A recurrent collateral pathway for presynaptic inhibition in the rat cuneate nucleus

BRAIN RESEARCH

63

A R E C U R R E N T C O L L A T E R A L PATHWAY FOR PRESYNAPTIC INHIBI-

TION IN THE RAT C U N E A T E NUCLEUS

N. DAVIDSON AND C A R O L A. SMITH

Department o/'Physiology, Marischal College, Aberdeen (Scotland) (Accepted March 8th, 1972)

INTRODUCTION

While many authors have described the production of presynaptic inhibition of cuneothalamic relay (CTR) cells and the associated depolarization of primary afferent terminals in the cuneate nucleus 2-4,a~,19 the central pathway responsible for primary afferent depolarization (PAD) is not known. Although PAD evoked from any of a number of inputs to the cuneate nucleus is accompanied by the activation of cuneate interneurones 2,4,5,7,9,10,2° there is no good evidence that all or any of these interneurones are necessarily part of the central pathway for presynaptic inhibition. In the experiments reported here it was discovered that antidromic invasion of the cuneate nucleus from the medial lemniscus can produce not only interneurone activity, as reported elsewhere5,7,15-17, but also PAD. Some of these interneurones were also activated by orthodromic stimulation which produced PAD in the cuneate nucleus. It is concluded that part of the presynaptic inhibitory mechanism in the rat cuneate nucleus is mediated by a recurrent collateral pathway from the medial lemniscus, consisting of at least two interneurones. METHODS

All experiments were performed on male rats (body weight 300-400 g) anaesthetized by intraperitoneal injection of a 1~o chloralose-10~ urethane solution (8 ml/kg). Rats were supported on an automatically controlled heating pad which maintained rectal temperature at 37 °C 24. Routine surgical procedures were employed to expose the dorsal column nuclei, the cerebral cortex contralateral to the cuneate nucleus to be studied and the ulnar and median nerves of the ipsilateral forelimb. In experiments where changes in afferent terminal excitability were measured the nerves were sectioned peripherally and mounted on bipolar platinum electrodes. All exposed tissues were protected from temperature changes and drying by warm mineral oil. Standard electrophysiological techniques were employed to make extracellular Brain Research, 44 (1972) 63-71

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N. DAVIDSON AND C. A. SMITH

recordings from single units in the cuneate nucleus, using glass micropipettes (tip diameter 2-4 #m) filled with 3 M CHzCOOK or NaC1.

Measurement of afferent terminal excitability The depolarization of primary afferent terminals which is responsible for presynaptic inhibition is difficult to measure directly. An alternative method of displaying PAD is to measure the increase in excitability of primary afferent terminals occurring during PAD using Wall's antidromic excitability testing technique3L In these experiments test stimuli were delivered to primary afferent terminals through a monopolar tungsten electrode (tip diameter 5 - 1 0 # m ) inserted into the cuneate nucleus about 1 mm caudal to the obex. The stimuli (5-20/~A) were negative-going, rectangular, 0.1 msec pulses and evoked submaximal antidromic potentials in the ipsilateral ulnar nerve. Conditioning stimuli used to evoke PAD were delivered either via bipolar platinum electrodes to the ipsilateral median nerve, percutaneously through bipolar pin electrodes to the contralateral forelimb or to the contralateral medial lemniscus via a stereotaxically oriented monopolar tungsten electrode (see below). These stimuli were supramaximal, negative-going, 0.1 msec pulses delivered either singly or in 10 msec trains at 350 c/sec. The increase in excitability o f t h e cuneate afferent terminals was measured as a percentage increase in the size of conditioned antidromic responses relative to a series of test antidromic responses.

Lemniscal stimulation With the aid of a stereotaxic atlas 14, a monopolar tungsten stimulating electrode was inserted into the contralateral medial lemniscus at various levels between the diencephalon and the medulla. It was possible, by monitoring cortical responses 21, to determine whether or not the electrode tip was correctly located in the medial lemniscus and whether current spread from the tip was confined to the lemniscus or was activating other neighbouring structures, particularly the corticospinal tracts. A single stimulus to the lemniscus evoked a response on the cortical primary somatosensory area (Fig. I B) similar to, but of shorter latency than, that evoked from the forelimb (Fig. I A). The early positive waves of the response in Fig. I A are less pronounced than in Fig. IB due to temporal dispersion of the afferent volley. Histological checks of the electrode positions confirmed that the lowest thresholds for the evoked cortical responses were obtained with the electrode in the medial lemniscus (see Fig. 4). If the electrode penetrated deeper than the medial lemniscus then the corticospinal fibres in the underlying cerebral peduncle were stimulated and a small short latency, positive response, the a-wave 21 (Fig. 1C) was evoked antidromicalty on the cerebral cortex. When stimulus current was large enough to activate both corticospinal fibres and the lemniscus the antidromic (Fig. I C) and orthodromic (Fig. 1B) responses were superimposed (Fig. ID). This technique provided a useful means of monitoring stimulus spread as well as a way of locating the medial lemniscus. Brain Research, 44 (1972) 63-71

