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Transmission of direct cortical responses
230
ELECTROENCEPHALOGRAPHY AND CLINICAL NEUROPHYSIOLOGY
TRANSMISSION
OF DIRECT CORTICAL RESPONSES x SIDNEY OCHS AND HISAO SUZUKI2
Department of Physiology, Indiana University Medical Center, Indianapolis 7, Ind. (U.S.A.) (Accepted for publication: February 17, 1965)
INTRODUCTION
With monopolar laminar stimulation of the cortex by means of a small electrode, characteristically different types of direct cortical responses (DCRs) were obtained at different depths: a negative (N) wave DCR from the surface layers; a positive-negative sequence D C R from the middle layers; an N wave D C R from the lowest layer (Suzuki and Ochs 1964). In chronic cortical islands, the N wave D C R was obtained unimpaired on surface layer stimulation while all underlying cortico-cortical axons were apparently degenerated. This indicated that an intracortical path between stimulating and recording sites was involved. The present study was done to determine the location of the neural elements subserving lateral transmission of the N wave DCR using micro-electrodes for laminar stimulation after making various types of cortical cuts between stimulating and responding sites. Gamma amino butyric acid (GABA) was used to determine the differences between the elements transmitting and those giving rise to the N wave DCR. A preliminary account of such experiments was previously presented (Ochs 1962).
lying white matter. Complete pailial cuts included section of the underlying white matter. Molecular layer preparations had occasionally all but the uppermost layer of the cortex cut but in most cases parts of the second layer were also left uncut. After completing the required cut and allowing 30-60 min for recovery from the effects of the cut, the recording electrodes were placed on the surface at 1-2 mm away from the cut, one being usually placed on the same side of the cut as the stimulating electrode, the other on the opposite side. By means of a micro-manipulator, the electrode used for laminar stimulation could be inserted at 0.2 n,m increments into the depths
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A total of 22 adult rabbits were studied. The techniques used for surgical preparation, microelectrode laminar stimulation and recording have been recently described (Suzuki and Ochs 1964). Cortical cuts were made in the visualsomesthetic area using knives described in an earlier study (Ochs 1956). Complete cortical cuts were made from just below the pial membrane extending down to, but not including the underI This study is supported by U.S. Public Health Grant Nos. NB 61993 and HM 04815. 2 Present address: Institute of Brain Diseases, Tohoku University, ~ndai, Japan.
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Fig. I Molecular layer preparation. A and B are examples of responses to 10minarstimulation with depth in mm shown by numbers m t:'~:h pair of tracings: upper one is from same side of cut as of stimulating electrode; lower one from across line of molecular layer preparation. In this and following figures, a negative deflection is in the upward direction. Calibrations: 2 mV, 2 msec.
Electroenceph. olin. NeuroplLvsioi., 1965, 19:230-236
231
TRANSMISSION OF CORTICAL RESPONSE
of the cortex. As in previous experiments, stimulation was monopolar with cathodal pulses 0.1--0.2 msec in duration at strengths of 100-200 /~A. Laminar recordings were accomplished with glass capillary or insulated metal micro-electrodes having tips of 1-2 ,u. To study the effects of GABA on either stimulated or responding sites, a drop of 1% solution in Ringer fluid was applied to the cortex via a fine glass capillary or to a narrow strip of filter paper placed between stimulated and responding sites over the line of the molecular layer cut. When the experiment was completed, a lethal dose of pentobarbital was injected intravenously, the brains removed and placed in 10% formalin. To determine the extent of the cuts, serial sections of the cortex were prepared with Nissl and silver stains.
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RESULTS
Molecular layer preparation and transmission In 5 different animals the N wave DCR was transmitted across the line of the cut where it appeared with similar form and amplitude (Fig. !). The responses had the same latency and with similar stimulus strengths as in the intact cortex, they were transmitted laterally to the same distance (approximately 5 mm). The fact that in some of these preparations only the upper part of the molecular layer remained uncut, suggests that not all of the layer is required for transmission. The micro-electrode was passed down to successively greater depths on one side of a molecular cut for laminar stimulation. When stimulating below the surface, negative wave DCRs of progressively smaller amplitude were obtained on the other side of the cut. On the stimulated side of the molecular cut, a positivenegative type of DCR, as previously found in the intact cortex ($-Jzuki and Ochs 1964), was induced by stimulation at the depth of 0.4-1.8 mm. However, these positive-negative responses were variable in size (probably reflecting alterations in cortical excitability which resulted from the cuts) particularly in their negative phase (cf. Fig. 1, A and B). Transmission of the N wave DCR elicited by deeper cortical stimulation also appeared to depend on the condition of the cortex. When the excitability was reduced, as
Fig. 2
Upper cortical cut and transmission.Transection through the upper three layers of the cortex. See Fig. 1 for explanation. Calibrations: 2 mV, 2 msec. suggested by the reduced negative phase of the positive-negative DCR obtained on the same side of the cut (see FIE'. 3, A), stimulation below the surface did not give rise to a transmitted N wave DCR. This reduced excitability did not seem to be due to a spreading depression since when the stimulating electrode was returned to the surface, the N wave DCR was present.
