- Email: [email protected]
Ultrastructural relation between cortical efferents and terminals containing enkephalin-like immunoreactivity in rat neostriatum
Regulatory Peptides, 8 (1984) 105-115
105
Elsevier RPT 00264
Ultrastructural relation between cortical efferents and terminals containing enkephalin-like immunoreactivity in rat neostriatum J.J. B o u y e r , R.J. M i l l e r a n d V . M . P i c k e l Laboratory of Neurobiology, Department of Neurology, Cornell University Medical College, 1300 York Avenue, New York, N Y 10021, U.S.A.
(Received9 August 1983; revisedmanuscript received19 December1983; accepted for publication 19 December1983)
Summary The interrelationships between cortical efferents and terminals containing enkephalin-like immunoreactivity (ELI) were examined by combining anterograde degeneration with electron microscopic immunocytochemistry in the adult rat neostriatum. Two days following unilateral removal of the cerebral cortex, the brains were fixed by aortic arch perfusion, then sectioned and processed for the immunocytochemical localization of an antiserum directed against methionine (Met 5)enkephalin. The observed relationships between the degenerating cortical efferents and immunocytochemically labeled terminals were of two types. In the first, the degenerating and ELI containing terminals converged on the same unlabeled dendrite or dendritic spine. In the second, terminal and preterminal axons of the ELI containing neurons had one surface directly apposed to the plasma membrane of a degenerating axon terminal. These findings support the concept that neurons containing opioid peptides and cortical efferents modulate the output of common recipient neurons and may also directly interact with each other through presynaptic axonal mechanisms in the rat neostriatum. neostriatum; ultrastructure; degeneration; enkephalin; immunocytochemistry
Introduction
The opioid peptides, methionine (MetS) - and leucine (LeuS)-enkephalin are immunocytochemically detectable in perikarya, dendrites and terminals in the Address all correspondence to: Dr. V.M. Pickel, Laboratory of Neurobiology,Cornell University Medical
College, 411 East 69th St., New York, NY 10021, U.S.A. Telephone(212) 472-5594. 0167-0115/84/$03.00 © 1984 ElsevierSciencePublishers B.V.
106 neostriatum [1,2]. A physiological function for these peptides is suggested by the high concentration of opiate receptors [3] and the depression of neuronal activity by enkephalin [4,5]. Furthermore the reduction in opiate receptor binding following chemical lesions of both afferents and endogenous neurons suggests that the inhibitory effects of enkephalin may include both pre- and postsynaptic sites of action [6,7]. Either of these types of interactions may be involved in the enkephalin induced depression of the excitatory effects produced by I_-glutamate [4], the presumed transmitter of most cortical efferents to the neostriatum [8,9]. Conceivably, the opiates may directly modulate the release of L-glutamate or may oppose the actions of L-glutamate on the same recipient neurons [10]. In the present study, we combined anterograde degeneration following unilateral cortical removal with electron microscopic immunocytochemical localization of an antiserum to MetS-enkephalin in order to determine the synaptic relations between cortical efferents and terminals showing enkephalin-like immunoreactivity (ELI) in the adult rat neostriatum. Materials and Methods
The cortex was unilaterally removed by suction in 15 male, Sprague-Dawley rats (120-150 g) which were anesthetized with Halothane. After a survival period of two days [11], the animals were sacrificed by aortic arch perfusion with 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer. Perfusion fixation of 6 min was followed by 1 h postfixation in the same aldehyde solution. The extent of the lesion was determined by visual examination and by histological observation of frozen sections stained with cresyl violet. There was almost complete unilateral removal of the medial and rostral cortex and more partial lesions in lateral and caudal regions. The sections used for histological verification of the lesion were cut in a coronal plane using a vibrating microtome. From eight animals showing the most complete decortication with minimum damage to underlying structures, alternate sections to those stained with cresyl violet were processed for immunocytochemistry. Antiserum directed against methionine MetS-enkephalin was produced in rabbits and tested for immunologic specificity by previously described methods [12]. The antiserum against MetS-enkephalin showed about 0.01% cross-reactivity with Leu 5enkephalin, but no cross-reaction with the closely related longer chain opiate-like peptide fl-endorphin could be detected. However, the antiserum may cross-react with other enkephalin precursors or structurally related peptides; thus the reaction product is referred to as enkephalin-like immunoreactivity (EL1). Control experiments revealed that ELI was completely abolished by the substitution of serum containing 50 /~g purified MetS-enkephalin in 1 ml of 1:400 diluted primary antiserum. In these studies, Vibratome sections were processed for the immunocytochemical localization of the specific and blocked antisera using Pickel et al.'s [1] modification of the peroxidase antiperoxidase (PAP) method of Sternberger [13]. The sections then were embedded in Epon 812, sectioned on an LKB ultramicrotome, and examined with a Philips 201 electron microscope.
107 In each of the eight animals processed for immunocytochemistry, the more superficial regions from at least 4 Vibratome sections were examined ultrastructurally. The thin sections were collected in both the medial and lateral portions of the dorsal half of coronal sections taken at the level of the anterior commissure. The frequency of associations between ELI containing and degenerating terminals was evaluated in randomly collected thin sections.
