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Immunohistochemistry of γ-aminobutyric acid in the cat nucleus tractus solitarius
364
Brain Research, 330 (1985) 364-368 Elsevier
BRE 20691
Immunohistochemistry of y-aminobutyric acid in the cat nucleus tractus solitarius BRUCE MALEY and BRUCE W. NEWTON Departmentof Anatomy, Universityof Kentucky Medical Center, Lexington, KY 40536-00849 (U.S.A.) (Accepted October 30th, 1984) Key words: GABA - - nucleus of solitary tract - - immunohistochemistry
Using a new antibody directed solely against the 7-aminobutyric acid (GABA) molecule, distribution of GABA was studied in the nucleus tractus solitarii of the cat. Both immunoreactive puncta and cell bodies had a homogenous distribution within the nucleus. The one exception was in the parvocellular subdivision where very little immunoreactive puncta, but numerous immunoreactive cell bodies, were found. Results of this investigation provide immunohistochemical evidence of GABA's localization in an autonomic nucleus involved in cardiovascular regulation.
The amino acid, 7-aminobutyric acid ( G A B A ) , has generally been regarded to be an inhibitory neurotransmitter within many types of neurons in both invertebrate and vertebrate central nervous systems9,12. It has been reported that G A B A plays a major role in the regulation of blood pressure1,4,13,19, decreases in heart rate1,2,4,10,19 and depression in respiration 4,10,21. While a number of investigators have reported the inhibitory effects of G A B A in the modulation of cardiovascular and respiratory functions, the precise sites of its actions are not clear. Bousquet et al. 2 proposed that it is situated in the forebrain, possibly in the hypothalamus; however, increasing evidence suggests that the medulla, and in particular the nucleus tractus solitarius (NTS), is one of the locations for G A B A ' s modulation of autonomic functions, The NTS receives its primary visceral input from the carotid bodies and carotid sinus, and is considered to be the site of the first synapse in the baroreceptor reflex16. Although a number of physiological and pharmacological data implicate G A B A in the regulation of autonomic functions, very little morphological information is available to substantiate G A B A ' s role. Dietrich et al.5 reported the presence of G A B A using histochemistry on tissue punches of the NTS, although they were unable to determine if G A B A was used as a transmitter or no more than an
intermediate in metabolic pathways in the nucleus. Immunohistochemical investigations have localized G A B A within the NTS using the marker enzyme, glutamic acid decarboxylase ( G A D ) , and it was shown to be preferentially distributed within cardiovascular and respiratory regions of the nucleus, clearly indicating possible involvement of the GABAergic system in neural control of cardiovascular and respiratory systems at the level of the NTS8,11. The use of G A D immunohistochemistryl2,20 or high affinity [ 3 H ] G A B A uptake 7,14 have been the most c o m m o n methods of studying the localization of G A B A ; however, both techniques have limitations. Since [3H]GABA uptake can occur in other structures such as glial cells in addition to presynaptic GABAergic neurons, it may not be an adequate marker for G A B A e r g i c systems 22. The G A D enzyme has been purified by a number of laboratories and used to make antibodies for its localization utilizing immunohistochemistry21,23; however, it has not been generally available and therefore its distribution within autonomic regions has not been extensively studied. In addition, G A D , as the biosynthetic enzyme for G A B A , is only an indirect marker for G A B A ; ideally an antibody for G A B A itself would be more specific and hopefully better suited for revealing the fine details of G A B A circuitry. It was the
Correspondence: B. Maley, Department of Anatomy, University of Kentucky Medical Center, Lexington, KY 40536-00849, U.S.A. 0006-8993/85/$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
365 intent of the present investigation to report on the localization of G A B A within the NTS using an antibody directed solely against G A B A . The antiserum used in the present study was produced in rabbits utilizing the procedure described by Storm-Mathisen et al. TM. Briefly, the G A B A was conjugated to bovine serum albumin with glutaraldehyde and then dialyzed in phosphate-buffered saline (PBS) and brought to a final dilution of 10 ~mol/ml. For the immunization procedure the conjugated G A B A was emulsified with Freund's complete adjuvant (1:1) and injected subcutaneously into rabbits. The animals were boosted every 2 weeks with the G A B A conjugate emulsified in Freund's incomplete adjuvant, and then 10 days following the third booster injection, blood was taken from each rabbit. Antiserum from one rabbit, when applied to sections of the cerebellum, a region known to have large amounts of G A B A , demonstrated immunostaining similar to that reported for G A D 12 and G A B A 15 (Fig. 1A). Within the cerebellar cortex G A B A immunoreactivity was contained within Golgi, Pur-
