0306-4522,'92 $5.00 + 0.00 Pergamon Press Ltd IBRO
Neuroscience Vol. 50, No. 4, pp. 907-920, 1992
Printed in Great Britain
G L Y C I N E - C O N T A I N I N G T E R M I N A L S IN THE RAT DORSAL VAGAL COMPLEX M. D. CASSELL,*tL. ROBERTS* and W. T. TALMAN+ *Department of Anatomy and :~Department of Neurology, VA Medical Center, University of Iowa, Iowa City, IA 52242, U.S.A. Abstract--The present study examined the distribution of glycine and glycine-receptorsin the dorsal vagal complex using pre-embedding immunocytochemiStry.Glycine-immunoreactiveterminals were present in moderate densities in the medial, intermediate, interstitial, commissural and ventrolateral subnuclei of the nucleus tractus solitarii. The dorsolateral nucleus tractus solitarii and the dorsal vagal motor nucleus contained only very few, scattered glycine-containing terminals. Glycine terminals appeared to be concentrated in regions of the dorsal vagal complex receiving primary vagal afferents, though previous studies have suggested that glycine is not present in these afferents. A conspicuously high concentration of glycine terminals was observed in the medial nucleus tractus solitarii where a population of cholinergic neurons has been identified previously. Ultrastructurally glycine immunoreactivity was principally associated with terminals containing flattened, pleomorphic vesicles and forming symmetrical synaptic contacts, mostly with dendrites. Glycine receptor immunoreactivity was present throughout the dorsal vagal complex with little evidence of subnuclear localization. With electron-microscopic examination, glycine receptor immunoreactivity was associated with dendritic membranes and was associated presynaptically with terminals containing flattened pleomorphic vesicles. Overall, the present data provide evidence consistent with a neurotransmitter role for glycine in the dorsal vagal complex. The presence of glycine in regions of the dorsal vagal complex receiving vagal afferents suggests a prominent role for this neurotransmitter in autonomic regulation.
Physiological evidence is accumulating showing that the amino acid glycine plays a role in medullary control of cardiovascular function.6,~4'24'35'36 In rat, microinjections of glycine into the caudal ventrolateral medulla elicit an increase in arterial pressure. 6 Similar injections into the dorsal vagal complex (DVC), including the nucleus tractus solitarii (NTS) and dorsal vagal motor nucleus (nX) produce changes in heart-rate and arterial pressure that vary with site of injection.36 With injections centered on the region of nX and ventral and medial parts of the NTS, blood pressure increases. 24'35 Injections involving the NTS dorsal, lateral and medial to the tractus solitarius produce depressor and bradycardiac responses. 35'36 Moreover, glycine injections into the NTS produce cardiovascular changes similar to those produced by analogous injections of the excitatory neurotransmitter glutamate, 36 which, however, does not increase arterial pressure or heart-rate when injected into the nX. While these data support site-specific actions of exogenous glycine in the DVC, the relationship between physiologically active sites and the distribution of endogenous glycine within the DVC remains unknown. It is known that the levels of glycine in the
?To whom correspondence should be addressed. Abbreviations: CHAT, choline acetyltransferase; DVC, dorsal vagal complex; NTS, nucleus tractus solitarii; nX, dorsal vagal motor nucleus; PBS, phosphate-buffered saline. NSC 50'4~F
907
medulla are high L2and are comparable to levels in the spinal cord, where glycine is a major inhibitory neurotransmitterJ° Several lines of evidence suggest that glycine is a neurotransmitter in the DVC. Potassium stimulation of the NTS causes calciumdependent release of glycine28 and a high-affinity uptake mechanism for glycine is present in the medulla. 9 The few immunocytochemical studies addressing glycine localization in the nervous system report numerous glycine-immunoreactiveelements in the medulla. 39 Glycine immunoreactivity has been localized to cells and terminals in the cochlear and superior olivary complexesfl Autoradiographic studies of the distribution of tritiated strychnine, a potent glycine-receptor antagonist, report a high density of binding in rat nX with much lower binding in the NTS and adjacent reticular formation. 42 Though this is suggestive of a heterogeneous distribution of glycine receptors in the DVC, similar studies in mouse ~3 report equivalent levels of [3H]strychnine binding in the NTS and nX. The present study has sought to determine the distribution of glycine-containing terminals and glycine-receptors in rat DVC using pre-embedding immunocytochemistry. Of particular interest was the relationship between glycine-containing terminals and the specific cytoarchitectonically defined subdivisions of the DVC. These subnuclei reflect differences in the origin and termination of cardiovascular and other visceral afferent and efferent connections.~'2°'25 Previous studies ~8'26'39 have associated glycine with