Results
At 2 days following cortical removal, many degenerating axon terminals were detected in the striatum homolateral to the decortication (Figs. 1-3). The degenerating terminals showed enhanced osmiophilia and dissolution of synaptic vesicles. These terminals were small (approximately 0.1-0.5 /tin in longest cross sectional diameter) and exhibited many bends and contortions. Occasionally a side twig (0.03-0.1 /~m in diameter) was seen in continuity with the degenerating terminal. The small twigs or side branches also contained electron dense material and sometimes formed contacts with other neuronal processes. At this short survival period, the degenerating terminals were not displaced from synaptic sites by intervening glial processes. Furthermore, there were no observable changes in dendrites to suggest transneuronal degeneration. The degenerating terminals formed asymmetric junctions with unlabeled dendrites and dendritic spines (Figs. 1 and 2). Direct appositions between the plasma membrane of degenerating terminals and other axons also were evident. The immunocytochemically labeled terminals were distinguished from the degenerating tei'minals by their greater electron density, granularity and intact synaptic vesicles (Figs. 1-3). The organelles within terminals with ELI included: (a) many small (40-60 nm), clear vesicles which were rimmed with peroxidase immunoreactivity, and (b) larger (80-100 nm) dense core vesicles. In addition, very large (100-150 nm), dense core vesicles and mitochondria were also present in certain labeled terminals (Fig. 3C). The terminals with ELI formed symmetric axo-dendritic contacts (Fig. 1) and appositions to other axon terminals (Fig. 2A). Interrelationships between the degenerating and ELI containing terminals were of two types. First, the degenerating and ELI containing terminals converged on a common dendrite or dendritic spine (Fig. 1). The immunocytochemically labeled terminal formed a symmetric synapse; and the degenerating efferent formed an asymmetric synapse with the recipient dendrite. Approximately 15% (92 out of 680) of the terminals showing ELI shared a common dendrite with a degenerating terminal in single sections. The second relationship involved possible axonal interactions between the degenerating and immunocytochemicallylabeled processes. In approximately half of the observed associations, the two types of terminals were in direct apposition to each other while synapsing with other dendrites and dendritic spines (Fig. 2). The remaining appositions between degenerating and ELI containing axons were seen within bundles of myelinated axons (Fig. 3A) and within more cellular regions where
108
Fig. 1. Convergence of degenerating (dg) cortical efferents and terminals showing ELI on a common dendritic spine (ds) in A or trunk (d) in B. Symmetric synapse of enkephalin labeled terminals = curved arrows; asymmetric synapse of degenerating terminal = straight arrow; dense core vesicles = dcv. Bar = 0.25/~m. r e c i p i e n t d e n d r i t e s c o u l d n o t b e i d e n t i f i e d (Fig. 3B, C). D e n s e r e a c t i o n p r o d u c t was d e t e c t e d in the r e g i o n o f the a p p o s i t i o n s w h i c h s h o w e d n o i n t e r v e n i n g glial processes. I n c e r t a i n cases, t h e E L I c o n t a i n i n g a x o n was in d i r e c t c o n t a c t w i t h a s m a l l side
109
Fig. 2. Degenerating (dg) and ELI containing terminals in close apposition to each other and to other terminals (t) while forming synapses with separate dendrites (d) and dendritic spines (ds). Dendritic synapse of degenerating terminals = straight arrows and enkephalin terminals = curved arrows. Bar = 0.25 p,m.
b r a n c h of t h e d e g e n e r a t i n g a x o n (Fig. 3D). H o w e v e r , t h e r e w a s n o e v i d e n c e of s y n a p s e s s u c h as m e m b r a n e s p e c i a l i z a t i o n s o r a c c u m u l a t i o n o f s y n a p t i c vesicles in a n y o f t h e a p p o s i t i o n s . O n l y a b o u t 8% (54 o u t o f 680) o f the E L I c o n t a i n i n g
110
Fig. 3. UItrastructrual relationship between degenerating axons and terminals or preterminal axons containing EL1. Within bundles of myelinated axons (A) and within more cellular portions (B-D) of the striatum. The degenerating (rig) and ELI containing processes show close contacts (arrows) at their apposed surfaces. Bar = 0.25 #m. terminals were in direct contact with a degenerating axon. I n c o n t r a s t to t h e d e n s e a c c u m u l a t i o n o f E L I i n a x o n t e r m i n a l s , p e r i k a r y a a n d dendrites were only lightly labeled and were sparsely distributed within the more
111 dorsal and medial portions of the striatum. Synaptic specializations between the cortical efferents and dendrites having ELI were not detected.
Discussion (-.4) Conuergence on c o m m o n dendrite
One of the primary findings of this study is that some of the cortical efferents and terminals showing ELI form synapses with the same dendrites within the neostriaturn. The presence of spines associated with dendrites receiving both cortical efferents and ELI containing terminals suggests that they may belong to medium spiny neurons [14]. These neurons are abundant in the neostriatum [14,15] and receive convergent excitatory input from cerebral cortex, thalamus and substantia nigra [16]. The terminals from the cerebral cortex and thalamus contain small clear synaptic vesicles and make asymmetric contacts with dendrites of the medium spiny neurons [17,18]. A second type of terminal which converges onto the same dendrites of spiny neurons has many similarities to the terminals showing ELI in the present study. In common with the ELI containing terminals, this second type of afferent to the spiny neuron contains large pleomorphic vesicles, forms symmetric synapses with dendrites and dendritic spines [14] and remains intact following surgical isolation of the striatum [19]. The fact that this type of terminal remains intact after surgical isolation, suggests that the terminals may belong to intrinsic, possibly spiny, neurons. The type of transmitter within the neuron receiving convergent cortical and ELI containing terminals can be postulated on the basis of other biochemical and immunocytochemical studies. One likely possibility is y-aminobutyric acid (GABA). GABA is an inhibitory transmitter found in many of the medium sized spiny neurons of the neostriatum [20,21]. Furthermore GABA has specific interactions with a variety of putative transmitters including L-glutamate in the basal ganglia [22]. A second possibility for the putative transmitter in the common recipient neurons is substance P which is also contained within the striato-nigral pathway [23]. These and other alternatives must be evaluated through additional double or triple labeling techniques. The relatively low number of observed interactions between cortical efferents and ELI containing terminals probably reflects certain methodological factors. For example, incomplete penetration of the antiserum limits the detection of ELI to the outer 1-2 ~m of the Vibratome section; whereas the degenerating terminals are recognizable throughout the tissue [37]. This factor tends to bias the sample in favor of the independent occurrence of degenerating terminals. In addition only single sections were analyzed in this study which eliminates the possibility of detecting interactions outside the plane of section. The present demonstration that cortical efferents form asymmetric densities and intrinsic ELI containing terminals make symmetric junctions is consistent with biochemical and electrophysiological studies correlating postsynaptic densities with excitatory (asymmetric) or inhibitory (symmetric) functions [24]. Symmetric junc-
112 tions were more numerous in this study than in our previous one [1]. Conceivably, there may be regional variations in the type of specializations formed by terminals containing ELI. In this study, all of the samples were taken from the more dorsal and medial neostriatum, whereas in the earlier report only limited sampling was done in this region. Symmetric junctions also have been reported for terminals showing ELI by other investigators [2]. Convergence of the ELI containing terminal on neurons receiving cortical input is analogous to convergence of enkephalin labeled terminals and primary afferents in the substantia gelatinosa of the spinal trigeminal complex [25]. In the latter region, collaterals of intrinsic neurons containing ELI terminate on the dendrites receiving primary afferents from the trigeminal ganglion [25]. Thus, neurons containing the opioid peptides may internally modulate the effects generated by afferents in at least two different regions of the CNS. However, in the striatum, the enkephalin containing neurons are known also to project to the globus pallidus [26,27]. This structural arrangement of synapses on intrinsic spiny neurons and their projections to the globus pallidus make it possible for neurons containing enkephalin to regulate the output of the basal ganglia through both intrinsic and extrinsic interactions. Feedback systems involving the neostriatal opiate containing neurons, cortical efferents and other dopaminergic efferents from substantia nigra are suggested by the present findings and the results of an earlier study combining the localization of tyrosine hydroxylase with anterograde degeneration following cortical ablation in the rat neostriatum [11]. The degenerating cortical efferents also shared the same dendrites with terminals showing immunoreactivity for tyrosine hydroxylase which are primarily dopaminergic in the neostriatum. Thus at least some of the neurons receiving cortical and nigral efferents may also be postsynaptic to the ELI containing terminals. This postulated interaction is supported by a number of biochemical and behavioral studies which suggest a relation between opiates and dopamine at postsynaptic receptors in the striatum [28,29].
