k'euroscience Vol. 37,No. 3,pp.693-707,1990 Printed in Great Britain

03064522/90$3.00+ 0.00 Pergamon Press plc 6 1990IBRO

SYNAPTIC CONNECTIONS OF DENTATE GRANULE CELLS AND HILAR NEURONS: RESULTS OF PAIRED INTRACELLULAR RECORDINGS AND INTRACELLULAR HORSERADISH PEROXIDASE INJECTIONS H. E. SCHARmAN,*f

D. D. KUNKEL~ and P. A. SCHWARTZKROIN$$

*Howard Hughes Medical Institute, Department of Neurobiology and Behavior, SUNY at Stony Brook, Stony Brook, NY 11794, U.S.A. IDepartment of Neurological Surgery, University of Washington, Seattle, WA 98195, U.S.A. §Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, U.S.A. Abstract-Simultaneous intracellular recordings were made in the dentate gyrus of rat hippocampal slices, from pairs of the following cell types: granule cells, interneurons located in the granule cell layer, hilar interneurons, and spiny hilar “mossy cells”. Granule cells were found to have strong excitatory effects on mossy cells and intemeurons. Intemeurons inhibited granule cells as well as other interneurons. No synaptic connections from mossy cells onto other cell types were found, within the confines of the slice, using intracellular recording methods. However, at the ultrastructur~l level, axon terminals of horseradish peroxida~-~11~ mossy cells were found making synaptic contacts in the hilus on dendrites of interneurons. These studies provide the first step towards determining the functional interactions of the various cell types in the fascia dentata.

For many years, studies of the fascia dentata have been focused on its principal cell type, the granule cell. To aid experimentation, the dentate circuitry has often been simplified to include only the major afferent system-the perforant path fibers from entorhinal cortex, and one efferent system-the projection of granule cells to Amman’s horn. However, several lines of investigation have indicated that the fascia dentata is much more than a relay station. In particular, there are many cell types in the fascia dentata in addition to granule cells, which receive afferent information and modulate granule cell output. Numerous local circuit cells, or “interneurons”, are located in the molecular layer, the granule cell layer, and the hilar region.‘,5.‘8,22.24,25,2*-30,43,*3 These inter_ neurons differ in their neurotransmitter content 5~‘8~24~25~28~30~43~53 and presumably their effects on targets; however, generally their axons remain within the local dentate region, where they contact granule cells’*~*’and other interneurons.# Another common cell type is the large, spiny “mossy cell” of the hilus,‘.62.46the axon of which projects both ipsilaterally and contralaterally to the inner molecular layer,3~7~‘9~2’~35~5”58 as well as ramifying in the area around the mossy cell soma.33,46 Numerous fiber systems originating in subcortical areas have also been shown to terminate in the fascia dentata (i.e.

tTo whom correspondence should be addressed. Abbreuiationr: AP, action potential; EPSP, excitatory postsynaptic potential; HRP, horseradish peroxidase; IPSP, inhibitory postsynaptic potential; R,,,input resistance; RMP, resting membrane potential.

originating in septum,‘4+16locus coeruleus,36 or raphe nuclei4’), where they may modulate granule cell activity directly, or indirectly via contacts on interneurons and/or mossy cells. Finally, the granule cell axon does not project only to the pyramidal cells of hippocampus; rather, it has been shown to arbor& profusely in the hilar region, 2*9where it innervates interneurons and/or mossy cells. These studies of the dentate have made it clear that the circuitry of the fascia dentata is quite complex. The data suggest that interactions of granule cells with other cell types may be extremely important for regulating granule cell output. Yet, very little is known about the fundamental synaptic interactions of granule cells with other cell types in the granule cell body layer and with hilar cells. We report here our initial studies of the interactions between granule cells, interneurons of the granule cell body layer and hilus, and hilar mossy cells, as examined by simultaneously recording from pairs of synaptically connected cells. Our experiments were aided by previous studies that described some of the axonal projections of the different cell types, providing hints about where one might expect synapses to occur. Firstly, since granule cell axons are known to arborize extensively within the hilus, we investigated connections between granule cells and hilar neurons. Secondly, since many interneurons contain the inhibitory transmitter GABA,5~‘8~28~30~43.53 and since numerous GABAergic, symmetric synapses exist on granule cells ‘8,27inhibitory/hyperpola~zing effects of interneurons on granule cells were sought. Thirdly, the synaptic interactions of mossy cells were examined; in

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particular, we studied mossy cell interactions with granule cells (via the mossy cell projection to the inner molecular layer), and with hilar interneurons (via mossy cell axon collaterals that branch within the hilus). EXPERIMENTAL PROCEDURES

Preparation qf slices and recording procedures

Hippocampal slices were made from 93 female adult Sprague-Dawley rats (I 25-175 g) as previously described.49 The animal was decapitated and the brain was removed and placed in oxygenated (95% 4-S% CO,), cold (4°C). modified Kreb’s Ringer (in mM): NaCl 126. KC1 5, CaCl, 2, MgSO, 2, NaHPO, 1.25, NaHCO, 26 and dextrose 10; pH = 7.2-7.4). Initial studies were conducted using slices cut with a standard, transverse orientation.4@‘” Subsequently (and for the majority of experiments) a different slicing orientation was used, as has been described and diagrammed elsewhere. 49 Firstly, a block of tissue containing the hippocampus was isolated from one of the hemispheres. The hemisphere was placed on a piece of moistened filter paper with the curved, lateral surface upward, and two straight, parallel cuts were made through the entire hemisphere with a razor blade: the first cut was oriented parallel to the longitudinal axis of the hippocampus (i.e. approximately 45°C from the coronal plane), just anterior to the most rostra1 areas of the hipp~ampus; the second cut was made parallel to the first cut, just posterior to the most caudal areas of the hippocampus.@ The resultant block of tissue contained the entire hippocampus. Subcortical and neocortical tissues were trimmed from the outer borders of the block, and the rostra1 cut surface of the block was glued with cyanoacrylate (Krazy glue) to a Vibratome stage (Vibroslice, Frederick Haer), submerged in cold (4”C), oxygenated buffer, and sliced into 4OOpm sections; each section was thus cut parallel to the rostra1 cut surface. This cutting procedure resulted in longitudinal slices such as the one illustrated in Fig. 1A. We recorded from areas of these slices that included both the dentate granule cells and the hilar region (Fig. IA). Slices were maintained in a warm (35°C) and oxygenated (95% 015% CO,) recording chamber, at an interface of gas and perfusate. Intracellular recordings were made with 80-120 MS2resistance electrodes pulled horizontally (Brown-Flaming puller, Sutter) from capillary-filled borosilicate glass (I .Omm outer diameter, 0.6 mm inner diameter; A&M systems), and were filled with 4 M KCH,COOH in 0.05 M KCl. Simultaneous intracellular recordings were made using a dual intracellular amplifier (Neurodata model IR-283) with an internal bridge for intra~lluiar stimulation. Data were recorded on tape (Neurodata DR-284) and analysed by computer (Noriand 300l-DMX). Calculation of input resistance (R,,) was based on responses at steady state to hyperpolarizing current pulses (O.l-l.OnA, 100 ms) delivered at the resting membrane potential (RMP) of the cell. R,R,,was calculated from the slope of the linear segment of the currentvohage relation (I--Y curve). For comparison of excitatory postsynaptic potentials (EPSPs) of mossy cells and interneurons, measurements of EPSP amplitude were made at the cells’ RMPs, which were comparable (see Results), and were measured from RMP to peak of the response. Identification of ceN types

