~ Pergamon Neuroscience Vol. 73, No. 2, pp. 317-334, 1996 IBRO

Copyright (C 1996 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain

0306-4522(95)00609-5 0306-4522/96 $15.00 + 0.00

D I F F E R E N T POPULATIONS OF VASOACTIVE INTESTINAL POLYPEPTIDE-IMM U N O R E A C T I V E

I N T E R N E U R O N S ARE SPECIALIZED TO CONTROL P Y R A M I D A L CELLS OR I N T E R N E U R O N S IN THE

HIPPOCAMPUS

L. ACS,&.DY,* T. J. G O R C S t and T. F. FREUND*:~

*Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest P.O.B. 67, H-1450, Hungary

tFirst Department of Anatomy, Semmelweis University, Medical School, Budapest, H-1450, Hungary

Abstract The postsynaptic targets of three vasoactive intestinal polypeptide-containing GABAergic interneuron types were examined in the rat hippocampus. Two of them showed remarkable target selectivity for other GABAergic neurons, while the third contacted the somata and proximal dendrites of pyramidal cells. Vasoactive intestinal polypeptide-positive interneurons innervating the stratum oriens/alveus border in the CA1 region were shown to establish multiple contacts with horizontal GABAergic interneurons immunoreactive for type 1 metabotropic glutamate receptor. Similarly, identified axons of vasoactive intestinal polypeptide-positive interneurons projecting to stratum radiatum were found to establish symmetrical synapses largely on GABAergic dendrites. The majority of these postsynaptic GABAergic neurons were shown to contain calbindin or vasoactive intestinal polypeptide. In contrast to the first two vasoactive intestinal polypeptide-containing cell populations, vasoactive intestinal polypeptide-positive interneurons arborizing in stratum pyramidale formed baskets around pyramidal cells.

These results revealed a new element in cortical microcircuits, interneurons which are specialized to innervate other GABAergic interneurons. The role of this new component may be the synchronization of dendritic inhibition, or an input-specific disinhibition of pyramidal cells in various dendritic domains. In contrast, vasoactive intestinal polypeptide-containing basket cells are likely to be involved in perisomatic inhibition of pyramidal neurons, and represents a new basket cell type different from that containing parvalbumin. Copyright ~ 1996 Published by Elsevier Science l td .

Key words: interneurons, GABA, VIP, hippocampus, synchronization, disinhibition.

In the h ippocampus , var ious types of in te rneurons were shown to selectively innervate different m e m b r a n e domains of the pyramidal cells, forming the ana tomica l basis of diverse inhibi tory func- tions. 8'17A9'33"49 Basket and axo-axonic cells innervate

the cell bodies and axon initial segments of the principal cells, where the threshold for sodium spike generat ion is the lowest, 54 thus, they are in ideal posi t ion to control the ou tpu t of large cell groups. 8'37 Other GABAerg ic neurons are specialized to innerv- ate different dendri t ic segments of the pyramidal cells, and probab ly influence dendri t ic electrogenesis. 37 The axon a r b o u r of some of the in te rneurons responsible for dendri t ic inhibi t ion were shown to coincide with

~:To whom correspondence should be addressed. Abbreviations: ABC, avidin biotinylated-horseradish per-

oxidase complex; CA, cornu Ammonis; CCK, cholecysto- kinin; DAB, diaminobenzidine; DAB-Ni, nickel-sulphate intensified DAB; mGluRla, type la metabotropic gluta- mate receptor; NGS, normal goat serum; O/A, border of strata oriens and alveus; PB, phosphate buffer; SPR, substance P receptor; TBS, Tris-buffered saline; VIP, vasoactive intestinal polypeptide.

the t e rmina t ion zone of certain excitatory afferents. In te rneurons innervat ing the en torh ina l t e rmina t ion zone have been described in the denta te gyrus 19 as well as in the CA3 and CA1 regions of the A m m o n ' s horn. 17'33 Othe r GABAerg ic cells (e.g., bistratified cells 8) were found to innervate the Schaffer collateral t e rmina t ion zone. Thus, these cells are able to control the effectiveness of a given excitatory input.

The different inhibitory interneurons so far described terminate largely on pyramidal cells but also innervate o ther in te rneurons at abou t the frequency of their occurrence. 17 This is similar to the t e rmina t ion pa t te rn of pyramidal cells, since the rat io of pa rva lbumin- conta in ing postsynapt ic targets, of a single CA3 pyramidal cell was similar to the ratio of parva lbumin- immunoreac t ive in te rneurons in the neuropil . 4s Simi- lar results were ob ta ined for the target d is t r ibut ion of pa rva lbumin-con ta in ing basket cells. 5° The bou ton d is t r ibut ion a long the axons of these intracellularly filled in te rneurons is similar to tha t of pyramidal cells. The bou tons are evenly spaced, the intervaricose distances show little variat ion, suggesting a quasi- r a n d o m postsynapt ic target selection.

317

318 L. Acsfidy et al.

In the accompanying paper 2 three vasoactive intes- tinal polypeptide- (VIP) containing interneuron types have been described in the rat hippocampus, all o f them containing GABA. One of the three types had a homogeneous axonal arbour in stratum pyramidale (VIP-positive basket cells); however, the other t w o - -

characterized on the basis of their projection pattern and correlated neurochemical features showed axonal characteristics different from the previously described interneurons. VIP-positive cells projecting to the stratum oriens/alveus (O/A) border densely innervated a narrow sublayer known to have no specific excitatory input. The termination zone of

VIP-positive axons at the O/A border showed a perfect overlap with the horizontal dendrites of a subset of GABAergic interneurons characteristically present in this zone. VIP-containing interneurons projecting to stratum radiatum had highly variable intervaricose distances, with grouping of boutons along radially running segments, similar to certain subcortical afferents, 13'14 shown to be selective for

GABAergic targets in the hippocampus. Thus, axonal features of both of these VIP-containing cell types suggested a possible target selectivity for GABAergic cells.

In this study we tested these anatomical predictions by identifying the postsynaptic elements of each three VIP-containing cell types.

EXPERIMENTAL PROCEDURES

Perfusion, preparation of tissue sections

Male Wistar rats (Charles River, 300 350 g) were deeply anaesthetized by Equithesin (chiornembutal, 0.3 ml/100 g), and perfused through the heart first with saline followed by a phosphate-buffered (PB, 0.1 M) fixative containing either 4% paraformaldehyde, 0.2% picric acid, 0.05% glutaralde- hyde (fixative A) for VIP-calbindin, VIP-parvalbumin and VIP-type la metabotropic glutamate receptor (mGluRla) double immunostaining, or 4% paraformaldehyde, 0.2% picric acid and 1% glutaraldehyde (fixative B) for pre- embedding VIP immunostaining combined with post- embedding immunogold staining for GABA. Coronal sections of 60 pm thickness were cut from the hippocampus on a Vibratome, washed in 0.1 M PB, cryoprotected in 30% sucrose in 0.1 M PB overnight, and freeze-thawed in aluminium-foil boat over liquid nitrogen to enhance penetration of the antisera.