RECURRENT PRESYNAPTICINHIBITION

A

65

B

10msec 500pVF

C

D

Fig. 1. Responses recorded from the surface of the primary somatosensory area of the cortex following stimulation of various sites. A, Response to stimulation of the ipsilateral forelimb. B, Shorter latency response to stimulation of the medial lemniscus in the mesencephalon, contralateral to the cuneate nucleus under study. C, Response to antidromic stimulation of corticospinal fibres lying below the medial lemniscus in the cerebral peduncle. D, Response to stimulation of the medial lemniscus as in B, but with a larger stimulus. Current spread has activated the corticospinal fibres and the resulting cortical response is a mixture of the responses in B and C. In this and other records arrows indicate stimulus artefacts and the convention of negative upwards is observed.

RESULTS

htentification of cell types Cuneothalamic relay (CTR) cells in the cuneate nucleus were identified by their brief, short latency response to ipsilateral forelimb stimulation, a short latency (1.5 msec) single spike response to antidromic stimulation of the contralateral medial lemniscus, a restricted ipsilateral peripheral receptive field and a following frequency to orthodromic stimulation of about 400 c/sec. These cells tended to lie more superficially (200-700 #m) in the nucleus than the interneurones (500-1200 #m). Longlasting inhibition of these ceils, like that reported elsewhere, could be produced following conditioning stimulation of the ipsilateral forelimb2,4,5,10, 20 and the contralateral forelimb10,19, e0. The long-lasting inhibition of spontaneously active CTR cells has also been reported elsewhere 15-17 Of the 197 cells in the cuneate nucleus activated from the ipsilateral forelimb, 61 were reliably identified as CTR ceils. The remaining 136 were regarded as interneurones and were characterized by a longer latency response to ipsilateral forelimb stimulation (Fig. 2A), a wider peripheral receptive field, a lower following frequency (up to 50 c/sec) than CTR cells and no sign of direct antidromic activation from the medial lemniscus. Contralateral, as well as ipsilateral forelimb stimulation was effective in exciting 46 of these cells. The contralaterally evoked response generally had a longer latency (Fig. 2B) and a lower following frequency than the ipsilaterally evoked response of the same cell (Fig. 2A). From the population of interneurones studied, 49, which responded to ipsilateral orthodromic stimulation, were also activated by stimulation of the contralateral

Brain Research, 44 (1972) 63-71

66

N. DAVIDSON AND C. A. SMITH lOmsec

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Fig. 2. Extracellular neurone responses in the cuneate nucleus of the rat. A, Response of a non-CTR interneurone to stimulation of ipsilateral forelimb. B, Response of same cell to stimulation of contralateral forelimb. C, Transsynaptic response of the same interneurone to antidromic stimulation of the medial lemniscus.

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Fig. 3. Graphs showing the time course of P A D in the cuneate nucleus evoked from different sites. A, Time course of P A D evoked by a single conditioning stimulus (C.S.) to the ipsilateral forelimb. B, As in A but with a repetitive conditioning stimulus (bar labelled C.S.) applied to the contrataterat forelimb. C, P A D evoked on repetitive stimulation of the contralateral medial lemniscusi In each case the insets illustrate successive specimen test records and conditioned antidromic potential records from which the graphs were constructed.

medial lemniscus (Fig. 2C). The response to lemniscal stimulation had a variable latency (from 4 to 15 msec) and a following frequency of about 30 c/sec, indicative of transsynaptic rather than direct antidromic activation. Seventeen of these interneurones also responded to stimulation of the contralateral forelimb (Fig. 2A-C). Orthodromically and antidromically evoked PAD

Conditioning stimuli delivered to both ipsilateral (Fig. 3A) and contralateral (Fig. 3B) forelimb evoked PAD orthodromically in the cuneate nucleus. In addition, PAD could be evoked antidromically by repetitive stimulation of the contralateral Brain Research, 44(1972) 63-71

