Medical Center, Washington, D.C.

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1 625 J. Physiol. (I955) I30, POTENTIALS RECORDED FROM THE SPINAL CORD WITH MICROELECTRODES By K. FRANK AND M. G. F. FUORTES From the Laboratory of Neurophysiology, National Institutes of Health, Bethesda, Md., and the Department of Neurophysiology, Walter Reed Army Medical Center, Washington, D.C. (Received 17 May 1955) The present article is the first of a series of papers designed to describe results obtained by recording the electrical activity of elements in the lumbar region of the spinal cord of cats by means of intracellular micropipettes. Results obtained applying this technique to the study of spinal reflexes have already been reported by Brock, Coombs & Eccles (1952, 1953), Eccles (1952), Eccles, Fatt & Koketsu (1954) and Woodbury & Patton (1952). While in the studies just quoted attention was focused on the abrupt responses elicitable in anaesthetized preparations, an effort was made in this research to analyse the activity of preparations presenting the sustained responses described by Sherrington. Whereas it was usually easy to determine whether the electrode was located in primary sensory fibres, interneurones or motoneurones, it was found difficult to establish whether the potentials were recorded from somata or axons of post-synaptic units, and whether they originated in 'normal' or in damaged units. The results to be described will show the importance of clarifying these problems, and may justify the rather extensive discussion of the techniques employed and of the criteria used for selecting results and for identifying somatic or axonal origin of the recorded potentials. METHODS The animal preparation. Spinal or decerebrate cats were used in most experiments, but some preliminary experiments and several dealing with activity of primary sensory fibres were performed under pentobarbitone sodium (Nembutal) anaesthesia. All animals were curarized with Intocostrin or tubocurarine, and were given artificial respiration with oxygen. The spinal cord was exposed either by conventional dorsal laminectomy or using a lateral approach. In the latter case, the animal was fixed on its side and laminectomy was extended to the transverse processes on one side. With this dissection the anterior horns could be reached by the 40 PHYSIO. CXXX

2 626 K. FRANK AND M. G. F. FUORTES micro-electrode, within 1 mm depth (P1. 1, fig. 1). Required dorsal and ventral roots were dissected free and mounted on platinum electrodes under paraffin oil. Cats prepared in this manner, maintained at a rectal temperature between 36 and 390 C, and given fluid occasionally, generally showed a systolic blood pressure between 100 and 150 mm Hg and good electrically recorded reflexes for a period of several hours. Fixation. Movement of the tip of the electrode relative to the spinal cord presents a serious problem. This was attacked by making both the cord and the microelectrode rigid with respect to the animal frame. The cat was suspended in a frame of parallel bars by pins in the crests of the ilium, a clamp on the spinous process just rostral to the laminectomy, and a head holder. Following a suggestion by Dr J. W. Woodbury, bilateral pneumothorax was performed and the lungs were inflated only out to the chest cage in order to eliminate transmission of respiratorv motions to the lumbar region. Rapid rate ventilation with 98% 02-2% CO2 was found to be necessary for adequate respiration in these conditions. With this method there is no visible movement. Even slight movement of the flanks in phase with respiration indicates either mucous clogging of the trachea or insufficient curare, and can be corrected. None of the commercially available micropositioners tried have been sufficiently free from vibration for use with this technique. However, both hydraulically and mechanically driven systems were constructed which were massive enough to give adequate rigidity. Relative movement due to pulse pressure is only occasionally severe enough to cause potential fluctuations; presumably this occurs near small arteries. Experimental arrangement. Text-fig. 1 A shows the apparatus in block diagram. A base rate generator controls the sweep frequency of the double-beam oscilloscopes B and C. Oscilloscope C is a slave of B used for visual observation. A pre-set counter controls the frequency of stimulation permitting selection over the range from 1 stimulus/sweep to 1 stimulus/100 sweeps. Isolation transformers and selector switches connect the stimulators to nerves and roots. Potentials from external electrodes on ventral or dorsal roots are amplified with capacitance coupling and displayed on the lower beams of oscilloscopes B and C. These beams were usually blanked when photographs were taken. The microelectrode is mounted on the preamplifier which is connected, through a direct-coupled amplifier, to oscilloscopes A, B and C, to the fourth channel of the direct-coupled paper recorder, and to a loud-speaker. The first three channels of the directwriting instrument record respectively blood pressure from a strain gauge bridge amplifier, microelectrode position in its axial direction, and the opening and closing of the two camera shutters. Camera B records the sweeps displayed on oscilloscope B while camera A records the microelectrode potential displayed on oscilloscope A with continuous film movement for time base (Text-fig. 2). Microelectrodes. Pyrex glass (No. 7740, redrawn) pipettes filled with 3M-KCI similar to those described by previous workers were used. Pipettes were drawn in a vertically mounted magnetic puller providing a tension which increases rapidly with elongation of the glass. The tips are less than 0-5 u and are not resolved by a visible-light microscope. The last few micra of length show a more rapid taper than the main drawn section, and this shape appears to be important for a fine tip with not too high a resistance. Earlier pipettes were filled by boiling in 3m-KCI for 2-3 hr. In later experiments, following a suggestion by Mrs I. Tasaki (Tasaki, Polley & Orrego, 1954), pipettes were first filled with methyl alcohol by boiling at reduced pressure. The alcohol was then replaced by 3M-KCI by simple diffusion in about 48 hr. Filled pipettesstored more than a week in 3M-KCI are rarely adequate for recording purposes. A platinum wire, silver plated and chlorided, was sealed into the open end with dental Sticky Wax and the pipette mounted inside the driven shield of the preamplifier. In some experiments the outside wall of the pipette was platinum plated, providing effective shielding up to the immediate neighbourhood of the microelectrode tip (Text-fig. 1B). This shielding is not needed when negative capacitance feed-back amplifiers are used, and was omitted in later experiments. Electrodes were selected for a resistance of MO when immersed in 3M-KCI; they showed two to five times higher resistance when inserted in the spinal cord.

3 POTENTIALS FROM SOMATA AND AXONS 627 Preamplifier. Five different preamplifiers have been designed and tested in connexion with this research. The last of these is a modification of a negative capacitance feed-back preamplifier developed by MacNichol & Wagner (1954) (see also Solms, Nastuk & Alexander, 1953; Woodbury, 1953), and appears to be the best circuit from a number of standpoints. Its approximate characteristics are: grid current over the dynamic range less than 10-12A, effective input A Cam. A B f^ Hg v KCI filled microelectrode Glass with Pt plating Stainless steel) Glass Pt-Ag-A C, Wa / A S~~~~~~~~~~~~~~~~ -X / / /Glass / H. / 3M-K t- -- Pt plating I,I Millimetres Text-fig. 1. A: diagram of arrangement used for stimulating and recording. B: drawing of mounted microelectrode. In later experiments its construction was simplified by eliminating platinum plating. capacitance of 1 pf, noise close to the theoretical noise of the input resistance (about 0*5 mv for 50 MD) and a dynamic range greater than 0-5 V. Direct current drift was less than 10 mv/hr. The feed-back of negative capacitance is designed to improve the frequency response of the preamplifier in the face of the high electrode resistance, and of the capacity between microelectrode and grounded spinal cord. For proper compensation, adjustment is required while the electrode is in the cord. Text-fig. 3A shows the method used for accomplishing this. A dummy electrode resistance Re and capacitance Ce of reasonable values (50 MQ and 10 pf) are connected to the input grid. A resistance R (20 MQ) can also be connected to the grid through the switch Sw-i. 40-2