908
M. D. CASSELL et at.
b o u t o n s c o n t a i n i n g flattened vesicles characteristic o f inhibitory synapses. Given that physiological data suggest t h a t glycine m a y exert "excitation-like" effects in the NTS, 36 evidence was sought for the presence of glycine in b o u t o n s o t h e r t h a n those characterized by flattened vesicles a n d symmetrical synaptic contacts. Parts of this study have a p p e a r e d in a b s t r a c t form. 29 EXPERIMENTAL PROCEDURES
Immunocytochemistry Fourteen adult, male Sprague-Dawley rats (Harlan Labs), weight 225-275g, were used for immunocytochemical studies. Animals were deeply anesthetized (Nembutal, 40 mg/kg) and perfused through the ascending aorta with 100ml of physiological saline followed by 500ml of either ice-cold 3% giutaraldehyde in 0.167M phosphate buffer, pH 7.3 (n = 6) or similarly buffered 4% paraformaldehyde and 0.5% glutaraldehyde (n = 8). After perfusion with the fixative for 20-30 min, animals were post-perfused with 500ml of ice-cold phosphate-buffered saline (PBS). Brains were removed and sectioned on a Vibratome (LKB) at 10/,m thickness. Sections were washed three times in 10raM PBS and approximately half were placed in 1% sodium borohydride for 30 min, followed by 5 min in Lugol's iodine and I min in 1% sodium thiosulfate. Following three washes in PBS, all sections were incubated in 40% normal goat serum containing 0.1% Triton-X (Mallenkrodt) for either 30 min (for electron microscopy) or 24 h (for light microscopy). Sections were then incubated in a 1:3000 dilution of rabbit anti-glycine (provided by Dr R. Wenthold, NIMH) for 18-24h. Sections were then washed in PBS and antibody binding detected using the avidin-biotin peroxidase method. Avidin-biotin reagents were provided by Vector Labs (Burlingame, CA). Peroxidase activity was detected using 3-3'-diaminobenzidine hydrochloride (Hach, Ames, IA). Sections for light microscopy were mounted on subbed slides, dehydrated, cleared and mounted in Permount. One half of the sections were counterstained with 0.5*/0 Cresyl Violet. For ultrastructural examination, sections were osmicated, dehydrated and flat embedded in Spurr's medium. Small pieces of the dorsal medulla, representing medial and lateral halves of the DVC, were cut from the sections using bevelled cuts to facilitate orientation. Ultrathin sections were mounted on uncoated grids and examined either unstained or counterstained with lead citrate and uranyl acetate. The rabbit anti-glycine antibody used in this study was an affinity-purified, polyclonal antibody. The antibody recognizes free glycine with a slight cross-reactivity with alanine and beta-alanine. Full details of the preparation, purification and immunostaining patterns of this antibody have
been provided elsewhere. 39,4° Control material for the present study included sections through the cochlear nuclei and cervical spinal cord (to allow comparison with previous studies of these areas); sections incubated in an antibody dilution series of 1: 500, 1: 1000, 1: 3000, I : 5000, I : I 0,000 and 1:15,000; sections incubated in pre-adsorbed anti-glycine (100 mg of glycine added to I0 ml of anti-glycine diluted 1: 5000); and sections incubated in 1% normal rabbit serum. The mouse anti-glycine-receptor antibody used here