Cortical cuts and laminar stimulation With the relatively discrete type of stimulation pro~,ided by the monopolar micro-electrode, the question arises as to whether there is transmission of responses when the upper layers have been cut through partially or completely. This was studied in 6 animals. In the example of Fig. 2, the upper three cortical layers were cut thr'~ugh. Then laminar stimulation on one side was performed as before. Stimulation at the surface gave a smaller response across the cut. A large positive-negative sequence was transmitted beyond the line of the ,:ut when stimulating at 1-2 mm in depth. On stimulation of the deeper layers there was a positive component mixed with the N wave DCR. In this example a relatively Electroenceph. clin. NeurophysioL, 1965, 19:230--236
S. OCHSANDH. SUZUKI
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Pallial cut and spread o f stimulus current When complete cuts were made in 2 animals through both the cortex and underlying white matter, transmission was completely blocked. This type of cut allowed a test to be made, in these two experiments, of the extent of the lateral spread of stimulating current; a spread which in our experimental conditions appeared to be remarkably small. Fig. 4 shows a response with the stimulating electrode 0.1 mm on the same side of the cut, a small response with the stimulating electrode placed over the line of the cut and no response when the electrode was placed just over the line of the cut on the opposite side. A
Fig. 3
B
C
Complete cortical cuts and transmission. Complete cortical cut down to white matter (A) and sparing the lowest layer (B). Responses from the electrode on same side of cut are shown respectively on lower (A) and upper (B) traces of each pair. Depths of stimulation in mm given by numbers. Calibrations: 2 mV, 2 msec. large stimulus artifact obscures the positive component. When all layers down to but not including the underlying white rr'atter were cut through as shown in the example of Fig. 3, A, transmission was much reduced. Here there was also evidence of reduced excitability as shown by the smaller DCRs ( btained on the same side of the cut. In B a smaller, mainly negative wave, preceded by one or more small spike-like fast waves, is recorded
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Fig. 4 Pailial cut. Linlited spread of stimulating current. Position of stimulating electrode on the same side of the cut (+0.1 ram), on the line of cut (0) and on the opposite side of the cut ( - 0. I ram). The downward displacement is an artifact. Calibrations: 2 mV, 2 msee.
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Fig. 5
Effec~. of GABA on DCR types. A: an N wave DCR is sho'L,n from intact cortex (top record); shortly after 1% GABA was topically applied and response hlo,'ked (middle record); recovery 10-20 min after washing surfitce (lower record). B: positive-negative seqt:~ence(upper record); shortly after GABA application (middle record); after recovery (bottom record). C: N wave DCR evoked by stimuli 2.2 mm deep (upper record); shortly after GABA (middle record); after recovery (bottom record). Calibrations: 2 msec, 2 mV. Effect o f GA BA on DCRs GABA acts rapidly on the responding site giving rise to surface negative responses. Th,is was shown in one experiment (Fig. 5) for all th,~: types of DCP~ responses: the N wave D C R obtained from the surface (A); the positive-negative sequence obtained from the middle layers (B); the negative wave D C R obtained by the lowest layer stimulation (C). The results were essentially similar to those earlier studied (lwama and Jasper 1957; Purpura et ai. 1959). The "inverted" phase seen after GABA was largest when the response was originally a positive-negative D C R (B). Eiectreenceph. c/in. Neuroph.vsiol , 1965, 19:230-236
233
TRANSMISSION OF CORTICAL RESPONSE A
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Fig. 6 GABA on stimulated and on responding site. Continuation of experiment of Fig. 2. A: Before GABA. B: Shortly after GABA applied to both recording position (same side) and stimulatea site but not to opposite side. C: Control after recovery from effect of GABA. D: Shortly after GABA placed on both recording sites and on stimulated site. Calibrations: 2 mV, 2 msec.
Whereas GABA is rapidly effective on the elements giving rise to the response, it was ineffective on the stimulated element or on those conducting the response. This is shown in a cortical cut preparation when GABA was placed on the stimulated site and the response transmitted to the other side of the cut was not blocked (Fig. 6). Also, when GABA was placed on the stimulated site of a molecular layer preparation it failed in three experiments to block the N wave /%,.._.,.. A
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DCR transmitted to the other side. When GABA was placed over the line of the cut of a molecular layer cut preparation between the stimulated and responding sites, it was ineffective in blocking transmission of the N wave DCR. Using 1-2/~ micro-electrodes for recording, in 2 complete experiments and in 3 more partial ones, a large negativity (sink) was not found present below the surface corresponding to the surface positivity occurring after GABA application. In laminar recordings m~.~e before GABA applications (Fig. 7, A), the responses below the surface were found to be smaller in amplitude, until at depths below 0.36-0.54 mm they were either very small or absent. There was no evidence of reversal of the N wave DCR at any depth, (see also Suzuki and Ochs 1964). Upon placing GABA on the surface (Fig. 7, t;), the surface responses became inverted and remained so throughout laminar recording. The inverted responses fell off rapidly in amplitude, until below 0.36-0.54 mm they were either small or absent. A small negativity indicative of a sink may be seen in the middle layers. However, no
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Fig. 7 Laminar recording with GABA. A: Micro-electrode (upper trace) and surface electrode recording (lower trace) from intact co~'tex. Micro-electrode recording from successively lower depths in mm (indicated by numbers). B: Same after GABA application and with inverted surface response. Calibrations: 2 mV, 2 msec.
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-3---. Fig. 8 Recovery cycle of DCR types. Column A: N wave DCR evoked by surface stimulation of intact cortex with two shocks at a series of different intervals between them. Column B: Positive-negative sequence evoked by stimulation at depth of 1.8 mm. Column C: N wave DCR evoked by stimulation at depth of 2.2 mm. Calibrations: 2 mV, 10 msec.