Pre-embedding immunocytochemistry

Following extensive washes and treatment with 1% sodium borohydride for 30 min (only for animals fixed with fixative B), the sections were incubated in solutions of the following antisera. For single VIP immunostaining rabbit anti-VIP, diluted 1:10,000, t6 was used as primary antiserum. The second layer was biotinylated anti-rabbit IgG (Vector, 1:200), followed by avidin biotinylated- horseradish peroxidase complex (ABC, Vector 1.5h, 1:150). The immunoperoxidase reaction was developed with 3,3'-diaminobenzidine (DAB, Sigma) as a chromogen. The sections were treated with 1% OsO 4 in 0.1 M PB for l h, dehydrated in ethanol and propylene oxide, and embedded in Durcupan (ACM, Fluka). During dehydra- tion the sections were treated with 1% uranyl acetate in 70% ethanol for 40rain. VIP-immunoreactive cells with

large axonal arbours were reconstructed with a drawing tube from serial 60#m thick sections. Varicose axon collaterals of identified VIP-positive neurons were re-embedded for further ultrathin sectioning.

Pre-embedding double immunostaining

The protocol was similar to that described for single immunostaining; however, in this case VIP was developed using ammonium nickel-sulphate intensified DAB (DAB- Ni) as a chromogen (black reaction product). After the first immunostaining the sections were treated with one of the following primary antisera: rabbit anti-calbindin D-28K (1:1500) 3 or rabbit anti-parvalbumin (1:1000) 3. This was followed by biotinylated anti-rabbit IgG (Vector, 1 : 200), and ABC (Vector 1.5 h, 1 : 150). The second immunoperoxidase reaction was developed with DAB as a chromogen (brown reaction product). Replacing the primary antisera with normal sera of the animals in which the primary antisera were raised resulted in lack of immunostaining. The sections were treated with 1% OsO 4 (also cqntaining 7% glucose to preserve colour differences between DAB-Ni and DAB end products) dehydrated and embedded in Durcupan as above.

In case of the mGIuRla-VIP double staining the sections were first incubated with rabbit anti-mGluRla (1:10,000), ~ and following the ABC method as above, the peroxidase reaction was developed with DAB-Ni as a chromogen. This was followed by immunostaining for VIP visualized with DAB. These sections were also pre- pared for electron microscopic examination as above. In several cases profiles labelled by DAB and DAB-Ni were identified on the basis of colour difference with the light microscope and the same time profiles were then examined with the electron microscope. In these correlated light and electron microscopic samples we confirmed that the two types of end product can be distinguished also with the electron microscope; the DAB-Ni precipitate was always more electron-dense and inhomogenous com- pared with DAB, frequently including coarse granules (Figs 1, 6).

Postembedding immunogold staining .for GABA

Blocks containing the identified VIP-positive axon segments were re-embedded, ultrathin sections were cut on an Ultramicrotome and mounted on nickel grids. Postembedding GABA immunostaining was carried out on the grids according to the protocol of Somogyi and Hodgson? 2 Incubations performed were on droplets of solutions in humid Petri dishes as described earlier. Briefly: 1% periodic acid (H5IO6) for 10 min; washed in distilled water; 2% sodium metaperiodate (NaIO4) for 10min; washed in distilled water; three-times 2min in Tris- buffered saline (TBS; pH 7.4); 30 min in 1% ovalbumin dissolved in TBS; three times 10min in TBS containing normal goat serum (NGS); I 2 h in a rabbit anti-GABA antiserum (1:1000 in NGS/TBS) s2 two times 10 min TBS; 10 rain in TB containing l% bovine serum albumin, 0.05% Tween 20, 1% NGS; 2h goat anti-rabbit lgG-coated colloidal gold (10 nm, Jackson, 1:20 or 15 nm Amersham, 1:10 in NGS/TBS) in the same solution as before; two times 5 min washed in distilled water; 30rain saturated uranyl acetate; washed in distilled water; stained with lead citrate; washed in distilled water.

Following GABA immunostaining, VIP-positive axons were searched for using the electron microscope (either randomly, or using correlated light microscopy) and their postsynaptic targets were identified. Profiles containing at least five-times higher density of gold particles than neighbouring asymmetrical (presumed glutamatergic) synaptic terminals in two to three serial sections were considered as GABA-positive. The electron micrographs were taken on a Hitachi 7100 electron microscope.

Specific connections between hippocampal interneurons 319

RESULTS

Ultrastructural features o f vasoactive intestinal polypeptide-containing neurons

The ultrastructural features of VIP-immunoreactive dendrites and somata were similar to those described in earlier electron microscopic studies, 29'3° and were reminiscent of the general characteristics of inter- neurons. Briefly, the cell bodies contained large invaginated nuclei, and were rich in cytoplasmic organelles including rough endoplasmatic reticulum and mitochondria. The dendrites and cell bodies were covered by a large number of both symmetrical and asymmetrical synapses.

As shown in the accompanying paper, 2 VIP-positive axons originate from three different populations of interneurons terminating in three different layers. In terms of basic ultrastructural features no differences were observed among the VIP-containing boutons, examined separately in strata radiatum, pyramidale and at the O/A border. The diameters of the boutons varied between 0.5 and 1 pm. They always contained mitochondria and large numbers of small clear vesicles. The majority of boutons contained large dense core vesicles as well (Figs 3, 4), which were more frequently encountered in boutons of stratum radiatum than in stratum pyramidale or at the O/A border. The DAB precipitate covered the outer surface of cytoplasmic organelles (e.g., clear vesicles and mito- chondria) while dense core vesicles often contained the end product inside. Nearly all VIP-positive boutons examined formed symmetric synaptic contacts (Figs 1, 3, 4, 6-8). In VIP-immunostained sections processed for postembedding immunogold staining for GABA, VIP-positive boutons in all three layers were found to be GABA-immunoractive (see Figs 1, 3, 8). This is in agreement with the light microscopic co-localization studies, 2 and demon- strates that indeed, all VIP-containing cell types are GABAergic. GABA labelling was evident only over the mitochondria of VIP-immunoreactive boutons. When a VIP-positive bouton did not contain a mito- chondrion in the plane of the section, the DAB precipitate masked GABA-antigenic sites, as reported previously.~3

Postsynaptic elements o f vasoactive intestinal polypeptide-positive interneurons projecting to the stratum oriens/alveus border

A population of VlP-containing interneurons was shown to innervate a narrow sublayer at the border of stratum oriens and alveus (O/A) in the CA 1 region (see accompanying paper2). This zone has been previously described to contain, among other cells, GABAergic interneurons with long horizontal dendrites. 5'33'42~56 Indeed, the band of horizontal dendrites immunostained for mGluRla - -known to label somatostatin-containing interneurons 5 - showed a perfect overlap with the plexus of VIP- immunoreactive axon collaterals at the O/A border

(Fig. 1A, B). Whether this specific co-distribution of axons and dendrites means specific synaptic connections between the two cell types was exam- ined using double immunostaining for mGluRla and VIP.