was obtained from Boehringer Mannheim and was used at a dilution of I:1000. Sections from four brains fixed with 4% paraformaldehyde and 0.5% glutaraldehyde were processed for immunochemical detection of glycine-receptor antibody binding by the same procedure used for glycine immunoreactivity. Preparation of material for ultrastructural analysis was also identical to that used for glycine. The distribution of glycine-immunoreactive terminals was mapped on representative counterstained sections through the DVC using a camera lucida at a final magnification of x 375. Subnuclear boundaries were demarcated using the cytoarchitectonic criteria established by Kalia and Sullivan3 ° For ultrastructural analysis, sections were oriented under low power ( x 800) according to the position of the bevelled cuts. The positions of subnuclei were approximated relative to these cuts, as well as relative to the tractus solitarius, hypoglossal nucleus, area postrema and ventricular surface. However, subsequent references in the present text to the positions of electron micrographs (e.g. "ventrolateral" NTS) should be taken as an indication of relative position to these landmarks and not necessarily as pertaining to a specific subnucleus of the DVC. For survey purposes, ultrathin sections were cut from each of three thick sections (representing roughly the rostral, mid- and caudal levels of the DVC) from the six glutaraldehyde-fixed animals. Single micrographs ( × l 7,000) were taken from the approximate location of each subnucleus, as determined by the lower power survey. Between five and 12 micrographs were taken from each thin section: a total of 630 micrographs were examined. For glycine-receptor immunoreactivity, five ultrathin sections were cut from two sections each from four animals. Micrographs (× 17,000 or ×25,000) were taken of receptor immunoreactivity associated with clear pre-synpatic terminals. Since light microscopic evaluation of glycine-receptor immunoreactivity revealed a uniform distribution across the DVC, no attempt was made to localize the immunoreactivity to specific subnuclei. RESULTS General immunocytochemical observations A t the light-microscopic level, glycine-immunoreactivity in the present material consisted largely of p u n c t a t e structures t h a t ultrastructural e x a m i n a t i o n revealed to be mostly terminal b o u t o n s (Fig. 1A--C).
Abbreviations used in the figures AP cc dps mlf mNTS MVe ncom nCu
area postrema central canal dorsal parasolitarius region medial longitudinal fasciculusn medial subnucleus of the NTS medial vestibular nucleus commissural subnuclens of NTS cuneate nucleus
ndl nGr ni nI nPr nvl nXII TS, ts
dorsolateral subnucleus of NTS gracile nucleus interstitial subnucleus of NTS intermediate subnucleus of NTS nucleus prepositus hypoglossi ventrolateral subnuclens of NTS hypoglossal nucleus tractus solitarius
Fig. 1. Low-power ( x 40) brightfield photomicrographs of coronal sections showing glycine immunoreactivity in the DVC at rostral (A), middle (B) and caudal levels of the dorsal medulla. Note the moderately dense immunoperoxidase labehng in the medial NTS and nX. Dotted lines represent the boundaries of the dorsal vagal motor nucleus.
Glyeine in the dorsal vagal complex
Fig. 1
909
910
M.D. CASSELLet al.
