THE JOURNAL OF COMPARATIVE NEUROLOGY 296:496-505 (1990)

Light Microscope Study of the Coexistence of GABA-Like and Glycine-Like Immunoreactivities in the Spinal Cord of the Rat ANDREW J. TODD AND ANNE C. SULLIVAN Department of Anatomy, University of Glasgow, Glasgow G12 SQQ, United Kingdom

ABSTRACT The distributions of GABA-like and glycine-like immunoreactivities in the rat spinal cord were compared by using postembedding immunohistochemistry on semithin sections. In laminae I, 11, and 111, the proportions of GABA immunoreactive cells were 28%, 31%, and 4696, respectively, whereas for glycine immunoreactive cells the proportions were 9 % , 1 4 % ,and 30%. Nearly all of the glycine immunoreactive cells in this area were also immunoreactive with the anti-GABA antiserum. In lamina 11, some Golgi-stained islet cells were glycine immunoreactive, whereas others were not. Immunoreactive cell bodies were also present in the remainder of the grey matter. Some of these reacted with anti-GABA or antiglycine antiserum; others showed immunoreactivity with both antisera. Immunoreactive axons were found in the ventral and lateral funiculi of the white matter. Many large axons reacted with antiglycine antiserum, whereas GABA-immunoreactive axons were mostly of small diameter. Some large and small axons showed both types of immunoreactivity. These results suggest that the inhibitory neurotransmitters GABA and glycine coexist within cell bodies and axons in the rat spinal cord. K e y words: y-aminobutyric acid, colocalization, immunohistochemistry, dorsal horn

There is much evidence that the amino acids GABA and glycine both function as inhibitory neurotransmitters within the spinal cord (reviewed by Nistri, '83; Young and Macdonald, '83) and are important in sensory processing within the dorsal horn. Strychnine (a glycine antagonist) and bicuculline (a GABA, receptor antagonist) block the inhibition of cells in laminae IV and V of the cat dorsal horn caused by stimulating myelinated fibres in peripheral nerves (Game and Lodge, '75), and when these compounds are injected into the lumbar subarachnoid space of rats, normally innocuous cutaneous stimuli appear to produce aversive responses (Yaksh, '89). GABA is thought to act a t both GABA, and GABA, receptors to inhibit the transmission of nociceptive information within the spinal dorsal horn, because agonists acting a t both types of receptor can suppress the responses to noxious stimulation (Cutting and Jordan, '75; Wilson and Yaksh, '78; Aanonsen and Wilcox, '89; Hwang and Wilcox, '89). The distribution of both compounds within the cord has been studied with immunohistochemistry. The original demonstration of GABA-containing structures was performed by using antiserum to the enzyme glutamic acid decarboxylase (GAD). Immunoreactivity was concentrated o 1 9 9 0 WILEY-LISS, INC.

within laminae 1-111 of the rat dorsal horn (McLaughlin et al., '75; Barber et al., '78; Hunt et al., '81),but was found in lower concentrations throughout the grey matter. When colchicine was injected into the spinal cord, immunoreactive cell bodies were found in all laminae except lamina IX (the motorneurone pools) (Barber et al., '82). More recently, antisera to GABA conjugates have been used on the spinal cord (Magoul et al., '87; Todd and McKenzie, '89). In these studies the distribution of terminal labelling was similar to that found with anti-GAD. Immunoreactive cell bodies were most frequent in laminae 1-111 where they constituted 25-33 % of the neuronal population, but were also found in deeper laminae and in particular around the central canal. Within lamina I1 the immunoreactive cell population included islet cells but not stalked cells (Todd and McKenzie, '89). Immunoreactive axons were found in all regions of the white matter but were particularly numerous in the dorsal part of the lateral funiculus. The distribution of glycine within the cord has been Accepted February 7,1990. Address reprint requests to Dr. A.J. Todd, Dept. of Anatomy, University of Glasgow, Glasgow G12 SQQ, U.K.