Vasoactive intestinal polypeptide-containing boutons in synaptic contact with type la metabotropic gluta- mate receptor-positive interneurons. Following double immunostaining, using DAB-Ni as a chromogen for mGluRla-posit ive dendrites and DAB for VIP- positive elements, the synaptic relationship between the two interneuron types was examined at the electron microscopic level. The DAB and DAB-Ni precipitates could be distinguished at the ultrastructural level (see Experimental Procedures). Immunostaining for mGluRla labelled only the dendrites and somata of horizontal interneurons at the O/A border of the CAI region, being very dense on the postsynaptic and mitochondrial membranes as reported previously? In agreement with previous findings, axonal staining was not observed in this layer of mGluRla- immuno- stained sections. In contrast, the vast majority of elements immunoreactive for VIP were boutons, largely belonging to the VIP-positive cell type that specifically projects to this sublayer. Occasionally, VIP-immunoreactive dendrites were also visible in this region; however, they were sparse compared with mGluRla-posit ive dendrites, and could be easily distinguished from them on the basis of less electron dense, homogeneous staining with DAB (Fig. IE).

VIP-immunoreactive boutons at the O/A border were frequently found to establish symmetrical synapses on small and medium calibre mGluRla- positive dendrites (Fig. 1 C, D). These dendrites, when followed in serial electron microscopical sections, were often seen to receive multiple synaptic contacts from VIP-positive boutons. Cell bodies were rarely contacted, but those were also positive for mGluRla . Unlabelled terminals often formed asymmetrical synapses on mGluRla-immunoreactive dendrites. In sections cut deeper in the block, postsynaptic dendrites of VIP-positive boutons were often found to be negative for mGluRla , which is likely to be due to the poor penetration of the mGluRla antiserum compared with the VIP antiserum. Therefore, it was impossible to establish what proportion of the postsynaptic elements of VIP-boutons are labelled by mGluRla. Nevertheless, based on samples from the surface of the Vibratome sections, we can confidently state that the overwhelming majority of the targets are mGluRla-posit ive dendrites of somatostatin cells.

Vasoactive intestinal polypeptide-positive boutons in synaptic contacts with calbindin D-28k-containing non-principal cells at the border o f strata oriens and alveus. A population of GABAergic neurons with horizontal dendrites containing calbindin D-28k was described at the O/A border, and shown to project to the medial septum. 56 According to its laminar distribution and dendritic tree this cell type may also

320 L. Acsfidy et al.

Fig. I.

Specific connections between hippocampal interneurons 321

be among the targets of VIP-positive cells projecting to this sublayer.

Following double immunostaining for VIP and calbindin D-28k, using DAB-Ni for VIP and DAB for calbindin D-28k as chromogens, horizontal dendrites of calbindin D-28k-containing neurons at the O/A border were frequently seen to receive multiple contacts from VIP-positive axons in a "climbing fibre-like" manner (Fig. 5A, E). Correlated electron microscopy of VIP-calbindin D-28k contacts con- firmed that they were conventional symmetrical synapses, as illustrated in Fig. 6, for VIP-positive neurons projecting to stratum radiatum (see below).

Vusoactit~e intestinal polypeptide-GABA double immunostaining at the border of strata oriens and ah~eus. Another way to demonstrate the selectivity of VIP-positive terminals for interneurons as targets in this sublayer is to stain ultrathin sections con- taining VIP-positive boutons for G A B A using the immunogold procedure. Since the majority of soma- tostatin-containing neurons, which give rise to the mGluRla-pos i t ive dendrites at the O/A border 5 are know to be GABAergic, 53 one would expect the targets to be positive for GABA. This is, indeed what has been observed: the majority of VIP-immunoreact ive boutons formed symmetrical synapses on GABA- immunoreactive dendrites and somata (Fig. 1F). The VIP-positive boutons themselves were always GABA- positive. However, among the postsynaptic elements of VIP-boutons GABA-negat ive dendrites were also found. This may be explained either by false-negative staining, or by the fact that some of the CB-containing and somatosta t in /NPY-containing neurons project to the medial septum, 56 and the level of G A B A in GABAergic neurons with distant projection is below the limit of immunocytochemical detection. 55 Densely spiny dendritic shafts showing characteristic ultra- structural features and orientation of pyramidal cell basal dendrites were not among the targets.

Postsynaptic targets c?f t,asoaetive intestinal polypeptide-containing interneurons projecting to stratum radiatum

Another type of VIP-containing interneuron was shown to innervate the stratum radiatum. The grouping of boutons along the radially oriented

collaterals of this cell type suggested that they may innervate irregularly spaced elements in the neuropil, similarly to certain subcortical pathways contacting GABAergic interneurons in the hippo- campus. 1'~3~4'~6'35'36 We examined the postsynaptic

target specificity of VIP-positive axons followed back to this cell type as well as a random sample of VIP-containing boutons in stratum radiatum, using postembedding G A B A immunogold staining.

Vasoactive intestinal polypeptide-immunoreaetiee interneurons selectieely innervate GABAergic cells in stratum radiatum. One subpopulation of VIP- containing interneurons with stratum radiatum pro- jections has a characteristic dendritic arbor restricted entirely to stratum lacunosum-moleculare. Three neurons of this type with profuse axon arbors were selected for correlated light and electron microscopy (two of them are shown in Fig. 2). The characteristics of these neurons were identical to those described in the accompanying paper, 2 their axons consisted of mainly radially oriented rows of boutons linked with long non-varicose segments (Figs 2, 3).

The postsynaptic targets were established for 36 boutons of three identified VIP cells using correlated electron microscopy of serial sections, which were immunoreacted for G A B A as well. Nearly all of these (35 out of 36) established symmetrical synapses with GABA-immunoreac t ive dendrites (Figs 3, 4), identified as such on the basis of consistent accumu- lation of colloidal gold particles over the positive profiles in several serial sections (see Experimental Procedures). In one case the identity of the post- synaptic target was questionable due to poor G A B A labelling. The ultrastructural characteristics of the postsynaptic GABAergic dendrites were similar to those of interneurons, i.e. a large number of mito- chondria, lack of spines, numerous GABA-negat ive asymmetrical synapses. Occasionally, GABAergic spiny neurons were also contacted by VIP-positive boutons in stratum radiatum. In addition to G A B A immunoreactivity, the structure of the spines (thick, stubby spines with more than one asymmetrical synapse) clearly showed that these dendrites belong to spiny interneurons of stratum radiatum rather than to pyramidal cells. VIP-positive boutons often formed multiple contacts on radially running

Fig. 1. Postsynaptic targets of VIP-immunoreactive interneurons projecting to the border of stratum oriens and alveus. (A, B) The plexus of VIP-immunoreactive axons shows a perfect overlap with dendrites of metabotorpic glutamate receptor- (mGluR 1 a) positive neurons at the border of str. oriens and alveus in the CA1 region. (C, D) High power electron micrographs of sections double-immunostained for mGluRla (Ni-DAB) and VIP (DAB). V1P-positive boutons are shown to establish multiple synaptic contacts (arrows) on small caliber dendrites (d) immunoreactive for mGluRla. The synapses formed by boutons labelled b~ in C, D are marked by arrows, whereas the synapses of boutons b 2 have been confirmed in adjacent sections (not illustrated). (E) The difference between DAB (dvjp) and Ni-DAB (dmciluR) precipitate is demonstrated on this high power electron micrograph of two adjacent dendrites. (F) A VIP-immuno- reactive bouton in the same layer is shown to form a symmetrical synapse on a GABA-positive soma (S6ABA). The accumulation of colloidal gold particles over the soma clearly indicates its GABA-immuno- reactivity. The V1P-containing bouton also shows GABA positivity in parts not filled by the DAB reaction product (i.e. over mitochondrion). Asterisk labels another GABAergic profile. Scale bars = 30/~m (A, B),

0.5 #m (C,D,F), 1 #m (E).

