Brain Research, 514 (1990) 171-174 Elsevier
171
BRES 24049
GABA-like immunoreactivity in type I glomeruli of rat substantia gelatinosa Andrew J. Todd and Valerie Lochhead Department of Anatomy, Universityof Glasgow, Glasgow (U.K.) (Accepted 2 January 1990)
Key words: ~-Aminobutyric acid; Presynaptic dendrite; Lamina II; Spinal cord; Presynaptic inhibition; Ultrastructure
A postembedding immunogold study of type I synaptic glomeruli in lamina II of rat dorsal horn was carried out using antiserum to y-aminobutyric acid (GABA). Gold particles were concentrated over some peripheral axons and vesicle-containing dendrites within these glomeruli and both types of profile were presynaptic to central axons. These results suggest that GABA is involved in presynaptic inhibition of unmyelinated primary afferents and is released by some presynaptic dendrites within lamina II.
Immunocytochemical studies using antisera directed against glutamic acid decarboxylase ( G A D ) or against conjugates of ~-aminobutyric acid ( G A B A ) have shown that G A B A is present in cell bodies and axon terminals in the dorsal horn of the rat spinal cord, and is concentrated in laminae I - I I I 1'2'9'11-13'24. Within this area, immunoreactive axons form symmetric synapses with dendrites, somata and other axons. It has been shown that primary afferent axons in laminae I - I I I (identified by dorsal root section) are postsynaptic to GAD-immunoreactive axons at axoaxonic synapses2; however, in that study it was not possible to recognize the types of degenerating primary afferent axon which were involved in such arrangements. In particular, it is not known whether unmyelinated primary afferents (which terminate mainly in laminae I and 11)21 are postsynaptic to GABAergic axons. Recently, we have shown that many islet cells within lamina II show GABA-Iike immunoreactivity (GABA-LI) 24. Islet cells are known to possess presynaptic dendrites 7'23 and yet GABA-immunoreactive presynaptic dendrites do not appear to have been identified within the superficial dorsal horn. Two types of synaptic glomeruli are present in lamina II of rat dorsal horn 16 and involve primary afferent axons 4. In both types peripheral axons and dendrites with and without vesicles are present. The central axons of type I glomeruli are almost certainly derived from unmyelinated afferents, since they are largely absent from animals treated with capsaicin as neonates 17. We have therefore carried out a postembedding immunocytochemical study of type I glomeruli in rat, using
antiserum to G A B A , in order to determine whether unmyelinated primary afferents are postsynaptic to GABAergic axons and also whether GABA-immunoreactive presynaptic dendrites are present in lamina II. Five male albino Swiss rats, aged between 9 and 12 weeks, were deeply anesthetised with pentobarbitone and fixed by intracardiac perfusion with 2.5% glutaraldehyde/l% formaldehyde in 0.1 M phosphate buffer. Lumbar spinal cord segments were removed, postfixed in 1% osmium tetroxide, block-stained in uranyl acetate, dehydrated in alcohol and embedded in Araldite or Durcupan. Ultrathin transverse sections of dorsal horn were cut and collected on uncoated nickel grids (300 mesh). The sections were reacted according to the method of Somogyi and Hodgson 19'2°. Briefly they were etched with 1% periodic acid (4-7 min) and treated with 1% sodium metaperiodate (4-8 min) to remove osmium. Non-specific reaction was blocked with 5% normal goat serum (NGS), and the sections were then incubated for 1.5-2 h in anti-GABA (diluted to 1:1000 or 1:2000 in 1% NGS) and for 1.5-2 h in goat anti-rabbit IgG coupled to colloidal gold (10 nm or 15 nm, Seralab; diluted 1:10 or 1:20 in 0.5% polyethylene glycol in Tris buffer). The grids were rinsed between each step and washed after the reaction. The sections were then stained with lead citrate and viewed with the electron microscope. The G A B A antiserum (GABA-98) was a gift from Dr. P. Somogyi. Control sections were reacted in the same way, but the primary antiserum was replaced by antiserum which had been incubated with G A B A coupled to polyacrylamide gel z2. This treatment abolished specific immunostaining.
