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Immunocytochemical evidence that GABA and neurotensin exist in different neurons in laminae II and III of rat spinal dorsal horn
Neuroscience Vol. 47, No. 3, pp. 685-691, 1992 Printed in Great Britain
0306-4522/92 $5.00 + 0.00 Pergamon Press plc © 1992 IBRO
IMMUNOCYTOCHEMICAL A N D N E U R O T E N S I N EXIST IN LAMINAE II A N D DORSAL
EVIDENCE THAT GABA IN D I F F E R E N T N E U R O N S III OF RAT SPINAL HORN
A. J. TODD,* G.RuSSELL and R. C. SPIKE Department of Anatomy, University of Glasgow, Glasgow, G12 8QQ, U.K. Abstraet--Pre-embedding immunocytochemistry with antiserum to neurotensin was combined with postembedding immunocytochemistry with GABA antiserum, in order to identify neurotensin- and GABAcontaining neurons in laminae 1711 of rat spinal dorsal horn. The distribution of cell bodies containing these two compounds was similar to that which has been described previously. None of the 88 neurotensin-immunoreactive neurons which were tested showed GABA-like immunoreactivity, which suggests that GABA and neurotensin exist in different cells in this region. Since both compounds are thought to be present in islet cells, it is likely that there are two neurochemically distinct populations of islet cells in lamina II of rat spinal cord.
The tridecapeptide neurotensin is present in the dorsal horn of the spinal cord and its distribution has been demonstrated in rat, cat and monkey in several immunocytochemical studies. 5,6,11,12'16,31'41'42 Neurotensin-immunoreactive axons are concentrated in laminae I and II, and within this region two distinct bands can be observed, one in lamina I and the dorsal part of lamina II and the other in the ventral part of lamina II. 6,11,31 Binding sites for neurotensin are also present in laminae I and II. 25'43 Little is known about the function of neurotensin in the superficial dorsal horn. When applied by intrathecal injection, neurotensin produces an apparent analgesia if the hot plate test is used, but has little or no effect on the tail-flick latency. 34,42 There are also conflicting reports concerning the actions of neurotensin applied to dorsal horn neurons by iontophoresis, since some studies have shown predominantly excitatory effects22,35 and others inhibitory ones. 9 After application of colchicine, numerous neurotensin-immunoreactive perikarya have been observed within the dorsal h o r n , 11'12'16'31'42 mainly within the ventral part of lamina II and in lamina III, but to a lesser extent in the dorsal part of lamina II and in l a m i n a I. 23'24 Neurotensin-immunoreactive cell bodies have also been observed in laminae II and III in animals which had not been treated with colchicineJ'6 By examining parasagittal sections from rats treated with colchicine, Seybold and Elde 31 were able to show that immunoreactive neurons in lamina II had small fusiform perikarya and dendrites which were elongated along the rostrocaudal axis and frequently *To whom correspondence should be addressed. Abbreviations: ABC, avidin biotin complex; -LI, -like
immunoreactivity.
had recurrent branches, thus resembling islet cells,7 while those in lamina III had rounded perikarya and gave rise to primary dendrites which had a dorsoventral orientation. Immunocytochemical studies have also provided evidence that GABA and its synthesizing enzyme glutamate decarboxylase are present in neurons in laminae I-III of rat dorsal horn, 1,11,18,39 and have indicated that GABA may co-exist with glycine in neurons in laminae I and 1140 and with glycine or acetylcholine in lamina III cells. 15,38 GABAergic neurons are thought to make up 31% of the total neuronal population in lamina II and 46% in lamina III. 4° In a combined Golgi and immunocytochemical study of lamina II neurons, Todd and McKenzie 39 found that while large islet cells were GABAimmunoreactive, a population of islet cells with small somata and more restricted dendritic trees were not immunoreactive. Because these small, nonimmunoreactive islet cells closely resembled the neurotensin-immunoreactive neurons found in lamina II by Seybold and Elde, 31 the present study was carried out in order to determine whether G A B A and neurotensin are present in different populations of cells in rat spinal dorsal horn. A pre-embedding immunocytochemical method was used to detect neurotensin-like immunoreactivity (neurotensin-LI) in Vibratome sections. Because the sensitive avidin-biotin complex (ABC) method was used, it was not necessary to apply colchicine in order to detect neurotensin-immunoreactive cell bodies. Semithin sections through the neurotensinimmunoreactive cells were then tested with antiserum to GABA, which strongly stains the nuclei of immunoreactive neurons in spinal cord, 39 in order to determine whether neurotensin-immunoreactive cells also showed GABA-LI.
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RESULTS
EXPERIMENTAL PROCEDURES
Eight adult male Albino Swiss rats (200-350 g; in house) were used in this study. They were anaesthetized with sodium pentobarbitone (60 mg, i.p.) and fixed by intracardiac perfusion with 1 1 of either 4% formaldehyde (two rats) or I% glutaraldehyde/l% formaldehyde (six rats). Both fixatives were made up in 0.1 M phosphate buffer, pH 7.4. Lumbar spinal cord segments were removed and stored in the corresponding fixative for 24 h (formaldehyde fixed material) or for 1~l h (glutaraldehyde/formaldehyde fixed material). Parasagittal or transverse sections 60 # m thick were cut from the L3 or L4 segments with a Vibratome. Sections from cords which had been fixed with glutaraldehyde were treated with 1% sodium borohydride 14 in phosphate-buffered saline for 30 min and then rinsed several times in phosphate-buffered saline.
