aspartate-like immunoreactivities in medullo-spinal pathways of rat and primate

MeuroScit?nce vat. 48, No. 3, pp. x-559, Printedin Great Britain

$5.00 i- 0.00 PergamonPress Ltd 0 1992 IERO

1992

03064522j92

SEROTONIN-, SUBSTANCE P- AND GLUTAMATE/ASPARTATE-LIKE IMMUNOREACTIVITIES IN MEDULLO-SPINAL PATHWAYS OF RAT AND PRIMATE A. P. NICHOLAS,*~ V. A. PIERHONE,* U. ARWXWN$and T. H~KFELT* *Department

of Histology and Neurobiology and fDepartment of Anatomy, Karolinska Institutet, Stockholm S-10401, Sweden

Abstrr&-Serotonergic neurons of the medulla obiongata have been proposed to play a role in the control of sensory, motor and autonomic cells in the spinal cord. Many of these raphe neurons have been shown to contain the und~~ptide substance P as well as the tripeptide thyrotropin-releasing hormone, but evidence for the presence of an excitatory amino acid in these pathways has not yet been documented. In ~lc~~cine-treats rats, we have used a ~mbination of retrograde tracing and tr&color immunohistofluorescence techniques to study co-localization of serotonin- and substance P- with glutamate- or aspartate-like immunoreactivities in medullary neurons and the possible spinal projections of these cells. In addition, the ~st~butions of serotonin-, substance P- and ~utamate-jmmunoreactive terminal fields in the dorsal, ventral and lateral horns of the spinal cord were examined with tri-color immunofluore~ce in the rat and the primate ~u~u&u~s~icu~~fa. In colchicine-treated rats, glutamate- and aspartate-like imm~noroa~ti~~ was found in practically all serotonin- and substance P-immunoreactive neurons of the Bl, B2 and B3 cell groups. Some of these neurons also contained wheat-germ agglutinin conjugated to inactivated horseradish peroxidase and colloidal gold particles retrogradely transported from the spinal cord. In the spinal cords of non-colchitine-treated monkeys and rats, striking co-localization of serotonin, substance P- and glutamate-like immunoreactivities was seen in large boutons, surrounding the dendrites and cell bodies of large alpha motor neurons in the ventral horn. These observations suggest the existence of spinally proj~ting serotonin~substan~e P neurons containing excitatory amino acids such as glutamate or aspartate. Thus, these bulbospinal neurons may utilize three types of messenger molecules, an excitatory amino acid, a biogemc amine and one or more peptides.

Studies on the anatomical, biochemical and functional diversity of scrotonin (S-IS) raphe neurons in the medulla oblongata have been a complex undertaking ever since their histochemical localization by Rahlstriim and Fuxe” over twenty-five years ago. In agreement with early neuroanatomical studies,‘” many of these neurons project to the spinal cord tl-1523,39,42,~,51.53,62,67,69.85 and they may play a role fn sensory, motor and autonomic functions. In fact, SHT-immunoreactive (IR) terminals have been found in the dorsal, ventral and lateral F~rth~~~~~, horns of the spinal cord. 12,17.2S.30,53,8? in several species these S-FIT neurons and their processes contain numerous other putative neurotransmitters, including the peptides substance P 2.3,12-15,18,32,36,399,43,47,51,M),62.63. thyrotro(SP), “_

I__-

pin-~ieas~n~ hQ~on~,2,32,~,51 enkephalin,33~46~*8~69~~ galanin4,& and, so far only shown in the monkey,

calcitonin gene-related peptide Moreover, some of the medullary 5-HT-IR neurons also contain the inhibitory amino acid GABA.‘0,67@ In the present study, a combination of retrograde tracing and tricoior ~mm~ohist~hem~stry was employed to examine the possible presence of further transmitter candidates, the excitatory amino acids ghrtamate or aspartate, in spinally projecting 5-HT/SP raphe neurons of the medulla oblongata in colchicine-treated rats. A preliminary report of these results in rat has been presented previousIy.74 In addition, tricolor immu~ohist~hem~st~ has been used to examine the distribution of 5-HT-IR, SP-IR and glutamate-IR terminal fields in the dorsal, ventral and lateral horns in the spinal cords of the rat and the primate Macaca

