by interneurones which are themselves inhibited by Renshaw cells, these

Transcription

1 J. Phyaiol. (1978), 285, pp With 9 text-ftgure Printed in Great Britain CROSSED DISYNAPTIC INHIBITION OF SACRAL MOTONEURONES BY ELZB1ETA JANKOWSKA, YVES PADEL* AND PETER ZARZECKIt From the Department of Physiology University of Gdteborg, Goteborg, Sweden (Received 31 March 1978) SUMMARY 1. Intracellular recording was made from motoneurones in lower sacral (S2 and S3) segments of the spinal cord in cats, to analyse the neuronal organization of the inhibition evoked in these motoneurones from contralateral afferents. 2. It was confirmed that stimulation of the lowest threshold afferents of contralateral dorsal roots evokes i.p.s.p.s with latencies similar to those of disynaptic i.p.s.p.s. evoked from group Ia muscle spindle afferents in limb motoneurones. 3. The crossed disynaptic i.p.s.p.s in sacral motoneurones were found to be mediated by interneurones which are themselves inhibited by Renshaw cells, these interneurones and Renshaw cells being activated from the dorsal and ventral roots respectively, on the side of the body opposite to the location of the inhibited motoneurones. 4. In unanaesthetized decerebrate preparations crossed recurrent facilitation of sacral motoneurones was evoked with a time course similar to that of recurrent facilitation of lumbar motoneurones. It was taken to indicate a tonic inhibition of sacral motoneurones by interneurones responsible for their crossed disynaptic inhibition, and a disinhibition following stimulation of contralateral ventral roots. 5. In anaesthetized preparations crossed recurrent inhibition appeared, instead of the recurrent facilitation, in more than one half of the tested motoneurones. 6. A comparison of the input from ipsilateral and contralateral afferents to identified motoneurones of tail muscles with the input to pudendal motoneurones led to the conclusion that crossed disynaptic inhibition is evoked specifically in tail motoneurones. 7. Intracellular staining of sacral motoneurones with horseradish peroxidase revealed that the tail motoneurones and others with crossed disynaptic inhibition differ from the pudendal motoneurones in their location and in a number of morphological features; tail motoneurones are larger, they have differently directed dendrites and they show more extensively branched initial axon collaterals which appeared to ramify only within the ventral and lateral parts of the ipsilateral ventral horn. 8. One Renshaw cell which was stained with horseradish peroxidase was found to project contralaterally, after giving a number of axon collaterals ipsilaterally. * Present address: Service de Neurophysiologie C.N.R.S., 31 Chemin Joseph-Aiguier, 139 Marseille, France. t Present address: Department of Physiology, Queen's University, Kingston, Ontario, Canada, K7L 3N6.

2 426 E. JANKOWSKA, Y. PADEL AND P. ZARZECKI INTRODUCTION Motoneurones located in the lower sacral (S2 and S3) segments of the cat spinal cord are inhibited with a very short latency following stimulation of contralateral afferents (Lloyd, 1941, 1944; Wilson & Lloyd, 1956; Lloyd & Wilson, 1959). On stimulation of the contralateral dorsal roots, inhibitory post-synaptic potentials, i.p.s.p.s, are evoked in motoneurones only about 7 msec later than monosynaptic e.p.s.p.s from the ipsilateral dorsal roots according to Curtis, Krnjevi6 & Miledi (1958), or S3-O7 msec later according to Frank & Sprague (1959). Since the segmental latencies of these i.p.s.p.s are within the same range as segmental latencies of the undoubtedly disynaptic i.p.s.p.s evoked in lumbar motoneurones they do preclude a polysynaptic coupling, but may not by themselves be sufficient to differentiate between a disynaptic and a direct coupling postulated by Lloyd (1941). For instance, if the inhibition were evoked via particularly thin or long collaterals of contralateral afferents, slowing of the conduction velocity along them (cf. Jankowska &A Roberts, 1972 and Wall & Werman, 1976) might introduce a considerable increase in the conduction time. However, the spatial facilitation and occlusion between i.p.s.p.s evoked from two separate contralateral dorsal roots (Curtis et al. 1958), similar to that shown for the disynaptic i.p.s.p.s in lumbar motoneurones (Eccles & Lundberg, 1958), practically exclude that these i.p.s.p.s. could be evoked monosynaptically. In the following they will therefore be referred to as 'crossed disynaptic i.p.s.p.s'. These i.p.s.p.s resemble disynaptic i.p.s.p.s evoked in motoneurones to limb muscles also in being evoked from lowest threshold afferents. It was therefore of interest to find out whether interneurones which mediate the crossed disynaptic i.p.s.p.s. are under control of Renshaw cells, or are uninfluenced by these cells. Previous studies have shown that interneurones which are interposed in the reflex path of the disynaptic Ia reciprocal inhibition of motoneurones, and apparently only those, are inhibited by Renshaw cells (Hultborn, Jankowska & Lindstr6m, 1971 a). Finding recurrent depression of the crossed disynaptic i.p.s.p.s evoked from the contralateral roots would thus indicate that the activity of the left and the right side muscles innervated by motoneurones receiving those i.p.s.p.s may be as closely related as that of the strict antagonists operating at the same joint of an extremity. The interpretation of the function of crossed disynaptic inhibition has been hampered by a lack of information as to the identity of the sacral motoneurones in which it is evoked. The second aim of this study was therefore to identify these motoneurones and evidence will be presented that they are among those innervating tail muscles. Crossed connexions within the sacral cord being different from those in the lumbar segments, it was also investigated whether any recurrent inhibition of motoneurones is evoked from the contralateral ventral roots. Since no crossed recurrent inhibition has so far been reported, except for preliminary observations of Ishikawa (1961) and such inhibition of parasympathetic neurones via a group of cells apparently different from the typical Renshaw cells (De Groat & Ryall, 1968; De Groat, 1976), its analysis became a third aim of this study.