GABA A N D GLYCINE I N RAT SPINAL CORD examined with antisera to glycine conjugates (Campistron et al., '86; Ottersen and Storm-Mathisen, '87; van den Pol and Gorcs, '88). Immunoreactive cell bodies were found in the dorsal horn (particularly ventral to lamina 11) and in the ventral horn, where they were smaller than motorneurons. Terminal labelling was present throughout the grey matter but was less dense in laminae I and 11. Immunoreactive axons were found throughout the white matter, although few were present in the dorsal funiculus. Monoclonal antisera to glycine receptor (Pfeiffer et al., '84) have also been used to determine the distribution of glycinergic synapses within the spinal cord. Van den Pol and Gorcs ('88) found that immunoreactivity was present throughout the grey matter with lower concentrations in laminae I and 11; Basbaum ('88) reported a generally similar distribution, although in this study labelling was almost absent from laminae I and I1 and was densest in laminae I11 and IV. Within the dorsal horn, therefore, GABA appears to be present at high concentration in somata and axon terminals within laminae 1-111, whereas glycine-containing cells and terminals are found in relatively low concentrations in laminae I and I1 but are present in larger numbers in lamina 111. This is in fairly good agreement with the findings of Ribeiro-da-Silva and Coimbra ('80) who studied the uptake of tritiated glycine and GABA into the rat dorsal horn in vivo. In that study, GABA was taken up into cell bodies in laminae I and 11, whereas the uptake of glycine was greatest in lamina 111.The lack of uptake of tritiated GABA into cells in lamina 111may have resulted from poorer diffusion of this compound. Recently coexistence of glycine-like and GABA-like immunoreactivity (glycine-LI and GABA-LI) has been found in rat cerebellar Golgi cells (Ottersen et al., '88) and in the dorsal cochlear nucleus of the guinea pig (Wenthold et al., '87); in the spinal ventral horn, GAD-immunoreactive axon terminals are frequently apposed to immunoreactive glycine receptors (Triller et al., '87). This suggests that some neurones may contain and release both transmitters. Since GABA and glycine appear to show different but overlapping distributions within the spinal cord, it is of interest to determine whether they exist within the same neurones. The present study has attempted to answer this by using resin-embedded semithin sections processed with antisera to GABA and glycine. In addition to allowing single cells to be tested with more than one antiserum, this method can also produce quantitative data, because the barriers to diffusion of immunochemicals are negligible. In this study we have concentrated on the cells of laminae 1-111; these are thought to be important in the transmission of nociceptive information through the dorsal horn (Basbaum, '88; Todd, '89), but in addition the distribution of immunoreactivity within other areas of grey matter and in the white matter is described.

METHODS Experimental material Twelve male Albino Swiss rats aged between 8 and 12 weeks were deeply anaesthetised with pentobarbitone and perfused through the left ventricle with 1,000 mls of fixative containing 2.5 93 glutaraldehydeh % formaldehyde in 0.1 M phosphate buffer. Lumbar spinal cord segments were removed and stored in the same fixative for 3 hours. The segments were then either osmicated, Golgi-stained, sectioned in the sagittal plane, gold-toned, and embedded in

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Araldite (as described previously, Todd and McKenzie, '89) or else cut into 100 pm sections in the transverse or sagittal plane with a Vibratome, osmicated (1% osmium tetroxide for 30 minutes), dehydrated, and embedded in Araldite. Semithin sections (0.5 pm thick) were cut and mounted on gelatinised slides for immunohistochemical processing.

Immunohistochemical methods Immunohistochemistry was carried out according to a modification of the method of Somogyi et al. ('85). The sections were etched in sodium ethanolate for 40 minutes to remove resin, rinsed in alcohol and then water, and treated with 1%sodium metaperiodate for 7 minutes to remove osmium tetroxide. After further rinsing they were incubated in primary antiserum (anti-GABA diluted 1:30,000-80,000 or antiglycine diluted 1:lOO) a t 4'C overnight and then treated according to the avidin-biotin (ABC) method (ABC Elite kit, Vector). Peroxidase activity was visualized by using diaminobenzidine. The reaction product was intensified with osmium tetroxide, after which the sections were dehydrated and mounted in DPX. The GABA antiserum (GABA-9) and glycine antiserum were gifts from Dr. P. Somogyi and Dr. R. Wenthold.

