BY KIRSTY GRANT AND GINETTE HORCHOLLE-BOSSAVIT ...

J. Phy8iol. (1983), 339, pp. 41-60 41With 9 text-figuresPrinted in Great Britain

CONVERGENCE OF TRIGEMINAL AFFERENTS ON RETRACTORBULBI MOTONEURONES IN THE ANAESTHETIZED CAT

BY KIRSTY GRANT AND GINETTE HORCHOLLE-BOSSAVITFrom the Laboratoire de Phy8iologie Nerveuse LA 204, C.N.R.S.,

91190 Gif 8ur Yvette, France

(Received 8 October 1982)

SUMMARY

1. Retractor bulbi motoneurones were identified by intracellular recording of theirantidromic invasion following stimulation of the motor axons.

2. Characteristics of excitatory post-synaptic potentials (e.p.s.p.s) evoked byelectrical stimulation of long ciliary nerves (corneal afferents), the supraorbital nerveand the ipsilateral or contralateral vibrissae were analysed.

3. Comparison ofthe orthodromic responses induced by supra-threshold stimulationof the four trigeminal inputs showed that the most powerful excitatory effect wasdue to corneal afferent stimulation.

4. Excitatory synaptic potentials were followed in some cases by a period ofhyperpolarization lasting 15-20 msec. It is suggested that this is an inhibitorypotential of post-synaptic origin.

5. Interaction between condition and test e.p.s.p.s evoked by long ciliary nerveand supraorbital nerve stimulation revealed a partial blocking of test e.p.s.p.s overa longer period (more than 30 msec), and it is suggested that inhibitory mechanismswithin the trigeminal nucleus may be in part responsible for the absence of facilitationat the level of the motoneurone.

INTRODUCTION

The retractor bulbi muscle has an anatomical organization which makes it ratherdifferent from the other extrinsic muscles of the eye. Phylogenetically, it appears inamphibians and reptiles. It is present in only some mammals, becomes vestigial inmonkeys, and is lacking in man. The retractor bulbi muscle is functionally associatedwith the presence of a nictitating membrane. This third eyelid is a retractable foldof skin containing a cartilaginous sheet and glandular cells (Arao & Perkins, 1968;Stibbe, 1928). In the cat, when the eye is open, the nictitating membrane is normallyactively retracted by the tonic activity of sympathetic neurones located in thesuperior cervical ganglion (Rosenblueth & Bard, 1932; Thompson, 1958). Protrusionof the nictitating membrane results from the retraction of the eyeball into the orbitby the contraction of the retractor bulbi muscles (Rosenblueth & Bard, 1932); themembrane then covers the cornea, being both protective and lubricating.

In different mammalian species, the retractor bulbi muscle shows a variety offorms

K. GRANT AND G. HORCHOLLE-BOSSA VIT

(Motais, 1885) and innervations (Hopkins, 1916; Spencer, Baker & McCrea, 1980).In the cat, it consists of four individual muscle slips, originating from a commontendon and inserting separately, symmetrically around the eyeball, behind andbetween the insertions of the four recti muscles. The motor innervation comes froma branch of the abducens (VI) nerve; these fibres are larger in diameter and have afaster conduction velocity than those going to the lateral rectus muscle which is alsoinnervated by the VI nerve (Steinacker & Bach-y-Rita, 1968; Batini, Buisseret-Delmas & Kado, 1979). These differences are consistent with the fact that the lateralrectus muscle contains three types of motor units: singly innervated slow and fasttwitch fibres and multiply innervated slow fibres (Goldberg, Lennerstrand & Hull,1976), whereas the retractor bulbi muscle contains a homogeneous population ofsingly innervated fast twitch fibres (Alvarado, Steinacker & Bach-y-Rita, 1967) whichdo not have high resistance to fatigue (Bach-y-Rita & Ito, 1965; Lennerstrand, 1974).

Recent studies using horseradish peroxidase labelling have 'rediscovered' thatretractor bulbi motoneurones are located in the accessory abducens nucleus (Grant,Gueritaud, Horcholle-Bossavit & Tyc-Dumont, 1979; Hutson, Glendenning &Masterton, 1979; Spencer et al. 1980). However, this nucleus had been described byseveral anatomists of the late 19th and early 20th centuries (Preziuzo, 1924; VanGehuchten, 1893; Terni, 1922a). Furthermore Shaner (1933) and Levi-Montalcini(1942) have shown that the accessory abducens nucleus is formed at an earlydevelopmental stage by the ventro-lateral migration of neuroblasts from the dorso-medially situated principal abducens nucleus. This migration has been cited by AriensKappers (1933) in support of the neurobiotaxis theory of development. Accordingto this hypothesis, the position of the accessory abducens nucleus would be linkedto the major influence from the descending trigeminal nucleus upon retractor bulbimotoneurones (Terni, 1922 b).The accessory abducens nucleus, constituted by the retractor bulbi motoneurones,

forms part of a ventrolateral motor column which also comprises the digastric andfacial (VII) motoneurones (Grant, Guegan & Horcholle-Bossavit, 1981). It mighttherefore be expected that the retractor bulbi motoneurones would receive atrigeminal input similar to that seen in the neighbouring facial motoneurones andin particular those innervating the orbicularis oculi muscle which is functionallysynergistic in the protective eye-closing reflexes. Recent intracellular data fromaccessory abducens motoneurones (Baker, McCrea & Spencer, 1980) are in agreementwith the absence of contralateral vestibular excitation observed in individual slipsof the retractor bulbi muscle (Guegan & Horcholle-Bossavit, 1981). In contrast, apowerful trigeminal excitation ofthe retractor bulbi motoneurones has been describedin the anaesthetized preparation (Baker et al. 1980, Guegan & Horcholle-Bossavit,1981).The present series of experiments was carried out in order to evaluate the relative

importance of trigeminal input from specific sources and to examine the convergenceof afferent information from the supraorbital, corneal and vibrissal pad regions onretractor bulbi motoneurones in ketamine anaesthetized cats. (Preliminary results ofthese experiments were presented to the Association des Physiologistes de la LangueFrangaise (Grant & Horcholle-Bossavit, 1980).)