E/ectroenceph. clin. Neurophysiol., 1965, 19:230-236
234
S. OCLIS AND H. SUZUKI
large sinks of potential were found below the surface layer. Recovery cycle for DCR types (see Fig. 8) Micro-electrodes were used to excite DCRs at the surface (A), middle cortex (B) and lowest cortex layer (C) and double shocks at different intervals were employed to study the recovery cycle of the superficial N wave DCR. positivenegative DCR and deep N wave DCR elicited at these different depths. The superficial N wave DCR showed a shorter period of depressed response with recovery completed in approximately 20-30 msec. The negative phase of the positive-negative DCR (B) showed a more prolong,.d depression, recovery taking several hundred millisecor.ds. The positive phase was not decreased in amplitude by the interactions, as previously described (Ochs 1962). The N wave DCR evoked from deep layer cortical stimulation also has a prolonged recovery phase of over 100 msec (~f. Suzuki and Taira 1959). In 2 complete experiments the results were essentially the same as those previously reported. In addition, spatio-temporal interaction studied between an N wave DCR transmitted across a molecular cut preparation and an N wave DCR on the same side showed an occlusive-like interaction (Suzuki and Ochs 1964).
layer preparations in layers below the surface where a fair sized N wave DCR remains. A similar simplification was produced in the chronic island preparation (Suzuki and Ochs 1964). The neural elements which transmit the response in the molecular layer could be the horizontal cells of Cajal, the afferent termination of axons in the cortex, the axons of stellate cells which have their cell bodies in lower cortical layers, the collaterals of pyramidal cells, or the apical dendrites themselves. The latter possibility is lessened by the relatively short lateral spread of apical dendrites (Sholl 1956) and the difference in action of GABA when placed either on the stimulated site or on the intervening transmission path or when placed on the responding site. Only in the latter event was there a rapid block, confirming lwama and Jasper (1957). This result supports the concept that the elements transmitting the response differ from those giving rise to the response (see below). The insensitivity of the transmitting elements to the action of GABA suggests their axonal nature (Curtis et al. 1959). That the transmitting elements are composed of afferent axons having a lateral spread was excluded by our previous studies of the chronic island where transmission of the response still remained present after degeneration of afferent
DISCUSSION
Transmission The present experiments suggest that elements in the molecular layer subserve the lateral cortical transmission of the N wave DCR excited by surface stimulation. There appears to be a fairly good number of these neural elements even in the uppermost part of the molecular layer. These experiments are the complement of those reported by Burns and Grafstein (1952) where block of transmission was found following very superficial cuts made down into the surface of the cortex for a distance of a few tenths of a ram. The cut used to make a molecular preparation blocks la'.eral transmission of the positive-negative responses and therefore prevents the complications caused by the addition of the positive wave of the positive-negative DCR to that of the N wave DCR. This is seen on laminar stimulation within the upper half of the cortex in molecular
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Fig. 9 Schematization of neurons possibly involved in transmission and generation of the N wave DCR. To left of molecular cut (C), stellate cells (S) and pyramidal cells (P) are shown with ~.xons passing up and laterally in molecular layer (M). Elements stimulated by electrode (St). Either one or both of these axons terminate on apical dendrites of pyramidal cells in which N wave DCR is generated and recorded by surface electrode (R). Gray matter (G); white matter (W).
Eiectroenceph. clin. Neurophysiol., 1965, 19:230-236
TRANSMISSION OF CORTICAL RESPONSE
terminations (Suzuki and Ochs 1964). Transmission via the horizontal cells of Cajal (Eccles 1957) seems a less likely possibility on the basis of their relative sparsity in the molecular layer and of our finding that laminar stimulation down through the upper half of the cortex in a molecular layer preparation can give rise to an N wave DCR. However, a more complex hypothesis involving excitation of deeper elements with synapses on Cajal cells which then transmit the response laterally cannot be excluded. When stimulating below the surface, response amplitudes were variable from experiment to experiment. In some cases very little response could be obtained whereas stimulation of the surface could give rise to N wave DCRs transmitted to the other side. This suggests that there may be a difference between deeper and surface transmitting elements. Two possible transmitting links are the recurrent collaterals of pyramidal cells and axons of stellate cells as shown in Fig. 9. Many steilate cells have cell bodies in lower layers with their axons passing upward and then laterally in the molecular layer (Sholl 1956). This could account for the lability found on stimulation below the surface if stimulating currents are exciting dendrites or cell bodies of the stellate cells, compared to excitation and transmission in axons passing laterally in the surface layer. The scheme shown in Fig. 9 is grossly simplified, lnterneurons in the deeper layers most likely could participate by transmitting excitation up to the laterally conducting elements. In addition a number of other transmission paths are available. This is shown in preparations where the superficial cortical layers are cut and to a lesser extent with complete cortical cuts when transmission of DCRs remains. These responses usually have smaller ampli',ude, a longer latency and are preceded by a positive phase having fast spikes at its onset. The transmission of responses by elements under the molecular layer involves either an intracortical or a cortico-cortic~! path (Ochs 1956; Ochs and Booker 1961), the latter pathway could account for DCRs transmitted to distances of 15 mm and more (Brooks and Enger 1959). Most likely the increased spatial summation afforded with the use of bipolar electrodes and stronger strengths of stimulation can result in
235
transmission over greater distances. Note should also be taken of the multiplicity of neuronal processes set into motion by deeper cortical stimulation. The prolonged depression of several hundred milliseconds seen in the interaction studies when a positive-negative response or deep N wave DCR is elicited compared to the shorter duration occlusion found with the surface N wave DCR, suggests an entry of additional neuronal processes with a more complex change of excitability. This aspect of the problem requires more extensive study. Electrogenesis o f the N **,ave D C R
Much of the evidence indicates the: the N wave DCR excited by superficial stimulation is a response of the apical dendrites. The response most likely takes place following synaptic excitation by the laterally distributed (axonal) elements in the molecular layer. The nature of the response in the membrane of the apical dendrites cannot be resolved on the basis of the present experiments. The rapid action of GABA to block and invert the N wave DCR can be explained by the superficial position of the apical dendrites. In accord with its primary action of the cortical negative waves, GABA was rapidly effective on the superficially and deep cortically evoked N wave DCR as well as on the negative phase of the positive-negative sequence. When an N wave DCR was inverted with GABA, the laminarly recorded inverted responses were found in the upper layers. The inverted responses decreased in amplitude with greater recording depth much as did the lami. narly recorded N wave DCRs. Whereas Purpura et al. (1959) considered GABA to selectively block EPSP activity, leaving an IPSP as the source of the hyperpolarization, Bindman et al. (1962) have recently given further evidence for a generalized depressive action of GABA on apical dendrites. An increased sink of activity in lower cortical layers might be expected after GABA application on the basis of a generalized action (Jasper et al. 1958). However, only a small negativity indicative of such an ~,ugmented sink was found in lower cortical layers corresponding with the inverted surface response. This can be due to either a more dispersed distribution of a sink in the lower layers or else the sink is so Electroeneeph. clin. NeurophysioL, 1965, 19:230-236
236
S. OCHS AND H. SUZUKI
close to the source near the surface that it could not be readily discriminated by our microelectrode recording. The pyramidal cells giving rise to the response pictured in Fig. 9 could therefore be considered to have their cell body in a layer in the cortex closer to the surface; perhaps in the second or third layer (Suzuki and Ochs 1964). Or, the responses in the upper apical dendrites have a decrementing or electrotonic spread down to the initial segments of cells in lower layers (Ochs 1962).
