Postsynaptic targets of somatostatin-immunoreactive interneurons in the rat hippocampus

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Neuroscience Vol. 88, No. 1, pp. 37–55, 1999 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00302-9

POSTSYNAPTIC TARGETS OF SOMATOSTATINIMMUNOREACTIVE INTERNEURONS IN THE RAT HIPPOCAMPUS I. KATONA, L. ACSA u DY and T. F. FREUND* Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O. Box 67, Budapest H-1450, Hungary Abstract––Two characteristic interneuron types in the hippocampus, the so-called hilar perforant path-associated cells in the dentate gyrus and stratum oriens/lacunosum-moleculare neurons in the CA3 and CA1 regions, were suggested to be involved in feedback circuits. In the present study, interneurons identical to these cell populations were visualized by somatostatin-immunostaining, then reconstructed, and processed for double-immunostaining and electron microscopy to establish their postsynaptic target selectivity. A combination of somatostatin-immunostaining with immunostaining for GABA or other interneuron markers revealed a quasi-random termination pattern. The vast majority of postsynaptic targets were GABA-negative dendritic shafts and spines of principal cells (76%), whereas other target elements contained GABA (8%). All of the examined neurochemically defined interneuron types (parvalbumin-, calretinin-, vasoactive intestinal polypeptide-, cholecystokinin-, substance P receptorimmunoreactive neurons) received innervation from somatostatin-positive boutons. Recent anatomical and electrophysiological data showed that the main excitatory inputs of somatostatin-positive interneurons originate from local principal cells. The present data revealed a massive GABAergic innervation of distal dendrites of local principal cells by these feedback driven neurons, which are proposed to control the efficacy and plasticity of entorhinal synaptic input as a function of local principal cell activity and synchrony.  1998 IBRO. Published by Elsevier Science Ltd. Key words: inhibition, non-pyramidal cells, neuropeptides, feedback inhibition, GABA, theta.

Interneurons in the cerebral cortex are known to play a crucial role in controlling the activity of large ensembles of principal cells. Recent electrophysiological and anatomical studies shed light on the specific function of various interneuron types in the regulation of population behaviour of principal cells at different nodes in the hippocampal network (for review see Ref. 22). Intracellular labelling of interneurons with biocytin in vitro and in vivo15,16,26,59,61 and immunostaining for selected neurochemical markers or their combinations are the most powerful tools to investigate the precise connectivity, and neurochemical features of an interneuron type. The former allows a direct correlation of electrophysiological properties with the axonal and dendritic arborization pattern of

a given cell, whereas the latter can visualize large populations of a specific cell type and allows correlation of neurochemical characteristics with certain morphological features. By using these methods, many distinct interneuron types were classified in the hippocampus. It was shown that perisomatic inhibitory neurons (i.e. basket and axoaxonic cells) contain either the calcium-binding protein parvalbumin35 or the neuropeptides cholecystokinin (CCK)51 and vasoactive intestinal polypeptide (VIP).1 Inhibitory neurons that predominantly target the dendrites of principal cells (i.e. dendritic inhibitory cells) include two major types that differ in their layer of termination and afferent input. Bistratified cells16 innervate pyramidal cell dendrites in strata radiatum and oriens (in conjunction with Schaffer collaterals) and a large proportion of them contains the calcium-binding protein calbindin.59,61 The other characteristic dendritic inhibitory neuron type has a dendritic tree restricted to those layers where local collaterals of principal cells of the given hippocampal subfield arborize, whereas it’s axon terminates on distal dendrites of principal cells in conjunction with entorhinal afferents. These interneurons are the so-called oriens/ lacunosum-moleculare (O-LM) cells in the CA147,61 and CA3 regions26 of the hippocampus and the hilar perforant path-associated (HIPP) cells in the hilus of the dentate gyrus.15,32,59

*To whom correspondence should be addressed. Abbreviations: ABC, avidin–biotin–horseradish peroxidase complex; ACPD, 1S,3R-aminocyclopentane dicarboxylic acid; BSA, bovine serum albumin; CCK, cholecystokinin; DAB, 3,3 -diaminobenzidine–4HCl; EPSP, excitatory postsynaptic potential; HIPP, hilar perforant pathassociated; IPSP, inhibitory postsynaptic potential; LTP, long-term potentiation; M2, type 2 muscarinic receptor; mGluR1á, type 1á metabotropic glutamate receptor; NGS, normal goat serum; NPY, neuropeptide Y; O-LM, strata oriens/lacunosum-moleculare; PB, phosphate buffer; SPR, substance P receptor; TBS, Tris-buffered saline; VIP, vasoactive intestinal polypeptide. 37

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Fig. 1.