Immunohistochemical controls Control sections were treated with primary antiserum that had been incubated with GABA or glycine coupled to polyacrylamide gel (Ternyck and Avrameas, '72; Hodgson et al., '85). These adsorbed antisera were used at 1:40,000 (anti-GABA) or 1:lOO (antiglycine). In addition, in some cases the primary antiserum was replaced with nonimmune rabbit serum.

Analysis The proportions of immunoreactive cells in laminae I, 11, and TI1 with each antiserum were estimated from parasagittal sections of the dorsal horn. In sets of three consecutive semithin sections, the first and third were processed with the anti-GABA and antiglycine antisera; the second was stained with toluidine blue. On the second section the laminar boundaries were identified (Todd and McKenzie, '89) and the positions of all neuronal nuclei with visible nucleoli in laminae 1-111 were plotted with a camera lucida. The first and third sections were then used to determine the proportions of nuclei that immunoreacted with each antiserum (Woodson et al., '89). Over 1,000cells each in laminae I1 and I11 and over 200 cells in lamina I were counted. Series of semithin transverse sections, including one-half of the cord, were cut from 2 blocks each from material obtained from 4 rats and processed with the 2 antisera in order to compare the distribution of immunoreactivities throughout the grey and white matter. Semithin parasagittal sections through the somata of 18 Golgi-stained neurones in lamina 11, which had previously been drawn with a camera lucida, were tested for glycine-LI according to the method of Somogyi and Hodgson ('85).

RESULTS Laminae 1-111 In transverse sections treated with the antiglycine antiserum, small immunoreactive cell bodies were present in laminae I, 11, and I11 and were evenly distributed across the mediolateral extent of the dorsal horn. They were rare in

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lamina I, most common in lamina 111, and were outnumbered in all 3 laminae by nonimmunoreactive cell bodies (Fig. 1).In transverse sections it was usually not possible to see processes emerging from labelled perikarya. Punctate labelling of the neuropil was light in laminae I and I1 and heavy in lamina 111. In parasagittal sections many immunoreactive cell bodies in all 3 laminae were fusiform and sometimes had stained primary dendrites emerging from the soma. The staining with anti-GABA was similar to that previously described in semithin sections of the rat dorsal horn (Todd and McKenzie, '89), with many small immunoreactive cell bodies and heavy punctate staining of the neuropil. The numbers of neurones in each lamina that were immunoreactive with either anti-GABA, antiglycine, or both are shown in Table 1. With the anti-GABA antiserum the proportions of immunoreactive cells in laminae I, 11,and I11 were 28%, 31 57 , and 46% ; with the antiglycine serum the proportions were 9 % , 14% , and 30 % . A comparison of the pairs of semithin sections showed that, with the exception of 4 cells in lamina 111, all of the cells examined within these laminae that displayed glycine-LI were also reactive with anti-GABA. In lamina I, 33% of GABA immunoreactive cells were also immunoreactive with antiglycine antiserum; in laminae I1 and I11 the proportions rose to 43 % and 64%, respectively. Examples of immunoreactive cells within laminae I1 and I11 are shown in Figure 2. In order to examine the morphology of glycine immunoreactive neurones in lamina II,18 Golgi-stained cells that had

Fig. 1. A transverse semithin section of the dorsal horn treated with antiserum to glycine. The border between laminae I1 and I11 is shown as a dashed line. Immunoreactive cell bodies (some of which are indicated

TABLE 1. Counb of Immunoreadive Cells in Laminae I-III

Lamina I I1 1J.I

Cells

Percentage of GABA-LI cells that alsoshowed GLY-LI

Number of cells counted

GABA-LI cells (total)

GLY-LI cells (total)

withonly

238 1,256 1,017

66(28%) 394(31%) 467(46%)

22(9%) 170(14%) 301(30%)

010%)

33% 43%

4(0%)

64%

GLY-LI O(O%)

previously been shown to be GABA-immunoreactive (Todd and McKenzie, '89; Figs. 3-5) were tested with the antiglycine antiserum. These included typical islet cells as well as cells showing features that were not typical of islet cells (e.g., ventrally directed dendrites). Eight of the cells showed glycine-LI and one of these is illustrated in Figure 3 A-D. There was no obvious relation between the morphology of the cells and their reaction to the antiglycine antiserum, since some islet cells were immunoreactive, whereas others were not (Fig. 3E,F).

