Electrophysiological studies of hippocampal neurons III. Responses of hippocampal neurons to repetitive perforant path volleys

ELECTROENCEPHALOGRAPHYAND CLINICAL NEUROPHYSIOI.(~Y

ELECTROPHYSIOLOGICAL HIPPOCAMPAL

353

STUDIES OF

NEURONS

III. RESPONSES OF H I P P O C A M P A L NEURONS TO REPETITIVE PERFORANT PATH VOLLEYS ..... P. Gt.o,3R, C. L. VERAx AND L. St~r~Tts Department of Neurology and Neurosurgery, McOili UniversPyand Montreal Neurological Institute, Montreal (Canada) (Accepted for publication: November 19, 1963)

The aim of the present paper is to describe the effects of repetitive afferent volleys mediated by the perforant path on the response characteristics of the hippocampus. MATERIAL AND METHOD

The AC- and DC-components of the hippocampal responses to repetitive stimulation were studied with single and multiple extraceUular micro-electrodes in 93 ligtttly anaesthetized cats. These were the same animals in which the observations reported in the two preceding articles of this series (Gloor et ai. 1963a and b) were made. in addition to those described in these two papers the following techniques were used: (I) In order to eliminate fast components from some of the DC records the output of the DC amplifier in some of the experiments was fed into a low pass filter reducing the output signal by 6 db at 70 c/see. This filter introduced no spurious DC changes. An unfiltered AC record, usually with a time constant of 0.2 sac, was always recorded simultaneously with the DC record. (2) In most experiments the frequency of repetitive stimulation applied to either the amygdais or the entorhinal cortex ranged between 1012/sec; in a few experiments, to investigate changes induced by low and rapid frequencies, the range extended from 0.1 to 100/see. First the t Parke and Davis Fellow. Presem address: Department of Neurosurgery, Catholic University, Santiago, Chile. s Bronfman Fellow in Neurophysiolosy. Present address: Istituto di Fisiologia Umana, Universit~t di Bologna, Bologna, Italy.

response to single shocks was studied. Subsequently repetitive stimulation was applied for a variable period of time and, immediately upon its cessation "single shock" stimulation, usually at l/sec, was resumed, in order to observe posttetanic changes. RESULTS

Amplitude changes induced by repetitive stimulation Repetive stimulation of perforant path afterants to the hippocampus led either to augmentation or diminution in the amplitude of the hippo. campal and dentate gyrus responses (Fig. 1). In general augmentation predominated at the beginning of repetitive stimulation and was followed by diminution especially with long trains of repetitive stimulation. Initial augmentation was three times as frequent as initial diminution with amygdaloid stimulation, whereas with entorhinal stimulation its predominance was only slight. In the previous papers of this series the hippo. campal responses to perforant path volleys had been subdivided into five components, depending upon their latency, form, duration and laminar distribution (see Fig. 3, A ofGloor et al. 1963a). These five components were found to vary independently in response to repetkive stimulation. The principal transient response, comported*. a To those of "recruitment" and "obliteration" employed earlier (Gloor 1955), we now prefer these noncommital terms, since "recruitment" implies an increase of the number of neurons responding to the incoming volleys, but this is by no means the only mechanism whereby the evoked response could incre~--~sein amplitude.

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FIg. I Characteristic changes induced in the hippocampus by repetitive perforant path volleys. A: Augmentation and post-tetaai¢ potentiation in the apical dendritic layer of the hippocampus; 12! .*ecamygdaioid stimulation (AC record, time constant 0. ! 5). From left to right: Single shook response; beginning and after 15 sac or repetitive stimulation; first post.t©tanio single shock response. O'he positive deflection following component ! of the pre-tetani¢ single shook response and of the first response at the start of repetitive stimulation is component V.) B: Records i'rom pyramidal layer of the hippocampus; 12/sec amy~laloid stimulation, All are records from the same micro-electrode: DC: AC with high pass filter and AC (T.C. 0,02). White line on top: DC reference level. FromJgit. to right same as in A. Note positive DC shift developing with repetitive stimulation (ph~lse~). C: Hippooampal responses to 12/seeentorhinal stimulation, Upper record: DC, ventricular surface: lower record: DC, apical dendritic layer (850 t~ deep). Diminution of the amplitude of component I with repetitive stimulation and disappearance of component II, Initial negative DC shift (phase A) in upper record only. D: Hippocampal responses to 20/s~ entorhinal stimulation. Both records from the same microo¢l~trode: DC with low pass filter above; AC (T.C. 0.2) below, Single shook response and onset of repetitive stimulation (see text), E: Hippoomnpal responses to 15/s~ entorhinal stimulation: upper records: DC from ventricular surface and apical dendritic layer (approximate depth: 60010; lower: AC with high pass filter from the same micro-electrode as in second beam. (Arrows on the right indicate initial DC level before start of stimulation) (see text). Calibration: amplitude, I mV; time, 50 m s ~ in A - E.