322 L. Acsfidy et al.

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Fig. 3. Postsynaptic targets of an identified VIP-containing interneurons (No. 1 in Fig. 3). (A) High power light micrograph of a bouton row originating from the identified neuron in stratum radiatum of the CA1 region. The synapses formed by boutons, bl, b2 and b6 are shown in this figure (B-E) while b5 can be seen in Fig. 4A. (B) Correlated low power electron micrograph of the boutons labelled as bl and b2 on A. The corresponding capillary (c) helps the identification. (C, E) The VIP-positive boutons of the identified neuron (bl and b2) establish symmetrical synaptic contacts (arrows) on the same dendrite, shown to be immunoreactive for GABA by the accumulation of gold particles (some of them labelled by small arrows). The terminals are also GABAergic. (D) High power electron micrograph of another bouton (b6) of the same cell, synapsing (arrow) on a GABA-positive beaded dendritic shaft. Asterisks label GABA-negative profiles, the open star in D shows a GABAergic terminal, while arrowheads point to dense core vesicles of the VIP-containing terminals. Scale bars =

10 ~m (A), 2 ~m (B), 0.5/~m (C-E).

324 L. Acs~dy et al.

Fig. 4.

Specific connections between hippocampal interneurons 325

GABAergic dendrites (Fig. 3C, E). Although postsynaptic GABAergic dendrites were not recon- structed systematically from serial electron micro- scopic sections, in some fortuitous cases it was apparent that long rows of boutons (eight to 10 boutons) contacted the same GABAergic dendrite. This was confirmed later by VIP-calbindin D-28k double staining (see below).

A random sample of 32 VIP-positive boutons in stratum radiatum were also examined, and they showed a similar target selectivity, 30 of them estab- lished symmetrical synapses on GABAergic dentrites or somata (Fig. 4B D). In two cases the GABA immunoreactivity of postsynaptic targets remained unidentified. Multiple contacts on single postsynaptic elements was the characteristic way of termination of these boutons as well. The VIP-containing boutons in both samples were also GABA-positive, providing further evidence for the GABAergic nature of VIP cells projecting to stratum radiatum. The vast majority of the 68 boutons examined formed single synapses.

GABAergic cell types in stratum radiatum inner- vated by ~'asoactive intestinal polypeptide-positive interneurons.

In order to further characterize the GABAergic cells innervated by VlP-positive interneurons, we performed double immunostaining for VIP and different neurochemical markers, known to label dis- tinct interneuron populations.

Interneurons containing calbindin D-28k are present in all layers, 56 and are known to innervate pyramidal cell dendrites. On the sections double- stained for VIP and calbindin D-28k, numerous calbindin D-28k-immunoreactive interneurons were found to receive multiple contacts from VIP-positive axons in the CAI and CA3 subfields (Fig. 5B, D). The cell bodies, as well as the proximal and distal dendrites were innervated by VIP-containing boutons in a "climbing fibre-like" manner. The innervation of one calbindin D-28k-positive inter- neuron was examined in more detail, and was found to be contacted by 64 VIP-positive boutons within a 60 #m thick section (Fig. 5D). Four VIP-positive axon collaterals were identified to give rise to the 64 boutons, innervating different parts of the calbindin D-28k-positive cell. The 20-60#m long segments of these collaterals before reaching the cell were free of varicosities. However, upon contacting the calbindin D-28k-positive interneuron, all four of them formed rows of boutons climbing along the

dendrites for a long distance. Correlated electron microscopic examination of a large sample of these contacts (15 out of the 64 were tested) revealed that each VIP-positive bouton established conventional symmetrical synapses on the calbindin D-28k- containing interneuron (Fig. 6). The ultrastructure of the calbindin D-28k-positive cell showed the characteristics of interneurons: invaginated nucleus, abundant cytoplasmic organelles and a large number of asymmetrical synapses on the dendrites and cell body. In the CA l region pyramidal cells are also immunoreactive for calbindin D-28k; however, different staining intensity, location, dendritic arbour and ultrastructural features distinguish them from interneurons. 3's6 VlP-immunoreactive boutons were not seen to form synapses (at electron microscopic level) or multiple contacts (at light microscopic level) with calbindin D-28k-immunoreactive dendrites of pyramidal cells in stratum radiatum, confirming the target selectivity of VIP-positive cells for interneurons.

Another calcium binding protein, parvalbumin, is present in basket and axo-axonic cells in the hippo- campus. 2L23 Following double-staining for VIP and parvalbumin in stratum radiatum the incidence of multiple VlP-positive contacts on the dendrites of parvalbumin-immunoreactive cells was low. These contacts consisted of not more than five to 10 boutons, and followed the dendrites for a relatively short distance (Fig. 5C).

VIP-positive axons frequently formed multiple contacts on other VIP-containing interneurons as de- scribed in the accompanying paper. 2 A VlP-positive cell in stratum pyramidale of CA1, innervated by another VIP-positive neuron in stratum radiatum was selected for correlated light and electron microscopy (Fig. 7). The VIP-positive boutons were found to make symmetrical synapses on the soma and proximal dendrite of the other VIP-containing interneuron.

Thus, in summary, VIP-containing cells projecting to stratum radiatum are also specialized to contact other GABAergic interneurons, including calbindin D-28k- and other VIP-containing cells, and occasionally parvalbumin-containing neurons.

Postsynaptic targets o f VIP-containing basket cells

A third population of VIP-positive cells was shown in the accompanying paper 2 to form basket-like arrays of boutons around pyramidal cell bodies in stratum pyramidale. Electron microscopic examination of stratum pyramidale in VIP-immunostained sections

Fig. 4. (A) Another bouton (b5) of the collateral shown in Fig. 3A is in synaptic contact (arrow) with a GABA-positive dendrite. (B-D) Postsynaptic targets of VlP-positive boutons randomly found in stratum radiatum. The VIP-containing boutons form symmetrical synaptic contacts with a GABA- immunoreactive soma (B) and a dendrite (C, D). The GABA labelling of the VIP-positive boutons are poor in these sections due to the dense DAB precipitate; however, it is evident in consecutive sections (not shown), where mitochondria in the boutons are larger. GABA-negative profiles are labelled by asterisks.

Scale bars = 0.5 #m (A-D).

326 L. Acsfidy et al.

VIP-CB

Fig. 5. Postsynaptic targets of VIP-positive interneurons at the O/A border (A, E) and in stratum radiatum (B-D) of the hippocampal CAI subfield. (A, E) High power light micrographs of VIP-containing axons (black) forming multiple contacts (arrows) with horizontal dendrites of calbindin-immunoreactive non-principal cells (brown) at the border of stratum oriens and alveus. (B) The soma and proximal dendrites of a calbindin-containing interneuron in stratum radiatum, receive multiple innervation (arrows) from VIP-positive boutons. (C) A parvalbumin-immunoreactive dendrite is also contacted in stratum radiatum, but typically by far fewer VIP-boutons than calbindin-positive cells. (D) Another calbindin- positive interneuron, near the border of stata radiatum, and pyramidale was found to receive 64 contacts from VIP-containing boutons in a single 60/~m thick section, some of which can be seen on this photo montage (arrows). Note that, beside the soma and proximal dendrites, distal dendrites are also contacted. Correlated electron microscopic pictures of synapses formed by boutons labelled bl and b4 can be seen

in Fig. 6. Scale bars = 20#m (A, B, D, E), 10/~m (C).