kinje, stellate and basket cells and numerous puncta in the molecular layer and puncta that surrounded glomeruli of the granular cell layer, as well as immunoreactive puncta. The G A B A antiserum, when pretreated for 24 h with 20/~g/ml of the conjugated G A B A - B S A or G A B A , resulted in a loss of G A B A immunoreactivity in the tissue (Fig. 1B). Pretreatment of the G A B A antiserum with a number of amino acids and peptides including glutamate, taurine, aspartate, glycine, alanine, tyrosine, enkephalin and substance P did not result in any loss of immunostaining in the tissue. To further test the specificity of the G A B A antiserum the same amino acids and peptides were coupled to cyanogen bromide-activated sepharose beads. The ability of the G A B A antiserum to bind to the beads was determined by fluorescein-labeled goat anti-rabbit IgG. Beads coupled to the amino acids or peptides did not possess any fluorescence, while beads coupled with G A B A and incubated with the G A B A antiserum were strongly fluorescent, suggesting this antiserum was specific for G A B A . In order to visualize the G A B A immunoreactivity
Fig. 1. A: GABA immunoreactivity in the cat cerebellum. Numerous GABA-immunoreactive puncta are visible in the molecular (M), Purkinje (P) and granular (G) layers. A Golgi cell (Go) can be seen at the open block arrow. Dashed line separates underlying white matter from cerebellar cortex. Final magnification 275 x. B: cat cerebellar cortex incubated in absorbed GABA antiserum note the absence of staining in the molecular (M), Purkinje (P) and granular (G) layers. Dashed line separates white matter from granule cell layer. Final magnification 275 x.
366 within the NTS and cerebellum the peroxidase-antiperoxidase (PAP) procedure was used17. The caudal brainstem containing the NTS of 8 cats was cut on a vibratome in the transverse plane, incubated in 3% normal sheep serum and placed in G A B A antiserum (1:4000 in PBS/0.3% Triton X-100) at room temperature overnight. The next day the tissue was rinsed several times and placed in sheep antirabbit IgG (1:600 in PBS/0.3% Triton X-100) for 1 h. Following a third set of rinses, the tissue was incubated in rabbit-PAP (1:1000 in PBS/0.3% Triton X-100). After 1 h the sections were rinsed several times, preincubated in 3,3'-diaminobenzidine (DAB; 0.5 mg/ml in Tris buffer) for 15 min, and then reacted with DAB containing 0.1% H20 2. The reaction was halted with several changes of buffer, and the sections were mounted in gelatin-coated slides, dehydrated and coverslipped. Adjacent sections of the NTS were incubated in G A B A antiserum absorbed with G A B A BSA conjugate (20#g/ml) and processed with the remainder of the tissue. These 'absorbed' sections did
not possess G A B A immunoreactivity within the NTS (Fig. 3B). G A B A immunoreactivity was distributed widely throughout the NTS of the cat as numerous puncta and scattered cell bodies. Similar to many peptidergic and catecholaminergic neurotransmitters in the NTS, G A B A immunoreactive puncta had a widespread distribution throughout all the major subdivisions of the nucleus. Most subdivisions contained moderate amounts of G A B A immunoreactivity with the exception of the parvocellular (PC) subdivision where very little GABA-like immunoreactivity was visible (Fig. 2A). In addition, within individual subdivisions the G A B A immunoreactive puncta appeared to be evenly distributed throughout the neuropil, and in many areas of the NTS they surrounded non-immunoreactive neurons (Fig. 2B). Preliminary electron microscopic evidence indicates that the G A B A immunoreactivity was located within numerous synaptic terminals which were presynaptic to cell bodies, dendrites and spines. Neurons immunoreactive for
Fig. 2. A: the homogenous distribution of GABA immunoreactivity in the cat NTS is evident with the exception of the parvocellular subdivision (PC). Also indicated are the medial (M) and dorsolateral (DL) subdivisions as well as the tract (T) of the NTS. Note the heavier staining in the dorsal motor nucleus of the vagus nerve (DVN). Final magnification 65×. B: numerous GABA immunoreactive puncta are visible (arrowheads) surrounding unstained NTS cell bodies (asterisks). Final magnification 170×.
