EXPERIMENTAL

NEUROLOGY

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35-57 (1975)

Physiological Studies of the Reciprocal Connections between the Hippocampus and Entorhinal Cortex SAM

h.

R.

DEADWYLER,JAMES

WEST,

CARL

\;li.

COTMAN,

ANDGARYLYNCH~

Drpavtwcnt

of Psychobiology, Uwiversity Ive~ine. Califorrzia 92664 Receiwd

March

of California,

7. 1975

Nemophysiological recordings were obtained from the hippocampus, entorhinal cortex, and dentate gyrus under conditions of controlled electrostimulation at interconnecting pathways in order to confirm their bidirectional nature as suggested by recent anatomical findings. The existence of a hippocampal to entorhinal pathway was confirmed physiologically by the presence of evoked field potentials and unitary driving of the entorhinal cortex following stimulation of the CA3 subfields of the ipsilateral and contralateral hippocampus. Activation of the entorhinal cortex by such procedures led to a subsequent excitation of the granule cells of the dentate gyrus through the axonal projections of the perforant pathway. The findings are discussed in the context of known anatomical circuitry which might provide the basis for such bidirectional interactions. The functional significance of the demonstrated physiological connections is indicated by the fact that the entorhinal cortex responds to hippocampal activation in a consistent manner and transmits that information back to the dentate gyrus; thereby completing an important three chain loop between three major components of limbic system circuitry.

INTRODUCTION Ramon y Cajal (11) was the first to suspect that the hippocampal formation was organized according to a plan in which events were initiated in the paleocortical entorhinal region, proceeded to the dentate gyrus, and from there to the pyramidal cells of the hippocampus proper. In addition, he showed that the pyramidal cells gave rise to the axons which constituted the massive fimbrial system which represented the output fibers of the hippocampus. Subsequent anatomical and physiological investigations 1 Supported by Grant NSF GB 39947 to S.A.D., NIMH Grants MH BMS 7202237-2 to G.L., and NIH Grant NS 08597-06 to C.W.C. 35 Copyright All rights

0 1975 by Academic of reproduction

Press, Inc. in any form reserved.

19793-04 and

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have proven the soundness of Cajal’s analysis and have provided a much more detailed description of the individual links in the entorhinal-dentate gyrus-hippocampal chain (5, 7, 9, 10, 16, 17, 21-24, 32). Recently, convincing evidence has been reported indicating the validity of the long held suspicion that the entorhinal-to-hippocampal connection is bidirectional. On the basis of silver degeneration techniques, HjorthSimonsen (20) concluded that the field CA3 of certain portions of the hippocampus projected caudally into the deep layers of the entorhinal cortex. While conclusions as to the origin and trajectories of this hippocampal-to-entorhinal projection require further support, the existence of the connection seems established. Such a circuit takes on added significance in the light of recent anatomical evidence in both the rat and monkey that large areas of the neocortex project to ,the entorhinal cortex (30) providing the basis for possible convergence and integration of hippocampal and cortical information. The results suggest that relations between retrohippocampal areas and the hippocampus itself are likely to be more complicated than the simple one-way circuit described by Cajal. The present paper reports studies designed to provide a preliminary analysis of the hippocampal-entorhinal projection, particularly as it relates to the operation of other intrahippocampal circuitry. Our initial goals were to identify the pathway using physiological techniques and to establish some of its characteristics. We then attempted to activate it by electrical stimulaion of the commissural and associational systems, axonal projections which arise and, in part, terminate in the CA3 pyramidal cell fields ( 10, 18, 19, 28, 33). Finally, we tested the possibility that entorhinal cells activated by the hippocampal formation were those that projected to the dentate gyrus. METHODS Forty male albino rats were anesthetized with a mixture of urethanechloralose (1 g/kg and 40 mg/kg) for acute neurophysiological experiments. Stimulation and recording techniques were as described (12, 27). Briefly, the stimulating electrodes were placed into either or both CA3c fields of the hippocampus in position to stimulate the monosynaptic associational pathway to the dentate gyrus ipsilaterally (18, 33) or the monosynaptic commissural projections contralaterally (10, 18, 19). These electrodes consisted of single strands of insulated 34-gauge stainless steel wire. Recording electrodes (31\6 NaCl filled micropipettes : l-5 Mohm) were oriented to either the entorhinal cortex, dentate gyrus or CA3c hippocampal subfield in the same hemisphere (Fig. 1). Procedures for localizing the electrodes have been described in detail elsewhere (12). Extracellular recordings of field potentials and simultaneous unit discharges

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VP

FIG. 1. Schematic showing arrangements for stimulating the commissural and associational pathways and recording from the dentate gyrus, entorhinal cortex and ipsilateral CA3 field of the hippocampus. A. Placement of commissural (Conim) and associational (Assoc) stimulating electrodes are designated by $. Recording electrode tract through the dentate gyrus (Ret: D,) is shown with commissural and associational terminal field marked by dots signifying the imler one-third of the molecular layer of the dentate gyrus. Recording locus within ipsilateral CA3 ( Ret : CA3) is approximately the same location as signified by the locus of the associational stimulating electrode. B. Rear view of the entorhinal cortex and recording electrode trajectory (Ret : En). RF-rhinal fissure ; LEn and REn-left and right entorhinal cortices respectively. were obtained from all three regions. Following completion of each experiment, stimulating and recording electrode tracts were verified using con-

ventional histological techniques. The animal was perfused with salineformalin, the brain removed and later sectioned (30 pm) for analysis of lesions and electrode placements. Lesions of the entorhinal cortex were made electrolytically through a stimulating electrode. RESULTS Activation Commissawe

of the Entorhixal and Associational

Cortex by Stimulation of the Hippocanzpal Pathways. Recordings from the entorhinal

cortex were obtained by inserting the recording electrode at a 10” angle away from the midline. This procedure allowed the traversal of large segments of entorhinal cortex along its dorsoventral axis (Fig. 1B). Histological reconstructions confirmed that the recording electrode passed through 1 to 3 mm of entorhinal cortex in a given electrode track. Both medial and lateral entorhinal areas were probed by this technique. Record-