Cell types were usually differentiated on the basis of their location and their electrophysiolo~cal properties. However, in some cases, mossy cells (n = 9) or interneurons (n = 7) were filled with Lucifer Yellow,46 and morphological information was also obtained to identify the cell. Granule cells were impaled in the granule cell layer, and were characterized by a variety of electrophysiological criteria, including

strong frequency adaptation, high RMP (- 75 to -80 mV), and low level of spontaneous activity.@’ Putative interneurons were impaled in both the granule cell layer and in the hilar region, and were characterized by weak spike frequency adaptation, and a large, fast afterhy~rpola~zation. Z.23~32-3447+5’ Interneurons that are referred to as interneurons “of the granule cell layer” were located either in stratum granulosum or at the border of stratum granulosum and the hilus. In the latter case the intracellular electrode was angled so that it passed into the granule cell layer as it was lowered through the slice; this impalement procedure avoided mistaking a “hilar interneuron” for a “granule cell layer interneuron.” When recording from hilar neurons, the intracellular electrode was located at least 50 pm from the border of stratum granulosum and the hilus, and the electrode was angled away from stratum granulosum (avoiding the possibility of mistaking granule cell interneurons for hilar interneurons). Pains were taken to differentiate these two types of interneurons because they may have different functional roles and connectivities. Studies have already demonstrated that interneurons of the fascia dentata do differ, not only in their histochemical identities,s~‘s~“~25~**~~~43~53.” but also in their electrophysiological characteristics.49,s2 Hilar mossy cells were easily distinguished from granule cells and interneurons by their electrophysiological properties.46 Mossy cells have a longer time constant, often discharge in bursts, have variable (usually depolarizing) afte~otentials, and very large, frequent spontaneous EPSPs. Since the firing pattern of mossy cells is somewhat similar to area CA3c pyramidal cells, impalements of mossy cells were made only in the region of the hilus that does not contain area CA3c pyramidal cells (i.e. less than 150pm from the base of the granule cell layer; see Fig. 1A and B). This restriction regarding the area where mossy cells were impaled was necessary because some mossy cells were not impaled with an electrode filled with Lucifer Yellow or horseradish peroxidase (HRP), and hence were not identified morphologically after the recording procedure. Morphological and histochemical data suggest that there are many subtypes of interneurons; I5,1*.?2.24,?5,28-30.43.53.54 simi_ larly, it is a simplification to group all of the large spiny cells of the hilus into the “mossy cell” category.’ However, since it is presently possible to differentiate these subpopulations only by detailed mo~ho1ogical and immunoh~stochem~cal techniques, we did not attempt to discriminate between subpopulations of mossy cells and interneurons in this study. Determination of synaptic connections

Pairs of cells were located less than 750,um apart, and typically less than 3OOpm apart. Pairs which included a granule cell and a hilar neuron were arranged so that the hilar neuron was in the area of the hilus closest to the blade in which the granule cell was impaled. One cell was stimulated with intracellular current injection while the second cell was held at various membrane potentials; subsequently the second cell was stimulated and the membrane potential of the first cell was manipulated. Changing membrane potential made it possible to distinguish between EPSPs and inhibitory postsynaptic potentials (IPSPs), although exact reversal potentials were often difficult to determine (see Results). Intracellular stimulation consisted of: (1) continuous d.c. to depolarize the cell so that discharge occurred spontaneously, or (2) depolarizing current pulses, (0. l-l .OnA) and durations of various amplitudes (IO-200 ms). A pair of cells was judged to be synaptically connected when a consistent membrane potential change occurred in the postsynaptic cell, immediately after discharge in the presynaptic cell. A consistent response, timelocked with the action potential of the presynaptic cell, differentiated synaptically coupled potentials from spontaneous postsynaptic potentials.

A

GC

C

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A mV

20ms

. MC Fig. I. Excitatory effects of granule cells on simultaneously impaled mossy cells. (A) A diagram is shown of the typical experimental arrangement for dual recording experiments in longitudinal hippocampal slices. Specifically, this illustration identifies the sites of recording during Lucifer Yellow injection of the mossy cell (MC) shown in B and the granule cell (GCkmossy cell dual recordings shown in C. Arrowheads in A and B point to the mossy cell axon. CAl, CA1 pyramidal cell layer; CA3, CA3 pyramidal cell layer; DG, dentate granule cell layer. (B) A mossy cell of the dentate hilus is shown following intracellular injection of Lucifer Yellow. The cell was impaled very close to the granule cell layer, which is outlined by white lines. The photograph is oriented as shown in A. Scale bar = 50 pm. (C) While recording from the mossy cell in A, a granule cell was impaled that appeared to be synaptically connected to the mossy cell; APs elicited in the granule cell (GC; top trace) by intracellular current injection (a OS-nA, IOO-ms depolarizing current pulse) were immediately followed by EPSPs or an AP in the mossy cell (MC; lower trace). Capacitive artifacts at the start and the end of the intracellular current pulse are clipped, and indicated by the dots near the granule cell trace. Capacitive artifacts of granule cell APs are indicated by the asterisks in the mossy cell trace. Arrowheads indicate spontaneous EPSPs of the mossy cell, unrelated to any AP activity of the simultaneously recorded granule cell. Granule cell RMP = -78 mV. Mossy cell RMP = 61 mV. (D) A granule cell and mossy cell recorded simultaneously in a different slice from the slice in parts A-C. A depolarizing current pulse (0.3 nA, 100 ms) evoked three APs in the granule cell. After each granule cell AP, an EPSP or one to two APs were recorded in the mossy cell. The granule cell was depolarized with d.c. to -65 mV during this recording; granule cell RMP = - 80 mV. Mossy cell RMP = - 65 mV.

