86 J. Phy8iol. (1961), 155, pp. 86-97With 6 text-ftgurewPrinted in Great Britain

FROG'S SPINAL CORD POTENTIALS GENERATED BYSTIMULATION OF CUTANEOUS NERVES

BY MARIA C. BRAVO AND A. FERNANDEZ DE MOLINAFrom the Departamento de Biofisica, (Centro de Investigaciones Biologica8,

C.S.I.C., Madrid, Spain

(Received 29 July 1960)

The potential changes recorded from the dorsal surface of the frog'sspinal cord after stimulation of a dorsal root have been analysed byBonnet & Bremer (1952) in spinal preparations. In 1953 Kolmodin &Skoglund investigated the distribution of the intramedullary potentialchanges evoked in isolated segments of the frog's cord by dorsal rootstimulation; they were able to record typical potential pictures from fourdifferent regions of the cross-section. Brookhart & Fadiga (1960) haveproduced evidence of monosynaptic activation of the motoneurones byboth dorsal-root and lateral-column stimulation in isolated frog's corddepressed with pentobarbital; by mapping the potential changes theyshowed that the post-synaptic responses were confined to a dorsal hornarea occupied by interneurones and motoneuronal dendrites, when evokedby dorsal-root stimulation, and to the ventral horn when motoneuroneswere excited by impulses in the lateral column. Dorsal-surface potentialselicited by stimulation of cutaneous and muscle nerves have been reportedin a short account by Marx (1959).The distribution of potential changes generated in the spinal cord of the

cat after stimulation of muscle and cutaneous afferent fibres has beeninvestigated by Eccles, Fatt, Landgren & Winsbury (1954), Coombs,Curtis & Landgren (1956) and Fernandez de Molina & Gray (1957); it waspossible to identify in certain areas groups of neurones which responded ina specific manner to activation of a particular group of afferent fibres.Such a study has not yet been made in the spinal cord of the frog: thepotential changes recorded by Kolmodin & Skoglund (1953) are complexbecause of the compound nature of the dorsal root volley. The presentinvestigation was undertaken in order to study the distribution of po-tential changes evoked in the spinal cord of the frog after selectiveactivation of cutaneous and muscle afferents. It is the purpose of thispaper to describe experiments dealing with activation of cutaneousafferents and designed to obtain information about the nature of the

POTENTIALS OF THE FROG'S SPINAL CORD 87

cord's responses and the location of the structures responsible for theirgeneration. A preliminary account of this work has already appeared(Bravo & Fernandez de Molina, 1960a).

METHODS

Preparations. The experiments were performed on frogs (Rana esculenta). After etheranaesthesia the vertebral canal was opened and the brain stem cut either above the opticlobes or, in a few experiments, at the caudal end of the medulla; the brain above the cut wasthen destroyed. The spinal cord was exposed from the VI segment down to the caudal tip.Nerves in one limb were prepared for stimulation, usually the dorsolateral pedicular or thelateral crural. The animal was then mounted in a brass frame, vertebral processes beingfixed by special clamps. The whole leg with dissected nerves was introduced into a paraffinpool. The white fat of the dura was removed and the arachnoid stripped from the dorsalsurface of the VIII, IX and X segments.

Stimulation. Stimuli were applied to the nerve through platinum wires from a square-wave stimulator having the output isolated from earth and of low impedance. The durationof the pulses was 02-0-4 msec.

Recording. Glass capillary electrodes filled with 4M-NaCl, and with a tip diameter of1-4 Z and a resistance of 2-4 MQl, were used to record the potential changes generated in thecord. The electrolyte solution was connected to the grid of a cathode follower through achlorided silver wire. The indifferent electrode, a small chlorided silver plate, was attachedto the back muscles and the frame was earthed. An RC-coupled differential amplifier witha time constant of 5 sec was employed and the potential changes were displayed on one beamof a double-beam oscilloscope and photographed. All the records shown in this paper wereobtained extracellularly with signal negativity of the micro-electrode tip as an upwarddeflexion. In some experiments the afferent volley was recorded from a filament of the dorsalroot and the reflex discharges from the ventral root by means of platinum wires; after suit-able amplification the potentials were displayed on the second beam of the oscilloscope.