fasciculata, tTo whom correspondence should be addressed at: Deuartment of Histoiogy and Neurobiology Box 6&t@, Karolinska institutet, Stockholm S-104 Of. Sweden. Ab6reuiufions: AB, acetate buffer; AMCA,’ ‘f-amino4 methylco~a~n-3-astir acid; FB, Fast Blue; FITC, Twenty-three male Sprague-Dawley rats (I%-3508) and fluorescein isothiocyanate; 5-IIT, 5-hvdroxvtrvutamine two adult male primates (Mucacu farciculutu) were used in (serotonin); IR, imktnoreactive; LI, hke imm&oreacthe present study. All animals were cared for according to tivity; LRHB, L&amine Rhodamine B: PBS obosguidelines approved by the Swedish ethical committ~ phaie-buffered saline; SP, substance P; W&A-apoif BP, Stockholms Norra Djurforsoksetiska Namnd. Nine of the wheat-germ a~Iutinin-horseradish peroxidase. rats were deeply anesthetized with intrape~toneal injections 545

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of chloral hydrate (400 mg/kg) and the spinal cord exposed (LRHB)-conjugated goat anti-guinea-pig (1: 40; Jackson at the intervertebral space between the C7 and Tl vertebrae. Immuno Research) and 7-amino-4-methylcoumarin-3-acetic Multiple, bilateral injections (2-3 ~1 total) of Fast Blue (FB; acid (AMCA)-conjugated donkey anti-rat (1: 20; Jackson n = 3) or a wheat-germ agglutinin-inactivated horseradish Immuno Research) antibodies in PBS containing Triton peroxidase (WGA-apoHRP; Sigma; n = 6) solution, preX-lOO,38sodium azide and Bacitracin at 37°C for 30min. viously conjugated to 7-lo-nm gold particles using the Secondary antisera were preabsorbed with IgG from nonmethod of Basbaum and Menetrey,* were made into the homologous species by the manufacturer. The sections were spinal cords of these animals through glass micropipettes. rinsed in PBS as before, coverslipped with glycerol-PBS After a five-day recovery period, two FB-injected and five (9: 1) containing 0.1% p-phenylenediamine,52~‘8 and examWGA-apoHRP-gold-injected animals, along with 12 other ined with epifluorescence using a Nikon Microphot-FX rats not injected with retrograde tracers, were deeply anesmicroscope. LRHB-fluorescent structures were visualized thetized as before, placed in a stereotaxic apparatus, and with a Nikon G-1B filter cube (excitation at 546 f. 5 nm with injected with 20 ~1 of a colchicine solution (6 pg/pl saline; a barrier filter at 590 nm), while FITC-fluorescent elements Sigma) into the lateral ventricle. Twenty-four hours later, were seen with a Nikon B-1E filter cube (excitation at these animals were reanesthetized with intraperitoneal pen480 k 10 nm with a bandpass emission filter passing tobarbital (35 mg/kg) and perfused transcardially with 50 ml 520-550 nm). AMCA-fluorescent structures were visualized of calcium-free Tyrode’s solution at 37°C then with 50ml with a Nikon UV-IA tilter cube (excitation at 365 k 5 nm of a mixture of 4% paraformaldehyde and 0.4% picric acid with a barrier filter at 420490nm). These filter combiin 0.16 M phosphate buffer (pH 6.9)“’ at 37°C followed by nations were used to avoid any possible “bleed-through” 350400 ml of the same fixative at 4°C. Two additional rats between fluorophores. Photographs were taken on blackand the two monkeys were perfused in the same manner, but and-white Kodak Tri-X (ASA 400) and color Kodak Highwithout previous colchicine treatments or spinal injections Speed Ektachrome (ASA 400) film. Colloidal gold particles of retrograde tracers. The rat brains and rat and monkey were photographed with transmitted light, with or without spinal cords were removed and postfixed in the phase-contrast; however, some sections previously photoparaformaldehyde-picric acid mixture at 4°C for 90 min and graphed for tricolor immunofluorescence were dehydrated - _ then stored in 10% sucrose in phosphate-buffered saline through graded ethyl alcohols and Histo-Clear (Hinze) and (PBS; pH 7.4) containing 0.01% sodium azide (Sigma) and coverslioned with Entellan (Merck). Colloidal gold oarticles 0.02% Bacitracin (Sigma) at 4°C from 48 h to two weeks. in these ‘sections were re-photographed undir dark-field At that time, 14-pm serial sections of the brainstems and illumination. spinal cords were cut on a cryostat (Dittes) and melted onto Control experiments were conducted by omitting one of gelatin-chrome alum-coated glass slides. Sections from the primary or secondary antisera86.98or by preabsorption of animals spinally injected with WGA-apoHRP-gold were antisera to homologous antigens on adjacent sections. For rinsed in acetate buffer (AB; pH 6.0) and processed for example, the glutamate antiserum was preabsorbed intensification of retrogradely transported gold particles by overnight with 0.01, 0.1 and 1.0 M of L-glutamate, asparbeing placed in a silver solution (Intense BL, Amersham) for tate, glycine, GABA and the conjugate hemocyanin. These 50-60min. Sections were removed when the retrogradely sections were subsequently processed for immunofluoreslabeled neurons were identified with the light microscope cence histochemistry as described above. Previous absorpand when “background” silver staining was minimal. This tion experiments with the glutamate and aspartate antisera tissue was briefly rinsed in AB, and the remaining silver fixed have been performed by Hepler et al.4’ and will be reviewed in a 2% solution of sodium thiosulfate in AB for 20 min. in the discussion. These sections then were rinsed twice in PBS (PH 7.4; 15 min each). All tissue was placed in humidified chambers and RESULTS processed for indirect single,*O doubleB or triple73,74.86,98 immunofluorescence staining by incubating the sections for Medulla oblongata-rat 2448 h at 4°C in the following primary antisera, diluted in PBS (pH 7.4) containing 0.3% Triton X-100, sodium azide In the medullary Bl, B2 and B3 cell groups of and Bacitracin: (i) 1:800-l : 1600 rabbit anti-glutamate,4’ (ii) Dahlstriim and Fuxe,” most SHT-IR (Figs IA, D, I:400 guinea-pig anti-5-HT,8’,95 and/or (iii) 1: 20 rat antiH; 2A, D, G, 3A, D, G, 4A) neurons in the nucleus SP.** Some medullary sections were also incubated in a raphe pallidus (Figs IA-G, 2A-Q the nucleus intercombination of 5-HT and SP antisera with 1: 800 rabbit anti-aspartate. 4’ After incubation in the primary antisera, fascicularis hypoglossi (Fig. 2D-J), the nucleus raphe the sections were rinsed twice in PBS for 15 min each, then obscurus (Fig. 3A-Q the nucleus raphe magnus transferred to a humid chamber and incubated with a (Figs lH-J, 3D-F) and the nucleus reticularis gigancombination of secondary antisera consisting of fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit (1: 40; tocellularis pars LX(Figs 3G-I, 4A-D) were also Jackson Immuno Research), Lissamine Rhodamine B immunoreactive for SP (Figs IB, E, I, 2B, E, H, 3B,