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Threshold for PAO pA 100

200

300

400

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400

600

800

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Fig. 4. Histological reconstruction of electrode track penetrating the rnesencephalon. The unbroken line in the graph shows the changes, at various depths, in the threshold stimulating current (top abscissa) required to produce PAD in the cuneate nucleus, using a repetitive stimulus. The lowest threshold coincides with the passage of the electrode through the medial lemniscus and it rises again as the electrode penetrates beneath the lemniscus. A second decrease in threshold for evoking PAD occurs as the electrode approaches the corticospinal fibres in the cerebral peduncle. The broken line indicates the stimulus current strength (bottom abscissa) for evoking the primary cortical response with a single stimulus and again the threshold is lowest at a Izoint corresponding to the medial lemniscus. Note the different scales of the two abscissae. ME, medial lemniscus; CP, cerebral peduncle; SN, substantia nigra; ZI, zona incerta; PF, parafascicular nucleus; lIl, origin of the oculomotor nerve.

medial lemniscus (Fig. 3C). Insets show specimen records successively o f test and c o n d i t i o n e d a n t i d r o m i c potentials. In o r d e r to ensure that P A D evoked by lemniscal stimulation was not caused by c u r r e n t spread to s u r r o u n d i n g structures the threshold for evoking P A D with a repetitive stimulus was d e t e r m i n e d at various depths as the local stimulating electrode descended t h r o u g h the brain. The results ( u n b r o k e n line) are shown in Fig. 4 plotted against a histological reconstruction o f the electrode track. It can be seen that the threshold c u r r e n t to evoke P A D was minimal (40/~A) at a d e p t h c o r r e s p o n d i n g to the medial lemniscus. The threshold rose again as the electrode p e n e t r a t e d beneath the lemniscus, but b e g a n to fall again as the electrode a p p r o a c h e d the corticospinal fibres in the cerebral peduncle. The two lowerings o f threshold are clearly separated. The b r o k e n line shows the changes in c u r r e n t strength required for a single stimulus to p r o d u c e a p r i m a r y cortical response 2t (see Fig. IB). The threshold was again minimal at the depth o f the medial lemniscus. W h e n the electrode penetrated deeper the response, typical o f a n t i d r o m i c corticospinal tract stimulation, was recorded at the cortex (see M e t h o d s a n d Fig. 1C). The results confirm that at threshold current, lemniscal s t i m u l a t i o n evoked P A D in the cuneate nucleus without activation o f neighb o u r i n g corticospinal fibres due to c u r r e n t spread. Both o r t h o d r o m i c a l l y and lemniscaIly evoked P A D survived r e m o v a l by suction o f the whole o f the c o n t r a l a t e r a l cerebral cortex. Brain Research, 44 (1972) 63-71

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N. DAVIDSON AND C. A. SMITH

DISCUSSION

It is generally agreed that at least two principal inhibitory mechanisms exist in the dorsal column nuclei 6,13. One is a postsynaptic inhibitory mechanism effected by a stabilization of the postsynaptic membrane, the other a presynaptic inhibition associated with a depolarization of primary afferent terminals. Both mechanisms can produce long-lasting inhibitions of CTR cellsL In the experiments reported here and elsewhere long-lasting inhibition of evoked or spontaneously active CTR cells was observed following stimulation of the ipsilateral forelimb2,4,~',~0,~0, the contralateral forelimb 10,~9,2° and the contralateral medial lemniscus 15-17. It is difficult to estimate how much of these inhibitions is postsynaptic in origin. However, from the properties of the interneurone population observed in the present experiments and since PAD was evoked from all 3 sites mentioned above, it can be concluded that part of these inhibitions are presynaptic in nature and that the pathways mediating PAD associated with the presynaptic inhibition have elements in common. From the population of 136 interneurones studied, 49 responded to ipsilaterat orthodromic stimulation and transsynaptically to stimulation of the contralateral medial lemniscus (Fig. 2A and C). There exists both anatomical z6 and electrophysiologicaV, 5,15-17 evidence of a collateral pathway from lemniscal fibres to interneurones in the cuneate nucleus but there are no published reports that this collateral pathway may be recurrent to cuneate primary afferent terminals thus providing a central pathway for PAD. In these experiments PAD was regularly evoked in the cuneate by stimulation of the contralateral medial lemniscus (Fig. 3C). It is well established that a corticofugal pathway running from the contralateral somatosensory cortex via the corticospinal tracts is responsible for the production of cell inhibition 22'2~,~s and large amounts of PAD in the cuneate nucleus a,lz. Monitoring of cortical responses (Figs. 1 and 4), however, indicated that at current strength required to evoke PAD from the medial lemniscus there were no signs of activation of the corticospinal tract. In addition, the plot of current threshold against depth (Fig. 4) clearly revealed two minima for evoking PAD in the cuneate nucleus, one corresponding to the medial lemniscus, the other to the corticospinal fibres in the underlying cerebral peduncle. The point corresponding to the medial lemniscus was also the point at which a primary cortical response (Fig. 1B) could be evoked at lowest threshold (Fig. 4). PAD evoked from the medial lemniscus survived removal by suction of the cerebral cortex, ruling out the possibility that stimulation of the medial lemniscus orthodromically activated the corticofugal projection back to the cuneate nucleus. Stimulation of the contralateral forelimb evoked PAD in the cuneate nucleus in the present experiments. This has been reported elsewhere 9,1°,19,2° but, apart from a few reports that the rostral pole of the gracile nucleus receives a small contralateral input16,18, 27, there is little evidence that interneurone activity in these nuclei can be evoked from the contralateral periphery. Forty-six of the cuneate interneurones studied here were found to be activated by both ipsilateral and contralateral forelimb Brain Research, 44 (1972) 63-71