Pre-embedding immunocytochemistry Sections were rinsed for 1 h in 10% normal goat serum, incubated overnight in rabbit anti-neurotensin antiserum (Peninsula) diluted 1:40,000 in 1% normal goat serum/0.3% Triton X-100 and then processed by the ABC method (ABC Elite kit, Vector), with diaminobenzidine as chromogen. The sections were dehydrated in acetone and flatembedded in Durcupan. Sections from five of the rats fixed with glutaraldehyde/formaldehyde were selected for postembedding immunocytochemistry. Cell bodies which displayed neurotensin-LI were drawn with a camera lucida and the Vibratome sections were then mounted onto blocks of cured resin. Serial 1-/~m semithin sections through these sections were cut with glass knives and mounted onto gelatinized slides. Selected semithin sections through the somata of the neurotensin-immunoreactive neurons were then processed for post-embedding immunocytochemistry.
Post-embedding immunocytochemistry Semithin sections through the nuclei of 88 neurotensinimmunoreactive neurons (69 in transverse sections and 19 in parasagittal sections) were reacted with antiserum to GABA according to a modification of the method of Somogyi et al. 33 described previouslyJ° The sections were etched with sodium ethanolate and then rinsed before being incubated in anti-GABA antiserum (diluted 1:40,000-1 : 50,000) at 4°C overnight and processed according to the ABC method. Peroxidase activity was demonstrated with diaminobenzidine and the reaction product was intensified with osmium tetroxide. The GABA antiserum (GABA-9 j°) was a gift from Dr P. Somogyi.
lmmunocytochemical controls Some Vibratome sections were processed by the preembedding protocol described above except that the primary antiserum was treated with synthetic neurotensin (Sigma; 10#g/ml diluted antiserum) for 1 h before the reaction. In addition, for some semithin sections the antiGABA antiserum was omitted or replaced with normal rabbit serum (diluted 1:40,000) or antiserum which had been treated with GABA conjugated with glutaraldehyde to bovine serum albumen. This was prepared by the method of Storm-Mathisen et al. 36 and 0.1 #1 of conjugate solution (5 mg protein/ml) was added to 50 #1 of primary antiserum (1:40,000) 1 h before use.
Analysis Because the sections were not osmicated it was not possible to identify the boundaries between the superficial laminae according to the distribution of myelinated axons. The ventral extent of the dense plexus of immunostained axons was therefore taken as the border between laminae II and III.
Neurotensin-like immunoreactivity N e u r o t e n s i n - L I showed a similar distribution in material treated with either o f the two fixatives, a l t h o u g h the intensity o f the reaction was moderately reduced by glutaraldehyde. Neurotensin-immunoreactive fibres were concentrated in laminae I a n d II; however, a few i m m u n o s t a i n e d axons with varicosities were present in all other dorsal h o r n laminae a n d these could often be traced ventrally from lamina 1I (Fig. 1). In b o t h transverse a n d parasagittal sections, n u m e r o u s i m m u n o r e a c t i v e cell bodies were present in the ventral p a r t of l a m i n a II and t h r o u g h o u t lamina III a n d these often a p p e a r e d to form a continuous b a n d along the b o r d e r between the two laminae (Fig. 1). The staining of these cells was restricted to the perikaryal cytoplasm a n d p r i m a r y dendrites, so t h a t their m o r p h o l o g y was n o t well seen; however, in parasagittal sections it was clear t h a t m o s t neurons h a d primary dendrites which initially passed in rostral a n d caudal directions (Fig. la), a l t h o u g h some n e u r o n s in l a m i n a III h a d dendrites which passed dorsally or ventrally (Fig. lb). The nuclei o f i m m u n o r e a c t i v e cells were invariably unstained. N o i m m u n o r e a c t i v e cells were seen in lamina I or deep to lamina III. In semithin sections cut from this material, i m m u n o r e a c t i v e cell bodies could be readily identified because o f the rim of i m m u n o s t a i n e d cytoplasm s u r r o u n d i n g the unstained nucleus a n d sometimes extending into primary dendrites (Fig. 2b, f).
Post-embedding immunosta&ing Semithin sections t h r o u g h 88 neurotensinimmunoreactive neurons were tested with antiG A B A antiserum. The quality of the p o s t - e m b e d d i n g reaction did n o t seem to be affected by the previous t r e a t m e n t a n d the distribution of G A B A i m m u n o r e a c t i v e n e u r o n s was the same as that described previously. 39'4° N o n e of the neurotensin-immunoreactive neurons showed G A B A - L I , although they were s u r r o u n d e d by n u m e r o u s G A B A i m m u n o r e a c t i v e cell bodies (Fig. 2a, e). The distribution o f the 69 n e u r o t e n s i n - i m m u n o reactive neurons examined in transverse sections is shown in Fig. 3.
