Evidence for autapses in the substantia nigra

Brain Research, 200 (1980) 467-473 © Elsevier/North-Holland Biomedical Press

467

Short Communications

Evidence for autapses in the substantia nigra

ATHANASIOS B. KARABELAS and DOMINICK P. PURPURA Department of Neuroscience and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N.Y. 10461 (U.S.A.)

(Accepted July 3rd, 1980) Key words. Autapses - - substantia mgra pars reticulata - - intracellular horseradish peroxldase - -

axon collaterals Intracellular recording and intracellular HRP staining were employed to trace the recurrent terminal plexus of cat substantia nigra pars reticulata neurons. Autaptlc neurons were labeled. The axon of an autaptic neuron was found to emit a recurrent collateral which distributed 'en passage' and terminal boutons contacting dendrites of the parent cell. Antldromic ventromedial thalamlc stimulation elicited a recurrent IPSP in the autaptic neuron. The term autapse was proposed by Van der Loos and Glaser 20 to describe a synapse between a neuron and a collateral of its own axon. Possible autaptic contacts have been sporadically observed in Golgi preparations of rabbit18, 20, dog 18, h u m a n 1~ and rat 5 cerebral cortex, cat spinal cord 16 and monkey neostriatumL The possibility of autapses on rat Purkinje cells 6 and striatal medium spiny cells 14, is supported by recent intracellular horseradish peroxidase (HRP) stud]es. In the substantia nigra, Golgi impregnation of pars reticulata neurons has shown 1-2 initial collateralsS,la, but has failed to reveal terminal arborizations despite Mirto's (1896) contribution quoted by Schwyn and Fox 17. During the course of a morphophysiological intracellular H R P exploration of the cat substantia nigra we observed axonal collaterals recurrent to the substantia nigra pars reticulata and completely revealed their terminal plexus. For the majority of HRP-stained substantia nigra pars reticulata single neurons, the clear separation between axonic and dendritic fields suggested that their recurrent terminals contacted dendrites of neighboring cells. However, in a few instances axonlc and dendritic overlap resulted in the identification of autaptic arrangements on dendrites of the parent cell. Data are derived from a series of experiments in which collicular and thalamic stimulation were employed to activate antidromically the nigrotectal2,7,11 and nlgrothalamic2,4, 7 pathways. Striatal stimulation was also used to evoke orthodromic synaptic activities m substantia nigra pars reticulata neurons 1°,21. The experiments were performed on cats anesthetized with sodium pentobarbltal (35 mg/kg), immobilized with gallamine trieth]odide and artificially respired. Bilateral pneumothorax, vertebral suspension and cisternal drainage were routinely

