132
Brain Research, 564 (1991) 132-137 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/911503.50 ,4DONIS 0006899391249180
BRES 24918
RoIMtonMt
hidr-
affMmlt axons and in
A.J.
T o d d 1, D . J .
Maxwell 2 and A.G.
Brown e
1Department of Anatomy, University of Glasgow, Glasgow (U. K.) and 2Department of Preclinical Veterinary Sciences, University of Edinburgh, Summerhall, Edinburgh (U. K.) (Accepted 13 August 1991) Key words: Glycine; Intra-axonal staining; Immunoeytochemistry; Primary afferent; Spinal cord
In order to identify synapses between hair-follicle afferent axons and glycine-containing structures in cat spinal cord, semithin sections containing physiologically identified primary afferent boutons which had been filled with horseradish peroxidase (HRP) were reacted with anti-glycine antiserum, while adjacent ultrathin sections were examined for synaptic contacts. Four axodendritic synapses between hair-follicle afferent boutons and glycine-immunoreactive dendrites and 4 axoaxonic synapses in which HRP-filled boutons were postsynaptic to immunoreactive axons were identified. These results suggest that glyeine is involved in the spinal processing of input from Aft hair-follicle afferent axons. The intra-axonal recording and injection technique has provided much information about the morphology and ultrastructure of large diameter (Aft) hair-follicle afferent fibres in the cat spinal cord. These axons give off collateral branches which arborize in laminae III and IV of the spinal dorsal horn 3. The boutons form asymmetric synapses, mainly with dendritic shafts, and are frequently postsynaptic at symmetric synapses to axons which often possess flattened synaptic vesicles 7'1°. Some presynaptic axons at these axoaxonic synapses are immunoreactive with antiserum to glutamic acid decarboxylase and it has therefore been concluded that hair-follicle afferents are under presynaptic control mediated by G A B A 8. Although the source of these presynaptic axons is not known, they are likely to be derived from local interneurones, since many dorsal horn neurones have been shown to possess GABA-Iike immunoreactivity in other species 1'4"6'16"19. Glycine-like immunoreactivity is particularly concentrated in laminae III and IV of the rat dorsal horn x8 and many neurones in this region appear to contain both G A B A and glycine ~7. The ultrastructural distribution of glycine-like immunoreactivity has been investigated by using pre-embedding immunocytochemistry TM. Immunoreactive axons were sometimes presynaptic at axoaxonic synapses and immunoreactive dendrites were frequently postsynaptic to large boutons which resembled those belonging to low-threshold mechanoreceptive primary afferents 9. Because the distribution of glycine corresponds
tO the areas where low-threshold mechanoreceptors terminate 2 it has been suggested that glycine may be important in the processing of information conveyed by these afferents TM. To investigate this, we have carried out a combined immunocytochemical and intra-axonal injection study in order to search for synapses between labelled hair-follicle afferent boutons and profiles containing glycine-like immunoreactivity. Postembedding immunocytochemistry was used to overcome the problem of penetration of antibodies into the tissue, which would restrict the likelihood of finding such synapses. The immunocytochemical procedure was carried out on semithin sections 12 since this is a very sensitive technique and it is frequently possible to detect immunoreactive cell bodies and dendrites, as well as axons 17. Adjacent ultrathin sections were then examined in order to confirm synaptic contacts. Two adult cats (2.9 and 2.2 kg) were anaesthetized with chloralose (70 mg/kg) and paralysed with gaUamine triethiodide. End-tidal CO 2, carotid arterial blood pressure and rectal temperature were monitored continuously. The level of anaesthesia was assessed from the blood pressure record and by examining the degree of pupillary constriction. The lumbosacral enlargement of the spinal cord was exposed and primary afferent axons in the dorsal columns were impaled with microelectrodes containing HRP. The conduction velocity of the afferents was measured by electrical stimulation of the sciatic nerve. Hair-follicle afferent axons, identified according
Correspondence: A.J. Todd, Department of Anatomy, University of Glasgow, Glasgow, G12 8QQ, U.K.
