Brain Research, 526 (1990) 73-80 Elsevier
73
BRES 15794
Fine structure of noradrenergic terminals and their synapses in the rat spinal dorsal horn: an immunohistochemical study S. Hagihira 1, E. Senba 2, S. Yoshida 1, M. Tohyama 2 and I. Yoshiya 1 Departments of 1Anesthesiology and 2Anatomy II, Osaka University Medical School, 1-1-50 Fukushima, Fukushima-ku, Osaka 553 (Japan) (Accepted 27 February 1990) Key words: Noradrenaline; Dopamine-fl-hydroxylase; Spinal cord; Pain transmission; Immunohistochemistry; Synaptoiogy
Noradrenergic fibers in the spinal dorsal horn originate from neurons in the A5-7 cell groups, and may participate in the modulation of pain. Here we studied the fine structure of noradrenergic terminals in the rat by immunohistochemistry using antiserum against dopamine-fl-hydroxylase (DBH). We also investigated the relationship between such terminals and primary afferent terminals. DBH-like immunoreactive terminals were found in lamina I and the outer layer of lamina II of the dorsal horn and they contained many clear round vesicles and some large granular vesicles. More than half of these terminals made synaptic contact with other neuronal elements with membrane specialization. Most of the postsynaptic structures of these terminals were small dendrites (69%); 28% were spines, and no synaptic contact was made with primary afferent terminals. These findings suggest that noradrenaline acts on the spinal dorsal horn neurons postsynaptically mainly via a direct synaptic mechanism. INTRODUC~ON Noradrenergic (NA) axons and terminals are present in the dorsal horn of the spinal cord 1'5'7. These NA axons are derived from N A cells in the nucleus locus coeruleus, nucleus subcoeruleus, and other pontine N A cell groups corresponding to the A 5 - 7 cell groups 27'33'37'45-48. Electrical stimulation of these regions has an analgesic effect in many species 12'17'21'23'35'49. Intrathecal administration of noradrenaline has the same effect 13'15'19'31'32'44'51. Thus, it is widely accepted that these descending NA projections modulate pain transmission. Pharmacological and physiological studies have suggested that the NA effect on pain transmission is mostly postsynaptic but partly presynaptic. First, noradrenaline and adrenergic agonists reduce the activity of dorsal horn neurons evoked by primary afferent stimulation 3"9-11'25'38'43. These findings suggest that noradrenaline acts on spinal neurons postsynaptically. Second, the local application of noradrenaline to the spinal dorsal horn inhibits the release of substance P from the sensory primary afferent terminals evoked by noxious-mechanical stimuli 2°. Noradrenaline increases the electrophysiological threshold for antidromic activation of primary afferent fibers 4'16. These findings suggest that noradrenaline acts on spinal neurons presynaptically. Therefore, the mechanism of
the effect of noradrenaline on the spinal dorsal horn is still unclear. For the analysis of the function of noradrenaline in the dorsal horn, an ultrastructural study of N A terminals is essential, and several attempts have been made. The fine structure of N A terminals in the central nervous system and spinal cord has been examined with the use of a fixative containing glyoxylic acid and potassium permanganate 34'36. Satoh et al. 34 reported that NA terminals in the rat spinal cord rarely formed typical synaptic contacts with other neuronal elements. Findings from a series of studies done with this fixative in other areas of the central nervous system were in agreement with those found in the spinal cord. Studies using a false transmitter 6'18'24'41'42 or autoradiography 2 in which conventional aldehyde-osmium fixation was used showed that most labeled terminals did not form typical synaptic contacts in the central nervous system; findings for the spinal cord have not been reported. Accordingly, it has been believed that N A terminals in the brain generally lack typical synaptic membranous specialization. Several experiments have been suggested. (1) Potassium permanganate fixation demonstrates synaptic membrane specialization poorly and is unsuitable for analysis of the synaptology of the N A terminals. (2) Labeling of NA terminals by false transmitters or autoradiography may
Correspondence: S. Hagihira, Department of Anesthesiology, Osaka University Medical School, 1-1-50 Fukushima, Fukushima-ku, Osaka, 553, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
74 n o t be specific. I m m u n o c y t o c h e m i c a l studies that use a n t i s e r a against the e n z y m e s i n v o l v e d in the synthesis of c a t e c h o l a m i n e or against c a t e c h o l a m i n e itself have s h o w n that t h e r e is typical s y n a p t i c c o n t a c t of c a t e c h o l a m i n e
iIi!/
t e r m i n a l s with o t h e r n e u r o n a l e l e m e n t s in the central n e r v o u s system 22'28'29. T h u s , t h i n k i n g a b o u t the synaptology of N A t e r m i n a l s is c o n f u s e d . H e r e we e x a m i n e d the s y n a p t o l o g y of N A t e r m i n a l s in the dorsal h o r n of the rat spinal cord b y i m m u n o c y t o c h e m i s t r y using a n a n t i s e r u m against d o p a m i n e - f l - h y droxylase, an e n z y m e i n v o l v e d in the synthesis of n o r a d r e n a l i n e , as a m a r k e r of this substance. We also e x a m i n e d the r e l a t i o n s h i p b e t w e e n N A t e r m i n a l s a n d s e n s o r y p r i m a r y afferent t e r m i n a l s by i m m u n o h i s t o c h e m istry c o m b i n e d with d e g e n e r a t i o n e x p e r i m e n t s , b e c a u s e a close r e l a t i o n s h i p b e t w e e n these two systems has b e e n suggested p h a r m a c o l o g i c a l l y , as described above.