42

RETRACTOR BULBI MOTONEURONES

METHODS

Experiments were performed on adult cats weighing from 1-8 to 35 kg. Initially, ketamineanaesthesia (Imalgene 500, Merieux) was used throughout the experiment, administered intra-muscularly for induction (20 mg/kg), followed by supplementary doses (10 mg/kg) given intra-venously every 30-60 min as required to maintain a state of catalepsy throughout surgery and thesubsequent recording period. Ketamine was chosen for these experiments because under thisanaesthetic, neuronal activity in the brain stem remains high. Furthermore, it has been stated byBlanks, Volkind, Precht & Baker (1977) that this anaesthetic agent does not alter the functionalstate of the oculomotor system. However, ketamine anaesthesia alone results in a generalizedmuscular hypertonus and an increase in arterial blood pressure, which renders dissection of the eyeparticularly difficult. In order to avoid this problem, in later experiments induction of anaesthesiawas obtained with halothane and maintained during tracheotomy and intubation, spinal sectionat the C1-C2 level and insertion of an intravenous catheter. Anaesthesia was then continued withketamine given intravenously at the rate of 10 mg/kg. hr throughout the experiment. After controlof the depth of anaesthesia, the animals were paralysed with Flaxedil and artificial respiration wasregulated to maintain end-tidal CO2 at 4%. Heart rate was monitored, but showed little variation,and body temperature was maintained at 37-5 0C by means of a heating blanket and cotton-woolinsulation.

For placement of the stimulating electrodes, the right orbit cavity was opened and the eyeballwas removed. Bipolar silver hook electrodes were placed under the lateral rectus and retractor bulbibranches of the VI nerve and the long ciliary nerves, all of which were cut distally; a bipolar silvercuff electrode was fixed around the supraorbital branch of the trigeminal nerve which was cutdistally, and two stainless-steel needle electrodes were inserted into the whisker pad. Since thebranch of the VI nerve innervating the retractor bulbi muscle is small and often situated very deepin the orbit, in some experiments, a silver-ball stimulating electrode was placed on the VI nerveat the branching point without separate dissection of the lateral rectus and retractor bulbi branches.In this case the nerves were not cut distally and the indifferent electrode was placed on the bellyof the lateral rectus muscle. This method had the disadvantage that lateral rectus and retractorbulbi axons could not be stimulated separately; in addition, antidromic action potentials were oftenfollowed by short-latency synaptic potentials, with little difference in stimulus threshold intensity,probably of trigeminal origin. The latter problem was avoided in other experiments by using amacro-electrode stereotaxically implanted in contact with the extra-cerebral, intracranial portionof the VI nerve. At this point the VI nerve contains both the lateral rectus and the retractor bulbiaxons, running along the ventral surface of the brain stem for several millimetres before enteringthe orbital foramen; the stereotaxic co-ordinates most usually used were the following: A2 L2-5H-8 to -10. Stimulating pulses of 05 or I 0 msec duration were provided by constant voltage,isolated stimulators; current measurements showed that in all cases threshold responses toperipheral stimulation were obtained for intensities in the range 50 to 100 ,uA.

Recordings were made in the abducens and accessory abducens nuclei using glass micro-electrodesfilled with 2 M-KC1 or 2 M-K citrate, with tips broken to 05-1 0 jam and resistances of 5-8 MCI. Thesewere introduced into the brain stem under stereotaxic control, inclined at an angle of 300 caudalto the vertical plane to avoid the tentorium; the cerebellum was left intact. The resistance andcapacitance of the recording electrodes were monitored continuously and compensated. Intracellulardepolarization or hyperpolarization was achieved using a constant current generator and currentsin the range 1-10 nA.At the end of recording, animals were killed with an overdose of ketamine or of sodium

pentobarbitone, given intravenously.

RESULTS

The accessory abducens nucleus consists of 100-200 motoneurones innervating theretractor bulbi muscle, which form a rostro-caudally orientated column 200-300 ,umwide, stretching over 700-1000 ,um parallel and approximately 2 mm lateral to theprincipal abducens nucleus (Guegan, Gueritaud & Horcholle-Bossavit, 1978) (Fig. 1 A).

43


B

A

C A LC so

H max

+ 250 ;Amm

Hernx NW^_

Hmax-250,Am

0

>Y-td

TA

Fig. 1. A, diagram of coronal section of brain stem at the stereotaxic level P6 showingseparation of abducens (6p) and accessory abducens (6acc) nuclei. B, diagram ofparasagittal section through brain stem at about L 3-5, showing the angle of electrodedescent. C, extracellular micro-electrode recordings offield potentials evoked by stimulationof the retractor bulbi nerve (A), the long ciliary nerves (LC), the supraorbital nerve (SO)and the ipsilateral vibrissal pad (Vi), at the centre of the accessory abducens nucleus(Hmax) and at 250 /sm above and below this point. Calibration: A, 1 mV/I msec; LC,SO and Vi, 1 mV/2 msec). Abbreviations: 5M, motor trigeminal nucleus; 6acc, accessoryabducens nucleus; 6p, abducens nucleus; 7, facial nerve; 7p, facial nucleus; FTL, lateraltegmental field; OS, superior olive; P, pyramid; S, solitary tract; V, 4th ventricle.

