Field potentials in rat hippocampus: Monosynaptic nature and heterosynaptic post-tetanic potentiation

EXPERIMENTAL

NEUROLOGY

Field

Potentials

Nature

and

21,

iti

de

Hippocampus:

IZQUIERDO

Monosynaptic

Post-Tetanic AND

BEATRIZ

VASQUEZ

Ciencias Quimicas, Universidad National Estafeta

Received

Rat

Heterosynaptic IVAN

Institute

133-146 (1968)

October

32, Cdrdbba,

23,1967;

Potentiation 1 de Cdrdoba,

Cba., Argentina

Revision

Received

February

19,1968

Subicular, commisural, and fornical stimulation evoked large field responses in the dentate gyrus and dorsal hippocampus of rats. Hippocampal responses were negative above and positive below the upper third of apical dendrites in CAl, CAZ, and CA3; the application of a pressing foot on the alveus resulted in a distortion of this polarity pattern. Since a compound action potential was found in the fornix during the rising phase of hippocampal field responses, it was concluded that the latter correlated with excitation of pyramidal cells. The following frequency of dentate and hippocampal potentials was high (up to 125/set) and similar for both; thus, it was concluded that both were monosynaptic responses. On the basis of excluding several impossible mechanisms, hippocampal field potentials were attributed to a flow of cations, through the extracellular clefts, from the stratum pyramidale down to the stratum radiale. Hippocampaland dentate-evoked potentials suffered post-tetanic potentiation; in the case of hippocampal responses, this was both homo- and heterosynaptic. The possible relevance of the latter to mechanisms of learning, and the possible genesis of both by (Kc), accumulation were discussed. Introduction

Electrical stimulation of the subiculum (or entorhinal cortex), of the contralateral alveus, or of the dorsal fornix, produces large evoked potentials in the dorsal hippocampus of diverse species (l-4, 8, 12, 17, 18, 20, 21, 24). These regions send fibers both to pyramidal (8, 9, 12, 18) and to dentate granule cells (1, 4, 5, 9, 12, 17, IS), and the latter are connected to the former by way of the “mossy” fibers (9, 12, 17, 18). In addition, pyramidal cell axons send collaterals to basket cells, which, in turn, have endings on pyramidal somata (2, 3, 9). In theory, then, hippocampal field responsesto the inputs mentioned above could be due to the activation of either mono- or disynaptic pathways, and indeed there are conflicting data, in the literature, in support of either possibility (l-5, 8, 12, 17, 18). This 1 Work done for the most part in the laboratories of the “Instituto Mercedes y Martin Ferreyra,” C&doba. Supported by a grant (NO 2389) of the Consejo National de Investigaciones ,Cientificas y Tecnicas, Argentina, to I. Izquierdo. CQ 1968 by Academic

Press Inc.

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paper treats of an attempt to clarify this point, and, further, includes a report on heterosynaptic post-tetanic potentiation (PTP) of hippocampal field responses. Methods Ninety-six adult albino rats of both sexes (180-540 g) were used, under deep urethane anesthesia (1.5-2.5 g/kg, ip). The preparation was an “open” one ; i.e., the dorsal hippocampus and fornix were exposed on both sides by suction of the overlying brain tissue, and electrodes were implanted with micromanipulators, under direct vision. Stimulating electrodes were pairs of 100 p stainless steel wires twisted together, with tips about 0.5 mm apart, and insulation scratched away for about 0.5 mm at the tips. Electrodes used for recording from the hippocampus and dentate gyrus were also 100 p stainless steel wires, and those used for recording from the fornix were 25 p stainless steel wires ; in both cases, they were insulated except for the flat section surface at the tips, and their impedance was of about 100 and 300 K, respectively. Recording was always “monopolar” ; the indifferent lead was a steel clip in the oral mucosa. Amplification and oscilloscope display (Tektronix 502) and filmation (Grass camera), as well as rectangular-wave stimulation (Tektronix 160-series and isolation transformers) were conventional. Results