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rnir~ee~l~~ar injection of ~orserad~hperoxidase For intracellular injection of HRP, recording electrodes were pulled identically to other recording electrodes, but backf;lled with a 4% solution of HRP VI (Sigma) in 0.5 M Tris-buffer and 0.5 M KCl. The shafts of recording electrodes were subsequently filled with 0.5 M KCl, and tips were broken (on the mesh holding the slices) to obtain a final electrode resistance of 100-150 MR. After a cell was identified by its ele~trophysiological characteristics, it was filled by injecting l-4 nA de~la~~ng pulses (20 ms duration) at 30 Hz for 2-min periods. Between each 2-min period of depolarizing pulses, the cell was hyperpolarized for 1045 s. Cells were processed for HRP if they could be held for at least four 2-min periods of HRP injection and if the cell RMP did not depolarize above -2OmV during that time. Microelectrodes were then slowly withdrawn from the cell, and the slice was fixed between two pieces of filter paper immersed in 2.5% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Light and electron microscopy of harseradi.~~l peroridasejilled neurons After 3-16 h in fixative, slices were rinsed in buffer and cryoprotected in 0.1 M sodium cacodylate buffer containing 30% sucrose (?--I6 h). Frozen sections (6~-80 Mm) were cut, and sections were rinsed in buffer. Sections were processed for HRP and examined according to previously described procedures.“.‘4,5* The sections containing HRP-tilled cells and processes were photographed using bright-field illumination on a Leitz Dialux 20 phosomicroscope. Trimmed sections containing the cells were further processed for electron microscopy using a routine schedule that included: post-staining in 1% 0~0, in 0.15 M sodium cacodylate (pH 7.4) for I h at room temperature, alcohol dehydration, and embedding in Medcast. All sections were stained with uranyl acetate and lead citrate before examination with a Philips 300 or 410 electron microscope. Serial thin sections were cut from cells injected with HRP in order to follow processes and synaptic contacts. Fifteen cells were processed for electron microscopy: in six of those, serial sectioning was used to follow synaptic profiles. Criteria for the identification of contacts have been previously described.‘0~“~5”

ol.

RESULTS Synaptically connected cell pairs were relatively rare. Out of 159 slices examined, in which 912 pairs of cells were recorded, only 44 pairs (less than 5%) were found to be synaptically connected (Table I). Nevertheless, the connections observed were consistent, and therefore provide a preliminary guide to the functional jnteractions between dentate neurons. Granule cells Synaptic efSects of granule cells on mossy cells. Of 694 pairs of simultaneously recorded mossy cells and granule cells, 694 different granule cells and 84 different mossy cells were impaled. Action potentials (APs) evoked by intracellular current injection in 36 of the granule cells were followed by EPSP-like events in 20 mossy cells (Table 1). In 25 out of the 36 pairs, the excited mossy cell was located within 200 pm of the granule cell; in the remaining cases the cells were separated (laterally in the slice) by a greater distance. Multiple granule cells in the same electrode track (up to three cells per electrode track), or multiple granule cells located in different electrode tracks that were within 50pm of each other, demonstrated an excitatory connection to the same mossy cell. Thus, convergence exists from neighboring granule cells to a single mossy cell. There also appears to be convergence from widely separated granule cells to a single mossy cell, since two granule cells separated by 600pm were both able to drive a single mossy cell. All of the effects of granule cells on mossy cells were strongly excitatory. Examples of the large mossy cell EPSPs that were evoked by single granule cell APs are shown in Fig. 1. At the mossy cell RMP, these EPSPs often triggered single or multiple APs

Table 1. Numbers of cell pairs that were tested for synaptic interactions Cell types paired Cell 1 Cell 2 GC GC GC HIL-INT HIL-INT GCL-INT MC GCL-INT HIL-INT

MC GCL-INT HIL-INT MC GCL-INT MC MC GCL-TNT HIL-INT Total

Number of pairs 694 50 69 36 5 28 22 3 6 913

Number of cells 1 2 694 50 69 36 5 28 22 3 6

84 14 21 26 5 24 22 3 6 Ill8

Number of connections 1+2 2-1 36

0

I

I

2 1

1 0 0 0 0 0 0 44

1 0 0 0 0

f-2 0 0

0 0 1 0 0 0 0

Smmttaneous recordings are categorized above according to the cell types (arbitrarily designated 1 or 2) in the pair. The Number of pairs refers to the total number of simultaneous recordings made of cell type 1 and cell type 2. For example, there were 694 cases where a granule cell and mossy cell were recorded simultaneously. In many experiments, one cell of a pair (Cell 1) was discarded and another cell was impaled, while maintaining the recording from the other cell (Cell 2) of the original pair; therefore, Number of pairs does not always equal Number of cells. For example, of the 694 pairs of simultaneously recorded granule cells and mossy cells, 694 different granule cells were examined, but only 84 mossy cells. The Number of connections found are also listed [cell 1 driving cell 2 (l-+2), cell 2 driving cell 1 (2-r I). or reciprocally connected pairs (1%2)]. GC, granule cell; MC, mossy cell; GCL-INT, interneuron located in the granule cell layer; NIL-INT, hilar interneuron.

Synaptic connections of dentate cells

(Figs l-4). Subthreshold responses of mossy cells to multiple granule cell APs often summated (Fig. 1C and D). The very strong connections between granule cells and mossy cells were evident in the bursts of mossy cell APs that could be driven from single granule cell APs (Fig. 2A) and in the one-to-one following seen even at high frequency of granule cell discharge (Fig. 2B). Large EPSPs were recorded in mossy cells even when the paired granule cell was injured (Fig. 2C). Often, as the granule cell recording electrode was slowly lowered into the granule cell layer, bursts of mossy cell APs were recorded, similar to the evoked bursts shown in Fig. 2A. These bursts increased in frequency as the granule cell recording electrode was lowered further, and ceased when that electrode was removed from the slice, suggesting that the induced mossy cell activity was a result of activating granule cells by injuring them with the electrode. In a few dual recordings, mossy cell excitatory events (postsynaptic potentials or APs) triggered immediately after a single granule cell AP were followed after several milliseconds by other excitatory events (Figs 3 and 4). These later postsynaptic potentials or APs were seen consistently in such pairs, and were of sufficiently different amplitude to be easily distinguished from spontaneously occurring EPSPs (see Fig. 3B).