MATERIALS AND METHODS
Animals Eight male Wistar rats weighing 100-150 g were used. Four untreated animals were killed for light microscopic (n = 1) and electron microscopic (n = 3) observations.
Dorsal root rhizotomy Animals (n = 2) were anesthetized with sodium pentobarbital (40 mg/kg i.p.) and dorsal roots were exposed by laminectomy of the L2 vertebra. L3-L 6 dorsal roots were cut unilaterally with a pair of fine scissors.
6-Hydroxydopamine study One animal was anesthetized with sodium pentobarbital (40 mg/kg i.p.) and 10~1 of 6-OHDA solution (100 k~g//Adistilled water with 0.1% ascorbic acid) was administered through a polyethylene tube first inserted into the subdural space at the mid-thoracic level and then advanced to the lumbar level. This tube was connected to a Hamilton's syringe. This animal together with one control animal was perfused 4 days after treatment.
Tissue preparation Animals were anesthetized with sodium pentobarbital (50 mg/kg i.p.) and perfused through the ascending aorta with 100 ml of ice-cold saline followed by 500 ml of modified Zamboni's fixative 5° (for light microscopy, 0.1 M phosphate buffer containing 2% paraformaldehyde and 0.2% picric acid; for electron microscopy, 0.05% glutaraldehyde was added to the fixative for light microscopy). Spinal cords were removed, immersed in the same fixative for 48 h at 4 °C, and soaked for an additional 48-72 h in phosphate-buffered saline (PBS), pH 7.4, containing 30% (w/v) sucrose at 4 °C. For light microscopy, frozen sections were cut at 20 /~m thickness with a cryostat. For electron microscopy, sections were cut at 40/~m thickness on a vibratome (Oxford).
lmmunocytochemical procedures for light microscopy Sections were first rinsed in 0.1 M PBS for 4 h at 4 °C. Then they were incubated with the primary antiserum (anti-DBH antiserum (Incstar Corp.) diluted 1:1000) for 48 h, the secondary antiserum (goat anti-rabbit IgG (Cappel) diluted 1:500) overnight, and rabbit peroxidase-antiperoxidase (diluted 1:1000) overnight. PBS containing 1% bovine serum albumin, 1% normal goat serum, and 0.3% Triton X-100 was used for dilution of these antisera and for rinsing of the sections between incubations. Sections were then rinsed with PBS and 0.05 M Tris-HCl buffer,
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! ~i~I¸!J!~%"
Fig. 1. Light photomicrograph showing the distribution of DBH-like immunoreactivity in the rat spinal dorsal horn (L 5 level). Bar = 200 tim.
pH 7.6, and put into 0.05 M Tris-HCl buffer containing 0.02% 3,3"-diaminobenzidine (DAB), 0.2% ammonium nickel(II) sulfate, and 0.005% hydrogen peroxide. Then sections were washed with 0.05 M Tris-HCl buffer, mounted on slides from gelatin solution, dehydrated, and coverslipped.
Immunocytochemical procedure for electron microscopy The sections were processed as described above, except that Triton X-100 was omitted from the solutions. They were then put in a solution containing 0.02% DAB and 0.005% hydrogen peroxide for 10 rain. After rinsing with Tris-HCl buffer, the sections were postfixed with 1% osmium tetroxide for 40 min, stained with 1% uranyl acetate, and dehydrated with a graded series of ethanol concentrations. After being flat-embedded in Epok 812, selected areas were cut and re-embedded in Epok 812. Ultrathin sections were cut on an ultramicrotome (Reichert-Jung). Lead-stained sections were observed under an electron microscope (Hitachi H-7000).
DBH specificity test To check that fibers and terminals labeled by the antiserum were NA, the effect of 6-OHDA was examined. 6-OHDA destroys NA terminals and cells. Samples treated with 6-OHDA and control sections were first rinsed in 0.02 M PBS for 1 h at 4 °C. Then the sections were incubated with the primary antiserum (anti-DBH antiserum diluted 1:1000) for 48 h and with the second antibody (anti-rabbit IgG conjugated with fluorescein isothio-cyanide (Cappel) diluted 1:1000) overnight. After being rinsed with PBS, sections were mounted on a slide glass, coverslipped with glycelinPBS (1:l), and observed under a fluorescence microscope (Nikon). RESULTS
(1) Light microscopy D B H - l i k e i m m u n o r e a c t i v e a x o n t e r m i n a l s were f o u n d m a i n l y in l a m i n a I a n d the o u t e r layer of l a m i n a II (Fig. 1). T h e r e were a few i m m u n o r e a c t i v e fibers in d e e p e r layers ( l a m i n a e III, IV). T h e n u m b e r s of these fibers were fewer after 4 days of 6 - O H D A t r e a t m e n t (Fig. 2).