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ings were obtained largely from the more dorsal regions of the entorhinal cortex due to the inaccessability of the ventral entorhinal areas. Hence, all recordings bracketed an area of approximately 1.5 to 2 mm mediolateral and approximately 1 to 3.5 mm dorsoventrally. Stimulation of both the ipsilateral and contralateral CA3 regions was employed to generate activation of the entorhinal cortex. The presentation of results will follow a pattern in which the ipsilateral CA3-to-entorhinal functional connections will be described first. Next, the activation of this ipsilateral circuit by stimulation of the contralateral CA3 region will be demonstrated. The results show that direct electrostimulation of the ipsilateral CA3-to-entorhinal circuit produces effects identical to those obtained when this same region is driven synaptically by the hippocampal commissural projections from the opposite CA3 region.

FIG. 2. A & B. Entorhinal unit discharges (bottom trace) and evoked field potential (top trace) to hippocampal commissural stimulation. C. dentate gyrus field potentials (early and “late”) evoked by commissural stimulation (top trace) recorded from the molecular layer 100 pm above the granule cells; bottom trace entorhinal potential. D. Entorhinal field potential and unit discharge (separate traces) faster sweep. E. Field potential from CA3 and same unit discharge in entorhinal cortex. F. Similar to C only lower trace shows entorhinal unit discharge (two superimposed sweeps). Stimulus at sweep onset. Calibration A-C 500 aV upper traces; 20 pV lower traces and 10 msec ; D-F 500 rV upper ; 20 JLV lower and 5 msec.

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Entorhinal field potentials to CA3 stimulation were generally positive and monophasic, averaging between 500 PV to 2.0 mV in peak amplitude and S-16 msec in duration. The latency to onset of the entorhinal potentials was dependent upon the hemisphere stimulated. Entorhinal unit discharges were often recorded coinciding with some or all of the components of the evoked potential and in the filtered records were easily distinguished from background noise by their waveform and amplitude. The latency to onset of these unit discharges was never shorter than 1 msec following the onset of the positive evoked potential. Figure 2 illustrates some of the characteristics of the entorhinal evoked potential to commissural stimulation. In Figures 2A and B the entorhinal potential is shown as predominantly positive (sometimes with an early negative deflection) with unit discharges coinciding with the positive slope. Figure 2C shows the relationship between the entorhinal evoked potential and the extracellular field potentials recorded simultaneously from the dentate gyrus molecular layer. Figure 2D shows another example of an entorhinal evoked potential and unit discharge from a different experiment. We could find no significant difference between field potentials recorded from the medial vs lateral entorhinal cortex. Figures 2E and F show the relationship between the entorhinal unit discharges and the ipsilateral CA3 and dentate gyrus field potentials. These earlier potentials (preceding the entorhinal unit activity shown on the bottom trace) were elicited by contralateral CA3 stimulation and have been described elsewhere as reflections of the monosynaptic inputs to these areas via the commissural pathways (3, 4, 12, 27). The CA3 and dentate potentials occurred within 5 msec of stimulus onset. The entorhinal activation followed approximately 3-10 msec later (Fig. ZD). Stimulation of the ipsilateral CA3 region evoked the shortest latency field potentials in the entorhinal cortex (8-15 msec). No other stimulated region of the ipsilateral hippocampus evoked either unit discharges or field potentials in the entorhinal cortex with latencies shorter than 10 msec. Figures 3A and B show the monosynaptic cell layer (CL) and dendritic (ML) ipsilateral association path responses (see below) in the dentate gyrus recorded simultaneously with the differentiated entorhinal field potentials. As can be seen in the lower traces, the onset of neuronal activity in the entorhinal cortex begins 15 msec after stimulus onset. Activation of the entorhinal cortex was also achieved through stimulation of the contralateral CA3 field. Figures 3C and D show the earlier monosynaptic commissural potentials recorded in the cell and molecular layers of the dentate gyrus, and the subsequent differentiated entorhinal activity which occured between 20-25 msec after stimulus onset. Comparison of the differences in latency of activation of the entorhinal cortex from the two

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of associational and commissural stimulation in the activation FIG. 3. Comparison of the entorhinal cortex and subsequent reactivation of the dentate gyrus. A. Associational stimulation. Recordings from granule cell layer (CL) field potentials and differentiated entorhinal potentials (En). B. Field potentials recorded from 100 pl above granule cells (ML) ; note early extracellular synaptic wave. C. Cell layer positivity to commissural stimulation and late positivity (upper trace) subsequent to entorhinal activation shown in the lower trace. D. Extracellular synaptic waves recorded 100 @rn above the granule cell layer evoked by commissural stimulation (upper trace). Late synaptic negativity is also shown subsequent to entorhinal activation [lower trace (En) J. Calibration : 5 msec ; 1 mV for field potentials, 20 pV for differentiated lower traces. Stimulus at sweep onset.