Occasional fibrous structures resembling axons and dendrites were observed, as were a few cell bodies. The most intense, and most consistent, immunostaining was obtained from brains perfused with 3% glutaraldehyde. Hence, all the following descriptions and the illustrations were obtained from material fixed with this concentration of glutaraldehyde. The portion of sections from brains additionally processed with sodium borohydride and Lugol's iodine were only marginally superior to those left untreated. Immunostaining in sections from brains fixed in 4% paraformaldehyde and 0.5% glutaraldehyde was markedly inferior; only the hypoglossal nucleus was consistently stained in this material. Optimal staining, in terms of contrast with nonspecific background staining, was found in sections incubated in antiserum diluted 1:3000. At a dilution of 1 : 10,000, only the hypoglossal nucleus was stained, and staining was virtually non-existent at an antibody dilution of 1:15,000. Sections incubated in normal rabbit serum showed no immunostained structures. Similarly, sections incubated in antiserum pre-adsorbed with excess glycine (10 mg/ml to a 1:5000 dilution of anti-glycine) showed no immunoreactivity, though stained structures were seen with antisera preadsorbed with lower concentrations of glycine (e.g. 0.1-2 mg/ml). The general pattern of glycine-immunoreactivity in the rat brainstem and spinal cord was broadly similar to that reported previously. Glycine-immunoreactive cell bodies were identified in the dorsal and ventral cochlear nuclei, the lateral superior olive, the nucleus of the trapezoid body, dorsal parvicellular reticular formation, gracile and cuneate nuclei and the ventral (laminae VIII and IX) and dorsal (laminae II and III) horns.7,39,4o
Distribution of glycine-immunoreactive terminals in the dorsal vagal complex The distribution of glycine-immunoreactive terminals at three levels of the DVC is shown in Figs 2-4. Sections used for mapping terminal distributions were counterstained with Cresyl Violet and the location and boundaries of the various subfields of DVC were identified according to the cytoarchitectonic criteria of Kalia and Sullivan. 2° These authors note that some subdivisions of the DVC are only clearly discernible in celloidin-embcdded material. In the present material, we were unable to identify a dorsal subdivision of the NTS, nor were we able to clearly identify the boundaries of the dorsal and ventral parasolitarius regions and, in some sections, the commissural subdivision of the NTS. Accordingly, these boundaries have not been included in Figs 2-4. At rostral levels (Figs 2, 3), the most dense concentrations of glycine-immunoreactive terminals were observed in the medial and intermediate subdivisions of the NTS. The density of terminals appeared to be
less than in the subjacent hypogiossal nucleus, though the appearance of the terminal immunoreactivity in the NTS was of a much finer texture than the rather coarse, granular appearance of the terminal staining in the hypoglossal nucleus (Fig. 1C). Staining in the intermediate subdivision was largely confined to the cellular area of the subdivision. In the medial NTS, terminal staining extended medially and laterally for about 100 # m outside the boundaries of the mNTS. The ventricular surface of the medulla at this level, as well as areas dorsal and ventral to the mNTS, were essentially devoid of glycine-immunoreactive terminals (Fig. 3). Moderate to low densities of immunoreactive terminals were present in interstitial and ventrolateral subdivisions of the NTS at these levels (Fig. 3). Scattered terminals and fiber-like structures were stained within the tractus solitarius (Figs 2, 3). Virtually no glycine-immunoreactive terminals were present in the dorsolateral subdivision of the NTS or the nX at rostral levels (Figs 2, 3). The almost complete absence of staining in nX contrasted sharply with the moderately dense staining in the hypoglossal nucleus and the mNTS (Fig. 3) at these levels. At the level of the obex (Fig. 4), the pattern of staining density was similar to that observed at rostral levels. Moderate densities of glycineimmunoreactive terminals were present in the medial, intermediate, interstitial and commissural subnuclei (in order of decreasing density). Glycine-immunoreactive terminals were virtually absent from the dorsolateral NTS, a situation contrasting with the dense, coarse-grained staining present in the adjacent cuneate and gracile nuclei (Fig, 4). The most important differences in staining density occurred in the nX and in the ventral subnucleus of the NTS. In the former, glycine-immunoreactive terminals were largely absent from the medial two-thirds of the nX at the level of the obex. Modest numbers of terminals were present in the lateral one-third of nX at this level and, at progressively more caudal levels, glycine-immunoreactive terminals were found scattered throughout this nucleus. At caudal levels, the density in nX was less than that in the surrounding commissural nucleus (Fig. 4) but was clearly greater than in the nX at more rostral levels. An even more conspicuous regional difference in terminal density was observed in the ventral subnucleus of the NTS, both at the level of the obex and caudal to it (Fig. 4). Compared to rostral levels, the density of glycine-immunoreactive terminals in the ventral subnucleus of the NTS appeared much greater, and was continuous with an equally dense terminal staining in the region immediately ventral to this subnucleus (Fig. 4). This ventral region appears to be similar to the ventral parasolitarius region of Kalia and Sullivan, 2° but this could not be confirmed in the absence of reliable cytoarchitectonic criteria in the present material.