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ELECTROPHYSlOLOGY OF HIPPOCAMPUS. 111

I, showed optimal augmentation in response to pcrforant path volleys at stimulation frequencies of 10-30]sec (Fig. 2, A). Very exceptionally some augmentation was seen at frequencies below 10/see and no change or diminution at lO]sec as ~ n in Fig. 2, B. Augmentation developed more rapidly with higher than with slower frequencies. Abnve 50/see the evoked response would not follow regularly the repetitive stimuli rate and at 100/see it was absent. Augmentation seemed to depend upon intervals between successive stimuli rather than upon the number of stimuli. Thus when repetitive stimulation was applied at a rate at which each successive stimulus fell within the optimal range for augmentation, each stimulus added about the same increment to the response. In the example shown in Fig. 3, the intervals of 67-100 msec (lO15/see stimulation) were optimal and the curves for 10, 12 and 15/see rose together when augmentation was plotted as a function ofthe number of stimuli, although when plotted as a function of time, the 15/see curve showed a small lead (Fig. 3). Below the critical range of intervals there was no augmentation, or the same would be delayed (e.g., for 7 c/see, implying that after some delay the critical interval lengthened to about

140 msec). Especially with long trains or relatively high frequencies of repetitive stimulation (Fig. I, D; 2, A and C; 6, A and B) secondary diminution foUowed upon the initial augmentation. This was sometimes followed by a second period of augmentation (Fig. 2, C). The mechanisms re° sponsible for augmentation and diminution are probably coexistent and competitive. Fig. 3 shows that the maximum augmentation with 15/see stimulation was less than with 10/sec and even less than the delayed augmentation with 7/see stimulation. This would suggest that the mechanisms responsible for diminution began to act before augmentation had a chance to fully develop. Diminution of amplitude could occur as the exclusive or initial response to repetitive stimulation (Fig. !, C and 2, B; 10/see). This was seen in a third of the experiments done with amygdaloid stimulation and in slightly less than half the experiments done with entorhinal stimulation. The factors which determined the direction of the

355

change remained elusive. Augmentation or diminution of amplitude were also observed ibr components II, III and IV of the response elicited with 10-15/see stimulations. In the case of component II, augmentation (Fig. 5, B and C) was slightly more frequent than diminution (Fig. 6, A), whereas for III the reverse was the case and for IV augmentation was exceptional. Component V always disappeared shortly following the beginning of repetitive stimulation (Fig. I, A). The type of amplitude change of component I was unrelated to that of the other components. Fig. 1, D, e.g., shows an example in which with 20/see stimulation, component I, after a brief transient diminution of amplitude, augmented whereas component 1V steadily diminished as repetitive stimulation progressed, (see also Fig. 2, B, 3/see). An example of a particular case of amplitude diminution is shown in Fig. 5, A where upon repetitive stimulation the negative response (cornpone)it I.~ in the apical dendritic layer diminished in amplitude, became iso-potential and then positive, assuming the form which characterizes the evoked response of the pyramidal cell layer and the stratum oriens. These changes were recorded in four animals ant] only at the junction between the apical dendritic a,d pyramidal layers, probably because the isoelectric zone moved past the recording electrode position in the dendritic layer, in the course of this shift the evoked potential representing a stationary dipole must perforce diminish, reach zero and then augment again with a reversed electrical sign. Within the hilus of the dentate gyrus, due to the overlapping and competition of the two tields of opposite orientation, created by the dipole layers of the hippocampal pyramidal neurons and the granule cell neurons of the dentate gyrus, amplitude and polarity changes were of~'en complex; furthermore, these two types of neurons responded with different and variable amplitudes to repetitive stimulation. Thus, in the example shown in Fig. 4, component i, in the hilus of the dentate gyrus, elicited by single shocks applied to the amygdala was positive (A). During repetitive stimulation (B-H), the response polarity shifted from positive to negative and back to positive with transient phases of isoelectricity, augmentation and diminution. ~ Electroenceph. clin. NeurophysioL. 1964, 17:353-370

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Fig. 3 Augmentation ofthe response in the apical dendritic layer to various frequencies of repetitive amygdaioid stimulation, A and B plot the same data; in A the changes in amplitude (ordinate) are plotted as a function of number of stimuli (abscissa); in,B they are plotted as a function of time. (Curve for ! 2/s¢c mentioned in text. not reproduced in figure) (see texO.

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These terms are used here to indicate the excitation o f a previously silent neuron and, respectively, for its opposite, the siler~cing of a previously active neuron, i.e., the eiicitation or abolition of an all-or-none type of propagated action potential. Recruitment, both in the pyramidal layer and the granule cell layer was more frequently encountered than arrest o f unit firing and was usually associated with an augmentation of component I, o f component II or o f both (Fig. 5, B, C, D and 6). Recruitme,t could, however, take z This sequence is thought to result from the competition between the positive field of the granule cells and the negative field of the more distant ~icai 0,.~ndrites of the hippocampus proper. In the course of repetitive stimulation the electrical sign shifted, depending upon whether the algebraic sum of the two fields was positive or negative at the recording site. The fluctuation of the amplitude of component I and of the DC level shown in Fig. 5, D can be explained in an analogous way.

Fig. 2 Changes in the hippocampal response induced by repetitive amy~laioid stimulation. (Graphs A, B and C obtained from different animals), A ' Augmentation (I) and post.tetanio potentiation (2) of component I recorded in the apical dendritic layer or the dentate gyrus with repetitive amygdalold stimulation at various frequenci0s applied for I0, 20 and 30 sac, Vertical bars on the left indicate range of amplitude of pre.tetanic single shock response. Curves take off`at the average point. B: Changes induced in component 1 (solid line) and component IV (dotted line) of the apical dendritic response of the hippocampus elicited by l/see, 3/see, and 10/seeamygdalold stimulation. C: Curves of terminal augmentation and maximum post-tetanic potentiation of component I of the hippocampal response, recorded in the pyramidal layer with various durations of 10/see an~'gdaloid stimulation. The vertical bar (R) indicates the range of amplitudes of the pre.tetanic single shock r~ponses. The thick dashed-and-dotted line connects the amplitude values measured at the end of successive periods of repetitivestimulation of increMing duration (indicated in seconds on the abscissa),The thin lined curves indicate the amplitude changes of post.tetanic single shock responses elicitedat a rate of I/see following ~uation of a number of trainsof repetitivestimulation. Numbers at the ends of these curves indicatethe duration in sac of the preceding period of repetitivestimulation used to elicitthe post-tetanicchanges. The thick dotted curve interconnectsthe maximal values of the potentiated responses, Numbers along this curve indicate the duration of the preceding period of repetitivestimulation used to induce the post-tetanicchanges, (These points are sometimes shifted to the right with relation to the corresponding numbers on the abscissabecause maximum potentiation is sometimes delayed; e,g,, curve for 20 sec). Thin vertical dotted lines interconnect the terminal amplitude values measured at the end of repetitive stimulation and that of the firstpost=tetanic response, Since the latterwas elicitedwithin ! sac after the end of repetitivestimulation the two were lined up along a verticalline,as ifthey had been simultaneous,

Electroenceph. clin. Neurophysiol.. 1964, 17:353-370

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p. OLOOR et al.