133 to s t a n d a r d criteria 3 and with c o n d u c t i o n velocities in the
M p h o s p h a t e b u f f e r at 37 °C and a f u r t h e r 2 1 of t h e
Actfl r a n g e , w e r e l a b e l l e d by i o n o p h o r e t i c e j e c t i o n of
s a m e fixative at 4 °C. B l o c k s of spinal c o r d w e r e re-
H R P . A t the e n d o f t h e e x p e r i m e n t the animals w e r e
m o v e d and s t o r e d in the s a m e fixative for at least 8 h.
p e r f u s e d with 0.9% saline f o l l o w e d by 1 1 of fixative con-
T r a n s v e r s e sections (40 # m ) w e r e cut with a V i b r a t o m e ,
taining
2.5% g l u t a r a l d e h y d e and 1% f o r m a l d e h y d e in 0.1
r e a c t e d with d i a m i n o b e n z i d i n e
(DAB)
to r e v e a l the
iiiiii! ;iii~; iiiiiiiiiUiz~iiiiiiiiiii?!iiiiiiii!i~!iiiiii!!iii ii~! ¸¸ s!!~i ;ili:il
Fig. 1. An axodendritic synapse between an HRP-filled hair-follicle afferent bouton and a glycine-immunoreactive dendritic shaft, a: a semithin section through the labelled bouton (arrow). b: the same semithin section after reaction with anti-glycine antiserum. The HRP-filled bouton is still visible (arrow) and lies next to a strongly immunoreactive profile (between arrowheads), e: a low magnification electron micrograph from a section adjacent to the semithin shows the HRP-labelled bouton (HFA) and the strongly immunoreactive profile present in b is seen to be a large dendritic shaft (D). d: in a nearby ultrathin section the labelled bouton (HFA) forms a synapse (between arrowheads) with the same dendritic shaft (D). Bars = 10/~m a; 1 /tm e; 0.5/2m d.
134 presence of HRP, osmicated, d e h y d r a t e d and flat-emb e d d e d in D u r c u p a n . Sections containing labelled boutons were then m o u n t e d o n t o blocks of cured resin and successive series, each consisting of 4 o r 5 ultrathin sections followed by a single semithin section (0.35-0.5 Urn) were cut with d i a m o n d or glass knives. Ultrathin sections were collected on grids and stained with uranyl acetate and lead citrate, while semithin sections were m o u n t e d on gelatinised slides. The semithin sections were searched for labelled boutons, which were p h o t o g r a p h e d (Figs. l a . 2a) and slides containing these b o u t o n s were then i m m u n o s t a i n e d with anti-glycine antiserum as described previously tT. The resin was etched in sodium ethoxide and the sections were t r e a t e d with sodium m e t a p e r i o d a t e to r e m o v e osm i u m tetroxide, incubated overnight in anti-glycine antiserum 2° (a gift from D r R. W e n t h o l d : diluted 1:100) and processed according to the a v i d i n - b i o t i n ( A B C ) m e t h o d ( A B C Elite kit. Vector). Peroxidase activity was visualized by using D A B and the reaction p r o d u c t was intensified with o s m i u m tetroxide. A f t e r the reaction the HRP-filled hair-follicle afferent boutons were still visible, but were now present amongst n u m e r o u s i m m u n o stained profiles (Figs. t b . 2b). Ultratin sections i m m e d i a t e l y preceding and following the i m m u n o s t a i n e d semithin sections were then examined with the electron microscope, in o r d e r to identify profiles which were in synaptic contact with the labelled hair-follicle afferent boutons, and p h o t o g r a p h s of these b o u t o n s and the surrounding neuronal profiles were taken. It was frequently possible to identify the same profiles in b o t h light and electron micrographs. In 4 cases a labelled hair-follicle afferent b o u t o n f o r m e d an asymmetric synapse onto a large o r medium-sized dendritic shaft which could be identified on the adjacent semithin section and which showed glycine-like immunoreactivity (Figs. 1, 2b,e). In addition, at 4 axoaxonic synapses involving hair-follicle afferent b o u t o n s the presynaptic axon could be identified on the semithin section and was strongly i m m u n o r e a c t i v e (Fig. 2b,d). In each case the presynaptic axon contained flattened vesicles and f o r m e d a symmetric synapse o n t o the H R P - l a b e l l e d bouton. In one case a triadic a r r a n g e m e n t was found