Glycine in the dorsal vagal complex
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Neuroscience Vol. 50, No. 4, pp. 907-920, 1992
Printed in Great Britain
G L Y C I N E - C O N T A I N I N G T E R M I N A L S IN THE RAT DORSAL VAGAL COMPLEX M. D. CASSELL,*tL. ROBERTS* and W. T. TALMAN+ *Department of Anatomy and :~Department of Neurology, VA Medical Center, University of Iowa, Iowa City, IA 52242, U.S.A. Abstract--The present study examined the distribution of glycine and glycine-receptorsin the dorsal vagal complex using pre-embedding immunocytochemiStry.Glycine-immunoreactiveterminals were present in moderate densities in the medial, intermediate, interstitial, commissural and ventrolateral subnuclei of the nucleus tractus solitarii. The dorsolateral nucleus tractus solitarii and the dorsal vagal motor nucleus contained only very few, scattered glycine-containing terminals. Glycine terminals appeared to be concentrated in regions of the dorsal vagal complex receiving primary vagal afferents, though previous studies have suggested that glycine is not present in these afferents. A conspicuously high concentration of glycine terminals was observed in the medial nucleus tractus solitarii where a population of cholinergic neurons has been identified previously. Ultrastructurally glycine immunoreactivity was principally associated with terminals containing flattened, pleomorphic vesicles and forming symmetrical synaptic contacts, mostly with dendrites. Glycine receptor immunoreactivity was present throughout the dorsal vagal complex with little evidence of subnuclear localization. With electron-microscopic examination, glycine receptor immunoreactivity was associated with dendritic membranes and was associated presynaptically with terminals containing flattened pleomorphic vesicles. Overall, the present data provide evidence consistent with a neurotransmitter role for glycine in the dorsal vagal complex. The presence of glycine in regions of the dorsal vagal complex receiving vagal afferents suggests a prominent role for this neurotransmitter in autonomic regulation.
Physiological evidence is accumulating showing that the amino acid glycine plays a role in medullary control of cardiovascular function.6,~4'24'35'36 In rat, microinjections of glycine into the caudal ventrolateral medulla elicit an increase in arterial pressure. 6 Similar injections into the dorsal vagal complex (DVC), including the nucleus tractus solitarii (NTS) and dorsal vagal motor nucleus (nX) produce changes in heart-rate and arterial pressure that vary with site of injection.36 With injections centered on the region of nX and ventral and medial parts of the NTS, blood pressure increases. 24'35 Injections involving the NTS dorsal, lateral and medial to the tractus solitarius produce depressor and bradycardiac responses. 35'36 Moreover, glycine injections into the NTS produce cardiovascular changes similar to those produced by analogous injections of the excitatory neurotransmitter glutamate, 36 which, however, does not increase arterial pressure or heart-rate when injected into the nX. While these data support site-specific actions of exogenous glycine in the DVC, the relationship between physiologically active sites and the distribution of endogenous glycine within the DVC remains unknown. It is known that the levels of glycine in the
?To whom correspondence should be addressed. Abbreviations: CHAT, choline acetyltransferase; DVC, dorsal vagal complex; NTS, nucleus tractus solitarii; nX, dorsal vagal motor nucleus; PBS, phosphate-buffered saline. NSC 50'4~F
907
medulla are high L2and are comparable to levels in the spinal cord, where glycine is a major inhibitory neurotransmitterJ° Several lines of evidence suggest that glycine is a neurotransmitter in the DVC. Potassium stimulation of the NTS causes calciumdependent release of glycine28 and a high-affinity uptake mechanism for glycine is present in the medulla. 9 The few immunocytochemical studies addressing glycine localization in the nervous system report numerous glycine-immunoreactiveelements in the medulla. 39 Glycine immunoreactivity has been localized to cells and terminals in the cochlear and superior olivary complexesfl Autoradiographic studies of the distribution of tritiated strychnine, a potent glycine-receptor antagonist, report a high density of binding in rat nX with much lower binding in the NTS and adjacent reticular formation. 42 Though this is suggestive of a heterogeneous distribution of glycine receptors in the DVC, similar studies in mouse ~3 report equivalent levels of [3H]strychnine binding in the NTS and nX. The present study has sought to determine the distribution of glycine-containing terminals and glycine-receptors in rat DVC using pre-embedding immunocytochemistry. Of particular interest was the relationship between glycine-containing terminals and the specific cytoarchitectonically defined subdivisions of the DVC. These subnuclei reflect differences in the origin and termination of cardiovascular and other visceral afferent and efferent connections.~'2°'25 Previous studies ~8'26'39 have associated glycine with