Correspondence: A.J. Todd, Department of Anatomy, University of Glasgow, Glasgow G12 8QQ, U.K. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
172 Type I synaptic glomeruli, with dark central axons containing vesicles of varying size and few mitochondria, were present in lamina 1116. They were surrounded by various profiles, some of which contained vesicles. These could usually be identified as axons, with numerous small agranular vesicles, which were often oval or flattened, or as vesicle-containing dendrites, which usually had fewer vesicles, often contained stacks of smooth endoplasmic reticulum and occasionally possessed ribosomes 5'16"1s. Gold particles were concentrated over many of the peripheral vesicle-containing profiles in type I glomeruli, but never over the central axons (Figs. 1-3). In labelled profiles they were associated with agranular vesicles and mitochondria. Some of the immunoreactive profiles identified as vesicle-containing dendrites were postsynaptic to the central axon at asymmetric synapses (Fig. 2). Since ribosomes are rare in the peripheral profiles of type I glomeruli 5 and peripheral axons are not postsynaptic to the central axons 16'18, this was found to be the best criterion for definitively identifying immunoreactive vesicle-containing dendrites. Immunoreactive profiles of both types were presynaptic to central axons (Figs. 1 and 3) and to dendrites without vesicles (Fig. 2) at symmetric synapses. One example of a reciprocal axodendritic
synapse involving a labelled dendrite was observed (Fig. 2) and this dendrite also took part in a synaptic triad. Dendrites which did not contain vesicles were seldom labelled. A quantitative study of 45 type I glomeruli (15 each from 3 animals) was made. Each of these was photographed in 2 adjacent sections and from the micrographs, the peripheral profiles around each glomerulus were identified and any synaptic contacts noted. The densities of gold grains over the central axon and each peripheral profile were then determined using a Bioquant image analysis system. Profiles for which the density of gold grains exceeded that over the central axon (which was taken as the background density) by more than 4 times on both of the serial sections were counted as immunoreactive. A total of 237 peripheral profiles was examined in the 45 glomeruli. Of these, 85 were identified as vesicle-containing dendrites and 33 as axons. Twentynine (34%) of the vesicle-containing dendrites and 26 (79%) of the axons were found to be immunoreactive using these criteria. Nine of the immunoreactive profiles classified as vesicle-containing dendrites were postsynaptic to the central axon, confirming their identity as dendrites. Eight of the axons and 5 of the dendrites were
Fig. 1. A central axon (C) is postsynaptic to a gold-labelled axon (a) at a symmetric synapse (between arrowheads). The central axon is also presynaptic to two dendrites (d) at asymmetric synapses. Bar = 0.5 pm. Fig. 2. a: an immunoreactive vesicle-containing dendrite (v) is postsynaptic to a central axon (C) and presynaptic to a dendrite (d) which is itself postsynaptic to the central axon, thus forming a synaptic triad, b: in an adjacent section the asymmetric axodendritic synapse (between arrowheads)-and the symmetric dendrodendritic synapse (arrow) are clearly seen. In addition, there is a cluster of vesicles in the dendrite (v) and a reciprocal dendroaxonic synapse is formed (curved arrow). Bars = 0.5 pm.
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Fig. 3. A central axon (C) is postsynaptic at a symmetric synapse (between arrowheads) to an immunoreactive profile (v) which contains a cluster of vesicles and two sacs of smooth endoplasmic reticulum (SER). This is therefore identified as a dendroaxonic synapse. A nearby axon (a) contains more synaptic vesicles and a higher density of gold grains. The inset shows the same section tilted and at higher magnification: the synaptic specialization is seen and the sacs of SER (arrows) are clearly visible. Bar = 0.5/tm.
difficult to distinguish b e t w e e n axons and vesicle-containing dendrites in single sections. In this study the identification of presynaptic dendrites was possible because in type I synaptic glomeruli of the rat, only dendrites a p p e a r to be postsynaptic to the central axon at asymmetric synapses 16'18. Occasional asymmetric axoaxonic synapses have been r e p o r t e d in lamina II of m o n k e y dorsal horn3'1°; however, in a serial section analysis no examples of such synapses were seen in synaptic glomeruli involving ' d a r k sinuous axon' terminals, which are thought to be the equivalent of type I glomeruli in the rat 1°. A n o t h e r reason why i m m u n o r e activity in presynaptic dendrites m a y have been missed is that since these dendrites usually contain fewer synaptic vesicles than axonal boutons, they m a y have a lower concentration of G A B A (and also of G A D ) . In the present material, the density of gold grains over vesiclecontaining dendrites was often quite low and the proportion which are i m m u n o r e a c t i v e m a y therefore have been u n d e r e s t i m a t e d . This study also provides evidence that some unmyelinated p r i m a r y afferent terminals are subject to presynaptic inhibition at G A B A e r g i c axoaxonic and dendroaxonic synapses. T h e r e is a l r e a d y indirect evidence suggesting that these terminals are postsynaptic at G A B A e r g i c synapses: Ribeiro-da-Silva and C o i m b r a 15 showed that tritiated G A B A a p p l i e d to the cord dorsum was c o n c e n t r a t e d in the p e r i p h e r a l axons of glomeruli in laminae II and III, and some of these a p p e a r to be of the type I, which they subsequently described 16. In addition, G A B A B - b i n d i n g sites are dense in lamina II of rat dorsal horn and are r e d u c e d by 4 0 - 5 0 % after administration of capsaicin to neonates 14. This suggests that some of the G A B A e r g i c profiles r e l a t e d to u n m y e l i n a t e d p r i m a r y afferents m a y be associated with G A B A B receptors and this m a y be i m p o r t a n t in explaining the analgesic actions of the G A B A B agonist baclofen 6.
