J. Physiol. (1983), 343, pp. 341-359 341With 7 text-ftgure8Printed in Great Britain

POST-SYNAPTIC POTENTIALS IN A POPULATION OFMOTONEURONES FOLLOWING ACTIVITY OF SINGLE

INTERNEURONES IN THE CAT

BY E. BRINK*, P. J. HARRISON, E. JANKOWSKA, D. A. McCREAtAND B. SKOOG

From the Department of Physiology, University of Giteborg, P.O. Box 33031,S-400 33, Giteborg, Sweden

(Received 22 February 1983)

SUMMARY

1. The technique of recording post-synaptic potentials from a population ofmotoneurones, by recording from ventral roots perfused with isotonic sucrose, hasbeen applied to investigate the action of single last-order interneurones; the targetmotoneurones were in either caudal L7 or S1 segments.

2. Using spike-triggered averaging, the inhibitory action of 70 % of previouslyidentified last-order interneurones (Renshaw cells and lamina VII Ia inhibitoryinterneurones) has been detected.

3. Previous observations had suggested that interneurones mediating disynapticnon-reciprocal inhibition from group I muscle afferents should be characterized by(i) location in laminae V-VI, (ii) monosynaptic group I input and (iii) ascendingcollateral axonal projection to upper lumbar segments. 65% of interneurones withthese characteristics were found to inhibit motoneurones.

4. In addition, spike-triggered averaging from this group of laminae V-VIinterneurones sometimes revealed a depolarizing potential which preceded theinhibitory potential evoked by the interneurone. The depolarizing potential isinterpreted as being due to the action of some presynaptic fibres which branch toinnervate both the investigated interneurones and motoneurones.

INTRODUCTION

Further progress in studies of the organization of the spinal cord may depend onto what extent we are able to recognize neurones in various spinal pathways andanalyse their properties and circuitry. Of great help in identifying the last-orderinterneurones in the pathways to motoneurones have been techniques, involvingsimultaneous recording from single cells and their potential target motoneurones,which allow one to determine whether these interneurones mediate inhibition or

* Present address: Neurologische Klinik der Technischen Universitat Muinchen, Mohistrasse 28,D-8000 Munchen 80, F.R.G.

t Present address: Department of Physiology, University of Manitoba, 770 Bannatyne Street,Winnipeg, Manitoba, R3E OW3, Canada.

342 E. BRINK AND OTHERS

excitation. However, screening the actions of single interneurones on individualmotoneurones is not always successful. We have, therefore, recently investigated thepossibility of using post-synaptic potentials (p.s.p.s) of a population of motoneuronesfor this purpose (Brink, Jankowska, McCrea & Skoog, 1981). P.s.p.s electrotonicallyconducted along motor axons were recorded from a ventral root (Brooks, Eccles &Malcolm, 1948; Eccles, 1946) perfused with an isotonic sucrose solution (Roberts &Wallis, 1978; Liischer, Ruenzel, Fetz & Henneman, 1979) in order to reduce theshort-circuiting by the extracellular fluid. Spike-triggered averaging (Mendel &Henneman, 1971; Jankowska & Roberts, 1972b; Kirkwood & Sears, 1973) was usedto improve further the signal-to-noise ratio of the responses evoked by singleinterneurones.The technique appeared to be sufficiently sensitive to demonstrate the action of

single lamina VII interneurones mediating reciprocal inhibition of motoneurones (theIa inhibitory interneurones). We have now supplemented our observations on theseinterneurones and extended them to Renshaw cells and a group of laminae V-VIinterneurones which are expected to mediate non-reciprocal inhibition of moto-neurones by group I afferents. The I a inhibitory interneurones and Renshaw cellshave already been identified as the last-order interneurones in inhibitory pathwaysto motoneurones, both indirectly (Eccles, Fatt & Koketsu, 1954; Hultborn,Jankowska & Lindstrom, 1971), and directly (Jankowska & Roberts, 1972b; vanKeulen, 1981). These two groups of interneurones could thus be used to test thereliability of the method. The identification of the inhibitory laminae V-VI inter-neurones has, on the other hand, still lacked a direct demonstration of their actionson motoneurones. Recent studies of the non-reciprocal inhibition from group Iamuscle spindle afferents and group I b tendon organ afferents have revealed a numberof characteristic features of the involved interneurones (Lundberg, Malmgren &Schomburg, 1977; Fetz, Jankowska, Johannison & Lipski, 1979; Jankowska, McCrea& Mackel, 1981; Jankowska & McCrea, 1983; Harrison, Jankowska & Johannisson,1983; Brink, Jankowska & Skoog, 1983b; Rudomin, Jimenez, Solodkin & Duenas,1983). We also have strong indications that interneurones mediating inhibition ofmotoneurones, but not other interneurones with group I input, send an axoncollateral to Clarke's column (Hongo, Jankowska, Ohno, Sasaki, Yamashita &Yoshida, 1983a, b). One of the main aims of this study was therefore to investigatewhether the laminae V-VI interneurones with the ascending projection to the levelofClarke's column do indeed inhibit motoneurones, do not include interneurones withexcitatory actions upon motoneurones, and can thereby be identified as inter-neurones mediating non-reciprocal inhibition of motoneurones. Some of thepreliminary observations of this study have been published (Brink et al. 1981; Brink,Harrison, Jankowska, McCrea & Skoog, 1982; Harrison, Jankowska & Skoog, 1983).

METHODS

Preparation. The experiments were performed on sixteen cats anaesthetized with chloralose(initial dose 40-50 mg/kg after ether anaesthesia during surgery, followed by two to threesupplementary doses of 10-20 mg/kg). The cats were paralysed with gallamine triethiodide (Mayand Baker) and artificially ventilated. End-tidal CO2 was kept at about 4% and blood pressureabove 90 mmHg. The following hind-limb muscle nerves were dissected and prepared for stimula-

POPULATION P.S.P.S EVOKED BY INTERNEURONEStion: quadriceps, anterior biceps-semimembranosus, posterior biceps-emitendinosus, medialgastrocnemius, lateral gastrocnemius-soleus, plantaris and flexor digitorum longus. In someexperiments the nerves to triceps surae and plantaris were dissected in continuity to allow selectiveactivation of group Ib afferents after raising the threshold of Ia afferents by muscle vibration(Coppin, Jack & McLennan, 1970; Fetz et al. 1979). The laminectomy exposed the spinal cord fromL4-L6 segment caudally, and at Th 12-13 level. Four cats were spinalized at Th 13. The L6-S1ventral roots were cut distally. The L7 ventral root was carefully divided into two equal parts andeither the caudal part of this root or the whole Si ventral root was used for recording populationp.s.p.s. Otherwise the proximal cut ends of the ventral roots were mounted for stimulation to aidin the identification of I a inhibitory interneurones and Renshaw cells, and to evoke recurrentp.s.p.s.