Fig. 1. Color photomicrographs of 5-HT-IR (A, D, H, K), SP-IR (B, E, I, L) and glutamate (GLU)-IR (C, F, J, M) neurons in the rat medulla oblongata (A-J) and terminal boutons in the primate spinal cord (K-M). 5-HT-IR structures were demonstrated in red representing LRHB-conjugated secondary antisera, SP-IR structures appeared blue representing AMCA-conjugated secondary antisera, and glutamate-IR structures fluoresced green representing FITC-conjugated secondary antisera. Examples of spinally projecting cells were seen in bright-field microscopy (G) as neurons containing black, silver-intensified colloidal gold particles conjugated to WGA-apoHRP. Examples of co-localization of 5-HT, SP and glutamate (solid arrows) were demonstrated in neurons of the rat nucleus raphe pallidus (A-C, DF), in which some neurons (one labeled with a solid arrow) also projected to the spinal cord (G). 5-HT/SP glutamate co-existence was also apparent in neurons of the rat nucleus raphe magnus (H-J). Examples of 5-HT-IR glutamate-IR, non-SP-IR neurons (large arrowheads), soley glutmate-IR neurons (small arrowheads) and soley 5-HT neurons (open arrows) were also demonstrated. The three immunoreactivities were co-localized in terminals (arrows) of the monkey in the ventral horn of the spinal cord (K-M). Scale bar=50pm (A=B=C=D=E=F=G=H=I=J; K=L=M).

Fig. 1. 547

rrg.