Experimental procedure and histological checking. These have been described in a previouspaper (Fernandez de Molina & Gray, 1957). Owing to the small size of the frog's cord nomore than two tracks could usually be made in each transverse plane. In some experimentsthe insertion of the micro-electrode was made through the lateral surface of the cord in orderto reach the motoneurones more easily.

RESULTS

Dorsal surface potentialsFigure 1 shows three records obtained from the dorsal surface of the

cord at the level of the IX segment in different experiments, after stimu-lation of the dorsolateral pedicular nerve with a strength above thresholdfor polysynaptic reflex discharge. The potential sequence is made up offour deflexions. The first deflexion (S) is the action potential of the in-coming sensory fibres, consisting of a small positive followed by a largenegative deflexion; the positive component was better observed when thepia was opened and the micro-electrode tip placed in the dorsal column orin the dorsal horn, as is the case in record (c) on the left in Fig. 6. Thisinitial spike has a latency which corresponds to the time for transmissionof the volley in the largest cutaneous fibres.

88 MARIA C. BRAVO AND A. FERNANDEZ DE MOLINAThe deflexions which follow the primary spike we shall call phases of

activity (Fernandez de Molina & Gray, 1957). The first phase reaches itspeak 1-8-2 msec after the negative peak of the incoming volley and has atotal duration of 12-14 msec (Fig. 5a). The latency of this phase, countedfrom the reversal point of the initial positivity of the spike to the onset ofthe potential (Fig. 6c, left column, between arrows) averages 1-2 +0-065 msec (n = 21), a time which is in agreement with that reported byBrookhart & Fadiga (1960) for synaptic delay in the frog's spinal cord. Thesecond phase rises from the descending limb of phase I, reaching its peak

S I

Fig. 1. Records from different experiments to show, S the afferent primary spike,and I, II and III the three phases of cord activity. Time marker, 10 msec; voltagecalibration in 0.4 mV steps.

3*5A4 msec after the negative peak of the primary spike; the amplitude ofthis phase at the surface of the cord is usually bigger, in a few experimentsequal, but never smaller than that of phase I. Its total duration cannot bedetermined with accuracy because it becomes obscured by the rising limbof the third phase, although it is of a similar order to that of the phase I,as can be seen when the third phase is abolished by stimulating with ahigher frequency (see below). Phase III has a much slower time course, itspeak being reached 20-25 msec after the negative peak of the 'initial spikeand its time of decay being about 200-250 msec.

WThen two cutaneous nerves, the dorsolateral pedicular (P) and lateralcrural (C), were maximally stimulated in such a way that both afferentvolleys arrived simultaneously at the cord, the three phases of activityshowed occlusion. Typical figures in arbitrary units were, for phase I,P = 14, C = 9, P±+C =17; for phase II, P = 17, C = 13, P+C = 20;and for phase III, P II1, C = 7 and P+C = 12. The occlusion forphase III was almost complete.

POTENTIALS OF THE FROG'S SPINAL CORD

Intramedullary distributionWhen recording at various depths along tracks in a dorsoventral

direction a characteristic pattern of distribution of the three phases ofactivity was observed. Figure 2 illustrates two series of records obtainedwith different sweep speeds in one of such tracks, passing along the dorsalhorn, intermediary region and ventral horn as is shown by the line A inFig. 4. The figure to the left of each record gives the depth in millimetresbelow the dorsal surface of the cord at which it was obtained. In this

s

0-25

0.50

E0-75E0

0.95

1.20 _

1-40 __

Fig. 2. Series of records obtained with different sweep speeds to show the focalpotentials recorded at the surface (S) and at the indicated depths (ordinate) alongthe track shown by line A in Fig. 4 after stimulation of the dorsolateral pedicularnerve with a strength 5 times threshold. Time, 10 and 30 msec intervals for the leftand right series respectively; voltage calibration 1 mV.At depth 0-25mm, phase I maximal; 0-50mm, phase II maximral, phase I

decreasing; 0 75 mm, phase I reversed, phase II considerably decreased andphase III maximal; 0-95mm, phases I and II reversed, phase III decreased;1-4 mmn, phase III completely reversed.