4 628 [ K. FRANK AND M. G. F. FUORTES.m 'I [J _, -I [ s - l 5. L L ~.. 3 ; _ ~~~B A 1.4! :.. I I IPW I -.-- f X _#,,,,,4 Text-fig. 2. Records taken simultaneously from the 4-channel direct-writing recorder and from the two cameras. 1, carotid blood pressure. 2, movement of the microelectrode. The limit of deflexion of the instrument is reached by a movement of 200,. After this, the pen jumps back and begins recording of further movement in the same way. An upward deflexion on the record indicates increased penetration. 3, signals from shutters of the two cameras. Upward deflexion indicates single-frame camera shutter open; downward deflexion indicates running-film camera shutter open. 4, record of microelectrode potential relative to reference electrode on vertebral column. Note potential oscillations when electrode is moved and steady negativity when it is, presumably, inside the membrane of a unit. Insert A shows responses of the penetrated unit to stimulation of a ventral root, as photographed by the single-frame camera. Insert B shows a strip of record taken by the moving-film camera at the time indicated by the two arrows. Note the different display of the same events in these photographs and in the direct records. Calibrations: channel 1, mm Hg; channel 2, 200 u; inserts A and B and channel 4, 50 mv. Time: 60 sec for direct records.

5 POTENTIALS FROM SOMATA AND AXONS 629 A low impedance square wave source is connected either to Re or B by the switch Sw-2, the other being grounded. With the square wave fed to the grid through Re and with Sw-i in position 1, the feed-back is adjusted for minimum distortion at the output. Reversing Sw-2 and throwing Sw-I to position 2 will, without C, require less feed-back for optimum response. C is therefore adjusted until the amount of feed-back required is the same with the switches in either of the two positions. If Ce and Re are now varied within the range to be expected with microelectrodes, it is noted that (once C is adjusted as indicated) the feed-back giving best response to a square pulse introduced through B is still quite similar to that required when the pulse is introduced through Re. There is, therefore, good assurance that adjustment of the feed-back through R will be approximately correct also for real microelectrodes having values of Re and Ce within the tested range (3-10 pf; MQ). By using this method it is soon found that an approximate adjustment of feed-back can be made by setting the control a little below the level producing 'ringing'. Repeated comparisons show that the recorded action potential height, using this approximation, rarely differs as much as 10% from that obtained using the more elaborate method described above. A B Driven shield Ii Sw-i 3 o~sw-i 3 Ce Re RTC Re R Sw-2 TE - ILJ Text-fig. 3. A: circuit diagram illustrating method used for adjusting the negative capacitance feed-back. B: circuit used for measuring the total input resistance when the electrode is inserted in the cord. Explanations in text. It is essential to measure the total input resistance from time to time while in the spinal cord to show breakage or clogging of the micropipette. A method for doing this is illustrated in Text-fig. 3 B. The battery E represents the sum of several constant potentials. These may include the junctional potentials of microelectrode and reference electrode, the membrane potential of any penetrated element, and the compensating potential used to bring the sum of these within the dynamic range of the preamplifier. Because of E, a deflexion V., is produced in the recording instrument when Sw-I is moved from 1 to 3. A smaller deflexion, V, will result if Sw-i is moved from 1 to 2. The electrode resistance Re is determined from the relative sizes of Vg and V, regardless of the value of E: Re,=R The amplitude of a nerve impulse may be used as the potential V. for a measurement of the input resistance with the electrode inside a neurone during the peak of its action potential. In this case V, is the reduction of spike height observed after moving Sw-I from 1 to 2 and Re is determined by the relation given above. Criteria for selection of results The experiments on which this and the following papers are based were performed between October 1951 and January 1955, and involved 150 cats. It is estimated that over 5000 units have been penetrated by the microelectrode during this series of experiments. Of these, photographic records have been taken from approx. 600, and only about 250 have been thoroughly analysed. These figures show that extensive selection was exerted not only during analysis of the records, but also during performance of the experiments,

6 630 K. FRANK AND M. G. F. FUORTES inasmuch as activity not possessing certain features was not recorded. The dangers of selecting results are particularly great when the features of the activity of any one unit are described as representative of a whole population, and it is important to clarify the criteria on the basis of which some units were chosen for analysis and others were discarded as abnormal. In the early stages of the research, technical failings of the equipment constituted a major source of faulty results. Movement of the electrode relative to the cord and unsuitable characteristics of the amplifying system have been mentioned by other authors as possible sources of error. Besides these, it was found that the microelectrode had to have certain features in order to penetrate cells without obvious damage. KCI solution leaks from some apparently fine and from most coarse or broken electrodes. The leakage can be observed under the microscope as a growth of solid substance (presumably KCI crystals)from the microelectrode tip (Moore & Cole, 1954). Fine electrodes can be inserted under microscopic observation in unfixed slices of spinal cord stained with methylene blue, and can be seen to puncture cells without evident damage (P1. 1, fig. 2). If a coarse (1 u) pipette is used instead, a yellow-brown colour soon appears in the penetrated cell and leaks along the electrode track when the pipette is withdrawn from the unit. When these coarse electrodes are used for recording, membrane and spike potentials can be held, with progressive deterioration, for a short time only, and the spikes present an approximately exponential falling phase of characteristic appearance. Elements presenting these features have been considered possibly damaged and have been discarded. Fine electrodes not presenting observable leakage of the filling solution do not seem always to inifict rapid damage on the penetrated unit. In several units, constant value of potentials and responsiveness to stimuli were maintained for over an hour. Ultimately, however, all units present signs of deterioration starting with decrease and ending with complete loss of membrane and spike potentials. Therefore, results obtained long after penetration were accepted with caution and discarded if they differed considerably from results obtained earlier in the same unit. Other results have been discarded because they were assumed to be vitiated by poor general conditions of the preparations. In previous work (Brooks & Fuortes, 1952), it was found useful to correlate the electrical discharges recorded at any given time from roots or rootlets with the movements elicitable at the same time by sensory stimulation. For instance, when studying the electrical activity accompanying flexor reflexes, it was checked from time to time that the preparation still retained the ability to withdraw the limb when this was touched or pricked, and only in the affirmative case was the discharge assumed to be representative of 'normal' reflex activity (see Matthews, 1953a). Since this criterion cannot be resorted to in curarized preparations, during the present experiments it was often found necessary to evaluate the actual conditions of the preparations from the features of the electrical records led off from ventral roots. When the composite reflex discharge elicited by sensory or electrical stimulation appeared abnormal, the unitary discharges led off from the microelectrode were presumed to be also abnormal and were disregarded. An effort was made not to select results on the basis of agreement with findings obtained by other authors using similar technique. Thus absolute values of membrane or spike potentials, spike duration, presence of overshoot were not used as criteria for choosing the units to be analysed. Because large potentials are more suitable for recording and analysing, the photographic records heavily favour elements yielding large electrical potentials. Small spikes were, however, not altogether discarded, and a large number of them was recorded or carefully observed. RESULTS Potentials observed when the electrode is moved through the cord P1. 1, fig. 1, is a photograph of a section of the lumbar region of the spinal cord of a cat. The lines across the photograph can be used as an indication of the structures which the electrode is likely to cross in a track through the cord.