Controls P r e t r e a t m e n t o f the neurotensin antiserum with synthetic n e u r o t e n s i n completely abolished the pree m b e d d i n g reaction (not illustrated). The poste m b e d d i n g reaction was abolished by omitting the p r i m a r y a n t i s e r u m or replacing it with n o n - i m m u n e serum or with a n t i s e r u m which h a d been pretreated with G A B A conjugated to bovine serum a l b u m e n (Fig. 2f).
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Fig. 1. (a) The appearance of neurotensin-like immunoreactivity in a parasagittal Vibratome section of rat dorsal horn, fixed with 4% formaldehyde. Immunoreactive cell bodies (two of which are marked with large arrows) are seen near the border between laminae II and III. Lamina II contains a dense plexus of immunoreactive axons, which gives it a dark appearance that contrasts with the much lighter staining in lamina III. A few immunoreactive fibres with varicosities are seen passing into lamina III (two of these are marked with small arrows). (b) In another parasagittal section from the same segment a neuron with its cell body (large arrow) in lamina III possesses a primary dendrite which passes ventrally (small arrow). Scale bar (for both parts) = 50/~m. DISCUSSION
The immunocytochemical techniques The neurotensin-LI seen in this study showed a similar distribution to that previously reported in studies which used a variety of antisera 5,6,11'12'16'31'41'42 and the staining was completely abolished by incubation of the antiserum with synthetic neurotensin. The G A B A antiserum has been extensively characterized 1°'33 and appears to be specific for neurons that contain G A B A . In this study a combination of pre- and postembedding immunocytochemistry 38 was used since this is particularly suitable for comparing the distribution of an antigen which is present only in the
cytoplasm of neurons (for example, an enzyme or neuropeptide) with that of G A B A or glycine, which can be detected in the nuclei of immunoreactive cells. 26'27'33'a° This method has the additional advantage that the primary antisera used for the pre- and post-embedding reactions can be derived from the same donor species. F o r these experiments it was important to optimize the immunostaining with the G A B A antiserum in order to prevent false negative results and for this reason a fixative containing 1% glutaraldehyde was used, followed by treatment with sodium borohydride. TM With this fixative, the distribution of G A B A - L I was identical to that seen previously in rat dorsal horn. 39'a° Although the use of glutaraldehyde resulted in some reduction in
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Fig. 2. (a) A semithin section through a parasagittal Vibratome section of rat dorsal horn which had previously been reacted with antiserum to neurotensin. This semithin section has been reacted with anti-GABA serum by using post-embedding immunocytochemistry. Two neurotensin-immunoreactive neurons (1 and 2, marked with large arrows) are present on the section and both are non-immunoreactive with GABA antiserum, although other GABA-immunoreactive neurons are present (asterisks). (b) In an adjacent semithin section which has not been further treated, the reaction product resulting from the pre-embedding reaction with neurotensin antiserum can be seen in the cytoplasm of both neurons. This is obscured by the post-embedding reaction product in a. c and d show the appearance of the two cells (1 and 2, respectively) which were photographed after the pre-embedding reaction and before the semithin sections were cut. (e) A semithin section from a transverse Vibratome section that had previously been reacted for neurotensin-LI. After the semithin section was reacted with GABA antiserum the neurotensinimmunoreactive neuron (arrow) was unstained, while other cells showed GABA-LI (one marked with an asterisk). (f) An adjacent semithin section was treated in the same way, except that the GABA antiserum had been pre-adsorbed with GABA conjugated to bovine serum albumen. This completely abolished the post-embedding reaction, so that the reaction product in the cytoplasm of the neurotensin-immunoreactive neuron can now be seen (arrow). Two capillaries (c) are marked as reference points. Scale bar = 20 #m.