Thus it represents a small target for micro-electrode recordings and for this reason,

in these experiments the accessory abducens nucleus was located stereotaxicallyby reference to the principal abducens nucleus. The principal abducens nucleus wasidentified by the field potential evoked antidromically following stimulation of thelateral rectus nerve and the point at which the field potential amplitude was maximalwas located. The recording electrode was then moved 2 mm laterally and penetrationswere continued caudally in the new parasaggital plane at intervals of 100-200 jsmand similarly at 100 or 200 gm lateral or medial to this plane. Retractor bulbi

Vi

AMN

nl

A|^

44

..Mass

a a


motoneurones were activated antidromically by stimulation of the retractor bulbibranch of the VI nerve, or by stimulating the whole VI nerve intracranially and theantidromic field potential was recorded. The dendritic arborizations of the accessoryabducens motoneurones spread widely across the reticular formation (Spencer et al.1980) and some isolated somata may be found along the trajectory of the retractorbulbi axons, in the reticular formation between the accessory and principal abducensnuclei (Grant et al. 1981). Thus it was not infrequent to make intra-axonic,intra-dendritic or intra-somatic recordings of retractor bulbi motoneurones outsidethe area delimited by an antidromic field potential.

Antidromic and orthodromic field potentialsWhen electrode tracks were made at an angle of 300 posterior to vertical, an

accessory abducens antidromic field potential could be recorded over approximately750 ,um of the descent (Fig. 1 B, C). Fig. 1 C illustrates the antidromic field recordedat its maximum and at 250 ,um above and below this point, evoked by intracranialstimulation of the VI nerve.Orthodromic field potentials evoked by stimulation of the long ciliary nerve, the

supraorbital nerve and the vibrissal pad could be recorded over a wider region(Fig. 1 C)'(Baldissera, Broggi & Mancia, 1967). Long ciliary and supraorbital fieldpotential amplitudes were increased by double shock stimulation, with an optimuminter-shock interval of2 msec. These orthodromic fields were large immediately aboveand within the accessory abducens nucleus (Fig. IC). The orthodromic field evokedby vibrissal stimulation was seen over a still greater part of the electrode descent andits form varied little within the region of the accessory abducens nucleus. Doubleshock stimulation ofthe vibrissal pad changed the form ofthe field potential, reducingthe amplitude of the second (larger) positive peak and increasing the amplitude ofthe following negative wave.

Intracellular identification of retractor bulbi motoneuronesRetractor bulbi motoneurones, identified antidromically were not spontaneously

active and showed no injury discharge on penetration. Membrane potentials rangedfrom -45 mV to -80 mV. Direct stimulation of the motoneurones by depolarizingpulses required high current intensity (up to 10 nA) and pulses of long duration (morethan 1 msec). Increasing the duration or the amplitude of depolarization rarelyresulted in repetitive spike generation. In many cases, somato-dendritic invasion bythe antidromic action potential became blocked for some time after penetration andit was only possible to obtain full antidromic invasion by simultaneously applyinga depolarizing potential via the recording micro-electrode.

Intracellularly recorded antidromic action potential amplitudes were in the range45-80 mV. Latencies ranged from 0-3 to 1'2 msec, with a marked peak at 0-4-0'6 msec.Assuming a conduction distance of 35 mm (measured in a cat weighing 2-8 kg), theselatencies correspond to conduction velocities of 116-29. m/sec, compatible with therelatively large size of the retractor bulbi motoneurone soma and axons (Batini etal. 1979; Spencer et al. 1980).

Representative intracellular recordings from two retractor bulbi motoneurones areillustrated in Fig. 2. In the first cell (Fig. 2A1-4) a characteristic inflexion appears

45

46 K. GRANT AND G. HORCHOLLE-BOSSA VIT

-55mV 75mV

* *0

A3pJ83/

**

A 4 \ \B4

Fig. 2. Antidromic activation of retractor bulbi motoneurones. A 1-4, high-gain (uppertrace) and low-gain (lower trace) records of the antidromic action potential showing afollowing delayed depolarization (arrow Al). In this neurone, with a membrane potentialof -55 mV, there was no overshoot ofthe resting membrane potential following the actionpotential. Double-shock stimulation (A2-4) shows, as the inter-stimulus interval isreduced, the increased inflexion on the rising phase of the spike between i.s. and s.d.components A2 lower trace), followed by failure of the s.d. component (A3 lower trace)and finally failure of the i.s. component, revealing an M spike (arrow head, A4). B 1-4in a second motoneurone, with a membrane potential of -75 mV, the antidromic actionpotential is followed by an after-hyperpolarizing potential. Arrow in BI indicates adelayed depolarizing potential. At reduced gain and a slower time base, B2 showssummation of after-hyperpolarizations following double-shock stimulation. This effect isabsent if the second action potential fails (B3). The full time course of the after-hyperpolarization is shown in B4. Calibration: a square pulse of 1 mV/1 msec is shownat the beginning of each trace.