7 POTENTIALS FROM SOMATA AND AXONS 631 Not more than three or four motoneurone bodies are crossed by each line, but numerous small cells and innumerable fibres will always be in the path of the electrode. In a number of experiments the microelectrode was continuously moved through the cord at an approximately constant speed. The potentials recorded under these conditions are illustrated in Text-fig. 4. It is seen that the potential of the microelectrode with respect to the reference electrode tends to return recurrently to one extreme value, emphasized in the figure by the /llaa 1 1 ti11/71/ 24j Text-fig. 4. Records of channels 2 and 4 of direct-writing instrument (see Text-fig. 2). The records were taken during continuous movement of the electrode from dorsal to ventral surface of cord for a depth of about 3 mm. The large downward swings in the lower trace indicate probable penetrations of neural elements. The dashed horizontal line emphasizes the stability of potential level measured when the electrode is presumably extracellular. Calibrations: channel 2, 200,u; channel 4, 50 mv. Time: 60 sec. dashed horizontal line: this potential value is taken as the adjusted zero and is assumed to be recorded when the electrode is in the extracellular space. The large downward swings, indicating microelectrode negativity, are assumed to reveal penetration of cellular membranes. Smaller negative potentials are recorded for a considerable percentage of time and are less easily interpretable. Some of these small potentials must originate in nervous elements because, on frequent occasions, they are recorded together with nerve impulses. Others may be due to potential gradients in the extracellular fluid either occurring naturally or due to electrode penetration of nearby membranes, or may represent the membrane potentials of non-neural elements, such as glia cells. The potential recorded when the electrode is presumably in the extracellular fluid has constant value at all depths in the cord. Thus the finding (G6pfert, 1953; Matthews, 1953b), that in frogs or rats the anterior horns are considerably negative with respect to the dorsal regions of the cord, was not confirmed in the preparation used in this study.

8 632 K. FRANK AND M. G. F. FUORTES Not infrequently large sudden jumps in potential to a steady level up to 80 mv (microelectrode negative) are observed. Stimulation of previously prepared roots or nerves may produce no spikes or other changes in these potentials. In fact, experiments in which stimulating shocks are delivered through the microelectrode while simultaneously recording from it (to be described in a later paper), indicate that when these potentials occur, no response is elicited by shocks more than adequate to stimulate cells and fibres. They may be, therefore, electrically inexcitable neural elements, or non-neural elements with large and stable membrane potentials. On several occasions it was observed that a unit could be punctured several times without severe loss of membrane and spike potentials (Text-fig. 5). In agreement with previous findings (Nastuk & Hodgkin, 1950; Draper & Weidmann, 1951) this observation leads to the conclusion that the damage produced by impalement is not necessarily very drastic. Obvious damage occurs, however, quite frequently. In some cases, immediately after penetration, spike and membrane potentials had a high value, but within a few seconds fell to a lower value which remained stable for some time. However, minute movements of the microelectrode frequently resulted in increase and stabilization of the potentials of a penetrated element. Both observations can be explained on the assumption that the amplitude of the recorded potentials depends on the completeness of the 'sealing' of the membrane around the shaft of the pipette. Whenever sealing is incomplete potentials smaller than normal should be recorded, and deterioration of the unit might be expected. Conversely, small movements or other events favouring the process of sealing may increase the amplitude of the recorded potentials and prevent further deterioration. The patterns of impulse responses to afferent excitation, obtained early after penetration from units producing full-size spikes, quite often did not change if the spike size later decreased. On several occasions deterioration of spike height from 80 to 1 mv was not accompanied by changes of these impulse patterns. This observation was at first somewhat surprising, but could be interpreted on the basis of results to be mentioned later on. Membrane and spike potentials can be obtained not only when the electrode is pushed down into the cord, but also when it is pulled back (Text-fig. 6). Large potentials can be recorded on withdrawal if the electrode can perforate both sides of the cell and be pulled back in it without evoking serious damage. Smaller potentials can be obtained in a similar way or also if the electrode, having ruptured the cell's membrane, is withdrawn into a region of extracellular fluid which, being close to the hole, reflects the potential changes occurring inside the cell. On some occasions penetration by the electrode evoked repetitive firing. In the majority of these cases, the firing had the typical features of the injury

9 POTENTIALS FROM SOMATA AND AXONS 633 2~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \jbcijd0 *W ab c d I _ I. 1. I..I... Text-fig. 5. Records from channels 2 and 4 of direct-writing instrument (see Text-fig. 2), and sections of photographic record taken simultaneously on running film. A ventral root was stimulated every 0-5 sec. The records start with the electrode presumably inside a motoneurone. At a in all records, the electrode is withdrawn from the motoneurone by upward movement of about 170 t. It is then moved down for approximately the same distance and repenetrates the unit at b. c and d indicate a similar withdrawal and repenetration. The photographic record was interrupted at e, but the same manoeuvre was again recorded on the direct writer. The regularly spaced vertical lines on channel 4 are the waves of post-spike hyperpolarization seen in the photographs. (The action potentials themselves are too fast to be recorded on the direct-writing instrument.) Calibrations: channel 2, 200,u; channel 4 and photographic record, 50 mv. Time: 60 sec for direct records, 1 sec for photographic record.

10 634 K. FRANK AND M. G. F. FUORTES b c d 2 L 3 1 Text-fig. 6. Records from channels 2, 3 and 4 of direct-writing instrument and sections of photographic records taken at the same time. Pairs of stimuli were applied to a ventral root at every 075 sec for purposes of identification (see p. 643 and Text-fig. 13). Record of channel 4 shows three successive penetrations, each followed by repetitive firing appearing in the record as a solid black band. Note that all penetrations occurred when the electrode was pulled back (see p. 632), and that between a and b the electrode slipped out without intentional movement. As shown in channel 3, running film record was started just after the first penetration and again just before the second penetration. a through e indicate corresponding times in photographic and direct records. Insert is a photograph with fast sweep (time marks 1 msec) of the response of the unit to the pair of ventral root stimuli. Calibrations: 50 mv. Time: 60 sec for direct records, 1 sec for photographic record.

11 POTENTIALS FROM SOMATA AND AXONS 635 discharge described by Adrian & Matthews (1934): an initially high frequency of discharge rapidly decreasing and ending within a few seconds with death of the unit. In other instances, the firing was of moderate frequency and subsided soon after penetration, leaving no sign of damage of the penetrated unit (Text-fig. 6). These instances suggest the possibility that the microelectrode may alter the rhythmical properties of the impaled unit. There is no way of determining if rhythmical discharges recorded from interneurones are normal or abnormal. In primary afferent fibres or motoneurones, however, discharges recorded with microelectrodes can be compared with those recorded from roots or from the periphery. Since no difference is found between patterns of discharges recorded with the two methods, it can be concluded that the rhythmical properties of tissues are not fundamentally changed by the microelectrode. Alterations too subtle to be detected by this comparison have to be disregarded at this stage of the research. Nearly maximal positive spikes are sometimes recorded with essentially no membrane potential. Tasaki (1952) has reported similar findings when the microelectrode tip was inside the myelin but outside the axis-cylinder of a medullated nerve fibre. He states (p. 93) that: 'The myelin sheath or a layer of myelin shows a high resistance to direct currents, but transient current pulses can flow through this layer.' The pure positive nature of this spike requires that the time constant of this capacitative coupling be large in comparison with the duration of the spike. Saltatory conduction permits another explanation. With some simplification it can be considered that the region inside the myelin but outside the membrane of an internode is coupled with the internodal axoplasm through a battery E (the membrane potential of the internode), which remains unchanged during activity. A microelectrode in this region will record the sum of the potential of the internodal axis cylinder and the steady potential E. At rest this sum is zero, while during activity it will give rise to a positive spike equal to that of the axoplasm but rising from zero rather than from the negative potential of the internodal axoplasm. Since during activity there is a small outward current through the myelin (Huxley & Staimpfli, 1949) a microelectrode inserted part way through this layer will record a positive spike proportional in amplitude to the depth of penetration. Measurement of membrane and spike potential Text-figs. 5 and 6 show that when the electrode is moved presumably from the extracellular fluid to the inside of a unit, or vice versa, the potential drop does not necessarily occur suddenly, but may take place either gradually or in several steps. Because of this, it is difficult to choose the correct reference potential if one considers only the potential changes occurring immediately before and after penetration. In this research, membrane potentials have