neurotensin-immunostaining, the distribution o f immunoreactive fibres and cell bodies was the same as that seen in the material which was fixed without glutaraldehyde. In m o s t previous immunocytochemical studies o f neurotensin, 11'12'16'23'24'31'42colchicine has
been applied to the spinal cord in order to raise the level o f peptide in neuronal cell bodies to detectable levels; however, with m o r e sensitive techniques (e.g. peroxidase--antiperoxidase and ABC) it has been possible to detect the peptide in somata o f untreated
Neurotensin- and GABA-containing neurons in rat spinal dorsal horn
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b Fig. 3. (a) The distribution of the 69 neurotensinimmunoreactive neurons from transverse sections which were tested for GABA-LI. The solid line represents the boundary between grey and white matter, while the dashed line corresponds to the border between laminae II and III. (b) For comparison, this drawing shows the distribution of all the GABA-immunoreactive neuronal nuclei which were present in a single semithin section. animals.5'6 In previous studies, the distribution of immunoreactive neurons in laminae II and III has been similar whether or not colchicine has been used; however, neurons in lamina I have only been immunostained after colchicine treatment. 11A6'23'24,31 This may be because some lamina I neurons normally produce neurotensin but have extremely low levels of the peptide within the perikaryon, or else because colchicine has caused abnormal synthesis of the peptide. 13,29 GABA and neurotensin in dorsal horn neurons
The main finding of this study is that neurotensinimmunoreactive neurons do not possess GABA-LI. It is unlikely that this result is due to interference between the pre- and post-embedding methods, since in a study using the same technique it was shown that the great majority of choline acetyltransferaseimmunoreactive neurons in lamina III were also GABA-immunoreactive?8 While it is possible that there are GABAergic neurons containing low concentrations of neurotensin in the dorsal horn which were not detected in this study, the present results suggest that GABA and neurotensin exist in different cells within laminae II and III of the rat dorsal horn. The morphology of neurons in laminae II and III of the spinal dorsal horn of several species has been examined in numerous studies either with the Golgi technique2'7,19,2°,3°,37 or with intracellular staining methods. 3,8,17,21,28Although there is great variation in
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the shapes of cells which have been described in this area, most reports have identified a population of neurons in both laminae which have dendrites that are elongated along the rostrocaudal axis (often with branches which curve back towards the cell body) and axons which arborize locally. These cells were classified as islet cells in the spinal trigeminal nucleus7 and similar neurons are present in lamina II of the spinal cord. 8 Two major morphological types of neuron have been identified in lamina III. 2°,21 Many neurons are similar to lamina II islet cells and have dendrites that remain within the lamina, while other cells have dendrites that pass dorsally or ventrally and cross laminar boundaries. Seybold and Elde 31 reported that neurotensinimmunoreactive cells in lamina II resembled islet cells, while those in lamina III had primary dendrites which projected dorsoventrally as well as mediolaterally. The dendritic staining in the present material was more limited, since colchicine was not used, but the findings (Fig. 1) were entirely consistent with those of Seybold and Elde. 31 In a combined Golgiimmunocytochemical study of lamina II neurons, Todd and McKenzie39 reported that while many islet cells possessed GABA-LI, some others did not. These non-immunoreactive islet cells had shorter dendritic trees and significantly smaller cell bodies than those which were immunoreactive. The islet cells which were not GABA-immunoreactive in that study (Todd and McKenzie, 39 Fig. 6a-d) closely resemble the neurotensin-immunoreactive cells illustrated by Seybold and Elde 31 (their Figs 2-4) Taken together, these findings suggest that there are two neurochemicalty distinct populations of islet cells within lamina II of rat dorsal horn, one consisting of GABAergic neurons and the other of cells which do not use GABA and which may contain neurotensin. Both types share certain morphological features, for example the orientation of their dendritic trees and the presence of recurrent dendrites, but the GABAergic neurons have larger cell bodies and dendrites which extend further along the rostrocaudal axis. In a recent study of lamina III neurons in the rat, we have found that many of the cells with rostrocaudally elongated dendrites were GABA-immunoreactive, although some of these cells and all of those with dorsally- or ventrally-directed dendrites were not immunoreactive.27a It is therefore likely that some of the lamina III cells with dendrites that projected dorsoventrally and which were not GABAimmunoreactive correspond to the neurotensinimmunoreactive cells described in lamina III by Seybold and Elde. 31 Function of neurotensin-containing neurons
Neurotensin-containing axons in dorsal horn are thought to be derived from intrinsic neurons, since neurotensin is not affected by dorsal root section or by spinal hemisection.25,42 In addition, neurotensinimmunoreactive neurons in dorsal horn do not
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a p p e a r to project to supraspinal levels. ~6 It is therefore likely t h a t the dense plexus of i m m u n o r e a c t i v e axons seen in laminae I a n d II is derived from the i m m u n o r e a c t i v e n e u r o n s seen in laminae II a n d III. N e u r o t e n s i n - i m m u n o r e a c t i v e axon terminals in laminae I a n d II have been s h o w n to form synapses o n t o dendrites a n d cell b o d i e s ) ,32 The b o u t o n s c o n t a i n p r e d o m i n a n t l y a g r a n u l a r vesicles and a few dense-cored vesicles a n d f o r m asymmetric synapses, a l t h o u g h symmetric synapses have also been described? 2 Since asymmetric synapses in the dorsal h o r n 4 are often associated with glutamate, it is possible t h a t n e u r o t e n s i n - c o n t a i n i n g axons also contain glutamate, in which case the n e u r o n s from which they are derived are likely to have a n excitatory
function. Some i m m u n o r e a c t i v e axons in the present material passed ventrally a n d generated b o u t o n s in deeper laminae o f the dorsal h o r n (Fig. la). If any o f these ventrally-projecting axons are derived from l a m i n a II neurons, they m i g h t represent a p a t h for forward c o n d u c t i o n from this lamina, as previously suggested by Light a n d Kavookjian. 17 However, it is clear t h a t the great majority of the o u t p u t from these n e u r o n s remains within the superficial laminae of the dorsal horn. Acknowledgements--We are very grateful to Dr P. Somogyi
for the generous gift of GABA antiserum and to Mr R. Kerr, Miss M. Hughes and Miss C. Morris for technical assistance. The work was supported by the Wellcome Trust and the Scottish Home and Health Department.
