally by a line extending from the nucleus ambiguus (NA) to the ventral pole of the spinal trigeminal tract (spS) and medially by a line to the midpoint of the ventral surface of the medulla between the pyramidal tracts (py). C: a histogram showing the proportion (mean + S.E.M., n = 3) of all bnlbospinal neurons that contain PAG immunoreactivity in the area shown in the inset extending 600 gm caudally from the facial nucleus. This histogram is derived from 1772 bulbospinai neurons in 3 rats. D: a histogram showing the proportion (mean + S.E.M., n = 3) of PAG-immunomactive bnlbospinal neurons that also contain either PNMT immunomactivity or serotonin immunoreactivity in the same area as C. This histogram is derived from 1379 PAG-immunoreactive bulbospinal neurons in 3 rats.
Fig. 2. Colocalization of PAG immunoreactivity (granular grey reaction product) with either PNMT- or serotonin immunoreactivity (diffuse amber reaction product) and retrogradely transported colloidal gold (black punctate deposits) in the rostral ventral medulla. PNMT immunoreactivity is shown in A-D, serotonin immunoreactivity is shown in E-G. A: immunoreactive neurons in the rostral ventrolateral medulla containing PAG only (single black arrows) and PAG colocalized with PNMT (double black arrows) and bulbospinal neurons (gold labelled) containing PAG only (single white arrows) or bnlbospinal neurons containing PAG colocalized with PNMT (double white arrows); bar = 50 #m. B: 3 neurons in the rostral ventrolateral medulla - - a bnlbospinal, non-immunoreactive neuron (tailed arrow), a PAG-immunoreactive, non-bulbospinal neuron (black arrow) and a bulbospinal neuron immunoreactive for PAG and PNMT (double white arrow); bar -- 10 #m. C: a bulbospinal neuron immunoreactive for PAG and PNMT; bar = 20/~m. D: 3 neurons in the rostral ventrolateral medulla - - a PAG-immunoreactive, non-bulbospinal neuron (black arrow), a neuron immunoreactive for both PAG and PNMT, but not bulbospinal (double black arrow) and a bulbospinal neuron immunoreactive for PAG and PNMT (double white arrow); bar = 20/~m. E: immunoreactive neurons in the rostral ventral medulla at the lateral edge of the pyramidal tract, containing PAG only (single black arrows) and PAG colocalised with serotonin (double black arrows) and bulbospinal neurons (gold labelled) containing either PAG only (single white arrow) or containing PAG colocalized with serotonin (double white arrow); bar = 50 /~m. F: the bulbospinal PAG- and serotoninimmunoreactive neuron seen in E (double white arrow) at a higher magnification; bar = 10/~m. G: neurons in the rostral ventral medulla, a neuron immunoreactive for PAG and serotonin (double black arrow), a bulbospinal neuron immunoreactive for PAG only (single white arrow) and a bulbospinal neuron immunoreactive for PAG and serotonin (double white arrow); bar = 20 gm.
anti-PNMT serum, displayed none of the specific staining normally associated with the particular antibody. A quantitative analysis of retrogradely labelled and immunoreactive neurons in the rostral ventral medulla was c a r i e d out in 3 rats. The area of the ventral medulla examined extended 600/am caudally from the caudal pole of the facial nucleus and was bounded laterally by a line extending from the compact portion of the nucleus ambiguus 5 to the ventral pole of the spinal trigeminal tract and medially by a line extending from the nucleus ambiguus to the midpoint of the ventral surface of the medulla between the pyramidal tracts (Fig. 1 inset). This area of the rostral ventral medulla included the retrofacial portion of the lateral paragigantocellular reticular nucleus 1 and medially parts of the gigantocellular reticular nucleus and encompassed the region where bulbospinal neurons with presumed vasomotor activity have been electrophysiologically identified 11,3~,4°. One in every 4 30-/am sections was reacted to visualise P N M T immunoreactivity and a second in every 4 sections was reacted to visualise serotonin immunoreactivity. The area analysed in this study therefore included the rostral PNMT-containing neurons of the C1 adrenaline group ~2 and the serotonin-containing neurons of the lateral divisions of the B3 and the rostral B1 serotonin groups 39. Retrogradely transported, silver-intensified colloidal gold was visualised as black punctate deposits within the cytoplasm (Fig. 2). P A G immunoreactivity was observed as grey granular deposits within the cytoplasm, while PNMT- or serotonin immunoreactivity was observed as a diffuse amber staining within the cytoplasm (Fig. 2). PAG-immunoreactive neurons were observed in the area of the rostral ventral medulla (Fig. 1A), as well as being scattered throughout the entire medulla, confirming other studies 15,16,37. Most of the PAG-positive neurons displayed only labelling for P A G , although a small proportion of neurons with P A G immunoreactivity were also immunoreactive for P N M T (Fig. 2A-D) or serotonin
(Fig. 2E-G). In contrast, all of the PNMT-immunoreactive C1 neurons and all of the serotonin-immunoreactive B1 and B3 neurons also contained P A G immunoreactivity. A n important and interesting observation, although outside the scope of the present study, was that all the PNMT-containing neurons of the medulla, i.e. the C1 neurons extending through the rostral portions of the ventrolateral medulla and the C2 and C3 neurons of the dorsal medulla, contained P A G immunoreactivity. This complete colocalisation of P A G within the adrenalinecontaining and the serotonin-containing neurons of the rostral ventral medulla is in contrast to the previous study of P A G colocalisation within these neurons 16, where P A G immunoreactivity was observed in approximately 80% of the C1, C2 and C3 neurons and in many, but not all, of the serotonin neurons of several different groups including the raphe pallidus (B1) nucleus. Since