Polarity of Hippocampal Field Potentials. If hippocampal field responses to subicular, commisural, and fornical stimulation were due to recurrent activation of basket cells, they should be positive at the stratum pyramidale, as was indeed reported by Andersen, Eccles and L$yning (2, 3). Basket cells inhibit pyramidal cells and this should cause outward current at the somata of the latter (3). This was found by us not to be the case in normal conditions. In fact, field responses in CAI, CA2, or CA3, to stimulation of any of the three inputs, were, as described by several authors in other species (8, 12, 20, 21), negative above, and positive below the upper third of stratum radiale (Fig. 1, left). If the overlying cortex was not sucked way, and electrodes were implanted stereotaxically, the polarity and phase reversal of hippocampal responses were also in this fashion. When, however, a glass rod was placed gently pressing the alveus of the recording side, evoked responses changed their polarity to one like that reported by Andersen, Eccles, and L$yning (2, 3) (Fig. 1, right). Parenthetically, this change was also observed when blood clots were allowed to accumulate on the hippocampus, or when records were taken too soon after the operation or after a seizure, or when too many electrode penetrations were made (usually, one or two per experiment).

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FIG. 1. Hippocampal field responses to subicuiar stimulation recorded at various depths (in micra, middle column) from the alveus, in CA2 For reference, the depth of the stratum pyramidale is from about 200~ to 400~. Records to the left (positive up) were in the absence, and those to the right (positive down) in the presence of a pressing foot (see text). Calibration : 3 mv, 30 msec.

Consequently, we conclude that the use of a pressing foot on the alveus, useful as it may be to reduce pulsation and permit intracellular recordings (1-5, 26)) introduces a distortion of the extracellular currents responsible for hippocampal field responsesin normal conditions, and that these cannot be explained by outward current at the stratum pyramidale, thus ruling out the recurrent circuit via basket cells. Excitatory Nature of Hippocampal Field Potentialsi At this point, it was still possible that hippocampal responseswere envelopes of inhibitory unit activity of pyramidal cells brought about by some pathway other than that of basket cells. In fact, both excitatory and inhibitory postsynaptic potentials have been recorded from these cells, upon stimulation of the

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inputs used by us, and within the same latency of our evoked potentials (l-5, 11,26). We did, then, simultaneous recording from the fornix and the hippocampus of the same side. Upon subicular and commisural stimulation, a sharp negative wave was found in the fornix, in synchrony with the rising phase of hippocampal field responses (Fig. ZA). When records were taken at successive distances along the fornix, this negative wave was found to suffer apparent cephalad travel at a rate of 3-10 m/set (Fig. ZB). On one hand, this rate was probably too high for electrotonic propagation, but suitable for conduction velocity by medullated fibers of a caliber such as those of the rat fornix (see Fig. 2A of Ref. 19). On the other hand, the

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m set FIG. 2. A: upper beam, fornix compound action potential (negative down, see text) ; lower beam,hippocampal field response recorded from the alveus (negative up), both upon subicular stimulation. Calibration : beam duration, 200 msec, hippocampal potential 1 mv high. This amplitude corresponds to 90 pv in the upper beam. B: latency to the peak of the fornix compound action potential (abscissae) versus approximate distance from the anterior fimbrial border (ordinates) in the same experiment in which records in A were taken.