3 CROSSED DISYNAPTIC INHIBITION METHODS The experiments were performed on twelve cats, six under chloralose anaesthesia (5-7 mg/ kg) and six decerebrated at amideollicular level, the dissection being done underether anaesthesia. Laminectomy was made from L5 to the first coccygeal vertebrae. In the main series of experiments on six cats, dorsal roots from SI to the first coccygeal and ventral roots from L6 to the first coccygeal, or a part thereof, were sectioned bilaterally and prepared for stimulation or recording. In the remaining cats dorsal roots were prepared bilaterally and the corresponding ventral roots contralaterally to the side of recording. S2-S3 ventral roots which were left intact on the side of recording were placed on bipolar silver hook electrodes for stimulation in continuity. Motoneurones projecting via these roots to the pudendal nerve or to tail muscles could then be identified. In these experiments the pudendal nerve was stimulated in the ischiorectal fossa, and two or three bipolar needle electrodes were inserted into those tail muscles contracting on stimulation of S2 and S3 ventral roots. That these needle electrodes were activating axons of tail motoneurones was confirmed by the observation that the muscle contractions evoked from them were eliminated by the administration of gallamine. Intracellular records were taken from motoneurones in 82 and S3, either on one or on both sides. Ipsilateral and contralateral dorsal roots were stimulated to evoke post-synaptic potentials in the motoneurones and when short-latency i.p.s.p.s of contralateral origin appeared, the effects of conditioning stimulation of various ventral roots were tested on them. In three cats the effects of stimulation of contralateral dorsal and ventral roots were tested on monosynaptic reflexes. These were recorded from S2 or S3 ventral roots and were evoked by stimulation of the corresponding dorsal roots. Intracellular recording from motoneurones was done with micro-electrodes filled with 2 m-k citrate solution. In three experiments micro-electrodes filled with 2% horseradish peroxidase (HRP) in -3 m-nacl were used as described previously (Czarkowska, Jankowska & Sybirska, 1976a). The recorded potentials were photographed from the oscilloscope directly or after averaging with a Hewlett Packard averager (type 548 A). When conditioned and nonconditioned i.p.s.p.s were compared they were fed alternately to two parts of the average's memory. RESULTS depression by volleys in motor axon Crossed inhibition of sacral motoneurones and its collateral Short-latency i.p.s.p.s were evoked on stimulation of contralateral dorsal roots (S2, S3 or coccygeal) in sixty-seven out of 127 motoneurones in S2 segment and in thirty-five out of forty motoneurones in S3 segment. The i.p.s.p.s appeared with segmental latencies ranging between 1-5 and 1-6 msec. Their amplitudes varied considerably but were in most cells between 1 and 3 mv when evoked from the most effective dorsal root. As described previously (Curtis et al. 1958; Frank & Sprague, 1959), and illustrated in Fig. 1, they were evoked from the lowest threshold afferents and usually reached a maximal amplitude at stimulus strengths times threshold. Stimulation of ventral roots ipsilateral to the stimulated dorsal roots and contralateral to the motoneurones under investigation, markedly depressed the i.p.s.p.s subsequently evoked. The effect was strongest on submaximal i.p.s.p.s and when the intervals between the conditioning ventral root stimuli and the test dorsal root stimuli were between 5 and 15 msec, as shown in Figs. 1 and 2 respectively. Under such conditions the depression of the i.p.s.p.s was found in all motoneurones, being practically complete in more than half of them. The depression usually followed stimulation of S2 and S3 as well as of coccygeal ventral roots, although stimulation of one of them was often more effective than of the others. It was 427

4 428 E. JANKOWSKA, Y. PADEL AND P. ZARZECKI maximal when the stimulation was about 2 times threshold. No relation was found between the location of the motoneurones or dorsal roots which gave rise to the tested i.p.s.p.s, and the level from which these i.p.s.p.s were most effectively depressed by conditioning stimulation of the ventral roots. This holds true for effects evoked from S2 and S3 dorsal and ventral roots. No depressant effect from SI was A thr B 1.1 C 1.2 D 13 E 1.5 co S3 DR _ 7 mv 1+5 msec F 1.5 G 1.1 H 1.15 / 1-25 J 155 co S2 VR+ co S3 D.R.....-i... co S3 DR a : ^ Test pulse5mv K L 1+5 msec X 8-8-.~6-6-. E o!. 2 ' *2 13 1*4 1.5 X thrdr V X thrdr Fig. 1. Crossed disynaptic i.p.s.p.s and their depression by conditioning stimulation of the contralateral ventral roots (VR). In A-E upper traces are intracellular records from a motoneurone in the S2 segment while lower traces are records from the surface of the spinal cord close to the dorsal root (DR) entry zone. Figures above the records indicate strength of stimulation of a contralateral (co) dorsal root in multiples of threshold. In F-J all the records (averaged) are from a motoneurone in S3. Lower traces show i.p.s.p.s evoked by stimulation of a contralateral dorsal root (second arrow) alone; in the upper records this followed a conditioning stimulation of a contralateral ventral root (first arrow). In K and L the normalized amplitudes of the i.p.s.p.s from this cell are plotted against the strength of stimulation of the dorsal roots. observed on i.p.s.p.s evoked from S2 or S3 (cf. Fig. 3H and I),when tested in about twenty motoneurones and on monosynaptic reflexes, and no disynaptic i.p.s.p.s appeared in S2 motoneurones on S1 dorsal root stimulation (tested in about thirty motoneurones). The effects of the conditioning ventral root stimulation alone differed in different motoneurones (see below). The depression of the tested i.p.s.p.s was, however, independent of whether it was combined with a depolarization (Fig. 1F-J, Fig.