Fig. 2. Strong excitatory effects of granule cells on mossy cells. (A) Simultaneous recording of a granule cell (GC; top) and a mossy cell (MC; bottom) illustrating the strong excitatory effects of granule cell APs (elicited in response to a 0.4-nA, lOO-ms depolarizing current step) on a mossy cell. Note that even single granule cell APs could evoke a burst of mossy cell APs. Granule cell RMP = 70 mV. Mossy cell RMP = -68 mV. (B) Simultaneous recording of a different granule cell (top) and mossy cell (bottom). A train of APs was triggered in the granule cell by a l.O-nA, lOO-ms depolarizing current pulse. The mossy cell fired APs after almost every granule cell AP. Granule cell RMP = -66 mV. Mossy cell RMP = -61 mV. (C) Mossy cell activity (MC; bottom) was recorded while a second electrode impaled a granule cell and produced injury discharge (GC; top). During the initial injury discharge of the granule cell, each granule cell AP was followed by an EPSP in the mossy cell. Note, however, the variability in EPSP amplitude and waveform. All APs are clipped. Mossy cell RMP = - 67 mV. NSC 37i3-E

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Fig. 3. Long-lasting excitatory effects of granule cells on mossy cells. (A) Simultaneous recordings are shown from a granule cell (GC; top) and mossy cell (MC, bottom). A single AP was triggered in the granule cell by a 0.3-t&, IOO-msdepolarizing current pulse. Two APs occurred in the mossy cell immediately after the granule cell AP, and an additional pair of APs occurred several milliseconds later. At no other time during the paired recording (which lasted approximately 10 min) did such doublets of mossy cell APs occur spontaneously, but this pattern of discharge was seen in a high proportion of cases in which granule cell APs were triggered. The granule cell was depolarized with d.c. to -62 mV; granule cell RMP = - 75 mV. Mossy cell RMP = - 70 mV. (B) A different granule cell (GC; top) and a mossy cell (MC; bottom) were recorded simultaneously and a depolarizing current pulse was injected intracellularly in the granule cell. The mossy cell response consisted of a burst of APs. Note that EPSPs and APs continued well after the last AP of the granule cell. Such a high frequency of mossy cell EPSP and AP occurrence was only evident when the granule cell was activated. Lower trace shows a sample of spontaneous mossy cell activity. Same pair of cells as shown in Fig. 2A.

Determination of the reversal potentials of evoked EPSPs was complicated by the variability in amplitude of EPSPs observed at fixed membrane potentials (Fig. 4). The same granule cell could evoke a small EPSP at one point during the recording and a large EPSP a few seconds later; in addition some EPSPs appeared to be multiphasic (Figs 4 and 5). Triggering granule cell APs at low frequency did not reduce this variability. Further, extreme polarization of the cell was difficult with potassium acetate-filled electrodes (i.e. in the absence of potassium channel blockers), and cell depolarization was not very useful since mossy cells fired at a high rate when membrane potential was even slightly depolarized from RMP. Given the variability, and the difficulty recording at polarized membrane potentials, extrapolation of measured EPSP amplitudes to reversal potential was uncertain. As would be expected of an EPSP, however, mossy cell EPSPs did increase in amplitude as the cell was hy~rpolari~d and decreased with mossy cell depola~zation (Fig. 5). There were some instances of apparent “failure” of synaptic transmission, when a granule cell AP did not effect a detectable membrane potential change in the mossy cell (Figs 4 and 5). The incidence of failures

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Fig. 4. Variability in granule cell excitation of mossy cells. From left to right are four traces from simultaneous recordings of a granule cell (GC; top), and a mossy cell (MC; bottom). An AP was evoked in the granule cell by intracellular depolarizing current pulses (0.3 nA, IO0 ms. at 0.3 Hz). Immediately after the granule call AP, the mossy cell produced (from left to right): a typical EPSP, an AP, a very large, multiphasic EPSP (upon which one AP occurred), or no response. Excitatory events that occurred with a long delay after the granule ceil AP are indicated by the small arrows. Same pair of cells as in Fig. 3A.

granule cell, which varied between 0.5 and 0.05 Hz. The variability in response and the number of failures did not appear to depend on the RMP or R, of the granule cell or mossy cell. Many of the EPSPs ehcited in mossy cells foliowing granule cell APs were very similar, in wavefo~ and amplitude, to spontaneous EPSPs observed in mossy cells (Fig. 6A). This was also the case for granule cell-driven and spontaneous EPSPs recorded in interneurons (Fig. 6B; see Synaptic effects of granule cells or interneurons), and for interneuron-driven and

spontaneous IPSPs recorded in granule cells (Fig. 8B; see Synaptic effects on granule cells). Synaptic effects of granule cells on interneurons. In three cases, granule cells had excitatory effects on simultaneously recorded interneurons. Of 69 granule cells that were recorded simultaneously with 21 hilar interneurons, two granule cells were found to have excitatory effects on two different hilar interneurons. Of 50 granule cells that were recorded simultaneously with 14 interneurons located in the granule ceil layer, one granule cell had an excitatory effect on one of the interneurons (Table 1). The synaptic effects of granule cells on interneurons were similar to the synaptic

Fig. 5. Mossy cell EPSPs evoked by granule cells increase in amplitude with hy~rpolarization. Simultaneously recorded granule cell (GC; top trace) and mossy cell (MC; bottom three traces). A depolarizing current pulse (0.5 nA, 100 ms) was delivered every 2 s and evoked four granule cell APs each time. Mossy cell membrane potential was manipulated with d.c. As membrane potential of the mossy cell was hyperpolarized, EPSPs increased in amplitude. An apparent failure of transmission occurred at the arrow. Granule cell RMP = -72 mV. Mossy cell RMP = -68 mV.