(Fig. 2) in which a hair-follicle afferent b o u t o n was postsynaptic to a glycine-immunoreactive axon and presynaptic to a large p r o x i m a l d e n d r i t e that was also immunoreactive. The i m m u n o r e a c t i v e axon was also presynaptic to the dendrite. Control semithin sections from the same material were processed according to the same immunocytochemical p r o c e d u r e , except that the primary antiserum was replaced with antiserum that had b e e n incubated for 1 h with glycine or G A B A conjugated to bovine serum alb u m i n ( B S A ) 13 (0.05 btl of conjugate solution containing 5 mg proteirdml a d d e d to 50 ~1 of antiserum at 1:100 dilution). A d d i t i o n of glycine conjugated to B S A completely abolished irnmunostaining, while addition of a conjugate of G A B A h a d no effect (Fig. 3). The c o m b i n a t i o n of irnmunocytochemistry carried out on semithin sections, with intraceUular staining is possible because the H R P reaction p r o d u c t resulting from the injection is not affected by the immunostaining procedure. The quality of the ultrastructure in the adjacent ultrathin sections is not c o m p r o m i s e d by the procedure. thus allowing identification of synapses. This a p p r o a c h is particularly suitable for the study of synapses involving large o r medium-sized dendritic shafts, since these can readily be identified on adjacent sections (Figs. 1 . 2 b . c ) and are m o r e likely to be i m m u n o s t a i n e d with this m e t h o d than with the i m m n n o g o l d procedure. H o w e v e r . it was also possible to identify immunoreactive axons. especially when these were large r e l a t i v e to the thickness of the semithin section. The antiserum used in this study has b e e n well-characterized a n d shows only a very weak cross-reactivity with G A B A 2°, In this material staitfing was abolished by incubation of the antiserum with conjugated glycine, but was unaffected by t r e a t m e n t with G A B A and this. together with the staining p a t t e r n seen within the dorsal horn 17. m a k e s it likely t h a t the antiserum was detecting fixed glycine. T h e present results therefore suggest that hair-follicle afferent b o u t o n s provide a direct synaptic input to inhibitory n e u r o n e s in the dorsal h o r n which use glycine as a transmitter, and that they are themselves postsynaptic to axons which contain glycine. Since there is evidence that at least some of the axons presynaptic
Fig. 2. A synaptic triad involving a HRP-filled hair-follicle afferent bouton and an axon and dendrite both of which show glycine-like immunoreactivity, a: a semithin section through the HRP-labelled bouton (arrow) which lies close to a e a p ~ (C). b: the same section after reaction with anti-glycine antiserum. The hair-follicle afferent bouton (arrow) lies next to a large proximal dendrite (D), which is moderately immunoreactive, and also next to a smaller strongly reactive profile (small arrow), c: on the last ultrathin section cut before the semithin section shown on a and b, the hair-follicle afferent bouton (HFA), the capillary (C) and the large proximal dendrite (D) are visible. The other profile next to the HRP-filled axon is a smaller bouton (b), which corresponds to the small strongly immunoreactive profile marked with the small arrow in b. d: a higher magnification of this section shows that the bouton (b) contains a cluster of vesicles (arrow) and is presynaptic to the hair-follicle afferent bouton (HFA). e: on the previous section the hair follicle afferent bouton (HFA) is presynaptic to the large dendrite (D) between arrowheads, f: on the second ultrathin section after the semithin, bouton (b) is larger and is presynaptic to the dendrite (D) at a symmetric synapse (arrow). Bars = 10 um a: 1 um c: 0.5 am d. d-f are at the same magnification.
/I
136 ons H.
Since glycine and
acetylcholine
appear
to be
p r e s e n t in d i f f e r e n t p o p u l a t i o n s of G A B A e r g i e n e u r o n e s in l a m i n a I I I o f rat dorsal h o r n ~5 it is p o s s i b l e t h a t acetylcholine is p r e s e n t in s o m e a x o n s w h i c h are p r e s y n a p tic to hair,follicle a f f e r e n t b o u t o n s , a n d glycine i s p r e s e n t in o t h e r s . E v i d e n c e for t h e i n v o l v e m e n t of glycine in spinal som a t o s e n s o r y p r o c e s s i n g has b e e n o b t a i n e d b y u s i n g the a n t a g o n i s t strychnine.