stimulus loci indicates that direct excitation of the ipsilateral CA3 field produced a response in the ipsilateral entorhinal area 3-7 msec earlier than stimulation of the contralateral CA3 field. Differentiation by high pass filtering of the evoked entorhinal field potentials proved to be an effective means of comparing differences in onset latencies between the two forms of stimulation (lower traces in Figs. 3A and C and Figs. 3B and D). Stimulation of the contralateral CA3 field also activated the monosynaptic projections to the ipsilateral dentate gyrus and CA3 field. Threshold stimulation currents were generally higher for eliciting entorhinal field potentials than for monosynaptic activation of the contralateral CA3 and dentate gyrus regions. The latency to activation of the ipsiIatera1 CA3 region was 3-5 msec and was similar to that demonstrated previously (3, 4, 12). This region (ipsilateral CA3) was the most effective stimulation site for eliciting entorhinal potentials. In addition, the latency difference between the onset of the contralateral vs. ipsilateral generated entorhinal potentials was roughly equivalent to the latency to Thereactivate the ipsilateral CA3 region via the commissural pathway. fore, activation of the entorhinal cortex from contralateral CA3 stimulation

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was probably mediated by the monosynaptic excitation of the ipsilateral CA3 field. Supportive evidence for this hypothesis was obtained from experiments demonstrating no difference between stimulation of either the CA3a and CA3c hippocampal loci for entorhinal activation. Stimulation of the CA3a field did not activate the monosynaptic connections to the dentate gyrus (see below) but was effective in generating entorhinal potentials which were similar to those elicited hy CA3c stimulation. The entorhinal response to hippocampal stimulation can therefore be categorized as a long-latency positive. sometimes negative-positive, field potential. The latency to onset of these potentials depended upon the hemisphere which was stimulated. The distribution of the field potentials was rather broad, persisting for up to 3mm in dorsoventral excursions of the recording electrode. Thus, it appears that both the lateral and medial segments of the entorhinal cortex respond to hippocampal stimulation. The significance of these entorhinal events for subsequent activation of the dentate gyrus will he considered in the next section. Latrnf Activation of the Dcnfafc Gyms via tlzc Entorhlal Cortex. In a previous report (12) we briefly described the appearance of a “late” evoked potential in the dentate gyrus following suprathreshold commissural stimulation. The onset of this late potential was between 18-25 msec (Figs. 2C and F). Its laminar profile resembled that of field potentials elicited by direct stimulation of the entorhinal cortex and perforant path axons. Figure 4 is a comparison of laminar profiles of extracellular field potentials recorded from the dentate gyrus. In Figs. 4A and C the laminar profile of two different field potentials elicited by commissural stimulation are shown for two different stimulus frequencies (l/set and 4/set). Figure 4B shows the laminar profile of the field potential elicited by entorhinal stimulation. Each of the profiles was constructed by measuring the amp15 tude of the respective field potentials 1 msec after onset to avoid contamination by other potentials not related to the extracellular synaptic current (5, 12. 24). It is clear that in Fig. 4C the “late” potential profile matches closely the profile of the potential generated by direct stimulation of the entorhinal cortex (Fig. 4B). In addition, the late potential was present in both the dorsal and ventral “blades” of the dentate gyrus and, in all cases,could be shown to reverse from negative to positive in the granule cell layer (Figs. 4B and 7). The late potential remained positive when the electrode was moved through the hilus. From these findings it is unlikely that the late potential was generated outside of the dentate gyrus since the negative to positive reversal of that potential was resricted to a total distance of 300-350 pm which corresponds closely to the dendritic and somal portions of the dentate granule cells (as verified by marking the

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FIG. 4. “Online” laminar profiles of dentate field potentials elicited by commissural stimulation (A & C) and stimulation of the entorhinal cortex (B). A. Field potential profile of monosynaptic commissural potential in the dentate gyrus measured 1 msec after onset (at TX) following threshold stimulation of contralateral CA3c. Note negative peak approximately 100 pm above the granule cell layer (GC). B. Field potential profile of entorhinal potentials in dentate gyrus measured 1 msec after onset (T,) following threshold stimulation of the ipsilateral entorhinal cortex. Note peak of negativity at 200 em above granule cell layer (BC). C. Field potential profile of the “late” dentate gyrus potential elicited by suprathreshold commissural stimulation also measured 1 msec after onset (Tl). Note negative peak 200 pm above granule cell layer (GC) similar to the profile in B from direct stimulation of the entorhinal cortex. Note also difference in amplitude of the late potential vs commissural potential in the insert. Record taken 200 I/m above g cell layer. Calibration: A & B 1 msec 500 pV; C 5 msec 250 I.LV. Arrow indicates stimulus onset. Traces are redrawn for illustrative purposes. HF-hippocampal fissure.

A 2“-

Tl

. . ...