Records from interneurones and spike triggered averaging. Interneuronal activity was recordedextracellularly with glass micro-electrodes filled with 1-3 M-sodium glutamate, or 1 M-homocysteicacid, with tips of about 1-5-2 0 ,um diameter and resistance of 8-20 MCI. The electrodes werepositioned so that the recorded action potentials (evoked by ionophoretic application of glutamateor homocysteate) were large enough for reliable triggering of the averager (Nicolet 1170), butwithout risk of unintentional penetration of the cell. The records from the interneurones werehigh-pass filtered to optimize the signal-to-noise ratio in order to secure stable triggering by thespikes. A pre-trigger delay of at least 2 msec was used to allow the onset of the interneuronal spikesto be seen, so that any changes in the averaged ventral root potentials could be related to them(see Fig. 2C and F); longer pre-trigger delays (up to 100 msec), together with a slower time base,were used to assess the presence or absence of slow potential changes unrelated to the spikes andthe occurrence of any average common excitation (a.c.e.) potentials (Kirkwood & Sears, 1978). Ofthe slowly developing potentials, only those which showed a distinct onset and were reproduciblewere accepted as p.s.p.s. Caution was observed that the interneurones were not firing with a shorterinterspike interval than the duration of the recorded population post-synaptic potential, as in thiscase the potential would not be a true reflexion of the action of each interneuronal spike actingalone. In practice this was difficult to achieve as (i) some interneurones would fire repetitively atvery high frequencies and it was not always possible to adjust the frequency to an appropriate oneby altering the ionophoretic current and (ii) some interneurones (notably Renshaw cells) wouldoften fire in doublets. In order to define the entire time course of potentials following single spikes(which was not our main aim) averaging only after those spikes which were not preceded or followedby other spikes within a certain interval (cf. van Keulen, 1981) would have been preferable, but theensuing errors were considered to be unimportant for the purposes of this study.

Ascending collateral axonal projection to the upper lumbar segments of the laminae V-VIinterneurones was judged from their antidromic activation following stimulation of the lateralfuniculus in the rostral part of L4 segment but not in Th 13. The L4 stimuli were applied via atungsten electrode inserted to a depth of 2-0-2-5 mm from the surface of the funiculus, close to itsborder with the dorsal columns. The position of the electrode was adjusted during the experimentto activate axons of the tested interneurones with stimulus intensities of 100lA or less. Theantidromic responses were identified with the collision test, as illustrated in Fig. 1. The stimuli wereapplied within the lateral funiculus rather than Clarke's column, to allow activation of ascendingaxonal collaterals of a maximal number of interneurones.

Use of a sucrose-gap for recording population post-synaptic potentials from motoneurones' axons inthe ventral roots. The conditions satisfactory for displaying post-synaptic potentials from a singleinterneurone electrotonically conducted along the ventral roots have been tested by recordingactions of Ia inhibitory lamina VII interneurones (Brink et al. 1981). Population inhibitorypost-synaptic potentials (i.p.s.p.s) evoked by single such interneurones were found to be detectablewhen the SI or the caudal half of the L7 ventral root was threaded through a 4-5 mm-long tubeperfused with 0-3 M-sucrose into a 0 9 % KCl-filled tube which contained a Ag-AgCl electrode (Fig.1 A; see also fig. 1 of Brink et al. 1981). The electrodes were similar to those described by Luscheret al. (1979) and were modified only to allow easy access to the spinal cord with other electrodes.Sucrose flow was kept at 0-05-0-1 ml./min and sucrose collecting around the spinal cord wasremoved by continuous suction. Compound monosynaptic excitatory post-synaptic potentials(e.p.s.p.s) evoked by stimulation of a muscle nerve increased 3-5 times in amplitude after the onsetof sucrose perfusion, and averaging the effects of 500-1000 interneuronal spike potentials, in somecases even 50-100, was sufficient to reveal the p.s.p.s which followed them.

Terminology. In order to avoid confusion between the intracellulary recorded post-synaptic

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potentials and their counterparts recorded from the ventral roots, the following terminology willbe used:unitary p.8.p: the intracellular potential evoked by a single neurone,compound p.s.p: the intracellular potential evoked by a population of neurones,unitary population p.s.p: the potential recorded from the ventral root, due to the action of a singleneurone onto a population of motoneurones,compound population p.s.p: the potential recorded from the ventral root, due to the action of apopulation of neurones onto a population of motoneurones.

Laminae V-VI int.Lamina VII int.Renshaw cell It

L4 lateral L.g.-s.Motoneurones funiculus + L4

Sucrose"WIVentral root 3

KCI

1 msecFig. 1. Experimental arrangement for parallel recording from single interneurones andfrom axons of motoneurones. The interneurones included I a inhibitory interneurones inlamina VII, Renshaw cells, and laminae V-VI interneurones characterized by their inputfrom group I afferents and antidromic activation following stimuli applied in the lateralfuniculus in L4. Further explanations are in the text. Records illustrate selectivity ofrecording from the investigated interneurones and the collision test used to ascertain thatspike potentials evoked by L4 stimuli in laminae V-VI were due to the antidromicinvasion. Note that the critical interval at which the second spike potential disappearedwas equal to twice its latency, minus 0-2 msec of the latent period for generation of thesespikes by electrical stimuli, plus 0-7 msec to account for the axonal refractory period.L.g.-s., lateral gastrocnemius-soleus.