W h e n s t r y c h n i n e i s a p p l i e d by
i o n o p h o r e s i s , it partially blocks the inhibition of neur o n e s in l a m i n a e I V a n d V which results f r o m electrical s t i m u l a t i o n o f m y e l i n a t e d p r i m a r y a f f e r e n t s 5. I n t r a t h e c a l a d m i n i s t r a t i o n o f strychnine in rats a p p e a r s to cause a h y p e r a l g e s i a , in which the animals b e c o m e particularly Fig. 3. Immunoeytoehemieal controls, a: a semithin section reacted with anti-glyeine antiserum shows an immunoreactive soma (S) as well as other stained profiles (2 of which are marked with arrows). b: pretreatment of the antiserum with glycine conjugated to BSA completely abolishes the i m m u n ~ n g , c: pretreatment with G A B A conjugated to B S A has no effect. Bar = 10/*m.
sensitive to s t i m u l a t i o n of hairs 2~. T h i s effect is t h o u g h t to b e specific for low t h r e s h o l d m e c h a n o r e c e p t i v e afferents a n d it has b e e n s u g g e s t e d t h a t this m a y he d u e eit h e r to p r e s y n a p t i c i n h i b i t i o n of t h e s e afferents by glycine, o r else result f r o m specific a c t i v a t i o n of glycinergic i n h i b i t o r y i n t e r n e u r o n e s by low t h r e s h o l d afferents. T h e
tO hair-follicle a f f e r e n t b o u t o n s c o n t a i n G A B A 8 and that
p r e s e n t results suggest that e i t h e r o r b o t h of t h e s e m e c h -
many dorsal horn
anisms m a y o c c u r .
neurones
in the rat c o n t a i n b o t h
G A B A a n d glycine 17 it is likely that t h e s e t w o transmitters coexist w i t h i n s o m e o f t h e a x o n s t h a t f o r m axoaxo n i c synapses. C h o l i n e a c e t y l t r a n s f e r a s e - l i k e i m m u n o r e -
at t h e s e synapses m a y i n c l u d e hair-follicle a f f e r e n t ax-
We are grateful to Dr R. Wenthold for the generous gift of antiglycine antiserum and to Mrs H. Anderson, Mrs W.M. Christie, Mrs P. Taggart and Miss C.A. Morris for technical assistance. The work was supported by the MRC.
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 Brown, A.G., Fyffe, R.E.W., Rose. P.K. and Snow. PJ.. Spinal cord collaterals from axons of type II slowly adapting units in the cat, J. Physiol., 316 (1981) 469-480. 3 Brown, A.G., Rose, P.K. and Snow, P.J.. The morphology of hair follicle afferent collaterals in the spinal cord of the cat. J. Physiol., 272 (1977) 779-797. 4 Carlton. S.M. and Hayes, E.S.. Light microscopic and ultrastructural analysis of GABA-immunoreactive profiles in the monkey spinal cord. J. Comp. Neurol.. 300 (1990) 162-182. 5 Game. C.J.A. and Lodge, D.. The pharmacology of the inhibition of dorsal horn neurones by impulses in myelinated cutaneous afferents in the cat. Exp. Brain Res., 23 (1975) 75-84. 6 Magoul, R.. Onteniente, B.. Geffard. M. and Calas, A., Anatomical distribution and ultrastruetural organization of the GABAergic system in the rat spinal cord. An immunocytochemical study using anti-GABA antibodies, Neuroscience. 20 (1987) 1001-1009. 7 Maxwell, D.J., Bannatyne, B.A., Fyffe, R.E.W. and Brown. A.G., Oltrastructure of hair-follicle afferent fibre terminations in the spinal cord of the cat, J. Neurocytol.. 11 (1982) 571-582. 8 Maxwell, D.J. and Noble, R., Relationships between hair-follicle afferent terminations and glutamic acid decarboxylase-containing boutons in the cat's spinal cord, Brain Research. 408 (1987) 308-312. 9 Maxwell, D.J. and Rethelyi, M.. Ultrastructure and synaptic connections of cutaneous afferent fibres in the spinal cord, Trends Neurosci.. 10 (1987) 117-123. 10 Ralston. H J . , Light, A.R., Ralston, D,D. and Perl, E.R.,