[:lace in spite of a diminution or even disappearance ofcomponent II (Fig. 6, A). A similar recruiting process was also often observed in the apical dendritic layer of the hippocampus. In such cases (Fig. 7), in the course of repetitive stimulation, the "dendritic" spike arose as a new element on top of the primary apical dendritic post-synaptic response (component !). Very infrequently arrest of firing followed upon initial recruitment; it was not necessarily associated with a diminution of either component I or !!. Such arrest during repetitive stimulation could bc observed in units which were driven (Fig. 8, A) or in "spontaneously" active, but not driven pyramidal and CA4 units (Fig. 8,

B). Our definition of recruitment would also apply to the appearance during repetitive stimulation, of a previously absent response component (e.g., I!, I!1, IV, V), if this can be attributed to the elaboration of an efferent discharge by a previously silent hippocampal interneuron. This recruitment was very commonly observed for component !1 (Fig. 5, B, C and D), seldom for component 111 and IV and never for component V. Conversely, disappearance of these tempo. hunts in the course of repetitive stimulation could be taken as evidence suggesting at rest of

firing. This invariably occucred with component V (Fig. 1, A), was very common for component IV(Fig. 2, B; 5; 7, A, 10, E-G), but less so for component Ill (Fig. 5, D) and II (Fig. 1, C). Post-tetanic potentiation Repetitive stimulation of the amygdala or entorhinal cortex also produced changes in the excitability of hippocampal neurons which outlasted the end of repetitive stimulation in the form of post-tetanic (or post-activation) potentiation (Fig. 1, A and B; 2, Aeand C; 4; 5, Band C; 6, A and 7). This potentiation was assumed to be present each time single shock responses re. corded after the end of a train of repetitive stimulation were of higher amplitude than the largest pre-tetanic single shock response or if a recruited response persisted after the end of repetitive stimulation. Post-tetanic potentiation was at its peak immediately or within a few seconds after termination of repetitive stimulation, whereupon the amplitude gradually fell to pre-tetanic levels within about 10 sec (Fig. 2, A2 and C). The delay of full development of post-tetanic potentiation was seen most often after long periods of repetitive stimulation (Fig. 2, C) or with stimulation at high frequencies. All components of the evoked hippoc~t,npal

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Fig. $ ,4: Polarity r e v e r e ~ hippocampal response during ! S/s~ entorhinal stimulation probably due to shirt of isoclcctric zone in junctional area between pyramidal and apical dendritic layer. Upper record: dentate ~rus (hilus); lower record: stratum radiatum near the junction between apical dendritic and pyramidal layer of the hippocampus. DC recording. B: Recruitment of unit discharge in the pyramidal layer of' the hippocampus with 12/see amygdaloid stimulation, In each section, the three records are from the same micro.electrude: DC; AC (T.C. 0.15); AC record with high pass filter, from top to bottom. (I) Spontaneous activity and two single shock responses. (2) First inset: oeginning of repetitive stimulation. No unit discharge; second inset: 4.5 sec later; inconstant recruitment of unit discharge with component Ii. (3) First inset: 10 sec later; unit now firing steadily with component Ill second inset: 7 sec later; end of repetitive sti,nulation with first potentiated response (unit fires). (4) 4th, 5th and 9th post-tetanic response, unit ceases to fire, but there is still residual potentiation. Positive DC shift (,phaseB) with augmentation of positive component I. C: Recruitment of hiplx~tmpal pyramidul unit with 12/secamygdaloid stimulation. Both records from the same microelectrode: upper: AC (T.C. 0.15); lower: AC with high passfilter. (1) single shock response,small positive component I, no unit spike; (2) 3 sec alter beginning of repetitive stimulation; augmentation of component 1; component !! (negalive) begins to appear; (3) 11 sec later during repetitive stimulation; recruitment of unit spike discharge on top of (augmented) component ! I; (4) 2nd post.tctanic single shock response; potentiation of component i ! and unit discharge. D: Responses recorded in the granule cell layer of the dentate 8yrus with single shock and ! 3/secemorhinal stimulation. Three records from the same micro-electrode: DC with low pass filter; AC (T.C. 0.2); AC with high pass filter from top to bottom. (I) Single shock response. (2) 12 sec after start of repetitive stimulation; component I! befins to develop on downgoing limb of component I in spite of the diminution of amplitude of the iatter. (.3) 3 se¢ later, component I 1 augments and begins to be assoc'.ated with recruitment of granule cell discharge (3rd beam). (4) 2 sec later, further augmentation of component il and unit recruitment. (5) 10 see later, further augmentation of component IS and unit recruitment; augmentation of component I. Note that the DC level shifts to the negative side whencomponent I diminishes, to the positive side when it augments. E/ectroenceph. clin. NeurophysioL, 1964, 17:353-370