Fig. 6. Similarity of spontaneous EPSPs to EPSPs driven by granule cells. (A) Simultaneous recording of a granule cell (top) and a mossy cell (bottom). The granule cell was depolarized with d.c. so that it fired an AP spontaneously; an EPSP was evoked in the mossy cell by the granule cell discharge. Other EPSPs that occurred in the mossy cell inde~ndent of APs in this granule cell (arrow) were very similar in amplitude, duration, and waveform. Granule cell RMP = -78 mV. Mossy cell RMP = -60 mV. (B) Simultaneous recording of a granule cell (top) and an interneuron (bottom). A depolarizing current pulse (0.5 nA, 100 ms) evoked one AP in the granule cell; the AP evoked an EPSP in the interneuron. EPSPs that were not triggered by granule cell APs (arrow) were similar in amplitude and waveform. Granule cell RMP = - 81 mV. Interneuron RMP = - 65 mV.

was unrelated to the rate at which intracellular depolarizing current pulses were delivered to the

Synaptic connections of dentate cells

effects of granule cells on mossy cells, in that they were excitatory, and EPSPs recorded in interneurons often triggered APs at their peaks (Fig. 7). In one pair, the EPSPs evoked in the interneuron were as large as some mossy cell EPSPs (Fig. 7); however, in the other two pairs, the interneuron EPSPs were neither as large nor as fon~lastin8 as most mossy cell EPSPs (Fig. 6B). As was the case for mossy cells, the amplitudes of evoked EPSPs within a given interneuron varied, but the variability was less than that for mossy cells (Fig. 7). It also appeared that interneurons were not as sensitive to granule ceil discharge as mossy cells; for example, trains of EPSPs or APs that were recorded in mossy cells while impaling granule cells, or whife lowering a microelectrode through the granule cell layer, were never recorded in interneurons, The mean RMP of mossy cells was not different from the mean RMP of interneurons (mossy cell w + S.E.M. = -66.2 f 2.0 mV; interneurans, - 68.0 14.1 mV; t-test, P > 0.05), nor did the input resistances of these two cell groups differ significantly (mossy cells, .%?= 98.6 Ifr:16 MC&interneurons, 109 + 21 MR f-test, P > 0.05).

Interneurons were located either in the granule cell layer (n = 53 cefls), or in the hiius (n = 74). Paired recordings were made from granute cell layer interneurons and one of the follawing cell types: granule cells {n = 50 pairs), mossy cells (n = 28), hilar interneurons (n = 5), or other granule cell layer interneurons (n = 3). Paired ~~ordings involving hilar interneurons were made with granule cells (n = 691, mossy cells (n = 36), interneurons of the granule cell layer (n = 51, or other hilar interneurons fn = 6). The

Fig. 7. Excitatory effects of a granule cell on an iute~euron. Two consecutive responses of a granule cell (GC; top) to a depolarizing current p&e (0.75 nA, IO0 ms current pulse. delivered at 0.5 Hz), and the simultaneously recorded responses from an interneuron in the hifus (INT, bottom) are shown. An EPSP OF an AP occurred in the interneuron after all granule cell APs. The granule cell was depolarized with d-c. to - 55 mV during this recording. Granule cell RMP = -80 mV. Inte~euron RMP = - 50 mV. In the second trace, a spontaneous AP occurred in the interneuron (arrow) prior to granule cell discharge.

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-73 -8.5

Fig. 8. Inhjbitory efI&ts of intern~urons on granule cells. (A) Simultaneo~ recording from an int~neuron located in the hilus. (HIL INT, top) and a granule cell (GC, bottom). During a depolarizing current pulse (0.5 nA, 100 ms) to the interneuron, a hyperpolarization occurred in the granule cell. A spontaneous AP (clipped) occurred in the granule cell following the hyperpolarization. The granule cell was depolarized with d.c. to - 54 mV during this recording so that it fired spontaneously; granule cell RMP = -80 mV. Interneuron RMP = -72 mV. (B) An interneuron, located on the border of the granule cell layer and the hilus, was activated by a depolarizing current pulse (0.3 nA, 100 ms). The interneuron responses were similar each time the depolarizing pulse was triggered. Responses of a simuftaneously recorded granule cell were observed at various membrane potentials during interneuron activation. Arrows point to spontaneous events recorded in the granule cell that were not related to APs of the interneuron in B. Note reversal of the evoked and spontaneous IPSPs at about the same membrane potential (approx. -70 mV). Intemeuron RMP = -53 mV. Granule cell RMP = -77 mV.

total number of paired recordings was 197, and evidence for interneuron effects on other dentate cells was found in tive pairs (Table 1). Synaptic effem on grade c&v. Two paired recordings demonstrated an inhibitory effect of an interneuron on a granule cell. One of these pairs involved a hilar intemeuron, and the other pair involved an intemeuron in the granule cell layer. In these two connections, the synaptic event was negligible or depolarizing at the granule cell RMP; however, when the granule cell was depolarized with d.c., interneuron APs were followed by hyperpolarizations in the granule cell (Fig. 8). The effects of multiple interneuron APs summed to produce larger hyperpolarizations (Fig. 8). IPSPs elicited by single granule cell APs were typically l-5 mV. Maximal hyperpolarizations, produced by triggering multiple interneuron APs and by depolarizing the granule cell to approximately -SO mV, were as small as 3 mV in some cases but as large as 10 mV in others. Many failures were observed, where interneuron APs failed to produce a measurable membrane potential change in the granule ceil (Fig. 8). Failures followed a pattern in that the failure always followed a large IPSP. Typically, the first or second AP in a train of interneuron APs would elicit a large IPSP; subsequent intemeuron APs would not elicit further hyperpolari~tions for a “refractory”-like period (Figs 8

700

H.E. SCHARFMAN~~ al

and 9). As was true for the granule cell-to-hilar cell synapse, variability made it dithcult to determine the precise reversal potential of the synaptic events elicited by interneuron APs. However, manipulation of membrane potential did show that the synaptic potential reversed polarity positive to the RMP of the granule cell, which was -80 mV in one cell and -78 mV in the other cell (Fig. 8B). Connections between interneur~~~. Evidence for synaptic interactions between interneurons was recorded from two different pairs of simultaneously recorded interneurons. The interactions of interneurons were inhibitory (Fig. 9); in one case there was a reciprocal inhibitory connection (Fig. 9B; Table 1). In both pairs, hiiar interneurons were able to hyperpolarize intemeurons located in the granule cell layer; in the case of the reciprocal connection, a hilar interneuron and an interneuron in the granule cell layer were able to hyperpolarize each other. For both pairs, the cells were located I SO-200 pm apart. As was the case with the other interneuron connections described above, there was considerable variability in the amplitude of evoked postsynaptic potentials, and failures occurred frequently. The hy~rpoIarizations evoked in the granule cell interneurons could be reversed when the granule cell layer interneuron was