(2) Fine structure of D B H - L I fibers and their synapses D B H - l i k e i m m u n o r e a c t i v e a x o n t e r m i n a l s were usually b e t w e e n 0.5 a n d 2.0/~m in d i a m e t e r . T h e y c o n t a i n e d
75
Fig. 2. Fluorescence photomicrographs showing DBH-like immunoreaetivity in the spinal dorsal horn, from a control rat (A) and a rat treated with 6-OHDA (B). Bar = 100/zm.
many small, clear, round vesicles (about 35 nm across), some large granular vesicles (about 80 nm across) and some mitochondria. Immunoreactive products were usually associated with both the small vesicles and the large ones (Fig. 3). A number of immunoreactive terminals formed synaptic contacts. For example, in some uitrathin sections, in which a total of 372 DBH-like immunoreactive terminals were observed, 36% of the terminals seemed to make synaptic contacts with membrane specializations. Observing serial sections, more than half of the DBH-like immunoreactive terminals made synaptic contacts. These synapses were asymmetric and had a
wide area of contact. The postsynaptic structures (Table I) were small dendrites in 69.1% or spines in 27.7% (Fig. 3A,B,E,F). These dendrites sometimes contained mitochondria, but never contained synaptic vesicles. Some 3% of these terminals made synaptic contact with large dendrites (more than 1.5/~m in diameter) and 0.2% made synaptic contacts with cell somas (Fig. 3C,D). No DBH-like immunoreactive terminals were seen that made axo-axonic synaptic contacts, and none participated in the formation of synaptic glomeruli. A very few DBH-like immunoreactive terminals were seen in glomeruli, but they did not make synaptic contacts.
76
Fig. 3. Electron photomicrographs showing DBH-like immunoreactive terminals making synaptic contacts (arrowheads) with a small dendrite (A,E), spines (B,F), a large dendrite (C), and a cell soma (D) in lamina I (A-C) and in the outer layer of lamina II (D-F). S, spines; D, dendrites. Bar = 0.5/~m.
77
Fig. 4. Electron photomicrographs showing degenerating primary afferent terminals (d) in the center of glomeruli. DBH-like immunoreactive terminals (DBH) appose primary afferent terminals, but they do not make synaptic contacts (B). Bar = 0.5/~m. (3) Dorsal root rhizotomy At 48 h after dorsal root rhizotomy, there were many degenerating primary afferent terminals in the superficial layers of the dorsal horn, especially the outer layer of
TABLE I Synaptic targets of DBH-like immunoreactive terminals in the rat spinal dorsal horn Target
Number
%
Spine Small dendrites (1.5/~m) Cell soma Total
103 257 11 1 372
28 69 3 0.2 100
lamina II. There are primary afferents in lamina I, but we did not find any degenerating terminals there. This may be because the processes of degeneration of primary afferent fibers are different. The morphology of the degenerating terminals and associated synaptic structures was well preserved. Most such terminals were in the center of glomeruli made up of several dendrites, spines, and axon terminals (Fig. 4). In the experiment that combined dorsal root rhizotomy with immunocytochemistry, degenerating primary afferent terminals did not appose DBH-like immunoreactive terminals in 773 of 776 instances. Three of these 776 terminals did appose the DBH-like immunoreactive terminals, but synaptic contact was not apparent between these terminals when serial ultrathin sections were
78 examined (Fig. 4). So, none of the DBH-like immunoreactive terminals made direct synaptic contact with primary afferent terminals. DISCUSSION There are several techniques used to identify monoaminergic axon terminals, including the autoradiographic demonstration of tritiated amine taken up by terminals 2, fixation with potassium permanganate 34'36, and labeling with false transmitters such as 5-hydroxydopamine6' 18,24,41,42 Using these techniques, several investigators have studied the morphology of NA terminals in various areas of the brain, and it is now widely believed that monoaminergic axon terminals do not make synaptic contacts with distinct membrane specialization. Monoamines seem to be released non-synaptically in the central nervous system, influencing not only adjacent but also more distant neurons. However, with the use of different methodology, other investigators have found that monoaminergic axon terminals do form synapses characterized by synaptic vesicle aggregation and specialized membrane thickening 18'22"24'28'29. Olschowka et al. 28 have reported that more than half of the varicose fibers stained by DBH form synapses with specialized junctional appositions in the rat diencephalon, cerebellum, and limbic cortex. In immunocytochemical study using antibody against noradrenaline, Papadopoulos et al. 29 have reported that at least 90% of the labeled terminals in rat cerebral cortex form synapses with specialized junctional appositions. Here, we identified NA neurons by the presence of DBH-like immunoreactivity, because DBH is the final enzyme in the biosynthetic pathway of noradrenaline, and it is found only in NA or adrenergic neurons. Adrenergic neurons and fibers are not found in the spinal dorsal horn TM. Thus, in the spinal dorsal horn, almost all the terminals containing DBH are considered as NA. The antibody we used was specific; the distribution of DBH-like immunoreactive neurons and terminals coincided with that reported previously 1'5'7'4° and DBH-like immunoreactive terminals in the spinal dorsal horn REFERENCES 1 Anden, N.E., DahlstrOm, A., Fuxe, K., Larsson, K., Olson, L. and Ungerstedt, U., Ascending monoamine neurons to the telencephalon and diencephalon, Acta Physiol. Scand., 67 (1966) 313. 2 Beaudet, A. and Descarries, L., The monoamine innervation of rat cerebral cortex: synaptic and nonsynaptic axon terminals, Neuroscience, 3 (1978) 851-860. 3 Belcher, G., Ryall, R.W. and Schaffner, R., The differential effects of 5-hydroxytryptamine, noradrenaline and raphe stimulation on nociceptive and non-nociceptive dorsal horn interneurons in the cat, Brain Research, 151 (1978) 307-321.