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Fig. 6 A: Hippocampal responses to single shock and 15/see entorhinal stimulation. DC records: above apical dendritic layer of hippocampus (depth 700 p); below pyramidal layer of hippocampus (exact depth unknown). (1) Single shock response and beginning (I); during (2)and end of rCl~titivestimulation (3); single shock responses in the post-tetani¢ phase (4. 5, 6). Time scale in (6): 10 mseo (.seetext). B: Responses from the pyramidal layer to single shock and repetitive entorhinal stimulation. All three records from the same micro.electrode: AC, with high pass filter; DC, with low pass filter; AC (T.C. 0.2), from top to bottom. (1) Repetitive stimulation leads to a negative DC shift (phase A), augmentation and diminution of component 1 at the plateau of phase A. Recruitment of a short latency spike-like component li from the 2nd response on and development of a longer latency second component II at the height of the negative DC shift, almost consistently associat~i wiith unit discharge (lst beam). (2) Continuation of 1. The DC shift becomes positive (phase 13), anti component ! waxes and wanes.

and dentate gyrus response to perforant path volleys underwent potentiation, including component V. Unit discharges and dendritic spikes recruited duri.g repetitive stimulation often per-

sisted as an expression of post-tetanic potentiation (Fig. 5, B and C; 7, A). The phenomenon was rarely absent, even when during stimulation there had been diminution of a response compoElectroenceph. clin. Neurophysiol., 1964, 17:353-370

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Fig. 7 A: Development of "dendritic spike" in the apical dendritic layer of the hippocampus in the course of amygdaloidstimulation, The two records are from the same micro-electrode: AC (T.C. 0.5) and AC with high pass filter. (I) Single shock response; (2) 12/see repetitive stimulation I I sec after its start. Arrow points to the "recruited.... dendritic spike"; (3) first potentiated post-tetanic response with "dendritic spike".

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B: "Dendritic spikes" appearing during post-tetanic potentiation in the apical dendritic layer of the hippocampus ahcr a period of 20/see repetitive entorhinal stimulation. Upper record from v©ntricular surface; lower record from dendritic layer of the hippocampus, (l) Beginning and (2) end of repetitive stimulation ? sec later, O) and (4) Post-tetanic potentiated responses 1, 2, 4.5 and 8 sec after end of repetitive stimulation,

nent, this component was as a rule potentiated in the post-tetanic phase. Potentiation could still be elicited beyond a 50 sec period of repetitive stimulation, or after high frequency tetanic volleys when any trace of augmentation had disappeared. Nevertheless a certain relationship with the augmenting mechanism was apparent. Thus, potentiation was most pronounced within the optimal frequency range for maximum augmentation and the two curves with trains of repetitive stimulation of increasing length were roughly congruous (Fig. 2, A arid C). Generally, the potentiated response differed from the pre-tetanic single shock response or the responses to repetitive stimulation only in amplitude, but some components, especially the dendritic spike, sometimes appeared only in the post-tetanic phase (Fig. 7, B).

The state of raised excitability characteristic for the post-tetanic phase, could be maintained at a steady or even augmenting level over a fairly long period, if stimulation in the post-tetanic phase was at a moderately rapid rate (see Fig. 9). Although this was only observed in two experiments, it was a very reproducible phenomenon in these two animalsY

DC c,~anges induced with repetitive stimulation These were most constant and can be divided into two phases, an initial short phase A, consisting of a negative displacement of the baseline in all layers of the hippocampal formation, and a longer lasting subsequent phase B, consisting of The phenomenon was not systematically looked for in this experimental series.

Electroenceph. clin. NeurophysioL. 1964, 17:353-370

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F~. 8 A: Arrest of unit discharge in the pyramidal layer of the hippocampus,developing during 10/see entorhinal stimulation. From top to bottom: records taken with three micro.electrodesfrom yentricular surface of hippocampus (DC); apical dendritic layer of the hi ppocampus (DC) and pyramidal layer of the hippocampus (AC, with high pass filter). Units firin8 "spontaneously" are driven in con. junction with component I durin8 the first four repetitive responses; thereafter, except for a few isolated instances, unit firing disappears. B: Responsesin urea CA4 to sinsle shock and repetitive amy[daloid stimulation. The two records are

from the same micro.electrode: top: AC (T.C. 0.~); bottom: AC with high pass filter. (I) Singleshock responses(unit firingindependentofeomponent I); beginningof 12/seestimulation; (2) 12se~after I; (3) Ist, 4th and 5th potentiated post.tetanic responses. Spontaneous unit firing is almost completely arrested during repetitivestimulation,but recovers in the post-tetanicphase. DC shifts gradually building up with a radial "dipolar" profile acro~ the two culv©d neuronal layers of the hippocampus and of the dentate gyrus. Thus, the pyramidal h,jer and stratum oriens of the hippocampus as well as the granule cell layer and hilus of the dentate 8yrus became ©lectropositive, whereas the apical dendritic layers of the hippocampus and of the dentate gytus became elcctronegative. These DC shifts ranged from a few hundred /~V to a maximum ofabout 2 inV. In reference to the pre-stimulation baseline. Phase B DC shifts were generally larger than those of phase A. Phase A had a close relationship with compo-

nent IV (Fig. 1, Dand 6). This is also suggested by the rare cases of a positive phase A shift, when component IV was positive (see Fig. 7 of Oloor et al. 1963b) and by the fact that phase A ended when component IV disappeared (Fig. 6, B and 10). Fig. 10 shows these relationships particularly clearly for a response with an unusually large component IV (probably fused with component Ill). Whereas with stimulation below 3/see each subsequent stimulus occurred after the end of component IV, with rates above 3/see each subsequent respon~ f©ii upon a remnant of th© p~ceding component IV. Consequently, the residual negativities of component IV summed to Electroenceph. clin. Neurophysiol., 1964, 17:353-370