RESULTS

A. Population p.8.p.8 evoked by Ia inhibitory lamina VII interneuronesInterneurones mediating reciprocal inhibition of motoneurones from group Ia

muscle spindle afferents are easy to identify by their activation by low thresholdmuscle afferents and by their inhibition following ventral root stimulation (Hultbornet al. 1971). The majority of those with input from quadriceps send a descendingcollateral to posterior biceps or semitendinosus motoneurones which have axons inthe L7 and S1 ventral roots (Jankowska & Roberts, 1972a). Population i.p.s.p.s werefound to follow spike activity of fourteen out of twenty (70 %) such lamina VIIinterneurones in five cats. Examples of these unitary population i.p.s.p.s are shownin Fig. 2D and F, together with compound i.p.s.p.s evoked by stimulation ofquadriceps I a afferents and recorded intracellularly from an antagonist motoneurone(Fig. 2A), or similarly from axons of a population of motoneurones (in Fig. 2B, Cand E).The unitary population i.p.s.p.s (0-5-2-0 1sV) were 10-50 times smaller than the

compound population i.p.s.p.s recorded in the sucrose-gap. Simple division of the

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POPULATION P.S.P.S EVOKED BY INTERNEURONES 345

compound population i.p.s.p.s by the mean unitary population i.p.s.p.s shouldprovide an estimate of the number of interneurones mediating reciprocal inhibition.However, this estimate (ten to fifty) may be an underestimate in view of the previousdata relating to intracellularly recorded i.p.s.p.s (Jankowska & Roberts, 1972 b). Since

A Q. 1-212

B Q. 1-4 CE%

C j-Q.14 Q. 1-4 1-5mVAC ~~~~E32pVB8

D 16 MV C,ED F

I05 pVVD7 i ~~~~~~~~~~1-0yV F

1 msec A,8 2 msec EF2 msec CD

Fig. 2. Comparison of compound and unitary population reciprocal i.p.s.p.s. In A, B, Cand E are examples of compound i.p.s.p.s evoked by stimulation of group Ia afferentsin the quadriceps (Q.) nerve. They were recorded intracellularly from a posterior bicepsor semitendinous motoneurone (A) or from a ventral root (B, C and E). I.p.s.p.s followingspike activity of single I a inhibitory interneurones induced by electrophoretic applicationof glutamate are shown in D and F (upper traces, interneuronal spikes recordedsimultaneously being displayed underneath). The unitary population i.p.s.p.s wererecorded with the same time base as the compound i.p.s.p.s immediately above, in thesame experiment (C and D), or in another (E and F). Note that the unitary populationi.p.s.p.s showed a similar or slower time-to-peak than the compound i.p.s.p.s. Theillustrated unitary i.p.s.p.s are among the fastest ones, while the compound i.p.s.p.srepresent the fastest (B) and the slowest ones (C and E). The filled upward and downwardarrowheads indicate onset of the afferent volleys in quadriceps nerve and the synchronousactivity of interneurones subsequently activated; the latter coincided in time with spikepotentials of Ia inhibitory interneurones. The open upward arrow head in D indicatespresynaptic potentials in axon collaterals of the investigated interneurones. The diagramshows the site of recording (from the motoneurone or from a ventral root) of records A-Fby the position of the respective letters. The letters to the left are for compound p.s.p.s(evoked by nerve stimulation) while those to the right are for unitary p.s.p.s followingactivity of an interneurone (black) induced by glutamate or homocysteate. The sameformat is used in diagrams of Figs. 3-5.

in that study individual interneurones evoked unitary i.p.s.p.s 10-200 (mean 69)times smaller than the compound ones and were expected to influence one fifth of thepopulation of motoneurones, these data would indicate approximately 350 inter-neurones. This discrepancy may be due to failure to detect the smallest unitarypopulation i.p.s.p.s and/or that in the present study the stimuli used to evoke thecompound population i.p.s.p.s were submaximal for firing all the interneurones.

E. BRINK AND OTHERS

The compound population i.p.s.p.s were 50-100 times smaller than the compoundi.p.s.p.s recorded intracellularly. Considering a mean decrement of the recordedpotentials of about 10,000 (see below and the Discussion) these figures indicate thatthe potentials were from at least 100 motoneurones.The unitary population i.p.s.p.s had somewhat longer times-to-peak: about 1.5

times longer than times-to-peak of compound population i.p.s.p.s and about 5 timeslonger than times-to-peak of both unitary and compound i.p.s.p.s in intracellularrecording. In Figs. 6A and B a number of unitary i.p.s.p.s recorded in singlemotoneurones (using the data ofJankowska & Roberts, 1972b) are superimposed forcomparison with similarly superimposed unitary population i.p.s.p.s. They have beenaligned so that the onsets coincide, the amplitudes being normalized. The rise timesfor p.s.p.s recorded under these conditions are given in Table 1 and the possiblereasons for the differences will be taken up in the Discussion. In contrast to the timecourse, the latencies of the unitary population i.p.s.p.s and of the unitary i.p.s.p.srecorded intracellularly (measured with respect to the presynaptic spike potentials)did not show major differences. They were 0-30-0 80 and 0-28-0-42 msec, respectively.The corresponding latencies measured from the onset ofthe interneuronal spikes were0-70-1P70 and 0-40-0 75 msec.

B. Population p.8.p.s. evoked by Renshaw cellsEight Renshaw cells were tested for their effects on motoneurones in two

experiments; the cells were located in the middle or in the rostral part of L7 segmentswhen the motoneuronal responses were recorded from the caudal part of L7 ventralroot, and in the caudal part of L7 segment when the records were taken from SIventral root. Spike activity of all but two most rostrally located cells was followedby unitary population i.p.s.p.s.Examples of unitary population i.p.s.p.s following activity of two Renshaw cells