5 CROSSED DISYNAPTIC INHIBITION 429 2A-E, Fig. 3 F, J), weak hyperpolarization (Fig. 4) or no detectable effect on the motoneurones' membrane potential. A similar result was obtained in the case of the depression of the disynaptic i.p.s.p.s evoked from group I muscle spindle afferents in lumbar motoneurones (Hultborn et al a). The depression is, therefore, attributed to the effects of Renshaw cells activated by volleys in the contralateral ventral roots upon interneurones interposed between the contralateral dorsal roots and the motoneurones. A B C D E cos3vr+' co S3 DR ' f co S3 DR F d Q. 6- I V V~~~~~~~~~~~~~~~ 1-..., i msec test conditioning-interval 5+25 msec Fig. 2. The time course of recurrent depression of crossed disynaptic i.p.s.p.s In A-E are sample intracellular records from a motoneurone in S2 following stimulation of a contralateral dorsal root (co S3 DR, second arrow), alone in lower traces, or combined with stimulation of a contralateral ventral root (co S3 VR, first arrow) in upper traces. Increasing time intervals between the two stimuli from A to E. Calibration pulse: 1 msec, 2 #etv. Note that the conditioning stimulation of the ventral roots evoked recurrent depolarization (recurrent facilitation) of the motoneurone and that the i.p.s.p.s were superimposed on it. The recurrent depression of the i.p.s.p.s (amplitudes of the conditioned i.p.s.p.s normalized) as a function of the interval between the test and the conditioning stimuli is plotted in F. The time course of this depression may be compared with the time course of the recurrent facilitation in Fig. 3 J. The depression of the i.p.s.p.s due to inhibition of interneurones mediating them could not be tested with conditioning stimulation of the ipsilateral ventral roots because such stimulation evoked too strong a recurrent inhibition in the tested motoneurones (see Fig. 4A, E, K), and the hyperpolarization associated with this could by itself account for a decrease in amplitude of the test i.p.s.p.s. Crossed recurrentfacilitation Motoneurones innervating hindlimb muscles may become depolarized following volleys in ipsilateral motor axon collaterals and Renshaw cells when these volleys evoke their disinhibition (Wilson, 1959; Wilson, Diecke & Talbot, 196; Wilson &

6 43 E. JANKOWSKA, Y. PADEL AND P. ZARZECKI Burgess, 1962). The phenomenon was attributed to inhibition by Renshaw cells of tonically active interneurones producing a steady hyperpolarization of motoneurones (Wilson & Burgess, 1962); these interneurones were subsequently identified as the ones that mediate the Ia reciprocal inhibition ofmotoneurones (Hultborn, Jankowska, Lindstr6m & Roberts, 1971 d). Since interneurones which mediate the crossed inhibition of sacral motoneurones appear also to be inhibited by volleys in motor axon A i S2 I VR 8 is3dr C is3vr _a_ I,. Ị I I I J msec (A, B, D, E,) D co S3DR E co S2 DR, F co S3 VR 1+5 msec (G-I) 1+5 msec (C, F) 2+1 msec (J) G a L- - co S3 DR H cos1 VR+ co S3 DR / cos3vr+ co S3 DR J co S3 VR I 2 mv (A-F) 1 mv (G-I) I control K 2-. QE. *._ O. U a 1- * *- *._ -t * * S Crossed recurrent facilitation Fig. 3. For legend see facing page. 3- mv

7 CROSSED DISYNAPTIC INHIBITION 431 collaterals, stimulation of the contralateral ventral roots should likewise evoke a recurrent facilitation under conditions when these interneurones are tonically active. This facilitation was indeed seen in practically all sacral motoneurones that were tested in unanaesthetized decerebrated preparations; in these preparations the tonic activity of the Ia inhibitory interneurones is most pronounced (Hultborn, Jankowska & Lindstr6m, 1971 b). Illustrations of this phenomenon are in Fig. 2A-E (upper records) and in Fig. 3F andj. The amplitude of the recurrent depolarization could be expected to depend on the A is2vr C is3vr E is2vr G is3vr i is2vr K is3vr I~ ~~~~ B co S2 VR D co S3 VR F co S2 VR H co S3 VR J co S2 VR L co S3 VR pv Control Control 2OmV(C),2mV(G,I,Kj,1 mv(a,b,d,j,) 1+5 msec Fig. 4. Crossed recurrent inhibition. Nembutal anaesthetized animal. The three groups of records A-D, E-H and I-L, are each from a different S3 motoneurone. In each case the upper record shows membrane potential changes. Where the lower traces are labelled 'control', these are intracellular records without stimulation; elsewhere the lower traces are records from the surface of the lateral funiculus at the level of recording. The illustrated potentials were evoked from ipsilateral (i) and contralateral (co) S2 and S3 ventral roots as indicated. Note that only i.p.s.p.s were evoked from i ventral roots, but that either recurrent facilitation (F) or recurrent inhibition (D, H, J, L) was evoked from co ventral roots and that the latency of i.p.s.p.s evoked from both sides was similar. Calibration pulse in averaged records is 1 msec and 1 1sV(for E, F, H) or 2 1uV (for L). Fig. 3. Crossed recurrent facilitation. In A-F and G-J are records from two motoneurones in S2; upper traces are intracellular records, lower traces being from the surface of the spinal cord. Antidromic spike potential (A), monosynaptic e.p.s.p. (B) and recurrent i.p.s.p (C) were evoked by stimulation of ipsilateral (i) S2 ventral root, S3 dorsal root and S3 ventral root respectively, as indicated. Disynaptic i.p.s.p. (D), a most likely disynaptic e.p.s.p. (E) and recurrent facilitatory potential (F) were evoked in the same motoneurone from contralateral S3 dorsal, S2 dorsal and S3 ventral roots. Note the similar latency and time course of the recurrent inhibition and facilitation. In G-I is shown a crossed disynaptic i.p.s.p. from contralateral (co) S3 dorsal root and its depression following conditioning stimulation of contralateral S3 ventral but not of SI ventral root, only the former evoking the recurrent facilitation of the motoneurone by itself. A total time course of the recurrent facilitation with DC recording (averaged record) is shown on a slower time scale in J. In K, amplitudes of the crossed recurrent facilitation evoked in twenty-four motoneurones from a most effective ventral root are plotted against amplitudes of the maximal crossed disynaptic i.p.s.p.s similarly evoked from the most effective dorsal root.