468 performed to reduce pulsation. Following cramotomy the superior colliculu~ was exposed by removal of overlying cortex and hlppocampus to enable insertion ol the recording mlcroelectrode at an angle towards the substanUa nigra. Concentric bipolar stimulating electrodes were positioned stereotaxlcally in the ventromedlat and ventrolateral thalamlc nuclei. Stimulating electrodes were also placed under visual control m the intermediate layer of the superior colhculus and in the exposed caudate. Beveled glass recording microelectrodes were filled with 7 °,o H R P (Sigma VI) solution i11 50 mM Trls buffer and 500 mM KCI (pH 7.4) and had DC resistances of 30-60 Mf~. Once a substantla nlgra pars reticulata neuron was impaled and Identified physiologically, HRP was injected intracellularly with depolarizing pulses (10-15 nA, 600 msec duration l/sec) dehvered through the microelectrode for 30 sec to 3 mln. One to six hours elapsed between staining and perfuslon with 1 ° o paraformaldehyde and 1.25 ~,, glutaraldehyde m phosphate buffer (pH 7.4). Brains were removed, blocked and kept in 20 °/o sucrose solution until they sank. Serial parasaglttal sections were cut at 100/~n~ on a freezing microtome. The H R P reaction product was intensified by placing the sections in a 5 3o cobalt chloride solution t prior to reaction with diaminobenzldme tetrahydrochlorlde Two autapt~c substantia nigra pars retlculata neurons were labeled. One had twenty autapses and the other had one autapse. An estimation of the frequency distribution of autapt~c neurons Is possible only by comparison with 14 HRP-stamed substantia mgra pars retlculata neurons whose recurrent non-autaptlc axon terminal plexus was completely revealed by the enzyme injection Both autaptic neurons were situated in the most rostral parts of the substant~a mgra pars reticulata. Although their somadendmIc features were impressively similar, the cells did not specifically characterize autaptlc neurons, since non-autapt~c substantia nlgra pars reticulata neurons exhiNted similar somadendrit~c features. Their elongated fusiform cell body (Fig. 1A) measured 35 ,urn m the long ax~s - - not including the dendritic trunks. Two primary dendrites emerged from each dendritic trunk. The neuron illustrated m Fig. 1A and B had a fifth primary dendrite artslng from the medial side of the soma. Most of the dendrites coursed ventrally and occupied a roughly mangular space (Fig. IB). Splne-hke short (1 #m) dendritic protrusions (Figs. 1A and 2F) were distributed particularly on secondary and ternary dendrites either in patches or sequentially arranged (22 per 50/~m). Some 4-6/~m long pedunculated spines were also encountered. Axons of the autaptlc neurons emerged from a dorsal primary dendrite (Fig. 1A). A short dendritic branch arose prior to the axon hillock (Fig. IA). Axons had a diameter of 1.5/~m. Within the substantla mgra, axons collaterahzed and assumed a caudal and eventually dorsal trajectory. The main axon was not followed to ~ts termination but after branching and exiting from the substantia nigra it pursued a dorsal course, presumably towards the superior colliculus. Seven collaterals (CI-Cv) arose from the axon of the autaptlc neuron reconstructed and shown in Fig. 1B. These originated at distances of 90-1400 #m from the axon hillock. The second autaptlc neuron had 4 collaterals arising at distances of 330-1300/~m from the axon hillock. Possible autaptlc contacts were established by the first collateral of both

469

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Fig. 1. Morphophysiological profile of an autapttc neuron. A: partial reconstruction delineating the autaptic loop. Axonal system is drawn by solid line. Free arrows point to autaptm contacts, stars indicate non-autaptie terminals and two thick arrows point to the extent of the 'en passage' branch winding around a primary dendrite. B: complete somadendritic reconstruction and partial illustration of the axonal system. Seven collaterals are labeled Ct-Cr. Main axon CeC~, and C7 have been traced for longer distances. C: recurrent graded IPSP evoked by ventromedJal (VM) thalamic stimulation (upper trace) and extracellular control (lower trace). Scales equal 50/~m in A and 500 ttm in B. Orientation is shown for A and B.