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location of the potentials through the tip of the recording electrode ; 27). Field potentials generated within the overlying CA1 region could not have contributed to this potential, since no responses occurred in this region with a latency greater than 10 msec (12, 24). Latent activation of the dentate gyrus occurred shortly after the field potentials and unit discharges were recorded from the entorhinal cortex. Simultaneous recordings from the entorhinal cortex and dentate gyrus showed that late potentials in the dentate molecular layer were usually preceded by entorhinal unit discharges (Fig. 2F and below). The latency between the onset of unit discharges in the entorhinal cortex and the late negative wave in the dentate molecular layer was between 2.7 and 3 msec. Occurrences of late potentials were seldom detected in the absence of entorhinal unit bursts, however, in such instances where no unit discharges were recorded at a particular location the entorhinal electrode could often be moved a short distance (0.1 mm) into an area which did show unit activation. Presumably the entorhinal cells giving rise to the perforant path axons were stimulated indirectly by the CA3c stimulation. Lomo (24) has shown a similar latency to activation of the dentate gyrus through stimulation of the angular bundle. In the latter circumstance, disynaptic activation of the dentate gyrus occurs through excitation of afferent projections to the entorhinal cortex traveling in the angular bundle which synapse upon the cells of origin of the perforant path. Ipsilateral CA3c stimulation activated the recently described associational pathway which originates in the CA3c/CA4 region and terminates in the same dendritic zone as the commissural projections to the dentate molecular layer (18, 33). There have been no prior reports of dentate gyrus activation through this pathway. However, our experiments have confirmed that such stimulation elicits a dendritically located negative extracellular synaptic wave whose maximum is restricted to the inner onethird of the molecular layer. This is the same region in which commissural stimulation elicits a maximally negative extracellular synaptic response ( 12, 27). The “associational” potentials were generally 0.5 to 1 mV larger than the commissural potentials and occurred with a latency of 0.5 to 2 msec (Fig. 3). Thus, associational stimulation activated the granule cell dendrites approximately 1 to 1.5 msec earlier than commissural stimulation. An important consideration in delineating the characteristics of the ipsilateral associational input to the dentate gyrus was the possible contamination by antidromic invasion through the mossy fibers of the dentate granule cells. Careful analyses however revealed that antidromic dentate gyrus field potentials were negative at the cell layer and positive in the molecular regions. In addition, the duration of the negative antidromic potentials was approximately half that of the orthodromic extracellular

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synaptic potentials recorded from the inner molecular region. The antidromic negative wave has been described as a “population spike” resulting from the synchronous excitation of a large number of granule cells (8). Stimulus intensities necessary to elicit such antidromic population spikes from the ipsilateral CA3c locus were generally an order of magnitude higher than those necessary to produce the extracellular synaptic wave and unit driving in :he dentate gyrus through the orthodromic associational pathway. We have previously reported that commissural stimulation does not elicit “population spikes” from the dentate granule cell layer (12). This was also true for ipsilateral CA3c stimulation. Only two or at the most three individual units could be driven through stimulation of the associational projections. On the basis of these criterion it was therefore assumed that ipsilateral CA3c hippocampal stimulation activated the associational pathway and thereby orthodromically excited the granule cells of the ipsilateral dentate gyrus. Suprathreshold or higher frequency stimulation of the associational pathnegative potentials in the outer way also elicited “late” extracellular molecular layer (Fig. 3). These late potentials were very similar to those previously described for commissural stimulation with the following important exceptions : (a) the latency to onset of the associational generated late potentials was 15-18 msec, approximately 3-5 msec earlier than commissural elicited late potentials (Fig. 3B vs. D) ; (b) correspondingly the latency to activation of dentate granule cells (see below) was 20 msec (vs 23 msec for commissural stimulation) ; and (c) the associational late potentials were somewhat smaller in amplitude than those produced by commissural stimulation (Fig. 3A vs B). Thus, the major difference between the two forms of stimulation was in the latency to activate the entorhinal cortex and subsequently reactivate the dentate gyrus through the perforant path. In close agreement with the laminar profile of the late potential, unit recordings showed corresponding discharges when the electrode was located within the dentate granule cells. Figure 5 shows four sets of recordings of single unit discharges : A and E are instances in which weak commissural and associational (ipsilateral CA3c) stimulation elicited a single unit discharge following monosynaptic activation of these fiber pathways. Figures 5B and D show that with higher frequency stimulation a second “late” unitary discharge occurred after an appreciable latency (15-18 msec) corresponding closely to the onset of the late positive field potential. Although in both cases the early and late unit discharges were similar in waveform and amplitude, it was not possible to establish whether they were in fact one and the same unit. These unit discharges could be enhanced by potentiation of the late field potentials with higher frequency commissural or associational stimulation.

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FIG. 5. Comparison between commissural and associational stimulation of evoked unit discharges and positive field potentials recorded from the dentate gyrus granule cell layer. A & C are instances in which stimulation of either pathway at 0.5 Hz fired a single granule cell with appropriate latency differences (bottom trace). Field potentials from the same electrode are shown in the upper traces. Note that unit discharges appear as “notches” in the field potential recordings (upper records), but as differentiated spikes in the filtered records below. Stimulus onset is coincidental with onset of sweep. B & D show the recordings from the same electrode after stimulation of each pathway [associational (B) or commissural (D) ] at 4 Hz with the same stimulus intensity. Note the “late” granule cell discharge elicited by both forms of stimulation. See text for further discussion. Calibration-5 msec; 1 mV for field potentials, 20 pV for units. Stimulus at sweep onset.

The recordings show that the late negative field potentials obtained from the middle and outer molecular regions (Fig. 4) were accompanied by individual or groups of unit discharges within the dentate granule cell layers (Fig. 5). Such observations are in full agreement with prior investigations of direct perforant path elicited dentate gyrus evoked potentials and unit discharges (5, 6, 12, 24). The time of appearance of the late potential and late unit driving in the dentate gyrus following commissural and associational stimulation, paralleled the latency differences for the activation of the entorhinal cortex by the two respective stimulus locations (Fig. 3). Since the latency difference between the two forms of stimulation for the onset of the late negative wave in the dentate molecular layer remained at approximately 3 to 5 msec, it is unlikely that additional