Remainder of grey matter Many immunoreactive cells were found in the grey matter ventral to lamina I11 with both antisera (Fig. 4), particularly in the medial parts of the deep dorsal horn and ventral horn. In general, with both antisera, immunoreactive cells were less intensely stained than in laminae 1-111, although some small cells around the central canal were strongly stained

with arrows) are common in laminae 111 and IV, where the punctate staining of the neuropil is also more dense. Scale bar = 100 pm.

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Fig. 2. Immunoreactive cells seen in parasagittal sections of laminae I1 and 111. A, B. One neurone showing GABA-LI (arrowhead) and another that reacted with both antisera (arrow) are seen lying close to a capillary (C) in lamina 11. Note the light staining of the neuropil with

antiglycine antiserum. C, D. Neurones reactive with anti-GABA (arrowhead) and with both antisera (arrow) within lamina I11 are illustrated. In both laminae some cells (asterisks) are not immunoreactive with either antiserum. Scale bar = 10 pm.

with the GABA antiserum (Fig. 4D). Some cells were immunoreactive with both antisera; others reacted only with antiglycine or with anti-GABA, and many cells did not react with either antiserum. Within the ventral horn, GABALI was found in small cells, some of which also showed glycine-LI (Fig. 4E,F). Many slightly larger cells reacted only with the glycine antiserum, whereas the largest cells (which were probably motorneurones) were not significantly immunoreactive. The distribution of immunoreactive cells whose nuclei were present in a single transverse semithin section of the L4 segment is shown in Figure 5. Glycine-LI within the neuropil was most abundant in laminae I11 and IV, but was densely distributed throughout the grey matter ventral to lamina 11. Many large immunoreactive axons coursed through the grey matter, particularly in the ventral horn. GABA-LI was concentrated in the superficial 3 laminae of the dorsal horn, present in moderate amounts in the medial part of the deep dorsal horn (in the area bordering the dorsal columns) and in the area around the central canal, and more sparsely distributed in the remainder of the dorsal horn and in the ventral horn. With both antisera punctate staining adjacent to neuronal cell bodies was seen. These puncta, which are presumably boutons, contacted neurones in both dorsal horn and ventral horn. In the ventral horn they were much more common in sections treated with the glycine antiserum and were present on both small and large neurones.

out the ventral and lateral funiculi and in Lissauer’s tract, but were much less frequent in the dorsal columns, except in their ventral part. Many large myelinated axons showed glycine-LI (Fig. 6A), and these were particularly numerous in the deeper parts of the ventral and lateral funiculi, adjacent to the grey matter. A few of these large axons were also immunoreactive with GABA antiserum (Fig. 6B). Small immunoreactive axons were found throughout the white matter. Many showed GABA-LI and some of these were also immunoreactive with antiglycine antiserum. These small axons were present in large numbers in the dorsal part of the lateral funiculus and in Lissauer’s tract.

White matter Many immunoreactive axons were present within the white matter with both antisera. These were found through-

Controls Pretreatment of the antisera with the corresponding amino acid coupled to polyacrylamide gel virtually abolished specific staining (Fig. 7A,C,D,F). The normal staining pattern was retained following treatment with the other amino acid (Fig. 7B,E); however, treatment of antiglycine with GABA resulted in a slight reduction of staining intensity (Fig. 7A,B). No specific staining was seen when nonimmune rabbit serum was substituted for the first antiserum.