12 636 K. FRANK AND M. G. F. FUORTES been taken with respect to the recurring positive potential of the direct-writing recorder (upper horizontal line in Text-fig. 4). Selecting the units in which the values could be measured more accurately (a procedure which strongly favoured the larger values), a number of measurements of membrane and spike potentials were taken. In Text-fig. 7 the spike potential is plotted against the corresponding membrane potential. The points situated above the line of unit slope correspond to units which present overshoot and form the majority of the population considered. Points from units presenting small membrane and spike potentials could have been included and would have filled the lower left corner of the figure. They have been discarded through the selection processes already discussed, although they constituted the majority of the units seen. mv 100 * Vs X 0 ; 0.: 50 * I,,... I V 100 mv Vm Text-fig. 7. Plot of spike amplitude against corresponding steady voltage (measured as described in text) of the units penetrated and recorded in fourteen unselected experiments. Units represented by circles were identified as primary afferent fibres. Units represented by dots were not so identified. One hundred and sixty-seven' units are included in the figure and only twenty-three of these fall below the unit slope line. Potentials recorded from primary sensory fibres Features of membrane and spike potentials. Contrary to assumptions by some authors (Brock et al. 1953, p. 453), and in agreement with others (Tasaki, 1952; Woodbury, 1952; Woodbury & Patton, 1952), fibres can be penetrated by microelectrodes. In these experiments it was found that microelectrodes can easily be introduced in root fibres lifted from the cord, or altogether severed from the animal and placed in a nerve trough. When the electrode is slowly moved through a lifted dorsal root, as many as twelve fibres can be penetrated in 1 mm. The electrode can be held in fibres as long as in other elements of the cord, and in favourable conditions can repenetrate them several times without great loss of membrane or spike potentials.

13 POTENTIALS FROM SOMATA AND AXONS 637 When the electrode is inserted in the cord, a penetrated unit can still be identified as a primary afferent fibre by measuring the latency of its response to a dorsal root shock. It is reasonable to assume that responses occurring with less than 05 msec latency are led off from primary fibres. Size and time course of spikes recorded from primary sensory fibres is the same in roots as within the cord. Apparently presence of a larger or smaller volume conductor around these fibres does not alter their recorded action potential, unless a large number of the root fibres are synchronously excited and the root is lifted into an insulating medium. Text-fig. 8 illustrates the effect of an insulating medium on the potentials recorded from a root fibre when a synchronous Text-fig. 8. Effect of volume conductor on potentials recorded by intracellular electrodes. The microelectrode is inserted in a fibre of a ventral root. In A and B the root is surrounded by paraffin oil; in C and D the oil is replaced by Ringer's fluid. A and C, stimulus just subthreshold for penetrated fibre; B and D, maximal stimulus. Calibration: 50 mv. Time: 1 msec. volley travels along the root. The upper records show the potentials recorded from a high threshold fibre due to activity in many of its neighbours, when the root is in oil. When the penetrated fibre is also excited, its spike is added to the extrinsic wave, which, in this limited volume conductor, is a considerable part of the recorded potential. The lower records show that a much smaller extrinsic potential is recorded from a similarly high threshold fibre when the root is surrounded by conducting fluid. If only a small number of impulses are synchronous in the neighbourhood of a microelectrode, very little extrinsic activity is recorded even in a limited volume conductor. It can be concluded that extrinsic potentials do not appreciably distort the potentials recorded by microelectrodes inserted in elements of roots or spinal cord, except in the presence of large synchronous volleys in a limited-volume conductor.

14 638 K. FRANK AND M. G. F. FUORTES Spikes from dorsal root fibres are always short-lasting. The distribution of spike duration for root fibres is shown in Text-fig. 9A, in which the values obtained from two experiments are presented without selection. Since damage to the fibre usually increases spike duration, it can be assumed that A B OL C VUJ U.0 V-7 1 * 1*' 1U A.1 msec Text-fig. 9. Charts illustrating distribution of duration of spikes recorded from different structures. Abscissae: spike duration in msec; ordinates: number of spikes within 0 3 msec groups. A, spikes recorded from dorsal and ventral root fibres; B, spikes recorded from motoneurones presenting signs of block of antidromic conduction (presumably motoneurone somata or dendrites-see p. 643); C, spikes recorded from post-synaptic elements of the cord other than those of A and B.

15 POTENTIALS FROM SOMATA AND AXONS 639 the asymmetry of the distribution curve is in part due to inclusion in the table of results obtained from damaged fibres. Despite this, the distribution curve presents a peak at 0-6 msec with a standard deviation of msec. Spikes of similarly short duration have been recorded in spinal cord units by Woodbury & Patton (1952), but have not been reported in work from Eccles's laboratory. Dorsal root reflexes. Toennies (1938, 1939) and Barron & Matthews (1938b, c) found that, following dorsal root stimulation, some fibres of the stimulated or of an adjacent dorsal root conduct antidromic impulses: the dorsal root reflex. As already reported by Frank (1953), these antidromic impulses can be picked up by microelectrodes. Knowledge of their features is important for determining the pre- or post-synaptic origin of certain discharges recorded from units in the cord. When a microelectrode is located in one of the more excitable fibres of a dorsal root, a weak stimulus evokes only a short-latency orthodromic response, but stronger shocks may elicit one or more antidromic, 'reflex' impulses. Conversely, when the penetrated unit does not respond readily to the applied electrical stimuli, its threshold is reached only by stimuli which are suprathreshold for a large number of the surrounding elements. For such fibres ' reflex' responses may appear first, and the direct orthodromic response will be elicited only by stronger stimulation. A minimal change in stimulus strength is sufficient to include or exclude excitation of the penetrated fibre. Presence or absence of a direct response does not alter the latency of the 'reflex' response in these conditions (Text-fig. 10). Apparently a certain quantity of impinging impulses is necessary for generation of dorsal root reflexes in any one fibre but participation of the fibre itself in the impinging volley does not necessarily alter the features of its ' reflex' response. In this research, the shortest latency observed between stimulus to a dorsal root and first antidromic impulse was 2-5 msec and the longest about 15 msec. Dorsal root reflexes often consist of more than one but rarely of more than three impulses following a single shock to a nerve or to a dorsal root. Dorsal root reflexes have been recorded with the present technique, not only following dorsal root stimulation, but also following stimulation of cutaneous or mixed nerves. When the electrode was inserted in a fibre of a peripherally cut dorsal root, dorsal root reflexes could be identified also following sensory stimulation. The increase in dorsal root reflexes at subnormal temperature (Barron & Matthews, 1938b, c) has been confirmed. Although it cannot be stated whether dorsal root reflexes occur in normal conditions, they are common enough in experimental preparations to justify some speculations on their possible functional effects. If they occur following normal activation of receptors, they will have two main consequences: by collision they will prevent impulses initiated in certain receptors from reaching the spinal cord, and, when they do not collide, by antidromically impinging on the receptive