908
M. D. CASSELL et at.
b o u t o n s c o n t a i n i n g flattened vesicles characteristic o f inhibitory synapses. Given that physiological data suggest t h a t glycine m a y exert "excitation-like" effects in the NTS, 36 evidence was sought for the presence of glycine in b o u t o n s o t h e r t h a n those characterized by flattened vesicles a n d symmetrical synaptic contacts. Parts of this study have a p p e a r e d in a b s t r a c t form. 29 EXPERIMENTAL PROCEDURES
Immunocytochemistry Fourteen adult, male Sprague-Dawley rats (Harlan Labs), weight 225-275g, were used for immunocytochemical studies. Animals were deeply anesthetized (Nembutal, 40 mg/kg) and perfused through the ascending aorta with 100ml of physiological saline followed by 500ml of either ice-cold 3% giutaraldehyde in 0.167M phosphate buffer, pH 7.3 (n = 6) or similarly buffered 4% paraformaldehyde and 0.5% glutaraldehyde (n = 8). After perfusion with the fixative for 20-30 min, animals were post-perfused with 500ml of ice-cold phosphate-buffered saline (PBS). Brains were removed and sectioned on a Vibratome (LKB) at 10/,m thickness. Sections were washed three times in 10raM PBS and approximately half were placed in 1% sodium borohydride for 30 min, followed by 5 min in Lugol's iodine and I min in 1% sodium thiosulfate. Following three washes in PBS, all sections were incubated in 40% normal goat serum containing 0.1% Triton-X (Mallenkrodt) for either 30 min (for electron microscopy) or 24 h (for light microscopy). Sections were then incubated in a 1:3000 dilution of rabbit anti-glycine (provided by Dr R. Wenthold, NIMH) for 18-24h. Sections were then washed in PBS and antibody binding detected using the avidin-biotin peroxidase method. Avidin-biotin reagents were provided by Vector Labs (Burlingame, CA). Peroxidase activity was detected using 3-3'-diaminobenzidine hydrochloride (Hach, Ames, IA). Sections for light microscopy were mounted on subbed slides, dehydrated, cleared and mounted in Permount. One half of the sections were counterstained with 0.5*/0 Cresyl Violet. For ultrastructural examination, sections were osmicated, dehydrated and flat embedded in Spurr's medium. Small pieces of the dorsal medulla, representing medial and lateral halves of the DVC, were cut from the sections using bevelled cuts to facilitate orientation. Ultrathin sections were mounted on uncoated grids and examined either unstained or counterstained with lead citrate and uranyl acetate. The rabbit anti-glycine antibody used in this study was an affinity-purified, polyclonal antibody. The antibody recognizes free glycine with a slight cross-reactivity with alanine and beta-alanine. Full details of the preparation, purification and immunostaining patterns of this antibody have
been provided elsewhere. 39,4° Control material for the present study included sections through the cochlear nuclei and cervical spinal cord (to allow comparison with previous studies of these areas); sections incubated in an antibody dilution series of 1: 500, 1: 1000, 1: 3000, I : 5000, I : I 0,000 and 1:15,000; sections incubated in pre-adsorbed anti-glycine (100 mg of glycine added to I0 ml of anti-glycine diluted 1: 5000); and sections incubated in 1% normal rabbit serum. The mouse anti-glycine-receptor antibody used here