EFFECT OF FORNICAL STIMULATION UPON THE CA1 AND CA2 APICAL OF RABBIT'S

Yasuichiro FUJITA* AND Yoshio NAKAMURA**

Section of Neurophysiology, Institute of Brain Research, School of Medicine University of Tokyo

Since GREEN and ADEY7) found a post-synaptic response in hippocampus following fornical stimulation in 1956, the fact has been re-affirmed by von EULER et al.5). These workers all seem to regard this response as excitatory arising in or near the of the . However, the authors of the present paper think that there have been no definite proofs to support such conclusion. FUJITA and SAKATA6)found a spike potential originating in the apical dendrite of the pyramidal cell of rabbit's hippo- campus following stimulation of CA3, CA4 or the Schaffer collaterals. On the other hand, by stimulating the neural structure within 200 micra from the alveus, FUJITA and NAKAMURArecorded a positive potential of 15 to 35 msec in duration in the apical dendrite layer, which inhibited the spike of the apical dendrite. The aims of the present paper are (1) to analyse precisely this inhibitory slow potential and (2) to know the nature of the postsynaptic response evoked by fornical stimulation, taking the spike of the apical dendrite as an indicator of the excitability change.

METHODS Thirty two rabbits were used. The experimental methods were nearly the same as those described in the separate paper6). The chief difference was that in this experiment the hipocampus was usually not exposed, because the hipocampus easily succumbed to depression, if exposed. As the readings of a micromaniplator is not always reliable owing to many reasons, the change in the time course or polarity of the potential evoked by stimulation of the collaterals of deeply or laterally placed pyramidal cells as the recording electrode was moved along the apical dendrite of the pyramidal cells in the medial part of the Ammon's horn, was taken as an indicator of the position of the tip of the recording electrode (FIG. 1). Neural structures which generated this evoked potential was examined histologically in the separate paper6). For immobilization, curare or nembutal was used. As far as the present experi-

Received for publication January 30, 1961 **藤 田安一郎,中 村嘉 男 *Present address: Departrnent of Physiology , Nippon Medical School, Bunkyoku, Tokyo.

357 358 Y. FUJITA AND Y. NAKAMURA

FIG. 1. Laminar analysis of EPSP and of the apical dendrite evoked by stimulation of the Schaffer collaterals. Downward deflection denotes negativity. Zero point is arbitrary. Numbers on the left show distances from zero point in micron. The pyramidal cell layer is considered to be located at 200 micra. A: weak stimulation; B: strong stimulation. Note the largest spike in the apical dendrite layer.

ment was concerned, no essential difference was found between curarised rabbit and nembutalized one. Liquor cerebro-spinalis was sucked out to minimize the disturbance by respiratory movement. The stimulating and recording electrodes were 3 molar KCL filled mcro-pipettes with 10 to 40 micra and I micron in tip diameters respectively. Usually a negative pulse of 1 msec duration, the intensity of which was strong enough to generate a spike in the apical dendrite, was delivered to the Schaffer collaterals. For stimulation of the fornix and intrahippocampal structure which evoked an inhibitory process in the apical dendrite, the same pulse of different intensity was used. After the experiment was over, the structure surrounding the stimulating electrode for the fornix was gently sucked out, and by exposing the fornix, the localization of the tip was ascertained, under direct vision. CR coupled amplifiers with time constant of 0.1 and 0.3sec respectively were used to record electrical changes. Cathode followers were constructed as the input stages of the amplifiers. Photographs were taken with a long recording camera. The upward deflection in the records denotes positive sign. All records in this paper are superimposition of four to six traces. FORNICAL STIMULATION AND HIPPOCAMPUS 359