particular exrperiment the dorsolateral pedicular nerve was stimulated butthe pattern of distribution was essentially the same when the lateralcrural or the posterior femoris were stimulated and records obtained atthe optimum segmental level.The incomingvoley could be recorded at the dorsum of the cord and

dorsal hoi, then became smaller and later could no longer be seen (fromfourth record downwards). In this experiment phase I reached its maxi-mum amplitude at 0p3 mm, then diminished in size, and at0s 6 mm

89

90 MARIA C. BRA VO AND A. FERNANDEZ DE MOLINAreversed to a positive potential of similar time course, which is seen in allthe deeper records. Phase II had its maximum more ventral than phase I,at about 0 5 mm, then decreased in amplitude and at 0-8 mm began toreverse to a positive potential which was well developed from the depthof 1 mm downwards. Phase III reached its maximum even more ventrally,at a level (0-8 mm) at which phase I had completey reversed and phase IIhad begun its reversal. This maximum was situated at the level of theintermediary region. More ventrally it diminished in amplitude and nearthe level of the motoneurone pool it reversed to a positive potential with ashorter time course.

a

1 p

b _

Fig. 3. Motoneurone and ventral-root responses. a, simultaneous recording fromthe IX ventral root (upper trace) and within the motoneuronal pool (lower trace)after stimulation of the dorsolateral pedicular nerve at the level of the lower halfof the IX cord segment. b, another experiment, record obtained from a positionslightly medial to the motoneurones (upper limit of the IX cord segment) afterstimulation of the same nerve. In both experiments the micro-electrode wasinserted through the lateral surface of the cord. Time marker, 10 msec; voltagecalibration applies to all records: a, upper trace 500 pV, lower trace 800 ZVand b, 700 puV.

In the track just described the electrode passed medial to the moto-neurone pool. Figure 3 illustrates two records obtained at the level of themotoneurones in two different experiments in which the micro-electrodewas inserted through the lateral surface of the cord. When the micro-electrode tip was placed within the motoneuronal pool the record obtainedconsisted of two initial positivities, which correspond in time course todorsal negativities (phases I and II), followed by a complex negative wavewith some spikes superimposed on it (Fig. 3a, lower record). These spikeshave a latency of a similar order to that of the reflex discharges recordedfrom the ventral root (Fig. 3a, upper record). Record (b) was obtained in

POTENTIALS OF THE FROG'S SPINAL CORDa position slightly medial to the motoneurones and the same sequence ofpotential changes can be seen; but in this record the ventral horn nega-tivity is interrupted by the development of the third positivity. In a stillmore medial position this late positivity is recorded without being maskedby the ventral negative wave (bottom records in Fig. 2).

05 I 0 0*5mm

Fig. 4. Distribution of phases of cord potential, all ipsilateral. Right side:dotted region, phase I, C1 maximum of phase I; hatching, phase II, A maximumfor phase II; crows-marked and interrupted lines are reversal levels for phases Iand IIrespectively. Left side: 0 maximum for phase III; dash-dot line is the reversallevel of this response, which was recorded in the grey substance above this line andin the dorsal column. A gives course of the track along which the potentialsshown in Fig. 2 were recorded. Lateral columns were not explored.

The distribution of the three phases of activity, as deduced from seriesof tracks made in different experiments, is shown in Fig. 4; the right siderefers to phases I and II (dotted and single-hatched zones respectively) andthe left side to phase III. The two first phases have their maxima in thedorsal horn, that of phase I being more dorsal than that of phase II. Thelateral positions of these optima could not be determined accuratelybecause usually no more than two tracks could be made in each trans-

91

92 MARIA C. BRAVO AND A. FERNANDEZ DE MOLINAverse plane, but both phases tend to be greatest at about 0 4 mm from themiddle line, near the line of entrance of the dorsal root. Cross-marked andinterrupted lines show the levels of reversal for phases I and II respec-tively. Phase III has the best defined maximum level and this correspondsto the intermediary region near the ependymal canal; dash-dot line showsthe reversal level for this phase.