Specific connections between hippocampal interneurons 327

Fig. 6. Correlated light and electron micrographs of the calbindin-immunoreactive interneuron shown in Fig. 5D in colour. (A) Black and white reproduction of a part of the colour montage shown in Fig. 5D. This calbindin-positive neuron was shown to receive 64 VIP-positive boutons in a 60 ~ m thick section. (B) Correlated low power electron micrograph of bouton b l -b3 . The corresponding capillary (c) and the relative position of boutons (bl b3), help the correlation. (C) High power electron micrograph of the VIP-positive bouton (bl) in synaptic contact with (arrows) the calbindin-immunoreactive dendrite (dCB). (D) Another bouton, see also in Fig. ID (b4) synapses (arrow) on the same dendrite more distally from

the soma. Scale bars = 20 p m (A), 4 # m (B), 0.5 # m (C, D).

328 L. Acsfidy et al.

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Fig. 7.

Specific connections between hippocampal interneurons 329

Fig. 8. Postsynaptic targets of VIP-containing basket cells. (A) Two VIP-immunoreactive boutons (bl, b2) are shown around the soma (S) of a pyramidal cell in stratum pyramidale. (B) The VIP-positive bouton (bl) establishes a symmetrical synapse (arrow) on the pyramidal cell body. (C) Postembedding GABA immunostaining shows that the VIP-containing, GABA-positive terminal synapses (arrow) on a GABA- negative pyramidal cell (SP) in stratum pyramidale. Open star labels another GABAergic profile. Scale

bars = 0.5 #m (A C).

revealed that VIP-positive boutons form symmetrical synapses with the somata and proximal dendrites of presumed pyramidal cells (Fig. 8). Single VIP-positive boutons were often found to establish two synapses when sandwiched between two cell bodies. Post- embedding immunogold staining for G A B A showed that the vast majority of postsynaptic targets in this layer were GABA-negat ive, thus, most likely pyramidal cells. All VIP-positive basket cell boutons, if containing mitochondria (see above), were shown to be GABA-posit ive. In stratum pyramidale of sections double-stained for VIP and parvalbumin, VIP-positive boutons were occasionally seen to con-

tact the somata of parvalbumin-positive basket (or chandelier) cells, which suggests that VIP-containing basket cells may innervate also other basket cells.

DISCUSSION

The present study of VIP-immunoreact ive inter- neurons revealed a new component in hippocampal networks, GABAergic interneurons, which are special- ized to control other interneurons. They should be considered as a third component , in addition to principal cells and other GABAergic interneurons, since the predominant targets of all interneurons

Fig. 7. Correlated light and electron micrographs ofVIP VIP contacts in the CAI region. (A) High power light micrograph of a VIP-containing interneuron ($2) in stratum radiatum, innervating another VIP- positive cell (S1) via multiple contacts (e.g., bl b3). Arrowhead labels the axon initial segment. (B) Three boutons (bl-b3) of cell $2 can be seen attached to the soma and proximal dendrite of cell SI in this correlated low power electron micrograph. The VIP-immunoreactive soma displays characteristics of interneurons: invaginated nucleus, a large number of cytoplasmic organelles. (C, D) High power electron micrographs show that the VIP-positive boutons (b2, b3) form symmetrical synapses (arrows) with the

VIP-positive interneuron (S1). Scale bars = 10/~m; (A), 2 #m; (B), 0.5 #m (C, D).

330 L. Acsfidy et al.

s. /ac-mo/.

s.rad.

T

Perforant path

Schaffer collaterals

Local WP-GABA

s.pyr.

s.ori.

L

A

s. lac-mol.

Perforant path

Schaffer collaterals

s.rad.

s.pyr.

s.ori.

B Fig. 9. Schematic diagrams of the connectivity of different VlP-containing cell types. (A) Basket cells (left) receive mainly Schaffer collateral input, and innervate the perisomatic region of pyramidal cells. Neurons projecting to the O/A border (right) have a dendritic tree spanning all strata but with an extensive arbor in the layer where entorhinal afferents terminate. They are often contacted by local VIP-positive neurons, and, in turn, innervate horizontal interneurons (mgluRl a-SOM) known to exert feed-back inhibition on distal dendrites of the pyramidal cells in stratum lacunosum-moleculare. (B) VIP-containing interneurons with a projection to stratum radiatum innervate other V1P-positive cells and GABAergic interneurons responsible for dendritic inhibition of pyramidal cells in the Schaffer collateral termination zone. One subtype is likely to receive afferent excitation exclusively from the entorhinal cortex (second from left), while the others have dendrites in all layers (left). Symbols: white cells, VlP-containing GABAergic cells; hatched cells, GABAergic cells innervated by VlP-cells and contacting pyramidal cells; black cells,

pyramidal cells; white dots, GABAergic terminal; black triangles, excitatory terminals.

Specific connections between

described earlier in any cortical areas were principal cells.

The postsynaptic targets of three distinct, VIP- containing interneuron types were established. Two of them were shown to selectively innervate other GABAergic neurons in the hippocampus, whereas the third contacted pyramidal cell bodies and proximal dendrites. VIP-positive cells, projecting to the O/A border, innervated the horizontal dendrites of mGluRla-immunoreactive (somatostatin and calbindin-containing) interneurons, which are known to be responsible for dendritic inhibition of pyramidal cells. VIP-containing cells projecting to stratum radiatum also innervated GABAergic interneurons selectively, in particular those which contain calbindin D-28k, SPR, VIP and occasionally parvalbumin.

Horizontal cells at the border of strata oriens and alveus innervated by vasoactive intestinal polypeptide- containing interneurons

Double immunostaining for VIP and mGluRla demonstrated that the striking parallel distribution of VIP-positive axons and mGluR I a-immunoreactive dendrites at the O/A border of the CAI region indeed reflected specific synaptic relationship between the two interneuron populations. The high incidence of mGluRla-posit ive dendrites among the targets of VIP-containing terminals suggests significant target selectivity. The poor penetration of the mGluRla antiserum is likely to account for a large proportion of the otherwise small number of mGluRla-negative postsynaptic dendrites. Calbindin-positive horizontal neurons may be the source of further mGluRla- negative targets, since the overlap between calbindin and somatostatin or calbindin and mGluRla- immunoreactive neurons in this sublayer is not perfect (unpublished observations).

In a recent study, Baude et al. 5 provided evidence that immunostaining for mGluRla labels the same interneuron population as antisera against somato- statin. Based on the extensive dendritic staining by mGluRla it is concluded that in the CAI region the overwhelming majority of mGluRla/somatostat in containing cells have a dendritic tree restricted to the O/A border. Intracellular labelling showed that axons of these cells innervated stratum lacunosum- moleculare both in CA133'5° and in CA3.17 This is supported also by the presence of a somatostatin- containing axon plexus in the s a m e layer, 4'7'2°'41'44 and the strong mGluRla-mediated excitation of interneurons projecting to stratum lacunosum- moleculare? 3 Several authors demonstrated that somatostatin-containing neurons are GABAergic, 24"46"53 suggesting an inhibitory role for this cell population.