367
Fig. 3. A: many GABA-immunoreactive neurons (arrowheads) are evident in the NTS as well as several unstained neurons (asterisks). The tract (T) of the NTS is unstained. Final magnification 125x. B: section of the NTS containing the medial (M) and parvocellular (PC) subdivisions which was incubated in GABA antiserum pretreated with GABA antigen 48 h prior to the PAP reaction. Note the lack of immunoreactivity. Final magnification 130 x.
G A B A were scattered t h r o u g h o u t all regions of the NTS with the greatest numbers located within the parvocellular subdivision (Fig. 3A). These neurons were small averaging 12-15 p m in d i a m e t e r and occasionally had one or two short p r i m a r y dendrites. This immunohistochemical study reports the presence of G A B A - i m m u n o r e a c t i v e puncta and cell bodies within the cat NTS using a new a n t i b o d y directed against the G A B A molecule. Tests to d e t e r m i n e the staining capabilities of this G A B A antiserum d e m o n strate that it recognizes the G A B A antigen, and no other amino acids or peptides which are located in the central nervous system. In addition, its patterns of localization are very similar to that previously rep o r t e d for G A D immunoreactivity both in the cerebellum 12 and NTSS, n. The G A B A staining p a t t e r n is also consistent with the concept that the NTS contains G A B A binding sites 6. The presence of G A B A i m m u n o r e a c t i v e puncta and neurons in cardiovascular subdivisions (medial, commissural and dorsolateral) of the cat NTS sug-
1 Antonaccio, M. J. and Taylor, D. G., Involvement of central GABA receptors in the regulation of blood pressure and heart rate of anaesthetized cats, Europ. J. Pharmacol., 46 (1977) 283-287. 2 Bousquet, P., Feldman, J., Bloch, R. and Schwartz, J., The
gests that at least a portion of the G A B A within the NTS may play a role in the regulation of this system. This is s u p p o r t e d by the reports of Bousquet et al.3 that d e m o n s t r a t e d the release of G A B A in the NTS is responsible for elevations in b l o o d pressure, while bicuculline, a G A B A antagonist, produces an opposite effect. Interestingly, the only regions of the NTS where this occurs are in i n t e r m e d i a t e levels of the nucleus, which coincides with cardiovascular subdivisions. The mechanism in which G A B A exerts its influence is not fully u n d e r s t o o d ; however, it has been suggested that G A B A m a y be involved in the inhibition of the b a r o r e c e p t o r reflex at the level of the NTS. Results of this study d e m o n s t r a t e significant amounts of G A B A - i m m u n o r e a c t i v e puncta and cell bodies in cardiovascular regions of the NTS. W h e t h er they are responsible for the m o d u l a t i o n of the cardiovascular system awaits further investigation. This study was s u p p o r t e d by N I H G r a n t HL30702 to B.M.
central hypotensive action of baclofen in the anaesthetized cat, Europ. J. Pharmacol., 76 (1981) 193-201. 3 Bousquet, P., Feldman, J., Bloch, R. and Schwartz, J., Evidence for a neuromodulatory role of GABA at the first synapse of the baroreceptor reflex pathway. Effects of
368
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5
6
7
8
9
10
11
12
GABA derivaties injected into the NTS, NaunynSchmiedeber g's Arch. Pharmacol., 319 (1982) 168-171. DeFeudis, F. V., GABA: an inhibitory neurotransmitter that is involved in cardiovascular control, Pharmacol. Res. Commun., 14 (1982) 567-576. Dietrich, W. D., Lowry, O. H. and Loewy, A. D., The distribution of glutamate, GABA and aspartate in the nucleus tractus solitarius of the cat, Brain Research, 237 (1982) 254-260. Gale, K., Hamilton, B. L., Brown, S. C., Norman, W. P., Dias Souza, J. and Gillis, R. A., GABA and specific GABA binding sites in brain nuclei associated with vagal outflow, Brain Res. Bull., 2 (1980) 325-328. Hokfelt, T. and Ljungdahl, /~., Autoradiographic identification of cerebral and cerebellar cortical neurons accumulating labelled gamma-aminobutyric acid ([3H]GABA), Exp. Brain Res., 14 (1972) 354-362. Hwang, B. H. and Wu, J.-Y., Ultrastructural studies on catecholaminergic terminals and GABAergic neurons in nucleus tractus solitarius of the rat medulla oblongata, Brain Research, 302 (1984) 57-67. Krnjevic, K., Inhibitory action of GABA and GABA-mimetic on vertebrate neurons. In: E. Roberts, T. N. Chase and D. R. Towers (Eds.), GABA in the Nervous System Functions, Raven Press, New York, 1976, pp. 269-286. Lalley, P. M., Evidence suggesting a role for GABA in cardiovascular and respiratory reflexes initiated by baroreceptor and cardiopulmonary afferents in Problems in GABA Research from brain to bacteria. Y. Okada and E. Roberts eds. Excerpta Medica, Amsterdam (1982) 137-146 pp. Maley, B., Eide, R. P., Oertel, W. H. and Schmechel, D. E., The localization of glutamic acid decarboxylase (GAD) immunoreactivity in the nucleus of the solitary tract, Soc. Neurosci. Abstr., 9 (1983) 1158. Oertel, W. H., Schmechel, D. E., Mugnaini, E., Tappaz, M. L. and Kopin, I. J., Immunocytochemical localization of glutamate decarboxylase in rat cerebellum with a new antiserum, Neuroscience, 6 (1981) 2715-2735.