(B) Axo-axonic interrelationships The present observation of appositions between terminals showing ELI and degeneration following cortical removal suggests that there may be additional axo-axonal interrelationships. Studies of opiate receptor binding support the concept of axonic interactions [30]. There is considerable pharmacological evidence for the existence of opiate receptors on afferent axons in the neostriatum and dorsal horn of the spinal cord [30-32]. While most of the existing studies indicate an interaction between opiates and opiate receptors on dopaminergic afferents to the striatum [33,34], lesion experiments suggest that there also may be opiate receptors on other afferent axons [7]. Neurotoxic lesions employing 6-hydroxydopamine for destruction of the dopaminergic nigrostriatal projection combined with kainic acid lesions of intrinsic striatal neurons fail to completely abolish the opiate receptor binding in the striatum [7,30]. Thus, the remaining opiate receptors could be located on nondopaminergic, possibly cortical efferents to the striatum. The absence of morphological evidence of synapses [35] between axons limits our interpretation of the existence or directionality of interrelations between the apposed
113 degenerating and ELI containing terminals. In the present study, dense membrane specializations could be masked by the accumulation of immunoreactivity or degenerative changes. However, even in conventionally stained materials axo-axonic synapses are found infrequently in the striatum [35,36]. Conceivably the postsynaptic densities which usually characterize synapses are not a necessary prerequisite for interactions between the cortical and ELI containing terminals. The postsynaptic densities are associated with calmodulin and cAMP-activated protein kinase [24], thus interactions without observable densities could indicate receptor mediated events not associated with protein phosphorylation. Alternatively, the membrane specializations may be relatively narrow or located along contorted portions of the axon which cannot be readily observed in single or even multiple serial sections. In favor of this latter possibility is the twisted and convoluted shape of the cortical axons which were observed in this and earlier studies [35,36]. The configuration of the cortical efferents as well as the superficial localization of ELI probably also contribute to the relatively infrequent observation of axonal pairs as considered in the above discussion of dendritic interactions.
(C) Cortical efferents and EL1 containing perikarya and dendrites Whether the perikarya and dendrites of ELI containing neurons receive cortical innervation could not be evaluated in the present study. This limitation was due to the small amount of detected immunoreactivity within the perikarya and dendrites. In non-colchicine treated animals, immunoreactivity for many peptides is low in neuronal perikarya [37]. However, using the same antiserum, we have previously shown ELI in perikarya and dendrites within the normal rat striatum [1]. Thus, the absence of ELI within the perikarya and dendrites in the neostriatum of decorticated animals probably reflects other variables such as fixation. Slightly longer post-fixation periods (60 as opposed to 30 min) were used in the present study in order to clearly visualize degenerating terminals. Longer fixation or higher concentrations of glutaraldehyde are known to reduce the immunocytochemical labeling for most antisera [37].
(D) Functional implications The observed associations between cortical efferents and ELI containing neurons include convergence on a single postsynaptic dendrite and direct appositions between t h e respective axon terminals. Convergence on a common dendrite could modulate the output from the neostriatum through either independent or synchronous actions on the recipient neuron. In contrast, the axonal interactions are more likely to be associated with the release of neurotransmitter substances from cortical efferents or the ELI containing terminals.
Acknowledgements This research was supported by a Grant from NSF (BNS-8023914) and from N I H (HL 18974). Virginia M. Pickel also holds a Research Career Development Award (MH 00078). Jean-Jacques Bouyer is a Charg6 de Recherche INSERM.