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stimulation. The pathway for this contralateral input is not yet known, although the longer latency and lower following frequency of contralateral responses indicates more synapses than on the ipsilateral input to the same interneurone. Of particular interest is the observation that 17 of the interneurones which responded to contralateral forelimb stimulation, also responded to stimulation of the ipsilateral forelimb and the contralateral medial lemniscus. This suggests a final common pathway for the 3 inputs to the cuneate nucleus, all of which in turn can mediate PAD in cuneate afferent terminals. The simplest explanation of these observations is that there exists a collateral pathway from the contralateral medial lemniscus recurrent to the primary afferent terminals in the cuneate nucleus causing depolarization of these terminals. Such a pathway is shown in Fig, 5. The latencies of interneurone responses were found to vary widely and, therefore, it is difficult to determine the precise number of interneurones in the pathway. A minimum of two cell types is suggested. Interneurone A is representative of those cells responding both to ipsilateral afferent volleys and to volleys from the medial lemniscus, while interneurone B represent the cells which responded, in addition, to volleys from the contralateral forelimb. Interneurone B is shown terminating in an axo-axonic synapse of the type described in the cuneate nucleus by Valverde e9 and Walberg 3°. It is suggested that interneurone B is responsible for the depolarization of primary afferent terminals and the associated presynaptic inhibition of CTR cells. This is not intended to exclude the possibility of such a neurone also mediating postsynaptic inhibition. It may well be that pre- and postsynaptic inhibition possess the same neural substrates. In support of this suggestion is the pharmacological similarity of pre- and postsynaptic inhibition in the cuneate nucleus8,11, 23 and the observation by Walberg 30 that in the cuneate nucleus the same interneurone terminal can form both axo-axonic and axo-dendritic contacts. SUMMARY

Stimulation of the ipsilateral forelimb, the contralateral forelimb and the contralateral medial lemniscus produced long-lasting inhibition of cuneothalamic relay (CTR) cells, primary afferent depolarization (PAD) and interneurone activity in the cuneate nucleus of the chloralose-urethane anaesthetized rat. Lemniscal stimulation was controlled by monitoring of sensorimotor cortical responses and histological identification of stimulus sites from which minimal threshold responses could be evoked. With low stimulus strengths there was no evidence of current spread to the underlying corticospinal tract. Two groups of cuneate interneurones were of special interest. Group A was activated both by stimulation of the ipsilateral forelimb and the contralateral medial lemniscus. Group B was activated by ipsilateral and contralateral forelimb stimulation and by stimulation of the contralateral medial lemniscus. It is concluded that a recurrent collateral pathway from the medial lemniscus, involving successively A- and B-type interneurones, forms a possible central pathway Brain Research, 44 (1972) 63-71

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N. DAVIDSON AND C. A. SMITH

IPSlLATERAL DORSAL COLUMN

c

\

CONTRALATERAL PATHWAY

MEDIAL LEMNISCus

Fig. 5. Diagrammatic representation of a proposed recurrent collateral presynaptic inhibitory pathway in the cuneate nucleus. X represents a CTR cell whose axon contributes to the medial lemniscus. A recurrent collateral of such an axon innervates interneurone A. This, in turn innervates interneurone B which impinges via an axo-axonic synapse on the primary afferent terminal and produces PAD. lnterneurone B can be activated not only from the ipsilateral periphery and the medial lemniscus but also from the contralateral periphery. It presents, therefore, a final common path for both orthodromically and lemniscaUy evoked PAD in the cuneate nucleus.

for the p r o d u c t i o n o f presynaptic inhibition and the associated P A D in the rat c u n e a t e nucleus. It is also c o n c l u d e d t h a t i n t e r n e u r o n e B provides a c o m m o n p a t h for the c o n t r a l a t e r a l p a t h w a y p r o d u c i n g these effects in the cu n eat e nucleus. ACKNOWLEDGEMENT C.A.S. has been s u p p o r t e d by a Science Research C o u n c i l Studentship.

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