on the rising phase of the antidromic action potential (Fig. 2A 1, low gain),corresponding to the invasion of the somato-dendritic region following spike propa-gation in the initial segment (i.s.). This inflexion is accentuated (Fig. 2A2, low gain)when a second antidromic action potential is generated at a short following interval,and somato-dendritic invasion fails altogether at shorter inter-stimulus intervals(Fig. 2A3, low gain). The latter gives the refractory period of the somato-dendritic(s.d.) region. With progressively shorter inter-stimulus intervals, the i.s. spike becamereduced in amplitude and duration, in a fashion which was not all-or-none; this wasa common feature of the refractory behaviour of retractor bulbi motoneurones.Failure of the i.s. component of the antidromic action potential revealed the M spike(Fig. 2A4, high gain), corresponding to the invasion of the first myelinated segmentof the axon. Separation of the s.d., i.s. and M components of the antidromic spikecould also be obtained by hyperpolarizing the impaled neurone (Grant et al. 1979).The refractory behaviour described above was used to identify somatic penetrations

and to distinguish these from recordings of motor axons, in which no inflexion is seenon the rising phase of the action potential, and where the antidromic spike is anall-or-none phenomenon (Grant et al. 1979). However, results from experiments inwhich horseradish peroxidase was used to label motoneurones by intracellularionophoresis suggest that this identification may be insufficient. In several cases,histological reconstruction has shown that micro-electrode penetration was in theaxon, sometimes several hundred microns from the soma, despite apparently somaticrefractory behaviour during recording. In addition, synaptic potentials underlyingorthodromic action potentials have been recorded in retractor bulbi axons, identifiedby intracellular injection of horseradish peroxidase, as far away as the principalabducens nucleus (2-3 mm) (Durand, Grant, Gueritaud, Horcholle-Bossavit & Tyc-Dumont, 1980).

After-potentials were frequently seen following retractor bulbi antidromic actionpotentials but these evolved with time after penetration. The antidromic spike wasusually followed by a delayed depolarization which occurred initially when the fallingphase of the action potential overshot the resting membrane potential (Fig. 2B1).After several minutes of penetration, the return to the resting membrane potentialbecame longer and the delayed depolarization occurred at a point on the falling phaseof the action potential more positive than the resting membrane potential (Fig. 2A 1).The delayed depolarization was generally associated with the occurrence of an s.d.spike and was not present when s.d. invasion failed (Fig. 2A3). Variations in theamplitude of the delayed depolarization were also observed (Fig. 2A1-4).The after-hyperpolarizing potential varied in amplitude and in form. Maximum

after-hyperpolarization amplitudes of 2-5 mV were seen between 9 and 15 msec afterthe stimulus pulse and the total duration of the return to the resting membranepotential was from 35 to 40 msec. Fig. 2B2-4 illustrates the summation ofafter-hyperpolarizations following two successive antidromic spikes; this effectincreased when the inter-stimulus interval was reduced and was no longer observedwhen the interval was greater than about 25 msec.When care was taken to isolate the peripheral nerve stimulation, no synaptic

potentials were observed following antidromic activation of retractor bulbi moto-neurones. However, when the retractor bulbi nerve was stimulated using a silver-ball

47


electrode without previous dissection, synaptic potentials were observed following theantidromic action potential at similar threshold intensities, with a latency of2-3 msec.This was most probably due to stimulus spread to surrounding structures innervatedby the trigeminal nerve (see Baker et al. 1980).

B SO

A

O A

2

3

2

C vi

A

i '

G

D Vc

140

0

H

0

1 so

A °A 0o _

Fig. 3. E.p.s.p.s evoked by trigeminal afferent stimulation. A-D: e.p.s.p.s evoked bystimulation of A, the long ciliary nerve (LC); B, the supraorbital nerve (SO); C, theipsilateral vibrissae (Vi) and D, the contralateral vibrissae (Vc) at increasing intensity.B3 and C3 show the facilitatory effect ofdouble-shock stimulation. E-H: e.p.s.p.s recordedin the same retractor bulbi motoneurone, illustrated at two sweep speeds for a comparisonof amplitude, form and total duration of the response evoked from each stimulation site.Amplitude calibration: A-F, 2 mV; 0, H, 1 mV. Time calibration: A-El, Fl, GI, H1,4 msec; E2, F2, G2, H2, 10 msec.

Excitatory trigeminal post-synaptic potentialsThe excitatory post-synaptic potentials (e.p.s.p.s) evoked in retractor bulbi

motoneurones from each of the stimulation sites were characteristic and differed inform, rise time, duration and efficacity for spike generation.Long ciliary nerve. The e.p.s.p. evoked by long ciliary nerve stimulation, recorded

in the same motoneurone at three stimulus intensities, is shown in Fig. 3A. Close to

48

pow

A LC

1

A A A


threshold (A 1) the latency was 2-6 msec and the rising phase was slow, reaching a peakafter 3-4 msec. Increasing the stimulus intensity (A2) reduced the latency, increasedthe e.p.s.p. amplitude and decreased and time to peak. At supra-maximal intensities,the long ciliary e.p.s.p. was evoked with a latency of 2-0 msec and rose rapidly toa peak (A3). This was followed by a plateau maintained during 8-10 msec. Thus,stimulation of the long ciliary nerves gave a powerful, long-lasting excitation of theretractor bulbi motoneurones.

Supraorbital nerve. Fig. 3B1-3 shows e.p.s.p.s evoked by stimulation of thesupraorbital nerve, at the three stimulus intensities. The e.p.s.p.s are characterizedby a short onset and rapid initial rise. Close to threshold, the latency of the e.p.s.p.(Fig. 3B1) was 2-6 msec. and this did not change with increasing stimulus intensity.The initial peak of the supraorbital e.p.s.p. lasted 4-5 msec. but this was followed,even at near threshold stimulation, by a later plateau of smaller amplitude (Fig. 3B),giving a total duration of 8-10 msec.As stimulus intensity was increased, the supraorbital e.p.s.p. increased in amplitude

by the addition of longer latency components, clearly distinct on the rising phase(Fig. 3B2). Thus, with increasing stimulus intensity, the time to peak became longer.The duration and amplitude of the plateau phase of the e.p.s.p. changed little withstimulus intensity. The addition of a second stimulating shock had a markedfacilitatory effect (Fig. 3B3) increasing the peak amplitude of the e.p.s.p.