decreased in number after treatment with 6 - O H D A which causes the destruction of NA terminals and cells 8'26'44. DBH is present in the synaptic vesicles of NA terminals. So our method seemed to detect NA neurons and terminals. Synapses made by NA axon terminals were found to have membrane specialization, in agreement with the reports for the central nervous system by Olschowka et al. 28 and Papadopoulos et al. 29. More than half of the NA terminals made synaptic contacts with distinct membrane specializations and these synapses had a wide area of contact, suggesting that the action of noradrenaline is mediated via a synaptic mechanism, although it may also act non-synaptically as well. Noradrenaline acted on spinal neurons mostly postsynaptically, because NA fibers made synaptic contact mostly with small dendrites and spines. Satoh et al. 34 reported that 28% of NA terminals in the spinal dorsal horn make axo-axonic synaptic contacts in experiments done with potassium permanganate as the fixative, but we found no such contacts. Using an immunohistochemical technique, Olschowaka et al. 28 reported finding no axo-axonic contacts involving N A terminals in several regions of the rat brain. They reported that more than 90% of the synapses made by NA terminals are axodendritic or axo-spinous; our results for the spinal dorsal horn are in agreement with theirs. We could not find synaptic contacts between degenerating primary afferent terminals and DBH-like immunoreactive terminals. Primary sensory afferent terminals were usually located in the center of glomeruli, as other investigators have already reported 3°'39, and DBH-like immunoreactive terminals were rarely found in the glomeruli. So, synaptic transmission between primary afferent terminals and NA terminals seems to be rare in the dorsal horn. More than half of the NA terminals in the rat spinal dorsal horn made synaptic contact with membrane specialization and the targets of these NA terminals were mainly neurons existing in the spinal cord. Noradrenaline may act via a synaptic mechanism, like other many neurotransmitters. 4 Calvillo, O. and Ghignone, M., Presynaptic effect of clonidine on unmyelinated afferent fibers in the spinal cord of the cat, Neurosci. Lett., 64 (1986) 335-339. 5 Carlsson, A., Faick, B., Fuxe, K. and Hillarp, N.A., Cellular localization of monoamines in the spinal cord, Acta Physiol. Scand., 60 (1964) 112-119. 6 Chiba, T. and Kato, M., Synaptic structures and quantification of catecholaminergicaxons in the nucleus tractus solitarius of the rat: possible modulatory roles of catecholamines in baroreceptor reflexes, Brain Research, 151 (1978) 323-338. 7 Dahlstr6m, A. and Fuxe, K., Evidence for existence of monoamine-containing neurons in the central nervous system II. Experimentally induced changes in the intraneuronal amine
79 levels of bulbospinal neuron systems, Acta Physiol. Scand., 64 (1965) 5-36 (Suppl. 247). 8 Descarries, L. and Saucier, G., Disappearance of the locus coeruleus in the rat after intraventricular 6-hydroxydopamine, Brain Research, 37 (1972) 310-316. 9 Engberg, L. and Ryall, R.W., The inhibitory action of noradrenaline and other monoamines on spinal neurones, J. Physiol., 185 (1966) 298-322. 10 Fleetwood-Walker, S.M., Hope, P.J., Iggo, A., Mitchell, R. and Molony, V., The effects of ionophoretically applied noradrenaline on the cutaneous sensory responses of identified dorsal horn neurons in the cat, J. Physiol., 342 (1983) 63P-64P. 11 Fleetwood-Walker, S.M., Mitchell, R., Hope, P.J., Molony, V. and Iggo, A., An a 2 receptor mediates the selective inhibitionby noradrenaline of nociceptive responses of identified dorsal horn neurons, Brain Research, 334 (1985) 243-254. 12 Gebhart, G.F., Sandkiihler, J., Thalhammer, J.G. and Zimmermann, M., Inhibition of spinal nociceptive information by stimulation in midbrain of the cat is blocked by lidocaine microinjected in nucleus raphe magnus and medullary reticular formation, J. Neurol., 50 (1983) 1446-1459. 13 Headley, P.M., Duggan, A.W. and Griersmith, B.T., Selective reduction by noradrenaline and 5-hydroxytryptamine of nociceptive responses of cat dorsal horn neurons, Brain Research, 145 (1978) 185-189. 14 H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Research, 66 (1974) 235-251. 15 Hylden, J.L.K. and Wilcox, G.L., Pharmacological characterization of substance P-induced nociception in mice: modulation by opioid and noradrenergic agonists at the spinal level, J. Pharmacol. Exp. Ther., 226 (1983) 398-404. 16 Jeftinija, S., Semba, K. and Randi6, M., Norepinephrine reduces excitability of single cutaneous primary afferent C-fibers in the cat spinal cord, Brain Research, 219 (1981) 456-463. 17 Jones, S.L. and Gebhart, G.E, Quantitative characterization of ceruleospinal inhibition of nociceptive transmission in the rat, J. Neurophysiol., 56 (1986) 1397-1410. 18 Kristt, D.A., Development of neocortical circuitry: quantitative ultra structural analysis of putative monoaminergic synapses, Brain Research, 178 (1979) 69-88. 19 Kuraishi, Y., Harada, Y. and Takagi, H., Noradrenaline regulation of pain-transmission in the spinal cord mediated by a-adrenergic receptors, Brain Research, 174 (1979) 333-336. 20 Kuraishi, Y., Hirota, N., Sato, Y., Kaneko, S., Satoh, M. and Takagi, H., Noradrenergic inhibition of the release of substance P from the primary afferents in the rabbit spinal dorsal horn, Brain Research, 359 (1985) 177-182. 21 Mayer, D.J. and Liebeskind, J.C., Pain reduction by focal electrical stimulation of the brain: an anatomical and behavioral analysis, Brain Research, 68 (1974) 73-93. 