RESULTS

Usually, two stimulating electrodes were inserted into the lateral part of the Ammon's horn. One was used for stimulating the Schaffer collaterals or deeper pyramidal cell layer. The other was used to stimulate the neural structure, which was located within 300 micra from the alveus. A recording electrode was in- troduced into the medial part (CA1) of the Ammon's horn. I. Stimulation of the neural structure within 300 micra from the alveus. When the stimulating electrode was placed at a point, which was located within 300 micra from the alveus, a positive potential of 15 to 35msec in duration was recorded in the apical dendrite layer (FIG. 3B). As the recording electrode was slowly withdrawn, the potential reversed its polarity and became negative in sign in the cell layer. Usually, the positive deflection in the apical dendrite layer was larger in amplitude than the negative deflection in the cell layer. The phase reversal took place 100 to 200 micra above the point, at which the phase- reversal of EPSP of the apical dendrite evoked by the Schaffer collaterals occurred. This potential inhibited the spike of the apical dendrite evoked by stimulation of the Schaffer collaterals (FIG. 2A, FIG. 3F). The degree of the inhibition was strongest approximately at the peak of the slow potential, though sometimes dis- crepancy of slight degree occurred. Considering from the fact that the slow potential was of a long duration and showed summation on double shocks, it is apparent that the inhibitory potential is of postsynaptic nature.

A

B

FiG. 2. Slow potential of the apical dendrite evoked by intrahip- pocampal stimulation. A: two stimuli were delivered at various time intervals. Note inhibition of the spike by slow potential. B: a spike evoked by stimulation of the Schaffer collaterals (Right) and slow potential evoked by intrahippocampal stimulation (Left). 360 Y. FUJITA AND Y. NAKAMURA

A B

FIG. 3. Laminar analysis of slow potential evoked by intrahippocampal stimulation. Zero point is arbitrary. The pyramidal cell layer is considered to be located at 200 micra. A: spike of the apical dendrite evoked by stimulation of CA4. B: slow potential of the apical dendrite evoked by intrahippocampal stimulation. C: a spike evoked by stronger stimulation of the same point as in the case of B. Note that the spike is larger in the apical dendrite layer than in the cell layer and that the spike in the cell layer is diphasic i.e. initially positive and later negative. Also note that slow negative deflection of the cell layer in C is much smaller than that in B. D: a typical spike of the apical dendrite recorded at 500 micra. Note a notch on the rising phase and after-positivity. E: slow potential evoked by intrahippocampal stimulation. F: inhibition of the spike by the slow potential.

As described above, stimulation of the neural structure which was situated within 300 micra from the alveus, evoked a slow positive potential in the apical dendrite layer, together with a negative deflection of the same duration in the cell layer. The latter was explained as a sink of the inhibitory postsynaptic potential of the apical dendrite. Therefore, no matter how the intensity of the stimulus may be increased, no spike should be generated from the slow negative wave seen in the cell layer. However, if stronger stimulus was delivered to the FORNICAL STIMULATION AND HIPPOCAMPUS 361 same point, there arose spike potentials from the cell layer down to the deeper part of the apical dendrite (FIG. 3C). In the apical dendrite layer the spike was seen superimposed on slow positive potential, while the spike potential above the phase-reversal point was observed superimposed on the slow negative potential. The amplitude of the spike was larger in the apical dendrite layer as compared with that above the phase-reversal point. The most remarkable facts were that the amplitude of the slow negative potential in the cell layer was decreased as the intensity of the stimulus was increased, and the spike potential seen in the cell layer was usually diphasic; that is, initially positive and later negative. From these observations, it was quite apparent that the spike potentials seen on these occasions were similar to those evoked by stimulation of the Schaffer collaterals. Therefore, it was concluded that by increasing the intensity of the stimulus, the Schaffer collaterals or some other fibres afferent to the apical dendrite were stimulated owing to the current spread. It was noteworthy that the increase in the intensity of the stimulus brought a reduction in the amplitude of the slow negative wave, and that the diphasic spike potential in the cell layer was initially positive and later negative. This definitely points to a conclusion that the negative wave in the cell layer is not the precursor to the spike potential. More-