Somatostatin-immunoreactive interneurons in hippocampus

In a recent study, Maccaferri and McBain44 showed that induction of long-term depression at Schaffer collateral–CA1 pyramidal cell synapses can increase the probability of long-term potentiation (LTP) generation at entorhinal afferent-CA1 pyramidal cell distal dendritic synapses. The network mechanism of this peculiar interaction was suggested to be feedback inhibition of distal dendrites by O-LM cells. This is supported by available anatomy demonstrating that the vast majority of excitatory input to O-LM cells derives from local CA1 pyramidal cells.14 The finding that O-LM cells can be discharged by Schaffer collateral stimulation only if it evokes a population spike in CA1, and excitatory postsynaptic potentials recorded from O-LM cells always occur after this population spike, also supports the exclusive feedback drive for this cell type.44 O-LM cells, in turn, terminate in stratum lacunosum-moleculare, but whether their major targets are pyramidal cells or interneurons, also having extensive dendritic arbors in this layer,1,40 is still unknown. Similar circuitry features hold true for HIPP cells in the dentate gyrus. Their dendrites are limited to the hilus, where local collaterals of granule cell axons (mossy fibres) terminate, and have no access to feedforward excitatory input, which terminate in stratum moleculare. In contrast, their axons arborize in the outer two-thirds of the molecular layer in conjunction with entorhinal afferents, similar to O-LM cells in the Ammon’s horn.31,32,59 Here again, whether the major targets are granule cell dendrites is still an open question. One may argue that the postsynaptic elements of O-LM cells in stratum lacunosum-moleculare have to be distal dendrites of pyramidal cells (or of granule cells in case of HIPP cells), because these are far the most abundant available targets in these layers. However, this reasoning has already proved to be wrong in the case of calretinin- and VIP-positive axon arbors at the stratum oriens-alveus border, where similarly pyramidal dendrites are the most abundant elements in the neuropil, nevertheless this GABAergic projection terminates selectively on

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GABAergic targets in this layer, and ignores pyramidal cell dendrites.2,24 A direct analysis of postsynaptic elements by immunocytochemical double-staining techniques could provide the answer. For such a study a neurochemical marker is required, which selectively visualizes the axon terminals of O-LM and HIPP cells. Interneurons immunoreactive for the neuropeptide somatostatin have been reported in the hippocampal formation in a number of species, for example in rat,21,36,50,56 mouse,21 rabbit,39 guinea-pig,21 monkey55 and human.8,20 Somatostatin-immunoreactive structures have the same laminar and regional distribution as O-LM and HIPP cells: i.e. the perikarya and dendritic trees are mainly located in the hilus of the dentate gyrus and stratum oriens of the CA1 and CA3 subfields and dense somatostatinimmunoreactive axon terminal fields are present in the outer molecular layer of the dentate gyrus and in the stratum lacunosum-moleculare of CA1 and CA3, where entorhinal inputs terminate. In the present study, using a new antiserum raised against prosomatostatin,41 we aimed to provide further evidence that SOM-positive cells indeed correspond to O-LM and HIPP cells. Doubleimmunostaining was employed to investigate the postsynaptic targets in the dentate molecular layer and in stratum lacunosum-moleculare of the Ammon’s horn according to their GABA content or immunoreactivity for other interneuron markers. EXPERIMENTAL PROCEDURES

Perfusion and preparation of tissue sections Eight male Wistar rats (300–350 g, two-months-old; Charles River, Budapest, Hungary) were deeply anaesthetized by Equithesin (chlornembutal, 0.3 ml/100 g), and perfused through the heart first with saline followed by a phosphate-buffered (PB, 0.1 M) fixative containing 4% paraformaldehyde, 0.15% picric acid and 0.05% glutaraldehyde in the case of series A (n=5) for single somatostatinimmunostaining and for somatostatin–parvalbumin, somatostatin–CCK, somatostatin–calretinin, somatostatin– VIP, somatostatin–subtance P receptor (SPR) double