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Boostingeffectupon hippocampalexcitabilityby a short train of 60/seeamysdaloidstimulation.The two records are from the same micro.electrodein the pyramidallayer of the hippocampus:AC with highpass filter; AC (T.C. 0.02); from top to bottom. A: 6/sac stimulationand beginningof train of 60/secstimulation. B: End of 60/see and resumption of 6/se~stimulation.C: (3.5 sac later): End of 6/sac and heginninl of 3/seestimulation;D: Continualion of C. £: 2.5 seo after D. Insets(a), (b), (¢) are enlargedsamplesfrom A, B and C as indicated by the correspondingletters. Units not lockedin with responsein (a), fire withcomponent I Ii in (b) and re). Amplitudecalibration I mV, timecalibration 50 rnsec. a negative DC shift, With a frequency of 15/see, subsequent responses fell upon the peaks of the preceding components IV and the greatest DC shift resulted. The rate of rise of phase A was maximal with the first few stimuli; then the DC potential rapidly levelled off', as component IV diminished. With further repetitive stimulation both disappeared and phase B began. In keeping with what was stated a~ove, during this phase the DC shift reversed its direction in the pyramidal layer and stratum oriens of th~ hippocampus as well as in the granule cell layer and hilus of the dentate gyrus, whereas in the apical dendritic layers the negative DC shift became intensified (Fig. I, B, D and E; 5; 6, and 10, E-G). The slowly developing phase B shift

(Fig. I, E) showed some relationship with component I, yet did not seem to result from a summation of successive components I, since it developed with interstimulus periods exceeding the duration of the component I. The possibility of this DC shift resulting from residual depolarization of component I ill apical dendrites is suggested, e.g., by Fig. 6, A. It was certainly unrelated to component IV, which did not show a "dipolar" laminar profile (Gloor et al. 1963b). Also phase B developed when component IV had disappeared. Whereas the magnitude of the phase A of the DC shift was clearly related to that of component IV, phase B DC shifts could take place even in association with a diminished compon¢,nt l[ Electroenceph. clin. Neurophysiol., 1964, 17:353-370

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G 25 C/sec Fig. I0 Relationship between phase A of the DC shift and frequency of repetitive stimulation. Records from the hippo~mpal pyramidal layer (depth 650/+), repetitive stimulation applied to the entorhinal c.orrex with frequencies from 3-25/sec. The three records ere from the same micro-electrode: DC with low p~s filter: AC with high pass filter: AC (T.C. 0,~) from top to bottom. The dotted horizontal line indicates the reference DC level.(Seetext). (Fig. 6, A) and a very steadily progressing dipolar shift was sometimes present while component I waxed and waned (Fig. 6, B).

Phase B of the DC shift outlasted the end o f repetitive stimulation for only a few seconds. This residual D C shift bore no relationship to Electroenceph. din. Neumphysiol., 1964, 17:353-370

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the presence or the duration of potentiation; the latter sometimes persisted when the DC leve| had already reached its "resting" value (Fig. 6, A). The second response in the train of repetitive stimulation, supcrimi~oscd upon phase A of the DC shift, often showed a conspicuous change in amplitude which bore no consistent relationship with the subsequent amplitude changes. The positive response of the pyramidal layer or of the granule cell layer of the dentate gyrus could be increased and the negative response of the apical dendritic layers reduced, or vice versa (Fig. 1!).

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~150 msec Fig. I I Amplitudechanges of the 2nd responseto repetitivestim. ulation superimposed upon phase A of the DC shift, shown in three samples. Upper sample: DC records taken from the hilus of the dentate 8yrus(above)and from the apical dendritic layer of the hippocampus (below) at the beginning of 15/see entorhinal stimulation. The 2nd response in the hilus of the dentate gyrus is diminishedin amplitude, the 2rid response in the apical dendritic layer is augmented. Middle sample: Three records from the pyramidal layer of the hippocampusat the beginningof 10/see entorhinal stimulation. (Same micro-electrode):DC with low pass filter; AC with high pass filter; AC (T.C. 0.2) from top to bottom. The second response (component !, positive) superimposed on the early DC shift (phase A) shows an augmentedamplitude. Lower sample: These records from the apical dendritic layer of the hippocampus during the early phase of 13/sec entorhinal stimulation (same micro-electrode):AC with high pass filter; DC with low pass filter; AC (T.C. 0.2), from top to bottom. The 2rid response in the dendritic layer superimposedon the early negativeDC shift (phase A) is diminished.

Latency changes Repetitive stimulation of the amygdala and, to a lesser degree, of the entorhinal cortex could produce shifts in the latency of the hippocampai response. Both decreases and increases in the latent period, already described by one of us (Gloor 1955), were not investigated in detail in the present series of experimeats. DISCUSSION

Changes of post-synaptic responses induced by repetitive stimulation The remarkable augmentation and occasional diminution of hippocampal responses with repetitive afferent bombardment has been reported earlier by one of us (Gloor 1955) and subsequently by Green and Adey (1956), Campbell and Sutin (1959), Andersen (1960a,b), Morillo et ai. (1962) and more recently in man by Brazier (1964). Our earlier study, carried out with amygdaloid stimulation only, suggested that within the amygdaloid projection system, this phenomenon of augmentation occurred only with multi-synaptically relayed responses. The hypothesis was then proposed that augmentation was the result of the recruitment of elements in the subliminal fringe of interneurons, thus increasing the population of cells transmitting the signals initially set up at the site of stimulation. A similar hypothesis was used to explain the post-tetanic potentiation observed in this system. Our present observations show that even if hippocampal neurons are excited by a direct pathway as, e.g., by stimulating the perforant orfimbrial path (Green and Adey 1956; Campbell and Sutin 1959 and Andersen 1960a, b), augmentation and post-tetanic potentiation still occur. Since no internuncials are involved in this situation several hypothetical mechanisms, other than that mentioned above, can be considered: (I) Pre-synaptic blocks in the terminal plexus of the perforant path may exist under conditions of sing!e shock or low frequency stimulation, but may be overcome by repetitive stimulation. Although this mechanism cannot be entirely eliminated, studies on other systems suggest that repetitive stimulation induces rather than relieves such pre.synaptic blocks (Lloyd 1957; Krnjevi6 and Miledi 1958). Electroenceph, clin. NeurophysioL, 1964, 17:353-370