114
References 1 Pickel, V.M., Sumal, K.K., Beckley, S.C., Miller, R.J. and Reis, D.J., lmmunocytochemical localization of enkephalin in the neostriatum of rat brain: a light and electron microscopic study, Brain Res., 189 (1980) 721-740. 2 Difiglia, M., Aronin, N. and Martin, J.B., Light and electronmicroscopic localization of immunoreaction leu-enkephalin in the monkey basal ganglia, Neuroscience, 2 (1982) 303-320. 3 Atweh, S.F. and Kuhar, M.J., Autoradiographic localization of opiate receptors in rat brain. I11. The telencephalon, Brain Res., 134 (1977) 393-405. 4 Fry, J.P., Zieglgansberger, W. and Herz, A., Development of acute opioid tolerance and dependence in rat striatal neurons, Naunyn Schmiedeberg's Arch. Pharmacol., 313 (1980) 145-149. 5 Frederickson, R.C.A. and Norris, F.H., Enkephalin-induced depression of single neurons in brain areas with opiate receptors-antagonism by naloxone, Science, 194 (1976) 440-442. 6 Malfroy, B., Swerts, J.P., Llorens, C. and Schwartz, J.C., Regional distribution of high-affinity enkephalin-degrading peptidase (enkephalinase) and effects of lesions suggest localization in the vicinity of opiate receptors in brain, Neurosci. Lett., 11 (1979) 329-334. 7 Pollard, H., Llorens, C., Schwartz, J.C., Gros, C. and Dray, F., Localization of opiate receptors and enkephalin in the rat striatum in relationship with the nigrostriatal dopaminergic system: lesions studies, Brain Res., 151 (1978) 392-398. 8 Spencer, H.J., Antagonism of cortical excitation of striatal neurons by glutamic acid diethyl ester: evidence for glutamic acid as an excitatory transmitter in the rat striatum, Brain Res., 102 (1977) 91-101. 9 McGeer, P.L., McGeer, E.G., Scherer, I. and Singh, K., A glutamatergic cortico-striatal path?, Brain Res., 128 (1977) 369-373. 10 Kitai, S.T., Kocsis, J.D., Preston, R.J. and Sugimori, M., Monosynaptic inputs to caudate neurons identified by intracellular injection of horseradish peroxidase, Brain Res., 109 (1976) 601-606. 11 Bouyer, J.J., Park, D.H., Joh, T.H. and Pickek V.M., Structural and chemical analysis of the relation between cortical efferents and tyrosine hydroxylase-containing terminals in rat neostriatum, Brain Res., (1984) in press. 12 Miller, R.J., Chang, K.J., Cooper, R. and Cuatrecasas, P., Radioimmunoassay and characterization of enkephalins in rat tissue, J. Biol. Chem., 253 (1978) 531-538. 13 Sternberger, L.A., lmmunocytochemistry, Prentice Hall, Englewood Cliffs, N.J., 1974. 14 Wilson, C.J. and Groves, P.M., Fine structure and synaptic connections of the common spiny neuron of the rat striatum: A study employing intracellular injection of horseradish peroxidase, .1. Comp. Neurol., 194 (1980) 599-615. 15 Difiglia, M., Pasik, P. and Pasik, T., A Golgi study of neuronal types in the neostriatum of monkeys, Brain Res., 114 (1976) 245-256. 16 Kocsis, J.D., Sugimori, M. and Kitai, S.T., Convergence of excitatory inputs to caudate spiny neurons, Brain Res., 124 (1977) 403-413. 17 Kemp, J.M. and Powell, T.P.S., The site of termination of afferent fibers in the cat caudate nucleus, Phil. Trans. R. Soc. L6nd., 262 (1971) 413-427. 18 Hattori, T., McGeer, G.E. and McGeer, P.L., Fine structural analysis of the cortico-striate pathway, J. Comp. Neurol., 185 (1979) 347-353. 19 Tennyson, V.M. and Marco, L.A., Intrinsic connections of caudate neurons. 11. Fluorescence and electron microscopy following chronic isolation, Brain Res., 53 (1973) 307-317. 20 Ribak, C.E., Vaughn, J.E. and Roberts, E., The GABA neurons and their axon terminals in rat corpus striatum as demonstrated by GAD immunocytochemistry, J. Comp. Neurol., 187 (1979) 261 284. 21 Bernardi, G., Marcini, M.G., Stanzione, P., Cherubini, E., and Mercuri, N., Evidence in favour of GABA as an inhibitory transmitter in rat striatum. In E. Costa and P. Greengard (Eds.), Advances in Biochemical Pharmacology, Vol. 30, Raven Press, New York, NY, 1981, pp. 69-78. 22 Bartholini, G., Scratton B., Worms, P., Zivkovic, B. and Lloyd, K.G., Interactions between GABA, dopamine, acetylcholine and glutamate containing neurons in the extrapyramidal and limbic systems. In E. Costa and P. Greengard (Eds.), Advances in Biochemical Pharmacology, Vol. 30, Raven Press, New York, NY, 1981, pp. 119-128.
115 23 Kanazawa, 1., Emson, P.C. and Cuello, A.C., Evidence for existence of substance P containing fibers in striato-nigral and pallido-nigral pathway in rat brain, Brain Res., 119 (1977) 447-453. 24 Cohen, R.S., Carlin, R.K., Grab, D.J. and Siekevitz, P., Phosphoproteins in postsynaptic densities, Prog. Brain Res., 56 (1982) 49-76. 25 Sumal, K.K., Pickel, V.M., Miller, R.J. and Reis, D.J., Enkephalin-containing neurons in substantia gelatinosa of spinal trigeminal complex: Ultrastructure and synaptic interaction with primary afferents, Brain Res., 248 (1982) 223-236. 26 Cuello, A.C., Evidence for a long leu-enkephalin striopallidal pathway in rat brain, Nature. 271 (1978) 178-180. 27 Preston, R.J., Bishop, G.A. and Katai, S.T., Medium spiny neurons projection from the rat striatum: An intracellular horseradish peroxidase study, Brain Res., 183 (1980) 253-263. 28 Clay, G.A, and Brougham, L.R., Haloperidol binding to an opiate receptor site, Biochem. Pharmacol., 24 (1975) 1363-1367. 29 Govoni, S., Kamakura, K., Spano, P.F., Tonon, G.C. and Trabucchi, M., Interaction of narcotic analgesics with dopamine receptors in rat brain, Pharmacol. Res. Commun., 7 (1975) 96-100. 30 Pollard, H., Llorens-Cortes, C. and Schwartz, J.C., Enkephalin receptors on dopaminergic neurons in rat striatum, Nature, 268 (1977) 745-747. 31 Snyder, S.H. and Simantov, R.J., The opiate receptor and opioid peptides, Neurochemistry, 28 (1977) 13-20. 32 Lamotte, C., Pert, C.B. and Snyder, S.H., Opiate receptor binding in primate spinal cord: distribution and changes after dorsal root section, Brain Res., 112 (1976) 407-412. 33 Chesselet, M.F., Cheramay, A., Reisine, T.D. and Glowinski, J., Morphine and opiate agonists locally stimulate in vivo dopamine release in cat caudate nucleus, Nature, 291 (1981) 320-322. 34 Lubetzki, C., Chesselet, M.F. and Glowinski, J., Modulation of dopamine release in rat striatal slices by delta opiate agonists, J. Pharmacol. Exp. Ther., 222 (1982) 435-440. 35 Bak, I.J., Choi, W.B., Hassler, R. and Usunoff, G.K., Fine structural synaptic organization of the corpus striatum and substantia nigra in rat and cat. In D.B. Calne, T.N. Chase and A. Barbeau (Eds.), Advances in Neurology, Vol. 9, Raven Press, New York, 1975, pp. 25-41. 36 Kemp, J.M. and Powell, T.P.S., The synaptic organization of the caudate nucleus, Phil. Trans. R. Soc. Lond. B, 262 (1971) 403-412. 37 Pickel, V.M., Immunocytochemical Methods. In L. Heimer and M.J. Robards (Eds.), Neuroanatomical Tract Tracing Methods, Plenum Press, New York, 1981, pp. 483-509.