Morphology and synaptic relationships of physiologically identified low-threshold dorsal root axons stained with intra-axonal horseradish peroxidase in the cat and monkey, J. Neurophysiol., 51 (1984) 777-792. Ribeiro-da-Silva. A. and Cuello. A.C.. Choline acetyltransferase-immunoreactive profiles are presynaptic to primary sensory fibers in the rat superficial dorsal horn. J. Comp. Neurol.. 295 (1990) 370-384. Somogyi, P., Hodgson, A J . . Chubb. I.W.. Penke, B. and Erdei. A., Antisera to y-aminobutyric acid. II. Immunocytochemical application to the central nervous system. J. Histochem. Cytochem., 33 (1985) 240-248. Storm-Mathisen, J., Leknes, A.K.. Bore, A.. Vaaland. J.L.. Edminson, P., Haug, F.M.S and Ottersen. O.P,. First visualization of glutamate and GABA in neurones by immunocytochemistry, Nature, 301 (1983) 517-520. Todd, A.J.. An electron microscope study of glycine-like lmmunoreaetivity in laminae I-III of the spinal dorsal horn of the rat, Neuroscience, 39 (1990) 387-394. Todd. A.J.. Immunohistoehemical evidence that ace~ylchotine and glycine exist in different populations of GABAergic neutones in lamina III of rat spinat dorsal horn. Neuroscience. in press. Todd. A.J. and McKenzie. J.. GABA-immunoreactive neutones in the dorsal horn of the rat spinal cord, Neuroscience. 31 (1989) 799-806. Todd. A.J. and Sullivan, A.C., Light microscope study of the coexistence of GABA-Iike and glycine-like immunoreactivities in the spinal cord of the rat. J. Comp. NeuroL, 296 (1990) 496505. van den Pol, A. and Gores. l.. Glycine and glycine-receptor
activity is p r e s e n t in s o m e p r e s y n a p t i c a x o n s at a x o a x o n i c synapses in rat d o r s a l h o r n a n d t h e p o s t s y n a p t i c targets
11
12
13
14
15
16 17
18
137 immunoreactivity in brain and spinal cord, J. Neurosci., 8 (1988) 472-492. 19 Waldvogel, H.J., Faull, R.L.M., Jansen, K.L.R., Dragunow, M., Richards, J.G., Mohler, H. and Streit, E, GABA, GABA receptors and benzodiazepine receptors in the human spinal cord: an autoradiographic and immunohistochemical study at the light and electron microscopic levels, Neuroscience, 39 (1990) 361-385.
20 Wenthold, R.J., Huie, D., Altschuler, R.A. and Reeks, K.A., Glycine immunoreactivity localized in the cochlear nucleus and superior olivary complex, Neuroscience, 22 (1987) 897-912. 21 Yaksh, T.L., 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 (1989) 111-123.
Brain Research, 564 (1991) 132-137 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/911503.50 ,4DONIS 0006899391249180
BRES 24918
RoIMtonMt
hidr-
affMmlt axons and in
A.J.
T o d d 1, D . J .
Maxwell 2 and A.G.
Brown e
1Department of Anatomy, University of Glasgow, Glasgow (U. K.) and 2Department of Preclinical Veterinary Sciences, University of Edinburgh, Summerhall, Edinburgh (U. K.) (Accepted 13 August 1991) Key words: Glycine; Intra-axonal staining; Immunoeytochemistry; Primary afferent; Spinal cord
In order to identify synapses between hair-follicle afferent axons and glycine-containing structures in cat spinal cord, semithin sections containing physiologically identified primary afferent boutons which had been filled with horseradish peroxidase (HRP) were reacted with anti-glycine antiserum, while adjacent ultrathin sections were examined for synaptic contacts. Four axodendritic synapses between hair-follicle afferent boutons and glycine-immunoreactive dendrites and 4 axoaxonic synapses in which HRP-filled boutons were postsynaptic to immunoreactive axons were identified. These results suggest that glyeine is involved in the spinal processing of input from Aft hair-follicle afferent axons. The intra-axonal recording and injection technique has provided much information about the morphology and ultrastructure of large diameter (Aft) hair-follicle afferent fibres in the cat spinal cord. These axons give off collateral branches which arborize in laminae III and IV of the spinal dorsal horn 3. The boutons form asymmetric synapses, mainly with dendritic shafts, and are frequently postsynaptic at symmetric synapses to axons which often possess flattened synaptic vesicles 7'1°. Some presynaptic axons at these axoaxonic synapses are immunoreactive with antiserum to glutamic acid decarboxylase and it has therefore been concluded that hair-follicle afferents are under presynaptic control mediated by G A B A 8. Although the source of these presynaptic