presynaptic to the central axon at symmetric synapses. T h e s e results strongly suggest that G A B A is contained in some presynaptic dendrites and released at dendrodendritic and d e n d r o a x o n i c synapses within lamina II. This is not surprising, since islet cells in this lamina show GABA-L124 and possess presynaptic dendritesT'23; however, a p a r t from a single r e p o r t that presumptive G A B A ergic dendrites were present in synaptic glomeruli 13, this a p p e a r s to be the first description of G A B A - i m m u n o r e active presynaptic dendrites. O n e possible reason why these have not previously been r e p o r t e d is that it is often
We are very grateful to Dr. P. Somogyi for providing the anti-GABA antiserum and to Miss M. Hughes, Miss C. Morris and Mr. R. Kerr for technical and photographic assistance. This work was supported by the Scottish Home and Health Department and by the Medical Research Funds of the University of Glasgow.
1 Barber, R.P., Vaughn, J.E. and Roberts, E., The cytoarchitecture of GABAergic neurons in rat spinal cord, Brain Research, 238 (1982) 305-328. 2 Barber, R.P., Vaughn, J.E., Saito, K., McLaughlin, B.J. and Roberts, E., GABAergic terminals are presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord, Brain Research, 141 (1978) 35-55. 3 Carlton, S.M., McNeill, D.L., Chung, K. and CoggeshaU, R.E., Organization of calcitonin gene-related peptide-immunoreactive terminals in the primate dorsal horn, J. Comp. Neurol., 276 (1988) 527-536. 4 Coimbra, A., Ribeiro-da-Silva, A. and Pignatelli, D., Effects of dorsal rhizotomy on the several types of primary afferent
terminals in laminae I-III of the rat spinal cord, Anat. EmbryoL, 170 (1984) 279-287. 5 Coimbra, A., Sodr6-Borges, B.P. and Magalhaes, M.M., The substantia gelatinosa Rolandi of the rat. Fine structure, cytochemistry (acid phosphatase) and changes after dorsal root section, J. Neurocytol., 3 (1974) 199-217. 6 Cutting, D.A. and Jordan, C.C., Alternative approaches to analgesia: baclofen as a model compound, Br. J. Pharmacol., 54 (1975) 171-179. 7 Gobel, S., Falls, W.M., Bennett, G.J., Abdelmoumene, M., Hayashi, H. and Humphrey, E., An EM analysis of the synaptic connections of horseradish peroxidase-filled stalked cells and islet cells of the substantia gelatinosa of adult cat spinal cord, J.