results were discussed with respect to the distribution of the cells in the cortex giving rise to the N wave DCR. The authors wish to thank Dr. Morio Matsunaga for his assistance in some of these experiments. Thanks are also due to Mrs. Catherine Henderson for making the histological preparations and to Mr. J. Glore and his staff for the illustrations. REFERENCES BINDMAN,L. J., LIPPOLD,O. C. J. and REDFEARN,J. W. T.
SUMMARY
I. Micro-electrodes were used to laminarly excite direct cortical responses (DCRs) in rabbit cortices where various cuts were made between stimulating and responding sites to study the neural transmission link involved. 2. The negative wave type of response (N wave DCR) excited by stimulation of the upper layers is transmitted by neuronal elements present in the molecular layer. This was shown by the use of a molecular layer preparation where the cortical layers below the uppermost layer and, in addition, the cortico-cortical fiber paths below the cortex were cut. Such cuts did riot block transmission of the N wave DCR. Another type of transmission link was shown after making cuts through the upper cortical layers or through the whole of the cortex. The deeper links were more dependent on the excitability of the cortex. The responses transmitted were positive-negative DCRs or negative wave DCRs with a longer latency and preceded by small fast spike wave. 3. Laminar stimulation below the surface on one side of a molecular layer preparation also excited N wave DCRs which were transmitted to the other side of the cut via the molecular layer. Topical application of G A B A was ineffective on the element in the molecular layer transmitting the N wave D C R or in exciting the response while it caused the usual rapid block or inversion of the response in the responding area. 4. Micro-electrode laminar recordings in the area of G A B A inversion showed evidence of only a small sink in layers below the surface. The
The non-selective blocking actior~ of F-aminobutyric acid on the sensory cerebral cortex of the rat. J. PhysioL (Lond.), 1962, 162: 105-120. BROOKS,V. B. and ENG~R,D. S. Spread of directly evoLed responses in the cat's cerebral cortex. J. gen. Physiol., 1959, 42: 761-777. BURNS, B. D. and GRAFSTEIN, B. The function and structure of some neurons in the cat's cerebral cortex. J. Physiol. (Lond.), 1952, 118: 412-433. CURTIS,D. R., PHILLIS,J. W. and WATKINS,J. C. The depression of spinal neurones by 3,-amino-nbutyric acid and/I-alanine. J. Physiol. (Lond.), 1959, 146: 185-203. ECeLES,J. C. The physiology of nerve cells. Johns Hopkins, Baltimore, 1957, 270 p. IWAMA,K. and JASPER,H. H. The action of gamma aminobutyric acid upon cortical electrical activity in ~he cat. J. Physiol. (Lond.), 1957, 138: 365-380. JASPEn,H.,GoNZALEZ,S. and ELLIOT,K. A. C. Action of F-aminobutyric acid (GABA) and strychnine upon evoked responses of cerebral cortex. Fed. Proc., 1958, 17: 79. Octts, S. The direct cortical response. J. Neurophysiol., 1956, 19: 513-523. POllS, S. Analysis of cellular mechanisms of direct cortical responses. Fed. Proc., 1962, 21: 642-647. OCHS,S. and BOOKER,H. Spatial and temporal interaction of direct cortical responses. Exp. NeuroL, 1961, 4: 70-82. PURPURA, D. P., GIRADO, M., SMITit, T. G., CALLAN, D.
A. and GRUNDVEST,H. Structure activity determinants of pharmacological effects of amino acids and related compounds on central synapses. J. Neurochem., 1959, 3: 238-268. SHOLL, D. A. The organization of the cerebral cortex. Wiley, New York, 195t~ 125 p. SUZUKI, H. and OCHS, S. Laminar stimulation for direct cortical responses from intact and chronically isolated cortex. Electroenceph. clin. NeurophysioL, 1964, 17: 405-4 13. SUZUKI, H. andT,,,mA,N, An analysis ofthe direct cortical response. Tohoku J. exp. Med., 1959, 70: 1-10.
Reference: OcHs, S. and SUZUKZ,H. Transmission of direct cortical responses. Electroenceph. clin. Neurophysiol., !_965__~ 19" 230--236.