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FIG. 6. Comparison of late dentate potentials elicited by stimulation of either CA3a field or CA3c field of the contralateral hippocampus. Dotted line in drawing marks the tract of the stimulating electrode. Traces are redrawn from photos of individual responses. Break in the trace indicates the onset and offset of the stimulus. Re-recording locus in contralateral dentate gyrus. Note early commissural monosynaptic potential from CA3c stimulation but not from overlying CA3a field. Total trace is 60 msec. Amplitude of late potential in dentate from CA3c stimulation is 1 mV. Stimulus shown as gap in trace.

delays other than the time required to activate the ipsilateral CA3 region (via commissural projections) contributed to the difference in latency between the late dentate responses. In support of this conclusion, generation of the late potential in the dentate gyrus was also accomplished by stimulation of the CA3a subfields both ipsilaterally and contralaterally in a manner similar to that observed for the activation of the entorhinal cortex (see above). Figure 6 shows the results of an experiment in which the stimulating electrode was systematically lowered through the CA3a field into CA3c. Recordings in this case were from the contralateral dentate gyrus. Note that in the top trace, the late dentate field potential appears following stimulation of the CA3a subfield in the absence of an earlier monosynaptic (3-5 msec) negative dendritic potential (lower trace CA3c stimulation). Late potentials, however, were never recorded unless the stimulating electrodes were in close proximity (within 50 pu) to the pyramidal cell bodies. These findings demonstrate that prior monosynaptic activation of the dentate gyrus is not required for either activation of the entorhinal cortex or for the subsequent generation of late dentate potentials. However, if the stimulating electrode was moved to the CA1 region (stratum radiatum and pyramidale) late potentials were not recorded from the dentate gurus and the entorhinal potentials were longer in latency and reduced in amplitude. Although a complete investigation of other hippocampal regions which might activate the entorhinal cortex has not been completed, the above results indicate that excitation of the ipsilateral CA3 subfield is a highly effective means of synaptically driving both the entorhinal cortex and its subsequent projection to the dentate gyrus.

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Eliwaination of the Late Dentate Gyms Potential Following Destruction of the Ipsilateral Entorhinal Cortex. In order to test the hypothesis that the late dentate gyrus potentials in fact originated in the ipsilateral entorhinal cortex, we performed a number of experiments in which lesions were made in the entorhinal cortex during and at various times prior to stimulation of the contralateral CA3c field. In most cases, the stimulation could be left on during the surgical procedure for observation of its immediate effects upon late potentials. Figure 7A shows records obtained immediately before, 90 min, and 3 days after the lesion. In all cases, the early commissural monosynaptic potential was unaffected by the lesion. However, the late potentials (arrows) were completely eliminated following the ipsilateral entorhinal lesion. The late potential could be seen to disappear usually within 10 to 15 set after the application of the lesioning current. In some cases the late potential would appear 10 to 20 min later but at considerably reduced amplitude, requiring larger stimulus currents to elicit it. A second lesion with the electrode relocated approximately 1.5 mm more ventral and/or 0.5 mm more lateral to the original locus was then found sufficient for complete elimination of the late potential. Histological analyses of these lesions revealed a complete destruction of, or considerable damage to, the dorsal entorhinal region projecting to the recording site (Fig. 7B). In all cases the effects of the lesion were permanent. Animals tested over time periods as long as one year following the lesion had no recovery of the late potential to either associational or commissural stimulation even though stimulus voltages were increased to five times prelesion levels. It appears conclusive therefore that destruction of the ipsilateral entorhinal cortex completely eliminates the late potential in the dentate gyrus. To ascertain whether the ipsilateral entorhinal lesion destroyed the cells of origin of the late potential or merely interrupted fibers of passage from other possible anatomical pathways, we performed a number of control lesions. Neither destruction of the contralateral entorhinal cortex, septum (medial, or lateral or both), nor sectioning the dorsal psalterium altered the driven entorhinal potentials or subsequent late potentials in the dentate gyrus. We have found that as long as the ipsilateral entorhinal cortex remains intact (including the perforant path), the late dentate gyrus potential is unaltered by destruction of these other extrahippocampal regions. Lesions of the retrohippocampal region were not attempted. Such lesions would almost certainly interrupt the perforant path or other angular bundle fibers, thus making the result uninterpretable with respect to the specific locus responsible for the elimination of the “late” potential. HOWever, careful histological examination of effective ipsilateral entorhinal

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FIG. 7. A. Elimination of the late dentate gyrus potential by destruction of the ipsilateral entorhinal cortex (see B). Field potentials evoked by commissural stimulation. Scale at left indicates distance from granule cell layer (CL) from which potentials were obtained. Left panel-Shows late potentials prior to ipsilateral entorhinal lesion (see arrows). Middle panel-Potentials recorded 90 min after entorhinal destruction (same electrode tract). Right panel-Recordings made 3 days later from same dentate region after stimulating electrode was cemented to skull. Note: Elimination of late potentials shown in right panel (arrows) subsequent to lesion but persistence of monosynaptic commissural potentials. All traces are averages of 8 individual potentials. Stimulus occurs at sweep onset. Traces are redrawn from original records. B. Horizontal section of rat entorhinal area showing two lesions on