8 432 E. JANKOWSKA, Y. PADEL AND P. ZARZECKI degree of the tonic inhibition of motoneurones and on the effectiveness of Renshaw cells in depressing the activity of interneurones which produce this inhibition. In confirmation, we have found that the largest recurrent depolarization was evoked by stimulation of the same ventral root from which the most effective depression of the disynaptic i.p.s.p.s was produced. The amplitude of the largest recurrent depolarization was also roughly related to the amplitude of the largest disynaptic i.p.s.p.s, as indicated by the graph in Fig. 3. When the onset of the recurrent depolarization was distinct enough to allow measurements, the latencies were between 1-4 and 2- msec (mean + S.D. = ; n = 25). These were within the same range as in lumbar motoneurones which is compatible with the proposed mechanism of disinhibition of motoneurones (cf. Hultborn et al d). A is2dr B cos2vr+ C cos2 DR+ D cos2vr+ i S2 DR i S2 DR co S2 DR+ S2 DR -~~~~~~~~~~~~~~~rrmlrrml 1+5 msec Fig. 5. Crossed inhibition and disinhibition of monosynaptic reflexes. Nembutal anaesthetized animal. A, test monosynaptic reflex in S2 ventral root evoked by stimulation of S2 dorsal root. B, recurrent inhibition of the monosynaptic reflex by conditioning stimulation of co S2 ventral root. C, crossed inhibition of the test monosynaptic reflex by a preceding stimulation of co S2 dorsal root; the arrows indicate the arrival of the two incoming volleys. D, disinhibition evoked by a preceding stimulation of the Co S 2 ventral root. The time to peak and the duration of the recurrent depolarization (about 5-1 and about 5-7 msec respectively) also corresponded with the optimal and the longest intervals between the conditioning stimulation of the ventral roots and the test stimulation of the dorsal roots at which the depression of the disynaptic i.p.s.p.s was observed, as shown in Fig. 2. Crossed recurrent inhibition In preparations in which recurrent facilitation was abolished by Nembutal (cf. Hultborn et al d), stimulation of the contralateral ventral roots sometimes evoked an i.p.s.p. These recurrent i.p.s.p.s were found in sixteen out of twenty-eight tested motoneurones and are illustrated in Fig. 4 with records from three motoneurones in S3 (A-D, E-H, and I-L respectively). These records are typical in showing that the crossed recurrent i.p.s.p.s were usually of smaller amplitudes than the recurrent i.p.s.p.s evoked from the same side; they were between -1 and -7 mv (mean ± S.D. = ; n = 16) as compared with the amplitude of recurrent

9 CROSSED DISYNAPTIC INHIBITION 433 i.p.s.p.s evoked from a neighbouring ipsilateral root which ranged between.5 and 4-5 mv (mean + S.D. = ; n = 54). With only two exceptions in fourteen tests, the amplitudes of crossed i.p.s.p.s did not exceed 4 % of the ipsilateral i.p.s.p. evoked in the same motoneurones from a neighbouring ventral root. The comparison could not be made with recurrent i.p.s.p.s evoked from the same segment, which would be expected to be even larger (cf. Eccles, 1961), because hyperpolarization following antidromic activation of the motoneurones was superimposed on most of these i.p.s.p.s. The latencies of the i.p.s.p.s evoked in these same motoneurones (n = 14) on stimulation of the opposite side and of the same side were, on the other hand, within the same range, i.e. 1'3-2' msec (mean + S.D. = ) and msec (mean + S.D. = ) respectively. Crossed recurrent inhibition could also be demonstrated by its effect on monosynaptic reflexes. In Fig. 5B contralateral ventral root stimulation reduced the test monosynaptic reflex by about 3 %, the most pronounced effect observed being by more than 6 %. The same figure shows also other phenomena detected by intracellular recording, including a pronounced inhibition of the test monosynaptic reflex on stimulation of a contralateral dorsal root (Fig. 5 C) and a much weaker inhibition when the same dorsal root stimulation was preceded by conditioning stimulation of a contralateral ventral root (Fig. 5D). The identity of sacral motoneurones in which disynaptic inhibition is evoked from contralateral dorsal roots Of three major groups of motoneurones in S2 and S3, (i) those innervating tail muscles, (ii) those with axons in the pudendal nerve, innervating urethra, penis and rectum, and (iii) those with axons in the pelvic nerve, the most likely candidates for receiving crossed disynaptic inhibition appeared to be the tail motoneurones. One of the main reasons for this supposition was the fact that crossed disynaptic inhibition is evoked in a high proportion of motoneurones in S3 (see above and Table 1), where motoneurones of the tail muscle sacrococcygeus superior (Sherrington, 1892) are present, while pudendal motoneurones appear only occasionally according to Oliver, Bradley & Fletcher (197) and pelvic c-motoneurones were not reported (Ryall & Piercey, 197). The possibility remained, however, that crossed disynaptic inhibition might appear in more than one type of motoneurone. It seemed unlikely, on the other hand, that a major proportion of motoneurones receiving crossed disynaptic inhibition were cells of origin of non-parasympathetic fibres of the pelvic nerve with conduction velocities above 15 m/sec since the number of these fibres was estimated to constitute not more than 9 % of fibres in the S2 ventral root (Ryall & Piercey, 197), and at least some of them were afferent. Therefore, in order to determine if crossed disynaptic inhibition is evoked both in tail and pudendal or only in tail motoneurones, a series of experiments was performed with recording from identified motoneurones of these two kinds. Tail motoneurones were identified by their antidromic invasion from intact S2 or S3 ventral roots as well as following electrical stimulation of the tail (see Methods). Table 1 shows that the crossed disynaptic i.p.s.p.s were found in all tail motoneurones (n = 1); all of these i.p.s.ps were depressed following preceding stimulation of the ventral roots on the side opposite to motoneurone location.