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Comp. Neurol., 194 (1980) 781-807. 8 Hodgson, A.J., Penke, B., Erdei, A., Chubb, I.W. and Somogyi, P., Antisera to y-aminobutyric acid. I. Production and characterization using a new model system, J. Histochem. Cytochem., 33 (1985) 229-239. 9 Hunt, S.P., Kelly, J.S., Emson, EC., Kimmel, J.R., Miller, R.J. and Wu, J.-Y., An immunohistochemical study of neuronal populations containing neuropeptides or ~,-aminobutyrate within the superficial layers of the rat dorsal horn, Neuroscience, 6 (1981) 1883-1898. 10 Knyihar-Csillik, E., Csillik, B. and Rakic, P., Periterminal synaptology of dorsal root glomerular terminals in the substantia gelatinosa of the spinal cord in the rhesus monkey, J. Comp. Neurol., 210 (1982) 376-399. 11 Magoul, R., Onteniente, B., Geffard, M. and Calas, A., Anatomical distribution and ultrastructural organization of the GABAergic system in the rat spinal cord. An immunocytochemicai study using anti-GABA antibodies, Neuroscience, 20 (1987) 1001-1009. 12 McLaughlin, B.J., Barber, R., Saito, K., Roberts, E. and Wu, J.-Y., Immunocytochemical localization of glutamate decarboxylase in rat spinal cord, J. Comp. Neurol., 164 (1975) 305-322. 13 Merighi, A., Polak, J.M., Fumagalli, G. and Theodosis, D.T., Ultrastructural localization of neuropeptides and GABA in rat dorsal horn: a comparison of different immunogold labeling techniques, J. Histochem. Cytochem., 37 (1989) 529-540. 14 Price, G.W., Wilkin, G.P., Turnbull, M.J. and Bowery, N.G., Are baclofen-sensitive GABA B receptors present on primary afferent terminals of the spinal cord?, Nature (Lond.), 307 (1984) 71-74. 15 Ribeiro-da-Silva, A. and Coimbra, A., Neuronal uptake of [3H]GABA and [3H]glycine in laminae I-III (substantia gelati-
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nosa Rolandi) of the rat spinal cord. An autoradiographic study, Brain Research, 188 (1980) 449-464. Ribeiro-da-Silva, A. and Coimbra, A., Two types of synaptic glomeruli and their distribution in laminae I-III of the rat spinal cord, J. Comp. Neurol., 209 (1982) 176-186. Ribeiro-da-Silva, A and Coimbra, A., Capsaicin causes selective damage to type I synaptic glomeruli in rat substantia gelatinosa, Brain Research, 290 (1984) 380-383. Ribeiro-da-Silva, A., Pignatelli, D. and Coimbra, A., Synaptic architecture of glomeruli in superficial dorsal horn of rat spinal cord, as shown in serial reconstructions, J. Neurocytol., 14 (1985) 203-220. Somogyi, P. and Hodgson, A.J., Antisera to 7-aminobutyric acid. III. Demonstration of G A B A in Go|gi-impregnated neurons and in conventional electron microscope sections in cat striate cortex, J. Histochem. Cytochem., 33 (1985) 249-257. Somogyi, P. and Soltesz, I., Immunogold demonstration of GABA in synaptic terminals of intracellularly recorded, horseradish peroxidase-filled basket cells and clutch cells in the cat's visual cortex, Neuroscience, 19 (1986) 1051-1065. Sugiura, Y., Lee, C.L. and Perl, E.R., Central projections of unmyelinated (C) afferent fibres innervating mammalian skin, Science, 234 (1987) 358-361. Ternyck, T. and Avrameas, S., Polyacrylamide-protein immunoadsorbents prepared with glutaraldehyde, FEBS Left., 23 (1972) 24-28. Todd, A.J., Electron microscope study of Golgi-stained cells in lamina II of the rat spinal dorsal horn, J. Comp. Neurol., 275 (1988) 145-157. Todd, A.J. and McKenzie, J., GABA-immunoreactive neurons in the dorsal horn of the rat spinal cord, Neuroscience, 31 (1989) 799-806.
171
BRES 24049
GABA-like immunoreactivity in type I glomeruli of rat substantia gelatinosa Andrew J. Todd and Valerie Lochhead Department of Anatomy, Universityof Glasgow, Glasgow (U.K.) (Accepted 2 January 1990)
Key words: ~-Aminobutyric acid; Presynaptic dendrite; Lamina II; Spinal cord; Presynaptic inhibition; Ultrastructure
A postembedding immunogold study of type I synaptic glomeruli in lamina II of rat dorsal horn was carried out using antiserum to y-aminobutyric acid (GABA). Gold particles were concentrated over some peripheral axons and vesicle-containing dendrites within these glomeruli and both types of profile were presynaptic to central axons. These results suggest that GABA is involved in presynaptic inhibition of unmyelinated primary afferents and is released by some presynaptic dendrites within lamina II.