22 Milner, T.A., Morrison, S.E, Abate, C. and Reis, D.J., Phenylethanolamine n-methyltransferase-containing terminals synapse directly on sympathetic preganglionic neurons in the rat, Brain Research, 448 (1988) 205-222. 23 Mokha, S.S., McMillan, J.A. and Iggo, A., Descending control of spinal nociceptive transmission. Actions produced on spinal multireceptive neurones from the nuclei locus coeruleus (LC) and raphe magnus (NRM), Exp. Brain Res., 58 (1985) 213-226. 24 Molliver, M.E. and Kristt, D.A., The fine structural demonstration of monoaminergic synapses in immature rat neocortex, Neurosci. Lett., 1 (1975) 305-310. 25 North, R.A. and Yoshimura, M., The actions of noradrenaline on neurones of the rat substantia gelatinosa in vitro, J. Physiol., 349 (1984) 43-55. 26 Nygren, L.G. and Olson, L., On spinal noradrenaline receptor supersensitivity: correlation between nerve terminal densities and flexor reflexes various times after intracisternal 6-hydroxydopamine, Brain Research, 116 (1976) 455-470. 27 Nygren, L.G. and Olson, L., A new major projection from locus
coeruleus: the main source of noradrenergic nerve terminals in the ventral and dorsal columns of the spinal cord, Brain Research, 132 (1977) 85-93. 28 Olschowka, J.A., Molliver, M.E., Grzanna, R., Rice, EL. and Coyle, J.T., Ultrastructural demonstration of noradrenergic synapses in the rat central nervous system by dopamine/5-hydroxylase immunocytochemistry, J. Histochem. Cytochem., 29 (1981) 271-280. 29 Papadopoulos, G.C., Parnavelas, J.G. and Buijs, R., Monoaminergic fibers form conventional synapses in the cerebral cortex, Neurosci. Leg., 76 (1987) 275-279, 30 Ralston III, H.J. and Ralston, D.D., The distribution of dorsal root axons in laminae I, II and III of the macaque spinal cord: a quantitative electron microscope study, J. Comp. Neurol., 184 (1979) 643-684. 31 Reddy, S.V.R., Maderdrut, J.L. and Yaksh, T.L., Spinal cord pharmacology of adrenergic agonist-mediated antinociception, J. Pharmacol. Exp. Ther., 213 (1980) 525-533. 32 Reddy, S.V.R. and Yaksh,T.L., Spinal noradrenergic terminal system mediates antinociception, Brain Research, 189 (1980) 391-401. 33 Satoh, K., Tohyama, M., Yamamoto, K., Sakumoto, T. and Shimizu, N., Noradrenaline innervation of the spinal cord studies by the horseradish peroxidase method combined with monoamine oxidase staining, Exp. Brain Res., 30 (1977) 175-186. 34 Satoh, K., Kashiba, A., Kimura, H. and Maeda, T., Noradrenergic axon terminals in the substantia gelatinosa of the rat spinal cord, Cell Tissue Res., 222 (1982) 359-378. 35 Segal, M. and Sandberg, D., Analgesia produced by electrical stimulation of catecholamine nuclei in the rat brain, Brain Research, 123 (1977) 369-372, 36 Senba, E., Tohyama, M., Shiosaka, S., Takagi, H., Sakanaka, M., Matsuzaki, T., Takahashi, Y. and Simizu, N., Experimental and morphological studies of the noradrenaline innervations in the nucleus tractus spinalis nervi trigemini of the rat with special reference to their fine structure, Brain Research, 206 (1981) 39-50. 37 Stevens, R.T., Apkarian, A.V. and Hodge Jr., C.J., Funicular course of catecholamine fibers innervating the lumbar spinal cord of the cat, Brain Research, 336 (1985) 243-251. 38 Sullivan, A.E, Dashwood, M.R. and Dickenson, A.H., a 2Adrenoceptor modulation of nociception in rat spinal cord: location, effects of interactions with morphine, Eur. J. Pharmacol., 138 (1987) 169-177. 39 Sumal, K.K., Pickel, V.M., Miller, R.J. and Reis, D.J., Enkephalin-containingneurons in substantia gelatinosa of spinal trigeminal complex: ultrastructure and synaptic interaction with primary sensory afferents, Brain Research, 248 (1982) 223-236. 40 Swanson, L.W. and Hartman, B.K., The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-fl-hydroxylase as a marker, J. Comp. Neurol., 163 (1975) 467-506. 41 Swanson, L.W., Connelly, M.A. and Hartman, B.K., Further studies on the fine structure of the adrenergic innervation of the hypothalamus, Brain Research, 151 (1978) 165-174. 42 Tennyson, V.M., Heikkila, R., Mytilineou, C., C6t6, L. and Cohen, G., 5-Hydroxydopamine 'tagged' neuronal boutons in rabbit neostriatum: interrelationship between vesicles and axonal membrane, Brain Research, 82 (1974) 341-348. 43 Todd, A.J. and Miilar, J., Receptive fields and responses to iontophoretically applied noradrenaline and 5-hydroxytryptamine of units recorded in laminae I-III of cat dorsal horn, Brain Research, 288 (1983) 159-167. 44 Ungerstedt, U., 6-Hydroxydopamine induced degeneration of central monoamine neurons, Eur. J. Pharmacol., 5 (1968) 107-110. 45 Westlund, K.N. and Coulter, J.D., Descending projections of the locus coeruleus and subcoeruleus/medial parabrachial nuclei
80 in monkey: axonal transport studies and dopamine-fl-hydroxylase immunocytochemistry, Brain Res. Rev., 2 (1980) 235-264. 46 Westlund, K.N., Bowker, R.M., Ziegler, M.G. and Coulter, J.D., Origins of spinal noradrenergic pathways demonstrated by retrograde transport of antibody to dopamine-fl-hydroxylase, Neurosci. Lett., 25 (1981) 243-249. 47 Westlund, K.N., Bowker, R.M., Ziegler, M.G. and Coulter, J.D., Noradrenergic projections to the spinal cord of the rat, Brain Research, 263 (1983) 15-31. 48 Westlund, K.N., Bowker, R.M., Ziegler, M.G. and Coulter, J.D., Origins and termination of descending noradrenergic projections to the spinal cord of monkey, Brain Research, 292
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73
BRES 15794