10 434 E. JANKOWSKA, Y. PADEL AND P. ZARZECKI Pudendal motoneurones were identified by their antidromic invasion following stimulation of the common pudendal nerve in the ischiorectal fossa. In agreement with the results of Romanes (1951) and Oliver et al. (197) these were found only in S2 or SI segments but not in S3. Records from Si, S2 and S3 ventral roots likewise showed that either all, in two of the three cats in which it was tested, or a great majority of pudendal fibres passed along Si and S2 ventral root fibres. As summarized in Table 1, in none of twenty-three pudendal motoneurones in S2 could any TABLE 1. Distribution of monosynaptic e.p.s.p.s from ipsilateral afferents and of disynaptic i.p.s.p.s from contralateral afferents in sacral motoneurones in which effects of stimulation of S2 and S3 dorsal roots from both sides were tested. Tail motoneurones; those antidromically activated following stimulation of the tail. Non-pudendal motoneurones; those antidromically activated from ventral roots but not from the pudendal nerve. Unidentified motoneurones; those tested only for invasion from ventral roots. Pudendal motoneurones; those antidromically activated from the pudendal nerve InS2 -~~~~A, f InS3 Motoneurones i e.p.s.p.s co i.p.s.p.s i e.p.s.p.s co i.p.s.p.s Tail 6/6 6/6 4/4 4/4 Non-pudendal 44/6 44/6 11/11 1/1 Unidentified 4/52 27/52* 15/15 1/15* Pudendal 4/23 /23 * Thirteen of the motoneurones from S2 and all fifteen motoneurones from S3 were tested under deep Nembutal anaesthesia in contrast to the remaining ones tested in unanaesthetized decerebrate preparation or under chloralose anaesthesia with only small supplementary doses of Nembutal. disynaptic i.p.s.p.s be detected following stimulation of the contralateral S2 and S3 dorsal roots. Considering the possibility that the i.p.s.p.s could have been masked by concurrent e.p.s.p.s or be difficult to detect because of their small amplitude, all the pudendal motoneurones were tested while varying their membrane potential. No disynaptic i.p.s.p.s appeared, however, even when 3-4 na of current were used to depolarize them, while the amplitude of i.p.s.p.s recorded in non-pudendal motoneurones increased considerably with currents of only 5-1 na. Among motoneurones antidromically invaded following stimulation of the S2 and S3 ventral roots but not of the pudendal nerve ('non-pudendal' motoneurones), crossed disynaptic i.p.s.p.s were found in forty-four of sixty recorded in S2 and in all ten recorded in S3. The majority of the non-pudendal motoneurones were recorded in experiments in which no stimulation of the tail was used and undoubtedly included motoneurones innervating the tail. Tail motoneurones also might have been among the non-pudendal motoneurones in the experiments in which the tail was stimulated because this stimulation was always submaximal in order to avoid risk of spread of current from the tail electrodes to the pudendal nerve. (In control records from the pudendal nerve as well as from penetrated pudendal motoneurones it was ascertained that, at the stimulus intensities used, there was no spread of current to the pudendal nerve.) In order to evaluate to what extent the observations made on our samples of tail and pudendal motoneurones could be generalized, a comparison was made of the

11 CROSSED DISYNAPTIC INHIBITION 435 peripheral input, axonal conduction velocity and location of tail, pudendal, nonpudendal and unidentified motoneurones with and without crossed disynaptic inhibition. The presence of crossed disynaptic i.p.s.p.s was associated with the presence of distinct monosynaptic e.p.s.p.s from lowest threshold ispilateral afferents, as summarized in Fig. 6 (first three upper histograms). Of ninety motoneurones with crossed disynaptic i.p.s.p.s all showed e.p.s.p.s with segmental latencies of 45- *75 msec and all but one of these e.p.s.p.s had amplitudes exceeding 1 mv; in more than half of them the e.p.s.p.s were > 3 mv with stimulus strengths about 1-5 times threshold for afferents in the dorsal roots. On the other hand, in the majority (87 %) FA d cl._ C, -C.51 d. n n 15 1 Presence of and latency of e.p.s&p.s Tail nlo msec no msec Non-pudendal Unidentified n n44 n36 2- fl ~~~~~~~~~~15 gin ~ ~~ I I am 1 _ ~~~~~~~~~~~~5 no msec no Latency of antidromic invasion Tail nlo Non-pudendal Unidentified n Pudendal nib fln3l 15 n33 1 Non-pudendal n44-3 msec msec Non-pudendal n msec no no now > 1 msec msec msec.:.. P.p s.p )-1 mv v/, e&pp.sp. MV msec Fig. 6. Distribution of monosynaptic e.p.s.p.s from ipsilateral dorsal roots and latencies of antidromic invasion in various groups of motoneurones. The latencies were measured with respect to the incoming afferent volleys and to the peak of the positive cord surface potential following stimulation of S2 and S3 ventral roots (also in tail and pudendal motoneurones). Motoneurones with e.p.s.p.s > 1 mv, e.p.s.p.s < 1 mv and without e.p~s.p.s are indicated by A, 3, and E] respectively. * This group of motoneurones was recorded under Nembutal anaesthesia; they were located mainly in S2. Further explanations in the text. of pudendal motoneurones and of motoneurones identified as 'non-pudendal', in which there were no crossed disynaptic i.p.s.p.s, e.p.s.p.s with segmental latencies of < 1- msec appeared to be lacking (first two lower histograms of Fig. 6). The amplitudes of all those found (four pudendal motoneurones and one in a non-pudendal motoneurone) were below 1 mv. We may, however, have failed to detect some e.p.s.p.s with amplitudes of less than -2 mv because extracellular field potentials were not taken for all the motoneurones and for some of them it was impossible to decide a podteriori if small potentials following dorsal root stimulation represented field or postsynaptic potentials. Monosynaptic e.p.s.p.s were lacking in pudendal motoneurones with membrane potential < 4 mv (those with membrane potentials