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0306-4522/92 $5.00 + 0.00 Pergamon Press plc © 1992 IBRO
IMMUNOCYTOCHEMICAL A N D N E U R O T E N S I N EXIST IN LAMINAE II A N D DORSAL
EVIDENCE THAT GABA IN D I F F E R E N T N E U R O N S III OF RAT SPINAL HORN
A. J. TODD,* G.RuSSELL and R. C. SPIKE Department of Anatomy, University of Glasgow, Glasgow, G12 8QQ, U.K. Abstraet--Pre-embedding immunocytochemistry with antiserum to neurotensin was combined with postembedding immunocytochemistry with GABA antiserum, in order to identify neurotensin- and GABAcontaining neurons in laminae 1711 of rat spinal dorsal horn. The distribution of cell bodies containing these two compounds was similar to that which has been described previously. None of the 88 neurotensin-immunoreactive neurons which were tested showed GABA-like immunoreactivity, which suggests that GABA and neurotensin exist in different cells in this region. Since both compounds are thought to be present in islet cells, it is likely that there are two neurochemically distinct populations of islet cells in lamina II of rat spinal cord.
The tridecapeptide neurotensin is present in the dorsal horn of the spinal cord and its distribution has been demonstrated in rat, cat and monkey in several immunocytochemical studies. 5,6,11,12'16,31'41'42 Neurotensin-immunoreactive axons are concentrated in laminae I and II, and within this region two distinct bands can be observed, one in lamina I and the dorsal part of lamina II and the other in the ventral part of lamina II. 6,11,31 Binding sites for neurotensin are also present in laminae I and II. 25'43 Little is known about the function of neurotensin in the superficial dorsal horn. When applied by intrathecal injection, neurotensin produces an apparent analgesia if the hot plate test is used, but has little or no effect on the tail-flick latency. 34,42 There are also conflicting reports concerning the actions of neurotensin applied to dorsal horn neurons by iontophoresis, since some studies have shown predominantly excitatory effects22,35 and others inhibitory ones. 9 After application of colchicine, numerous neurotensin-immunoreactive perikarya have been observed within the dorsal h o r n , 11'12'16'31'42 mainly within the ventral part of lamina II and in lamina III, but to a lesser extent in the dorsal part of lamina II and in l a m i n a I. 23'24 Neurotensin-immunoreactive cell bodies have also been observed in laminae II and III in animals which had not been treated with colchicineJ'6 By examining parasagittal sections from rats treated with colchicine, Seybold and Elde 31 were able to show that immunoreactive neurons in lamina II had small fusiform perikarya and dendrites which were elongated along the rostrocaudal axis and frequently *To whom correspondence should be addressed. Abbreviations: ABC, avidin biotin complex; -LI, -like
immunoreactivity.
had recurrent branches, thus resembling islet cells,7 while those in lamina III had rounded perikarya and gave rise to primary dendrites which had a dorsoventral orientation. Immunocytochemical studies have also provided evidence that GABA and its synthesizing enzyme glutamate decarboxylase are present in neurons in laminae I-III of rat dorsal horn, 1,11,18,39 and have indicated that GABA may co-exist with glycine in neurons in laminae I and 1140 and with glycine or acetylcholine in lamina III cells. 15,38 GABAergic neurons are thought to make up 31% of the total neuronal population in lamina II and 46% in lamina III. 4° In a combined Golgi and immunocytochemical study of lamina II neurons, Todd and McKenzie 39 found that while large islet cells were GABAimmunoreactive, a population of islet cells with small somata and more restricted dendritic trees were not immunoreactive. Because these small, nonimmunoreactive islet cells closely resembled the neurotensin-immunoreactive neurons found in lamina II by Seybold and Elde, 31 the present study was carried out in order to determine whether G A B A and neurotensin are present in different populations of cells in rat spinal dorsal horn. A pre-embedding immunocytochemical method was used to detect neurotensin-like immunoreactivity (neurotensin-LI) in Vibratome sections. Because the sensitive avidin-biotin complex (ABC) method was used, it was not necessary to apply colchicine in order to detect neurotensin-immunoreactive cell bodies. Semithin sections through the neurotensinimmunoreactive cells were then tested with antiserum to GABA, which strongly stains the nuclei of immunoreactive neurons in spinal cord, 39 in order to determine whether neurotensin-immunoreactive cells also showed GABA-LI.
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RESULTS
EXPERIMENTAL PROCEDURES
Eight adult male Albino Swiss rats (200-350 g; in house) were used in this study. They were anaesthetized with sodium pentobarbitone (60 mg, i.p.) and fixed by intracardiac perfusion with 1 1 of either 4% formaldehyde (two rats) or I% glutaraldehyde/l% formaldehyde (six rats). Both fixatives were made up in 0.1 M phosphate buffer, pH 7.4. Lumbar spinal cord segments were removed and stored in the corresponding fixative for 24 h (formaldehyde fixed material) or for 1~l h (glutaraldehyde/formaldehyde fixed material). Parasagittal or transverse sections 60 # m thick were cut from the L3 or L4 segments with a Vibratome. Sections from cords which had been fixed with glutaraldehyde were treated with 1% sodium borohydride 14 in phosphate-buffered saline for 30 min and then rinsed several times in phosphate-buffered saline.