DISCUSSION Coexistence of GABA- and glycine-like immunoreactivities The results presented here suggest that significant concentrations of GABA and glycine are both present within many neurones in the spinal cord and also in some axons in the

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GABA AND GLYCINE IN RAT SPINAL CORD

Fig. 4. These micrographs illustrate immunoreactive cells in deeper laminae. A,B.One cell in lamina IV displays onlyglycine-LI (arrowhead); another cell is also weakly reactive with anti-GABA (arrow). Other cells are not reactive with either antiserum (asterisks). C,D.In the region around the central canal (CC), 4 small cells show strong GABA-LI. One of these is weakly reactive with antiglycine (arrows); the other 3 are not

Fig. 3. The reaction of 2 Golgi-stained lamina I1 islet cells to antiglycine antiserum. The cell illustrated in A-D was immunoreactive. B shows a semithin section through the soma treated with the antiserum; the section shown in C was treated with antiserum that had been preadsorbed with glycine coupled to polyacrylamide heads. This abolished the immunostaining and rendered the gold deposit from the Golgi reaction more visible. D. A photomicrograph through the Golgi-stained cell body and primary dendrites taken before the neurone was sectioned. E. An islet cell that was not immunoreactive. F. A semithin section through the soma of the cell drawn in E does not react with the antiglycine antiserum. This cell was heavily stained with gold from the Golgi reaction and this can be seen within the cytoplasm, but the nucleus (n), which is devoid of gold, is clearly immunonegative. Scale bars for A and E = 100 pm; for B, C, D, and F = 10 pm.

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(arrowheads).E, F. In the ventral horn, 3 small cells are immunoreactive with anti-GABA. Two also show glycine-LI (arrows); the other does not (arrowhead). Note the large nonimmunoreactive neurones (asterisks) and the much higher density of staining of the neuropil with antiglycine antiserum. Scale bars for A-D = 20 pm; for E, F = 50 pm.

spinal white matter. I t is therefore necessary to exclude the possibility that the results are due to a cross reaction, with both antisera recognizing the same antigen. This is unlikely for a number of reasons. Both antisera have been extensively characterised and found to show considerable specificity in their reactions with fixed amino acids. The antiGABA serum does not cross-react with fixed glycine (Hodgson et al., '85; Ottersen et al., '88), whereas the antiglycine serum shows only minor reaction with conjugated GABA (Wenthold et al., '87). The slight reduction in staining seen when antiglycine antiserum was pretreated with GABA may result from this weak cross reaction. However, a similar result was found by Ottersen et al. ('88) and attributed t o a nonspecific interaction between antibod-

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A

0

A

A I A 8

I A

8

A A A

8

I 8

A

0

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A

A

Fig. 5. This drawing illustrates the positions of all of the immunoreactive cells whose nuclei appeared in a single semithin transverse section through one side of the L4 segment. Squares represent GABA immuno-

reactive cells, triangles glycine immunoreactive cells, and circles cells that were reactive with both antisera. The approximate positions of the laminar boundaries are shown.

ies and amino acid complexes. Because, in the present study, cell bodies or axons could be strongly immunoreactive with one antiserum and not reactive with the other, it is unliklely that major cross reaction was occurring in this material. The distributions of the two types of immunoreactivity within

the neuropil are in good agreement with the distribution of GABA- and glycine-containing axon terminals predicted using different methods. Thus immunostaining with antiserum to GAD is most intense in laminae 1-111 and present in moderate concentrations in the medial part of the deep

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Fig. 6. These micrographs illustrate immunoreactivity within the white matter. With the glycine antiserum many large myelinated immunoreactive axons are seen within the ventral funiculus, and some

of these also display GABA-LI (large arrows). Small axons with only GABA-LI (arrowheads) or with both GABA- and glycine-LI (small arrows) are also seen. Scale bar = 20 sm.

dorsal horn (McLaughlin et al., '75), which matches the distribution of GABA-LI in the neuropil seen here, whereas the staining seen after the application of monoclonal antibodies to glycine receptor is weak in laminae I and I1 but present throughout the remainder of the grey matter (van den Pol and Gorcs, '88) with the highest concentration in laminae I11 and IV (Basbaum, '88),which is very similar to the distribution of glycine-LI found in the present study. The distribution of immunoreactive cells reported here is similar to that described in studies using antisera to GABA and glycine from different sources (Campistron et al., '86; Magoul et al., '87; Ottersen and Storm-Mathisen, '87; van den Pol and Gorcs, '88); the distribution of glycine immunoreactive cells within laminae 1-111 matches the distribution of cells that

take up tritiated glycine applied to the cord dorsum in vivo (Ribeiro-da-Silva and Coimbra, '80).