AB C

FIG. 4. EPSP and spike of the apical dendrite. Zero point is arbitrary. The pyramidal cell layer is considered to be located at zero. Stimulus was delivered to the super- ficial layer of CA2 and it was considered that near the soma were activated. A: spike of the apical dendrite evoked by stimula- tion of CA3. B: EPSP arised near the soma. C: spike evoked by stimulation of the super- ficial layer. 362 Y. FUJITA AND Y. NAKAMURA over, the slow wave inhibits the spike of the apical dendrite. The diphasic spike potential seen in the cell layer was considered as the spike of the soma which was propagated from the deeper part of the apical dendrite. Sometimes, by increasing the intensity of the stimulus, a monophasic spike was seen in the cell layer, together with spike potentials along the apical dendrite (FIG. 4C). On such occasions, in the deeper part of the apical dendrite, the spike was usually diphasic, that is, initially positive and later negative. Measurement of the peak latency showed that the spike was conducted from the soma to the deeper part of the apical dendrite (FIG. 5). In such cases, the slow wave, which was recorded as negative and positive deflections in the cell layer and apical dendrite layer respectively was considered as EPSP of the soma or basal dendrite of the pyramidal cells. II. Effect of fornical stimulation. As described by GREEN and ADEY7)for the first time, a single shock delivered to the dorsal fornix, evoked a postsynaptic response in the hippocampus. The response is recorded as negative and positive deflections in the cell layer and apical dendrite layer respectively. According to the present experiment, the positive deflection recorded in the apical dendrite layer was in most cases larger than the negative deflection recorded in the cell layer. The duration of the evoked potential was varied from 15 to 40 msec, and its latency ranged from 6 to 15 msec. The potential evoked by fornical stimulation

FIG. 5. Conduction of a spike along the apical dendrite. The superficial layer was stimulated. Antidromic conduction is observed. Ordinate: distance in micron from an arbitrary point. The pyramidal cell layer was considered to be located at 300 micra. Abscissa: time in msec. FORNICAL STIMULATION AND HIPPOCAMPUS 363

A

B

FIG. 6. Slow potential of the apical dendrite evoked by stimulation of the dorsal fornix recorded at the apical dendrite layer. A: single shocks to the fornix and Schaffer collaterals were delivered at various time intervals. Note inhibition of the spike by the slow potential. B: control responses; Spikes of the apical dendrite immediately before (left) and after (right) the double-shock experiment, and slow potential of the apical dendrite (middle).

A B C

FIG. 7. Laminar analysis of the slow potential of the apical dendrite evoked by fornical stimulation. Zero point is arbitrary. The pyramidal cell layer is considered to be located at 200 micra. A: spike of the apical dendrite evoked by stimulation of the Schaffer collaterals. B: slow potential of the apical dendrite evoked by fornical stimulation. C: single shocks to the fornix and Schaffer collaterals were delivered at a definite time interval. Note inhibition of the spike at all levels. 364 Y. FUJITA AND Y. NAKAMURA inhibited the spike of the apical dendrite generated by stimulation of the Schaffer collaterals (FIG. 6A, FIG. 7C, FIG. 8E, H). In most cases, the inhibition was maximum approximately at the peak of the slow wave. The slow potential inhibited the conducting spikes of the apical dendrite at all levels. For example, at 400 micra FIG. 7, the slow potential which was recorded there as negative deflection, inhibited the spike potential which was conducting toward the cell body. As described above, the slow potential evoked by fornical stimulation was recorded as positive and negative deflections in the apical dendrite and cell layers respectively. However, when the intensity of the stimulus was increased, spike potentials were seen along the apical dendrite (FIG. 8C). The spikes below the phase reversal point were seen superimposed on slow positive potential, while those above the phase reversal point was observed superimposed on negative potential quite similarly as in the case of stimulation of the neural structure described in I. The amplitude of the spikes in the apical dendrite layer were larger than those above the phase reversal point, and in the cell layer, the spike potential were usually diphasic (initially positive and later negative) (FIG. 8D). The reduction in the amplitude of the negative potential recorded in the cell layer was also observed together with generation of spikes along the apical dendrite, as the intensity of the stimulus was increased. These spikes were quite similar to those evoked by stimulation of the Schaffer collaterals in that firstly the largest spike was seen in the deeper part of the apical dendrite and secondly in the cell layer the spike was diphasic with initially positive deflection. Therefore, it was assumed that the spikes seen along the apical dendrite were evoked by antidromic stimula- tion of the of the pyramidal cells in CA3 or CA4; that is, the spikes originated in these axons propagated antidromically and excited the Schaffer collaterals, the spikes of the collaterals activating the axodendritic synapses there. In some cases, fornical stimulation evoked a slow diphasic potential (FIG. 8B), which was initially negative and later positive in the apical dendrite layer. By increasing the intensity of the stimulus, a spike arose from the negative phase of the slow potential recorded in the apical dendrite layer. Moreover, if EPSP of the apical dendrite evoked by stimulation of the Schaffer collaterals was superimposed on this negative phase, a spike potential was generated (FIG. 8K). Therefore, the negative phase was considered as EPSP of the apical dendrite, and as mentioned above, it was assumed that the apical dendrite was activated by antidromic stimulation of axons of deeply placed pyramidal cells by way of the Schaffer collaterals. FORNICAL STIMULATION AND HIPPOCAMPUS 365