Relation of the cord potentials to the afferent volleyIn six experiments the afferent volley was recorded from a filament of

the dorsal root simultaneously with the dorsal surface potentials. Figure 5shows three pairs of records obtained in one such experiment when thedorsolateral pedicular nerve was stimulated with progressively increasingstrengths. With a strength 1-2 times threshold (a) the phase I is well

a I

Cl

Fig. 5. Stimulus strength. In all records, top beam, afferent volley in a dorsal rootfilament and bottom beam, cord potentials. a, b and c stimulus strength: 1-2, 2 and5 times threshold. Cord potentials were recorded from the dorsal surface. Timemarker 10 msec; voltage calibration, 1-3 mV for top beam and 0-6 mV for bottombeam.

developed together with a small component of phase III and one spike canbe seen in the dorsal-root action potential; the conduction velocity for thisgroup of fibres is in the range of 25-28 m/sec. As the strength of stimu-lation is increased (2T) the second phase rises from the descending limbof the first one, the third phase increases in amplitude and simultaneously

POTENTIALS OF THE FROG'S SPINAL CORDa second spike develops in the action potential recorded from the dorsalroot (b); this second group of fibres has a conduction velocity of 16-20 m/sec. The record (c) was obtained with 5T strength; both spikes in thedorsal root record have reached their maximum amplitude and the threephases are completely developed. It is of interest to note that the timebetween the first afferent spike and the peak of the first phase is of anorder similar to that between the second afferent spike and the peak of thesecond phase. With the intensities and pulse durations employed in theseexperiments we were not able, even when using higher amplification, todetect a further component in the action potential recorded from thedorsal-root filament.

It might be thought that the second spike in the dorsal-root actionpotential could be due to repetitive activity in the same group of fibresresponsible for the generation of the first spike; this explanation seemsunlikely to us for several reasons. In order to obtain a better estimate ofthe conduction velocity of the pedicular nerve fibres activated, the actionpotential was recorded both in the sciatic nerve and in the dorsal root; inthese experiments the interval between the two spikes was shorter at thelevel of the sciatic nerve than in the dorsal root, a result which is not tobe expected if the same group of fibres were responsible for the generationof both waves in the afferent volley. We have also to consider the lowstrength of stimulation required to obtain the second afferent spike (insome experiments 1-5 x T only), the consistency of its appearance in allthe experiments and the regalarity of the interval between the spikesdurng each experiment.

Effect of repetitive 8timulation and barbituratesThe three phases of cord activity were affected in different ways by

repetitive stimulation and by Nembutal (pentobarbitone; Abbott Labora-tories) administration. Figure 6 illustrates the effect of various frequenciesof stimulation on the amplitude of the phases of the cord potential.Records in the left column were taken when stimulating with a frequencyof 10/min and a strength five times threshold; records (a) and (b) aresingle traces and record (c) was made by superimposing five sweeps.Records in the right column were made as follows: (a) is a single trace, thethird of a series made with a repetition rate of 15/min; (b) and (c) are eachthe second of a series taken when stimulating at frequencies of 20/sec and50/sec respectively; as the burst of stimuli had a duration of 1 sec there are20 and 50 traces superimposed on each record respectively. The burst ofstimulation was repeated every 3 sec. It can be seen that repetition ratesof 15/min suppressed phase III and reduced phases I and II by 10-15 %.A frequency of 20/sec reduced both phases I and II by 60%, and these

93

94 MARIA C. BRAVO AND A. FERNANDEZ DE MOLINA

phases were almost abolished by a repetition rate of 50/sec. The adminis-tration of 2 mg of Nembutal (total amount) abolished phase III within5 min of the injection and reduced the amplitudes of phases I and II by20%.

a - _

bw4

C

I I IL *,*,I,,

Fig. 6. Effect of repetitive stimulation. Left column: frequency of stimulation,10/min; a and b single traces, c five superimposed sweeps. Right column: a, singletrace, frequency of stimulation 15/min; b, 20/sec and c, 50/sec; in b and c there are20 and 50 superimposed sweeps respectively. In a, cord potentials were recordedfrom the dorsal surface; in b and c (another experiment), the records were obtainedat 0-4 mm depth and 0 3 mm from the mid line in the dorsal horn. Time markerunder b, 10 msec; under c, 10 msec for row a, and 1 msec for row c. Voltagecalibration in 450 /AV steps.