Fig. 7. These micrographs show the effect of pretreating the antisera with GABA or glycine coupled to polyacrylamide beads. A-C. Three serial sections treated with antiglycine antiserum. In A (treated with untreated antiserum) an immunoreactive neurone in lamina I1 is seen (arrow). B. Treatment of the antiserum with GABA results in a reduction of staining intensity, but the neurone is still clearly visible. C.

Treatment with glycine results in almost complete loss of staining. D-F. Three serial sections treated with anti-GABA antiserum. Two immunoreactive neurones (arrows) are seen after treatment with untreated anti-GABA (D) or anti-GABA pretreated with glycine (E). F. Pretreatment of the antiserum with GABA virtually abolishes staining. Scale bar = 10 sm.

GABA and glycine within laminae 1-111 Todd and McKenzie ('89), using the peroxidase antiperoxidase (PAP) method, found that many cells in laminae 1-111 displayed GABA-LI and that the population of immunoreactive neurones in lamina I1 included islet cells but not stalked cells. The proportions of GABA-immunoreactive cells in laminae I and I1 found in that study (24% and 29%, respectively) are close to those obtained in the present experiments; however, Todd and McKenzie ('89) found that only 33% of lamina I11 cells were immunoreactive, instead of the 46% seen here. This difference almost certainly

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reflects the greatly improved sensitivity and specificity of the avidin-biotin method. Many of the GABA-immunoreactive cells in lamina I11 in this study were of low or intermediate staining intensity (although still being clearly distinct from nonimmunoreactive cells) and may not have been seen using the less sensitive PAP technique. The presence of few glycine immunoreactive cells in laminae I and I1 and many in lamina I11 is in agreement with previous reports (Ribeiro-da-Silva and Coimbra, '80; van den Pol and Gorcs, '88), and a t least some of the immunoreactive cells in lamina I1 can be identified as islet cells. An unexpected finding of the present study was that essentially all of the glycine immunoreactive cells in the superficial 3 laminae also showed GABA-LI. This suggests that many of the neurones with somata in laminae I, 11, and I11 are inhibitory interneurones containing GABA, with or without glycine. There are 2 reasons that suggest that these cells may be the major source of the immunoreactive axon terminals within the superficial dorsal horn. Within each lamina the density of staining within the neuropil with each antiserum matched the frequency of immunoreactive cell bodies, and in addition, some of the immunoreactive neurones were islet cells whose axons arborize locally (Gobel, '78; Todd, '88). It is not yet known whether the 2 types of immunoreactivity coexist within some axon terminals in the dorsal horn, as has been proposed for the ventral horn (Triller et al., '87); however, this is likely to be the case, because some islet cells are immunoreactive with both antisera. The inhibitory interneurones within laminae 1-111 are thought to be important in controlling the flow of sensory information through the dorsal horn (Wall, '80) and may have a specific role in regulating the primary afferent input to cells in deeper laminae (Todd, '89). The neurotransmitters contained within the cells of laminae 1-111 that were not immunoreactive in the present study cannot yet be definitely identified. Acetylcholine is probably present in some lamina I11 cells (Barber et al., '84), but it may coexist with GABA in some of these (Kosaka et al., '88). Many cells in laminae I and I1 accumulate tritiated Daspartate applied to the dorsal horn (Rustioni and Cuenod, '82), and in addition many are immunoreactive with antiserum to fixed glutamate (Miller et al., '88), suggesting that there are excitatory interneurones using glutamate and/or aspartate within these laminae. It remains to be seen whether these cells form a different (and complementary) population to those that were immunoreactive in the present study. If this is the case, it would provide support for the suggestion by Gobel ('78) that lamina I1 islet cells are inhibitory (using GABA with or without glycine) and stalked cells excitatory (using glutamate and/or aspartate).

ACKNOWLEDGMENTS We thank Drs. P. Somogyi and R. Wenthold for the generous gifts of anti-GABA and antiglycine antisera. We are grateful to Miss M. Hughes, Miss C. Morris, and Mr. R. Kerr for technical assistance.