Ipsilateral vibrissae. The e.p.s.p. evoked in retractor bulbi motoneurones bynear-threshold stimulation of the vibrissal pad (Fig. 3C) resembled that evoked bylong ciliary nerve stimulation at threshold. Latencies varied from 1-6 to 2-8 msec, witha marked peak at 1-8 msec. Fig. 3C1 shows the ipsilateral vibrissal e.p.s.p., close tothreshold, with a long rising phase and a similar decay. Increasing stimulus intensity(Fig. 3C2) gave a sharper onset, due to a shorter initial rise time, and an increasedamplitude. Double-shock stimulation increased the e.p.s.p. amplitude and reducedthe time to peak (Fig. 303).

Contralateral vibris8ae. Stimulation of the contralateral vibrissal pad also gaveexcitation of retractor bulbi motoneurones (Fig. 3D). The latency of contralateralvibrissal e.p.s.p.s was rather variable, ranging from 3-5 to 6-0 msec in differentmotoneurones. E.p.s.p. amplitudes were comparatively smaller than for ipsilateralstimulation; the initial rising phase was generally long but sometimes a more rapidonset was observed (Fig. 4C). E.p.s.p. amplitudes increased and latencies decreasedwith increasing stimulus intensity.For comparison of their form and amplitude at supra-maximal stimulation

intensities, Fig. 3E-H shows e.p.s.p.s evoked from each of the stimulation sites,recorded in the same motoneurone. The latency histograms (not illustrated) fore.p.s.p.s of different trigeminal origin show a remarkable similarity in latencydistribution and peak values. Latencies ranged from 1-6 to 2-8 msec, with in eachcase a marked peak between 2-0 and 2-25 msec.

Orthodromic action potentialsLong ciliary e.p.s.p. The large-amplitude, long-lasting e.p.s.p. evoked by stimulation

of the long ciliary nerve was extremely powerful in terms of orthodromic actionpotential generation. Fig. 4A illustrates spikes generated in response to stimulation

49


at increasing intensities. One or two orthodromic action potentials were generatedat near threshold intensity for the e.p.s.p. (Fig. 4A1). At higher stimulus intensitythe e.p.s.p. remained above the firing threshold for several milliseconds and gave riseto a train ofspikes, increasing in number with stimulus intensity reaching a maximumof five. In some records, the somatic component or the orthodromic action potential

A LC B SO C Vi D Vc

A 00

A 0000 AA

A~ bub moonurne

Fig. 4. Action potential responses evoked by four trigeminal afferents in the same retractorbulbi motoneurone. A 1-3, increasing number of action potentials evoked at increasingintensity stimulation, applied to long ciliary nerve (LC). B1-3, responses evoked atincreasing intensity stimulation applied to supraorbital nerve (SO). Note the second actionpotential and stable latency of the first action potential at maximal intensity. C1-3,responses evoked at increasing intensity stimulation applied to ipsilateral vibrissae (Vi).D1-3, responses evoked at increasing intensity stimulation applied to contralateralvibrissae (Vc). Note longer and variable latencies. Amplitude calibration: 4 mV, 10 mV.Time calibration: 4 msec.

became blocked soon after penetration. This frequently revealed partial spikes oflarge amplitude arising from the e.p.s.p. (Fig. 5A-D), presumably of dendritic origin,which also increased in number with increasing stimulus intensity.

Supraorbital e.p.s.p. The fast-risinge.p.s.p. evoked by supraorbital nerve stimulationreadily generated an orthodromic action potential, even at near-threshold stimulationintensities (Fig. 4B). Increasing stimulus intensity often resulted in the generationof a second spike rising from the beginning of the plateau phase of the e.p.s.p.(Fig. 4B3). Longer trains of spikes, similar to those generated by the long ciliarye.p.s.p., were not seen following stimulation of the supraorbital nerve. Partial spikesevoked by supraorbital nerve stimulation are shown in Fig. 5E.

Ipsilateral vibrissal e.p.s.p. Stimulation of the ipsilateral vibrissal pad generated

50

RETRACTOR BULBI MOTONEURONES5

full orthodromic action potentials in retractor bulbi motoneurones at near thresholdintensities. Close to threshold for the e.p.s.p., the latency of the orthodromic actionpotential was variable (Fig. 4C1). As stimulus intensity was increased (Fig. 4C2, 03),the latency of the first orthodromic spike became stable and sometimes a secondaction potential occurred (Fig. 4C3).

A LC D LC

_~I I

C

Fig. 5. Partial spike responses in retractor bulbi motoneurones. A-D, partial spikes evokedat increasing intensity stimulation applied to long ciliary nerve (LC). In D, high- andlow-gain traces at low sweep speed to show the full time course of the synaptic potentials.E, a similar partial spike response is evoked by stimulation applied to the supraorbitalnerve. Amplitude calibration: A, B, C, 2 mV; D, E, 2 mV-10 mV. Time calibration: A,B, C, 5 msec; D, E, 10 msec.

Contralateral vibrissal e.p.s.p. The rather variable e.p.s.p. evoked by stimulation ofthe contralateral vibrissal pad could also be sufficient to generate orthodromic actionpotentials, especially in motoneurones in which the initial rising phase of the e.p.s.p.was rapid. Fig. 4D illustrates an example in which the e.p.s.p. showed two small peaksat low stimulus intensities and an orthodromic action potential could be generatedfrom either of these. At higher stimulus intensity, generation of the orthodromicaction potential was stabilized from the first peak (Fig. 4D3).