was obtained from Boehringer Mannheim and was used at a dilution of I:1000. Sections from four brains fixed with 4% paraformaldehyde and 0.5% glutaraldehyde were processed for immunochemical detection of glycine-receptor antibody binding by the same procedure used for glycine immunoreactivity. Preparation of material for ultrastructural analysis was also identical to that used for glycine. The distribution of glycine-immunoreactive terminals was mapped on representative counterstained sections through the DVC using a camera lucida at a final magnification of x 375. Subnuclear boundaries were demarcated using the cytoarchitectonic criteria established by Kalia and Sullivan3 ° For ultrastructural analysis, sections were oriented under low power ( x 800) according to the position of the bevelled cuts. The positions of subnuclei were approximated relative to these cuts, as well as relative to the tractus solitarius, hypoglossal nucleus, area postrema and ventricular surface. However, subsequent references in the present text to the positions of electron micrographs (e.g. "ventrolateral" NTS) should be taken as an indication of relative position to these landmarks and not necessarily as pertaining to a specific subnucleus of the DVC. For survey purposes, ultrathin sections were cut from each of three thick sections (representing roughly the rostral, mid- and caudal levels of the DVC) from the six glutaraldehyde-fixed animals. Single micrographs ( × l 7,000) were taken from the approximate location of each subnucleus, as determined by the lower power survey. Between five and 12 micrographs were taken from each thin section: a total of 630 micrographs were examined. For glycine-receptor immunoreactivity, five ultrathin sections were cut from two sections each from four animals. Micrographs (× 17,000 or ×25,000) were taken of receptor immunoreactivity associated with clear pre-synpatic terminals. Since light microscopic evaluation of glycine-receptor immunoreactivity revealed a uniform distribution across the DVC, no attempt was made to localize the immunoreactivity to specific subnuclei. RESULTS General immunocytochemical observations A t the light-microscopic level, glycine-immunoreactivity in the present material consisted largely of p u n c t a t e structures t h a t ultrastructural e x a m i n a t i o n revealed to be mostly terminal b o u t o n s (Fig. 1A--C).
Abbreviations used in the figures AP cc dps mlf mNTS MVe ncom nCu
area postrema central canal dorsal parasolitarius region medial longitudinal fasciculusn medial subnucleus of the NTS medial vestibular nucleus commissural subnuclens of NTS cuneate nucleus
ndl nGr ni nI nPr nvl nXII TS, ts
dorsolateral subnucleus of NTS gracile nucleus interstitial subnucleus of NTS intermediate subnucleus of NTS nucleus prepositus hypoglossi ventrolateral subnuclens of NTS hypoglossal nucleus tractus solitarius
Fig. 1. Low-power ( x 40) brightfield photomicrographs of coronal sections showing glycine immunoreactivity in the DVC at rostral (A), middle (B) and caudal levels of the dorsal medulla. Note the moderately dense immunoperoxidase labehng in the medial NTS and nX. Dotted lines represent the boundaries of the dorsal vagal motor nucleus.
Glyeine in the dorsal vagal complex
Fig. 1
909
910
M.D. CASSELLet al.