LITERATURE CITED Aanonsen, L.M., and G.L. Wilcox (1989) Muscimol, gamma-aminobutyric acid A receptors and excitatory amino acids in the mouse spinal cord. J. Pharmacol. Exp. Ther. 248:1034-1038. Barber, R.P., J.E. Vaughn, and E. Roberts (1982) The cytoarchitecture of GABAergic neurons in rat spinal cord. Brain Res. 238.305-328.

Barber, R.P., J.E. Vaughn, K. Saito, B.J. McLaughlin, and E. Roberts (1978) GABAergic terminals are presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord. Brain Res. 141.35-55. Barber, R.P., P.E. Phelps, C.R. Houser, G.D. Crawford, P.M. Salvaterra, and J.E. Vaughn (1984) The morphology and distribution of neurons containing choline acetyl transferase in the adult rat spinal cord: An immunocytochemical study. J. Comp. Neurol. 229:329-346. Basbaum, A.I. (1988) Distribution of glycine receptor immunoreactivity in the spinal cord of the rat: Cytochemical evidence for a differential glycinergic control of lamina I and lamina V nociceptive neurons. J. Comp. Neurol. 278t330-336. Campistron, G., R.M. Buijs, and M. Geffard (1986) Glycine neurons in the brain and spinal cord. Antibody production and immunocytochemical localization. Brain Res. 376:400405. Cutting, D.A., and C.C. Jordan (1975) Alternative approaches to analgesia: Baclofen as a model compound. Brit. J. Pharmacol. 54:171-179. Game, C.J.A., and D. Lodge (1975) The pharmacology of the inhibition of dorsal horn neurones by impulses in myelinated cutaneous afferents in the cat. Exp. Brain Res. 23:75-84. Gobel, S. (1978) Golgi studies of the neurons in layer I1 of the dorsal horn of the medulla (trigeminal nucleus caudalis). J. Comp. Neurol. 180:395-414. Hodgson, A.J., B. Penke, A. Erdei, I.W. Chubh, and P. Somogyi (1985) Antisera to y-aminobutyric acid. I. Production and characterization using a new model system. J. Histochem. Cytochem. 33.929-239. Hunt, S.P., J.S. Kelly, P.C. Emson, J.R. Kimmel, R.J. Miller, and J-Y. Wu (1981) An immunohistochemical study of neuronal populations containing neuropeptides or a-aminobutyrate within the superficial layers of the rat dorsal horn. Neuroscience 6:1883-1898. Hwang, AS., and G.L. Wilcox (1989) Baclofen, gamma-aminobutyric acid B receptors and substance P in the mouse spinal cord. J. Pharmacol. Exp. Ther. 248:1026-1033. Kosaka, T., M. Tauchi, and J.L. Dahl (1988) Cholinergic neurons containing GABA-like and/or glutamic acid decarboxylase-like immunoreactivities in various brain regions of the rat. Exp. Brain Res. 70:605-617. McLaughlin, B.J., R. Barber, K. Saito, E. Roberts, and J-Y. Wu (1975) Immunocytochemical localization of glutamate decarboxylase in rat spinal cord. J. Comp. Neurol. I64:305-322. Magoul, R., B. Onteniente, M. Geffard, and A. Calas (1987) Anatomical distribution and ultrastructural organization of the GABAergic system in the rat spinal cord. An immunocytochemical study using anti-GABA antibodies. Neuroscience 2O:lOOl-1009. Miller, K.E., J.R. Clements, A.A. Larson, and A.J. Beitz (1988) Organization of glutamate-like immunoreactivity in the rat superficial dorsal horn: Light and electron microscopic observations. Synapse 2:2%36. Nistri, A. (1983) Spinal cord pharmacology of GABA and chemically related amino acids. In R.A. Davidoff (ed): Handbook of the Spinal Cord, Vol. 1. New York: Marcel Dekker, pp. 45-104. Ottersen, O.P., and J. Storm-Mathisen (1987) Distribution of inhibitory amino acid neurons in the cerebellum with some observations on the spinal cord An immunocytochemical study with antisera against fixed GABA, glycine, taurine, and 0-alanine. J. Mind. Behav. 8~503-518. Ottersen, O.P., J. Storm-Mathisen, and P. Somogyi (1988) Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: A postembedding light and electron microscope study. Brain Res. 450t342-353. Pfeiffer, F., R. Simler, G. Grenningloh, and H. Betz (1984) Monoclonal antibodies and peptide mapping reveal structural similarities between the subunits of the glycine receptor of rat spinal cord. Proc. Natl. Acad. Sci. USA 81:7224-7227. Ribeiro-da-Silva, A,, and A. Coimbra (1980) Neuronal uptake of [3H]-GABA and F3H]-g1ycine in laminae 1-111 (substantia gelatinosa Rolandi) of the rat spinal cord. An autoradiographic study. Brain Res. 188:449-464. Rustioni, A., and M. Cuenod (1982) Selective retrograde transport of D-aspartate in spinal interneurons and cortical neurons of rats. Brain Res. 236:143-155. Somogyi, P., and A.J. Hodgson (1985) Antisera to y-aminobutyric acid. 111. Demonstration of GABA in Golgi-impregnated neurons and in conventional electron microscope sections of cat striate cortex. J. Histochem. Cytochem. 33:249-257. Somogyi, P., A.J. Hodgson, I.W. Chubb, B. Penke, and A. Erdei (1985) Antisera to y-aminobutyric acid. 11. Immunocytochemical application to the central nervous system. J. Histochem. Cytochem. 33.240-248. Ternyck, T., and S. Avrameas (1972) Polyacrylamide-protein immunoadsorbents prepared with glutaraldehyde. FEBS Lett. 23:24-28.