In the light of the present findings, and those summarized above, we propose that VIP-positive cells projecting to the O/A border and innervating the horizontal dendrites of mGluRla-posit ive (somato- statin-containing) neurons exert a GABAergic control over feedback inhibition in the entorhinal termination

hippocampal interneurons 331

zone (Fig. 9A). The dendritic distribution of VIP- positive cells of this type (see accompanying paper 2) suggests that they may be excited by all afferent pathways present in the region, with some bias for entorhinal afferents.

The postsynaptic targets of vasoactive intestinal polypeptide-positive cells in stratum radiatum

In stratum radiatum VIP-positive boutons with identified origin, and also those of a random sample, established synaptic contacts predominantly on GABA-immunoreactive profiles, suggesting that both VIP-containing subtypes projecting to stratum radiatum (i.e. those with dendrites confined to stratum lacunosum-moleculare and those with dendrites span- ning all layers, see accompanying paper 2) innervate mostly, if not exclusively inhibitory cells.

Three subpopulations of GABAergic interneurons were found to receive multiple innervation from VIP-containing cells in stratum radiatum: calbindin D-28k-positive, VIP-positive, and occasionally parvalbumin-positive interneurons. Apical dendrites of calbindin-positive pyramidal cells--which are numerous in this layer in the CA 1 region3--were never seen to receive multiple contacts from VIP-positive axons. Calbindin-positive neurons of stratum radi- atum were shown to innervate pyramidal dendrites whereas parvalbumin-containing cells contact the perisomatic region of pyramidal cells. 2~'23 In addition to other interneurons, VIP-containing cells also innervate each other by multiple synapses. Thus, the VIP-containing cells projecting to the stratum radiatum are mutually connected, and specifically influence dendritic inhibition via calbindin D-28k- containing interneurons, and, to a smaller extent, somatic inhibition via parvalbumin-containing inter- neurons (Fig. 9B). The major source of excitatory drive is different in the two subtypes, since VIP- positive cells with dendrites in stratum lacunosum- moleculare are driven mostly if not exclusively by entorhinal afferents, while the other subtype with dendrites spanning all layers is likely to receive considerable Schaffer collateral input as well. In addition to the GABAergic cells mentioned above, other interneuron populations may also be contacted by VIP-positive axons. Spiny GABA-positive elements were also found to be innervated by VIP-positive boutons in the CA1 region, but neither calbindin D-28k- nor parvalbumin-containing cells possess significant numbers of spines.

Vasoactive intestinal polypeptide-containing basket cells

In contrast to the first two VIP-containing cell types, VIP-positive basket cells innervate the cell bodies and proximal dendrites of pyramidal cells. Basket cells are anatomically and electrophysio- logically well characterized and were shown to con- tain the calcium binding protein parvalbumin. 21'235° However, VIP-positive basket cells do not contain

332 L. Acsfidy et al.

parvalbumin (or either of the other two calcium binding proteins known to label interneurons: calbindin and calretinin, see accompanying paper2), but co-localize another neuropeptide, cholecystokinin (CCK). This represents direct evidence that basket cells are neurochemically heterogeneous, confirming earlier suggestions based on the presence of large numbers of parvalbumin-negative synapses on pyramidal and granule cell bodies. 43 Whether this neurochemical difference is associated with unique electrophysiological characteristics and/or actions on the postsynaptic pyramidal cells remains to be established by paired intracellular recordings and combined immunostaining.

VIP applied in ~itro was shown to have direct effects on pyramidal cells, 18 as well as indirect modulatory actions on their response to noradrenalin ~1~32 and acetylcholine. ~u~47 The modulation of cholinergic effects is of interest in the light of our observation since the distribution of acetylcholine receptors overlaps with that of VIP-positive axon terminals. Both alpha- bungarotoxin binding, ~ and muscarinic type II recep- tor immunostaining 3~ label the horizontal dendrites of O/A interneurons, suggesting that VIP and acetyl- choline may interact on this cell type. Furthermore, type II muscarinic receptors are located on boutons of basket cells in stratum pyramidale) ~ where VIP- positive baskets also terminate, thus allowing pre- synaptic interaction of acetylcholine and VIP on GABAergic terminals. The combined effect of VIP and G A B A 47 needs to be studied before a reasonable hypothesis can be formed for the functional differences between VIP- and parvalbumin-containing basket cells.

Implications q / ' a selective interneuronal control o[ hTterneurons

Although GABAergic interneurons were shown to receive input from local GABAergic sources 22'25 _,~,39,4s,59 the origin and specificity of these connections is still poorly understood. GABA-A and GABA-B inhibitory postsynaptic potentials were recorded from inter- neurons in stratum pyramidale 22'2s and at the border of strata radiatum and lacunosum-moleculare 22'28"3~59

showing the convergence of pharmacologiclly differ- ent inhibitory inputs to interneurons. Hyperpolarized membrane potential was shown to affect the mode of firing in stratum lacunosum-moleculare interneurons, 5'~ while the cessation of the hyperpolarizing current pulse often resulted in anodal breakdown response. 22'5'~ In intracellularly recorded pairs of interneurons the activation of stratum lacunosum-moleculare or O/A interneurons hyperpolarized the membrane potential of stratum pyramidale interneurons and inhibited

their spontaneous and evoked activity. 262~ Similar results were shown in pairs of interneurons in the dentate gyrus. 45 Although in these studies the identity of these interneurons has not been established by intracellular labelling, the results indicate that GABA- ergic cells have pronounced inhibitory effects on each other. According to these data, VlP-containing cells, specialized to control other interneurons, are likely to disinhibit the dendritic region of pyramidal cells via inhibiting GABAergic neurons that terminate on pyramidal dendrites. This effect appears to be layer specific, i.e. VIP-positive cells projecting to the O/A border may cause disinhibition in the entorhinal termination zone via horizontal mGluRla-pos i t ive cells, whereas VIP-containing cells projecting to stratum radiatum may disinhibit the Schaffer collateral termination zone via calbindin-positive interneurons. The blockade of feed-forward dendritic inhibition may increase the probability of N-methyl-I)-aspartate receptor activation, ~°58 and/or the opening of voltage- gated Ca 2~ channels, 3v thus may facilitate long-term potentiation of afferent synapses. Indeed, in a recent study, the activity of certain interneurons was found to be suppressed in the hippocampus as the animal entered a new environment, 6° i.e. when synaptic plasticity is expected to be enhanced.

However, disinhibition is not the only possible inter- pretation of GABAergic innervation of interneurons. Interneurons in stratum pyramidale were shown to excite O/A interneurons, 26 and in slices perfused with 4-amino-piridin, interneurons were shown to syn- chronize their activity via GABAergic transmission. ~4'3'~ In a recent study 5v synchronized, G A B A - A receptor mediated inhibitory postsynaptic potentials oscillating at 40 Hz were evoked in pyramidal cells in vitro. High- frequency network oscillations modulated by syn- chronized interneuronal activity were also described in different frequency ranges (from 40 to 200 Hz) in both extra- and intracelllular studies 6'~5~< in z'ivo.