are shown in Figs. 3D, F and H. They are shown together with the compoundrecurrent i.p.s.p.s evoked by ventral root stimulation, recorded intracellularly (Fig.3A) in the neighbouring segment and from the adjacent ventral root (B, C, E andG). The four pairs of potentials (AB, CD, EF and GH) were recorded simultaneouslyor in quick succession. As in the case of Ia inhibitory interneurones the potentialsshowed considerable differences in time course. The time-to-peak of the unitarypopulation i.p.s.p.s (Fig. 3 and D, F and H) was either longer than or similar to thatofthe compound population i.p.s.p.s (B, C, E and G) but longer than the time-to-peakof either compound (Fig. 3A) or unitary i.p.s.p.s recorded from intracellularly (seeTable 1). In Figs. 6C and D are superimposed records oftwo unitary i.p.s.p.s recordedintracellularly by L. van Keulen, reproduced from records published in Fig. 2 ofBaldissera, Hultborn & Illert (1981) and of six unitary population i.p.s.p.s obtainednow.The population i.p.s.p.s showed a time-to-peak which was similar to that of the

slower intracellularly recorded i.p.s.p of Fig. 6C and some 6 times longer than of thefaster unitary potential. As in the case of the Ia inhibitory interneurones nosignificant differences have been found between latencies (05-2-0) of the unitarypotentials produced by Renshaw cells recorded under different conditions (cf. vanKeulen, 1981).

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POPULATION P.S.P.S EVOKED BY INTERNEURONES

The unitary population i.p.s.p.s (1-4 1sV) were up to about 20 times smaller thanthe compound population i.p.s.p.s, which in turn were some 100 times smaller thanrecurrent i.p.s.p.s recorded intracellularly. Since the ratio between amplitudes ofunitary and compound population i.p.s.p.s appeared to give an underestimate of thenumber of Ia inhibitory interneurones, the conclusion that only twenty Renshawcells were responsible for the recorded compound population recurrent i.p.s.p.s maybe justified.

A

B A

I, ~~~~~BDC FE 'FG ~

C _ E G

D F H

t3mVA2 msecA,B 32p4 msec CD 16pV CEGBmsecEF1VEG16 msec G,H 4 AV D,F

2 gV H

Fig. 3. Comparison of compound and unitary population recurrent i.p.s.p.s. In A-C, Eand G are examples of i.p.s.p.s evoked by stimulation of the rostral part of L7 ventralroot. They were recorded intracellularly from a posterior biceps-semitendinosus moto-neurone in caudal L7 (A) or from the caudal part of L7 ventral root, as indicated in thediagram. i.p.s.p.s following activity of single Renshaw cells induced by electrophoreticapplication ofglutamate are shown in D, F and H. A and B were recorded simultaneously;the other pairs of records were taken one after another in quick succession. Note in C,E and G the depolarizing potential following the recurrent inhibition. This may correspondto the recurrent facilitation of motoneurones.

C. Population p.s.p.8 evoked by laminae V-VI interneuronesSatisfactory parallel records from single laminae V-VI interneurones and from

ventral roots were obtained for thirty-six interneurones with group I input. Thesewere located in L6-L7 segments at a depth 2-0-2-5 mm from the surface, in the regionwith group I field potentials (Eccles, Fatt & Landgren, 1956) typical for laminae V-VIofRexed, and where the majority ofinterneurones projecting to Clarke's column havebeen found (Hongo et al. 1983a). Twenty-two interneurones (the main group) wereantidromically activated on stimulation of the lateral funiculus in L4 segments.Nineteen of these interneurones were monosynaptically excited by group I afferentsof triceps surae and plantaris or flexor digitorum longus and three interneurones bygroup I afferents of quadriceps or hamstring. The fourteen other interneurones

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E. BRINK AND OTHERS

(control interneurones) differed from the main group only in lacking antidromicactivation following stimulation of the lateral funiculus.

Interneurones with group I input and an axon collateral ascending to L4. Populationp.s.p.s associated with potentials of these interneurones fell into three categories. Thefirst category (n = 6), exemplified in Fig. 4A and B and Fig. 5D, were populationi.p.s.p.s with latencies 1P1-2 1 msec from the onset of the spike; these latencies wereas required for i.p.s.p.s evoked monosynaptically from the respective distances (cf.

A -Off I D J G

B E

c FpV

20 msec

Fig. 4. Examples of unitary population p.s.p.s related to the activity of laminae V-VIinterneurones. Upper traces, averaged records of spike potentials of the interneurones.Lower traces (A-F) records from a ventral root. A-F, under chloralose anaesthesia. E,following a dose of sodium pentobarbitone (20 mg/kg). F, following a dose of pentobar-bitone and a dorsal rhizotomy. The diagram illustrates a possible origin of the depolarizingpotentials preceding the i.p.s.p.s (see text).

Jankowska & Roberts, 1972b; Brink, Jankowska, McCrea & Skoog, 1983a). Thesecond category (n = 8) consisted of depolarizing potentials, the onset of whichpreceded the appearance of the interneuronal spikes, and were interrupted by apopulation i.p.s.p which followed the spike (Fig. 4C-F and Fig. 5E); such populationi.p.s.p.s showed a rather sharp onset and latencies similar to potentials in the firstcategory. The third category (n = 8), consisted of depolarizing potentials precedingthe interneuronal spikes, as in the second category, but these were not followed byany distinct i.p.s.p.s. The depolarizing potentials are interpreted as being due to aparallel excitation of both the motoneurones and the interneurones. Such excitationhas been described in detail for respiratory motoneurones and is referred to asaveraged common excitation (a.c.e) potential (Kirkwood & Sears, 1978; Kirkwood,Sears, Tuck & Westgaard, 1982). Spike-triggered averaging using spike activityevoked by electrophoretically applied glutamate or homocysteate (instead of spon-taneous discharges) was expected to obviate complications due to common excitation.However, since a.c.e. potentials were present it appeared that glutamate or homo-cysteate must have often acted on a background of synaptic depolarization of theinterneurones and that the frequency of discharges of the interneurones was thenmodulated by impulses from this unknown source. It was expected that deepeningthe anaesthesia by adding 20 mg/kg of sodium pentobarbitone and/or reducing theinput to the interneurones by spinalization and/or sectioning the dorsal roots at the

348

POPULATION P.S.P.S EVOKED BY INTERNEURONES

level of the interneurone location would abolish the a.c.e. potentials, but this was notthe case (cf. Kirkwood et al. 1982).The presence of a.c.e. potentials associated with ionophoretically evoked firing of

the interneurones emphasizes the interpretative problems when using spike-triggeredaveraging and illustrates the difficulties of being confident that the recordedpotentials are evoked by the tested interneurones and not simply correlated with theiractivity. However, the observations illustrated in Fig. 5 increase our confidence that