16 640 K. FRANK AND M. G. F. FUORTES terminals, they may condition some receptors to subsequent natural stimuli. It is interesting to note in this respect that sensitization of skin receptors has been observed to occur following antidromic bombardment (Lewis, 1942; Habgood, 1950). Receptor discharges. When the dorsal roots are not severed from the periphery, receptor discharges are often recorded by the microelectrode. Stimulation of A B C a b 4 D C u i 2 )0 -g-o b d Successive stimuli Text-fig. 10. Dorsal root reflexes. A, impulses recorded from a dorsal root fibre following stimulation of that root. Dorsal root stimulus is threshold for the penetrated fibre in a and just subthreshold in b. Presence of the direct impulse does not affect latency of the 'reflex' spike. Strength of dorsal root stimulus decreased further in c and d. B, impulses recorded from a different dorsal root fibre following strong a, medium b and weak c stimuli to the same root. Note double 'reflex' response to strong stimuli. C, same fibre as B: 'reflex' impulses evoked by stimulation of a neighbouring root. In A-C, calibration: 50 mv. Time: 1 msec. D, latency of first dorsal root reflex of a fibre following successive root stimulations at threshold strength for the penetrated fibre. Stimulus evoked orthodromic firing in eight cases (solid circles) and only 'reflex' firing in six (open circles), but latency remained approximately constant throughout. the intact dorsal root can still be performed in order to permit (on the basis of latency of response) identification of the penetrated element as a primary sensory neurone. Trains originating in receptors are strikingly regular in comparison with those originating in post-synaptic units (Text-fig. 11). No new features of organization of sensory discharges have been revealed

17 POTENTIALS FROM SOMATA AND AXONS 641 during this research, and some of the properties already found by other authors have been confirmed. Among these, resetting of the rhythm of the sensory train could be demonstrated to occur followingsbombardment of the receptor with an antidromic impulse. A B C D t Text-fig. 11. Primary afferent fibre responses to touch recorded in the spinal cord. Light touch begins at, increases progressively and terminates abruptly at A. Traces are contiguous. Throughout records fibre is stimulated by dorsal root shocks every 0-85 sec. Latency of response to these shocks permits identification of unit as primary afferent. Calibration: 50 mv. Time: 1 sec. Receptor discharges have been recorded when the electrode was situated up to 4-5 mm deep in the cord. When the electrode penetrates a primary sensory fibre deep in the cord, latencies of up to 0-4 msec were measured between an electrical shock to a dorsal root and appearance of the direct spike. In these cases, the impulses must have been recorded from already subdivided branches of primary fibres. Still, sensory discharges picked up from the depth of the cord maintained their usual features. Intermittence of the rhythmical trains, such as that found to occur after conduction along the posterior columns (Barron & Matthews, 1935), or other evidence of presynaptic inhibition, was not observed in the experimental situations employed. 41 PHYSIO. CXXX

18 642 K. FRANK AND M. G. F. FUORTES Dorsal root potentials. When impulses enter the spinal cord by way of a dorsal root, a slow potential change develops between two conventional electrodes located on the root of entry or on a neighbouring root, one close to the cord and another more distally (Barron & Matthews, 1938a). These potentials have been called dorsal root potentials. They are supposed to propagate electrotonically along the root. Following strong stimulation of a dorsal root, a small potential change similar in its time course to the dorsal root potentials sometimes develops between a microelectrode inserted in a dorsal root fibre and a distant point. A B Text-fig. 12. A and B, slow potentials from a microelectrode in a dorsal root; A just before, and B just after penetration of a fibre. Shock just below threshold for fibre. High gain, condenser-coupled trace shows no change in potentials from A to B. Low gain, direct-coupled trace has a different sweep speed, but indicates about 70 mv membrane potential. Calibrations show 50 mv (short bar) for low gain, and 10 mv for condenser-coupled record. Time: 1 msec. C, dorsal root fibre action potential showing negligible slow potentials. Calibration: 50 mv. Time 10 msec. This slow wave cannot, however, be interpreted as a potential drop across the membrane of the fibre considered because: (1) it is equally large and has the same polarity when the electrode is outside the fibre as when it is inside (Text-fig. 12A, B); (2) it becomes negligibly small when the root is placed in a pool of conducting fluid instead of oil, or when the microelectrode is inserted in a similar fibre inside the cord (Text-fig. 12 C). Dorsal root potentials are also not recorded following sensory stimulation (Text-fig. 11). The reasons for the failure to record dorsal root potentials by means of microelectrodes cannot be explained without the help of assumptions, and these will be discussed later.

19 POTENTIALS FROM SOMATA AND AXONS 643 Potentials recorded from motoneurones Membrane potentials and spikes recorded from ventral root fibres are similar to those seen in dorsal root fibres. The spikes have short duration and are not accompanied by slow potentials. When the microelectrode is inserted in the spinal cord, two types of short-latency responses to ventral root stimulation are recorded from different units. Both types of units respond to a ventral root shock by discharging a single impulse within 0f2 msec and are thereby defined as motoneurones. Some of these units present spikes as short as those recorded from ventral root fibres, and are not accompanied by slow potentials (Text-fig. 13 B). Other units instead present spikes of longer duration followed by long-lasting hyperpolarization (Text-fig. 13A). These two types of units differ also in the nature of their response to pairs of ventral root shocks. Units producing long-duration spikes respond to double ventral root stimuli in the manner already described by Brock et at. (1953). As the stimulus interval is decreased, a clear inflexion appears in the rising phase of the second spike, the duration of which is prolonged as a consequence. The inflexion becomes more pronounced following further reduction of stimulus interval, and finally the response falls from the point of inflexion. In most cases, the height of the small response at this critical stimulus interval is 30-40% of the height of the full spike (Text-fig. 13 A). In agreement with the conclusions by Brock et al. (1953), the events just described can be interpreted by assuming that conduction from the point of stimulation to the point of recording is blocked for some time after passage of one impulse. The non-medullated section of the motoneurone axon or the axon hillock itself are likely points of block. When a penetrated unit produces short-duration spikes, as the stimulus interval is decreased below 2-3 msec, the size of the second spike decreases progressively, then fails altogether (Text-fig. 13 B, C). Reduction of the second spike can be ascribed to reduced responsiveness in the relatively refractory period, and its failure to the absolute refractoriness of the stimulated region of the fibre following discharge of the first impulse. If one accepts that the block occurs at the axon hillock, then, since only elements with long-duration spikes show evidence of conduction block, it must be concluded that long spikes originate upstream from the axon hillock, i.e. in the cell bodies or dendrites, and short spikes originate in axons. The distribution of spike durations from presumed motoneurone somata indicating such a conduction block is shown in Text-fig. 9B. The peak of the distribution is at about 1-4 msec. The mean, however, is 1-57 msec with standard deviation of msec. If the spike of the axon hillock can be detected by a microelectrode in the soma, one would expect that, conversely, the spike of the soma could be 41-2

20 644 K. FRANK AND M. G. F. FUORTES A B 70 _0 C -0 60H mv _ 10 _ 10 OK. I_I _.1I I msec Text-fig. 13. Antidromic conduction block in motoneurones. Electrodes inserted in the cord may pick up two types of responses to pairs of ventral root shocks. In unit of column A the response to the second of a pair of shocks suddenly drops to 30-40% when the shock interval is reduced below a critical stimulus interval (about 10 msec in this case). Conduction block must occur near microelectrode since blocked impulse is visible there. Unit of column B shows instead a smooth decrease in height of second response as the stimulus interval is decreased. Calibration: 50 mv. Time in both columns: 1 msec. (Note different sweep speed in A and B.) Only units like that of column B are found when the electrode is inserted in ventral roots. They have shorter duration spikes than those of column A for either ortho- or antidr9mic excitation. C, height of second response plotted as function of interval between two ventral root shocks for two different units. Open circles, type A unit; solid circles, type B unit. Increase in critical stimulus interval for type A unit occurred with lapse of time and is interpreted as a loss in safety factor for conduction.