Immunocytochemical studies using antisera directed against glutamic acid decarboxylase ( G A D ) or against conjugates of ~-aminobutyric acid ( G A B A ) have shown that G A B A is present in cell bodies and axon terminals in the dorsal horn of the rat spinal cord, and is concentrated in laminae I - I I I 1'2'9'11-13'24. Within this area, immunoreactive axons form symmetric synapses with dendrites, somata and other axons. It has been shown that primary afferent axons in laminae I - I I I (identified by dorsal root section) are postsynaptic to GAD-immunoreactive axons at axoaxonic synapses2; however, in that study it was not possible to recognize the types of degenerating primary afferent axon which were involved in such arrangements. In particular, it is not known whether unmyelinated primary afferents (which terminate mainly in laminae I and 11)21 are postsynaptic to GABAergic axons. Recently, we have shown that many islet cells within lamina II show GABA-Iike immunoreactivity (GABA-LI) 24. Islet cells are known to possess presynaptic dendrites 7'23 and yet GABA-immunoreactive presynaptic dendrites do not appear to have been identified within the superficial dorsal horn. Two types of synaptic glomeruli are present in lamina II of rat dorsal horn 16 and involve primary afferent axons 4. In both types peripheral axons and dendrites with and without vesicles are present. The central axons of type I glomeruli are almost certainly derived from unmyelinated afferents, since they are largely absent from animals treated with capsaicin as neonates 17. We have therefore carried out a postembedding immunocytochemical study of type I glomeruli in rat, using
antiserum to G A B A , in order to determine whether unmyelinated primary afferents are postsynaptic to GABAergic axons and also whether GABA-immunoreactive presynaptic dendrites are present in lamina II. Five male albino Swiss rats, aged between 9 and 12 weeks, were deeply anesthetised with pentobarbitone and fixed by intracardiac perfusion with 2.5% glutaraldehyde/l% formaldehyde in 0.1 M phosphate buffer. Lumbar spinal cord segments were removed, postfixed in 1% osmium tetroxide, block-stained in uranyl acetate, dehydrated in alcohol and embedded in Araldite or Durcupan. Ultrathin transverse sections of dorsal horn were cut and collected on uncoated nickel grids (300 mesh). The sections were reacted according to the method of Somogyi and Hodgson 19'2°. Briefly they were etched with 1% periodic acid (4-7 min) and treated with 1% sodium metaperiodate (4-8 min) to remove osmium. Non-specific reaction was blocked with 5% normal goat serum (NGS), and the sections were then incubated for 1.5-2 h in anti-GABA (diluted to 1:1000 or 1:2000 in 1% NGS) and for 1.5-2 h in goat anti-rabbit IgG coupled to colloidal gold (10 nm or 15 nm, Seralab; diluted 1:10 or 1:20 in 0.5% polyethylene glycol in Tris buffer). The grids were rinsed between each step and washed after the reaction. The sections were then stained with lead citrate and viewed with the electron microscope. The G A B A antiserum (GABA-98) was a gift from Dr. P. Somogyi. Control sections were reacted in the same way, but the primary antiserum was replaced by antiserum which had been incubated with G A B A coupled to polyacrylamide gel z2. This treatment abolished specific immunostaining.
Correspondence: A.J. Todd, Department of Anatomy, University of Glasgow, Glasgow G12 8QQ, U.K. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
172 Type I synaptic glomeruli, with dark central axons containing vesicles of varying size and few mitochondria, were present in lamina 1116. They were surrounded by various profiles, some of which contained vesicles. These could usually be identified as axons, with numerous small agranular vesicles, which were often oval or flattened, or as vesicle-containing dendrites, which usually had fewer vesicles, often contained stacks of smooth endoplasmic reticulum and occasionally possessed ribosomes 5'16"1s. Gold particles were concentrated over many of the peripheral vesicle-containing profiles in type I glomeruli, but never over the central axons (Figs. 1-3). In labelled profiles they were associated with agranular vesicles and mitochondria. Some of the immunoreactive profiles identified as vesicle-containing dendrites were postsynaptic to the central axon at asymmetric synapses (Fig. 2). Since ribosomes are rare in the peripheral profiles of type I glomeruli 5 and peripheral axons are not postsynaptic to the central axons 16'18, this was found to be the best criterion for definitively identifying immunoreactive vesicle-containing dendrites. Immunoreactive profiles of both types were presynaptic to central axons (Figs. 1 and 3) and to dendrites without vesicles (Fig. 2) at symmetric synapses. One example of a reciprocal axodendritic