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fornix wave was unaltered by filtering out frequencies under SO cycle/set, which was done using a Tektronix 122 amplifier, but hippocampal responses were abolished by this procedure. In consequence, the fornix wave was not an electrotonic propagation of hippocampal responses, but, rather, a compound action potential. Therefore, hippocampal field potentials were not envelopes of inhibitory unit responses of pyramidal cells, but were, instead, synchronous with excitation of these. Extracellular spikes have been reported, of pyramidal somata, as occurring mostly at a similar phase angle of evoked responses ( 11,20). Monosynaftic Nature of Hippocampal Field Responses. Having thus established that hippocampal field potentials were coincident with excitation of pyramidal cells, it still had to be solved whether they were monosynaptic responses, or secondary to a granule-cell relay ( 12, 18). Indeed, stimulation of the three inputs mentioned above evoked field potentials that reversed polarity along electrode tracks in the dentate gyrus (Fig. 3B), and these responses were smaller and had an earlier peak than hippocampal potentials. The start of the rising phase of dentate responses was, however, about synchronous with that of hippocampal potentials. This, plus the similar polarity of both types of response at the stratum radiale, very often made it difficult to detect the dentate wave there (Fig. 3A, lower). In more superficial records, where the hippocampal response became negative, the dentate potential was seen as a notch or as a delay in the rising phase of the former (Fig. 3A, upper). In rabbits, a sharp negative wavelet attributed to granule cell firing has been described instead of the dentate response as reported here (12,18,24). The latency of the peak of denate responses was of 2.60 .-c 0.11 msec (n = 31) for fornical stimulation; of 2.57 + 0.38 msec (n = 31) for subicular stimulation, and of 2.77 -t- 0.09 msec (n = 31) for commisural stimulation. (Means + standard error; differences not significant at the p = 0.05 level.) The latency of the peaks of hippocampal responses, measured at the stratum radiale, was of 9.30 ,+ 0.30 msec (a = 38) for fornical stimulation ; of 8.55 & 0.31 msec (n = 47) for subicular stimulation, and of 9.27 & 0.38 msec (n = 44) for commisural stimulation. Here, again, differences between the three means were not significant, but the three were significantly larger than the corresponding values for dentate responses. This was not, however, necessarily an indication that hippocampal field potentials were secondary to a granule cell relay. On one hand, phase reversal of hippocampal responses was identical in CAl, CA2, and CA3 (21), whereas mossy fibers do not reach CAl. On the other hand, it was found that both hippocampal and dentate responses followed with no

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recorded from the alveus FIG. 3. A: hippocampal responses to fornical stimulation (upper record) and from 800 a below it (lower record ; positive down) ; in the superficial recording, the arrow indicates a wavelet which reversed phase in the dentate gyrus (see B) ; in the deeper recording, this wave is lost into the larger and also positive hippocampal response. Calibration : 0.5 mv, 5 msec. B: phase reversal of the wavelet marked by an arrow in A (positive down) ; the more positive record was from a depth of 950 p from the alveus and succeeding records, in the order of lesser positivity, were each from a point 100 p deeper than the preceding one. The more negative record (depth, 1350 p ) IS . retouched. CaIibration: 0.2 mv, 5 msec. decrement (in fact, often with some potentiation: 12, IS), and for over frequencies of up to 63/set to any of the three inputs ; 15 set, stimulation and followed with decrement, for at least I set, stimulation frequencies of up to 125/set. Furthermore, in each individual animal, the following frequencies of hippocampal and of dentate responses were about identical. Consequently, and in view of the fact that reflexes other than monosynaptic would not be expected to follow these rates of stimulation, it can be concluded that hippocampal and dentate potentials were all monosynaptic, and, of course, synaptically unrelated. Aftereffects of Tetanic Stimulation. After 1-15 set of tetanic stimulation (4, 5, 6.3, 10, 20, 31.2, 50, 63, 100, 200. 312 or 5OO/sec) to any of the

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three inputs, there was 4A, B ; SA ; 6) and the ever, potentiation was (8)) but it extended to 4A, B; 5A). Heterosynaptic PTP

PTP of responses in both the hippocampus (Figs. gyrus dentatus (Fig. 6). In the hippocampus, hownot restricted to responses to the stimulated input the responses to the other two inputs as well (Figs. in the hippocampus

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-v--‘v FIG. 5. A : potentials evoked by subicular (left) and commisural (right) stimuli ; shock artifacts, too small to be seen; upper record, 1 set before tetanus; middle record, 1 set after a lOO/sec, 5 set tetanus to subiculum; lower record, 7 set after tetanus : note seizure activity. B : potentials evoked by fornical (left) and subicular (right) stimulation; upper record, 1 set before; middle record, 1 set after a SO/set, 2 set tetanus to subiculum; lower record, 10 set after tetanus. Calibration for A and B : one beam lasts 200 msec; the first potential to the left in the upper record of A is 0.9 mv high.

pairing of stimuli. It occurred when the input to which tetani were given was either the first or the second of a pair at SO-500 msec intervals between both, or when stimulation to inputs other than the one tetanized was either maintained at a l/set rate, or cut off during the tetanus. Heterosynaptic PTP has been reported before, both in the hippocampus (13, 14) and elsewhere (13, 14, 23, 34)) but for responsesclearly identiiied as poly synaptic. The responsesfor which heterosynaptic PTP was reported before in the hippocampus were to amygdaloid, fornical, and commisural stimulation (13, 14) ; they were, however, of longer latency than those described in this paper; and, fui-thermore, they often were double responsesand only the second, undoubtedly polysynaptic peak suffered heterosynaptic PTP (14). Such long latency, double potentials were occasionally encountered by us upon very strong stimulation, more often in CA3 than elsewhere. They were found not to follow stimulus frequencies over 5 or lO/sec.