12 JANKOWSKA, Y. PADEL AND P. ZARZECKI < 2 mv were discarded) as well as in those with membrane potentials > 4-5 mv. An additional indication that unsatisfactory recording conditions were not a factor in the failure to observe monosynaptic e.p.s.p.s in most pudendal motoneurones is the observation that even in the most poorly recorded of the above neurones, some e.p.s.p.s were evoked from higher threshold (2-1 times threshold) ipsilateral or contralateral afferents, or with latencies of msec. If such weak monosynaptic input is a general feature of pudendal motoneurones, as indicated also by observations of R. Mackel (personal communication), as well as of pelvic motoneurones, large monosynaptic e.p.s.p.s evoked from ipsilateral dorsal roots in sacral motoneurones with crossed disynaptic i.p.s.p.s might define them as motoneurones of tail muscles. Lack of disynaptic i.p.s.p.s was not always accompanied by a lack of monosynaptic e.p.s.p.s, as shown in the third lower histogram of Fig. 6. However, the results summarized in this histogram may not be quite comparable with the remaining ones. All nineteen motoneurones with monosynaptic e.p.s.p. without the corresponding disynaptic i.p.s.p.s were recorded in one experiment under relatively deep Nembutal anaesthesia, which was used to abolish the crossed recurrent facilitation and to disclose the crossed recurrent inhibition. Consequently the excitability of interneurones mediating the crossed disynaptic inhibition was lower than in other experiments. In addition in only two of these nineteen motoneurones was it also ascertained that i.p.s.p.s were undetectable after their depolarization. The presence or absence of crossed disynaptic i.p.s.p.s. was also correlated with the presence or absence of ipsilateral recurrent inhibition. The latter was seen in thirty-one of thirty-two tested motoneurones with crossed i.p.s.p.s, while it was undetectable in all seventeen pudendal motoneurones. The conditions of testing were not optimal for pudendal motoneurones because the effects from ventral roots were analysed in them at submaximal stimulus strengths (below threshold for antidromic activation of a given motoneurone), S2 and S3 ventral roots being stimulated together, or when the antidromic invasion of the cells became blocked after their deterioration. Consequently, one cannot exclude that under better conditions some recurrent inhibition might have been detected in at least some of the pudendal motoneurones. Control records of recurrent inhibition in non-pudendal motoneurones in the same experiments and under the same conditions do not, however, lend support to this possibility. A difference in conduction velocities along axons of motoneurones with and without crossed disynaptic inhibition is indicated by longer latencies of antidromic invasion of the latter (Fig. 6) but in view of overlap between the latencies found in different groups of motoneurones this feature could hardly be used to distinguish between the tail and pudendal motoneurones. The locations of these two groups of motoneurones were clearly different. This has been established for five tail motoneurones, four pudendal motoneurones and twenty-one non-pudendal motoneurones showing crossed disynaptic i.p.s.p.s, which were stained by the intracellular injection of horseradish peroxidape (Snow, Rose & Brown, 1976) as described previously (Czarkowska et al. 1976a) and for at least twice as many motoneurones of each kind penetrated in the closest neighbourhood of the stained ones. As expected from the observations of Oliver et al. (197), Sato, Mizuno and Konishi (1978) and R. Mackel (personal communication), the location of pudendal motoneurones (filled contours in Fig. 7A) was more lateral than the location of identified tail (arrows in Fig. 7A, B) and non-pudendal motoneurones (open contours in Fig. 7A, B), which occupied the same part of the ventral horn. The tail and other motoneurones with crossed disynaptic inhibition were generally larger than the pudendal motoneurones. This holds true both for those stained and for the whole population of neurones at the two locations. The dendrites of the stained tail and non-pudendal motoneurones extended mainly in the lateral and medial

13 CROSSED DISYNAPTIC INHIBITION 437 directions, covering a half-moon shaped area along the borders of the ventral horn. Several of the dendrites directed medially crossed the midline and projected over about one-third of the width of the contralateral ventral horn. Dendrites of pudendal motoneurones extended more symmetrically around their somata in the lateral parts of the ventral horn. Fig. 7C and D show also the distribution of initial axon collaterals, and their branches, within the ventral horn. All appeared to ramify within the ventral part of the ipsilateral ventral horn, more laterally than medially. A S2 a S3 D 5 im Fig. 7. Locations of motoneurones and initial axon collaterals. A and B, location of motoneurones innervating tail muscles (open contours identified by arrows), nonpudendal motoneurones with crossed disynaptic i.p.s.p.s (open contours) and pudendal motoneurones (filled contours) in S2 and S3. Only somata of the motoneurones are indicated, C and D, ramifications of initial axon collaterals of five non-pudendal, four tail and one pudendal motoneurone (five in S2 and five in S3). Locations of large cells within the same parts of the ventral horn in which tail and pudendal motoneurones were found, in ten successive sections 5,tm thick, are indicated to show the extent of the corresponding motor nuclei. Only those cells with nucleus and nucleolus on a given section were taken into account. The mid line is to the right of each diagram. It should be mentioned that axon collaterals were given off not only by axons of tail or non-pudendal motoneurones but also by one of the four well stained pudendal motoneurones (lateral most axon in Fig. 7 C). Since no recurrent inhibition was seen in pudendal motoneurones a termination of the axon collaterals of these neurones upon other motoneurones may be considered, as described for lumbar motoneurones