L.

glutamate (GLu)-LI (C, F, I) in : to project to the spinal cord (J; long solid arrow), while others did not contain retrograde tracer (J; short solid arrow). Silver-enhancedcolloidal gold particles conjugated to WGA-apoHRP were photographed with phase-contrast microcopy (J). In nucleus raphe paIlidus (A-C), and nucleus in~~~c~~s hypoglossi (I-F, G-J) many small, solely glutamate-IR neurons (small arrowheads) were seen in the inferior olive (IO), sometimes between the fascicles of the hypoglossal nerve rootlets (stars), while near the ventral medullary surface (A-C, G-J), ~HT-IR~~u~a~-iR, non-SP-IR cells (large arrowheads; A-C) were sometimes observed. Spinally prajecting, solely 5-HT-IR neurons (open arrows) were also observed in this area (J). Scale bars = 50 pm (A = B = C; D = E = F; G = H = I = J). 548

Fig. 3. Black-~d-~~te p~ot~~~~~a~s of w-existence of 5-HT-LI IA, D, G}, SP-I.2 (B7 E, H) and glutamate-IJ (C, F, r) in neurons (solid arrows) of the B2 091 group in nucleusraphe obscurus (A-CJ and of the I33 cell group in nucleus rapbe magnus (D-F) and in nucleusreticularis gigantocelluladspars cz(G-I). Solely &&mate (GI.X&IRcells (small arrowheads; G-I) were again seen in the inferior olive (A-C, lower left mner), whiie S-HT-IR/SF-IR, non-glutamate-IR(openarraws) and 5-HT-IR/glutamat~ IR, non-SFIR (large arrowhcctds) neurons were also occasionally observed. !&ale bar = 50pm (A=B=c; D=E=F; G=H=I). 549

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E, H, 4B). Glutamatelike immunoreactivity (LI) was observed in cell bodies of the medulla oblongata in the inferior olivary nucleus, the ventrolateral medullary reticular formation, the substantia gelatinosa of the spinal trigeminal nucleus, the dorsal motor nucleus of the vagus, the nucleus tractus solitarii and the nucleus commissuralis, as well as

in the nucleus raphe pallidus (Figs lC, F, 2C), the nucleus interfascicularis hypoglossi (Fig. 2F, I), the nucleus raphe obscurus (Fig. 3C), the nucleus raphe magnus (Figs lJ, 3F) and the nucleus reticularis gigantocellularis pars a (Figs 31, 4C). In the medulla oblongata, glutamate-11 was demonstrated uniformly in all serotonergic cell groups, without a

Fig. 4. In the nucleus reticularis gigantocellularis pars OT, which consists of B3 cells that extend over the pyramids (PYR) to the ventral medullary surface, examples of spinally projecting S-HT-IR/SPIR/glutamate-IR neurons (solid arrows) were seen. Gccasionally, spinally projecting S-HT-IR/glutamateIR, non-SP-IR neurons (large arrowheads) were observed above the pyramids, as well as at the ventral medullary surface (open arrows). Silver-enhanced colloidal gold particles conjugated to WGA-apoHRP were photographed with dark-field illumination (D) following dehydration of sections previously photographed for immunofluorescencc (A-C). Note many neuronal processes containing retrograde tracer (D; small arrowheads), representing a colchicine effect. Scale bar = 50 pm (A = B = C = D). GLU,

glutamate.

GLU/ASP-LI

in bulbospinal 5-HT/SP pathways

rostral-to-caudal or medial-to-lateral preference. In these regions, practically all 5-HT-IR/SP-IR cells contained glutamate-11 (Figs 14). Also in these areas, a few neurons containing only glutamate-11, or glutamate-11 with either S-HT- or SP-LI were seen (Figs l-4). Aspartate-LI was also observed uniformly in all medullary serotonergic cell groups, and examples of co-localization of 5-HT-, SP- and aspartate-LI were often seen (Fig. 5). In the retrograde tracing experiments, both untreated and colchicine-treated rats were examined and two tracers, FB and WGA-apoHRP-gold, were tested. FB appeared diffusely in the cytoplasm, whereas WGA-apoHRP-gold had a granular appearance. In untreated rats, both tracers labeled numerous and approximately the same number of neurons. In colchicine-treated rats, FB-labeled neurons were present in similar numbers and had similar morphologies as was seen in untreated rats, whereas the WGA-apoHRP-gold-positive neurons were fewer in number, and the granules were often dislocated into neuronal processes (Fig. 4D). These processes most likely are dendrites, since WGA-apoHRP has been shown to be taken into lysosomes,37 and colchicine has been shown to cause lysosomes to be dislocated into the dendrites of neurons.34~35**1~*2 However, colchicine pretreatment greatly improved somatic glutamate-L1 and was essential to demonstrate somatic SP-LI. Furthermore, since FB and AMCA cannot be differentiated in the fluorescence microscope due to similar excitation/emission wave lengths, only WGA-apoHRP-gold could be combined with triplefluorescence staining Using this procedure, several triple-immunolabeled neurons were found to contain WGA-apoHRP-gold particles (Figs lG, 21, 4D). Also in these medullary areas, small subpopulations