Fig. 9. inhibition between interneura~s of the hilus and the granule cell layer. (A) Simultaneous recording of a hilar interneuron (HIL INT; top) and an interneuron located in the granule cell layer (GCL INT; bottom). During currentinduced (0.5 nA, 100 ms) activation of the hilar interneuron, the granule cell layer interneuron hyperpolarized; a spontaneous AP then occurred that was not correlated with the activity of the hilar interneuron. The granule cell layer interneuron was depolarized to -5OmV with d.c. during this recording. Granule cell layer interneuron RMP = - 69 mV. Hilar interneuron RMP = - 72 mV. (B) Simultaneous recording of another pair of interneurons, one located in the hilus (HIL INT; top), and one located in the granule cell layer (GCL INT, bottom). During current-induced (0.25 nA, 100 ms) activation of the hilar interneuron, a hyperpolarization was recorded in the granule cell layer interneuron. During spontaneous APs in the granule cell layer interneuron, hyperpolarizations (arrows) were observed in the hilar interneuron. Both interneurons were depolarized with d.c. Hilar interneuron membrane cell potential = - 65 mV; RMP = -70 mV. Granule layer interneuron membrane potential = - 55 mV; RMP = -60 mV.

Fig. 10. Excitation of a mossy cell by an interneuron. Simultaneous recording of an interneuron located in the hilus (INT; top), and a mossy cell (MC; bottom). During a depolarizing current-induced (0.3 nA, 100 ms) activation of the interneuron, a depolarization was seen in the mossy cell. The mossy cell was depolarized to - 54 mV during this recording, thus making it unlikely that the evoked depolarization was an inverted IPSP. Mossy cell RMP = - 70 mV. Interneuron RMP = - 57 mV.

hyperpolarized to potentials more negative than - 70 mV. However, the hyperpolarizations that occurred in the hilar interneuron following stimulation of a granule ceil layer interneuron could not be reversed; as the cell was hyperpolarized the hyperpolarizations became smaller, but never reversed polarity. Synaptic effects of interneurons on mossy cells. A possible synaptic effect of an interneuron on a mossy cell was observed in one pair, where interneuron APs appeared to cause depolarizations in the mossy cell (Fig. 10; Table 1). This was an intriguing observation, since excitatory interneurons are rare in hippocampus. The excitation was observed even when the mossy cell was depolarized (Fig. lo), so the effect could not have been due to a “reversed” IPSP. At depolarized membrane potentials, spontaneous APs of the interneuron appeared to trigger mossy cell APs, which further supports the conclusion that the effect of the interneuron was excitatory (Fig. 10).

Mossy cells did not have effects on 36 simultaneously recorded hilar interneurons, 28 interneurons located in the granule cell layer, or -694 granule cells (Table 1). We found no evidence for any connections between different mossy cells that were recorded simultaneously (22 pairs), nor was there any synchronicity in the spontaneous activity recorded from simultaneously impaled mossy cells; although pairs of simultaneously recorded mossy cells had many spontaneous EPSPs, none was exactly synchronous in onset. Anatomical approaches were used to investigate the connections of mossy cells with other cells of the fascia dentata further. In these experiments, HRP was injected into electrophysiologically identified mossy cells (Fig. II), and synaptic contacts of the HRP-filled axon of six cells were subsequently examined with electron microscopy. The axon of each

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Fig. 12. An HRP-filled mossy cell axon terminal located on an interneuron dendrite in the hilus. An HRP-filled mossy cell terminal profile (solid arrow) is shown contacting a dendrite in the hilus. The lack of dendritic spines, slight varicosities, and numerous synaptic contacts along its shaft (open arrows) suggested that the dendrite was from an interneuron. Scale bar = I pm.

HRP-filled mossy cell collateralized within the hilus, and sent a process through area CA3c, into stratum oriens (where it became myelinated), and finally into the alveus. We assume that this axon projected via the commissural system to the contralateral dentate. Ipsilaterally, axon collaterals of HRP-filled mossy cells could be followed to the border of the granule cell layer and the hilus, but axonal processes were never found in the granule cell layer or the molecular layer. Synaptic contacts were found from four of the HRP-filled mossy cells on interneuron dcndritic processes in the hilus (Fig. 12). Interneuron dendrites were identified by their aspinous appearance and high density of presynaptic terminals contacting the dendritic shaft. In one case, postsynaptic dendrites could be traced to an interneuron cell body located in the hilus. In other cases, somata associated with the mossy cell-to-interneuron dendrite synapses were not found; the cell bodies could have been located in the hilus, in the granule cell layer, or even in the area CA3c pyramidal cell body layer. Serial section analysis showed that the contacts of HRP-filled mossy cells on interneurons had presynaptic vesicles (partially obscured by the HRP reaction product), closely apposed pre- and postsynaptic membranes with cleft material, and a thin postsynaptlc specialization (Fig. 13). DISCUSSION

The results of paired recording, and of electron microscopic analysis of HRP-filled neurons, support

a view of local circuitry in the fascia dentata as illustrated in Fig. 14. We have shown directly that granule cell APs evoke large EPSPs in hilar cells, both interneurons and mossy cells. In addition, our results demonstrate that interneurons produce IPSPs in both granule cells and in other interneurons; interneuronal inhibition may be reciprocal in some cases. Finally, there appear to be synaptic interactions of hilar interneurons and mossy cells, since mossy cells make synaptic contacts on interneurons. The degree of generalization that can be drawn from the connections demonstrate thus far is unclear, since we do not know how representative these interactions might be. We present these results, not as definitive description, but rather as essential first steps in unraveling the complex circuitry of the fascia dentata. Connections qf granule cells Our studies are consistent with much of the pre-existing data concerning cell circuitry in the fascia dentata, and certainly support the long-held conviction that granule cells are excitatory.4.‘0~62 However, various aspects of the EPSPs elicited by granule cells were not necessarily predictable based on previous studies. For example, single granule cell APs produced extremely large excitatory synaptic events in both interneurons and mossy cells. The large amplitude of these EPSPs may be due to the nature of the mossy fiber bouton--- large, often with more than one release site, and densely packed with vesicles.2~“”

Fig. 13. Serial sections through a synaptic contact of a HRP-filled mossy ce11 axon. Three serial sections (A-C) are shown of two HRP-fiiled mossy cell terminals (A-C, solid arrows) contacting an interneuron dendrite. The interneuron dendrite was followed in other serial sections to reveal further regions of interneuron-like morphology. Both mossy cell terminals made synaptic contacts (B, asterisks) that had clearly apposed membranes containing cleft material, and thin postsynaptic membrane specializations, suggesting that the contacts were symmetric. An adjacent synaptic contact from a cell that was not tilled with HRP (A and B, open arrows) exemplifies an asymmetric contact. Scale bars = 0.5 pm. 703

704

H.E.