13 Persson, B., A hypertensive response to baclofen in the nucleus tractus solitarii in rats, J. Pharm. Pharmacol., 33 (1981) 226-231. 14 Schon, F. and Iverson, L. L., The use of autoradiographic techniques for the identification and mapping of transmitter-specific neurons in the brain, Life Sci., 15 (1974) 157-175. 15 Seguela, P., Geffard, M., Buijs, R. M. and LeMoal, M., Antibodies against 7-aminobutyric acid: specificity studies and immunocytochemical results, Proc. nat. Acad. Sci. U.S.A., 81 (1984) 3888-3892. 16 Seller, H. and Illert, M., The localization of the first synapse in the carotid sinus baroreceptors reflex pathway and its alteration of the afferent input, Pfliiger's Arch. ges. Physiol., 306 (1969) 1-19. 17 Sternberger, L. A., Immunocytochemistry, Prentice-Hall, Englewood Cliffs, NJ, 1979. 18 Storm-Mathisen, J., Leknes, A. K., Bore, A. T., Vaaland, J. L., Edminson, P., Haug, F.-M. S. and Ottersen, O. P., First visualization of glutamate and GABA in neurons by immunocytochemistry, Nature (Lond.), 301 (1983) 517-520. 19 Williford, D. J., Hamilton, B. L., Dias Souza, J., Williams, T. P., DiMicco, J. A. and Gillis, R. A., Central nervous system mechanisms involving GABA influence arterial pressure and heart rate in the cat, Circular. Res., 47 (1980) 80-88. 20 Wu, J.-Y., Lin, C. T., Brandon, C., Chan, D. S., Mohler, H. and Richards, J. G., Regulation and immunocytochemical characterization of GAD. In S. Palay and V. ChanPalay (Eds.), Cytochemical Methods in Neuroanatomy, Alan R. Liss, New York, 1982, pp. 279-294. 21 Yamada, K. A., Hamosh, P. and Gillis, R. A., Respiratory depression produced by activation of GABA receptors in the hindbrain of the cat, J. appl. Physiol., 51 (1981) 1278-1286. 22 Zucker, C., Yazulla, S. and Wu, J.-Y., Non-correspondence of [3H]GABA uptake and GAD localization in goldfish amacrine cells, Brain Research, 298 (1984) 154-158.
Brain Research, 330 (1985) 364-368 Elsevier
BRE 20691
Immunohistochemistry of y-aminobutyric acid in the cat nucleus tractus solitarius BRUCE MALEY and BRUCE W. NEWTON Departmentof Anatomy, Universityof Kentucky Medical Center, Lexington, KY 40536-00849 (U.S.A.) (Accepted October 30th, 1984) Key words: GABA - - nucleus of solitary tract - - immunohistochemistry
Using a new antibody directed solely against the 7-aminobutyric acid (GABA) molecule, distribution of GABA was studied in the nucleus tractus solitarii of the cat. Both immunoreactive puncta and cell bodies had a homogenous distribution within the nucleus. The one exception was in the parvocellular subdivision where very little immunoreactive puncta, but numerous immunoreactive cell bodies, were found. Results of this investigation provide immunohistochemical evidence of GABA's localization in an autonomic nucleus involved in cardiovascular regulation.