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The PTP of hippocampal (filled circles) and dentate responses (white to fornical stimulation (tetani : lO/sec, 3 set). This is a plot of five condeterminations, at 3-min intervals, in the same animal.

In the hippocampus, but not in the gyrus dentatus, there was occasionally post-tetanic depression, homo- and heterosynaptic (Figs. 4B ; 5B). Not infrequently, seizure activity occurred superimposed upon, instead of, or after PTP or depression (Fig. 5A). Discussion

Hippocampal field potentials 2 cannot be explained by currents massively entering or leaving neuronal membranes at any given layer. For reasons already exposed, disynaptic pathways via basket (Fig. 7A) or granule cells (Fig. 7B) may be ruled out. Massive firing and blocking at pyramidal somata may also be ruled out; for one thing, such firing is neither massive (21) nor blocked at the somata (Fig. 2A) ; for another, it is restricted only to the rising phase of evoked potentials (21) (Fig. 7C). Since the effect of the inputs here studied on pyramidal cells was an excitatory one, the somatal negativity of field responses could certainly not be correlated 2 Hippocampal responses from under a pressing foot will not be discussed here. Andersen, Eccles and L&ning (2, 3) have interpreted them as due to outward currents from pyramidal somata ; though this obviously does not apply to what we chose to call “normal” responses, it may certainly be a suitable explanation for their potentials, inasmuch as it fits with their other experimental data (2, 3).

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FIG. 7. A: inhibitory endings of basket cells causing outward current from pyramidal somata (2, 3) cannot account for our hippocampal potentials, which were negative at the cell body layer. B : if all inputs acted on a granule cell relay, the layer of mossy fiber endings would not be far from that of maximum evoked potential negativity (X2), but the following frequency of these, as well as anatomical considerations, preclude them from being disynaptic (see text). C: massive firing of pyramidal cells would cause large inward current at the stratum pyramidale, but could not account for evoked responses for reasons discussed in the text. D : excitatory terminals in the dendrites (such as those of the perforant path schematized here) would cause localized inward currents and relative cation depletion in the neighbouring extracellular space. Cations would then flow to this region down a concentration gradient, from upper layers such as the stratum pyramidale. If this flow were entirely through the extracellular clefts, and these were narrow enough, a voltage gradient would be produced of the same polarity as hippocampal field potentials.

with the dendritic location of the corresponding synapses (1, 4-6, 9, 17), as was done by Andersen and others (1, 4, 5). It is to be noted that these authors had found evoked potentials of different polarity than ours, and that they have usedpressing feet ( 1,4,5). Thus, the only explanation we find that fits the peculiar polarity of hippocampal field potentials, is that of a flow of cations from the stratum pyramidale to, mainly, the stratum radiale, through the extracellular clefts (Fig. 7D). The trigger for this ionic current would be the cation depletion in the space around excitatory terminals that presumbably occurs upon local inward synaptic currents. These currents would take place principally in the stratum radiale, since the axons corresponding to the inputs we stimulated terminate mostly there (6, 9, 17). In the case of commisural responses, some flow upwards to the stratum oriens would have to be assumedadditionally in evoked responses (6)) which is not at all incompatible with this explanation, since the negativity of evoked responses