in preliminary studies we observed that P A G immunoreactivity was particularly sensitive to small variations in fixation and staining protocols, it seems likely that these observed differences in the incidence of P A G colocalisation in the two studies reflect differences in the tissue fixation and immunohistochemical procedures used. Small bilateral CTB-gold injection sites were localised in all 3 rats to the area of the intermediolateral cell column. In the area defined as the rostral ventral medulla (Fig. 1 inset), a total of 591 + 40 (mean + S.E.M., n = 3) neurons were found to contain CTB-gold and hence were bulbospinal. These bulbospinal neurons were located throughout the ventral medulla (Fig. 1A,B). Seventy-eight percent of the total bulbospinal neuron population contained P A G immunoreactivity (71, 81 and 83% in the 3 rats examined; Fig. 1C). Of the bulbospinal PAG-immunoreactive neurons, 23% of the bulbospinal P A G neurons also contained P N M T immunoreactivity (bulbospinal neurons with colocalised P A G and PNMT/ total bulbospinal P A G neurons = 71/260, 51/212 and 45/261; Fig. 1D) and another 12% contained serotonin
330 immunoreactivity (bulbospinal neurons with colocalised P A G and serotonin/total bulbospinal P A G neurons = 19/218, 31/196 and 30/232; Fig. 1D). Other immunoreactive bulbospinal neurons contained P A G immunoreactivity alone (64%; Fig. 1D). These findings provide the first anatomical evidence suggestive of a glutamatergic projection from the rostral ventral medulla to the area of the sympathetic preganglionic neurons in the intermediolateral cell column of the thoracic spinal cord. Glutamate immunoreactivity has been found within neurons that form axodendritic synapses with neurons in the intermediolateral cell column 32, but the only evidence to date that this input might be from a descending pathway is that glutamate immunoreactivity is reduced in the intermediolateral cell column caudal to a spinal transection in the upper thoracic spinal cord 32. The bulbospinal P A G neurons identified in the present study are located in areas previously identified as sympathoexcitatory 4'9-11'27'31'36. That there are separate, chemically identified populations of bulbospinal neurons in the rostral ventral medulla is consistent with anatomical, pharmacological and electrophysiological studies that have identified a predominant group of non-catecholaminergic bulbospinal neurons and another group of PNMT-containing bulbospinal neurons 11'27"28'4°. That the bulbospinal neurons of the rostral ventral medulla might use glutamate as a transmitter is consistent with our previous findings, where the intrathecal administration of excitatory amino acid receptor antagonists attenuated the pressor responses to stimulation of the rostral ventral medulla in the C1 area of the PNMT-containing neurons 23'25 and in the B1/B3 area of the serotonincontaining neurons 24'25. That glutamate is colocalised with serotonin in the B3 neurons is supported by other findings where the pressor response to stimulation of the B3 area was abolished by the prior destruction of the bulbospinal serotonin neurons with the neurotoxin 5, 7-dihydroxytryptamine 13,27'34.Moreover, preliminary micro1 Andrezik, J.A., Chan-Palay, V. and Palay, S.L., The nucleus paragigantoceilularis lateralis in the rat, Anat. Embryol., 161 (1981) 355-371. 2 Bacon, S. and Smith, A.D., Preganglionic sympathetic neurones innervating the rat adrenal medulla: immunocytochemical evidence of synaptic input from nerve terminals containing substance P, G A B A or 5-hydroxytryptamine, J. Auton. Nerv. Syst., 24 (1988) 97-122. 3 Bacon, S., Zagon, A. and Smith, A.D., Electron microscopic evidence of a monosynaptic pathway between cells in the caudal raph6 nuclei and sympathetic preganglionic neurons in the rat spinal cord, Exp. Brain Res., 79 (1990) 589-602. 4 Barman, S.M. and Gebber, G.L., Axonal projection patterns of ventrolateral medullospinal sympathoexcitatory neurons, J. Neurophysiol., 53 (1985) 1551-1566. 5 Bieger, D. and Hopkins, D.A., Viscerotopic representation of the upper alimentary tract in the medulla oblongata in the rat: the nucleus ambiguus, J. Comp. Neurol., 262 (1987) 546-562.
dialysis studies in our laboratory have demonstrated an increase in glutamate release from the lateral column area of the upper thoracic spinal cord after electrical stimulation of the rostral ventrolateral medulla x7. Spinally projecting, glutamate-immunoreactive neurons have been identified in the rostral ventrolateral medulla 3s. However, unlike the bulbospinal neurons identified in the present study, these glutamate-immunoreactive neurons project to the phrenic motor neuron pool in the ventral horn of the mid-cervical spinal cord and are likely to mediate respiratory drive 3s. The proportion of these bulbospinal neurons containing glutamate immunoreactivity (approximately 80%3s), was similar to the incidence of P A G immunoreactivity observed in the present study. Together these results suggest that glutamate is likely to be a major transmitter in different bulbospinal pathways which mediate different autonomic functions. In conclusion, PAG-immunoreactive (glutamate-synthesising) neurons have been identified in the rostral ventral medulla. These neurons have bulbospinal axons that provide a glutamatergic input to the area of the intermediolateral cell column in the thoracic spinal cord. Subgroups of these bulbospinal PAG-immunoreactive neurons either contain P N M T immunoreactivity and form part of the C1 adrenaline cell group or contain serotonin immunoreactivity and form part of the B1 and B3 serotonin cell groups. This study provides further evidence of the chemical heterogeneity of the bulbospinai neurons of the rostral ventral medulla that might act in the regulation of blood pressure.
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