Hyperpolarizing potentialsIn many recordings, trigeminally evoked e.p.s.p.s were followed by a phase of

hyperpolarization, 1-8 mV in amplitude, beginning 8-10 msec after stimulation andlasting for 10-30 msec. This pattern of events was particularly clear following thelong ciliary e.p.s.p. and to a lesser extent, the supraorbital e.p.s.p. Fig. 6 illustratesthe hyperpolarization which followed long ciliary nerve stimulation. At near-thresholdstimulation a small hyperpolarization was already present, beginning at about

51


8 rnsec after stimulation. With increasing stimulus intensity (Fig. 6B, C) the hyper-polarization increased in amplitude and its exact onset became masked by the increasein the preceding e.p.s.p. and action potentials. The maximum amplitude observed forthe hyperpolarization was about 8 mV and its total duration was 25-30 msec.Attemptsto inverse the hyperpolarization bypassage ofcurrent via the micro-electrode

AXLCLLC DCLC

-80 my

J~~~~~~~~ ~~~~~~~~i:woo

B E H

-90 mV

AF AF -lO~~~~mV I~~so I

L 0*

Fig. 6. Hyperpolarizing potentials in retractor bulbi motoneurones. A-C, hyperpolarizingpotential following the excitatory response evoked by stimulation applied to long ciliarynerves (LC) at increasing stimulus intensity. Resting membrane potential -70 mV. D-F,current injection via the micro-electrode induces a membrane hyperpolarization. E.p.s.p.amplitude is increased, action potential generation is blocked and the amplitude of thefollowing hyperpolarization is reduced. Partial inversion is seen at -100 mV. G,H,complex synaptic potentials evoked in two different retractor bulbi motoneurones. Notedifferences in apparent duration of e.p.s.p.s and following hyperpolarization. I, hyper-polarizing potential following the excitatory response evoked by stimulation applied tothe supraorbital nerve. Amplitude calibration: A, B, C, 4 mV; D-I, 4 mV, 10 mV. Timecalibration: A, B, C, 10 msec; D-I, 20 msec.

were never wholly successful. Fig. 6D-F illustrates the effect of membrane hyper-polarization on the synaptic potentials evoked by stimulation of the long ciliarynerve. The resting membrane potential was -70 mV. At -80 mV to -100 mV, thee.p.s.p. amplitude was increased and orthodromic action potential generation wasblocked but no clear reversal ofthe following hyperpolarization was obtained. Fig. 6Gillustrates the example of another motoneurone in which partial spikes, presumed

52


to be of dendritic origin, evoked by long ciliary nerve stimulation, were also absentduring the period of hyperpolarization. In certain motoneurones a similarhyperpolarization occurred following the excitation evoked by supraorbital nervestimulation, as shown in Fig. 61. (The synaptic potentials evoked in the samemotoneurone by long ciliary nerve stimulation are illustrated for comparison inFig. 6H.)

A B CI

D E F

Fig. 7. Original intracellular records of post-synaptic potentials following conditioningstimulation of the long ciliary nerves (A) and test stimulation of the retractor bulbi nerve(@) (A-C) or test stimulation of the supraorbital nerve (0) (D-F). The amplitude of theantidromic spike is reduced (A), or the s.d. component is blocked during the period10-30 msec after stimulation of the long ciliary nerve (B). The supraorbital e.p.s.p. isreduced in amplitude and in duration during the period ofhyperpolarization (D) followingthe conditioning long ciliary e.p.s.p. Control records of the antidromic action potentialand of the supraorbital e.p.s.p. are shown in C and F respectively. Calibrations: A, B,C, 10 mV/5 msec; D, E, F, 2 mV/10 msec.

The inhibitory nature ofthe hyperpolarization was investigated usingtwo condition-test procedures. Firstly, the amplitude of the antidromic action potential recorded inthe soma was reduced when antidromic activation was preceded by long ciliary nervestimulation (Fig. 7 A-C) with condition-test intervals of 15-30 msec. This effect wasmaximal with condition-test intervals of 15-25 msec, which corresponds to the periodin which hyperpolarization evoked by long ciliary nerve stimulation was also greatest.The upper curve of the graph in Fig. 8 shows the amplitude of the antidromic actionpotential evoked at various time intervals after a conditioning stimulation ofthe longciliary nerve, as a percentage of its control amplitude. (Controls were recorded beforeand after each condition-test trial.) Original records showing the same condition-testprocedure in another motoneurone are shown in Fig. 7 A, B and C. With condition-test

53


intervals of 10-20 msec, somato-dendritic invasion of the antidromic action potentialwas delayed, shown by the accentuated inflexion on the rising phase (Fig. 7A), orblocked (Fig. 7B).The interaction between two orthodromic inputs was tested in a second procedure.

Long ciliary nerve stimulation preceded supraorbital nerve stimulation with varying

100- - Control\-AAmplitude

80-

A Antidromic a.p.60- * E.p.s.p.