A L.g. 12 E

C i L.g.13 F

D E+F E-F

1 msec A,B 4 msec EF I -4mVAv msecCD 0.6 pV D

Fig. 5. Comparison of compound and unitary non-reciprocal i.p.s.p.s. In A, B and C areexamples of i.p.s.p.s evoked by stimulation of lb afferents in a peripheral nerve (lateralgastrocnemius, l.g.) after raising threshold of a great majority of Ia afferents by aprolonged muscle vibration (Coppin et al. 1970; see also Fetz et al. 1979). They wererecorded intracellularly in a lateral gastrocnemius motoneurone (A) or from ventral roots(B) in the same preparation (B simultaneously with A), as indicated in the diagram. Thei.p.s.p.s are preceded by small e.p.s.p.s since the vibration block was not complete. Controlrecords of population e.p.s.p.s evoked by near-threshold muscle stretch showed that thepopulation i.p.s.p.s were genuine. In D-F are population p.s.p.s. following activity of twolaminae V-VI interneurones. The i.p.s.p.s in D seem to be preceded by a presynaptic volley(arrow). For comments on records E-F see text.

this group of interneurones with axon collaterals to L4 and group I input evokeinhibition in motoneurones. The Figure shows two population potentials recordedfollowing activity of such an interneurone, first when the electrode was in anextracellular position (Fig. 5E) and then after the penetration of the interneurone(Fig. 5F). After penetration the interneurone showed an approximately similar rateof spike-like responses but these had deteriorated to only a few mV amplitude. Suchsmall potentials were most likely local responses which were not conducted along theaxon. The population a.c.e potential had then a much longer time course and no shortlatency i.p.s.p interrupted it. The i.p.s.p recorded before the penetration of theinterneurone (Fig. 5E) appears thus to have been evoked by this particular

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E. BRINK AND OTHERS

interneurone; it is shown as the difference between records E and F. Unfortunatelywe have obtained such records only in this case.

In total, fourteen of the twenty-two tested interneurones, or 65 %, evoked shortlatency population i.p.s.p.s in motoneurones, with or without a preceding a.c.e.potential. Unitary population i.p.s.p.s following activity of six laminae V-VIinterneurones had times-to-peak of 6-10 msec. The times-to-peak of the remainingi.p.s.p.s were 10-60 msec (n = 8). Those with the shortest and longest times-to-peak

A > C E

B D F

G

2 msec A,BE-G3 msec CD

Fig. 6. Comparison of intracellularly recorded unitary i.p.s.p.s and of population i.p.s.p.sfollowing activity of La inhibitory interneurones, Renshaw cells and laminae V-VIinterneurones. A, superimposed traces of unitary i.p.s.p.s evoked by single lamina VII I ainhibitory interneurones with input from quadriceps, in posterior biceps or semitendinosusmotoneurones (unpublished data of E. Jankowska and W. Roberts). B, superimposedtraces of population i.p.s.p.s evoked by single la inhibitory interneurones, as in A butrecorded from a SI ventral root with sucrose gap. C, superimposed traces ofi.p.s.p.s evokedby single Renshaw cells in two motoneurones (data of L. van Keulen 1981, reproducedfrom Baldissera et al. 1981, with permission): D, superimposed traces ofpopulation i.p.s.p.sfollowing spikes of all tested Renshaw cells. E, superimposed traces ofthree i.p.s.p.s evokedby single Ib afferents in lumbar motoneurones (reproduced from Watt et al. 1976 withpermission). F, superimposed traces of two i.p.s.p.s evoked by single lb afferents, as inE, but recorded from ventral roots (reproduced from Luischer et al. 1979, with permission).0, superimposed traces of six fastest population i.p.s.p.s evoked by single laminae V-VIinterneurones with projections to rostral L4. The amplitudes of all the records have beennormalized in such a way that the i.p.s.p.s crossed common points corresponding to themean time-to-peak of the respective potentials (cf. Table 1).

are illustrated in Figs. 5D and 4A and B respectively. Fig. 5 also shows that thetime-to-peak ofthe unitary i.p.s.p.s was at least twice as long as that ofthe compoundpopulation i.p.s.p.s evoked by nerve stimulation (compare D with B and C) whichin turn was estimated as being about twice that of i.p.s.p.s recorded intracellularly(compare B with A). The latter comparison has been made for i.p.s.p.s recorded infive motoneurones, in the same preparation in which the population i.p.s.p.s wereobtained and for i.p.s.p.s recorded in twenty-five motoneurones in a preceding seriesof experiments (see Table 1). All were evoked by selective stimulation of I b afferents(see Fetz et al. 1979) or by stimulation involving only a small number of Ia afferents,as was the case of records in Fig. 5A-C. In both cases the stimulation was nearthreshold for I b afferents which precluded comparison ofamplitudes ofthe compoundand unitary (0 5-5 ,uV) population i.p.s.p.s. Fig. 6E and G shows the difference in time

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POPULATION P.S.P.S EVOKED BY INTERNEURONES 351

course of the unitary population (G) and unitary intracellularly recorded (E) i.p.s.p.s;the latter following activity of single Ib afferents (reproduced from Watt, Stauffer,Taylor, Reinking & Stuart, 1976). On the other hand, there is very good correspon-dence between our records of unitary population i.p.s.p.s (Fig. 6G) and thosepopulation p.s.p.s which followed activity of single I b afferents (F) as illustrated byLuscher et al. (1979).A number of other interneurones were also tested for their action upon moto-

neurones. Of fourteen laminae V-VI interneurones with group I input, but withoutantidromic activation following simulation of the lateral funiculus at L4 level, onlyone was found to evoke population i.p.s.p.s and two to evoke population e.p.s.p.s.The i.p.s.p.s resembled the fastest unitary population i.p.s.p.s described above. Thee.p.s.p.s were much smaller (about 0- 4-2 ItV). The activity ofthe other interneuroneswas not followed by any distinct averaged population p.s.p.s.