21 POTENTIALS FROM SOMATA AND AXONS 645 detected by an electrode inserted in the region of the hillock. In the experiment illustrated in Text-fig. 14, the spikes recorded following two ventral root stimuli presented an inflexion in the falling phase which disappeared from the second spike if the stimulus interval was less than 3 or 4 msec. The late hump in this spike can be considered to be the reflexion of the firing of the motoneurone soma, and its disappearance would reveal that conduction is blocked at the axon hillock. In a few cases when this type of response was observed, the spike duration in the absence of the late hump was about 10 msec. This appears to be in agreement with Lloyd's (1951 a, b) conclusion that the spike of the non-medullated segment has longer duration than that of the medullated part of motoneurone axons. Text-fig. 14. Evidence of antidromic conduction block. Microelectrode is presumed to be in motoneurone, just distal to soma. At critical stimulus interval hump on falling phase of second response disappears. Interpretation in text. Calibration: 50 mv. Time: 1 msec. The critical stimulus interval is considerably shorter for orthodromic than for antidromic conditioning (Text-fig. 15). Since impulses of antidromic origin are followed by long-lasting hyperpolarization, while impulses evoked by orthodromic stimulation are not, it is likely that the soma hyperpolarization contributes to maintaining the block of antidromic conduction as suggested by Brock et al. (1953, p. 432). The results obtained by Takeuchi & Tasaki (1942) on medullated nerve fibres offer adequate basis for interpreting this, by showing that the safety factor of conduction is decreased by currents flowing inside the axis cylinder in the direction of propagation. Facilitation of antidromic invasion following orthodromic excitation was also demonstrated by a slightly different experiment. Two ventral root stimuli were so timed that the impulse evoked by the second stimulus regularly failed

22 646 K. FRANK AND M. G. F. FUORTES to invade the motoneurone soma. When appropriate sensory stimulation was applied, the second impulse consistently invaded the soma. Relations between this orthodromic facilitation and soma depolarization (postulated by Brooks & Eccles, 1948; Brock et al. 1953), and between the hyperpolarization following antidromic invasion and inhibition of the motoneurone, will be discussed in a later paper. A R C E F G H Text-fig. 15. Effect of orthodromie firing on antidromic conduction block in a motoneurone. A, ventral root stimulus; B and C, dorsal root stimulus; D, two ventral root shocks at less than critical stimulus interval; E, through H, dorsal then ventral root shocks showing that critical stimulus interval is shorter following ortho- than following antidromic excitation. Calibration: 50 mv. Time: 1 meec. Potentials recorded from other somata and axons Identifiwation of interneurones. Interneurones can be defined as postsynaptic spinal cord units which do not send their axons to ventral roots. In order to establish the post-synaptic nature of a unit, it is not sufficient to show that its response to orthodromic stimulation is delayed by a period

23 POTENTIALS FROM SOMATA AND AXONS 647 longer than the expected conduction time, since delayed responses to orthodromic stimulation can occur in primary fibres (dorsal root reflexes). Knowledge of the properties of dorsal root reflexes is necessary in order to distinguish these from post-synaptic responses. As mentioned before, dorsal root reflexes never have central times shorter than 2*5 msec and do not present more than four impulses (Frank, 1953). Discharges with central times of less than 2-5 msec but longer than conduction time, or responses consisting of more than four impulses, can therefore be considered to arise from post-synaptic elements. Moreover, dorsal root reflexes are unchanged during slow frequency repetitive stimulation and decrease during stimulation at higher frequency (Frank & Fuortes, unpublished), but never show progressive increase (build-up). Build-up is therefore a useful criterion for identification of a discharge as post-synaptic. Since some interneurones are known to respond to ventral root stimulation (Renshaw, 1946; Eccles et al. 1954) the properties of the response of a unit to ventral root stimuli have to be analysed before defining it as a motoneurone. The definition of a motoneurone given above (p. 643) requires that the response to a single ventral root shock consists of a discharge of a single impulse after not more than 02 msec. Units not responding at all to ventral root stimuli, or units responding in a different manner were accepted as interneurones if their post-synaptic nature was otherwise established. Elements defined as interneurones were often penetrated in locations other than the anterior horns and frequently presented modes of activity different from those of known motoneurones. These will be described in a following paper. Duration of spikes from interneurones. Charts showing the distribution of impulse duration in known fibres and in presumed motoneurone somata have already been presented (Text-fig. 9A, B). Text-fig. 9C shows the distribution of impulse duration recorded from interneurones and presented without selection, other than that already described. The curve appears to be made up of many short duration spikes like those seen from known fibres plus fewer long duration spikes like those recorded from presumed motoneurone somata. It would appear reasonable to make the assumption that the short duration spikes in the cord are from axons, while the long duration spikes are from cell bodies. Text-fig. 16 illustrates typical spikes recorded from interneurones. The spike of Text-fig. 16A (presumably recorded from a soma) lasts about 1 msec and is preceded by a slow potential similar to those recorded by Brock et al. (1952) from motoneurones, whereas the spike of Text-fig. 16 B (presumably from an axon) rises abruptly and lasts only about 03 msec.

24 648 K. FRANK AND M. G. F. FUORTES A B Text-fig. 16. Action potentials from two interneurones. A, spike duration about 1 msec after pre-spike potential, presumably a soma response. B, spike duration about 0 3 msec with no pre-spike potential, presumably from an axon. Brief post-spike hyperpolarization is a frequent but not a constant finding. Calibration: 50 mv. Time: 1 msec. Strychnine potentials Although this paper is not intended to describe in full discharges recorded from spinal cord elements under strychnine poisoning, mention of some of their features will be made with the purpose of illustrating the different behaviour of the two classes of units interpreted as cell bodies and axons respectively. When the microelectrode is inserted in the spinal cord of a strychninized preparation, some of the penetrated units present discharges such as that illustrated in Text-fig. 17A, consisting of very large slow potentials (up to 40 mv) with superimposed long-lasting spikes. Other units in the spinal cord present similar impulse organization, but much smaller underlying slow potentials. These later units discharge spikes of shorter duration (Textfig. 17 B). The two types of discharges are found in both inter- and motoneurones. When the electrode is inserted in the ventral root fibres rather than in elements of the cord, only negligibly small slow potentials are recorded. It appears from these results that strychnine emphasizes the distinction between a group of spinal cord elements discharging long duration spikes with slow potentials, and a second group discharging spikes of short duration and small or no slow potentials. Since elements of this second group only are found in ventral root fibres, the results are consistent with the assumption mentioned above that potentials of the first type are recorded from cell bodies and potentials of the second type from axons.