axons is not known, they are likely to be derived from local interneurones, since many dorsal horn neurones have been shown to possess GABA-Iike immunoreactivity in other species 1'4"6'16"19. Glycine-like immunoreactivity is particularly concentrated in laminae III and IV of the rat dorsal horn x8 and many neurones in this region appear to contain both G A B A and glycine ~7. The ultrastructural distribution of glycine-like immunoreactivity has been investigated by using pre-embedding immunocytochemistry TM. Immunoreactive axons were sometimes presynaptic at axoaxonic synapses and immunoreactive dendrites were frequently postsynaptic to large boutons which resembled those belonging to low-threshold mechanoreceptive primary afferents 9. Because the distribution of glycine corresponds
tO the areas where low-threshold mechanoreceptors terminate 2 it has been suggested that glycine may be important in the processing of information conveyed by these afferents TM. To investigate this, we have carried out a combined immunocytochemical and intra-axonal injection study in order to search for synapses between labelled hair-follicle afferent boutons and profiles containing glycine-like immunoreactivity. Postembedding immunocytochemistry was used to overcome the problem of penetration of antibodies into the tissue, which would restrict the likelihood of finding such synapses. The immunocytochemical procedure was carried out on semithin sections 12 since this is a very sensitive technique and it is frequently possible to detect immunoreactive cell bodies and dendrites, as well as axons 17. Adjacent ultrathin sections were then examined in order to confirm synaptic contacts. Two adult cats (2.9 and 2.2 kg) were anaesthetized with chloralose (70 mg/kg) and paralysed with gaUamine triethiodide. End-tidal CO 2, carotid arterial blood pressure and rectal temperature were monitored continuously. The level of anaesthesia was assessed from the blood pressure record and by examining the degree of pupillary constriction. The lumbosacral enlargement of the spinal cord was exposed and primary afferent axons in the dorsal columns were impaled with microelectrodes containing HRP. The conduction velocity of the afferents was measured by electrical stimulation of the sciatic nerve. Hair-follicle afferent axons, identified according
Correspondence: A.J. Todd, Department of Anatomy, University of Glasgow, Glasgow, G12 8QQ, U.K.
133 to s t a n d a r d criteria 3 and with c o n d u c t i o n velocities in the
M p h o s p h a t e b u f f e r at 37 °C and a f u r t h e r 2 1 of t h e
Actfl r a n g e , w e r e l a b e l l e d by i o n o p h o r e t i c e j e c t i o n of
s a m e fixative at 4 °C. B l o c k s of spinal c o r d w e r e re-
H R P . A t the e n d o f t h e e x p e r i m e n t the animals w e r e
m o v e d and s t o r e d in the s a m e fixative for at least 8 h.
p e r f u s e d with 0.9% saline f o l l o w e d by 1 1 of fixative con-
T r a n s v e r s e sections (40 # m ) w e r e cut with a V i b r a t o m e ,
taining
2.5% g l u t a r a l d e h y d e and 1% f o r m a l d e h y d e in 0.1
r e a c t e d with d i a m i n o b e n z i d i n e
(DAB)
to r e v e a l the
iiiiii! ;iii~; iiiiiiiiiUiz~iiiiiiiiiii?!iiiiiiii!i~!iiiiii!!iii ii~! ¸¸ s!!~i ;ili:il
Fig. 1. An axodendritic synapse between an HRP-filled hair-follicle afferent bouton and a glycine-immunoreactive dendritic shaft, a: a semithin section through the labelled bouton (arrow). b: the same semithin section after reaction with anti-glycine antiserum. The HRP-filled bouton is still visible (arrow) and lies next to a strongly immunoreactive profile (between arrowheads), e: a low magnification electron micrograph from a section adjacent to the semithin shows the HRP-labelled bouton (HFA) and the strongly immunoreactive profile present in b is seen to be a large dendritic shaft (D). d: in a nearby ultrathin section the labelled bouton (HFA) forms a synapse (between arrowheads) with the same dendritic shaft (D). Bars = 10/~m a; 1 /tm e; 0.5/2m d.