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of retrogradely labeled and non-retrogradely labeled neurons were immunoreactive for only one or two of the putative neurotransmitters (Figs l-4). There were also retrogradely labeled neurons which were not immunoreactive (Figs IG, 21, 4D). Spinal cord-rat

and monkey

In the ventral horn of both rat and primate, striking examples of co-localized 5-HT-, SP- and glutamate-11 were observed in large terminals, outlining the dendrites and cell bodies of large alpha motor neurons (Figs IK-M, 6A-F). This co-existence was particularly distinct in primate thoracic cord (Figs lK-M, 6B, D, F), while in the rat, triple-labeled terminals in the ventral horn were seen with about equal frequency in the thoracic and lumbar levels. In the dorsal horn of both rat and primate, small, individual glutamate-IR somata were seen in the substantia gelatinosa. Even though there seemed to be partial overlap of SP-, 5-HT- and glutamate-IR terminals, the high density and the intensity of SPand glutamate-IR staining in the dorsal and lateral horns of both species made it impossible to differentiate between the three labels at the light microscopic level. Control experiments

Specificity experiments employing preabsorption of 5-HT,*7,95SP,**glutamate9~26,4’*73*91 and aspartate4’.91 antisera to homologous and heterologous antigens have been described previously. In the medulla oblongata of the rat, glutamate-L1 was observed in neurons of the nucleus raphe obscurus, the nucleus raphe pallidus and the inferior olivary nucleus in sections incubated overnight in 1: 1600 dilution of glutamate antiserum (Fig. 7A). This immunoreactive staining

Fig. 5. Black-and-white photomicrographs of co-existence of 5-HT-LI (A), SP-LI (B) and aspartate-11 (C) in neurons (arrows) of the B3 cell group in nucleus raphe magnus. 5-HT-IR/aspartate-IR, non-SP-IR neurons (large arrowheads), and small, solely aspartate-IR ceils (small arrowheads) also were seen in this area. Scale bar = 50 pm (A = B = C). ASP, aspartate.

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was totally blocked in an adjacent section that was incubated overnight in the same dilution of glutamate antiserum preabsorbed with 0.01 M L-glutamate (Fig. 7B), while preabsorption with 0.1 and 1.0 M of glycine, GABA and hemocyanin and 0.01 M of aspartate had no effect on glutamate-IR staining. Preabsorption of the glutamate antiserum with high concentrations of aspartate (0.1 and 1.O M) markedly reduced the intensity of glutamate-LI. The aspartate antiserum, on the other hand, was blocked with 0.01 M aspartate, but not with 0.01-1.0 M t-glutamate, Immunoreactive staining was blocked using 5-HT and SP antisera preabsorbed with 5HT and

SP, respectively. No evidence of “cross-reactivity” was observed between primary and secondary antibodies directed against non-homologous species. Staining patterns previously demonstrated in triplestained sections (Figs lM, 6F) were found to be similar to those seen in adjacent sections incubated in only one (Fig. 7C) or two (Fig. 7D, F) primary antisera. Also in triple-labeling experiments, omission of one of the secondary antisera did not alter staining patterns seen with the other two labels. Since many examples of co-localization of 5-HT and glutamate were observed in boutons of the ventral horn in the monkey (Fig. 6B, D, F),

Fig. 6. Examples of co-localization (arrows) of 5-HT (A, B), SP (C-D) and glutamate (E, F) in terminal boutons in the ventral horn of the spinal cord in rat (A, C, E) and primate (B, D, F). Scale bar = 50 pm (A = C = E; B = D = F). GLU, glutamate.