GABA A N D GLYCINE I N RAT SPINAL CORD Todd, A.J. (1988) Electron microscope study of Golgi-stained cells in lamina I1 of the rat spinal dorsal horn. J. Comp. Neurol. 275:145-157. Todd, A.J. (1989) Cells in laminae 111and IV of rat spinal dorsal horn receive monosynaptic primary afferent input in lamina 11. J. Comp. Neurol. 289:676-686. Todd, A.J., and J. McKenzie (1989) GABA-immunoreactive neurones in the dorsal horn of the rat spinal cord. Neuroscience 31:799-806. Triller, A., F. Cluzeaud, and H. Korn (1987) GABA-containing terminals can be apposed to glycine receptors at central synapses. J. Cell Biol. 104:947956. van den Pol, A,, and T. Gorcs (1988) Glycine and glycine-receptor immunoreactivity in brain and spinal cord. J. Neurosci. 8:472-492. Wall, P.D. (1980) The substantia gelatinosa. A gate control mechanism set across a sensory pathway. Trends in Neurosciences 3:221-224.

505 Wenthold, R.J., D. Huie, R.A. Altschuler, and K.A. Reeks (1987) Glycine immunoreactivity localized in the cochlear nucleus and superior olivary complex. Neuroscience 22897-912. Wilson, P.R., and T.L. Yaksh (1978) Baclofen is antinociceptive in the spinal intrathecal space of animals. Eur. J. Pharmacol. 51:323-330. Woodson, W., L. Nitecka, and Y. Ben-Ari (1989) Organization of the GABAergic system in the rat hippocampal formation: A quantitative immunocytochemical study. J. Comp. Neurol. 280.254-271. Yaksh, T.L. (1989) Behavioral and autonomic correlates of the tactile evoked allodynia produced by spinal glycine inhibition: Effects of modulatory receptor systems and excitatory amino acid antagonists. Pain 37:lll-123. Young A.B., and R.L. Macdonald (1983) Glycine as a spinal cord neurotransmitter. In R.A. Davidoff (ed): Handbook of the Spinal Cord, Vol. 1. New York Marcel Dekker, pp. 1 4 4 .

Light microscope study of the coexistence of GABA-like and glycine-like immunoreactivities in the spinal cord of the rat.

The distributions of GABA-like and glycine-like immunoreactivities in the rat spinal cord were compared by using postembedding immunohistochemistry on...
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