105
Elsevier RPT 00264
Ultrastructural relation between cortical efferents and terminals containing enkephalin-like immunoreactivity in rat neostriatum J.J. B o u y e r , R.J. M i l l e r a n d V . M . P i c k e l Laboratory of Neurobiology, Department of Neurology, Cornell University Medical College, 1300 York Avenue, New York, N Y 10021, U.S.A.
(Received9 August 1983; revisedmanuscript received19 December1983; accepted for publication 19 December1983)
Summary The interrelationships between cortical efferents and terminals containing enkephalin-like immunoreactivity (ELI) were examined by combining anterograde degeneration with electron microscopic immunocytochemistry in the adult rat neostriatum. Two days following unilateral removal of the cerebral cortex, the brains were fixed by aortic arch perfusion, then sectioned and processed for the immunocytochemical localization of an antiserum directed against methionine (Met 5)enkephalin. The observed relationships between the degenerating cortical efferents and immunocytochemically labeled terminals were of two types. In the first, the degenerating and ELI containing terminals converged on the same unlabeled dendrite or dendritic spine. In the second, terminal and preterminal axons of the ELI containing neurons had one surface directly apposed to the plasma membrane of a degenerating axon terminal. These findings support the concept that neurons containing opioid peptides and cortical efferents modulate the output of common recipient neurons and may also directly interact with each other through presynaptic axonal mechanisms in the rat neostriatum. neostriatum; ultrastructure; degeneration; enkephalin; immunocytochemistry
Introduction
The opioid peptides, methionine (MetS) - and leucine (LeuS)-enkephalin are immunocytochemically detectable in perikarya, dendrites and terminals in the Address all correspondence to: Dr. V.M. Pickel, Laboratory of Neurobiology,Cornell University Medical
College, 411 East 69th St., New York, NY 10021, U.S.A. Telephone(212) 472-5594. 0167-0115/84/$03.00 © 1984 ElsevierSciencePublishers B.V.
106 neostriatum [1,2]. A physiological function for these peptides is suggested by the high concentration of opiate receptors [3] and the depression of neuronal activity by enkephalin [4,5]. Furthermore the reduction in opiate receptor binding following chemical lesions of both afferents and endogenous neurons suggests that the inhibitory effects of enkephalin may include both pre- and postsynaptic sites of action [6,7]. Either of these types of interactions may be involved in the enkephalin induced depression of the excitatory effects produced by I_-glutamate [4], the presumed transmitter of most cortical efferents to the neostriatum [8,9]. Conceivably, the opiates may directly modulate the release of L-glutamate or may oppose the actions of L-glutamate on the same recipient neurons [10]. In the present study, we combined anterograde degeneration following unilateral cortical removal with electron microscopic immunocytochemical localization of an antiserum to MetS-enkephalin in order to determine the synaptic relations between cortical efferents and terminals showing enkephalin-like immunoreactivity (ELI) in the adult rat neostriatum. Materials and Methods
The cortex was unilaterally removed by suction in 15 male, Sprague-Dawley rats (120-150 g) which were anesthetized with Halothane. After a survival period of two days [11], the animals were sacrificed by aortic arch perfusion with 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer. Perfusion fixation of 6 min was followed by 1 h postfixation in the same aldehyde solution. The extent of the lesion was determined by visual examination and by histological observation of frozen sections stained with cresyl violet. There was almost complete unilateral removal of the medial and rostral cortex and more partial lesions in lateral and caudal regions. The sections used for histological verification of the lesion were cut in a coronal plane using a vibrating microtome. From eight animals showing the most complete decortication with minimum damage to underlying structures, alternate sections to those stained with cresyl violet were processed for immunocytochemistry. Antiserum directed against methionine MetS-enkephalin was produced in rabbits and tested for immunologic specificity by previously described methods [12]. The antiserum against MetS-enkephalin showed about 0.01% cross-reactivity with Leu 5enkephalin, but no cross-reaction with the closely related longer chain opiate-like peptide fl-endorphin could be detected. However, the antiserum may cross-react with other enkephalin precursors or structurally related peptides; thus the reaction product is referred to as enkephalin-like immunoreactivity (EL1). Control experiments revealed that ELI was completely abolished by the substitution of serum containing 50 /~g purified MetS-enkephalin in 1 ml of 1:400 diluted primary antiserum. In these studies, Vibratome sections were processed for the immunocytochemical localization of the specific and blocked antisera using Pickel et al.'s [1] modification of the peroxidase antiperoxidase (PAP) method of Sternberger [13]. The sections then were embedded in Epon 812, sectioned on an LKB ultramicrotome, and examined with a Philips 201 electron microscope.
107 In each of the eight animals processed for immunocytochemistry, the more superficial regions from at least 4 Vibratome sections were examined ultrastructurally. The thin sections were collected in both the medial and lateral portions of the dorsal half of coronal sections taken at the level of the anterior commissure. The frequency of associations between ELI containing and degenerating terminals was evaluated in randomly collected thin sections.