GLU/ASP-LI

in bulbospinal 5-HT/SP pathways

Fig. 7. Black-and-white photomicrographs of several control experiments. (A) Many glutamate-IR neurons were observed in the nucleus raphe obscurus (NRO), inferior olivary nucleus (IO) and nucleus raphe pallidus (NRP) in the medulla oblongata of the rat incubated overnight in 1: 1600 dilution of anti-glutamate. (B) This immunoreactive staining was totally blocked in an adjacent section that was incubated overnight in the same dilution of anti-glutamate pmabsorbed with 0.01 M @utamate (PYR, pyramid). (C) Single staining for glutamate-11 alone did not alter the staining pattern, i.e. many glutamate-IR boutons were observed outlining the profiles of alpha motor neurons (asterisks), as previously demonstrated in triple-stained adjacent sections (Figs lM, 5F). (D, F) To further examine the possibility of bleed-through phenomenon, i.e. intense 5-HT-IR (LRHB) being responsible for “false-positive” glutamate-11 staining, close observation of other regions of the primate spinal cord revealed examples of 5-HT/glutamate co-localization (arrows), as well as solely glutamate-IR (small arrowheads) and solely 5-HT-IR (large arrowhead) boutons. (E, G) In the reticular formation of the primate medulla oblongata, neurons (asterisks) outlined by intensely fluorescent 5-HT-IR boutons exhibited no glutamate-11 labeling, suggesting that LRHB bleed-through was not a confounding factor in these experiments. Scale bar = 50 pm (A = B; D = F; E = F). GLU, glutamate.

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additional control experiments were performed to determine if apparent glutamate-IR terminal staining was due to bleed-through of the LRHB (for 5-HTLI). Sections of primate spinal cord (Fig. 7D, F) and medulla oblongata (Fig. 7E, G) were incubated overnight with rabbit anti-glutamate (1: 800) and guinea-pig anti-5-HT (1: 400) followed by incubation in a combination of LRHB-conjugated goat antiguinea-pig (1: 40) and FITC-conjugated donkey antirabbit (1:40) antisera. In the intermediate gray matter of the spinal cord, close observation revealed examples of 5HT/glutamate co-localization, as well as solely glutamate-IR and solely 5-HT-IR boutons. In the primate medulla oblongata, many neurons that were outlined by strongly fluorescent 5-HT-IR boutons (Fig. 7E) had no such glutamate-11 labeling (Fig. 7G), suggesting that LRHB bleed-through was not a confounding factor in our experiments. DISCUSSION

During recent years, immunohistochemistry has been used to localize glutamate-11 at the light and Previous re_ electron microscopic level. 9,26.73-75,91.101 ports using the same glutamate antiserum4’ as in the present study, have documented glutamate-11, for example, in terminals of the spinal cord and in cell bodies of dorsal root ganglia’ and of the ventrolateral medulla oblongata. 73 In the latter study, glutamatepositive cells also contained tyrosine hydroxylase and SP, raising the possibility that one and the same neuron contains three types of messenger molecules: an amino acid, a biogenic amine and, at least, one peptide. In the present study, glutamate- and asparate-11 were seen in virtually all of the medullary 5-HT neurons as originally described by DahlstrGm and Fuxe,24 i.e. cells in the nucleus raphe pallidus, nucleus interfascicularis hypoglossi (Bl cell group), nucleus raphe obscurus (B2 cell group), nucleus raphe magnus and nucleus reticularis gigantocellularis pars CI(B3 cell group), and many of these neurons were also immunoreactive for SP. Technical considerations

The nature of the glutamate-11 is still unclear. Hepler et a1.4’ demonstrated in immunoblot tests that the glutamate antiserum used in the present study cross-reacts mainly with aspartate, and conjugates such as glycine-glutamate and aspartate-glutamate. However, when used at optimal concentrations in (peroxiimmunoabsorption/immunohistochemical dase-antiperoxidase method) experiments, no apparent cross-reactivity was observed. In the present study, the immunofluorescence method was employed and fairly high antiserum concentrations were used (1: 800 or 1: 1600 vs 1: 10,000). Also in present experiments, preabsorption of the glutamate antiserum with very high concentrations (0.1 and 1.OM) of aspartate, but not of glycine or GABA, reduced glutamate-11 in medullary sections. Using an aspar-

tate antiserum4’ that did not appear to cross-react with glutamate,41.91 practically all 5-HT-IR-SP-IR neurons in the medulla oblongata also exhibited a specific immunofluorescence for aspartate. Therefore, it cannot be excluded that aspartate or dipeptides including glutamate or aspartate contribute to the staining patterns described using the glutamate antibody. However, further support for glutamate in 5-HT neurons has recently been obtained, demonstrating immunoreactivity for phosphate-activated glutaminase, the proposed major synthetic enzyme of glutamate, in virtually all 5-HT-IR neurons of nucleus raphe pallidus and obscurus.54 Furthermore, it remains to be proven that the glutamate-11 in bulbospinal 5-HT-IR neurons reflects presence of glutamate as a neurotransmitter, since glutamate-11 could also represent that pool of glutamate used in normal cellular metabolism. Possibly, cells exhibiting a high metabolic activity could also exhibit glutamate-11. However, a high degree of correlation has been noted between those neurons exhibiting glutamate-11 and those using glutamate as a neurotransmitter, while some neurons exhibiting presumably high metabolic rates (i.e. Purkinje cells of the cerebellum) do not exhibit glutamate-11.” Glutamate-like immunoreactivity in substance Pjserotonin medullospinal pathways-anatomy