10 20 30

Inter-stimulus interval (msec)

Fig. 8. Graphic representation of test antidromic spike (A) and supraorbital e.p.s.p. (@)amplitudes, as a percentage of control amplitudes, following conditioning long ciliarynerve stimulation. Antidromic activation of the motoneurone is depressed during theperiod 10-30 msec after long ciliary nerve stimulation; orthodromic supraorbital excitationof the neurone is partially blocked from 15 msec to more than 40 msec after theconditioning long ciliary nerve stimulation. Ordinate: % of control amplitude; abscissa:time after conditioning stimulus.

intervals. Original records from the same motoneurone are shown in Fig. 7 D, E andF. When the test supraorbital e.p.s.p. was evoked just after the falling phase of thelong ciliary e.p.s.p., not only the peak amplitude of the supraorbital e.p.s.p. wasreduced, but its total duration was also noticeably affected. The plateau occurringon the falling phase was suppressed and thus the decay of the e.p.s.p. was faster(Fig. 7 D, E). This modification of the decay lasted for 10-20 msec and showed a timecourse shorter than that of the peak amplitude depression which lasted for more than40 msec, as shown in the curve in Fig. 8.These effects on the test supraorbital e.p.s.p. were seen in the absence of full action

potentials generated by long ciliary nerve stimulation. This is thus a furtherindication that the hyperpolarizing potential is of synaptic origin rather than linkedto the after-hyperpolarization which follows the somato-dendritic action potential.

Trigeminal premotor afferent fibre8Trigeminally excited axons, which were not antidromically activated by stimulation

of the VI nerve, were also recorded intracellularly in the region delimited by thepresence of an accessory abducens antidromic field potential. From their firingpattern in response to trigeminal stimulation, it is suggested that these axons maybe premotor afferent fibres. Fig. 9A, D illustrates the example of an intra-axonal

54


recording showing a burst ofspikes generated by stimulation ofthe long ciliary nerves,with a latency of2-7 msec and a duration of8-10 msec. Supraorbital nerve stimulation(Fig. 9B, E) evoked a different pattern of activity in the same axon. A burst of twoor three spikes with a latency of 1-4 msec was followed by a short silent period ofabout 4 msec and a second longer burst of four to six action potentials. Ipsilateralvibrissal stimulation (Fig. 9C, F) evoked only two action potentials in the same axon,

LC SO ViA B C

-WAA

FE

IL

AA

Fig. 9. Activity of a presumed pre-motor trigeminal axon recorded intracellularly in theaccessory abducens nucleus. In the same axon, stimulation at each of the three sites evokeda different pattern of activity. (A, long ciliary nerve; O, supraorbital nerve; A, ipsilateralvibrissae). Slower sweep traces in D-F show the absence of spontaneous activity and thefull duration of the different evoked activities. Calibration: A-C, 4 mV/4 msec; D-F,4 mV/8 msec.

the first with a latency of 2-7 msec. Thus three different discharge patterns were

recorded in the same axon in response to three different trigeminal stimuli. This showsthat some convergence has already taken place at the level of the trigeminal sensoryrelay.

DISCUSSION

The electrophysiological properties ofretractor bulbi motoneurones recorded in theaccessory abducens nucleus indicate notable differences between these neurones and

other extra-ocular motoneurones located in the III (Sasaki, 1963), the IV (Baker &Precht, 1972) or the VI (Baker, Mano & Shimazu, 1969; Remmel & Marrocco, 1975;Grantyn & Grantyn, 1978) nuclei. Intracellular recordings showed a fast axon

conduction velocity, a low security factor for antidromic invasion of the somato-

dendritic region and a high threshold to direct stimulation. Prolonged imposeddepolarization did not result in repetitive firing, unlike other extra-ocular moto-

neurones (Barmack, 1974; Remmel & Marrocco, 1975; Grantyn & Grantyn, 1978).

D

I

55

I _


The after-hyperpolarization following antidromic action potentials was similar intime course and in amplitude to that described in abducens motoneurones (Bakeret al. 1969; Grantyn & Grantyn, 1978) though longer than that reported in trochlearmotoneurones (Baker & Precht, 1972). After-spike depolarizations were presentfollowing antidromic invasion of retractor bulbi motoneurones with a form andlatency similar to those seen in abducens or trochlear motoneurones. No evidence wasfound for synaptic potentials following antidromic stimulation in agreement with theabsence of axon collaterals after intracellular staining with horseradish peroxidase(Grant et al. 1979; Spencer et al. 1980). The low security factor for antidromic invasionof the soma and the high threshold of the motoneurones to direct stimulation wouldperhaps indicate a low sensitivity to trans-synaptic excitation and the need for a highlevel ofconvergent depolarization to initiate spike generation. This may be in keepingwith the protective function of the retractor bulbi motoneurones, responding tosudden, intense stimulation of the face but activated only phasically.

Intracellular recordings of the synaptic potentials evoked by trigeminal afferentsrevealed that the retractor bulbi motoneurones receive excitatory inputs originatingfrom the perioral and periorbital areas as well as from the cornea. These afferentpathways involve fibres from both the ophthalmic and the maxillary branches of theV nerve. The latencies ofthe e.p.s.p. evoked by peripheral trigeminal stimulation werein the range for disynaptic connexions. The shortest latencies for trigeminal e.p.s.p.swere seen in response to vibrissal pad stimulation and this is consistent with theactivation of fast-conducting Aa fibres in the infraorbital nerve (Gogan, Gueritaud,Horcholle-Bossavit & Tyc-Dumont, 1981; Guegan & Horcholle-Bossavit, 1981).Although all e.p.s.p.s evoked from trigeminal origins reached large amplitudes andlong durations at maximum stimulus intensities, the forms of the e.p.s.p.s evokedfrom the three sites tested were characteristically different. Differences in the numberof components visible on the rising phase, in latency changes with increased stimulusintensity and in duration of a plateau phase, probably reflect not only differences inrecruitment thresholds and conduction velocities in the primary afferent fibres butalso different patterns of convergence onto the second order trigeminal neurones.This is illustrated by recordings from presumed pre-motor afferent fibres in thevicinity of the retractor bulbi motoneurones, showing a distinct discharge patternspecifically related to the origin ofthe stimulated afferent fibres. Recent studies usingboth anatomical and electrophysiological techniques have dealt with the centralprojections of the primary trigeminal fibres (Mosso & Kruger, 1973; Yokota &Nismikawa, 1980; Hu, Dostrovsky & Sessle, 1981; Marfurt, 1981; Panneton &Burton, 1981). These investigations show that the representation ofthe cornea is verylocalized and weak in comparison with the other sources of trigeminal afferents suchas the supra-orbital and infraorbital nerves, which have been described to projectalong the rostro-caudal axis of the trigeminal nuclear complex. Thus, it is interestingto observe that the few afferent fibres originating from the corneal plexus give riseto the most powerful synaptic effects in the retractor bulbi motoneurones. A similarstrong corneal reflex response has been described in the orbicularis oculi motoneurones(Hiraoka & Shimamura, 1977).The localization of the trigeminal neurones involved in the trigemino-retractor