Fine structure of noradrenergic terminals and their synapses in the rat spinal dorsal horn: an immunohistochemical study S. Hagihira 1, E. Senba 2, S. Yoshida 1, M. Tohyama 2 and I. Yoshiya 1 Departments of 1Anesthesiology and 2Anatomy II, Osaka University Medical School, 1-1-50 Fukushima, Fukushima-ku, Osaka 553 (Japan) (Accepted 27 February 1990) Key words: Noradrenaline; Dopamine-fl-hydroxylase; Spinal cord; Pain transmission; Immunohistochemistry; Synaptoiogy
Noradrenergic fibers in the spinal dorsal horn originate from neurons in the A5-7 cell groups, and may participate in the modulation of pain. Here we studied the fine structure of noradrenergic terminals in the rat by immunohistochemistry using antiserum against dopamine-fl-hydroxylase (DBH). We also investigated the relationship between such terminals and primary afferent terminals. DBH-like immunoreactive terminals were found in lamina I and the outer layer of lamina II of the dorsal horn and they contained many clear round vesicles and some large granular vesicles. More than half of these terminals made synaptic contact with other neuronal elements with membrane specialization. Most of the postsynaptic structures of these terminals were small dendrites (69%); 28% were spines, and no synaptic contact was made with primary afferent terminals. These findings suggest that noradrenaline acts on the spinal dorsal horn neurons postsynaptically mainly via a direct synaptic mechanism. INTRODUC~ON Noradrenergic (NA) axons and terminals are present in the dorsal horn of the spinal cord 1'5'7. These NA axons are derived from N A cells in the nucleus locus coeruleus, nucleus subcoeruleus, and other pontine N A cell groups corresponding to the A 5 - 7 cell groups 27'33'37'45-48. Electrical stimulation of these regions has an analgesic effect in many species 12'17'21'23'35'49. Intrathecal administration of noradrenaline has the same effect 13'15'19'31'32'44'51. Thus, it is widely accepted that these descending NA projections modulate pain transmission. Pharmacological and physiological studies have suggested that the NA effect on pain transmission is mostly postsynaptic but partly presynaptic. First, noradrenaline and adrenergic agonists reduce the activity of dorsal horn neurons evoked by primary afferent stimulation 3"9-11'25'38'43. These findings suggest that noradrenaline acts on spinal neurons postsynaptically. Second, the local application of noradrenaline to the spinal dorsal horn inhibits the release of substance P from the sensory primary afferent terminals evoked by noxious-mechanical stimuli 2°. Noradrenaline increases the electrophysiological threshold for antidromic activation of primary afferent fibers 4'16. These findings suggest that noradrenaline acts on spinal neurons presynaptically. Therefore, the mechanism of
the effect of noradrenaline on the spinal dorsal horn is still unclear. For the analysis of the function of noradrenaline in the dorsal horn, an ultrastructural study of N A terminals is essential, and several attempts have been made. The fine structure of N A terminals in the central nervous system and spinal cord has been examined with the use of a fixative containing glyoxylic acid and potassium permanganate 34'36. Satoh et al. 34 reported that NA terminals in the rat spinal cord rarely formed typical synaptic contacts with other neuronal elements. Findings from a series of studies done with this fixative in other areas of the central nervous system were in agreement with those found in the spinal cord. Studies using a false transmitter 6'18'24'41'42 or autoradiography 2 in which conventional aldehyde-osmium fixation was used showed that most labeled terminals did not form typical synaptic contacts in the central nervous system; findings for the spinal cord have not been reported. Accordingly, it has been believed that N A terminals in the brain generally lack typical synaptic membranous specialization. Several experiments have been suggested. (1) Potassium permanganate fixation demonstrates synaptic membrane specialization poorly and is unsuitable for analysis of the synaptology of the N A terminals. (2) Labeling of NA terminals by false transmitters or autoradiography may
Correspondence: S. Hagihira, Department of Anesthesiology, Osaka University Medical School, 1-1-50 Fukushima, Fukushima-ku, Osaka, 553, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
74 n o t be specific. I m m u n o c y t o c h e m i c a l studies that use a n t i s e r a against the e n z y m e s i n v o l v e d in the synthesis of c a t e c h o l a m i n e or against c a t e c h o l a m i n e itself have s h o w n that t h e r e is typical s y n a p t i c c o n t a c t of c a t e c h o l a m i n e
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t e r m i n a l s with o t h e r n e u r o n a l e l e m e n t s in the central n e r v o u s system 22'28'29. T h u s , t h i n k i n g a b o u t the synaptology of N A t e r m i n a l s is c o n f u s e d . H e r e we e x a m i n e d the s y n a p t o l o g y of N A t e r m i n a l s in the dorsal h o r n of the rat spinal cord b y i m m u n o c y t o c h e m i s t r y using a n a n t i s e r u m against d o p a m i n e - f l - h y droxylase, an e n z y m e i n v o l v e d in the synthesis of n o r a d r e n a l i n e , as a m a r k e r of this substance. We also e x a m i n e d the r e l a t i o n s h i p b e t w e e n N A t e r m i n a l s a n d s e n s o r y p r i m a r y afferent t e r m i n a l s by i m m u n o h i s t o c h e m istry c o m b i n e d with d e g e n e r a t i o n e x p e r i m e n t s , b e c a u s e a close r e l a t i o n s h i p b e t w e e n these two systems has b e e n suggested p h a r m a c o l o g i c a l l y , as described above.