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lesions showed that it was possible to eliminate the late potential without destroying any of the pre- or parasubicular regions (Fig. 7). As discussed below, it does not seem likely that the retrohippocampal regions could be directly responsible for the late potential in the dentate gyrus. However, it is possible that these regions could play an important mediational role in the activation of the entorhinal cortical cells whose axons project to the dentate gyrus. Frequemy Potentiation of Entorlzinal Cortical Potentials Produces Potentiation of the Dentate Late Potential. Simulation of either the ipsilateral associational pathway or the commissural pathway with frequencies between 4 and 15 Hz produced both frequency and posttetanic potentiation of the polysynaptically evoked entorhinal potentials. Frequency potentiation of the entorhinal potentials was often followed closely by frequency potentiation of the late potential in the dentate gyrus, the latter occurring only after entorhinal potentials reached about one half of their maximal potentiate amplitude. Figure 8 shows that both the entorhinal and dentate gyrus late potentials were increased up to five times their prepotentiated amplitudes. The most effective frequencies for potentiation were between 4 and 7 Hz, although higher frequencies were also adequate. Frequency potentiation of the entorhinal and late dentate gyrus potentials occurred in the absence of noticeable potentiation of the associational or commissural monosynaptic potentials. Recordings of CA3 potentials (cell layer positivity) showed a similar lack of potentiation to commissural stimulation (Fig. 8). We have found that these direct monosynaptic pathways are potentiated with much lower (nearer threshold) stimulus intensities than those required to potentiate the entorhinal and late dentate gyrus potentials. An interesting outcome of these experiments is demonstrated in Fig. 8J3. Associational stimulation, while producing as much frequency potentiation of the entorhinal potentials as commissural stimulation (Fig. SA), only effected slight increases in the amplitude of the late dentate gyrus potential. A similar result was obtained for granule cell unit discharges and the cell layer positivity. In these comparisons it was always the case that commissural stimulation was more effective potentiating dentate gyrus late potentials than associational stimulation. However, as can also be seen in Figs. SC and D, the earlier monosynaptic potentials in the dentate gyrus from associational stimulation were larger than those produced by commissural stimulation (see above). each side of the brain.

Right hemisphere lesions were contralateral to recording electrodes in Fig. 7A and had no effect on the late potential recorded in the dentate gyrus. Left hemisphere shows a typical lesion which is discussed under A. Regions of destruction can be seen as faint “rings” surrounding electrode holes. Short survival time did not allow complete deterioration of damaged tissue in this experiment.

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FIG. 8. Potentiation of entorhinal and late dentate gyrus field potentials. A. Top trace; Entorhinal field potentials potentiated by 7 Hz commissural stimulation (En). Bottom-Dentate gyrus molecular layer recordings of monosynaptic and late potential potentiation recorded simultaneously. Note : Only the late dentate potential shows from CA3 frequency potentiation. B. Top trace same as in A, Bottom-Recording field shows no potentiation (record obtained simultaneously). Calibration: 5 msec; 2.50 pV. C & D comparison of commissural elicited potentiation with associational stimulation at same frequencies (same recording arrangement as in A except entorhinal field potentials have been inverted). Note lack of potentiation from association stimulation in the late dentate wave and only slight potentiation of entorhinal field potentials. Calibration : 5 msec; 500 pV. E & F show simultaneous recording of dentate field potentials (molecular layer) and entorhinal unit discharges before (E) and immediately after (F) 7.0 Hz (10 set) commissural stimulation. Note increased unit discharge in entorhinal cortex following frequency potentiation. Also note increased amplitude and duration of late extracellular wave recorded from 200 p above dentate granule cells. Calibration : 10 msec; 1 pV (field potentials) 20 ,LV (units). Stimulus at sweep onset.

In addition to the frequency potentiation produced by associational and commissural stimulation of both entorhinal and late dentate gyrus field potentials, post-tetanic potentiation was also observed. Single pulses delivered immediately after the stimulus train elicited enhanced field potentials and unit discharges in both regions. Of particular interest was the correspondence between the enhanced entorhinal unitary discharge and the concomitatnt enhancement of the late negative synaptic potential in the dentate gyrus molecular layer. Figures 8E and F show the effects of single commissural stimulus pulses before and after a 5 set train of 7 Hz pulses. Following the stimulus train both the number of entorhinal units activated by a single pulse and the amplitude of the negative field potential (synaptic wave) in the granule cell dendrites were markedly increased (Fig. 8).

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DISCUSSION The above experiments have demonstrated that intense or repetitive stimulation of the hippocampus evoked polysynaptic field potentials in numerous regions of the entorhinal cortex. This excitation was produced by stimulation of both intrahippocampal (associational) and interhippocampal (commissural) circuitry. Whether or not such stimulation was a sufficient or necessary condition for entorhinal activation was only partly resolved by the demonstration that stimulation of the CA1 field did not produce similar activating consequences, Because of the long latency between the initial activation of hippocampal circuitry and the onset of the field potentials and unit discharges in the entorhinal cortex, it can be assumed that the neuronal pathway involved was not monosynaptic and very likely polysynaptic in nature. However, the facility with which entorhinal field potentials could be evoked by either suprathreshold or moderate frequencies of hippocampal stimulation illustrates that the polysynaptic hippocampal-to-entorhinal pathway may be one which reliably “tracks” the degree of hippocampal activity once transmission through the circuit is initiated. In addition to the ease with which driven entorhinal activity was obtained from stimulation of CA3, the extent to which these potentials appeared throughout both the lateral and medial aspects of the dorsal entorhinal areas 27 and 28 was surprising. Excursions of the microelectrode over a distance of 2-3 mm continuously yielded driven unit activity with little variance in field potential amplitudes. Under certain stimulation conditions the entorhinal potentials were potentiated by repetitive hippocampal stimulation (i.e., 4-7 Hz). This occurred in the absence of potentiated hippocampal responses. Both recruitment of additional units and increased amplitude of field potentials illustrated that the hippocampal-to-entorhinal circuit showed post-tetanic potentiation as well as frequency potentiation. In this regard, the entorhinal field potentials were potentiated by a stimulus which did not produce potentiation of hippocampal field potentials. Since the other elements in the hippocampal-to-entorhinal circuit are not known, we cannot ascertain at this time which synaptic junction may have been responsible for the enhancement of the polysynaptic potentials. It is possible, however, that the observed increases were the result of a decreased inhibitory influence which is normally active through other portions of this circuit. The question as to what exact anatomical connections are responsible for these polysynaptic potentials is not resolved by these experiments. Hjorth-Simonsen (20) maintained that the CA3-to-entorhinal hippocampal efferent pathway originates in the ventral or caudal hippocampus. If this circuit was responsible for the effects seen here, then some means of transmitting impulses from the dorsal or rostra1 hippocampus to the