Occasional fibrous structures resembling axons and dendrites were observed, as were a few cell bodies. The most intense, and most consistent, immunostaining was obtained from brains perfused with 3% glutaraldehyde. Hence, all the following descriptions and the illustrations were obtained from material fixed with this concentration of glutaraldehyde. The portion of sections from brains additionally processed with sodium borohydride and Lugol's iodine were only marginally superior to those left untreated. Immunostaining in sections from brains fixed in 4% paraformaldehyde and 0.5% glutaraldehyde was markedly inferior; only the hypoglossal nucleus was consistently stained in this material. Optimal staining, in terms of contrast with nonspecific background staining, was found in sections incubated in antiserum diluted 1:3000. At a dilution of 1 : 10,000, only the hypoglossal nucleus was stained, and staining was virtually non-existent at an antibody dilution of 1:15,000. Sections incubated in normal rabbit serum showed no immunostained structures. Similarly, sections incubated in antiserum pre-adsorbed with excess glycine (10 mg/ml to a 1:5000 dilution of anti-glycine) showed no immunoreactivity, though stained structures were seen with antisera preadsorbed with lower concentrations of glycine (e.g. 0.1-2 mg/ml). The general pattern of glycine-immunoreactivity in the rat brainstem and spinal cord was broadly similar to that reported previously. Glycine-immunoreactive cell bodies were identified in the dorsal and ventral cochlear nuclei, the lateral superior olive, the nucleus of the trapezoid body, dorsal parvicellular reticular formation, gracile and cuneate nuclei and the ventral (laminae VIII and IX) and dorsal (laminae II and III) horns.7,39,4o
Distribution of glycine-immunoreactive terminals in the dorsal vagal complex The distribution of glycine-immunoreactive terminals at three levels of the DVC is shown in Figs 2-4. Sections used for mapping terminal distributions were counterstained with Cresyl Violet and the location and boundaries of the various subfields of DVC were identified according to the cytoarchitectonic criteria of Kalia and Sullivan. 2° These authors note that some subdivisions of the DVC are only clearly discernible in celloidin-embcdded material. In the present material, we were unable to identify a dorsal subdivision of the NTS, nor were we able to clearly identify the boundaries of the dorsal and ventral parasolitarius regions and, in some sections, the commissural subdivision of the NTS. Accordingly, these boundaries have not been included in Figs 2-4. At rostral levels (Figs 2, 3), the most dense concentrations of glycine-immunoreactive terminals were observed in the medial and intermediate subdivisions of the NTS. The density of terminals appeared to be
less than in the subjacent hypogiossal nucleus, though the appearance of the terminal immunoreactivity in the NTS was of a much finer texture than the rather coarse, granular appearance of the terminal staining in the hypoglossal nucleus (Fig. 1C). Staining in the intermediate subdivision was largely confined to the cellular area of the subdivision. In the medial NTS, terminal staining extended medially and laterally for about 100 # m outside the boundaries of the mNTS. The ventricular surface of the medulla at this level, as well as areas dorsal and ventral to the mNTS, were essentially devoid of glycine-immunoreactive terminals (Fig. 3). Moderate to low densities of immunoreactive terminals were present in interstitial and ventrolateral subdivisions of the NTS at these levels (Fig. 3). Scattered terminals and fiber-like structures were stained within the tractus solitarius (Figs 2, 3). Virtually no glycine-immunoreactive terminals were present in the dorsolateral subdivision of the NTS or the nX at rostral levels (Figs 2, 3). The almost complete absence of staining in nX contrasted sharply with the moderately dense staining in the hypoglossal nucleus and the mNTS (Fig. 3) at these levels. At the level of the obex (Fig. 4), the pattern of staining density was similar to that observed at rostral levels. Moderate densities of glycineimmunoreactive terminals were present in the medial, intermediate, interstitial and commissural subnuclei (in order of decreasing density). Glycine-immunoreactive terminals were virtually absent from the dorsolateral NTS, a situation contrasting with the dense, coarse-grained staining present in the adjacent cuneate and gracile nuclei (Fig, 4). The most important differences in staining density occurred in the nX and in the ventral subnucleus of the NTS. In the former, glycine-immunoreactive terminals were largely absent from the medial two-thirds of the nX at the level of the obex. Modest numbers of terminals were present in the lateral one-third of nX at this level and, at progressively more caudal levels, glycine-immunoreactive terminals were found scattered throughout this nucleus. At caudal levels, the density in nX was less than that in the surrounding commissural nucleus (Fig. 4) but was clearly greater than in the nX at more rostral levels. An even more conspicuous regional difference in terminal density was observed in the ventral subnucleus of the NTS, both at the level of the obex and caudal to it (Fig. 4). Compared to rostral levels, the density of glycine-immunoreactive terminals in the ventral subnucleus of the NTS appeared much greater, and was continuous with an equally dense terminal staining in the region immediately ventral to this subnucleus (Fig. 4). This ventral region appears to be similar to the ventral parasolitarius region of Kalia and Sullivan, 2° but this could not be confirmed in the absence of reliable cytoarchitectonic criteria in the present material.
Glycine in the dorsal vagal complex
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