Pre-embedding immunocytochemistry Sections were rinsed for 1 h in 10% normal goat serum, incubated overnight in rabbit anti-neurotensin antiserum (Peninsula) diluted 1:40,000 in 1% normal goat serum/0.3% Triton X-100 and then processed by the ABC method (ABC Elite kit, Vector), with diaminobenzidine as chromogen. The sections were dehydrated in acetone and flatembedded in Durcupan. Sections from five of the rats fixed with glutaraldehyde/formaldehyde were selected for postembedding immunocytochemistry. Cell bodies which displayed neurotensin-LI were drawn with a camera lucida and the Vibratome sections were then mounted onto blocks of cured resin. Serial 1-/~m semithin sections through these sections were cut with glass knives and mounted onto gelatinized slides. Selected semithin sections through the somata of the neurotensin-immunoreactive neurons were then processed for post-embedding immunocytochemistry.
Post-embedding immunocytochemistry Semithin sections through the nuclei of 88 neurotensinimmunoreactive neurons (69 in transverse sections and 19 in parasagittal sections) were reacted with antiserum to GABA according to a modification of the method of Somogyi et al. 33 described previouslyJ° The sections were etched with sodium ethanolate and then rinsed before being incubated in anti-GABA antiserum (diluted 1:40,000-1 : 50,000) at 4°C overnight and processed according to the ABC method. Peroxidase activity was demonstrated with diaminobenzidine and the reaction product was intensified with osmium tetroxide. The GABA antiserum (GABA-9 j°) was a gift from Dr P. Somogyi.
lmmunocytochemical controls Some Vibratome sections were processed by the preembedding protocol described above except that the primary antiserum was treated with synthetic neurotensin (Sigma; 10#g/ml diluted antiserum) for 1 h before the reaction. In addition, for some semithin sections the antiGABA antiserum was omitted or replaced with normal rabbit serum (diluted 1:40,000) or antiserum which had been treated with GABA conjugated with glutaraldehyde to bovine serum albumen. This was prepared by the method of Storm-Mathisen et al. 36 and 0.1 #1 of conjugate solution (5 mg protein/ml) was added to 50 #1 of primary antiserum (1:40,000) 1 h before use.
Analysis Because the sections were not osmicated it was not possible to identify the boundaries between the superficial laminae according to the distribution of myelinated axons. The ventral extent of the dense plexus of immunostained axons was therefore taken as the border between laminae II and III.
Neurotensin-like immunoreactivity N e u r o t e n s i n - L I showed a similar distribution in material treated with either o f the two fixatives, a l t h o u g h the intensity o f the reaction was moderately reduced by glutaraldehyde. Neurotensin-immunoreactive fibres were concentrated in laminae I a n d II; however, a few i m m u n o s t a i n e d axons with varicosities were present in all other dorsal h o r n laminae a n d these could often be traced ventrally from lamina 1I (Fig. 1). In b o t h transverse a n d parasagittal sections, n u m e r o u s i m m u n o r e a c t i v e cell bodies were present in the ventral p a r t of l a m i n a II and t h r o u g h o u t lamina III a n d these often a p p e a r e d to form a continuous b a n d along the b o r d e r between the two laminae (Fig. 1). The staining of these cells was restricted to the perikaryal cytoplasm a n d p r i m a r y dendrites, so t h a t their m o r p h o l o g y was n o t well seen; however, in parasagittal sections it was clear t h a t m o s t neurons h a d primary dendrites which initially passed in rostral a n d caudal directions (Fig. la), a l t h o u g h some n e u r o n s in l a m i n a III h a d dendrites which passed dorsally or ventrally (Fig. lb). The nuclei o f i m m u n o r e a c t i v e cells were invariably unstained. N o i m m u n o r e a c t i v e cells were seen in lamina I or deep to lamina III. In semithin sections cut from this material, i m m u n o r e a c t i v e cell bodies could be readily identified because o f the rim of i m m u n o s t a i n e d cytoplasm s u r r o u n d i n g the unstained nucleus a n d sometimes extending into primary dendrites (Fig. 2b, f).
Post-embedding immunosta&ing Semithin sections t h r o u g h 88 neurotensinimmunoreactive neurons were tested with antiG A B A antiserum. The quality of the p o s t - e m b e d d i n g reaction did n o t seem to be affected by the previous t r e a t m e n t a n d the distribution of G A B A i m m u n o r e a c t i v e n e u r o n s was the same as that described previously. 39'4° N o n e of the neurotensin-immunoreactive neurons showed G A B A - L I , although they were s u r r o u n d e d by n u m e r o u s G A B A i m m u n o r e a c t i v e cell bodies (Fig. 2a, e). The distribution o f the 69 n e u r o t e n s i n - i m m u n o reactive neurons examined in transverse sections is shown in Fig. 3.