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(2) The post-synaptic membrane may increase its responsiveness in the course of repetitive stimulation. This mechanism remains purely hypothetical and actually specific examples seem to disprove its existence (Larrabee and Bronk 1947; Lloyd 1949; Hutter 1952). (3) Repetitive stimulation could lead to an increased efficiency of the pre-synaptic link in the synaptic transmissional process by two mechanisms which are not mutually exclusive: (a) Pre-synaptic fibres when activated repetitively at short intervals may discharge impulses of increased amplitude because they arise from a membrane that underwent hyperpolarization in the course of repetitive activation. A larger presynaptic impulse would then elicit a larger postsynaptic response. This mcchanism has been in= yoked by Lloyd(1949), Wall and Johnson (1958), and Eccles and Krnjevi6 (1959) to explain posttetanic potentiation in the spinal cord. (b) Increased mobilization and release of transmitter substance at the pre-synaptic terminals with each successive impulse arriving within a short interval after its predecessor should elicit an increased post-synaptic response (Liley 1956a, b; Curtis and Eccles 1960; Dudel and KutlIer 1961). Mechanisms (a) and (b) may be closely interrelated as suggested by the recent observations of Hubbard and Willis (1962} on the neuro-muscular junction. This last hypothesis seems to us the most acceptable to explain our results, Within the lower frequency ranges and with relatively short periods of repetitive stimulation, the degree ofaugmentation and potentiation were found to be closely related, suggesting a common underlying mechanism for both changes. However, above a certain frequency or beyond a certain duration of repetitive stimulation, as already pointed out by one of us (Gloor 1955), augmentation fails to occur or there may even be diminution, but post-tetanic potentiation persists. Similar observations were made on the stellate ganglion by Larrabee and Bronk (1947). Thus the mechanism for post-tetanic potentiation can become operant even when response transmission across the synapse has begun to fa~l during the preceding tetanic stimulation. This suggests that the changes responsible for potentiation take place in pre-synaptic elements. Such dissociation

between augmentation and post-tetanic potentiation could then be explained as follows: each presynaptic stimulus, as suggested by Liley (1956a, b), Curtis and Eccles (1960), and Dudel and Kuflier (1961) is followed by a relatively brief activation of the mechanism of transmitter release. This release mechanism has to draw on the available reserves oftransmitter substance. Under conditions of high frequency or pro!onged repetitive stimulation, the drain upon these reserves may be great enough to exhaust the available transmitter reserves in the pre-synaptic fibres. Even though the release mechanism as such may be highly active, theactualamount of transmitter that can be released thus be small and the post-synapfic response is diminished or fails to occur. At the end of repetitive stimulation the transmitter substan,:e re-accumulates and can then be released in increased quantities by the post-tetanic volley, due to the still persisting activation of the release mechanism. Full re-accumulation of the reserves may be sufficiently prolonged to explain the delayed development of potentiation after repetitive stimulation which is most often seen after prolonged periods or high frequency of repetitive stimulation. Thus reg',~titive stimulation induces concurrently both depression and facilitation and the two states compete with each other. Similar observations have also been made in other synaptic systo,ms by Liley (1956b), Beswick and Evanson (1957), Vera and Luco (1958), Curtis and Eccles (1960) and Dudel and Kufller (1961). Other observations also lend themselves to interpretation by this hypothesis. Thus augmentation appears to be a function of appropriate intervals between stimuli and not primarily a function of the number of stimuli (Fig, 3). Liley (1956a) has shown that in the neuromuscular junction the activation of the release mechanism by a single nerve volley, when measured by the increase in probability of occurrence of miniature end.plate potentials, subsides over a period of somewhat less than 200 msec although tetanic stimulation may increase the rate of release of these potentials for a period lasting up to 7 min. It would therefore be expected that if the stimuli were to follow upon each other at intervals exceeding 200 msec there would be no facilitation. This line of reasoning was used by Curtis and Eccles (1960) to account for the lack of such facilElectroenceph. clin. Newophysiol., 1964,/7:353-370

ELECTROPHYSIOL(X~Y OF HIPPOCAMPUS. Ill

itatory changes observed in the spinal cord with rates of repetitive stimulation below the optimal 50/see frequency. In the hippocampus, such optimal frequency lies around 10-20/see. Below about 7/sec, intervals between stimuli are too long to bring out evidence of residual facilitation induced by the preceding stimulus. This suggests that after an interval of about 50 to 100 msec following the stimulus, residual activation of the transmitter release mechanism can set free the maximal amount of transmitter substance, and that this amount is reduced at shorter intervals; at longer intervals the available supplies are probably adequate, but the activation of the release mechanisms has sufficiently declined to produce less than maximal release. The limitations imposed upon the efficiency of the activated release mechanism by the lack of large reserves of available transmitter is also suggested by observations (Fig. 3), where with 15/see stimulation frequency, the final augmentation of the response was less than that achieved with frequencies of 10/see and this in spite of the fact that in the early stages of stimulation the response had augmented with each stimulus by about the same amount at both 10 and 15/see frequencies. The less frequently observed initial diminution of response amplitude seen with repetitive stimulation probably results from a depletion of available transmitter reserves that could not be overcome by the activation of the release mechanism. (A similar diminution has been described by Vera and Luco (1958) for the monosynaptic reflex arc activated by dorsal roots undergoing WaUerian degeneratiola.) This interpretation is supported by the findings of Curtis and Eccles (1960) showing that in the spinal cord, facilitating and depressing effects always accompany repetitive stimulation and only on occasion the former can completely overcome the latter. The possible role of inhibition, either pre- or post-synaptic, as a determinant factor in the diminution in amplitude should also be kept in mind, since inhibitory neurons are known to exist in the hippocampus (Kandel et aL 1961; Andersen et aL 1963). Their response characteristics to repetitive stimulation are not clearly known, except for our observation that component V, which is an inhibitory potential (Gloor