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ventral or caudal hippocampus must have also been utilized. This would explain in part the long latency of the potentials recorded from the entorhinal cortex to dorsal hippocampal stimulation. In such a circuit at least two synapses would be interposed prior to activation of the entorhinal cells (i.e., one in the connection from dorsal to ventral hippocampus; and one from the ventral hippocampus to the entorhinal cortex). The only pathway which might provide such a connection is the longitudinal associational tract of Lorento de No (23, 26). This tract apparently originates within the CA3 fields and travels the entire longitudinal axis of the hippocampus in a septotemporal excursion. The fibers are assumed to traverse the length of the hippocampus via the stratum lacunosum, but their site of termination is presumed to be in stratum radiatum of the CA2 and CA3 fields (23). Two factors in the reported findings support the conjecture that the longitudinal tract mediated the hippocampal-to-entorhinal influences : stimulation of the CA3 commissural system activated the CA3c region of the contralateral hippocampus monosynaptically (latency = 3 msec) on every occasion when polysynaptic potentials were recorded in the entorhinal cortex (Fig. 2) ; and low voltage stimulation of the ipsilateral CA1 field did not evoke entorhinal field potentials of a similar latency or amplitude. These factors indicate that unless the ipsilateral CA3 neurones are activated, the entorhinal cortex does not respond to hippocampal stimulation. The rostra1 CA3 field must activate the caudal CA3 field in order to incorporate the hippocampal efferent connections to the entorhinal cortex shown by Hjorth-Simonsen (20). The caudal or “temporal” CA3-toentorhinal pathway could be activated through the longitudinal associational system of Lorente de No (26). Although these assumptions are highly speculative, they represent a likely means whereby stimulation of intraand interhippocampal fiber systems might participate in the activation of entorhinal neurons. Alternative anatomical pathways which must be considered in the context of a polysynaptic hippocampal-to-entorhinal circuit include the retrohippocamal structures, pre- and parasubiculum and the subicular cortex itself. Recent physiological evidence has shown that CA1 fibers project into these regions, primarily to the subiculum (9). However, anatomical evidence from this laboratory suggests that the subiculum proper projects to the septum and not to the entorhinal cortex (Geisert et al., in preparation). This leaves the presubicular and parasubicular complexes as possible candidates for mediation of the polysynaptic hippocampal influences upon the entorhinal cortex. Both of these regions send fibers to the entorhinal cortex and each receives projections from the limbic cortex and anterior thalamic nuclei as well as crossed connections from

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their contralateral counterparts (10, 26). Unfortunately, there is little evidence in support of a direct hippocampal projection to the pre- or parasubicular complexes. Hjorth-Simonsen (20) found no evidence for terminal degeneration in these regions following lesions of the various hippocampal subfields of the rodent. The functional nature of these retrohippocampal regions has only recently begun to receive the attention of neurophysiologists. Hence, the possibility of involvement of these regions in the hippocampal-to-entorhinal circuit is still a question for future analyses. In the present investigation, the results of the lesion experiments considerably reduce the likelihood of septal or thalamic mediation of hippocampal influences upon the entorhinal cortex. Massive septal lesions did not affect hippocampally driven entorhinal evoked potentials even during application of the lesion producing current. Furthermore, histological analyses revealed that extensive damage to the postcommissural fornix was evident in a majority of the septal lesions, thus decreasing further the likelihood of mediation via the anterior thalamic nuclei (13). A final question concerning the anatomical basis for these findings deals with the role of the CA3c commissural fibers which project to the contralateral hippocampus. The limited septotemporal dispersion of these fibers indicates that they could not be responsible for activating the contralateral temporal CA3 field directly. The furthest that the commissural fibers have been traced by autoradiographic and degeneration techniques is to mid septotemporal levels of the contralateral hippocampus ( 10, 19, 28). The above considerations have led us to conclude that the longitudinal associational pathway is the most likely candidate for mediation of the hippocampal-to-entorhinal influences. Since it has been shown that the “temporal” CA3 field projects to the more dorsal aspects of the entorhinal cortex in the rat (20), this would explain how activation of a ventral hippocampal efferent pathway (i.e., through the longitudinal associational system) might excite the dorsal regions of the entorhinal cortex as demonstrated in our experiments. In this respect the hippocampal efferent pathway is not “online” with either the direct perforant path projections to the hippocampus (21, 22) nor with the “lamellar” arrangement of intrinsic fiber pathways within the hippocampus (7, 24). The temporal CA3-toentorhinal pathway as described by Hjorth-Simonsen (20) conforms to a less discrete dorsoventral topographic arrangement of projections involving all levels of the entorhinal cortex. This would account for the rather wide dispersion of hippocampally generated evoked potentials recorded from the entorhinal cortex in the experiments reported here. The “late” potentials observed in the dentate gyrus to commissural and associational stimulation were obtained under stimulus conditions which