25 POTENTIALS FROM SOMATA AND AXONS I~~~~~~~~~~~~~~~~ A B _~~ ~~~~~~~~~~~~~~l _ I _~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 649 Text-fig. 17. Responses from two motoneurones during strychnine convulsion. Long duration spikes rise from large slow potential in A. Unit is presumably a soma. In B spikes have shorter duration and slow potentials are negligible. This unit is presumably an axon. Fractions of the moving film records are displayed also on sweeps for better time resolution. Calibrations: 50 mv, tall bar for vertical spikes and short bar for horizontal spikes. Time: 1 sec between sweeps for moving film records. DISCUSSION The variability of the results obtained has been described in this paper in order to emphasize the limitations of the method and to clarify the assumptions needed to restore consistency. Since the potentials recorded varied extensively in size and time course, and since slow potentials accompanied the spike discharges in some instances but not in others, some sort of classification is required for interpretation of the results. The aim of the present paper is to show that a reasonable consistency can be obtained classifying the recorded potentials in three groups: from damaged elements; from axons; and from somata or dendrites. The conclusion has been reached that damaged elements present small spikes and membrane potentials, axons present spikes of short duration and small, if any, slow potentials, and

26 650 K. FRANK AND M. G. F. F'UORTES somata present longer-lasting spikes usually accompanied by appreciable slow potentials. Woodbury & Patton (1952) made a somewhat different classification of responses picked up with microelectrodes from the spinal cord on the basis of responses to ventral or dorsal root stimuli without attempting to distinguish between responses from different parts of neurones. Judging by the results of the present research their type 1 units are probably primary afferent fibres; their type 2 units are either interneurone axons or somata, or primary afferents showing dorsal root reflexes; their type 3 units are either motoneurone somata or axons. Tasaki et al. (1954) have made a similar attempt to classify microelectrode potentials from the cat geniculate and striate cortex. These authors begin with the assumption that an intracellular electrode, '.. injures the cell seriously'. Extracellular recording gives instead,.... satisfactory, undistorted pictures of the function of the nerve fibres, cell bodies and dendrites in the central nervous system'. Despite this difference in approach the values they report for the duration of action potentials from axons and somata are only slightly larger than those presented here. However, in this research no third group of potentials was seen which could be interpreted as the intracellular counterpart of the long-lasting dendritic potentials reported by these authors and by Clare & Bishop (1955). Possible explanations for this difference are that microelectrodes cannot successfully penetrate dendrites and record their transmembrane action potentials, or that the action potentials of dendrites in the cord cannot be distinguished from those of axons or somata. In this research a unit was considered to have been seriously damaged when the potentials recorded from it decreased rapidly, when the spike was abnormally long and presented an exponentially falling phase, and when its response pattern differed from that obtained without microelectrode insertion. Minor damage cannot be recognized, and the degree of abnormality due to insertion of the microelectrode can only be postulated in a number of cases. The assumption that small spikes are recorded from damaged structures seems to be contradicted by the observation that elements producing small spikes may respond to orthodromic stimulation with normally organized patterns of impulses. However, once the assumption that cell bodies only can be penetrated is abandoned, this finding can be easily explained, since it may be thought that damage has occurred at the place of recording (axon) without involving the structures responsible for generation of the response (soma or dendrites). A surprising feature of records from root fibres was the absence of slow potentials comparable to the dorsal and ventral root potentials of conventional recording. It may be that: (1) dorsal and ventral root potentials are conducted

27 POTENTIALS FROM SOMATA AND AXONS 651 only in root fibres which cannot be penetrated by the microelectrode; (2) the microelectrode selectively destroys the properties of the fibre membrane which are necessary for conduction of slow potentials; (3) the major portions of root slow potentials as recorded with external leads do not reflect potential changes of the fibre membranes but are conducted extracellularly. No conclusive consideration could be found to support or refute any one of the proposed interpretations. Whatever causes failure to record slow potentials from root fibres is probably responsible also for the absence of slow potentials in axons of motoneurones. Among the slow potentials in the spinal cord the depolarization occurring in a post-synaptic unit following impingement of impulses and prior to its firing is of particular interest. This slow potential has been referred to as 'ventral root potential' by Barron & Matthews (1938a), 'synaptic potential' by Eccles (1946) and 'excitatory post-synaptic potential' by Brock et al. (1952). Its detection has led to the theory that local depolarization is a necessary step between pre-synaptic bombardment and post-synaptic firing. The finding that this depolarization could be recorded in only a few of the post-synaptic units penetrated in this research contradicts the mentioned theory unless one accepts the assumptions proposed so far: (1) that short spikes are recorded from axons only; and (2) that slow potentials are not recorded from axons. Detailed data on the correlations between spike duration and slow potentials will be furnished in a future article. In'this paper, two types of strychnine discharges have been described as a remarkable example of the different size of the slow potential recorded when the electrode was inserted in elements belonging to one or the other of the above-mentioned groups. The finding that axons in dorsal and ventral roots produce spikes of short duration, and motoneurone somata spikes of long duration, does not necessarily mean that all axons must present short- and all somata long-lasting spikes. However, until exceptions can be demonstrated it is perhaps best to assume that an unknown neural element is identified as soma or axon by the duration of its spike. It has just been mentioned that classification of structures based on this assumption has been useful in explaining the absence of synaptic potentials in some spinal cord elements. The conclusion of this discussion is that a great deal of the variability of results obtained in the present study was, very probably, due to the fact that both somata and fibres were penetrated, and that these two types of structures differ in several of their properties. Accepting the criteria outlined for identification of cell bodies and axons, it must be concluded that the majority of the units penetrated in this study were axons. In contrast with this, other authors have found that microelectrodes record potentials almost exclusively from somata. This contradiction may be due to some minor difference in the

28 652 K. FRANK AND M. G. F. FUORTES techniques employed, and possibly could be simply explained by difference of construction of the micropipette. If the assumptions discussed so far are accepted, the results obtained are not in conflict with previously established theories on synaptic excitation. SUMMARY 1. A method for inserting microelectrodes in elements of the spinal cord is described. 2. Despite all precautions, damage to the penetrated elements frequently occurs. Potentials or patterns of discharge obtained a short time before death of the unit are considered to originate in damaged elements. These include (1) small membrane and spike potentials; (2) small spikes presenting abnormally long duration and exponential course of the falling phase; (3) high-frequency discharges occurring on penetration (injury discharges). Transient repetitive firing evoked by penetration in the absence of signs of drastic damage is considered to reveal that the microelectrode may produce only minor alterations of the properties of the penetrated elements. 3. Penetrated units are considered to be essentially normal when the potentials they produce maintain similar values and time courses for a long time, and when the patterns of their firing are similar to those which can be recorded by means of external electrodes. These include (with few exceptions) spikes and membrane potentials of larger values, and spikes presenting a time course such as is illustrated in Text-figs Spikes recorded from primary sensory fibres, either in the roots or in the cord, are always of short duration and are accompanied by negligibly small slow potentials. In particular, dorsal root potentials are not recorded by microelectrodes inserted in dorsal root fibres. 5. Dorsal root reflexes can be recorded with microelectrodes. Following single volley impingement, they consist of discharge of one to four impulses. The minimum central latency of dorsal root reflexes is controlled by strength of stimulation and varies between 2-5 and about 15 msec. 6. Spikes recorded from structures of motoneurones located in the cord divide into two groups according to duration, distributed around durations of msec or msec. Only long-duration spikes are accompanied by slow potentials. Responses ascribable to block of antidromic invasion of the motoneurone soma are observed only when the electrode penetrates elements producing long-lasting spikes. 7. Criteria for identification of interneurones are outlined. Spikes recorded from interneurones also divide into two groups according to duration. These two groups coincide approximately with those of recognized axons and of motoneurone somata. Also in interneurones, slow potentials are associated. with longer-lasting spikes only.