The amino acid, 7-aminobutyric acid ( G A B A ) , has generally been regarded to be an inhibitory neurotransmitter within many types of neurons in both invertebrate and vertebrate central nervous systems9,12. It has been reported that G A B A plays a major role in the regulation of blood pressure1,4,13,19, decreases in heart rate1,2,4,10,19 and depression in respiration 4,10,21. While a number of investigators have reported the inhibitory effects of G A B A in the modulation of cardiovascular and respiratory functions, the precise sites of its actions are not clear. Bousquet et al. 2 proposed that it is situated in the forebrain, possibly in the hypothalamus; however, increasing evidence suggests that the medulla, and in particular the nucleus tractus solitarius (NTS), is one of the locations for G A B A ' s modulation of autonomic functions, The NTS receives its primary visceral input from the carotid bodies and carotid sinus, and is considered to be the site of the first synapse in the baroreceptor reflex16. Although a number of physiological and pharmacological data implicate G A B A in the regulation of autonomic functions, very little morphological information is available to substantiate G A B A ' s role. Dietrich et al.5 reported the presence of G A B A using histochemistry on tissue punches of the NTS, although they were unable to determine if G A B A was used as a transmitter or no more than an
intermediate in metabolic pathways in the nucleus. Immunohistochemical investigations have localized G A B A within the NTS using the marker enzyme, glutamic acid decarboxylase ( G A D ) , and it was shown to be preferentially distributed within cardiovascular and respiratory regions of the nucleus, clearly indicating possible involvement of the GABAergic system in neural control of cardiovascular and respiratory systems at the level of the NTS8,11. The use of G A D immunohistochemistryl2,20 or high affinity [ 3 H ] G A B A uptake 7,14 have been the most c o m m o n methods of studying the localization of G A B A ; however, both techniques have limitations. Since [3H]GABA uptake can occur in other structures such as glial cells in addition to presynaptic GABAergic neurons, it may not be an adequate marker for G A B A e r g i c systems 22. The G A D enzyme has been purified by a number of laboratories and used to make antibodies for its localization utilizing immunohistochemistry21,23; however, it has not been generally available and therefore its distribution within autonomic regions has not been extensively studied. In addition, G A D , as the biosynthetic enzyme for G A B A , is only an indirect marker for G A B A ; ideally an antibody for G A B A itself would be more specific and hopefully better suited for revealing the fine details of G A B A circuitry. It was the
Correspondence: B. Maley, Department of Anatomy, University of Kentucky Medical Center, Lexington, KY 40536-00849, U.S.A. 0006-8993/85/$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
365 intent of the present investigation to report on the localization of G A B A within the NTS using an antibody directed solely against G A B A . The antiserum used in the present study was produced in rabbits utilizing the procedure described by Storm-Mathisen et al. TM. Briefly, the G A B A was conjugated to bovine serum albumin with glutaraldehyde and then dialyzed in phosphate-buffered saline (PBS) and brought to a final dilution of 10 ~mol/ml. For the immunization procedure the conjugated G A B A was emulsified with Freund's complete adjuvant (1:1) and injected subcutaneously into rabbits. The animals were boosted every 2 weeks with the G A B A conjugate emulsified in Freund's incomplete adjuvant, and then 10 days following the third booster injection, blood was taken from each rabbit. Antiserum from one rabbit, when applied to sections of the cerebellum, a region known to have large amounts of G A B A , demonstrated immunostaining similar to that reported for G A D 12 and G A B A 15 (Fig. 1A). Within the cerebellar cortex G A B A immunoreactivity was contained within Golgi, Pur-
kinje, stellate and basket cells and numerous puncta in the molecular layer and puncta that surrounded glomeruli of the granular cell layer, as well as immunoreactive puncta. The G A B A antiserum, when pretreated for 24 h with 20/~g/ml of the conjugated G A B A - B S A or G A B A , resulted in a loss of G A B A immunoreactivity in the tissue (Fig. 1B). Pretreatment of the G A B A antiserum with a number of amino acids and peptides including glutamate, taurine, aspartate, glycine, alanine, tyrosine, enkephalin and substance P did not result in any loss of immunostaining in the tissue. To further test the specificity of the G A B A antiserum the same amino acids and peptides were coupled to cyanogen bromide-activated sepharose beads. The ability of the G A B A antiserum to bind to the beads was determined by fluorescein-labeled goat anti-rabbit IgG. Beads coupled to the amino acids or peptides did not possess any fluorescence, while beads coupled with G A B A and incubated with the G A B A antiserum were strongly fluorescent, suggesting this antiserum was specific for G A B A . In order to visualize the G A B A immunoreactivity
Fig. 1. A: GABA immunoreactivity in the cat cerebellum. Numerous GABA-immunoreactive puncta are visible in the molecular (M), Purkinje (P) and granular (G) layers. A Golgi cell (Go) can be seen at the open block arrow. Dashed line separates underlying white matter from cerebellar cortex. Final magnification 275 x. B: cat cerebellar cortex incubated in absorbed GABA antiserum note the absence of staining in the molecular (M), Purkinje (P) and granular (G) layers. Dashed line separates white matter from granule cell layer. Final magnification 275 x.