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et aL 1963b), invariably was reduced by repet-

itive stimulation in our experiments.

DC shifts The initial negative DC shift (phase A)produced by repetitive perforant volleys results from the summation of late negativities (component IV) of each evoked response. It r.~sembles the negative DC shift in the neocortical surface resulting from summation of after-negativities (Goldring and O'Leary 1951, 1957; Goldring et al. 1959). Elsewhere (Gloor et aL 1963b), reasons were given for interpreting component IV as summed EPSPs in apical and basal dendrites produced by hippocampal interneurons. These EPSPs could summate to a negative DC shift since dendritic responses do not seem to exhibit refractoriness (Clare and Bishop 1955, 1956). A similar mechanism has been invoked by Brookhart et el. (1958) for the "high frequency shift" of the cortical DC level induced by high frequency thalamic stimulation. The DC shift of phase B, though characterized by a "dipolar" distribution similar to that of component I of the response to afferent perforant path volleys, does not seem to result from direct summation of such compotlents. It is in this respect similar to DC shifts induced by low frequency stimulation of the thalamus (Clare and Bishop 1956; Brookhart e t a / . 1958). Yet it is possible that phase B of the DC shift results from the summation of residual depolarizations in apical dendrites, late remnants (often masked by later phenomena) of component 1. These in their turn may result from post-activation increase in the "spontaneous" release of transmitter quanta by the pre-synaptic impulse (Liley 1956a, b; Dudel and Kuffler 1961), possibly due to a persistence of pre-synaptic terminal depolarization (Liley 1956c; Wall 1958). Sometimes however, the isoelectric lines of the transient dipole of component I and of the "steady" dipole of phase B do not coincide (see e.g., Fig. 5, A; 6, A). Caspers (1959) has suggested that cortical dendritic potentials represent a modification of the cortical DC level, which determines their amplitude and polarity. He observed an inverse relationship between surface DC negativity and negativity of the cortical response that could lead to disappearance and even polarity reversal of Electroenceph. clin. NeurophysioL, 1964, 17:353-370

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the latter. Conversely, surface DC positivity was directly proportional to the amplitude of the negative cortical response. We did not observe such relationships in the hippocampus: in the apical dendritic layer of the hippocampus negative DC shifts were often associated with an increase of the negative dendritic response (component I) and only occasionally the latter showed a transient diminution during phase A of the DC shift (see e.g., Fig. 11). A polarity reversal with the development of a negative DC shift in the dendritic layer was never observed. Therefore, Casper's conclusions do not apply to the hippocampus and their general validity as to the electrophysiological properties of dendrites appears questionable, The DC shifts are probably unrelated to the phenomenon of post-tetanic potentiation, since the latter can outlast their duration. Recruitment

gradient between these two layers, and sufficiently intense outward currents may begin to flow toward the apical dendrites to depolarize the soma or initial segment of the axon to the critical firing level. A relationship between augmenting amplitude of a presumably apical dendritic response and the recruitment of cortical unit firing has also been demonstrated by Li et ai. (1956) for the cortical recruiting response. In this situation too, one finds augmentation of amplitude at slow rates of stimulation (Dempsey and Morison 1942; Jasper 1949) and a concomitant negative DC shift in the apical dendritic re. glen (Brookhart et al. 1958). Stefanis (1963) has recently shown, for neocortical cells recorded intraceUularly, similar relationships to those described here for the hippocampus. Mechanisms analogous to those leading to recruitment of unitary discharges from the cell somata were probably responsible for the recruitment of the "dendritic spike". Progresssive depolarization of the dendritic membrane might reach the critical level for the development of an action potential.likedischarge. The less frequently observed arrest of unit discharge induced by repetitive stimulation, with or without preceding recruitment, may at times be caused by a depletion of transmitter substance, but the possible role of inhibitory synaptic events must also be taken into consideration. The experiments here t~ported demonstrate that the many factors determining the final output of hippocampal cells are variably and differentially affected by repetitive stimulation, which results in phenomena of increased or decreased excitability. In general, however, the former prevail and the probability of discharge of propagated action potentials is increased. Synaptic junctions, which under conditions of low frequency stimulation may act as a barrier in the way of further transmission of impulses, may be opened up in consequence to the various changes induced by repetitive stimulation. Rapid, slow and very slow potential shifts seem to interact and bring about these changes.