29 POTENTIALS FROM SOMATA AND AXONS Strychnine discharges recorded from elements producing spikes of long duration originate from slow potential oscillations of great amplitude. These slow potentials are, instead, quite small in elements producing short-lasting spikes. 9. On the basis of these data, it is suggested that axons, both in the roots and inside the cord, produce short-lasting spikes unaccompanied by slow potentials, and somata or dendrites generate spikes of long duration accompanied by slow potentials. The authors are deeply indebted to Mrs Mary C. Becker, without whose devoted efforts in every phase of the performance of these experiments this paper would not have been possible. Thanks are also due to Dr Ichiji Tasaki for his encouragement and frequent valuable discussions. REFERENCES ADRIAN, E. D. & MATTHEWS, B. H. C. (1934). The interpretatibn of potential waves in the cortex. J. Phy8iol. 81, BARRON, D. H. & MATTHEWS, B. H. C. (1935). Intermittent conduction in the spinal cord. J. Phy8iol. 85, BARRON, D. H. & MATTHEwS, B. H. C. (1938a). The interpretation of potential changes in the spinal cord. J. Physiol. 92, BARRON, D. H. & MATTHEwS, B. H. C. (1938b). Dorsal root reflexes. J. Physiol. 94, 26-27P. BARRON, D. H. & MATTHEwS, B. H. C. (1938c). Dorsal root potentials. J. Physiol. 94, 27-29P. BROCK, L. G., COOMBS, J. S. & ECCLES, J. C. (1952). The recording of potentials from motoneurones with an intracellular electrode. J. Physiol. 117, BROCK, L. G., CooMBs, J. S. & ECCLES, J. C. (1953). Intracellular recording from antidromically activated motoneurones. J. Phy8iol. 122, BROOKS, C. McC. & ECCLES, J. C. (1948). Inhibitory action on a motor nucleus and focal potentials generated therein. J. Neurophysiol. 11, BROOKS, C. McC. & FUORTES, M. G. F. (1952). Electrical correlates of the spinal flexor reflex. Brain, 75, CLARE, M. H. & BISHOP, G. H. (1955). Properties of dendrites; apical dendrites of the cat cortex. Electroenceph. clin. Neurophysiol. 7, DRAPER, M. H. & WEIDMANN, S. (1951). Cardiac resting and action potentials recorded with an intracellular electrode. J. Phys8ol. 115, ECCLES, J. C. (1946). Synaptic potentials of motoneurones. J. Neurophysiol. 9, ECCLES, J. C. (1952). The electrophysiological properties of the motoneurone. Cold Spr. Harb. Symp. quant. Biol. 17, ECCLES, J. C., FATT, P. & KOKETSU, K. (1954). Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones. J. Physiol. 126, FRANK, K. (1953). Single fibre dorsal root reflexes. Abstr. XIX int. physiol. Congr. p GOPFERT, H. F. (1953). Steady potentials and slow potential changes in the spinal cord. J. Physiol. 122, 20-21P. HABGOOD, J. S. (1950). Sensitization of sensory receptors in the frog's skin. J. Physiol. 111, HUXLEY, A. F. & STAMPFLI, R. (1949). Evidence for saltatory conduction in peripheral myelin. ated nerve fibres. J. Physiol. 108, LEWIS, T. (1942). Pain. New York: MacMillan. LLOYD, D. P. C. (1951 a). Electrical signs of impulse conduction in spinal motoneurone. J. gen. Physiol. 35, LLOYD, D. P. C. (1951b). After-currents, after-potentials, excitability and ventral root electrotonus in spinal motoneurones. J. gen. Physiol. 35, MAcNIcHOL, E. F. Jr. & WAGNER. H. G. (1954). A high-impedance input circuit suitable for electrophysiological recording from micropipette electrodes. Nav. Med. Res. Inst. 12,

30 654 K. FRANK AND M. G. F. FUORTES MATrHEws, B. H. C. (1953a). The Spinal Cord (CIBA Foundation Symposium), pp Boston: Little, Brown and Co. MATTHEWS, B. H. C. (1953b). Current flow in the central nervous system. J. Physiol. 122, 22P. MOORE, J. W. & COLE, K. S. (1954). Membrane potentials of the squid giant axon in vivo. Nav. Med. Re8. Inst. (Memo Report no. 54-7). NASTUK, W. L. & HODGKIN, A. L. (1950). The electrical activity of single muscle fibres. J. cell. comp. Physiol. 35, RENSHAW, B. (1946). Central effects of centripetal impulses in axons of spinal ventral roots. J. Neurophy8iol. 9, SoLms, S. J., NASTUK, W. L. & ALEXANDER, J. T. (1953). Development of a high fidelity preamplifier for use in the recording of bioelectric potentials with intracellular electrodes. Rev. 8eCi. In8trum. 24, TAKEUCHI, T. & TASAKI, I. (1942). tbertragung des Nervenimpulses in der polarisierten Nervenfaser. Pfl1g. Arch. ges. Physiol. 246, TASAKI, I. (1952). Properties of myelinated fibres in frog sciatic nerve and in spinal cord as examined with micro-electrodes. Jap. J. Physiol. 3, TASAKI, I., POLLEY, H. & ORREGO, F. (1954). Action potentials from individual elements in cat geniculate and striate cortex. J. Neurophysiol. 17, TOENNIES, J. F. (1938). Reflex discharge from the spinal cord over the dorsal root. J. Neurophysiol. 1, TOENNIES, J. F. (1939). Conditioning of afferent impulses by reflex discharges over the dorsal roots. J. Neurophysiol. 2, WOODEBURY, J. W. (1952). Direct membrane resting and action potentials from single myelinated nerve fibres. J. cell. comp. Physiol. 39, WOODBURY, J. W. (1953). Recording central nervous system activity with intracellular ultramicroelectrodes: use of negative-capacity amplifier to improve transient response. Fed. Proc. i2, 159. WOODBURY, J. W. & PATTON, H. D. (1952). Electrical activity of single spinal cord elements. Cold Spr. Harb. Symp. quant. Biol. 17, EXPLANATION OF PLATE Fig. 1. Section of a cat's spinal cord at L6. Thionin stain to show cell bodies. The black lines show direction of electrode penetrations used in most experiments. Fig. 2. Methylene-blue stain of unfixed spinal cord showing KCl-filled microelectrode penetrating a motoneurone. The microelectrode disappears in the tissue a few,u below the motoneurone but presence of the tip inside the motoneurone could be observed by adjusting the focus of the microscope. Note added in proof. After this paper went to press an article by del Castillo, J. & Katz. B. (J. Physiol. i28, ) has appeared, in which it is reported that KCl-filled micropipettes can show sudden changes in junctional potential due to clogging. Since evidence of clogging (increased resistance) is often seen with electrodes inserted in the spinal cord, it is possible that such artifacts may account in part for exceptionally large membrane potentials (see Text-fig. 7) and for the sudden jumps in potential not associated with spike activity which are mentioned on pp. 631 and 632.

31 THE JOURNAL OF PHYSIOLOGY, VOL. 130, No. 3 PLATE I (Facing p. 654)