In the in vitro study 57 a computer simulation has also been performed, in which extensive interconnection of interneurons was required for the generation of 40 Hz oscillation. The present study provides structural evidence that such connections indeed exist in the hippocampus, and may underlie synchronous population events.

Acknowledgements We are grateful to Dr P. Somogyi for antisera against GABA and to Dr K. G. Baimbridge for antisera against calbindin and parvalbumin. The excellent technical assistance of Ms E. Bor6k, Ms I. Weisz, Ms A. Z61di and Mr G. Terstyfinszki is also acknowledged. This work was supported by the Human Frontier Science Pro- gram Organization, the Howard Hughes Medical Institute and OTKA (T5532 and T16962) Hungary.

REFERENCES

I. Acsady L., Halasy K. and Freund T. F. (1993) Calretinin is present in non-pyramidal ceils of the rat hippocampus--lII. Their inputs from the median raphe and medial septal nuclei. Neuroscience 52, 829-841.

Specific connections between hippocampal interneurons 333

2. Acsady L., Arabadizisz D. and Freund T. F. (1996) Correlated morphological and neurochemical features identify different subsets of VIP-immunoreactive interneurons in rat hippocampus. Neuroscienee 73, 299-315.

3. Baimbridge K. G. and Miller J. J. (1982) Immunohistochemical localization of calcium-binding protein in the cerebellum, hippocampal formation and olfactory bulb of the rat. Brain Res. 245, 223 229.

4. Bakst I., Morrison J. H. and Amaral D. G. (1985) The distribution of somatostatin-like immunoreactivity in the monkey hippocampal formation. J. comp. Neurol. 236, 423 442.

5. Baude A., Nusser S., Roberts J. D., Mulvihill E., McIlhinney R. A. and Somogyi P. (1993) The metabotropic glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction. Neuron 11, 771 787.

6. Bragin A., Jando G., Nadasdy Z., Hetke J., Wise K. and Buzsaki G. (1995) Gamma (40 100 Hz) oscillation in the hippocampus of the behaving rat. J. Neurosci. 15, 47-60.

7. Buckmaster P. S., Kunkel D. D., Robbins R. J. and Schwartzkroin P. A. (1994) Somatostatin-immunoreactivity in the hippocampus of mouse, rat, guinea pig, and rabbit. Hippocampus 4, 167-180.

8. Buhl E. H., Halasy K. and Somogyi P. (1994) Diverse sources of hippocampal unitary inhibitory postsynaptic potentials and the number of synaptic release sites [see comments]. Nature 368, 823-828.

9. Buzsaki G., Horvath Z., Urioste R., Hetke J. and Wise K. (1992) High-frequency network oscillation in the hippocampus. Science 256, 1025 1027.

10. Collingridge G. L., Herron C. E. and Lester R. A. (1988) Synaptic activation of N-methyl-D-aspartate receptors in the Schaffer collateral-commissural pathway of rat hippocampus. J. Physiol., Lond. 399, 283-300.

11. Ferron A., Siggins G. R. and Bloom F. E. (1985) Vasoactive intestinal polypeptide acts synergistically with norepinephrine to depress spontaneous discharge rate in cerebral cortical neurons. Proe. natn. Acad. Sci. U.S.A. 82, 8810-8812.

12. Freedman R., Wetmore C., Stromberg I., Leonard S. and Olson L. (1993) Alpha-bungarotoxin binding to hippocampal interneurons: immunocytochemical characterization and effects on growth factor expression. J. Neurosci. 13, 1965 1975.

13. Freund T. F. and Antal M. (1988) GABA-containing neurons in the septum control inhibitory interneurons in the hippocampus. Nature 336, 170-173.

14. Freund T. F., Gulyas A. I., Acsady L., Gorcs T. and Toth K. (1990) Serotonergic control of the hippocampus via local inhibitory interneurons. Proc. natn. Acad. Sei. U.S.A. 87, 8501 8505.

15. Gorcs T. J., Penke B., Boti Z., Katarova Z. and Hamori J. (1993) Immunohistochemical visualization ofa metabotropic glutamate receptor. NeuroReport 4, 283 286.

16. Gulyas A. I., Gorcs T. J., Freund T. F. (1990) Innervation of different peptide-containing neurons in the hippocampus by GABAergic septal afferents. Neuroscience 37, 31 44.

17. Gulyas A. I., Miles R., Hajos N. and Freund T. F. (1993) Precision and variability in postsynaptic target selection of inhibitory cells in the hippocampal CA3 region. Eur. J. Neurosci. 5, 1729 1751.

18. Haas H. L. and Gahwiler B. H. (1992) Vasoactive intestinal polypeptide modulates neuronal excitability in hippocampal slices of the rat. Neuroscienee 47, 273 277.

19. Han Z. S., Buhl E. H., Lorinczi Z. and Somogyi P. (1993) A high degree of spatial selectivity in the axonal and dendritic domains of physiologically identified local-circuit neurons in the dentate gyrus of the rat hippocampus. Eur. J. Neurosei. 5, 395 410.

20. Johansson O., H6kfelt T. and Elde R. P. (1984) Immunohistochemical distribution of somatostatin-like immuno- reactivity in the central nervous system of the adult rat. Neuroscience 13, 265 339.

21. Katsumaru H., Kosaka T., Heizmann C. W. and Hama K. (1988) Immunocytochemical study of GABAergic neurons containing the calcium-binding protein parvalbumin in the rat hippocampus. Expl Brain Res. 72, 347 362.

22. Kawaguchi Y. and Hama K. (1987) Two subtypes of non-pyramidal cells in rat hippocampal formation identified by intracellular recording and HRP injection. Brain Res. 411, 190 195.

23. Kosaka T., Katsumaru H., Hama K., Wu J. Y. and Heizmann C. W. (1987) GABAergic neurons containing the Ca2+-binding protein parvalbumin in the rat hippocampus and dentate gyrus. Brain Res. 419, 119 130.

24. Kosaka T., Wu J. Y. and Benoit R. (1988) GABAergic neurons containing somatostatin-like immunoreactivity in the rat hippocampus and dentate gyrus. Expl Brain Res. 71, 388 398.

25. Lacaille J. C. (1991) Postsynaptic potentials mediated by excitatory and inhibitory amino acids in interneurons of stratum pyramidale of the CAI region of rat hippocampal slices in vitro. J. Neurophysiol. 66, 1441 1454.

26. Lacaille J. C., Mueller A. L., Kunkel D. D. and Schwartzkroin P. A. (1987) Local circuit interactions between oriens/ alveus interneurons and CAI pyramidal cells in hippocampal slices: electrophysiology and morphology. J. Neurosci. 7, 1979 1993.

27. Lacaille J. C. and Schwartzkroin P. A. (1988) Stratum lacunosum moleculare interneurons of hippocampal CAI region--I. Intracellular response characteristics, synaptic responses, and morphology. J. Neurosei. 8, 1400 1410.

28. Lacaille J. C. and Schwartzkroin P. A. (1988) Stratum lacunosum-moleculare interneurons of hippocampal CA1 region |I. Intrasomatic and intradendritic recordings of local circuit synaptic interactions. J. Neurosci. 8, 1411 1424.