Controls P r e t r e a t m e n t o f the neurotensin antiserum with synthetic n e u r o t e n s i n completely abolished the pree m b e d d i n g reaction (not illustrated). The poste m b e d d i n g reaction was abolished by omitting the p r i m a r y a n t i s e r u m or replacing it with n o n - i m m u n e serum or with a n t i s e r u m which h a d been pretreated with G A B A conjugated to bovine serum a l b u m e n (Fig. 2f).
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Fig. 1. (a) The appearance of neurotensin-like immunoreactivity in a parasagittal Vibratome section of rat dorsal horn, fixed with 4% formaldehyde. Immunoreactive cell bodies (two of which are marked with large arrows) are seen near the border between laminae II and III. Lamina II contains a dense plexus of immunoreactive axons, which gives it a dark appearance that contrasts with the much lighter staining in lamina III. A few immunoreactive fibres with varicosities are seen passing into lamina III (two of these are marked with small arrows). (b) In another parasagittal section from the same segment a neuron with its cell body (large arrow) in lamina III possesses a primary dendrite which passes ventrally (small arrow). Scale bar (for both parts) = 50/~m. DISCUSSION
The immunocytochemical techniques The neurotensin-LI seen in this study showed a similar distribution to that previously reported in studies which used a variety of antisera 5,6,11'12'16'31'41'42 and the staining was completely abolished by incubation of the antiserum with synthetic neurotensin. The G A B A antiserum has been extensively characterized 1°'33 and appears to be specific for neurons that contain G A B A . In this study a combination of pre- and postembedding immunocytochemistry 38 was used since this is particularly suitable for comparing the distribution of an antigen which is present only in the
cytoplasm of neurons (for example, an enzyme or neuropeptide) with that of G A B A or glycine, which can be detected in the nuclei of immunoreactive cells. 26'27'33'a° This method has the additional advantage that the primary antisera used for the pre- and post-embedding reactions can be derived from the same donor species. F o r these experiments it was important to optimize the immunostaining with the G A B A antiserum in order to prevent false negative results and for this reason a fixative containing 1% glutaraldehyde was used, followed by treatment with sodium borohydride. TM With this fixative, the distribution of G A B A - L I was identical to that seen previously in rat dorsal horn. 39'a° Although the use of glutaraldehyde resulted in some reduction in
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Fig. 2. (a) A semithin section through a parasagittal Vibratome section of rat dorsal horn which had previously been reacted with antiserum to neurotensin. This semithin section has been reacted with anti-GABA serum by using post-embedding immunocytochemistry. Two neurotensin-immunoreactive neurons (1 and 2, marked with large arrows) are present on the section and both are non-immunoreactive with GABA antiserum, although other GABA-immunoreactive neurons are present (asterisks). (b) In an adjacent semithin section which has not been further treated, the reaction product resulting from the pre-embedding reaction with neurotensin antiserum can be seen in the cytoplasm of both neurons. This is obscured by the post-embedding reaction product in a. c and d show the appearance of the two cells (1 and 2, respectively) which were photographed after the pre-embedding reaction and before the semithin sections were cut. (e) A semithin section from a transverse Vibratome section that had previously been reacted for neurotensin-LI. After the semithin section was reacted with GABA antiserum the neurotensinimmunoreactive neuron (arrow) was unstained, while other cells showed GABA-LI (one marked with an asterisk). (f) An adjacent semithin section was treated in the same way, except that the GABA antiserum had been pre-adsorbed with GABA conjugated to bovine serum albumen. This completely abolished the post-embedding reaction, so that the reaction product in the cytoplasm of the neurotensin-immunoreactive neuron can now be seen (arrow). Two capillaries (c) are marked as reference points. Scale bar = 20 #m.