D. Attenuation and time course in sucrose-gap recordingRecording p.s.p.s from the ventral roots inevitably leads to distortion of the signals

due to the resistive-capacitative coupling between the site ofgeneration of the p.s.p.sand the recording site. In order to assess this more quantitatively the followingapproaches have been used. In the first instance square-wave current pulses,subthreshold for motoneurone firing, were applied intracellularly in an attempt tosee how they appeared in the ventral root recording. However, since no differencewas detected between pulses applied intracellularly and those applied extracellularlytheir interpretation was equivocal. Anexamination ofafterhyperpolarization followingaction potentials in single motoneurones appeared to be much more useful. When brief(0-1 msec) current pulses, suprathreshold for firing, were injected intracellularly tosingle motoneurones, action potentials could be recorded from the appropriateventral root. The fast component of the action potential could not be used for thepresent purpose since it would be actively conducted along the intraspinal part ofthe axon and therefore its record would not reflect events restricted to the soma.However, the long duration afterhyperpolarization following the spike could be usedsince its generation is restricted to the motoneurone cell body.

Fig. 7 A shows the afterhyperpolarization of a tibial motoneurone recorded fromthe ventral root, and Fig. 7B shows its intracellular record, the two being superimposedin C. Three further examples are shown in Fig. 7 D, E and F.

Since the size of these potentials recorded from the ventral root was small(03-2-0 ,uV), it was important to check that they were the electronically conductedcounterpart ofthe soma afterhyperpolarization and not due to, for example, amplifier' cross-talk' or other artifact. It was therefore ascertained that such potentials couldonly be recorded when the current pulses were large enough to fire the motoneuronesand when the motoneurone had its axon in the appropriate ventral root. Furthermore,the amplitude and shape of the potentials varied, as the afterhyperpolarization didfrom one motoneurone to the next, and while recording from one motoneurone asits amplitude and shape were changed with depolarizing and hyperpolarizing d.c.current. In addition, such a potential could not be evoked from a 'blocked' spike;a spike in which the soma-dendritic component (including the afterhyperpolarization)was lacking. This latter control also ruled out the possibility that the potentials could

352 E. BRINK AND OTHERS

be recurrent i.p.s.p.s, evoked by activity of Renshaw cells being made to dischargeby single action potentials ofa motoneurone. To further obviate this we selected tibialmotoneurones which are reported to lack recurrent axon collaterals (Cullheim &Kellerth, 1978).

SiA D

8 E

C F

4 msec

Fig. 7. Comparison of the afterhyperpolarization of single motoneurones when recordedfrom the ventral root (A) and intracellularly (B). The two records are superimposed inC. Similar superimpositions for three other motoneurones are shown in D-F. Calibrations:ventral root recording; 2 1sV for A and C; 1 1sV for D-F: intracellular; 6 mV for B,C and.E; 4 mV for D; 12 mV for F.

The decrement in amplitude observed was from a few millivolts in the intracellularrecording to a fraction of a microvolt in the ventral root recording and was of from1000 to 20,000 times. The differences in time course were more difficult to quantifyin view of the small amplitudes in the ventral root recording. However, anyprolongation ofthe time course ofthe afterhyperpolarization wasmuch less pronouncedthan in the cases of unitary i.p.s.p.s.

DISCUSSION

The interpretation of population post-synaptic potentialsP.s.p.s recorded from the ventral root are notably of small amplitude and long

duration. With regard to their small amplitude we can estimate the expecteddecrement from the length constant of the motoneurones' axons and from thedistance between the motoneurone soma and the ventral root electrode. Thus, if theaxonal length constant is about 1-7 mm (Gogan, Gueritaud & Tyc-Dumont, 1983) andthe ventral root recording electrode is about 15 mm along the motor axons from thesoma, the signal will decrement to exp (- 15/1-7) of its amplitude in the motoneuronesoma; which gives a decrement of about 7000 times. However, judging from the size

POPULATION P.S.P.S EVOKED BY INTERNEURONES

of compound e.p.s.p.s recorded before and after sucrose infiltration, the sucrose-gapincreases the amplitude ofthe potentials 3-5 times and thus should reduce the over-alldecrement to about 2000 times.The decrement along the motor axons can also be estimated, albeit approximately,

from the data relating to Ia e.p.s.p.s. Consider a single Ia afferent evoking 100 ,sVe.p.s.p.s in motoneurones of one ventral root (say 200 motoneurones, see first sectionof the Results). Then the 'aggregate' e.p.s.p. of a single I a afferent would be around20 mV (200 x 100 ,V) which decrements to a ventral root potential of about 1,sV(Lischer et al. 1979): a factor of 20,000 times.As a direct measure of the decrement, the afterhyperpolarizations of single

motoneurones were recorded both intracellularly, from the motoneurone soma, andfrom the ventral roots. The amplitude decrement observed ranged from 1000 to 20,000times. Since these three estimates of the decrement from intracellular potentials toventral root potentials are approximately to the same order of magnitude ourconfidence is increased that the technique is reliable. However, the perfusion of theventral roots with sucrose appeared to reduce the decrement of potentials along theiraxons much less substantially than might be expected from previous studies (Liischeret al. 1979). Since the potentials decrement by 1/e of their value in 1-7 mm, the 3-5times reduction in decrement achieved with the sucrose solution is not cost effective.Consequently, what is lost in having a longer length of ventral root in order to beable to perfuse the roots with sucrose is not offset by the gain achieved with sucrosesolution. This suggests that it may be better in future experiments to dispense withthe sucrose and simply record from the ventral roots that have been cut as short aspossible.With regard to the time course of ventral root potentials, we can expect a longer

time course due to electrotonic slowing as the potentials are decremented along themotoneurones' axons, and from Table 1 it is clear that all population p.s.p.s are oflonger duration than their intracellular counterparts. However, the intra-axonal timeconstant is very small (50 ,usec) and calculations indicate that the electrotonic slowingwithin the axons is small and certainly not enough to account for the observedprolongation of p.s.p.s (B. Gustafsson, personal communication). However, furtherprolongation may be expected from any sources of stray capacitance. For example,connective tissue or myelin interposed between the axoplasm and the potassiumchloride solution would prevent the latter two being in direct electrical contact andwould therefore increase the time constant.