FIG. 8. Slow potential of the apical dendrite evoked by fornical stimulation. Zero point is arbitrary. The pyramidal cell layer is considered to be located at 150micra. A: spike of the apical dendrite evoked by stimula- tion of the Schaffer collaterals. B: slow potential of the apical dendrite evoked by fornical stimulation. The slow potential evoked by fornical stimulation is biphasic. Namely, it is initially positive and later negative in the superficial layer, and in the apical dendrite layer polarity is perfectly reversed, that is, initially negative and later positive. The initial phase is considered to be EPSP of the apical dendrite, because a spike is generated if EPSP evoked by stimulation of the Schaffer collaterals (I) is superimposed on this phase. On the other hand, later phase is considered to be IPSP of the apical dendrite, because it inhibits the spike of the apical dendrite (E, H). C: spike evoked by stronger stimulation of the same point as in the case of B. Note that the spike is larger in the apical dendrite layer than in the superficial layer. In this respect, the spike is quite similar to that evoked by stimulation of axon collaterals of deeply or laterally placed pyramidal cell layer (FIG. 1, B: FIG. 8, A: FIG. 4, A: FIG. 7, A). The spike arises from the initial phase which is recorded as negative deflection in the apical dendrite layer. D: the same spike as that seen in the uppermost record in C was recorded with higher sweep velocity and amplification. Note that the spike is biphasic, i.e. initially positive and later negative in this region (nearly the pyramidal cell body layer). E: inhibition of the spike by the slow potential. Control responses of the spike and slow potential are recorded at 450 micra in A and B respectively. F: spike of the apical dendrite evoked by stimulation of CA4, recorded at 800 micra. The spike is inhibited (H) by the later phase of the biphasic slow potential (G) evoked by fornical stimulation. I, J, K, L: all recorded at 700 micra. These records show spacial summation of two EPSPs. I: EPSP evoked by stimulation of the Schaffer collaterals. J: EPSP evoked by fornical stimulation (initial negative phase).: two EPSPs were superimposed at definite time interval. Note the generation of a spike. L: the time interval of two stimuli was increased. No spike generation at this phase. 366 Y. FUJITA AND Y. NAKAMURA