DISCUSSION

Impulses in primary neurones from the skin of the frog can only berecorded in the dorsal half of the grey substance, as has also been shown inthe cat (Fernandez de Molina & Gray, 1957). This suggests that most ofthe cutaneous afferent fibres form synapses with other cells in that partof the cord.The three phases of activity that follow the primary spike have been

shown to be sensitive to repetitive stimulation and to Nembutal, and toexhibit occlusion, a result suggesting that these potential changes can beconsidered as originating from post-synaptic structures.The brief latency and time course of phase I as well as its relative resist-

ance to repetitive stimulation and to Nembutal seem to indicate that thispotential results from activity in second-order neurones located in thedorsal horn. The afferents that set up this response are the largest from theskin.The parallel development of phase II and of the second spike in the

action potential recorded from the dorsal root supports the view that thissecond component of the afferent volley activates neurones responsible forthe generation of the second phase of cord activity. The time between the

POTENTIALS OF THE FROG'S SPINAL CORDfirst spike of the afferent volley and the peak of phase I is similar to thatbetween the second afferent spike and the peak of phase II. Phases I andII also respond in a similar way to repetitive stimulation and to Nembutaladministration. It has been shown that the maximum amplitudes ofphases I and II occur at different levels in the cord, near the dorsalboundary of the grey matter and at the base of the dorsal horn respectively.All these facts are consistent with the inference that phase II originatesfrom activity in second-order neurones located in a more ventral level ofthe dorsal horn.The two groups of afferent fibres whose activity is responsible for the

generation of phases I and II might be related to the two types of cu-taneous receptors (A1 and A2) described by Fessard & Segers (1942) in theskin of the frog.The third phase had a longer latency and it was more sensitive to repeti-

tive stimulation and to Nembutal than phases I and II. It would seemthat this phase represents activity in at least third-order neurones. Itcould be obtained at stimulus strengths at which phase I was not maximaland continued to increase, with increasing stimulus strength, as thesecond phase rose from the descending limb of phase I. The maximumamplitude was recorded at the level of the intermediary region. It seems,then, that interneurones located at this level are likely to be activated byimpulses generated in interneurones of the dorsal horn, which are them-selves activated by impulses in the two groups of primary afferent fibres.However, it should be kept in mind that at this level of the cord a greatnumber of the more dorsal dendrites of motoneurones are also present andwe cannot exclude the possibility that activity generated in these dendritescontributed to phase III. This possibility must be considered, since thefacilitation of the antidromic response by a cutaneous afferent volley(Bravo & Fernandez de Molina, 1960b) begins to appear at the peak of thesecond phase, a fact which suggests that only one neuronal link existsbetween cutaneous primary fibres and these dendrites in the shortestpathway to motoneurones.The sequence of potential changes that we have recorded on the dorsal

surface differs from that reported by Marx (1959) in that this author doesnot mention our phase III. The most probable reason is the extremesensitiveness of this phase to frequencies of stimulation as low as12-15/min.The two first negative potentials recorded in the dorsal horn are

paralleled by positive potentials in the more ventral regions of the cord;it could be assumed that these positivities represent source activity fordorsal sinks, because the latencies are identical for both dorsal negativeand ventral positive potentials. It would seem then that axons from dorsal

95

96 MARIA C. BRAVO AND A. FERNANDEZ DE MOLINAhorn interneurones pass into the intermediary region and ventral horn butwe have no evidence from which to draw any conclusion about the furthercourse or termination of these axons.The complex negative potential and superimposed spikes which were

recorded at the level of the motoneurones, and which followed the firsttwo positivities, seem to represent asynchronous synaptic potentials andspike potentials generated in the motoneurones. The spike dischargesrecorded from the ventral root had a latency of a similar order to thoserecorded in the ventral horn.The third positivity which can be seen in records obtained at the ventral

horn level had a time course somewhat shorter than that of the latenegative potential (phase III). Its appearance here depends on the degreeof development of the ventral horn negativity. If the assumption is madethat phase III is due to activity of both intermediary neurones and dorsaldendrites of motoneurones, this third positivity could then be interpretedas being source activity for these two components. But the possibilitycannot be excluded that potentials of an inhibitory nature and of a di-synaptic order take part in the generation of this ventral positivity.Further research is required in order to clear up these problems.