Further factors contributing to the slow time course of population p.s.p.s. relateto the asynchrony that is expected of the action of individual presynaptic fibres ontotheir target motoneurones. Thus, because of differences in conduction velocity andlength of terminal branches of single presynaptic neurones, compound p.s.p.s evokedin a motoneurone by a population of presynaptic neurones should, as a consequenceof the resulting asynchrony, have a longer time constant than unitary p.s.p.s and thisis the observed result (Table 1). Differences in conduction velocity and length ofterminal branches of presynaptic neurones will have an additional, significantincrease in asynchrony when considering p.s.p.s evoked in a population of moto-neurones distributed over, for example, half a segment. Consequently, all populationp.s.p.s will have a longer time course than their intracellular counterparts.

PHY 343

353

12

354 E. BRINK AND OTHERS

TABLE 1. Comparison of time-to-peak of p.s.p.s recorded under various experimental conditions.A, mean values or ranges in msec. *, 10-90% rise-time values. [1] Jankowska & Roberts, 1972b;[2] Watt et al. 1976; [3] Curtis & Eccles, 1959; [4] present study; [5] van Keulen, 1981; [6] Eccleset al. 1954; [7] Sypert, Fleshman & Munson, 1980; [8] Burke, 1967; [9] Jankowska et al. 1977; [10]Luscher et al. 1979; [11] Stauffer, Watt, Taylor, Reinking & Stuart, 1976; [12] Munson et al. 1980.B, relative values of data in A.

A. Time-to-peak (mean values in milliseconds)

Unitaryintracellular

Compoundintracellular

Compoundpopulation

Unitarypopulation

lai.p.s.p.s

.085 [1]1-38 [2]

0-96[1]1-2 [3]

3-5 [4]

5-2 [4]

Recurrenti.p.s.p.s

3-0 [5]

3-10 [6]4.7 [4]

10-2 [4]

17-2 [4]

lbe.p.s.p.s

1.4 [2]

3-8 [4]

5-0 [4]

9.5 [4]

lae.p.s.p.s

1*0 [2]0-59 [7]

IIe.p.s.p.s

1-0 [11]0-65 [12]*

0-85 [8]-1-500-86 [9]

1-36 [4]

2-54 [10]* 3-17 [10]*

B. Time-to-peak (relative values)

Recurrenti.p.s.p.8

lbe.p.s.p.s

Unitaryintracellular

Compound 1.1intracellular

Compound 2-9population --3-6

Unitarypopulation

2 7 085

242 j13 1

3.7 2-5 2-9

1-7 1-9 1.9

Following this reasoning it is to be expected that compound population p.s.p.s are

of longer duration than unitary population p.s.p.s. However, Table 1 indicates theconverse. One likely explanation for this discrepancy is that since we did not alwaysexclude averages in which the interneurones fired in doublets or triplets, the secondand third action potentials would prolong the duration of the averaged unitarypopulation potential. Consequently, in these cases the compound population p.s.p.

would not be directly comparable to the unitary population p.s.p. Another possibleexplanation for the discrepancy is that the time course of unitary populationpotentials is often obscured by the presence of superimposed a.c.e. potentials. Thus,when the falling phase of the a.c.e. potential is occurring concurrently with the rising

Ia

i.p.s.p.sIa

e.p.s.p.sII

e.p.s.p.s

4.3* I4.9*

POPULATION P.S.P.S EVOKED BY INTERNEURONES

phase of the unitary population i.p.s.p.s, the apparent time course is artifactuallylengthened. This is illustrated by the records in Fig. 5 where it has been possible tosubtract the a.c.e. potential and reveal an i.p.s.p. of briefer rise time. With regardto any other possible explanations it may be significant that all unitary populationp.s.p.s, including monosynaptic e.p.s.p.s, appeared to show a proportionally similarprolongation when compared with compound population p.s.p.s; some factors mightthus be common for all of them.

Averaged common excitation potentialsThe presence of a.c.e. potentials associated with interneuronal activity implies

common excitation of the last-order interneurones and the motoneurones themselves,though in the present experiments it is not known whether the common excitationis to the same motoneurones as the motoneurones to which the interneurones project.Common excitation is generally thought to be due to synchronized synaptic activityonto the two groups of interneurones investigated. This may occur in either of twoconditions: (i) synchronization of activity of presynaptic neurones or (ii) the jointoccurrence of unitary p.s.p.s by branches of common stem presynaptic fibres. Thetime course of common excitation is one feature that may be used to infer which ofthese two conditions is operative. The characteristics of the common excitationpotential expected solely as a result of branching presynaptic fibres have been workedout in detail for single motoneurones (Kirkwood & Sears, 1978), and experimentallyobserved a.c.e. potentials can be directly related to them. However, the a.c.e.potentials reported here cannot be directly compared to those recorded intracellularlywithout prior consideration of additional factc-d. First, they are population a.c.e.potentials, reflecting the superimposed a.c.e. potentials of many motoneurones, whichwill lead to a more prolonged time course. Secondly, the time course of the presenta.c.e. potentials is further distorted by varying degrees of superimposed inhibition.Since the rise times of the most prominent components of the population a.c.e.potentials are about twice as long as those of the intracellular potentials of Kirkwood& Sears (1978; 12-20 msec compared to 4-16 msec) they follow time course relationssimilar to other population p.s.p.s when compared to their intracellular counterparts.They are thus likely to reflect an intracellular component as fast as those describedby Kirkwood & Sears (1978). The importance of emphasizing the correspondence ofthe fast component of the a.c.e. potentials is that such a component is compatiblewith the potentials being due to the actions of branching presynaptic axons, whereasin order for input synthrony to account for this component 'it must be on a verynarrow time scale and be strong' (Kirkwood & Sears, 1978). Consequently, it appearslikely that activity in branching presynaptic fibres accounts, at least partly, for thecommon excitation. However, a contribution of common excitation by synchronizedpresynaptic inputs cannot be excluded, although it appears rather unlikely sincepopulation a.c.e. potentials were present in deep anaesthesia and when the spinal cordwas intact, conditions which were apparently unfavourable for the occurrence ofpresynaptic synchrony in the work of Kirkwood et al. (1982).