MATERIALS AND METHODS
Animals Eight male Wistar rats weighing 100-150 g were used. Four untreated animals were killed for light microscopic (n = 1) and electron microscopic (n = 3) observations.
Dorsal root rhizotomy Animals (n = 2) were anesthetized with sodium pentobarbital (40 mg/kg i.p.) and dorsal roots were exposed by laminectomy of the L2 vertebra. L3-L 6 dorsal roots were cut unilaterally with a pair of fine scissors.
6-Hydroxydopamine study One animal was anesthetized with sodium pentobarbital (40 mg/kg i.p.) and 10~1 of 6-OHDA solution (100 k~g//Adistilled water with 0.1% ascorbic acid) was administered through a polyethylene tube first inserted into the subdural space at the mid-thoracic level and then advanced to the lumbar level. This tube was connected to a Hamilton's syringe. This animal together with one control animal was perfused 4 days after treatment.
Tissue preparation Animals were anesthetized with sodium pentobarbital (50 mg/kg i.p.) and perfused through the ascending aorta with 100 ml of ice-cold saline followed by 500 ml of modified Zamboni's fixative 5° (for light microscopy, 0.1 M phosphate buffer containing 2% paraformaldehyde and 0.2% picric acid; for electron microscopy, 0.05% glutaraldehyde was added to the fixative for light microscopy). Spinal cords were removed, immersed in the same fixative for 48 h at 4 °C, and soaked for an additional 48-72 h in phosphate-buffered saline (PBS), pH 7.4, containing 30% (w/v) sucrose at 4 °C. For light microscopy, frozen sections were cut at 20 /~m thickness with a cryostat. For electron microscopy, sections were cut at 40/~m thickness on a vibratome (Oxford).
lmmunocytochemical procedures for light microscopy Sections were first rinsed in 0.1 M PBS for 4 h at 4 °C. Then they were incubated with the primary antiserum (anti-DBH antiserum (Incstar Corp.) diluted 1:1000) for 48 h, the secondary antiserum (goat anti-rabbit IgG (Cappel) diluted 1:500) overnight, and rabbit peroxidase-antiperoxidase (diluted 1:1000) overnight. PBS containing 1% bovine serum albumin, 1% normal goat serum, and 0.3% Triton X-100 was used for dilution of these antisera and for rinsing of the sections between incubations. Sections were then rinsed with PBS and 0.05 M Tris-HCl buffer,
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! ~i~I¸!J!~%"
Fig. 1. Light photomicrograph showing the distribution of DBH-like immunoreactivity in the rat spinal dorsal horn (L 5 level). Bar = 200 tim.
pH 7.6, and put into 0.05 M Tris-HCl buffer containing 0.02% 3,3"-diaminobenzidine (DAB), 0.2% ammonium nickel(II) sulfate, and 0.005% hydrogen peroxide. Then sections were washed with 0.05 M Tris-HCl buffer, mounted on slides from gelatin solution, dehydrated, and coverslipped.
Immunocytochemical procedure for electron microscopy The sections were processed as described above, except that Triton X-100 was omitted from the solutions. They were then put in a solution containing 0.02% DAB and 0.005% hydrogen peroxide for 10 rain. After rinsing with Tris-HCl buffer, the sections were postfixed with 1% osmium tetroxide for 40 min, stained with 1% uranyl acetate, and dehydrated with a graded series of ethanol concentrations. After being flat-embedded in Epok 812, selected areas were cut and re-embedded in Epok 812. Ultrathin sections were cut on an ultramicrotome (Reichert-Jung). Lead-stained sections were observed under an electron microscope (Hitachi H-7000).
DBH specificity test To check that fibers and terminals labeled by the antiserum were NA, the effect of 6-OHDA was examined. 6-OHDA destroys NA terminals and cells. Samples treated with 6-OHDA and control sections were first rinsed in 0.02 M PBS for 1 h at 4 °C. Then the sections were incubated with the primary antiserum (anti-DBH antiserum diluted 1:1000) for 48 h and with the second antibody (anti-rabbit IgG conjugated with fluorescein isothio-cyanide (Cappel) diluted 1:1000) overnight. After being rinsed with PBS, sections were mounted on a slide glass, coverslipped with glycelinPBS (1:l), and observed under a fluorescence microscope (Nikon). RESULTS
(1) Light microscopy D B H - l i k e i m m u n o r e a c t i v e a x o n t e r m i n a l s were f o u n d m a i n l y in l a m i n a I a n d the o u t e r layer of l a m i n a II (Fig. 1). T h e r e were a few i m m u n o r e a c t i v e fibers in d e e p e r layers ( l a m i n a e III, IV). T h e n u m b e r s of these fibers were fewer after 4 days of 6 - O H D A t r e a t m e n t (Fig. 2).