DISCUSSION

1) IPSP of the apical dendrite. The present experiment showed that some kind of inhibition existed in the rabbit's hippocampus. As for the nature of the inhibitory slow potential, there are two possibilities. One is inhibitory postsynaptic potential of the apical dendrite. The other is afterhyperpolarization of the spike of the apical dendrite. As no spike was usually recorded immediately before the slow positive potential recorded in the apical dendrite layer following fornical stimulation or stimulation of the neural structure described in I. and the time course of its rising phase was clearly slower than that of the afterhyperpolarization, it was safely assumed that the slow positive potential recorded in the apical dendrite layer was the inhibitory postsynaptic potential of the apical dendrite. Inhibitory postsynaptic potential was initially discovered in the spinal moto- neuron1,3,4) and its ionic mechanism has been fairly well disclosed. However, the ionic mechanism of IPSP of the apical dendrite quite remains in the dark at the present moment. But, though the time course of IPSP of the apical dendrite differs from that of the spinal motoneuron, the authors consider that at least inhibitory and postsynaptic natures of the slow positive potential found in the apical dendrite layer are beyond any doubt. Hitherto known inhibitory are short-axon-cell type. If such fact is valid also for the hippocampus, the inhibitory fibres running in the fornix must change synapses somewhere in the hippocampus. The whereabout of the inhibitory is left to future investigation. 2) IPSP evoked by intrahippocampal stimulation. As described above, stimu- lation of the neural structure which was located within 300 micra from the alveus evoked IPSP of the apical dendrite. However, the nature of the neural structure, that is, what kind of fibres or cells were activated, is yet unknown, and it must wait further analysis. Since slow potentials (IPSP) evoked by fornical stimula- tion and stimulation of this intrahippocampal structure respectively are nearly the same in their time courses, phase reversal points, and inhibitory nature, it was assumed that fornical afferent fibres might enter the hippocampus from the lateral part of the Ammon's horn, at least concerning those converging into the pyramidal cells of CA1 and CA2. 3) Effect of fornical stimulation. Whenever the fornix is stimulated, the possi- bility that the pyramidal cell is antidromically stimulated should always be taken into account. In the Ammon's horn, the effect of antidromic stimulation is prac- tically the same as stimulation of the Schaffer collaterals, though the effectiveness is much smaller. It is noteworthy that von EULER et al.5) and CAMPBELL & SUTIN2) found the shifts of negative potential field from the cell layer toward the apical dendrite layer by repetitive stimulation of the fornix. Moreover, GREEN et al.7) and von EULER et al.5) found the same phenomena when the seizure discharge developed FORNICAL STIMULATION AND HIPPOCAMPUS 367 following fornical stimulation. These facts suggest that the effect of fornical stimultaion is a complex one and can not be explained easily as long as one consider only one kind of pathways from the fornix to the hippocampus.

According to the present investigation, the fornix contains at least two kinds of pathways running back to the hippocampus. One is the inhibitory pathway making synaptic contact with the apical dendrite of the Ammon's horn. The other is the excitatory pathway activating the axodendritic . The inhibitory pathway seems to correspond to that originally discovered by GREEN et al. in 1956. But, GREEN et al. and other investigators considered the function of this pathway as excitatory to the soma of the pyramidal cell changing synapse at the . However, the authors of the present paper think that such hypothesis could not explain the result of the present investigation, because (1) the post- synaptic response evoked by fornical stimulation inhibited the spike of the apical dendrite (2) stimulation of the intrahippocampal structure which was situated below the phase-reversal point could not evoke inhibitory slow potential in the apical dendrite layer. If any response was evoked by stimulation of the deeper part of the hippocampus, that is, the granule cell layer or deeper pyramidal cell layer, it was usually the same in nature as that evoked by stimulation of the