Since an examination of common excitation was subsidiary to the main aim of thisstudy an investigation of its origin has not been pursued, although we do have someindications ofit. Considering common sources ofmonosynaptic input to motoneuronesand to the laminae V-VI interneurones, the first possibility that we considered was

12-2

355

E. BRINK AND OTHERS

that it was due to the monosynaptic action from muscle spindle Ia afferents, sincethey are spontaneously active and have been shown to innervate both the presentpopulation of laminae V-VI interneurones (Jankowska & McCrea, 1983; Harrison etat. 1983) and motoneurones. However, since section of the dorsal roots was foundto give a negligible reduction of the a.c.e. potential, their contribution is concludedto be small relative to other sources. The possibility of descending origin seemedremote since the major descending tracts (cortico-, rubro- and vestibulospinal) wouldappear to terminate upon either the investigated interneurones or motoneurones, butnot both (for references see Baldissera et al. 1981). This was supported by theobservation that the a.c.e. potentials were unaffected by spinalization. The remainingpossibility, that the common excitation was of interneuronal origin, was somewhatdifficult to reconcile with the observations that the a.c.e. potentials were unaffectedby large doses of barbiturate which are known to have a depressant effect on neuronalactivity. Interneurones responsible for the common excitation should then be amongonly those which are spontaneously active even under deep anaesthesia. In addition,since Kirkwood & Sears' (1978) analysis predicts that common inhibition evokesqualitatively the same effect as common excitation, they may be either excitatoryor inhibitory interneurones. In the case of common inhibition the interneurones usedfor triggering will tend to fire in the absence of inhibition, which will correlate withthe absence of inhibition in motoneurones. Correspondingly an apparent excitation(or disinhibition) will result. With this in mind one can consider the possibility thatcommon background inhibition may be due not so much to genuine 'spontaneous'activity as to the residual effects of ionophoretically applied glutamate or homo-cysteate. If after investigating the action of an inhibitory laminae V-VI inter-neurone, the interneurone continued to discharge, it would have inhibited bothmotoneurones and other inhibitory interneurones (Brink et al. 1983 a), among whichwere the subsequently investigated ones. It is thus conceivable that such residualionophoretically induced interneuronal activity could be the sole origin of the a.c.e.potentials.The same mechanism could also operate in the case of occasionally recorded a.c.e.

potentials when Renshaw cells and lamina VII I a inhibitory interneurones were usedfor spike-triggered averaging. Since Renshaw cells inhibit both other Renshaw cellsand motoneurones, and Renshaw cells and Ia inhibitory interneurones inhibit bothIa inhibitory interneurones and motoneurones, it may be that these a.c.e. potentialsalso resulted from common inhibition. Records in Fig. 3C, E and G are particularlyinteresting in this context because they show that the recurrent i.p.s.p.s were evokedon a background of tonic inhibition of the motoneurones; the latter is evidenced byrecurrent facilitation following the inhibition.

Identification of the interneurones mediating non-reciprocal inhibition of motoneuronesThe principle aim of this study was to use the sucrose-gap recording from ventral

roots in order to identify the last-order interneurones mediating non-reciprocalinhibition of motoneurones from group I afferents. Initial attempts to achieve this,using simultaneous recording from single interneurones and from single motoneuronesexpected to be inhibited by them, were unsuccessful (E. Jankowska, D. McCrea &zB. Skoog, unpublished). In contrast to the case for target motoneurones of Iainhibitory interneurones (Jankowska & Roberts, 1972b), it appeared that the

356

POPULATION P.S.P.S EVOKED BY INTERNEURONES

probability of penetrating target motoneurones of laminae V-VI interneurones wasvery low. Consequently the synaptic actions of laminae V-VI interneurones wereassessed by recording p.s.p.s from a population of motoneurones' axons.

In order to test whether the technique was sufficiently sensitive to detect theactions of single interneurones, the actions of identified last-order interneurones werefirst used as a control. As the preceding (Brink et al. 1981) and the present reportsshow, single lamina VII Ia inhibitory interneurones and Renshaw cells evokesufficient inhibition in motoneurones for their action to be detected in the ventralroot recording. This is not to say that the technique can detect the action of alllast-order interneurones, as the inhibitory action of only 70% of the I a inhibitoryinterneurones and Renshaw cells was detected. Whether the remaining 30% sent theircollaterals to motoneurones with axons in other ventral roots, or the resolution of thetechnique was not high enough to detect the smaller i.p.s.p.s, cannot be decided.With regard to the laminae V-VI interneurones, 65% of those with an axonal

projection to upper lumbar segments were found to evoke short latency i.p.s.p.s. Sincenone of the tested interneurones of this group appeared to have an excitatory actionon motoneurones, our observations are well in keeping with the previous conclusionsbased on observations with a more indirect approach that all such interneuronesshould be inhibitory (Hongo et al. 1983 a,b; Brink et at. 1983 a). They are particularlyin agreement with intracellular records from hind-limb motoneurones in which onlyi.p.s.p.s were evoked upon stimulation of Clark's column, and thus fully confirm theoriginal hypothesis of this study (see the Introduction).Our data do not allow us to claim that only interneurones with ascending collaterals

mediate the non-reciprocal inhibition of motoneurones, since one interneurone of thecontrol group (with group I input but no antidromic activation) appeared to inhibitmotoneurones, although this neurone could also have had an ascending axoncollateral which may have run in the ventral funiculus (cf. Jankowska et al. 1981).On the other hand, since the proportion of laminae V-VI interneurones with theascending projection found to be inhibitory was similar to that of other identifiedinhibitory interneurones (I a inhibitory interneurones and Renshaw cells) we concludethat all laminae V-VI interneurones with group I monosynaptic input and ascendingprojection to upper lumbar segments may be considered as interneurones mediatingnon-reciprocal inhibition of motoneurones.

We wish to thank K. Danielsson for technical assistance. The study was supported by the SwedishMedical Research Council (project no. 05648). E. B. was supported by a N.I.H./Swedish MedicalResearch Council Postdoctoral Fellowship. P. J. H. was supported by a NATO PostdoctoralFellowship.

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