SCHARFMAN

These large EPSPs contrast with the small EPSPs evoked in area CA1 pyramidal cells of hippocampus following APs in CA3 pyramidal neurons.45 Both studies employed similar recording methods, so technical differences are not likely to account for the difference in EPSP amplitude observed. It is possible that the larger EPSP evoked by granule cells at its targets underlies a greater functional importance for faithful transmission of information at the individual synapse. Excitation of CA1 cells may depend more heavily on the number of CA3 cells that converge upon it, rather than on the strength of an excitatory signal produced by a single CA3 afferent fiber. EPSPs elicited in mossy cells by granule cell activation were different in several respects from comparable EPSPs recorded from interneurons. Firstly, EPSPs in mossy cells were often larger, longer lasting and more variable than the EPSPs elicited in hilar interneurons. Secondly, multiple APs could be elicited in mossy cells by single granule cell APs; at best, a single AP was driven in interneurons. Thirdly, summation of the EPSPs following multiple granule cell APs was greater and more prevalent in mossy cells than interneurons. It is unlikely that these differences are due to variation among granule cells, since granule cells are thought to be a homogeneous population with respect to their axonal projection.4.‘0.62It is more likely that the variation in EPSP amplitudes are due to differences between the targets-mossy cells and interneurons. Although RMPs and R,s of these two populations were not significantly different, there may be important differences in R,,at the site at which the granule cell bouton makes contact with the postsynaptic cell. For example, the tortuous spine complexes characteristic “thorny excrescences”-may of mossy cells-the

f

CA3

Fig. 14. Diagram of local circuitry in the fascia dentata. The following circuitry is supported by our results. (1) Granule ceils (G) form excitatory synaptic contacts on mossy cells (M) and interneurons of the granule cell layer and the hilus (I). (2) Interneurons of the granule ceil layer and the hilus form inhibitory synapses on granule cells, other interneurons, or both. (3) Excitatory interneurons also exist in the hilus, and appear to contact mossy cells. (4) Mossy cells synapse on interneuron dendrites that may have cell bodies in the hilus (as shown), or have somata elsewhere in the dentate region (such as in the granule cell layer). SM, stratum moleculare; GCL. granule cell layer; HIL, hilus. Solid lines outline cell bodiesand dendrites: Open triangles, excitatory synapses; closed triangles, inhibitory synapses. Question marks indicate synapses where the functional effect of transmitter release on the target cell is unclear.

et af.

present a significantly higher R,,to the synapsing bouton than the simple postsynaptic site on the smooth dendritic shaft of the interneuron. Differences in EPSP amplitude could also be due to the site on the dendritic tree at which the granule cell input occurs. Mossy fiber terminals of granule cells occur on proximal dendrites of mossy cells,‘~2~9~” from which little electrotonic decay would be expected. Another factor contributing to the large EPSP in mossy cells could be the number of synapses or release sites between a single granule cell and mossy cell. Serial section analysis of our electron micrographs show that the synapses of granule cell boutons onto thorny excrescences of HRP-filled mossy cells contain multiple release sites (data not shown), similar to the mossy fiber synapses onto CA3 pyramidal cells.’ Comparative data are unavailable for interneurons at this time. Of considerable interest in evaluating granule cellto-mossy cell and granule cell-to-interneuron EPSPs was the number of failures. Such failures may be due to the extreme collateralization of granule cell axons, increasing the likelihood of branch point failures. Thus, the APs produced by intracellular current injection may not invade, and cause transmitter release, at all axon terminals. Slice preparation in general may also underly failures, given that inferior energy supplies are available in slices compared with tissue in vivo; this condition could impair many processes fundamental to synaptic transmission, such as the ability of the terminal to respond to repetitive action potential invasion. Given the complexity of the granule cell axonal ramification, and of its terminals, present data are insufficient to support speculation about the reasons for the observed failures. Perhaps relevant to the high degree of granule cell axon collateralization was the observation of late or “secondary” excitation of mossy cells. Given extensive granule cell axon collateralization, such secondary excitation could have occurred by mossy cell APs activating other dentate cells (such as other granule cells, or interneurons) which in turn excited the same mossy cell. Also intriguing was the occurrence of spontaneous (i.e. independent of presynaptic discharge) EPSPs, with amplitude and waveform identical to the evoked EPSPs. On the one hand, these similarities suggest that at least some of the spontaneous EPSPs recorded in mossy cells, interneurons, and granule cells could be due to spontaneous release of transmitter (i.e. without AP discharge) from the same terminals that drove the evoked EPSP. On the other hand, “spontaneous” events may have resulted from discharge of other granule cells which produced similar PSP waveforms. This latter possibility is unlikely, since granule cells in our preparation have a very low probability of spontaneous discharge (healthy granule cells generally had very negative RMPs, and were relatively di~cult to activate; they almost never fired spontaneously). It is hard to imagine that even when

Synaptic connections of dentate cells summated across many converging cells, this low discharge level could account for the very high rate of spontaneous EPSPs seen in mossy cells. Connections of interneurons Our demonstration of inhibitory actions of interneurons are in agreement with previous studies that have shown that many interneurons of the hippocampus contain the inhibitory neurotransmitter and that physiolo~cally identi~ed GABA 3~18~2B-M~43J3 interneurons are inhibitory elsewhere in hippocampus,23,32.34,39 Our data are comparable with the effects of interneurons on area CA3 pyramidal cells, where large unitary events were recorded in pyramidal cells when interneurons were stimulated.39 In contrast, interneurons in area CA1 appear to produce small, slow hyperpolarizations in pyramidal cells.23,32.34 The basis for the difference is still unclear-whether it reflects a property of the presynaptic terminal or of the postsynaptic neuron. It is interesting, however, that both granule cell and CA3 pyramidal cell (and interneuron) R,,s are higher than the R,,s of CA1 cells; this feature, alone, could result in a significantly larger IPSP. Our recordings also provided preliminary support for the existence of two types of IPSPs in the fascia dentata. Interneuron activation appeared to produce one IPSP that reversed at potentials positive to - 80 mV, and one that did not reverse until more hyperpolarized potentials were reached. Evidence for the presence of two such types of IPSPs in the fascia dentata has been reported.j9 One type of IPSP, with a reversal potential near - 70 mV, is associated with chloride ions and is attributed to GABA, receptor activation; the other IPSP type, with a more negative reversal potential, may be mediated by GABA, receptors that are coupled (via G proteins) to potassium channels.42 Our finding that interneurons inhibit other interneurons is in agreement with studies showing that GABAergic terminals synapse onto GABA-containing interneurons of the fascia dentata,40 and is consistent with the inhibitory actions of interneurons in the CA1 region.23*32*34 Our data, showing failures (or decreases) in the unitary IPSP during a train of presynaptic (granule cell) APs, are also in agreement with the known lability of IPSPs, which appear to decrease greatly upon repetitive stimulation.6.37 The effects of GABA also appear to decrease with repeated application. ” We have hypothesized elsewhere48 that the decline in IPSPs in area CA1 could be due to co-release of somatostatin from GABAergic interneurons during high frequency firing of the interneuron; the same may be true in the fascia dentata, since many GABA-containing interneurons also contain somatostatin.30.s3 Another possibility is that GABA has an “autoinhibjtory” effect on subsequent GABA release.‘*@ Intriguing was the finding of an interneuron-driven excitatory effect in the dentate hilus. Although