14 438 E. JANKOWSKA, Y. PADEL AND P. ZARZECKI by Cullheim, Kellerth & Conradi (1977). The more dorsal and lateral projections of collaterals of the stained pudendal motoneurones, as compared to the collaterals of other motor axons, would be consistent with such a destination. In the lateral part of the ventral horn was located one Renshaw cell stained with horseradish peroxidase (Fig. 8). The cell responded with a typical burst of high frequency discharges to stimulation of one of the ipsilateral ventral roots. Its axon projected to the opposite side of the spinal cord after giving off a number of axon 2gm Mid line Fig. 8. Reconstruction of axonal projections of a Renshaw cell. Shaded are contours of large cells located on the same sections. Arrows indicate nodes of Ranvier from which no axon collaterals were given off. collaterals in the neighbourhood of motoneurones on the same side. The nodes of Ranvier (constrictions of the axon, cf. Czarkowska, Jankowska & Sybirska, 1976b) were seen at fairly regular intervals at sites from which axon collaterals were given off and at two sites indicated by arrows. The internodal length was 1-2 #sm for the stem axon and less for secondary and higher order branches as described previously (Czarkowska et al b). DISCUSSION Similarities between Ia reciprocal inhibition of motoneurones innervating limb muscles and crossed inhibition of sacral motoneurones The crossed inhibition of sacral motoneurones appeared to have many features in common with Ia reciprocal inhibition. Both are evoked from the lowest threshold afferents (Lloyd & Wilson, 1959; Curtis et al. 1958; Frank & Sprague, 1959), have a similar time course (Frank & Sprague, 1959), indicating synaptic contacts on the soma of the motoneurones, and are apparently mediated by the same kind of interneurones. As shown under Results, interneurones interposed in the pathway of the crossed disynaptic inhibition are very effectively inhibited by stimulation of the contralateral ventral roots and all the available evidence points to the conclusion that no interneurones other than those mediating inhibition of motoneurones from group Ia afferents are inhibited by Renshaw cells (Hultborn etal a, b; H. Hultborn, unpublished; E. Jankowska, unpublished), except Renshaw cells themselves (Ryall,

15 CROSSED DISYNAPTIC INHIBITION ). That the observed depression of the crossedi.p.s.p.sbystimulationoftheventral roots is mediated by Renshaw cells is indicated by the following observations: (1) this depression occurred with conditioning stimulation of the ventral roots at or not exceeding two times the threshold for motor axons, this being below the strength which might excite, e.g. any unmyelinated fibres within the ventral roots (Coggeshall, Coulter & Willis, 1974), (2) the latency of this depression was compatible with only two synaptic delays between the motor axon collaterals and the inhibited inhibitory interneurones (cf. Hultborn et al a) and its time course corresponded to the time course of other effects of Renshaw cells (cf. Hultborn et al a), and (3) cells with the same characteristics of firing as described for Renshaw cells have been found in the same sacral segments (E. Jankowska and Y. Padel, unpublished observations). Recurrent facilitation of lumbar motoneurones has been shown to be due to an arrest of their steady bombardment by inhibitory interneurones (Wilson, 1959) which were identified as the same interneurones which mediate the Ia reciprocal inhibition (Hultborn et al d). In lower sacral motoneurones the recurrent facilitation was found in parallel with their disynaptic inhibition; when the latter -was absent the recurrent facilitation was also absent and there was a rough relation between their amplitudes. Finally, the occurrence of the recurrent facilitation of sacral motoneurones only in unanaesthetized preparations indicates that the interneurones mediating the crossed disynaptic inhibition are tonically active and that their activity is reduced by barbiturates, similarly as that of the interneurones mediating the Ia reciprocal inhibition of lumbar motoneurones (Hultborn et al b, d). The only difference found so far between the disynaptic inhibition of lumbar and of sacral motoneurones, lies in their origin from ipsilateral and from contralateral afferents respectively. It remains, however, to be determined if the disynaptic inhibition of sacral motoneurones is evoked, as in the case for lumbar motoneurones, from group Ia spindle afferents from muscles with a strictly antagonistic function (cf. Eccles, Eccles & Lundberg, 1957) and also whether the recurrent depression of this inhibition is via impulses in motor axons to the same antagonistic muscles (Hultborn et al c). The present series of experiments did not aim at establishing these relations and strong recurrent inhibition of sacral motoneurones by impulses in ipsilateral ventral root fibres did not allow proper testing of their effects on the crossed inhibition. Crossed recurrent inhibition of sacral motoneurones Whatever function is ascribed to the recurrent inhibition of motoneurones (Renshaw, 1941; Eccles, Fatt & Koketsu, 1954; Granit, 1955; Wilson, 1959; Eccles, 1969; Hultborn, 1972) there is no doubt that it operates between motoneurones innervating synergistic but not antagonistic limb muscles (Hultborn et al b). If the same holds true for sacral motoneurones the occurrence of the crossed recurrent inhibition found in the present study might be correlated with the previously reported crossed monosynaptic excitation (Curtis et al. 1958; Frank & Sprague, 1959) and would indicate that some muscles on both sides of the body innervated by S2 and S3 motoneurones act as synergists.

16 44 E. JANKOWSKA, Y. PADEL AND P. ZARZECKI The crossed monosynaptic e.p.s.p.s could be mediated by primary afferents projecting to the opposite side of the spinal cord (Edisen, 1963) or by afferents contacting distal parts of the dendrites extending to the side of the afferents as proposed by Frank & Sprague (1959) (cf. also Sprague, 1958). Two similar possibilities may be considered in the case of the crossed recurrent inhibition. It could be evoked either by Renshaw cells located at the same side as their target motoneurones and excited Mid line Fig. 9. Diagram of proposed monosynaptic and disynaptic connexions within the lower sacral segments. Explanation in the text. via motor axon collaterals from the other side, or else by Renshaw cells located on the side of the axon collaterals through which they are excited and sending their axons across the mid line or terminating on dendrites projecting towards them. We favour the two latter alternatives because all Renshaw cells encountered in S2 and S3 were excited only from the ventral roots ipsilateral to them (E. Jankowska and Y. Padel, unpublished observations), and because no contralaterally projecting motor axon collaterals have been previously reported while cells in lower sacral segments with a similar location as Renshaw cells have contralaterally projecting axons (Scheibel & Scheibel, 1966). Our own morphological observations, summarized in Figs. 8 and 9 likewise show ipsilateral ramifications of initial motor axon collaterals and contralateral projection of the one HRP-filled Renshaw cell.