ELECTROENCEPHALOGRAPHY AND CLINICAL NEUROPHYSIOLOGY
TRANSMISSION
OF DIRECT CORTICAL RESPONSES x SIDNEY OCHS AND HISAO SUZUKI2
Department of Physiology, Indiana University Medical Center, Indianapolis 7, Ind. (U.S.A.) (Accepted for publication: February 17, 1965)
INTRODUCTION
With monopolar laminar stimulation of the cortex by means of a small electrode, characteristically different types of direct cortical responses (DCRs) were obtained at different depths: a negative (N) wave DCR from the surface layers; a positive-negative sequence D C R from the middle layers; an N wave D C R from the lowest layer (Suzuki and Ochs 1964). In chronic cortical islands, the N wave D C R was obtained unimpaired on surface layer stimulation while all underlying cortico-cortical axons were apparently degenerated. This indicated that an intracortical path between stimulating and recording sites was involved. The present study was done to determine the location of the neural elements subserving lateral transmission of the N wave DCR using micro-electrodes for laminar stimulation after making various types of cortical cuts between stimulating and responding sites. Gamma amino butyric acid (GABA) was used to determine the differences between the elements transmitting and those giving rise to the N wave DCR. A preliminary account of such experiments was previously presented (Ochs 1962).
lying white matter. Complete pailial cuts included section of the underlying white matter. Molecular layer preparations had occasionally all but the uppermost layer of the cortex cut but in most cases parts of the second layer were also left uncut. After completing the required cut and allowing 30-60 min for recovery from the effects of the cut, the recording electrodes were placed on the surface at 1-2 mm away from the cut, one being usually placed on the same side of the cut as the stimulating electrode, the other on the opposite side. By means of a micro-manipulator, the electrode used for laminar stimulation could be inserted at 0.2 n,m increments into the depths
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A total of 22 adult rabbits were studied. The techniques used for surgical preparation, microelectrode laminar stimulation and recording have been recently described (Suzuki and Ochs 1964). Cortical cuts were made in the visualsomesthetic area using knives described in an earlier study (Ochs 1956). Complete cortical cuts were made from just below the pial membrane extending down to, but not including the underI This study is supported by U.S. Public Health Grant Nos. NB 61993 and HM 04815. 2 Present address: Institute of Brain Diseases, Tohoku University, ~ndai, Japan.
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Fig. I Molecular layer preparation. A and B are examples of responses to 10minarstimulation with depth in mm shown by numbers m t:'~:h pair of tracings: upper one is from same side of cut as of stimulating electrode; lower one from across line of molecular layer preparation. In this and following figures, a negative deflection is in the upward direction. Calibrations: 2 mV, 2 msec.
Electroenceph. olin. NeuroplLvsioi., 1965, 19:230-236
231
TRANSMISSION OF CORTICAL RESPONSE
of the cortex. As in previous experiments, stimulation was monopolar with cathodal pulses 0.1--0.2 msec in duration at strengths of 100-200 /~A. Laminar recordings were accomplished with glass capillary or insulated metal micro-electrodes having tips of 1-2 ,u. To study the effects of GABA on either stimulated or responding sites, a drop of 1% solution in Ringer fluid was applied to the cortex via a fine glass capillary or to a narrow strip of filter paper placed between stimulated and responding sites over the line of the molecular layer cut. When the experiment was completed, a lethal dose of pentobarbital was injected intravenously, the brains removed and placed in 10% formalin. To determine the extent of the cuts, serial sections of the cortex were prepared with Nissl and silver stains.
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RESULTS
Molecular layer preparation and transmission In 5 different animals the N wave DCR was transmitted across the line of the cut where it appeared with similar form and amplitude (Fig. !). The responses had the same latency and with similar stimulus strengths as in the intact cortex, they were transmitted laterally to the same distance (approximately 5 mm). The fact that in some of these preparations only the upper part of the molecular layer remained uncut, suggests that not all of the layer is required for transmission. The micro-electrode was passed down to successively greater depths on one side of a molecular cut for laminar stimulation. When stimulating below the surface, negative wave DCRs of progressively smaller amplitude were obtained on the other side of the cut. On the stimulated side of the molecular cut, a positivenegative type of DCR, as previously found in the intact cortex ($-Jzuki and Ochs 1964), was induced by stimulation at the depth of 0.4-1.8 mm. However, these positive-negative responses were variable in size (probably reflecting alterations in cortical excitability which resulted from the cuts) particularly in their negative phase (cf. Fig. 1, A and B). Transmission of the N wave DCR elicited by deeper cortical stimulation also appeared to depend on the condition of the cortex. When the excitability was reduced, as
Fig. 2
Upper cortical cut and transmission.Transection through the upper three layers of the cortex. See Fig. 1 for explanation. Calibrations: 2 mV, 2 msec. suggested by the reduced negative phase of the positive-negative DCR obtained on the same side of the cut (see FIE'. 3, A), stimulation below the surface did not give rise to a transmitted N wave DCR. This reduced excitability did not seem to be due to a spreading depression since when the stimulating electrode was returned to the surface, the N wave DCR was present.