neurotensin-immunostaining, the distribution o f immunoreactive fibres and cell bodies was the same as that seen in the material which was fixed without glutaraldehyde. In m o s t previous immunocytochemical studies o f neurotensin, 11'12'16'23'24'31'42colchicine has
been applied to the spinal cord in order to raise the level o f peptide in neuronal cell bodies to detectable levels; however, with m o r e sensitive techniques (e.g. peroxidase--antiperoxidase and ABC) it has been possible to detect the peptide in somata o f untreated
Neurotensin- and GABA-containing neurons in rat spinal dorsal horn
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b Fig. 3. (a) The distribution of the 69 neurotensinimmunoreactive neurons from transverse sections which were tested for GABA-LI. The solid line represents the boundary between grey and white matter, while the dashed line corresponds to the border between laminae II and III. (b) For comparison, this drawing shows the distribution of all the GABA-immunoreactive neuronal nuclei which were present in a single semithin section. animals.5'6 In previous studies, the distribution of immunoreactive neurons in laminae II and III has been similar whether or not colchicine has been used; however, neurons in lamina I have only been immunostained after colchicine treatment. 11A6'23'24,31 This may be because some lamina I neurons normally produce neurotensin but have extremely low levels of the peptide within the perikaryon, or else because colchicine has caused abnormal synthesis of the peptide. 13,29 GABA and neurotensin in dorsal horn neurons
The main finding of this study is that neurotensinimmunoreactive neurons do not possess GABA-LI. It is unlikely that this result is due to interference between the pre- and post-embedding methods, since in a study using the same technique it was shown that the great majority of choline acetyltransferaseimmunoreactive neurons in lamina III were also GABA-immunoreactive?8 While it is possible that there are GABAergic neurons containing low concentrations of neurotensin in the dorsal horn which were not detected in this study, the present results suggest that GABA and neurotensin exist in different cells within laminae II and III of the rat dorsal horn. The morphology of neurons in laminae II and III of the spinal dorsal horn of several species has been examined in numerous studies either with the Golgi technique2'7,19,2°,3°,37 or with intracellular staining methods. 3,8,17,21,28Although there is great variation in
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the shapes of cells which have been described in this area, most reports have identified a population of neurons in both laminae which have dendrites that are elongated along the rostrocaudal axis (often with branches which curve back towards the cell body) and axons which arborize locally. These cells were classified as islet cells in the spinal trigeminal nucleus7 and similar neurons are present in lamina II of the spinal cord. 8 Two major morphological types of neuron have been identified in lamina III. 2°,21 Many neurons are similar to lamina II islet cells and have dendrites that remain within the lamina, while other cells have dendrites that pass dorsally or ventrally and cross laminar boundaries. Seybold and Elde 31 reported that neurotensinimmunoreactive cells in lamina II resembled islet cells, while those in lamina III had primary dendrites which projected dorsoventrally as well as mediolaterally. The dendritic staining in the present material was more limited, since colchicine was not used, but the findings (Fig. 1) were entirely consistent with those of Seybold and Elde. 31 In a combined Golgiimmunocytochemical study of lamina II neurons, Todd and McKenzie39 reported that while many islet cells possessed GABA-LI, some others did not. These non-immunoreactive islet cells had shorter dendritic trees and significantly smaller cell bodies than those which were immunoreactive. The islet cells which were not GABA-immunoreactive in that study (Todd and McKenzie, 39 Fig. 6a-d) closely resemble the neurotensin-immunoreactive cells illustrated by Seybold and Elde 31 (their Figs 2-4) Taken together, these findings suggest that there are two neurochemicalty distinct populations of islet cells within lamina II of rat dorsal horn, one consisting of GABAergic neurons and the other of cells which do not use GABA and which may contain neurotensin. Both types share certain morphological features, for example the orientation of their dendritic trees and the presence of recurrent dendrites, but the GABAergic neurons have larger cell bodies and dendrites which extend further along the rostrocaudal axis. In a recent study of lamina III neurons in the rat, we have found that many of the cells with rostrocaudally elongated dendrites were GABA-immunoreactive, although some of these cells and all of those with dorsally- or ventrally-directed dendrites were not immunoreactive.27a It is therefore likely that some of the lamina III cells with dendrites that projected dorsoventrally and which were not GABAimmunoreactive correspond to the neurotensinimmunoreactive cells described in lamina III by Seybold and Elde. 31 Function of neurotensin-containing neurons
Neurotensin-containing axons in dorsal horn are thought to be derived from intrinsic neurons, since neurotensin is not affected by dorsal root section or by spinal hemisection.25,42 In addition, neurotensinimmunoreactive neurons in dorsal horn do not
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a p p e a r to project to supraspinal levels. ~6 It is therefore likely t h a t the dense plexus of i m m u n o r e a c t i v e axons seen in laminae I a n d II is derived from the i m m u n o r e a c t i v e n e u r o n s seen in laminae II a n d III. N e u r o t e n s i n - i m m u n o r e a c t i v e axon terminals in laminae I a n d II have been s h o w n to form synapses o n t o dendrites a n d cell b o d i e s ) ,32 The b o u t o n s c o n t a i n p r e d o m i n a n t l y a g r a n u l a r vesicles and a few dense-cored vesicles a n d f o r m asymmetric synapses, a l t h o u g h symmetric synapses have also been described? 2 Since asymmetric synapses in the dorsal h o r n 4 are often associated with glutamate, it is possible t h a t n e u r o t e n s i n - c o n t a i n i n g axons also contain glutamate, in which case the n e u r o n s from which they are derived are likely to have a n excitatory
function. Some i m m u n o r e a c t i v e axons in the present material passed ventrally a n d generated b o u t o n s in deeper laminae o f the dorsal h o r n (Fig. la). If any o f these ventrally-projecting axons are derived from l a m i n a II neurons, they m i g h t represent a p a t h for forward c o n d u c t i o n from this lamina, as previously suggested by Light a n d Kavookjian. 17 However, it is clear t h a t the great majority of the o u t p u t from these n e u r o n s remains within the superficial laminae of the dorsal horn. Acknowledgements--We are very grateful to Dr P. Somogyi
for the generous gift of GABA antiserum and to Mr R. Kerr, Miss M. Hughes and Miss C. Morris for technical assistance. The work was supported by the Wellcome Trust and the Scottish Home and Health Department.
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