Results
At 2 days following cortical removal, many degenerating axon terminals were detected in the striatum homolateral to the decortication (Figs. 1-3). The degenerating terminals showed enhanced osmiophilia and dissolution of synaptic vesicles. These terminals were small (approximately 0.1-0.5 /tin in longest cross sectional diameter) and exhibited many bends and contortions. Occasionally a side twig (0.03-0.1 /~m in diameter) was seen in continuity with the degenerating terminal. The small twigs or side branches also contained electron dense material and sometimes formed contacts with other neuronal processes. At this short survival period, the degenerating terminals were not displaced from synaptic sites by intervening glial processes. Furthermore, there were no observable changes in dendrites to suggest transneuronal degeneration. The degenerating terminals formed asymmetric junctions with unlabeled dendrites and dendritic spines (Figs. 1 and 2). Direct appositions between the plasma membrane of degenerating terminals and other axons also were evident. The immunocytochemically labeled terminals were distinguished from the degenerating tei'minals by their greater electron density, granularity and intact synaptic vesicles (Figs. 1-3). The organelles within terminals with ELI included: (a) many small (40-60 nm), clear vesicles which were rimmed with peroxidase immunoreactivity, and (b) larger (80-100 nm) dense core vesicles. In addition, very large (100-150 nm), dense core vesicles and mitochondria were also present in certain labeled terminals (Fig. 3C). The terminals with ELI formed symmetric axo-dendritic contacts (Fig. 1) and appositions to other axon terminals (Fig. 2A). Interrelationships between the degenerating and ELI containing terminals were of two types. First, the degenerating and ELI containing terminals converged on a common dendrite or dendritic spine (Fig. 1). The immunocytochemically labeled terminal formed a symmetric synapse; and the degenerating efferent formed an asymmetric synapse with the recipient dendrite. Approximately 15% (92 out of 680) of the terminals showing ELI shared a common dendrite with a degenerating terminal in single sections. The second relationship involved possible axonal interactions between the degenerating and immunocytochemicallylabeled processes. In approximately half of the observed associations, the two types of terminals were in direct apposition to each other while synapsing with other dendrites and dendritic spines (Fig. 2). The remaining appositions between degenerating and ELI containing axons were seen within bundles of myelinated axons (Fig. 3A) and within more cellular regions where
108
Fig. 1. Convergence of degenerating (dg) cortical efferents and terminals showing ELI on a common dendritic spine (ds) in A or trunk (d) in B. Symmetric synapse of enkephalin labeled terminals = curved arrows; asymmetric synapse of degenerating terminal = straight arrow; dense core vesicles = dcv. Bar = 0.25/~m. r e c i p i e n t d e n d r i t e s c o u l d n o t b e i d e n t i f i e d (Fig. 3B, C). D e n s e r e a c t i o n p r o d u c t was d e t e c t e d in the r e g i o n o f the a p p o s i t i o n s w h i c h s h o w e d n o i n t e r v e n i n g glial processes. I n c e r t a i n cases, t h e E L I c o n t a i n i n g a x o n was in d i r e c t c o n t a c t w i t h a s m a l l side
109
Fig. 2. Degenerating (dg) and ELI containing terminals in close apposition to each other and to other terminals (t) while forming synapses with separate dendrites (d) and dendritic spines (ds). Dendritic synapse of degenerating terminals = straight arrows and enkephalin terminals = curved arrows. Bar = 0.25 p,m.
b r a n c h of t h e d e g e n e r a t i n g a x o n (Fig. 3D). H o w e v e r , t h e r e w a s n o e v i d e n c e of s y n a p s e s s u c h as m e m b r a n e s p e c i a l i z a t i o n s o r a c c u m u l a t i o n o f s y n a p t i c vesicles in a n y o f t h e a p p o s i t i o n s . O n l y a b o u t 8% (54 o u t o f 680) o f the E L I c o n t a i n i n g
110
Fig. 3. UItrastructrual relationship between degenerating axons and terminals or preterminal axons containing EL1. Within bundles of myelinated axons (A) and within more cellular portions (B-D) of the striatum. The degenerating (rig) and ELI containing processes show close contacts (arrows) at their apposed surfaces. Bar = 0.25 #m. terminals were in direct contact with a degenerating axon. I n c o n t r a s t to t h e d e n s e a c c u m u l a t i o n o f E L I i n a x o n t e r m i n a l s , p e r i k a r y a a n d dendrites were only lightly labeled and were sparsely distributed within the more
111 dorsal and medial portions of the striatum. Synaptic specializations between the cortical efferents and dendrites having ELI were not detected.
Discussion (-.4) Conuergence on c o m m o n dendrite
One of the primary findings of this study is that some of the cortical efferents and terminals showing ELI form synapses with the same dendrites within the neostriaturn. The presence of spines associated with dendrites receiving both cortical efferents and ELI containing terminals suggests that they may belong to medium spiny neurons [14]. These neurons are abundant in the neostriatum [14,15] and receive convergent excitatory input from cerebral cortex, thalamus and substantia nigra [16]. The terminals from the cerebral cortex and thalamus contain small clear synaptic vesicles and make asymmetric contacts with dendrites of the medium spiny neurons [17,18]. A second type of terminal which converges onto the same dendrites of spiny neurons has many similarities to the terminals showing ELI in the present study. In common with the ELI containing terminals, this second type of afferent to the spiny neuron contains large pleomorphic vesicles, forms symmetric synapses with dendrites and dendritic spines [14] and remains intact following surgical isolation of the striatum [19]. The fact that this type of terminal remains intact after surgical isolation, suggests that the terminals may belong to intrinsic, possibly spiny, neurons. The type of transmitter within the neuron receiving convergent cortical and ELI containing terminals can be postulated on the basis of other biochemical and immunocytochemical studies. One likely possibility is y-aminobutyric acid (GABA). GABA is an inhibitory transmitter found in many of the medium sized spiny neurons of the neostriatum [20,21]. Furthermore GABA has specific interactions with a variety of putative transmitters including L-glutamate in the basal ganglia [22]. A second possibility for the putative transmitter in the common recipient neurons is substance P which is also contained within the striato-nigral pathway [23]. These and other alternatives must be evaluated through additional double or triple labeling techniques. The relatively low number of observed interactions between cortical efferents and ELI containing terminals probably reflects certain methodological factors. For example, incomplete penetration of the antiserum limits the detection of ELI to the outer 1-2 ~m of the Vibratome section; whereas the degenerating terminals are recognizable throughout the tissue [37]. This factor tends to bias the sample in favor of the independent occurrence of degenerating terminals. In addition only single sections were analyzed in this study which eliminates the possibility of detecting interactions outside the plane of section. The present demonstration that cortical efferents form asymmetric densities and intrinsic ELI containing terminals make symmetric junctions is consistent with biochemical and electrophysiological studies correlating postsynaptic densities with excitatory (asymmetric) or inhibitory (symmetric) functions [24]. Symmetric junc-
112 tions were more numerous in this study than in our previous one [1]. Conceivably, there may be regional variations in the type of specializations formed by terminals containing ELI. In this study, all of the samples were taken from the more dorsal and medial neostriatum, whereas in the earlier report only limited sampling was done in this region. Symmetric junctions also have been reported for terminals showing ELI by other investigators [2]. Convergence of the ELI containing terminal on neurons receiving cortical input is analogous to convergence of enkephalin labeled terminals and primary afferents in the substantia gelatinosa of the spinal trigeminal complex [25]. In the latter region, collaterals of intrinsic neurons containing ELI terminate on the dendrites receiving primary afferents from the trigeminal ganglion [25]. Thus, neurons containing the opioid peptides may internally modulate the effects generated by afferents in at least two different regions of the CNS. However, in the striatum, the enkephalin containing neurons are known also to project to the globus pallidus [26,27]. This structural arrangement of synapses on intrinsic spiny neurons and their projections to the globus pallidus make it possible for neurons containing enkephalin to regulate the output of the basal ganglia through both intrinsic and extrinsic interactions. Feedback systems involving the neostriatal opiate containing neurons, cortical efferents and other dopaminergic efferents from substantia nigra are suggested by the present findings and the results of an earlier study combining the localization of tyrosine hydroxylase with anterograde degeneration following cortical ablation in the rat neostriatum [11]. The degenerating cortical efferents also shared the same dendrites with terminals showing immunoreactivity for tyrosine hydroxylase which are primarily dopaminergic in the neostriatum. Thus at least some of the neurons receiving cortical and nigral efferents may also be postsynaptic to the ELI containing terminals. This postulated interaction is supported by a number of biochemical and behavioral studies which suggest a relation between opiates and dopamine at postsynaptic receptors in the striatum [28,29].