134 presence of HRP, osmicated, d e h y d r a t e d and flat-emb e d d e d in D u r c u p a n . Sections containing labelled boutons were then m o u n t e d o n t o blocks of cured resin and successive series, each consisting of 4 o r 5 ultrathin sections followed by a single semithin section (0.35-0.5 Urn) were cut with d i a m o n d or glass knives. Ultrathin sections were collected on grids and stained with uranyl acetate and lead citrate, while semithin sections were m o u n t e d on gelatinised slides. The semithin sections were searched for labelled boutons, which were p h o t o g r a p h e d (Figs. l a . 2a) and slides containing these b o u t o n s were then i m m u n o s t a i n e d with anti-glycine antiserum as described previously tT. The resin was etched in sodium ethoxide and the sections were t r e a t e d with sodium m e t a p e r i o d a t e to r e m o v e osm i u m tetroxide, incubated overnight in anti-glycine antiserum 2° (a gift from D r R. W e n t h o l d : diluted 1:100) and processed according to the a v i d i n - b i o t i n ( A B C ) m e t h o d ( A B C Elite kit. Vector). Peroxidase activity was visualized by using D A B and the reaction p r o d u c t was intensified with o s m i u m tetroxide. A f t e r the reaction the HRP-filled hair-follicle afferent boutons were still visible, but were now present amongst n u m e r o u s i m m u n o stained profiles (Figs. t b . 2b). Ultratin sections i m m e d i a t e l y preceding and following the i m m u n o s t a i n e d semithin sections were then examined with the electron microscope, in o r d e r to identify profiles which were in synaptic contact with the labelled hair-follicle afferent boutons, and p h o t o g r a p h s of these b o u t o n s and the surrounding neuronal profiles were taken. It was frequently possible to identify the same profiles in b o t h light and electron micrographs. In 4 cases a labelled hair-follicle afferent b o u t o n f o r m e d an asymmetric synapse onto a large o r medium-sized dendritic shaft which could be identified on the adjacent semithin section and which showed glycine-like immunoreactivity (Figs. 1, 2b,e). In addition, at 4 axoaxonic synapses involving hair-follicle afferent b o u t o n s the presynaptic axon could be identified on the semithin section and was strongly i m m u n o r e a c t i v e (Fig. 2b,d). In each case the presynaptic axon contained flattened vesicles and f o r m e d a symmetric synapse o n t o the H R P - l a b e l l e d bouton. In one case a triadic a r r a n g e m e n t was found
(Fig. 2) in which a hair-follicle afferent b o u t o n was postsynaptic to a glycine-immunoreactive axon and presynaptic to a large p r o x i m a l d e n d r i t e that was also immunoreactive. The i m m u n o r e a c t i v e axon was also presynaptic to the dendrite. Control semithin sections from the same material were processed according to the same immunocytochemical p r o c e d u r e , except that the primary antiserum was replaced with antiserum that had b e e n incubated for 1 h with glycine or G A B A conjugated to bovine serum alb u m i n ( B S A ) 13 (0.05 btl of conjugate solution containing 5 mg proteirdml a d d e d to 50 ~1 of antiserum at 1:100 dilution). A d d i t i o n of glycine conjugated to B S A completely abolished irnmunostaining, while addition of a conjugate of G A B A h a d no effect (Fig. 3). The c o m b i n a t i o n of irnmunocytochemistry carried out on semithin sections, with intraceUular staining is possible because the H R P reaction p r o d u c t resulting from the injection is not affected by the immunostaining procedure. The quality of the ultrastructure in the adjacent ultrathin sections is not c o m p r o m i s e d by the procedure. thus allowing identification of synapses. This a p p r o a c h is particularly suitable for the study of synapses involving large o r medium-sized dendritic shafts, since these can readily be identified on adjacent sections (Figs. 1 . 2 b . c ) and are m o r e likely to be i m m u n o s t a i n e d with this m e t h o d than with the i m m n n o g o l d procedure. H o w e v e r . it was also possible to identify immunoreactive axons. especially when these were large r e l a t i v e to the thickness of the semithin section. The antiserum used in this study has b e e n well-characterized a n d shows only a very weak cross-reactivity with G A B A 2°, In this material staitfing was abolished by incubation of the antiserum with conjugated glycine, but was unaffected by t r e a t m e n t with G A B A and this. together with the staining p a t t e r n seen within the dorsal horn 17. m a k e s it likely t h a t the antiserum was detecting fixed glycine. T h e present results therefore suggest that hair-follicle afferent b o u t o n s provide a direct synaptic input to inhibitory n e u r o n e s in the dorsal h o r n which use glycine as a transmitter, and that they are themselves postsynaptic to axons which contain glycine. Since there is evidence that at least some of the axons presynaptic
Fig. 2. A synaptic triad involving a HRP-filled hair-follicle afferent bouton and an axon and dendrite both of which show glycine-like immunoreactivity, a: a semithin section through the HRP-labelled bouton (arrow) which lies close to a e a p ~ (C). b: the same section after reaction with anti-glycine antiserum. The hair-follicle afferent bouton (arrow) lies next to a large proximal dendrite (D), which is moderately immunoreactive, and also next to a smaller strongly reactive profile (small arrow), c: on the last ultrathin section cut before the semithin section shown on a and b, the hair-follicle afferent bouton (HFA), the capillary (C) and the large proximal dendrite (D) are visible. The other profile next to the HRP-filled axon is a smaller bouton (b), which corresponds to the small strongly immunoreactive profile marked with the small arrow in b. d: a higher magnification of this section shows that the bouton (b) contains a cluster of vesicles (arrow) and is presynaptic to the hair-follicle afferent bouton (HFA). e: on the previous section the hair follicle afferent bouton (HFA) is presynaptic to the large dendrite (D) between arrowheads, f: on the second ultrathin section after the semithin, bouton (b) is larger and is presynaptic to the dendrite (D) at a symmetric synapse (arrow). Bars = 10 um a: 1 um c: 0.5 am d. d-f are at the same magnification.