366 within the NTS and cerebellum the peroxidase-antiperoxidase (PAP) procedure was used17. The caudal brainstem containing the NTS of 8 cats was cut on a vibratome in the transverse plane, incubated in 3% normal sheep serum and placed in G A B A antiserum (1:4000 in PBS/0.3% Triton X-100) at room temperature overnight. The next day the tissue was rinsed several times and placed in sheep antirabbit IgG (1:600 in PBS/0.3% Triton X-100) for 1 h. Following a third set of rinses, the tissue was incubated in rabbit-PAP (1:1000 in PBS/0.3% Triton X-100). After 1 h the sections were rinsed several times, preincubated in 3,3'-diaminobenzidine (DAB; 0.5 mg/ml in Tris buffer) for 15 min, and then reacted with DAB containing 0.1% H20 2. The reaction was halted with several changes of buffer, and the sections were mounted in gelatin-coated slides, dehydrated and coverslipped. Adjacent sections of the NTS were incubated in G A B A antiserum absorbed with G A B A BSA conjugate (20#g/ml) and processed with the remainder of the tissue. These 'absorbed' sections did
not possess G A B A immunoreactivity within the NTS (Fig. 3B). G A B A immunoreactivity was distributed widely throughout the NTS of the cat as numerous puncta and scattered cell bodies. Similar to many peptidergic and catecholaminergic neurotransmitters in the NTS, G A B A immunoreactive puncta had a widespread distribution throughout all the major subdivisions of the nucleus. Most subdivisions contained moderate amounts of G A B A immunoreactivity with the exception of the parvocellular (PC) subdivision where very little GABA-like immunoreactivity was visible (Fig. 2A). In addition, within individual subdivisions the G A B A immunoreactive puncta appeared to be evenly distributed throughout the neuropil, and in many areas of the NTS they surrounded non-immunoreactive neurons (Fig. 2B). Preliminary electron microscopic evidence indicates that the G A B A immunoreactivity was located within numerous synaptic terminals which were presynaptic to cell bodies, dendrites and spines. Neurons immunoreactive for
Fig. 2. A: the homogenous distribution of GABA immunoreactivity in the cat NTS is evident with the exception of the parvocellular subdivision (PC). Also indicated are the medial (M) and dorsolateral (DL) subdivisions as well as the tract (T) of the NTS. Note the heavier staining in the dorsal motor nucleus of the vagus nerve (DVN). Final magnification 65×. B: numerous GABA immunoreactive puncta are visible (arrowheads) surrounding unstained NTS cell bodies (asterisks). Final magnification 170×.
367
Fig. 3. A: many GABA-immunoreactive neurons (arrowheads) are evident in the NTS as well as several unstained neurons (asterisks). The tract (T) of the NTS is unstained. Final magnification 125x. B: section of the NTS containing the medial (M) and parvocellular (PC) subdivisions which was incubated in GABA antiserum pretreated with GABA antigen 48 h prior to the PAP reaction. Note the lack of immunoreactivity. Final magnification 130 x.