(B) Axo-axonic interrelationships The present observation of appositions between terminals showing ELI and degeneration following cortical removal suggests that there may be additional axo-axonal interrelationships. Studies of opiate receptor binding support the concept of axonic interactions [30]. There is considerable pharmacological evidence for the existence of opiate receptors on afferent axons in the neostriatum and dorsal horn of the spinal cord [30-32]. While most of the existing studies indicate an interaction between opiates and opiate receptors on dopaminergic afferents to the striatum [33,34], lesion experiments suggest that there also may be opiate receptors on other afferent axons [7]. Neurotoxic lesions employing 6-hydroxydopamine for destruction of the dopaminergic nigrostriatal projection combined with kainic acid lesions of intrinsic striatal neurons fail to completely abolish the opiate receptor binding in the striatum [7,30]. Thus, the remaining opiate receptors could be located on nondopaminergic, possibly cortical efferents to the striatum. The absence of morphological evidence of synapses [35] between axons limits our interpretation of the existence or directionality of interrelations between the apposed
113 degenerating and ELI containing terminals. In the present study, dense membrane specializations could be masked by the accumulation of immunoreactivity or degenerative changes. However, even in conventionally stained materials axo-axonic synapses are found infrequently in the striatum [35,36]. Conceivably the postsynaptic densities which usually characterize synapses are not a necessary prerequisite for interactions between the cortical and ELI containing terminals. The postsynaptic densities are associated with calmodulin and cAMP-activated protein kinase [24], thus interactions without observable densities could indicate receptor mediated events not associated with protein phosphorylation. Alternatively, the membrane specializations may be relatively narrow or located along contorted portions of the axon which cannot be readily observed in single or even multiple serial sections. In favor of this latter possibility is the twisted and convoluted shape of the cortical axons which were observed in this and earlier studies [35,36]. The configuration of the cortical efferents as well as the superficial localization of ELI probably also contribute to the relatively infrequent observation of axonal pairs as considered in the above discussion of dendritic interactions.
(C) Cortical efferents and EL1 containing perikarya and dendrites Whether the perikarya and dendrites of ELI containing neurons receive cortical innervation could not be evaluated in the present study. This limitation was due to the small amount of detected immunoreactivity within the perikarya and dendrites. In non-colchicine treated animals, immunoreactivity for many peptides is low in neuronal perikarya [37]. However, using the same antiserum, we have previously shown ELI in perikarya and dendrites within the normal rat striatum [1]. Thus, the absence of ELI within the perikarya and dendrites in the neostriatum of decorticated animals probably reflects other variables such as fixation. Slightly longer post-fixation periods (60 as opposed to 30 min) were used in the present study in order to clearly visualize degenerating terminals. Longer fixation or higher concentrations of glutaraldehyde are known to reduce the immunocytochemical labeling for most antisera [37].
(D) Functional implications The observed associations between cortical efferents and ELI containing neurons include convergence on a single postsynaptic dendrite and direct appositions between t h e respective axon terminals. Convergence on a common dendrite could modulate the output from the neostriatum through either independent or synchronous actions on the recipient neuron. In contrast, the axonal interactions are more likely to be associated with the release of neurotransmitter substances from cortical efferents or the ELI containing terminals.
Acknowledgements This research was supported by a Grant from NSF (BNS-8023914) and from N I H (HL 18974). Virginia M. Pickel also holds a Research Career Development Award (MH 00078). Jean-Jacques Bouyer is a Charg6 de Recherche INSERM.