Cortical cuts and laminar stimulation With the relatively discrete type of stimulation pro~,ided by the monopolar micro-electrode, the question arises as to whether there is transmission of responses when the upper layers have been cut through partially or completely. This was studied in 6 animals. In the example of Fig. 2, the upper three cortical layers were cut thr'~ugh. Then laminar stimulation on one side was performed as before. Stimulation at the surface gave a smaller response across the cut. A large positive-negative sequence was transmitted beyond the line of the ,:ut when stimulating at 1-2 mm in depth. On stimulation of the deeper layers there was a positive component mixed with the N wave DCR. In this example a relatively Electroenceph. clin. NeurophysioL, 1965, 19:230--236
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Pallial cut and spread o f stimulus current When complete cuts were made in 2 animals through both the cortex and underlying white matter, transmission was completely blocked. This type of cut allowed a test to be made, in these two experiments, of the extent of the lateral spread of stimulating current; a spread which in our experimental conditions appeared to be remarkably small. Fig. 4 shows a response with the stimulating electrode 0.1 mm on the same side of the cut, a small response with the stimulating electrode placed over the line of the cut and no response when the electrode was placed just over the line of the cut on the opposite side. A
Fig. 3
B
C
Complete cortical cuts and transmission. Complete cortical cut down to white matter (A) and sparing the lowest layer (B). Responses from the electrode on same side of cut are shown respectively on lower (A) and upper (B) traces of each pair. Depths of stimulation in mm given by numbers. Calibrations: 2 mV, 2 msec. large stimulus artifact obscures the positive component. When all layers down to but not including the underlying white rr'atter were cut through as shown in the example of Fig. 3, A, transmission was much reduced. Here there was also evidence of reduced excitability as shown by the smaller DCRs ( btained on the same side of the cut. In B a smaller, mainly negative wave, preceded by one or more small spike-like fast waves, is recorded
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Fig. 4 Pailial cut. Linlited spread of stimulating current. Position of stimulating electrode on the same side of the cut (+0.1 ram), on the line of cut (0) and on the opposite side of the cut ( - 0. I ram). The downward displacement is an artifact. Calibrations: 2 mV, 2 msee.
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Fig. 5
Effec~. of GABA on DCR types. A: an N wave DCR is sho'L,n from intact cortex (top record); shortly after 1% GABA was topically applied and response hlo,'ked (middle record); recovery 10-20 min after washing surfitce (lower record). B: positive-negative seqt:~ence(upper record); shortly after GABA application (middle record); after recovery (bottom record). C: N wave DCR evoked by stimuli 2.2 mm deep (upper record); shortly after GABA (middle record); after recovery (bottom record). Calibrations: 2 msec, 2 mV. Effect o f GA BA on DCRs GABA acts rapidly on the responding site giving rise to surface negative responses. Th,is was shown in one experiment (Fig. 5) for all th,~: types of DCP~ responses: the N wave D C R obtained from the surface (A); the positive-negative sequence obtained from the middle layers (B); the negative wave D C R obtained by the lowest layer stimulation (C). The results were essentially similar to those earlier studied (lwama and Jasper 1957; Purpura et ai. 1959). The "inverted" phase seen after GABA was largest when the response was originally a positive-negative D C R (B). Eiectreenceph. c/in. Neuroph.vsiol , 1965, 19:230-236
233
TRANSMISSION OF CORTICAL RESPONSE A
B
C
D
Fig. 6 GABA on stimulated and on responding site. Continuation of experiment of Fig. 2. A: Before GABA. B: Shortly after GABA applied to both recording position (same side) and stimulatea site but not to opposite side. C: Control after recovery from effect of GABA. D: Shortly after GABA placed on both recording sites and on stimulated site. Calibrations: 2 mV, 2 msec.
Whereas GABA is rapidly effective on the elements giving rise to the response, it was ineffective on the stimulated element or on those conducting the response. This is shown in a cortical cut preparation when GABA was placed on the stimulated site and the response transmitted to the other side of the cut was not blocked (Fig. 6). Also, when GABA was placed on the stimulated site of a molecular layer preparation it failed in three experiments to block the N wave /%,.._.,.. A
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DCR transmitted to the other side. When GABA was placed over the line of the cut of a molecular layer cut preparation between the stimulated and responding sites, it was ineffective in blocking transmission of the N wave DCR. Using 1-2/~ micro-electrodes for recording, in 2 complete experiments and in 3 more partial ones, a large negativity (sink) was not found present below the surface corresponding to the surface positivity occurring after GABA application. In laminar recordings m~.~e before GABA applications (Fig. 7, A), the responses below the surface were found to be smaller in amplitude, until at depths below 0.36-0.54 mm they were either very small or absent. There was no evidence of reversal of the N wave DCR at any depth, (see also Suzuki and Ochs 1964). Upon placing GABA on the surface (Fig. 7, t;), the surface responses became inverted and remained so throughout laminar recording. The inverted responses fell off rapidly in amplitude, until below 0.36-0.54 mm they were either small or absent. A small negativity indicative of a sink may be seen in the middle layers. However, no
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Fig. 7 Laminar recording with GABA. A: Micro-electrode (upper trace) and surface electrode recording (lower trace) from intact co~'tex. Micro-electrode recording from successively lower depths in mm (indicated by numbers). B: Same after GABA application and with inverted surface response. Calibrations: 2 mV, 2 msec.
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-3---. Fig. 8 Recovery cycle of DCR types. Column A: N wave DCR evoked by surface stimulation of intact cortex with two shocks at a series of different intervals between them. Column B: Positive-negative sequence evoked by stimulation at depth of 1.8 mm. Column C: N wave DCR evoked by stimulation at depth of 2.2 mm. Calibrations: 2 mV, 10 msec.