synapse involving a labelled dendrite was observed (Fig. 2) and this dendrite also took part in a synaptic triad. Dendrites which did not contain vesicles were seldom labelled. A quantitative study of 45 type I glomeruli (15 each from 3 animals) was made. Each of these was photographed in 2 adjacent sections and from the micrographs, the peripheral profiles around each glomerulus were identified and any synaptic contacts noted. The densities of gold grains over the central axon and each peripheral profile were then determined using a Bioquant image analysis system. Profiles for which the density of gold grains exceeded that over the central axon (which was taken as the background density) by more than 4 times on both of the serial sections were counted as immunoreactive. A total of 237 peripheral profiles was examined in the 45 glomeruli. Of these, 85 were identified as vesicle-containing dendrites and 33 as axons. Twentynine (34%) of the vesicle-containing dendrites and 26 (79%) of the axons were found to be immunoreactive using these criteria. Nine of the immunoreactive profiles classified as vesicle-containing dendrites were postsynaptic to the central axon, confirming their identity as dendrites. Eight of the axons and 5 of the dendrites were
Fig. 1. A central axon (C) is postsynaptic to a gold-labelled axon (a) at a symmetric synapse (between arrowheads). The central axon is also presynaptic to two dendrites (d) at asymmetric synapses. Bar = 0.5 pm. Fig. 2. a: an immunoreactive vesicle-containing dendrite (v) is postsynaptic to a central axon (C) and presynaptic to a dendrite (d) which is itself postsynaptic to the central axon, thus forming a synaptic triad, b: in an adjacent section the asymmetric axodendritic synapse (between arrowheads)-and the symmetric dendrodendritic synapse (arrow) are clearly seen. In addition, there is a cluster of vesicles in the dendrite (v) and a reciprocal dendroaxonic synapse is formed (curved arrow). Bars = 0.5 pm.
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Fig. 3. A central axon (C) is postsynaptic at a symmetric synapse (between arrowheads) to an immunoreactive profile (v) which contains a cluster of vesicles and two sacs of smooth endoplasmic reticulum (SER). This is therefore identified as a dendroaxonic synapse. A nearby axon (a) contains more synaptic vesicles and a higher density of gold grains. The inset shows the same section tilted and at higher magnification: the synaptic specialization is seen and the sacs of SER (arrows) are clearly visible. Bar = 0.5/tm.
difficult to distinguish b e t w e e n axons and vesicle-containing dendrites in single sections. In this study the identification of presynaptic dendrites was possible because in type I synaptic glomeruli of the rat, only dendrites a p p e a r to be postsynaptic to the central axon at asymmetric synapses 16'18. Occasional asymmetric axoaxonic synapses have been r e p o r t e d in lamina II of m o n k e y dorsal horn3'1°; however, in a serial section analysis no examples of such synapses were seen in synaptic glomeruli involving ' d a r k sinuous axon' terminals, which are thought to be the equivalent of type I glomeruli in the rat 1°. A n o t h e r reason why i m m u n o r e activity in presynaptic dendrites m a y have been missed is that since these dendrites usually contain fewer synaptic vesicles than axonal boutons, they m a y have a lower concentration of G A B A (and also of G A D ) . In the present material, the density of gold grains over vesiclecontaining dendrites was often quite low and the proportion which are i m m u n o r e a c t i v e m a y therefore have been u n d e r e s t i m a t e d . This study also provides evidence that some unmyelinated p r i m a r y afferent terminals are subject to presynaptic inhibition at G A B A e r g i c axoaxonic and dendroaxonic synapses. T h e r e is a l r e a d y indirect evidence suggesting that these terminals are postsynaptic at G A B A e r g i c synapses: Ribeiro-da-Silva and C o i m b r a 15 showed that tritiated G A B A a p p l i e d to the cord dorsum was c o n c e n t r a t e d in the p e r i p h e r a l axons of glomeruli in laminae II and III, and some of these a p p e a r to be of the type I, which they subsequently described 16. In addition, G A B A B - b i n d i n g sites are dense in lamina II of rat dorsal horn and are r e d u c e d by 4 0 - 5 0 % after administration of capsaicin to neonates 14. This suggests that some of the G A B A e r g i c profiles r e l a t e d to u n m y e l i n a t e d p r i m a r y afferents m a y be associated with G A B A B receptors and this m a y be i m p o r t a n t in explaining the analgesic actions of the G A B A B agonist baclofen 6.
presynaptic to the central axon at symmetric synapses. T h e s e results strongly suggest that G A B A is contained in some presynaptic dendrites and released at dendrodendritic and d e n d r o a x o n i c synapses within lamina II. This is not surprising, since islet cells in this lamina show GABA-L124 and possess presynaptic dendritesT'23; however, a p a r t from a single r e p o r t that presumptive G A B A ergic dendrites were present in synaptic glomeruli 13, this a p p e a r s to be the first description of G A B A - i m m u n o r e active presynaptic dendrites. O n e possible reason why these have not previously been r e p o r t e d is that it is often
We are very grateful to Dr. P. Somogyi for providing the anti-GABA antiserum and to Miss M. Hughes, Miss C. Morris and Mr. R. Kerr for technical and photographic assistance. This work was supported by the Scottish Home and Health Department and by the Medical Research Funds of the University of Glasgow.