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does indeed decline from the stratum pyramidale upward (Fig. 1, left). This explanation rests upon three additional assumptions, none of which is, perhaps, unreasonable : (1) that synaptic currents are so strictly localized to the subsynaptic space and membrane, that it is not possible to record significant signs of them with gross electrodes ; (2) that the clefts are the only extracellular space available to ionic currents; (3) that the clefts impose such restrictions on ionic flow, that a cation concentration gradient along them would cause a voltage gradient. The first assumption may be extended perhaps to microelectrode recordings as well (12)) although with these there have been suggestions that postsynaptic potentials may be found extracellularly and that it is their sum which produces hippocampal evoked potentials (20). The second assumption is substantiated by rather overwhelming recent evidence (15, 25) that the extracellular space in nervous tissue involves nothing in addition to the clefts. The third assumption may be made to rely on the fact that the clefts are, in the hippocampus, seemingly narrower than elsewhere (17, 19, 26) and, indeed, very little ramified at and about the stratum pyramidale (19). There is indirect evidence that hippocampal clefts do restrict ionic flow ( 17,24,26). Since heterosynaptic PTP of monosynaptic reflexes seems to be, as is the narrowness of its extracellular space, a unique property of the hippocampus, it appears reasonable to try to correlate both. Heterosynaptic PTP of monosynaptic responses has been looked for, but not found elsewhere (13, 14, 23), with the one possible exception of the report by Libet (29) on the “late negative” ganglionar potential. The latency (0.2-0.4 set) and the uncommon mechanism of this ganglionar response (10) prevents one, however, from considering it as a monosynaptic response in the usual sense. There is indirect evidence that after repetitive stimulation, large K+ accumulations occur in the hippocampal clefts (17, 22, 24, 26). Since high ( K+)O is known to increase the amount of transmitter released per spike (16, 25,30), ob viously hippocampal PTP, both homo- and heterosynaptic, might be explained by a ( K+)O accumulation brought about by presynaptic repetitive firing during tetanic stimulation. In the case of heterosynaptic PTP, a spread of the high ( K+)O to other terminals, through the clefts, would have to he assumed, which is certainly not unreasonable (17, 22, 24). Also, since one other effect of high (K+),, might be to depolarize and to depress axonal firing, it might be supposed that it had this effect as predominant over the one on transmitter release in some instances, and thus post-tetanic depression would also be explained. The frequent associa-

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tion of PTP or depression with seizures (Fig. 4C) may be taken as further support for the K+ hypothesis (see 17, 22, 24). PTP has, indeed, been explained in the past by an accumulation of (I(‘-), (7, 31, 32). This explanation was, in later years, largely abandoned due to criticisms based on the presumed impossibility of a significant increase in (K+)” in in vitro PTP experiments, in which the extracellular space was thought to be continuous with the bathing solution, and thus very large (see 23). These criticisms are, however, no longer tenable since the convincing demonstrations of restricted 200-300 A spaces in excitable tissues immersed in baths (1525). Kandel and Taut (27, 28) have found that after pairing a shock to one nerve with a brief burst to another, there was heterosynaptic facilitation in certain Aplysia ganglion cells ; in one of these cells, strict pairing of the shock and the burst was not necessary (28). Clearly, this latter observation would be very similar to heterosynaptic PTP as described in this paper, were it not for the fact that it occurred only upon the use of very short bursts (two to ten stimuli) (28)) whereas we have found heterosynaptic PTP after tetani of various and longer durations, and within a wide range of frequencies (see Results). This range of frequencies included those of the medial septal unit firing, brought about by reticular stimulation which, when conducted to the hippocampus by the dorsal fornix, causes in it “theta” or faster electroencephalographic rhythms (17, 33). Since these rhythms are specially prominent and long-lasting during the acquisition phase of learning (see 17 for references), it is tempting to postulate that hippocampal heterosynpatic PTP may be a major factor in, if not the actual basis of, association learning. Indeed, there is some agreement that a heterosynaptic “transfer of facilitation” (13, 14, 23, 27), such as that of heterosynaptic PTP (13, 14) should be involved in association learning. Also, the evidence in favor of a crucial role of the hippocampus in learning, and in particular in the phase of acquisition, is great (17). However, since heterosynaptic PTP as described in this paper was not dependent on any specific pairing of stimuli, we feel more inclined to postulate it as a possible basis for states of increased (behavioral) performance. In fact, medial septal burst firing and hippocampal theta or faster rhythms are typical not only of learning, but also of other behavioral situations categorized by increased performance and “attention” ( 17, 33). Since, however, the acquisition phase of learning is characterized by an increased level of performance, and since heterosynaptic PTP does indeed involve, by definition, heterosynaptic transfer of facilitation (13, 14), it may be asked up to what extent high performance, acquisition, theta or faster rhythms, and heterosynaptic PTP are not interrelated, or at least inseparable phenomena.

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