It has been estimated that about 40% of the raphe-spinal neurons in the rat contain both 5-HT and SP.15 The present study demonstrates that practically all spinally projecting 5-HT-IR/SP-IR neurons in the medulla oblongata also exhibit glutamate-11. Medullary raphe stimulation causes 5-HT release in the ventral horn of the spinal cord,16 and anatomical studies show that these 5-HT neurons project to the dorsal, lateral and ventral horn of the spinal cord using anterograde transport of Phaseolus vulgarisleucoagglutinin.53,‘” Many of the glutamate_IR/5HT-IR/SP-IR neurons in the present study contained WGA-apoHRP conjugated to colloidal gold particles injected into the rostra1 thoracic spinal cord followed by colchicine injection. These neurons thus belong to the bulbospinal system. However, pretreatment with colchicine reduced the number of retrogradely labeled WGA-apoHRP-gold cells, resulting in low numbers of quadruple-labeled medullary cells. Therefore, no attempt was made to quantify the proportion of medullary neurons exhibiting 5-HT-, SP- and glutamate-L1 which also contained WGA-apoHRP-gold. Furthermore, quantification of bulbospinal 5-HT/SP glutamate projections could not be demonstrated in non-colchicine-treated animals, since this pretreatment is required for optimal visualization of somatic glutamate- and SP-LI (see Ref. 59). In the present study, the spinal cords of two non-colchicine-treated rats and two non-colchicinetreated monkeys were used to examine the distributions of 5-HT-, SP- and glutamate-IR terminal fields in the dorsal, lateral and ventral horns of the

GLU/ASP-LI in bulbospinal 5-HT/SP pathways spinal gray matter. This was done to draw a possible parallel between proposed medullospinal S-HT-, SPand glutamate-IR pathways in rats and primates. In the spinal cords of these animals, 5-HT, SP and glutamate could only clearly be demonstrated in terminals of the ventral horn. In fact, there is evidence that the 5-HT neurons projecting to the dorsal horn do not, or only to a limited extent, contain SP (or thyrotropin-releasing hormone), whether in rats9,‘W or in cat.3 In previous light microscopic studies, a high percentage of co-existence between 5-HT and SP-LI has been shown in terminal boutons that were in apparent contact with alpha motor neurons in the rat 99,‘00primate’* and cat* spinal cords. Electron mi&oscopic studies have also shown S-HT and SP co-localized in the same dense-core vesicles in terminals of the ventral horn in both rat77,*3and primate,83 and this was supported by subcellular distribution studies.** Also, our findings need to be verified in electron microscopic studies. Furthermore, electron microscopy may elucidate the subcellular location(s) of S-HT, SP and glutamate in synaptic vesicles of various types. Glutamate-like immunoreactivity in substance P/ serotonin medulla-spinal pathways-fictional implications The compounds analysed histochemically in this study have all been shown to exert effects on motor neurons. Thus, SP excites motor neurons when iontophoresed in the cat ventral hom”vs6 and when superfused over newborn rat spinal cord” or over individual rat motor neurons in the presence of tetrodotoxin and low-calcium media.‘05 S-HT also directly excites individual, neonatal rat spinal motor neurons in the presence of tetrodotoxin and low calcium in vitro.‘* Other studies suggest a modulatory role for this monoamine in motor neuron functioning.5~‘1~65~7’~76~80~‘02-‘04 Both intrathecally applied SPp2 and intraperitoneally applied 5-HT agonists or precursors’*’ activate the hindlimb electromyogram in paraplegic rats. The increased electromyographic activity produced by intrathecal SP in this model was enhanced by previous destruction of central 5-HT pathways with the neurotoxin 5,7-dihydroxytryptamine, suggesting that SP exerts postsynaptic effects on motoneurons and that destruction of spinal 5-HT/SP nerve endings by the neurotoxin induces a supersensitivity to SP. 92 It has also been proposed that SP may increase the transmission at 5-HT/SP synapses by blocking the inhibitory 5-HT autoreceptor in the ventral horn.” This is supported by the demonstration that SP reduces the affinity but increases the number of binding sites for [‘HIS-HT in spinal cord membranes.’ There is also evidence that the release of co-existing peptides and classical transmitters in some peripheral neurons is frequency dependent,27s48*6’and this may also be the case for 5-HT and SP in the ventral hom.49 Further analysis is needed to reveal if, under what ‘circumstances,