SUMMARY

1. This investigation is concerned with the potential changes generatedin the spinal cord of the frog after stimulation of cutaneous nerves.

2. When recording from the dorsal surface of the cord the primary spikewas followed by three phases of potential change.

3. Phase I reached a peak 1*8-2 msec after the primary spike and waselicited by activation of the lowest threshold afferent fibres, having aconduction velocity of 25-28 m/sec. Phase II reached its peak 1-8 msecafter the negative peak of a second component of the afferent volley, whichhad a conduction velocity of 16-20 m/sec. Phase III reached its peak20-25 msec after the primary spike and developed almost parallel withthe two first phases of activity.

4. The three phases were sensitive to repetitive stimulation and toadministration of pentobarbital, phase III being the most sensitive.Occlusion between two cutaneous nerves was found for all three phases.

5. The intramedullary distribution of these phases was analysed.Phases I and II had their optimum amplitudes in the dorsal horn, that forphase II being more ventral. Phase III showed its maximum at the levelof the intermediary region.

6. Three positive potentials occurred in the ventral horn. At the levelof the motoneurones a complex negative wave with superimposed spikesdeveloped after the second positivity; the latency of these spikes was of

POTENTIALS OF THE FROG'S SPINAL CORD 97

the same order as that of the reflex discharge recorded from the ventralroot.

7. The nature of these phases of activity has been discussed. It issuggested that phases I and II are due to post-synaptic activity in second-order neurones located at different levels in the dorsal horn. Phase IIIrepresents activity in higher-order neurones; the two possible componentsgenerating this phase were discussed.

REFERENCES

BONNET, V. & BREMER, F. (1952). Les potentiels synaptiques et la transmission nerveusecentrale. Arch. int. Phy8iol. 60, 33-93.

BRAVO, M. C. & FERNANDEZ DE MOLINA, A. (1960a). Frog's spinal cord potentials generatedby impulses in cutaneous afferent fibres. J. Physiol. 152, 14P-15P.

BRAVO, M. C. & FERNANDEZ DE MOLINA, A. (1960b). La activation de motoneuronas en lamedula espinal de la rana por aferentes cutaneos. Rev. esp. Fisiol. (In the Press.)

BROOKEHART, J. M. & FADIGA, E. (1960). Potential fields initiated during monosynapticactivation of frog motoneurones. J. Physiol. 150, 633-655.

COOMBS, J. S., CURTIS, D. R. & LANDGREN, S. (1956). Spinal cord potentials generated byimpulses in muscle and cutaneous afferent fibres. J. Neurophysiol. 19, 452-467.

ECCLES, J. C., FATT, P., LANDGREN, S. & WINSBURY, G. J. (1954). Spinal cord potentialsgenerated by volleys in the large muscle afferents. J. Physiol. 125, 590-606.

FERNANDEZ DE MOLINA, A. & GRAY, J. A. B. (1957). Activity in the dorsal spinal greymatter after stimulation of cutaneous nerves. J. Physiol. 137, 126-140.

FESSARD, A. & SEGERS, M. (1942). Dualite des r6cepteurs tactiles chez la grenouille. C.R.Soc. Biol., Paris, 136, 666-667.

KOLMODIN, G. M. & SKOGLUND, C. R. (1953). Potentials within isolated segments of thefrog's spinal cord during reflex activation and changes induced by cholinesterase inhibitorsand temperature variations. Acta phy8iol. 8cand. 29, Suppl. 106, 503-529.

MARX, CH. (1959). Actions centrales des influx d'origine cutanee et musculaire chez lagrenouille. I. Potentiel de racine ventrale. C.R. Soc. Biol., Paris, 153, 836-840.

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