E/ectroenceph. clin. Neurophysiol., 1965, 19:230-236
234
S. OCLIS AND H. SUZUKI
large sinks of potential were found below the surface layer. Recovery cycle for DCR types (see Fig. 8) Micro-electrodes were used to excite DCRs at the surface (A), middle cortex (B) and lowest cortex layer (C) and double shocks at different intervals were employed to study the recovery cycle of the superficial N wave DCR. positivenegative DCR and deep N wave DCR elicited at these different depths. The superficial N wave DCR showed a shorter period of depressed response with recovery completed in approximately 20-30 msec. The negative phase of the positive-negative DCR (B) showed a more prolong,.d depression, recovery taking several hundred millisecor.ds. The positive phase was not decreased in amplitude by the interactions, as previously described (Ochs 1962). The N wave DCR evoked from deep layer cortical stimulation also has a prolonged recovery phase of over 100 msec (~f. Suzuki and Taira 1959). In 2 complete experiments the results were essentially the same as those previously reported. In addition, spatio-temporal interaction studied between an N wave DCR transmitted across a molecular cut preparation and an N wave DCR on the same side showed an occlusive-like interaction (Suzuki and Ochs 1964).
layer preparations in layers below the surface where a fair sized N wave DCR remains. A similar simplification was produced in the chronic island preparation (Suzuki and Ochs 1964). The neural elements which transmit the response in the molecular layer could be the horizontal cells of Cajal, the afferent termination of axons in the cortex, the axons of stellate cells which have their cell bodies in lower cortical layers, the collaterals of pyramidal cells, or the apical dendrites themselves. The latter possibility is lessened by the relatively short lateral spread of apical dendrites (Sholl 1956) and the difference in action of GABA when placed either on the stimulated site or on the intervening transmission path or when placed on the responding site. Only in the latter event was there a rapid block, confirming lwama and Jasper (1957). This result supports the concept that the elements transmitting the response differ from those giving rise to the response (see below). The insensitivity of the transmitting elements to the action of GABA suggests their axonal nature (Curtis et al. 1959). That the transmitting elements are composed of afferent axons having a lateral spread was excluded by our previous studies of the chronic island where transmission of the response still remained present after degeneration of afferent
DISCUSSION
Transmission The present experiments suggest that elements in the molecular layer subserve the lateral cortical transmission of the N wave DCR excited by surface stimulation. There appears to be a fairly good number of these neural elements even in the uppermost part of the molecular layer. These experiments are the complement of those reported by Burns and Grafstein (1952) where block of transmission was found following very superficial cuts made down into the surface of the cortex for a distance of a few tenths of a ram. The cut used to make a molecular preparation blocks la'.eral transmission of the positive-negative responses and therefore prevents the complications caused by the addition of the positive wave of the positive-negative DCR to that of the N wave DCR. This is seen on laminar stimulation within the upper half of the cortex in molecular
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Fig. 9 Schematization of neurons possibly involved in transmission and generation of the N wave DCR. To left of molecular cut (C), stellate cells (S) and pyramidal cells (P) are shown with ~.xons passing up and laterally in molecular layer (M). Elements stimulated by electrode (St). Either one or both of these axons terminate on apical dendrites of pyramidal cells in which N wave DCR is generated and recorded by surface electrode (R). Gray matter (G); white matter (W).
Eiectroenceph. clin. Neurophysiol., 1965, 19:230-236
TRANSMISSION OF CORTICAL RESPONSE
terminations (Suzuki and Ochs 1964). Transmission via the horizontal cells of Cajal (Eccles 1957) seems a less likely possibility on the basis of their relative sparsity in the molecular layer and of our finding that laminar stimulation down through the upper half of the cortex in a molecular layer preparation can give rise to an N wave DCR. However, a more complex hypothesis involving excitation of deeper elements with synapses on Cajal cells which then transmit the response laterally cannot be excluded. When stimulating below the surface, response amplitudes were variable from experiment to experiment. In some cases very little response could be obtained whereas stimulation of the surface could give rise to N wave DCRs transmitted to the other side. This suggests that there may be a difference between deeper and surface transmitting elements. Two possible transmitting links are the recurrent collaterals of pyramidal cells and axons of stellate cells as shown in Fig. 9. Many steilate cells have cell bodies in lower layers with their axons passing upward and then laterally in the molecular layer (Sholl 1956). This could account for the lability found on stimulation below the surface if stimulating currents are exciting dendrites or cell bodies of the stellate cells, compared to excitation and transmission in axons passing laterally in the surface layer. The scheme shown in Fig. 9 is grossly simplified, lnterneurons in the deeper layers most likely could participate by transmitting excitation up to the laterally conducting elements. In addition a number of other transmission paths are available. This is shown in preparations where the superficial cortical layers are cut and to a lesser extent with complete cortical cuts when transmission of DCRs remains. These responses usually have smaller ampli',ude, a longer latency and are preceded by a positive phase having fast spikes at its onset. The transmission of responses by elements under the molecular layer involves either an intracortical or a cortico-cortic~! path (Ochs 1956; Ochs and Booker 1961), the latter pathway could account for DCRs transmitted to distances of 15 mm and more (Brooks and Enger 1959). Most likely the increased spatial summation afforded with the use of bipolar electrodes and stronger strengths of stimulation can result in
235
transmission over greater distances. Note should also be taken of the multiplicity of neuronal processes set into motion by deeper cortical stimulation. The prolonged depression of several hundred milliseconds seen in the interaction studies when a positive-negative response or deep N wave DCR is elicited compared to the shorter duration occlusion found with the surface N wave DCR, suggests an entry of additional neuronal processes with a more complex change of excitability. This aspect of the problem requires more extensive study. Electrogenesis o f the N **,ave D C R
Much of the evidence indicates the: the N wave DCR excited by superficial stimulation is a response of the apical dendrites. The response most likely takes place following synaptic excitation by the laterally distributed (axonal) elements in the molecular layer. The nature of the response in the membrane of the apical dendrites cannot be resolved on the basis of the present experiments. The rapid action of GABA to block and invert the N wave DCR can be explained by the superficial position of the apical dendrites. In accord with its primary action of the cortical negative waves, GABA was rapidly effective on the superficially and deep cortically evoked N wave DCR as well as on the negative phase of the positive-negative sequence. When an N wave DCR was inverted with GABA, the laminarly recorded inverted responses were found in the upper layers. The inverted responses decreased in amplitude with greater recording depth much as did the lami. narly recorded N wave DCRs. Whereas Purpura et al. (1959) considered GABA to selectively block EPSP activity, leaving an IPSP as the source of the hyperpolarization, Bindman et al. (1962) have recently given further evidence for a generalized depressive action of GABA on apical dendrites. An increased sink of activity in lower cortical layers might be expected after GABA application on the basis of a generalized action (Jasper et al. 1958). However, only a small negativity indicative of such an ~,ugmented sink was found in lower cortical layers corresponding with the inverted surface response. This can be due to either a more dispersed distribution of a sink in the lower layers or else the sink is so Electroeneeph. clin. NeurophysioL, 1965, 19:230-236
236
S. OCHS AND H. SUZUKI
close to the source near the surface that it could not be readily discriminated by our microelectrode recording. The pyramidal cells giving rise to the response pictured in Fig. 9 could therefore be considered to have their cell body in a layer in the cortex closer to the surface; perhaps in the second or third layer (Suzuki and Ochs 1964). Or, the responses in the upper apical dendrites have a decrementing or electrotonic spread down to the initial segments of cells in lower layers (Ochs 1962).