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glutamate is released in relation to other co-existing compounds, such as SP,9,26SP plus catecholamines73 and SP plus 5-HT. 74 In these conditions, glutamate presumably may be responsible for a fast component in signalling events at spinal synapses.% The present results indicate that the excitatory amino acids may interact with 5-HT and SP in medullospinal motor pathways. Several physiological studies also suggest a role for glutamate in motor control and even in interaction between glutamate and 5-HT in these events. Thus, glutamate alone has been shown to excite motor neurons.23~40~55~65~76,94,Lo2-‘W Glutamate released together with 5-HT and SP thus could act in a synergistic fashion with these signal substances. Moreover, glutamate and 5-HT have been shown to interact in many neuronal systems, such as in the locus coeruleus,‘9 the somatosensory cortex,72,97the cerebellar cortex,M and the deep cerebellar nuclei3’ More importantly, 5-HT has been shown to facilitate the glutamate-induced excitation of rat spinal’02-‘@ and faciaP5sN motor neurons and may be responsible for the plateau potentials seen in cat spinal motor neurons. 2’,45In the lamprey spinal cord, 5-HT also caused a marked depression in the late phase of the afterhyperpolarization of motoneurons.93*96 These observations have led to the hypothesis that 5-HT has a “gain-setting” function,57,65regulating the intensity and duration of the postsynaptic response elicited by a given excitatory such as glutamate or SP. input, 2’,45,57,65,93,94,96 Given that the physiologic effects of glutamate on motor neurons23@‘*55~65~76J02-‘~ are more pronounced than those of ~_~~,2’,45,65,‘6,88,93.94,96,‘0*-’~ wrhaps glu_ tamate should be viewed as the primary neurotransmitter in the raphe-spinal system, with 5-HT functioning primarily as a modulatory substance. This idea is supported by experiments showing that stimulation of the raphe pallidus evoked shortlatency, fast, excitatory potentials in motor neurons.29 These potentials are more reminiscent of those elicited by synapses utilizing an excitatory amino acid than by those using 5-HT or SP. In addition, stimulation of the raphe obscurus resulted in a biphasic excitation of phrenic motor neurons.43 In the latter study, the first fast excitation was not enhanced dramatically by higher frequency of stimulation and was not affected by systemic 5-HT receptor antagonists. On the other hand, the later, longerlasting excitation was dramatically enhanced with increased stimulation rates and was dramatically reduced by 5-HT receptor antagonists. Possibly, this first potential is mediated by glutamate or aspartate release from these raphe neurons, while the latter may be the result of 5-HT-SP release from these neurons. Thus, bulbospinal5-HT, SP and glutamate (and/or aspartate) may differentially influence motor outflow, and further physiological experiments are needed to understand the concomitant effects these compounds have on individual motor neurons.

Acknon,led~rmenr.s-This work was supported by an NRSA fellowship from the Heart, Lung and Blood Institute of the NIH (F32HL0849401Al) and The Jeane B. Kempner Foundation of the University of Texas Medical Branch at Galveston, TX, U.S.A. (to A.N.), the NSF (INT-8908720) and the Fogarty Fellowship Foundation (I F20 TWO 158601) (to V.P.), Anders Otto Swards Stiftelse. Goljes Minnesfond (to U.A.). the Swedish MRC (12X-6815 and 04X-2887) (to U.A. and T.H.) and the NIMH (MH-43230) (to T.H.). We are thankful for the generous supplies of

glutamate. 5-HT and SP antisera from (respectively) Dr A. Rustioni, University of Chapel Hill, NC, U.S.A., Dr A. A. J. Verhofstad, Catholic University, Nijmegen. The Netherlands, and the Hybridoma Technology Core of the Canadian Center of Excellence along with Dr A. C. Cuello, who is supported by the Canadian MRC and the Office of the Dean, McGill University, Montreal, Canada. For skilful technical assistance the authors also wish to thank MS K. Aman. MS W. Hiort and MS J. Fair-Nicholas.

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