114
References 1 Pickel, V.M., Sumal, K.K., Beckley, S.C., Miller, R.J. and Reis, D.J., lmmunocytochemical localization of enkephalin in the neostriatum of rat brain: a light and electron microscopic study, Brain Res., 189 (1980) 721-740. 2 Difiglia, M., Aronin, N. and Martin, J.B., Light and electronmicroscopic localization of immunoreaction leu-enkephalin in the monkey basal ganglia, Neuroscience, 2 (1982) 303-320. 3 Atweh, S.F. and Kuhar, M.J., Autoradiographic localization of opiate receptors in rat brain. I11. The telencephalon, Brain Res., 134 (1977) 393-405. 4 Fry, J.P., Zieglgansberger, W. and Herz, A., Development of acute opioid tolerance and dependence in rat striatal neurons, Naunyn Schmiedeberg's Arch. Pharmacol., 313 (1980) 145-149. 5 Frederickson, R.C.A. and Norris, F.H., Enkephalin-induced depression of single neurons in brain areas with opiate receptors-antagonism by naloxone, Science, 194 (1976) 440-442. 6 Malfroy, B., Swerts, J.P., Llorens, C. and Schwartz, J.C., Regional distribution of high-affinity enkephalin-degrading peptidase (enkephalinase) and effects of lesions suggest localization in the vicinity of opiate receptors in brain, Neurosci. Lett., 11 (1979) 329-334. 7 Pollard, H., Llorens, C., Schwartz, J.C., Gros, C. and Dray, F., Localization of opiate receptors and enkephalin in the rat striatum in relationship with the nigrostriatal dopaminergic system: lesions studies, Brain Res., 151 (1978) 392-398. 8 Spencer, H.J., Antagonism of cortical excitation of striatal neurons by glutamic acid diethyl ester: evidence for glutamic acid as an excitatory transmitter in the rat striatum, Brain Res., 102 (1977) 91-101. 9 McGeer, P.L., McGeer, E.G., Scherer, I. and Singh, K., A glutamatergic cortico-striatal path?, Brain Res., 128 (1977) 369-373. 10 Kitai, S.T., Kocsis, J.D., Preston, R.J. and Sugimori, M., Monosynaptic inputs to caudate neurons identified by intracellular injection of horseradish peroxidase, Brain Res., 109 (1976) 601-606. 11 Bouyer, J.J., Park, D.H., Joh, T.H. and Pickek V.M., Structural and chemical analysis of the relation between cortical efferents and tyrosine hydroxylase-containing terminals in rat neostriatum, Brain Res., (1984) in press. 12 Miller, R.J., Chang, K.J., Cooper, R. and Cuatrecasas, P., Radioimmunoassay and characterization of enkephalins in rat tissue, J. Biol. Chem., 253 (1978) 531-538. 13 Sternberger, L.A., lmmunocytochemistry, Prentice Hall, Englewood Cliffs, N.J., 1974. 14 Wilson, C.J. and Groves, P.M., Fine structure and synaptic connections of the common spiny neuron of the rat striatum: A study employing intracellular injection of horseradish peroxidase, .1. Comp. Neurol., 194 (1980) 599-615. 15 Difiglia, M., Pasik, P. and Pasik, T., A Golgi study of neuronal types in the neostriatum of monkeys, Brain Res., 114 (1976) 245-256. 16 Kocsis, J.D., Sugimori, M. and Kitai, S.T., Convergence of excitatory inputs to caudate spiny neurons, Brain Res., 124 (1977) 403-413. 17 Kemp, J.M. and Powell, T.P.S., The site of termination of afferent fibers in the cat caudate nucleus, Phil. Trans. R. Soc. L6nd., 262 (1971) 413-427. 18 Hattori, T., McGeer, G.E. and McGeer, P.L., Fine structural analysis of the cortico-striate pathway, J. Comp. Neurol., 185 (1979) 347-353. 19 Tennyson, V.M. and Marco, L.A., Intrinsic connections of caudate neurons. 11. Fluorescence and electron microscopy following chronic isolation, Brain Res., 53 (1973) 307-317. 20 Ribak, C.E., Vaughn, J.E. and Roberts, E., The GABA neurons and their axon terminals in rat corpus striatum as demonstrated by GAD immunocytochemistry, J. Comp. Neurol., 187 (1979) 261 284. 21 Bernardi, G., Marcini, M.G., Stanzione, P., Cherubini, E., and Mercuri, N., Evidence in favour of GABA as an inhibitory transmitter in rat striatum. In E. Costa and P. Greengard (Eds.), Advances in Biochemical Pharmacology, Vol. 30, Raven Press, New York, NY, 1981, pp. 69-78. 22 Bartholini, G., Scratton B., Worms, P., Zivkovic, B. and Lloyd, K.G., Interactions between GABA, dopamine, acetylcholine and glutamate containing neurons in the extrapyramidal and limbic systems. In E. Costa and P. Greengard (Eds.), Advances in Biochemical Pharmacology, Vol. 30, Raven Press, New York, NY, 1981, pp. 119-128.
115 23 Kanazawa, 1., Emson, P.C. and Cuello, A.C., Evidence for existence of substance P containing fibers in striato-nigral and pallido-nigral pathway in rat brain, Brain Res., 119 (1977) 447-453. 24 Cohen, R.S., Carlin, R.K., Grab, D.J. and Siekevitz, P., Phosphoproteins in postsynaptic densities, Prog. Brain Res., 56 (1982) 49-76. 25 Sumal, K.K., Pickel, V.M., Miller, R.J. and Reis, D.J., Enkephalin-containing neurons in substantia gelatinosa of spinal trigeminal complex: Ultrastructure and synaptic interaction with primary afferents, Brain Res., 248 (1982) 223-236. 26 Cuello, A.C., Evidence for a long leu-enkephalin striopallidal pathway in rat brain, Nature. 271 (1978) 178-180. 27 Preston, R.J., Bishop, G.A. and Katai, S.T., Medium spiny neurons projection from the rat striatum: An intracellular horseradish peroxidase study, Brain Res., 183 (1980) 253-263. 28 Clay, G.A, and Brougham, L.R., Haloperidol binding to an opiate receptor site, Biochem. Pharmacol., 24 (1975) 1363-1367. 29 Govoni, S., Kamakura, K., Spano, P.F., Tonon, G.C. and Trabucchi, M., Interaction of narcotic analgesics with dopamine receptors in rat brain, Pharmacol. Res. Commun., 7 (1975) 96-100. 30 Pollard, H., Llorens-Cortes, C. and Schwartz, J.C., Enkephalin receptors on dopaminergic neurons in rat striatum, Nature, 268 (1977) 745-747. 31 Snyder, S.H. and Simantov, R.J., The opiate receptor and opioid peptides, Neurochemistry, 28 (1977) 13-20. 32 Lamotte, C., Pert, C.B. and Snyder, S.H., Opiate receptor binding in primate spinal cord: distribution and changes after dorsal root section, Brain Res., 112 (1976) 407-412. 33 Chesselet, M.F., Cheramay, A., Reisine, T.D. and Glowinski, J., Morphine and opiate agonists locally stimulate in vivo dopamine release in cat caudate nucleus, Nature, 291 (1981) 320-322. 34 Lubetzki, C., Chesselet, M.F. and Glowinski, J., Modulation of dopamine release in rat striatal slices by delta opiate agonists, J. Pharmacol. Exp. Ther., 222 (1982) 435-440. 35 Bak, I.J., Choi, W.B., Hassler, R. and Usunoff, G.K., Fine structural synaptic organization of the corpus striatum and substantia nigra in rat and cat. In D.B. Calne, T.N. Chase and A. Barbeau (Eds.), Advances in Neurology, Vol. 9, Raven Press, New York, 1975, pp. 25-41. 36 Kemp, J.M. and Powell, T.P.S., The synaptic organization of the caudate nucleus, Phil. Trans. R. Soc. Lond. B, 262 (1971) 403-412. 37 Pickel, V.M., Immunocytochemical Methods. In L. Heimer and M.J. Robards (Eds.), Neuroanatomical Tract Tracing Methods, Plenum Press, New York, 1981, pp. 483-509.