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were effective for generating potentials in the entorhinal cortex. The late potentials were shown to constitute inputs from the entorhinal cortex by four separate criteria: (a) laminar profiles were the same as for potentials elicited monosynaptically through direct entorhinal stimulation ; 0) destruction of the ipsilateral entorhinal cortex eliminated the late potentials but did not influence the monosynaptically driven associational and commissural potentials; (c) late potentials were never observed in the absence of earlier occurring entorhinal evoked potentials ; and (d) potentiation of entorhinal field potentials resulted in a subsequent potentiation of dentate gyrus late potentials. From these results it can be tentatively concluded that the late potential is generated by entorhinal neurones through the axon terminals of the perforant path. The functional significance of these hippocampal-to-entorhinal connections is still not understood. Adey, Sunderland, and Dunlop (1) demonstrated that stimulation of the ipsilateral hippocampus in the marsupial phalanger elicited evoked potentials in the entorhinal cortex at a latency similar to that reported here (i.e., 10 msec). Destruction of the entorhinal cortex in this animal produces an extreme reduction in aggressiveness and a heightened curiosity (2). Destruction of the entorhinal cortex produces deficits in passive avoidance learning in cats (15) and rodents (31). Recordings of entorhinal EEG (14) and unit activity (29) during learning production indicate a close correspondence between hippocampal and entorhinal processes as a function of behavioral change. Perhaps these observations represent instances in which the bidirectional nature of these projections were observed. The set of experimental results presented in this paper provide the impetus for further analyses of hippocampal-entorhinal interactions. The chain of neuronal events initiated by stimulation of the hippocampal commissural system is a complicated one. In order of occurrence these events are as follows: (a) both the contralateral dentate granule cells and CA3 pyramidal cells are activated almost simultaneously within 3 msec; (b) the CA1 field is then activated 1 to 3 msec later (West et al., in preparation) ; (c) the entorhinal cortex is then excited 7 to 10 msec later; and finally (d) the dentate gyrus receives a volley from the entorhinal cortex 3 msec thereafter through the perforant pathway. If it is assumed that the longitudinal associational tract is activated by commissural stimulation, then activity persists within these intra-hippocampal and extrahippocampal circuits for up to 25 msec following a brief pulse to the contralateral CA3 field. Thus, the consequences of activating this single fiber system are multiplied both spatially and temporally into divergent neural information, incorporating most of the major hippocampal-entorhinal connections. The possible functional significance of such circuitry is not known. However,

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the “wrap around” time from initiation of the monosynaptic potentials in the dentate gyrus to the late potential arrival via the perforant path (20-25 msec) corresponds closely to the interstimulus interval which produces facilitation of the perforant path synapses (25). We have demonstrated in these experiments, a set of conditions whereby it is possible to study the characteristics of the entorhinal-to-dentate synaptic relationship in the absence of direct electrical stimulation of the presynaptic fibers. It is clear from experiments that controlled stimulation of either the ipsilateral or contralateral CA3 field is capable of activating large areas of the entorhinal cortex. This results in the subsequent excitation of perforant path projections to the dentate gyrus and the eventual driving of the dentate granule cells. Our preliminary analyses of these relationships suggest that repetitive stimulation of the CA3 fields (4-7 Hz) provides the optimal conditions for polysynaptic activation of the entorhinalto-dentate gyrus circuitry. The potentiation studies have indicated that an increased frequency of CA3 stimulation results in an increased input to the dentate gyrus from the entorhinal cortex. In this context we have shown that the frequency of neural impulse arrival within the ipsilateral CA3 region determines the probability with which information will be transmitted to the entorhinal cortex and subsequently relayed into the dentate gyrus. Thus the entorhinal-to-dentate gyrus projections appear to monitor the degree of hippocampal excitability. REFERENCES 1. ADEY, W. R., S. SUNDERLAND, and C. W. DUNLOP. 1957. The entorhinal area: Electrophysiological studies of its interrelation with rhinencephalic structures and the brain stem. Elcrtromccph. Clin. Ncurophysiol. 9: 309-324. 2. ADEY, W. R., N. C. R. MERRILIZES, and S. SUNDERLAND. 1957. The entorhinal area: Behavioral evoked potential and histological studies of its interrelationships with brain stem regions. Rra& 10: 414-439. 3. ANDERSEN, P. 1959. Intrahippocampal impulses: I. Origin, course and distribution in cat, rabbit and rat. rlrta Phgsiol. Scmd. 47: 63-90. 4. ANDERSEN, P. 1960. Intrahippocampai impulses : III. Basal dendritic activation of CA3 neurons. Acta Physiol. Sca)ld. 48: 209-230. 5. ANDERSEN, P., B. HOLMQUIST, and P. E. VOORHOEVE. 1966. Entorhinal activation of dentate granule cells. Arta Phgsiol. Stand. 66: 448-460. 6. ANDEKSEN, P., and T. LOMO. 1970. Mode of control of hippocampal pyramidal cell discharges, pp. 3-21. In “The Neural Control of Behavior.” R. E. Whalen, R. F. Thompson, M. Verzeano, and N. M. Weinberger [Eds.]. Academic Press, New York. 7. ANDERSEN, P., T. V. P. BLISS, and K. K. SKHEDE. 1971a. Lamellar organization of the hippocampal excitatory pathways. Exp. Brain Rrs. 13: 222-238. 8. ANDERSEN, P., T. V. P. BLISS, and K. K. SKREDE. 1971b. Unit analysis of hippocampal population spikes. E.zp. Brain Rcs. 13: 20%221.

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