470 neurons. The recurrent collateral of the autaptic neuron m F~g. 1A and B Is shown to arise from the axon m the photomicrograph of F~g. 2A. It turned ventrally towards the dendritic territory of the parent cell where it gave off: (I) a branch winding around a primary dendrite (F~gs. IA and 2B-E); and (2) s~de twigs terminating on a thin branch of another prmlary dendrite (Figs. I A and 2G), on the primary dendrite ~tself (Figs. IA and 2F) and on Its secondary branch (Figs. 1A, 2F and G) a fiber chmbmgon and along a primary dendrite (Fig. I A and partly in 2F). The winding "en passage' fiber emanated from the recurrent collateral at a &stance of 200/zm from the collateral origin. This axonal branching wrapped itself around a primary dendme &stributing 15 'en passage' boutons over 210 #m of dendritic length. The &stance between the first autaptic contact and the origin of the dendrite from the soma was 75/~m. After emitting the winding branch, the recurrent collateral continued more ventrally, becoming varicose (Fig. 2B, F and G), and running parallel to another primary dendrite (Figs I A and 2F). Autaptic contacts were established by 3 branchlets arising at acute angles and terminating on spines or spree-free dendritic surfaces (Figs I A, 2F and 2G). The beaded chmbmg fiber (Fig 1A, upper pomon of Fig. 2F) provided two more side twigs terminating on the primary dendrite. The distances of the autaptic contacts from the points of the origin of the collateral and dendrite ranged between 350-625 #m and 240--400/~m respectively. Non-autapt~c labeled terminals were also wsualized. The labeled boutons of the winding fiber closely correspond to the type III boutons described by Rinvik and Grofov/t ~5 in the cat substantm nlgra. They have an apparent 'en passage' character, similar shape (most often elhpsold) and size (largest diameter 1.6-3.0 /zm), shortest dmmeter 1.0-1.5 /~m) and contact large dendrites. Moreover, Rinvik and Grofov/t15 suggested an mtramgral origin of these boutons from coUateralized axons. Due to the hmitations of the light microscopy the possibility cannot be excluded that the recurrent terminals may contact closely apposed dendrites of non-stained neurons or other axonal terminals of extrinsic or intrinsic origin. The latter possibility is supported by the ultrastructural demonstration of occasional axo-axonic synapses m the cat substantm nigra 15. In th~s case, the labeled 'en passage' boutons may be postsynapt~c to boutons of extrinsic origin since Rinvik and Grofovfi~ have found that axo-axonic synapses are formed only between type I and type 1II boutons and type 1 boutons are of extrinsic (striatal) origin and invarmbly presynaptlc to type 111 boutons. Type II1 boutons are always engaged in symmemcal contacts 15 suggestive of inhibitory synapses. In view of recent physiological studiesS, 19 in&cating monosynaptlc inhibition of thalamic neurons after nigral stimulation, mhabitory synaptic action of the nigral recurrent terminals on mtranigral targets can be anticipated. Indeed, antidromic activation and recurrent inhibition of non-autapt~c substantia nigra pars reticulata neurons has been shown (unpublished personal observations). In the case of the autaptlc neuron illustrated in Fig. 1A and B, ventromedml thalamic stimulation elicited a graded recurrent IPSP with constant latency (10.2 msec) to stimuh of gradually increasing intensity (Fig. 1C) whereas colhcular and striatal stimulation pro-

471

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Fig. 2. Photographic details of autaptic relations of neuron m Fig. 1. A: the recurrent collateral (C~) arises from the main axon. B: the arrowhead points to the origin of the winding " e n passage" branch. C - D : successive focal planes of the dendrite shown in B. E: the same dendrite in an adjacent section. Arrows in B - E indicate "on passage" boutons. F: side twigs arising from the recurrent collateral and terminating on another dendrite at points indicated by downward arrows. The upward arrow points to a terminal provided by a beaded climbing fiber emanating in an acute angle from the recurrent collateral. G : contact of the recurrent collateral with a thin branch of the dendrite shown in F. Scale equals 10 pm. Scale in B applies for A - E and G. (All photomicrographas obtained with 100 × oil immersion objective).

472 ved meffective. T h e ascending collaterals Co. a n d C7 (Fig. 1B) o f this n e u r o n extended b e y o n d the rostral b o r d e r o f the s u b s t a n t i a m g r a but, unfortunately, were not traced for long distances t o w a r d s their targets p r e s u m a b l y including the v e n t r o m e d i a l t h a l a m i c nucleus. Therefore, it c a n n o t be determined from available d a t a whether the r e c u r r e n t I P S P was generated by possible actavation o f these collaterals at t h a l a m l c stimulating sites or by activataon o f n i g r o t h a l a m i c fibers o f other SN R neurons having recurrent terminals in c o n t a c t with the a u t a p t i c neuron. Whale the fmlure to observe a n t i d r o m i c spikes suggests i n v o l v e m e n t o f neaghbormg SN R neurons m the recurrent 1PSP (F,g. 1C) it is not possible to rule o u t impulse b l o c k a d e at the first collateral b r a n c h p o i n t o f the autaptac axon. F u r t h e r studies o f this assue revolving c o m b i n e d light a n d electron microscoptc tracing o f H R P - s t a m e d mgral neurons and electrop h y s i o l o g i c a l analysas o f recurrent activaties are m progress. It is sufficient here to call a t t e n t i o n to a possibly ~mportant role o f a u t a p t l c relations in the complex o p e r a t i o n s o f the s u b s t a n t m m g r a and n e u r o n s at o t h e r neuraxml s~tes.

This study was s u p p o r t e d by grants f r o m the N a t , o n a l Institutes o f H e a l t h (NS07512 a n d HD-01799).

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