29. Leranth C. and Frotscher M. (1983) Commissural afferents to the rat hippocampus terminate on vasoactive intestinal polypeptide-like immunoreactive non-pyramidal neurons. An EM immunocytochemical degeneration study. Brain Res. 276, 357 361.

30. Leranth C., Frotscher M., Tombol T. and Palkovits M. (1984) Ultrastructure and synaptic connections of vasoactive intestinal polypeptide-like immunoreactive non-pyramidal neurons and axon terminals in the rat hippocampus. Neuroscience 12, 531 542.

31. Levey A. I., Edmunds S. E., Koliatsos V., Wiley R. G. and Heilman C. J. (1995) Expression of ml-m4 muscarinic acetylcholine receptor proteins in rat hippocampus and regulations by cholinergic innervation. J. Neurosci. 15, 4077 4092.

32. Magistretti P. J. and Schorderet M. (1984) VIP and noradrenaline act synergistically to increase cyclic AMP in cerebral cortex. Nature 308, 280 282.

33. McBain C. J., DiChiara T. J. and Kauer J. A. (1994) Activation of metabotropic glutamate receptors differentially affects two classes of hippocampal interneurons and potentiates excitatory synaptic transmission. J. Neurosei. 14, 4433 4445.

334 L. Acsfidy et al.

34. Michelson H. B. and Wong R. K. (1991) Excitatory synaptic responses mediated by G A B A A receptors in the hippocampus. Science 253, 1420 1423.

35. Miettinen R. and Freund T. F. (1992) Convergence and segregation of septal and median raphe inputs onto different subsets of hippocampal inhibitory interneurons. Brain Res. 594, 263 272.

36. Miettinen R. and Freund T. F. (1992) Neuropeptide Y-containing interneurons in the hippocampus receive synaptic input from median raphe and GABAergic septal afferents. Neuropeptides 22, 185 193.

37. Miles R., T6th K., Gulyfis A. I., H~ijos N. and Freund T. F. (1996) Distinct functional roles for somatic and dendritic inhibition in hippocampus. Neuron (in press).

38. Misgeld U. and Frotscher M. (1986) Postsynaptic-GABAergic inhibition of non-pyramidal neurons in the guinea-pig hippocampus. Neuroseience 19, 193 206.

39. Muller W. and Misgeld U. (1990) Inhibitory role of dentate hilus neurons in guinea pig hippocampal slice. J. Neurophysiol. 64, 46 56.

40. Murphy P. C., Grieve K. L. and Sillito A. M. (1993) Effects of vasoactive intestinal polypeptide on the response properties of cells in area 17 of the cat visual cortex. J. Neurophysiol. 69, 1465 1474.

41. Naus C. C., Morrison J. H. and Bloom F. E. (1988) Development of somatostat in-containing neurons and fibers in the rat hippocampus. Brain Res. 468, 113 121.

42. Ramon y Cajal (1968) The Structure Of Ammon's Horn. Charles C. Thomas. 43. Ribak C. E., Nitsch R. and Seress L. (1990) Proportion of parvalbumin-positive basket cells in the GABAergic

innervation of pyramidal and granule cells of the rat hippocampal formation. J. comp Neurol. 300, 449 461. 44. Roberts G. W., Woodhams P. L., Polak J. M. and Crow T. J. (1984) Distribution of neuropeptides in the limbic system

of the rat: the hippocampus. Neuroscience I !, 35 77. 45. Scharfman H. E., Kunkel D. D. and Schwartzkroin P. A. (1990) Synaptic connections of dentate granule cells and hilar

neurons: results of paired intracellular recordings and intracellular horseradish peroxidase injections. Neuroscience 37, 693 707.

46. Schmechel D. E., Vickrey B. G., Fitzpatrick D. and Elde R. P. (1984) GABAergic neurons of manamalian cerebral cortex: widespread subclass detined by somatostat in content. Neurosci. Lett. 4T, 227 232.

47. Sessler F. M., Grady S. M., Walerhouse B. D. and Moises H. C. (1991) Electrophysiological actions of VIP in rat somatosensory cortex. PeptMes 12, 715 721.

48. Sik A., Tamamaki N. and Freund T. F. (1993) Complete axon arborization of a single CA3 pyramidal cell in the rat hippocampus, and its relationship with postsynaptic parvalbumin-containing interneurons. Eur. J. Neurosci. 5, 1719 1728.

49. Sik A., Ylinen A., Penttonen M. and Buzsaki G. (1994) Inhibitory CAI-CA3-hi lar region feedback in the hippocampus. Science 265, 1722 1724.

50. Sik A., Pentonncn M., Ylinen A. and Buzs~tki G. (1995) Hippocampal CA l interneurons: an in vitro intracellular labeling sludy. J. Neuroxci.

51. Soltesz 1. and Mody I. (1994) Patch-clamp recordings reveal powerful GABAergic inhibition in dentatc hilar neurons. J. Neurosci. 14, 2365 2376.

52. Somogyi P. and Hodgson A. J. (1985) Antisera to gamma-aminobutyr ic acid IIk Demonstrat ion of GABA in Golgi-impregnated neurons and in conventional electron microscopic sections of cat striate cortex. J. Histochem. Qvloehem. 33, 249 257.

53. Somogyi P., Hodgson A. J., Smith A. D., Nunzi M. G., Gorio A. and Wu J. Y. (1984) Different populations of GABAergic neurons in the visual cortex and hippocampus of cat contain somatostatin- or cholecystokinin-immunoreactive material. J. Neurosci. 4, 2590 2603.

54. Spruston N., Schiller Y., Stuart G. and Sakmann B. (1995) Activity-dependent action potential invasion and calcium influx into hippocampal CAI dendrites. Science 268, 297 300.

55. Toth K., Borhegyi Z. and Freund T. F. (1993) Postsynaptic targets of GABAergic hippocampal neurons in the medial septum-diagonal band of Broca complex. J. Neuros~i. 13, 3712 3724.

56. Toth K. and Freund T. F. (1992) Calbindin D28k-containing nonpyramidal cells in the rat hippocampus: their immunoreactivity for GAB A and projection to the medial septum. Neuroseienee 49, 793 805.

57. Whitt ington M. A., Traub R. D. and Jefferys J. G. R. (1995) Synchronized oscillations in interneuron networks driven by metabotropic glutamate receptor activation. Nature 373, 612 615.

58. Wigstrom H. and Gustafsson B. (1983) Facilitated induction ofh ippocampal long-lasting potentiation during blockade of inhibition. Nalure 301, 603 604.

59. Williams S., Samulack D. D., Beaulieu C. and Lacaille J. C. (1994) Membrane properties and synaptic responses of interneurons located near the s tratum lacunosum-moleculare/radiatum border of area CA I in whole-cell recordings from rat hippocampal slices. J. Neurophysiol. 71, 2217 2235.

6(1. Wilson M. A. and McNaughton B. L. (1993) Dynamics of the hippocampal ensemble code for space [see comments]. Science 261, 1055 1058.

61. Ylinen A., Bragin A., Nadasdy Z., Jando G., Szabo 1. and Sik A. (1995) Sharp wave-associated high-frequency oscillation (200 Hz) in the intact hippocampus: network and intracellular mechanisms. J. Neurosci. 15, 30 46.

(Accepted 12 December 1995)