Repetitive perforant path volleys often led to recruitment of pyramidal neurons of the hip. pocampus and of granule cells of the dentate gyrus, in general along with recruitment or augmentation of component II. The frequent asso. elation of this component with unit discharge, pointed out by Gloor et al. (1963b), presumably signals either the activity of the bas. ket cells and/or excitation of the mossy fibres or the development of a "dendritic spike". In the first case such increaxd activity would precede the recruitment of the hippocampal pyramidal cells, but this does not seem to be a necessary prerequisite for such recruitment, since pyram. idal firing can be initiated without augmentation of component II or even with its diminution or disappearance. Augmentation of component I and phase B of the DC shift might be important for the recruitment of pyramidal cell discharges. It was shown (GIoor et al. 1963b) that component I, when evoked by single shocks was rarely associated with unit discharge, probably because its amplitude failed to exceed the "resting" potential drop between the apical dendritic and cell layers. SUMMARY With repetitive stimulation, however, component I and phase B of the DC shift having the same In experiments performed in lightly anaespol~,-ity, summateo One c ~ thu~ obtain ~ t.ra::- thetized cats it was found that repetitive afferent sicn, rc;'czsa; of ~ e original "resting" potential volleys to the hippocampus mediated by the per-

Electroenceph. din. NeurophysioL, 1964, 17:353--370

ELECTROPHYSIOLOGYOF HIPP(~AMPUS. II! forant path and elicited by repetitive entorhinal or amygdaloid stimulation induced the following alterations of the hippocampal and dentate 8yrus response: 1. Components I and II of the evoked response were augmented or, less frequently, diminished in amplitude by relatively slow (10-20/ sac) repetitive stimulation. Components llI and IV were usually diminished and component V invariably disappeared during repetitive stimulation. Augmentation is most easily explained by assuming an increase in the efficiency of the presynaptic link of the synaptic transmissional process most likely due to increased release of transmitter substance. Diminution is thought to develop when the available transmitter reserw,~ are depleted. The two antagonistic mechanisms interact. 2. Post-tetanic potentiation was observed to follow upon cessation of repetitive stimulation. All response components were apt to be potentiated regardless of whether they had previously undergone augmentation or diminution. This is most easily explained in the light of the above hypothesis of an activation of transmitter release induced by repetitive stimulation. 3. Repetitive stimulation produced two types of DC shifts. The initial shift, phase A, was negative in all layers of the hippocampus and dentate gyrus and resulted from direct summation of components IV of the response. The second phase, phase B, followed upon phase A and showed the same laminar profile as the main transient component I. it is probable that it resulted from the summation of residual apical dendritic depolarizations consecutive to component I. 4. Repetitive perforant path volleys often lead to recruitment of hippocampal pyramidal and dentate granule cell discharges as evidenced by the elaboration of single cell action potentials or apical dendritic spikes. Augmentatien of component I and phase B oi the DC shift may cancel and reverse the "resting" potential gradient of the hippocampus and lead to sufficiently intense current flows to elicit firing of the pyramidal cell soma. In other instances unit firing seemed to be induced as a consequence of augmentation or recruitment of component II.

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Arrest of unit firing was also sometimes observed and confirms that mechanisms opposing the build-up of an increased excitatory state are concut'rentiy activated with excitatory changes by repetitive stimulation, although in most instances the latter prevail. REFERENCES ANDEP.SE~,P. Interhippocampal impulses. II. Apical dendritic activation of CA1 neurons. Acta physiol, stand., 1960, 48: i 78-208. ANDERSEN,P. lnterhippocampal impulses. IlL Basal dendritic activation of CAa neurons. Acta physiol, stand., !960, 48: 209-230. AND~aS~N,P., Ecct.~s, J. C. and LeYNmG,Y. Recurrent inhibition in the hippocampus with identification of the inhibitory cell and its synapses. Nature, 1963, 198: 540-542. BESWiCK, F. B. and EVANSON,J. M. Homosynaptic depression of the monosynaptic reflex, followingits activation. J. Physiol. ( Lond. ) , 195"/,135:400-41 I. BRAZieR, M. A. B. Evoked responses recorded from the depths of the human brain. Ann. N. Y. Acad. SeL, 1964, in press. BROOKHART,J. M., ARDUINI,A., MANCIA,M. and MoRUZZl, G. Thalamocortical relations as revealed by induced slow potential changes. J. Neurophysiol., 1958, 21 • 499-525. CAUPBI~I.L,B. and StrnN, J. Organization of cerebral cortex. IV. Post.tetanic potentiation of hippocampal pyramids. Amer. J. Physiol., 1959,196: 330-334. CASPEP~S,H. 0bar die lkziehungen zwischen DendritenI~tential und Gleichspannung an der Hirnrinde, Prefers Arch. gas. Physiol., 1959,269: ! 57 !8 I. CcAIt~, M. H. and lllstiop, G. H. Properties of dendrites; apical dendrites of the cat cortex. Electroenceph. c/in, Neurophysiol., 1955, 7: 85-98. CLAa~, M. H, and BIsHoP,G. H. Potential wave mechanism in cat cortex. Eleetro~'nceph. elln. Neurophysiol., 1956, 8: 583-602. CuRTIs, D. R. and Ecct,~, J. C. Synaptic action during and after repetitive stimulation. J. Physiol.(Lond.), 1960, 150: 374-398. DEMPSEV,E. W. and Mom~N, R. S. The production of rhythmically recurrent cortical potentials after localized thalamic stimulation. Amer. J. Physiol., 1942,135: 293-300. DUD~L,T. and KUVFLE~,S. W. Mechanismof facilitation at the crayfish neuromuscular junction. J. PhysloL ( Lond.), 1961, 155: 530-542. ECCLeS,J. C. and KRNJEVI~,K. Pre-synapticchangesassociated with post.tetanic potentiation in the spinal cord. d. Physiol. (Lond.), 1959, 149: 274-287. GLOOR, P. Electrophysiologicalstudies on the connections of the amygdaloid nucleus in the cat. !!. The electrophysiological properties of the amygdaloJd projoction system. Electroenceph. clin. Neurophysiol., 195[;, 7: 243-264. GLoom,P,, VER^,C. L. and SPERTI,L. ElectrophysiologiEleetroenceph. clin. NeurophysioL, 1964, 17:353-370

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