G A B A were scattered t h r o u g h o u t all regions of the NTS with the greatest numbers located within the parvocellular subdivision (Fig. 3A). These neurons were small averaging 12-15 p m in d i a m e t e r and occasionally had one or two short p r i m a r y dendrites. This immunohistochemical study reports the presence of G A B A - i m m u n o r e a c t i v e puncta and cell bodies within the cat NTS using a new a n t i b o d y directed against the G A B A molecule. Tests to d e t e r m i n e the staining capabilities of this G A B A antiserum d e m o n strate that it recognizes the G A B A antigen, and no other amino acids or peptides which are located in the central nervous system. In addition, its patterns of localization are very similar to that previously rep o r t e d for G A D immunoreactivity both in the cerebellum 12 and NTSS, n. The G A B A staining p a t t e r n is also consistent with the concept that the NTS contains G A B A binding sites 6. The presence of G A B A i m m u n o r e a c t i v e puncta and neurons in cardiovascular subdivisions (medial, commissural and dorsolateral) of the cat NTS sug-
1 Antonaccio, M. J. and Taylor, D. G., Involvement of central GABA receptors in the regulation of blood pressure and heart rate of anaesthetized cats, Europ. J. Pharmacol., 46 (1977) 283-287. 2 Bousquet, P., Feldman, J., Bloch, R. and Schwartz, J., The
gests that at least a portion of the G A B A within the NTS may play a role in the regulation of this system. This is s u p p o r t e d by the reports of Bousquet et al.3 that d e m o n s t r a t e d the release of G A B A in the NTS is responsible for elevations in b l o o d pressure, while bicuculline, a G A B A antagonist, produces an opposite effect. Interestingly, the only regions of the NTS where this occurs are in i n t e r m e d i a t e levels of the nucleus, which coincides with cardiovascular subdivisions. The mechanism in which G A B A exerts its influence is not fully u n d e r s t o o d ; however, it has been suggested that G A B A m a y be involved in the inhibition of the b a r o r e c e p t o r reflex at the level of the NTS. Results of this study d e m o n s t r a t e significant amounts of G A B A - i m m u n o r e a c t i v e puncta and cell bodies in cardiovascular regions of the NTS. W h e t h er they are responsible for the m o d u l a t i o n of the cardiovascular system awaits further investigation. This study was s u p p o r t e d by N I H G r a n t HL30702 to B.M.
central hypotensive action of baclofen in the anaesthetized cat, Europ. J. Pharmacol., 76 (1981) 193-201. 3 Bousquet, P., Feldman, J., Bloch, R. and Schwartz, J., Evidence for a neuromodulatory role of GABA at the first synapse of the baroreceptor reflex pathway. Effects of
368
4
5
6
7
8
9
10
11
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GABA derivaties injected into the NTS, NaunynSchmiedeber g's Arch. Pharmacol., 319 (1982) 168-171. DeFeudis, F. V., GABA: an inhibitory neurotransmitter that is involved in cardiovascular control, Pharmacol. Res. Commun., 14 (1982) 567-576. Dietrich, W. D., Lowry, O. H. and Loewy, A. D., The distribution of glutamate, GABA and aspartate in the nucleus tractus solitarius of the cat, Brain Research, 237 (1982) 254-260. Gale, K., Hamilton, B. L., Brown, S. C., Norman, W. P., Dias Souza, J. and Gillis, R. A., GABA and specific GABA binding sites in brain nuclei associated with vagal outflow, Brain Res. Bull., 2 (1980) 325-328. Hokfelt, T. and Ljungdahl, /~., Autoradiographic identification of cerebral and cerebellar cortical neurons accumulating labelled gamma-aminobutyric acid ([3H]GABA), Exp. Brain Res., 14 (1972) 354-362. Hwang, B. H. and Wu, J.-Y., Ultrastructural studies on catecholaminergic terminals and GABAergic neurons in nucleus tractus solitarius of the rat medulla oblongata, Brain Research, 302 (1984) 57-67. Krnjevic, K., Inhibitory action of GABA and GABA-mimetic on vertebrate neurons. In: E. Roberts, T. N. Chase and D. R. Towers (Eds.), GABA in the Nervous System Functions, Raven Press, New York, 1976, pp. 269-286. Lalley, P. M., Evidence suggesting a role for GABA in cardiovascular and respiratory reflexes initiated by baroreceptor and cardiopulmonary afferents in Problems in GABA Research from brain to bacteria. Y. Okada and E. Roberts eds. Excerpta Medica, Amsterdam (1982) 137-146 pp. Maley, B., Eide, R. P., Oertel, W. H. and Schmechel, D. E., The localization of glutamic acid decarboxylase (GAD) immunoreactivity in the nucleus of the solitary tract, Soc. Neurosci. Abstr., 9 (1983) 1158. Oertel, W. H., Schmechel, D. E., Mugnaini, E., Tappaz, M. L. and Kopin, I. J., Immunocytochemical localization of glutamate decarboxylase in rat cerebellum with a new antiserum, Neuroscience, 6 (1981) 2715-2735.
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