(2) Fine structure of D B H - L I fibers and their synapses D B H - l i k e i m m u n o r e a c t i v e a x o n t e r m i n a l s were usually b e t w e e n 0.5 a n d 2.0/~m in d i a m e t e r . T h e y c o n t a i n e d
75
Fig. 2. Fluorescence photomicrographs showing DBH-like immunoreaetivity in the spinal dorsal horn, from a control rat (A) and a rat treated with 6-OHDA (B). Bar = 100/zm.
many small, clear, round vesicles (about 35 nm across), some large granular vesicles (about 80 nm across) and some mitochondria. Immunoreactive products were usually associated with both the small vesicles and the large ones (Fig. 3). A number of immunoreactive terminals formed synaptic contacts. For example, in some uitrathin sections, in which a total of 372 DBH-like immunoreactive terminals were observed, 36% of the terminals seemed to make synaptic contacts with membrane specializations. Observing serial sections, more than half of the DBH-like immunoreactive terminals made synaptic contacts. These synapses were asymmetric and had a
wide area of contact. The postsynaptic structures (Table I) were small dendrites in 69.1% or spines in 27.7% (Fig. 3A,B,E,F). These dendrites sometimes contained mitochondria, but never contained synaptic vesicles. Some 3% of these terminals made synaptic contact with large dendrites (more than 1.5/~m in diameter) and 0.2% made synaptic contacts with cell somas (Fig. 3C,D). No DBH-like immunoreactive terminals were seen that made axo-axonic synaptic contacts, and none participated in the formation of synaptic glomeruli. A very few DBH-like immunoreactive terminals were seen in glomeruli, but they did not make synaptic contacts.
76
Fig. 3. Electron photomicrographs showing DBH-like immunoreactive terminals making synaptic contacts (arrowheads) with a small dendrite (A,E), spines (B,F), a large dendrite (C), and a cell soma (D) in lamina I (A-C) and in the outer layer of lamina II (D-F). S, spines; D, dendrites. Bar = 0.5/~m.
77
Fig. 4. Electron photomicrographs showing degenerating primary afferent terminals (d) in the center of glomeruli. DBH-like immunoreactive terminals (DBH) appose primary afferent terminals, but they do not make synaptic contacts (B). Bar = 0.5/~m. (3) Dorsal root rhizotomy At 48 h after dorsal root rhizotomy, there were many degenerating primary afferent terminals in the superficial layers of the dorsal horn, especially the outer layer of
TABLE I Synaptic targets of DBH-like immunoreactive terminals in the rat spinal dorsal horn Target
Number
%
Spine Small dendrites (1.5/~m) Cell soma Total
103 257 11 1 372
28 69 3 0.2 100
lamina II. There are primary afferents in lamina I, but we did not find any degenerating terminals there. This may be because the processes of degeneration of primary afferent fibers are different. The morphology of the degenerating terminals and associated synaptic structures was well preserved. Most such terminals were in the center of glomeruli made up of several dendrites, spines, and axon terminals (Fig. 4). In the experiment that combined dorsal root rhizotomy with immunocytochemistry, degenerating primary afferent terminals did not appose DBH-like immunoreactive terminals in 773 of 776 instances. Three of these 776 terminals did appose the DBH-like immunoreactive terminals, but synaptic contact was not apparent between these terminals when serial ultrathin sections were
78 examined (Fig. 4). So, none of the DBH-like immunoreactive terminals made direct synaptic contact with primary afferent terminals. DISCUSSION There are several techniques used to identify monoaminergic axon terminals, including the autoradiographic demonstration of tritiated amine taken up by terminals 2, fixation with potassium permanganate 34'36, and labeling with false transmitters such as 5-hydroxydopamine6' 18,24,41,42 Using these techniques, several investigators have studied the morphology of NA terminals in various areas of the brain, and it is now widely believed that monoaminergic axon terminals do not make synaptic contacts with distinct membrane specialization. Monoamines seem to be released non-synaptically in the central nervous system, influencing not only adjacent but also more distant neurons. However, with the use of different methodology, other investigators have found that monoaminergic axon terminals do form synapses characterized by synaptic vesicle aggregation and specialized membrane thickening 18'22"24'28'29. Olschowka et al. 28 have reported that more than half of the varicose fibers stained by DBH form synapses with specialized junctional appositions in the rat diencephalon, cerebellum, and limbic cortex. In immunocytochemical study using antibody against noradrenaline, Papadopoulos et al. 29 have reported that at least 90% of the labeled terminals in rat cerebral cortex form synapses with specialized junctional appositions. Here, we identified NA neurons by the presence of DBH-like immunoreactivity, because DBH is the final enzyme in the biosynthetic pathway of noradrenaline, and it is found only in NA or adrenergic neurons. Adrenergic neurons and fibers are not found in the spinal dorsal horn TM. Thus, in the spinal dorsal horn, almost all the terminals containing DBH are considered as NA. The antibody we used was specific; the distribution of DBH-like immunoreactive neurons and terminals coincided with that reported previously 1'5'7'4° and DBH-like immunoreactive terminals in the spinal dorsal horn REFERENCES 1 Anden, N.E., DahlstrOm, A., Fuxe, K., Larsson, K., Olson, L. and Ungerstedt, U., Ascending monoamine neurons to the telencephalon and diencephalon, Acta Physiol. Scand., 67 (1966) 313. 2 Beaudet, A. and Descarries, L., The monoamine innervation of rat cerebral cortex: synaptic and nonsynaptic axon terminals, Neuroscience, 3 (1978) 851-860. 3 Belcher, G., Ryall, R.W. and Schaffner, R., The differential effects of 5-hydroxytryptamine, noradrenaline and raphe stimulation on nociceptive and non-nociceptive dorsal horn interneurons in the cat, Brain Research, 151 (1978) 307-321.
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