1 Barber, R.P., Vaughn, J.E. and Roberts, E., The cytoarchitecture of GABAergic neurons in rat spinal cord, Brain Research, 238 (1982) 305-328. 2 Barber, R.P., Vaughn, J.E., Saito, K., McLaughlin, B.J. and Roberts, E., GABAergic terminals are presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord, Brain Research, 141 (1978) 35-55. 3 Carlton, S.M., McNeill, D.L., Chung, K. and CoggeshaU, R.E., Organization of calcitonin gene-related peptide-immunoreactive terminals in the primate dorsal horn, J. Comp. Neurol., 276 (1988) 527-536. 4 Coimbra, A., Ribeiro-da-Silva, A. and Pignatelli, D., Effects of dorsal rhizotomy on the several types of primary afferent
terminals in laminae I-III of the rat spinal cord, Anat. EmbryoL, 170 (1984) 279-287. 5 Coimbra, A., Sodr6-Borges, B.P. and Magalhaes, M.M., The substantia gelatinosa Rolandi of the rat. Fine structure, cytochemistry (acid phosphatase) and changes after dorsal root section, J. Neurocytol., 3 (1974) 199-217. 6 Cutting, D.A. and Jordan, C.C., Alternative approaches to analgesia: baclofen as a model compound, Br. J. Pharmacol., 54 (1975) 171-179. 7 Gobel, S., Falls, W.M., Bennett, G.J., Abdelmoumene, M., Hayashi, H. and Humphrey, E., An EM analysis of the synaptic connections of horseradish peroxidase-filled stalked cells and islet cells of the substantia gelatinosa of adult cat spinal cord, J.
174
Comp. Neurol., 194 (1980) 781-807. 8 Hodgson, A.J., Penke, B., Erdei, A., Chubb, I.W. and Somogyi, P., Antisera to y-aminobutyric acid. I. Production and characterization using a new model system, J. Histochem. Cytochem., 33 (1985) 229-239. 9 Hunt, S.P., Kelly, J.S., Emson, EC., Kimmel, J.R., Miller, R.J. and Wu, J.-Y., An immunohistochemical study of neuronal populations containing neuropeptides or ~,-aminobutyrate within the superficial layers of the rat dorsal horn, Neuroscience, 6 (1981) 1883-1898. 10 Knyihar-Csillik, E., Csillik, B. and Rakic, P., Periterminal synaptology of dorsal root glomerular terminals in the substantia gelatinosa of the spinal cord in the rhesus monkey, J. Comp. Neurol., 210 (1982) 376-399. 11 Magoul, R., Onteniente, B., Geffard, M. and Calas, A., Anatomical distribution and ultrastructural organization of the GABAergic system in the rat spinal cord. An immunocytochemicai study using anti-GABA antibodies, Neuroscience, 20 (1987) 1001-1009. 12 McLaughlin, B.J., Barber, R., Saito, K., Roberts, E. and Wu, J.-Y., Immunocytochemical localization of glutamate decarboxylase in rat spinal cord, J. Comp. Neurol., 164 (1975) 305-322. 13 Merighi, A., Polak, J.M., Fumagalli, G. and Theodosis, D.T., Ultrastructural localization of neuropeptides and GABA in rat dorsal horn: a comparison of different immunogold labeling techniques, J. Histochem. Cytochem., 37 (1989) 529-540. 14 Price, G.W., Wilkin, G.P., Turnbull, M.J. and Bowery, N.G., Are baclofen-sensitive GABA B receptors present on primary afferent terminals of the spinal cord?, Nature (Lond.), 307 (1984) 71-74. 15 Ribeiro-da-Silva, A. and Coimbra, A., Neuronal uptake of [3H]GABA and [3H]glycine in laminae I-III (substantia gelati-
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