705

excitatory interneuron actions have rarely been reported, such effects could certainly be mediated by one of several neuropeptides contained in interneurons.‘7~24~25~28~30~S3 Many neuropeptides are thought to be excitatory in the hippocampus.17,47 To our knowledge, this is the first demonstration of a functionally excitatory interneuron in the dentate region. Given the small sample in which in~meuron-d~ven effects have been found in the dentate, details about the inhibitory or excitatory connections of interneurons are still to be determined. Also uncertain is the relative importance-excitatory and inhibitoryof the connections described above. Connections of mossy cells We were surprised to find no electrophysiological evidence of synaptic connections from mossy cells onto other elements in fascia dentata, despite the use of slices that were cut with various orientations (i.e. both transverse and longitudinal). Mossy cells are thought to innervate the inner molecular layer of the dentate3,7,‘9~2’~‘5~s6~s8 but our study was unable to find physiologic synaptic connections between mossy cells and granule cells. One reason for our failure may be that the ipsilateral projections of mossy cells onto granule cells occur quite distal to the mossy cell ,,ody,3.56h3 and it is difficult to maintain such a long segment of axon within a slice. Maintaining connectivity would be especially difficult if the axon took a meandering course to reach its distant target. Consistent with these possibilities, axon collaterals of mossy cells that were injected with HRP were never found to cross into the granule celt layer or inner molecular layer of our slices. Further, although synapses of HRP-filled mossy cells were found on interneuron dendrites in the hilus, no such synapses were observed in the inner molecular layer. Surprisingly, the mossy cell synapses on interneuron dendrites in the hilus appeared to be symmetric; given the historical association between symmetric synapse morphology and inhibitory function, the effect of mossy cells on these interneurons might be interpreted to be inhibitory. However, since other studies have demonstrated that the majority of the commissural input to the dentate (arising largely from mossy cells) is excitatory,” and that large hilar cells (many of which are mossy cells) display immunoreactivity against the excitatory transmitter glutamate,‘3.55 it seems unlikely that mossy cells are inhibitory. However, this point remains somewhat confused; some studies have not found glutamate immunoreactivity in the hilus3* and many large hilar neurons are actually immunoreactive for neurotransmitters that may not be excitatory, such as GABA or various peptides.5~‘s~2S~zS~30~43~53 Taken together with the results of recent studies that have shown that hippocampal neurons may possess asymmetric as well as s~metri~ contacts,s and that “transitional” synapses (which are not clearly symmetric or asymmetric) may occur in hip~campus,2b our

H. E. SCHARFMANer ui

706

ultrastructural data are inadequate to determine the excitatory or inhibitory nature of mossy cell synapses. CONCLUSION

In summary, we have begun to describe several aspects of local circuitry in rat fascia dentata as a result of our study of synaptic interactions between three major interneurons, strong

dentate cell populations: granule cells, and mossy cells. Granule cells produced

excitatory

effects

on all other

cell types.

Inter-

neurons were predominantly inhibitory. The synaptic effects of mossy cells remain unclear on the basis of our eIectrophysiologica1 and morphological data. These studies provide a first step in gaining an understanding

of local synaptic interactions in the rat fascia dentata. work was supported by NIH postdoctoral training grant NS-01744 to H.E.S., and NINCDS grants NS-18895, NS-20482 to P.A.S. P.A.S. is a research affiliate of the Child Development and Mentai Research Center, University of Washington Acknowledgements-This

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A. and Laurberg S. (1977) Commissural connections of the dentate area of the rat. J. camp. Neural. 21. Hjorth-Simonsen 174, 591-606. Y. and Hama K. (1987) Fast-spiking non-pyramidal cells in the hippocampal CA3 region, dentate gyrus, 22. Kawaguchi and subiculum of rats. Brain Rex. 425, 351.,.355. P. A. (1981) Local circuit interactions in hippo~dmpal brain slices. J. .~~~~~~.~~~. 1, 23. Knowles W. D. and ~h~~artzkroin 318-322. analysis of vasoactive intestinal polypeptide (VIP)-like immunoreactive neurons in 24. Kohler C. (1983) A morphological the area dentata of the rat brain. J. camp. Neural. 221, 247-262. V. (1987) Co-localization of neuropeptide tyrosine and somatostatin 25. Kohler C., Eriksson I., Davies S. and Chan-Palay immunoreactivity in neurons of individual subfields of the rat hippocampal region. Neurosci. Lett. 78, 1.6. and synaptology of initial segment of the 26. Kosaka T. (1980) The axon initial segment as a synaptic site: ultrastru~ture pyramidal cell in the rat hippocampus (CA3 region). J. NeuroqTtuf. 9, 861-882. 21. Kosaka T., Hama K. and Wu J.-Y. (1984) GABAergic synaptic boutons in the granule cell layer of rat dentate gyrus. Brain Res. 293, 3533359. N., Wu J.-Y. and Hama K. (1985) GABAergic neurons 2x. Kosaka T., Kosada K., Tateishi K., Hamaka Y.. Yanaihara contain CCK-8 like and/or VIP-like, immunoreactivities in the rat hippocampus and dentate gyrus. 1. romp. Neurof. 239, 420-430. H., Hama K., Wu J.-Y. and Heizmann C. W. (1987) GABAergic neurons containing the 29 Kosaka T., Katsumaru calcium-binding protein parvalbumin in the rat hippocampus and dentate gyrus. Bruin Res. 419, 119-130.

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