Schaffer collaterals. There is a possibility that sharp spike with the shortest latency found by GREEN et al. and von EULER et al. in the deeper part of the hippocampus following fornical stimulation might be an antidromically activated spike of the axons or

soma of the deeply placed pyramidal cells. As for the excitatory pathway men- tioned above, the authors consider it as axons of deeply placed pyramidal cells , impulses of the axons running back to the apical dendrite of CA1 or CA2 by

way of the Schaffer collaterals and resulting in the activation of the axodendritic synapses. At least it is certain that there are two pathways in the fornix , one inhibiting the spike of the apical dendrite, and the other generating the spike,

though the possibility is not completely excluded the excitatory pathway may be not antidromic one, but afferent fibres. Stimulation of the reticular formation or

sciatic nerve evokes 0-rhythm activity in the hippocampus. It is generally assumed

that the impulses generating such responses may arrive in the hippocampus by way of the septum and dorsal fornix. In this connection, it is very interesting that

stimulation of the dorsal fornix inhibited the action potential of the apical dendrite . It seems to be worthy of consideration that the Į-rhythm seen in the hippocampus

in arousal state might be the summated IPSPs of the apical dendrite.

4) Functional organization of the pyramidal cell. Since the apical dendrite

is about 700 micra in length and 3 micra in diameter, the electrotonic current

flow in the cell body caused by a potential in the apical terminal would be probably negligible. This disability is covered by the transmission of impulses along the apical dendrite. In the apical dendrite of the hippocampus , an in- hibitory synapse exists at the level of the Schaffer collaterals , thus effectively 368 Y. FUJITA AND Y. NAKAMURA preventing the generation of impulses in the apical dendrite. On the other hand, the activation of the inhibitory synapse would have probably negligible influence upon the soma. Therefore, there is a possibility that by the activation of the inhibitory synapse, the activity of the apical dendrite can be selectively depressed, with negligible influence upon the soma. If these speculations were the fact, it may be considered that the pyramidal cell is functionally more differentiated than the cell without the apical dendrite, because in the latter a selective depression of the activity of one portion of the cell is almost impossible.

SUMMARY

1) A neural structure, stimulation of which evoked a postsynaptic potential in the apical dendrite of CA1 or CA2 was found within the rabbits hippocampus. The slow potential inhibited the spike of the apical dendrite. 2) It was found that the fornix contained at least two pathways to the hippo- campus, one exciting and the other inhibiting the activity of the apical dendrite. The former was considered as the antidromic pathway, generating spike in the apical dendrite by way of the Schaffer collaterals. The latter was regarded as the same as that found by GREEN et al. in 1956. But as for its function, quite contrary to GREEN et al. it was found that impulses running this pathway inhibited the spike of the apical dendrite by generating a slow positive potential. 3) The inhibitory pathway was considered entering directly into the Ammon's horn, without changing synapses at the granule cells.

The authors are deeply indebted to Professor Tokizane for his valuable suggestions and encouraging support throughout the present investigation. Many thanks are also due to the members of Tokizane school.

REFERENCES

1) BROCK,L. G., COOMBS,J. S. AND ECCLES, J. C. The recording of potentials from motoneurones with an intracellular electrode. J. Physiol., 1952, 117: 431-460. 2) CAMPBLL,B. ANDSUTIN, J. Organization of . IV. Posttetanic potentia- tion of hippocampal pyramids. Amer. J. Physiol., 1959, 196: 330-334. 3) COOMBS,J. S., ECCLES, J. C. AND FATT, P. The inhibitory suppression of reflex discharge from motoneurones. J. Physiol., 1955, 130: 396-413. 4) COOMBS,J. S., ECCLES,J. C. ANDFATT, P. The specific ionic conductances and the ionic movements across the motoneuronal membrane that produce the inhibitory postsynaptic potential. J. Physiol., 1955, 130: 326-373. 5) EULER,C. VON, GREEN,J. D. AND RICCI, G. The role of hippocampal in• evoked responses and after-discharges. Acta Physiol. Scand. 1958, 42: 87-111. 6) FUJITA, Y. AND SAKATA,H. Electrophysiological properties of the apical dendrite of rabbit's hippocampus. Unpublished. 7) GREEN, J. D. AND ADEY,W. R. Electrophysiological studies of hippocainpal connec- tions and excitability. EEG din. Neurophysiol., 1956, 8: 245-262.