/I
136 ons H.
Since glycine and
acetylcholine
appear
to be
p r e s e n t in d i f f e r e n t p o p u l a t i o n s of G A B A e r g i e n e u r o n e s in l a m i n a I I I o f rat dorsal h o r n ~5 it is p o s s i b l e t h a t acetylcholine is p r e s e n t in s o m e a x o n s w h i c h are p r e s y n a p tic to hair,follicle a f f e r e n t b o u t o n s , a n d glycine i s p r e s e n t in o t h e r s . E v i d e n c e for t h e i n v o l v e m e n t of glycine in spinal som a t o s e n s o r y p r o c e s s i n g has b e e n o b t a i n e d b y u s i n g the a n t a g o n i s t strychnine.
W h e n s t r y c h n i n e i s a p p l i e d by
i o n o p h o r e s i s , it partially blocks the inhibition of neur o n e s in l a m i n a e I V a n d V which results f r o m electrical s t i m u l a t i o n o f m y e l i n a t e d p r i m a r y a f f e r e n t s 5. I n t r a t h e c a l a d m i n i s t r a t i o n o f strychnine in rats a p p e a r s to cause a h y p e r a l g e s i a , in which the animals b e c o m e particularly Fig. 3. Immunoeytoehemieal controls, a: a semithin section reacted with anti-glyeine antiserum shows an immunoreactive soma (S) as well as other stained profiles (2 of which are marked with arrows). b: pretreatment of the antiserum with glycine conjugated to BSA completely abolishes the i m m u n ~ n g , c: pretreatment with G A B A conjugated to B S A has no effect. Bar = 10/*m.
sensitive to s t i m u l a t i o n of hairs 2~. T h i s effect is t h o u g h t to b e specific for low t h r e s h o l d m e c h a n o r e c e p t i v e afferents a n d it has b e e n s u g g e s t e d t h a t this m a y he d u e eit h e r to p r e s y n a p t i c i n h i b i t i o n of t h e s e afferents by glycine, o r else result f r o m specific a c t i v a t i o n of glycinergic i n h i b i t o r y i n t e r n e u r o n e s by low t h r e s h o l d afferents. T h e
tO hair-follicle a f f e r e n t b o u t o n s c o n t a i n G A B A 8 and that
p r e s e n t results suggest that e i t h e r o r b o t h of t h e s e m e c h -
many dorsal horn
anisms m a y o c c u r .
neurones
in the rat c o n t a i n b o t h
G A B A a n d glycine 17 it is likely that t h e s e t w o transmitters coexist w i t h i n s o m e o f t h e a x o n s t h a t f o r m axoaxo n i c synapses. C h o l i n e a c e t y l t r a n s f e r a s e - l i k e i m m u n o r e -
at t h e s e synapses m a y i n c l u d e hair-follicle a f f e r e n t ax-
We are grateful to Dr R. Wenthold for the generous gift of antiglycine antiserum and to Mrs H. Anderson, Mrs W.M. Christie, Mrs P. Taggart and Miss C.A. Morris for technical assistance. The work was supported by the MRC.
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