results were discussed with respect to the distribution of the cells in the cortex giving rise to the N wave DCR. The authors wish to thank Dr. Morio Matsunaga for his assistance in some of these experiments. Thanks are also due to Mrs. Catherine Henderson for making the histological preparations and to Mr. J. Glore and his staff for the illustrations. REFERENCES BINDMAN,L. J., LIPPOLD,O. C. J. and REDFEARN,J. W. T.
SUMMARY
I. Micro-electrodes were used to laminarly excite direct cortical responses (DCRs) in rabbit cortices where various cuts were made between stimulating and responding sites to study the neural transmission link involved. 2. The negative wave type of response (N wave DCR) excited by stimulation of the upper layers is transmitted by neuronal elements present in the molecular layer. This was shown by the use of a molecular layer preparation where the cortical layers below the uppermost layer and, in addition, the cortico-cortical fiber paths below the cortex were cut. Such cuts did riot block transmission of the N wave DCR. Another type of transmission link was shown after making cuts through the upper cortical layers or through the whole of the cortex. The deeper links were more dependent on the excitability of the cortex. The responses transmitted were positive-negative DCRs or negative wave DCRs with a longer latency and preceded by small fast spike wave. 3. Laminar stimulation below the surface on one side of a molecular layer preparation also excited N wave DCRs which were transmitted to the other side of the cut via the molecular layer. Topical application of G A B A was ineffective on the element in the molecular layer transmitting the N wave D C R or in exciting the response while it caused the usual rapid block or inversion of the response in the responding area. 4. Micro-electrode laminar recordings in the area of G A B A inversion showed evidence of only a small sink in layers below the surface. The
The non-selective blocking actior~ of F-aminobutyric acid on the sensory cerebral cortex of the rat. J. PhysioL (Lond.), 1962, 162: 105-120. BROOKS,V. B. and ENG~R,D. S. Spread of directly evoLed responses in the cat's cerebral cortex. J. gen. Physiol., 1959, 42: 761-777. BURNS, B. D. and GRAFSTEIN, B. The function and structure of some neurons in the cat's cerebral cortex. J. Physiol. (Lond.), 1952, 118: 412-433. CURTIS,D. R., PHILLIS,J. W. and WATKINS,J. C. The depression of spinal neurones by 3,-amino-nbutyric acid and/I-alanine. J. Physiol. (Lond.), 1959, 146: 185-203. ECeLES,J. C. The physiology of nerve cells. Johns Hopkins, Baltimore, 1957, 270 p. IWAMA,K. and JASPER,H. H. The action of gamma aminobutyric acid upon cortical electrical activity in ~he cat. J. Physiol. (Lond.), 1957, 138: 365-380. JASPEn,H.,GoNZALEZ,S. and ELLIOT,K. A. C. Action of F-aminobutyric acid (GABA) and strychnine upon evoked responses of cerebral cortex. Fed. Proc., 1958, 17: 79. Octts, S. The direct cortical response. J. Neurophysiol., 1956, 19: 513-523. POllS, S. Analysis of cellular mechanisms of direct cortical responses. Fed. Proc., 1962, 21: 642-647. OCHS,S. and BOOKER,H. Spatial and temporal interaction of direct cortical responses. Exp. NeuroL, 1961, 4: 70-82. PURPURA, D. P., GIRADO, M., SMITit, T. G., CALLAN, D.
A. and GRUNDVEST,H. Structure activity determinants of pharmacological effects of amino acids and related compounds on central synapses. J. Neurochem., 1959, 3: 238-268. SHOLL, D. A. The organization of the cerebral cortex. Wiley, New York, 195t~ 125 p. SUZUKI, H. and OCHS, S. Laminar stimulation for direct cortical responses from intact and chronically isolated cortex. Electroenceph. clin. NeurophysioL, 1964, 17: 405-4 13. SUZUKI, H. andT,,,mA,N, An analysis ofthe direct cortical response. Tohoku J. exp. Med., 1959, 70: 1-10.
Reference: OcHs, S. and SUZUKZ,H. Transmission of direct cortical responses. Electroenceph. clin. Neurophysiol., !_965__~ 19" 230--236.