17 CROSSED DISYNAPTIC INHIBITION 441 Proposed neuronal pathways of the crossed inhibition and disinhibition of sacral motoneurones Fig. 9 indicates the most likely mono- and disynaptic connexions within the lower sacral segments. The large circle to the left represents motoneurones innervating muscles on one side, tail motoneurones if our conclusions presented above and in the discussion to follow are correct, while two similar circles to the right represent motoneurones innervating their antagonists and synergists on the other side. The small circle labelled 'I' represents interneurones which mediate crossed disynaptic inhibition of motoneurones and are most likely excited by collaterals of primary afferents which terminate on motoneurones to the antagonists. No similar connexion has been indicated for ipsilateral disynaptic inhibition because no evidence for it has been reported. Small circles labelled A, B and C represent Renshaw cells which mediate ipsilateral recurrent inhibition, crossed recurrent inhibition and disinhibition of the motoneurones to the left respectively. By the heavily and lightly shaded vertical lines are indicated the more and the less likely positions of the mid line in relation to cells B and I. As discussed in the preceding paragraph we find it more likely that crossed recurrent inhibition is mediated by Renshaw cells located on the side of the spinal cord opposite the inhibited motoneurones. Of the two possible locations of the inhibitory interneurones on (i) the side of their target motoneurones or on (ii) the same side as the fibres that excite or inhibit them, we similarly favour the latter. The reasons are that these interneurones are effectively excited by lowest threshold afferents and as effectively inhibited by volleys in motor axons and Renshaw cells and that the crossed effects of primary afferents and of Renshaw cells are much less pronounced, at least on motoneurones. We assume that the same Renshaw cells (those labelled C) inhibit the inhibitory interneurones I and the motoneurones excited by the same afferents, in analogy with the pattern of inhibition and disinhibition of lumbar motoneurones (Hultborn et al c). Functional implications From a comparison of the input to pudendal motoneurones and to motoneurones positively identified as innervating tail or, in a negative way, to motoneurones identified as non-pudendal, we conclude that crossed disynaptic inhibition occurs in the latter two groups of motcneurones but not in the former and that there are indications that all motoneurones in these segments with crossed disynaptic inhibition innervate tail muscles. The reasons for these two conclusions are the following. (i) In all the recorded tail motoneurones disynaptic i.p.s.p.s were evoked from contralateral dorsal roots while no such i.p.s.p.s could be detected in any pudendal motoneurones. (ii) All the motoneurones in S2-S3 appeared to fall into one of two categories with features common to either tail or pudendal motoneurones. Their majority showed distinct (> 1 mv) monosynaptic e.p.s.p.s from lowest threshold ipsilateral afferents, disynaptic i.p.s.p.s from lowest threshold contralateral afferents, distinct ipsilateral recurrent inhibition and high conduction velocity. Other motoneurones were characterized by lack of (or only small, < 1 mv) ipsilateral monosynaptic e.p.s.p.s, lack of crossed disynaptic i.p.s.p.s, lack of ipsilateral recurrent i.p.s.p.s and a much larger range of conduction velocities. Some of the above differ-

18 442 E. JANKOWSKA, Y. PADEL AND P. ZARZECKI ences between pudendal and non-pudendal motoneurones have been found also by R. Mackel (personal communication), although the percentage of pudendal motoneurones with monosynaptic e.p.s.p.s from ipsilateral afferents is higher in his sample of those innervating muscles of external urethral sphincters in male cats. In our experiments on both male and female cats the motoneurones were identified on the basis of activation from the whole pudendal nerve. (iii) Tail motoneurones should be practically the only ones receiving crossed disynaptic inhibition in S3 where hardly any other motoneurones are to be taken into account. Both our own and previous data (Oliver et al. 197) indicate that the location of pudendal motoneurones is limited to SI and S2 and only exceptionally to the rostral part of S3 (see, however, Yamamoto, Satomi, Ise, Takatama & Takahashi, 1978). Neither were any fast conducting pelvic fibres found in S3 (Ryall & Piercey, 197), the parasympathetic motoneurones of pelvic nerve being excluded from our sample because all of those we recorded were conducting with a-fibre conduction velocity. (iv) The locations of tail and non-pudendal motoneurones with crossed disynaptic inhibition overlapped and was more medial than that of pudendal motoneurones in S2 which were found in the same part of the ventral horn as indicated by Oliver et al. (197) and R. Mackel (personal communication) (see, however, Yamamoto et al. 1978). Pudendal motoneurones appeared to be situated outside the area within which the distinct compound antidromic field potentials are evoked by stimulation of the ventral roots, and they were rather difficult to penetrate unless tracked for systematically. It seems therefore unlikely that any significant number of pudendal motoneurones would be impaled while trying to record from sacral motoneurones generally and being guided by antidromic field potentials to stimulation of ventral roots. The destination of axons of non-pudendal motoneurones of S2 without ipsilateral monosynaptic e.p.s.p.s or contralateral disynaptic i.p.s.p.s remains to be established. Crossed disynaptic inhibition of tail motoneurones under recurrent inhibitory control, together with the other features of the input to these motoneurones (strong ipsilateral monosynaptic excitation and recurrent inhibition) would indicate that the activity of the left and right side tail muscles may be as closely related as that of the strict antagonists operating at the same joint of a limb. Whether such close relations occur for other parts of the body as well remains to be established. A later paper (E. Jankowska & A. Odutola, in preparation) will describe the results of experiments on relations between motoneurones of back muscles. This study was supported by the Swedish Medical Research Council (project no. 94). We are indebted to Dr Robert Mackel for permission to refer to some of his unpublished observations on pudendal motoneurones and for helpful discussions. The technical assistance of Mrs Rauni Larsson is gratefully acknowledged. REFERENCES CoGGEsHAL, R. E., CouLTER, J. D. & Wiuuis, W. D. (1974). Unmyelinated axons in the ventral roots of the cat lumbosacral enlargement. J. comp. Neurol. 153, CULLHESIM, S., KLTTTERTm, J.-O. & CoNRADi, S. (1977). Evidence for direct synaptic interconnections between cat spinal a-motoneurones via the recurrent axon collaterals: a morphological study using intracellular injection of horseradish peroxidase. Brain Res. 132, 1-1.

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