bulbi motor reflex arc remains to be elucidated. Since anatomical studies have shown

56


that neurones of the trigeminal complex project to the thalamus (Burton & Craig,1979), the superior colliculus (Baleydier & Maugiere, 1978), the cerebellum (Ikeda,1979) and to the spinal cord (Matsushita, Okado, Ikeda & Hosoya, 1981), themotoneuronal excitation could be due to collaterals of distantly projecting neurones,similar to the activation offacial motoneuronesby collateralsofthe trigemino-thalamictract (Tanaka, Yu & Kitai, 1971). An alternative could be the presence of directprojections from specific trigeminal neurones with short axons. A recent report(Matsushita et al. 1981) suggested that direct trigemino-spinal projections arisingfrom neurones in the subnucleus oralis could subserve the trigemino-neck reflexes andin this context the close proximity of the accessory abducens nucleus and the parsoralis of the trigeminal nucleus should be noted. Furthermore, intratrigeminalconnexions such as the projections from subnucleus caudalis to the subnucleus oralis(Hu et al. 1981; Panneton & Burton, 1982) may be involved in polysynaptic pathwaysresponsible for the prolonged excitation seen in retractor bulbi motoneuronesfollowing peripheral trigeminal stimulation. Finally, the extension of the dendriticarborization of the retractor bulbi motoneurones into the trigeminal nucleus (Bakeret al. 1980) suggests the possibility of synaptic contacts beween distal dendrites andaxon terminals of trigeminal neurones. This could account for the occurrence ofdendritic spikes, observed in ketamine anaesthetized animals and also described insodium pentobarbitone anaesthetized preparations (Baker et al. 1980).

In ketamine anaesthetized cats, we observed that corneal afferent stimulationproduced complex post-synaptic effects in retractor bulbi motoneurones. The long-duration e.p.s.p. plateau, probably generated by polysynaptic pathways, induced aburst discharge of the motoneurone, corresponding to the complex contractionresponse which is similarly observed in the retractor bulbi muscle (Guegan et al. 1981).This powerful excitatory effect was followed by a hyperpolarizing potential of whichthe latency could not be measured precisely since its onset probably occurred duringthe falling phase of the e.p.s.p. Within this period of temporal overlap, partialinversion of the i.p.s.p. may contribute to the increase in e.p.s.p. amplitude observedduring hyperpolarization of the motoneurone membrane. Similar e.p.s.p. - i.p.s.p.sequences have been described in spinal motoneurones following stimulation ofcutaneous afferents (Rosenberg, 1970; Pinter, Burke, O'Donovan & Dunn, 1982). Thenature of this short-latency inhibitory mechanism was investigated in several ways.Hyperpolarization of the motoneurone membrane reduced or abolished the hyper-polarizing potential but complete inversion of the i.p.s.p. was not obtained. Thissuggests that post-synaptic inhibition may occur at sites distant from the recordingelectrode, presumably on the distal dendritic region of the motoneurone membrane.However, before becoming blocked, the amplitude of the spike generated byantidromic invasion during the hyperpolarizing potential was reduced, indicating areduction in membrane resistance at this time, as has been described in trigeminalmotoneurones (Chandler, Chase & Nakamura, 1981). The changes in form of thesupraorbital e.p.s.p. evoked during this hyperpolarizing potential affected both itsduration and its peak amplitude. The decreased peak amplitude could be due to theabsence of presynaptic facilitation. Indeed, the time course of this amplitudedepression outlasts the effect on the rate of decay of the supraorbital e.p.s.p., whichshows the same time course as the effects on antidromic invasion. Thus, these two

57


events could be due to a post-synaptic inhibitory mechanism, while the peak e.p.s.p.amplitude depression could result from a presynaptic inhibition or lack of facilitationin the trigeminal nucleus where both presynaptic and post-synaptic inhibition havebeen reported (Baldissera et al. 1967; Stewart, Scibetta & King, 1967; Ishimine,Hikosaka & Nakamura, 1980).The origin of the post-synaptic inhibition, which is similar to that described in

trigeminal motoneurones (Takatori, Nozaki & Nakamura, 1981), may involve thebulbar reticular formation since trigeminal afferents are known to project to reticularneurones (Nord & Ross, 1973; Yokota & Nishikawa, 1980). The functional role of thisinhibition could be important in situations where the nictitating membrane has to berapidly retracted, for instance after a lateral wiping movement, when the tonicsympathetic activity which is required to keep the membrane retracted might not besufficient to oppose its active extension.

This work was supported by a grant from the C.N.R.S. (ATP no. 126).

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