Fig. 1. (A) Low power light micrograph showing a specific laminar distribution of somatostatinimmunostaining in CA1 and in the dentate gyrus. Cell bodies and dendrites (arrows) are confined to stratum oriens and the hilus, whereas the main axon terminal fields are located in stratum lacunosummoleculare and in the outer part of stratum moleculare. Arrowheads indicate a typical main axon emerging from stratum oriens crossing stratum pyramidale and stratum radiatum towards stratum lacunosum-moleculare. (B) At higher magnification a dense network of varicose axon collaterals is visible in stratum lacunosum-moleculare. Arrowheads point to an axon entering from stratum radiatum to stratum lacunosum-moleculare. (C) Main axons crossing the hippocampal fissure (i.e. the border of stratum moleculare and stratum lacunosum-moleculare) were often observed (arrowheads). (D) High power light micrograph of a somatostatin-immunoreactive neuron located in stratum oriens of CA1. Note the axon emerging from a proximal dendrite, crossing stratum pyramidale and bifurcating in stratum radiatum. The two main branches were partially reconstructed as shown in Fig. 2 (Cell 1). They reached stratum lacunosum-moleculare where they extensively arborized, suggesting that they are conventional O-LM neurons. (E–F) Somatic and dendritic spines were often observed on somatostatin-immunoreactive neurons. Some of these spines are depicted by arrows. (G) The axon collaterals of Cell 4 bore several drumstick-like boutons. Two of them are indicated by arrows. ais, axon initial segment; h., hilus; s.g., stratum granulosum; s.m., stratum moleculare; s.l-m., stratum lacunosum-moleculare; s.o., stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum. Scale bars: (A)=100 µm; (B)=30 µm; (C)=80 µm; (D)=15 µm; (E–F)=10 µm; (G)=5 µm.

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Fig. 2.

Somatostatin-immunoreactive interneurons in hippocampus immunostaining. In series B (n=3 animals) the fixative contained 4% paraformaldehyde, 0.15% picric acid and 1% glutaraldehyde dissolved in 0.1 M PB (pH 7.4) for preembedding somatostatin-immunostaining combined with postembedding immunogold staining for GABA. Brains were removed from the skull, blocks of the hippocampus and overlying neocortex were dissected and coronal sections of 60 µm thickness were cut on a Vibratome. After extensive washes the sections were cryoprotected in 30% sucrose in 0.1 M PB overnight, and freeze-thawed in an aluminium foil boat over liquid nitrogen to enhance the penetration of antisera without destroying the ultrastructure of sections. The sections prepared for light microscopic reconstruction of somatostatin-immunoreactive neurons were treated with 0.5% Triton X-100 diluted in 0.05 M Tris-buffered saline (TBS) also containing 5% bovine serum albumin (BSA). Pre-embedding immunocytochemistry Following extensive washes and treatment with 1% sodium borohydride for 30 min (only for animals perfused with fixative B), the sections were incubated first in 5% BSA and then in solutions of the following antisera: for single somatostatin-immunostaining rabbit anti-somatostatin, diluted 1:20,000,41 was used as primary antiserum (48 h). The second layer was biotinylated anti-rabbit IgG (Vector, 2 h, 1:400) followed by avidin–biotin–horseradish peroxidase complex (Elite ABC, Vector, 1.5 h, 1:400). The immunoperoxidase reaction was developed using 3,3 -diaminobenzidine (DAB; Sigma) intensified with ammonium nickel–sulphate (DAB–Ni) as a chromogen (black reaction product). The sections were treated with 1% osmium tetroxide in 0.1 M PB for 1 h, dehydrated in ethanol and propylene oxide, and embedded in Durcupan (ACM, Fluka). During dehydration the sections were treated with 1% uranyl acetate in 70% ethanol for 45 min. Reconstruction of somatostatin-immunoreactive neurons and their axonal and dendritic processes Selected somatostatin-immunoreactive cells were drawn with the aid of a camera lucida from 60-µm-thick serial Vibratome sections. The dendrites and axons of each cells were reconstructed using 100 oil immersion objective and were followed as long as it was possible to distinguish them from other immunoreactive processes. On the surface of sections, capillaries near the cut ends of dendrites and axons were also drawn, to serve as landmarks. In the adjacent sections the same dendrites and axons were identified by matching the capillaries. Pre-embedding double immunostaining After the first immunostaining for somatostatin, the sections were blocked by the ABC blocking kit, then incubated in solutions of one of the following antisera: rabbit anti-parvalbumin (1:1500);11 rabbit anti-calretinin (1:5000);55 rabbit anti-VIP (1:10,000);23 rabbit anti-CCK

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(1:3000);23 and rabbit anti-substance P receptor (SPR) (1:300)58 for 48 h. This was followed by biotinylated antirabbit IgG (Vector, 2 h, 1:400) and Elite ABC (Vector, 1.5 h, 1:400). This second immunoperoxidase reaction was developed with DAB as a chromogen (brown reaction end product). The sections were treated with 1% osmium tetroxide also containing 7% glucose to preserve colour difference between DAB–Ni and DAB end products, dehydrated and embedded in Durcupan as above. The specificity of the primary antisera have been tested by the laboratories of origin (see references above). In double-stained sections each antisera gave the same staining pattern as if applied in single staining. Although the primary antisera in both cycles were raised in rabbit, the end product of the first immunoperoxidase reaction masked the immune complex so that the antisera of the second cycle could not bind to the first. Replacing the primary antisera with normal rabbit serum resulted in the lack of specific immunostaining, only a faint nuclear background staining was present on the surface of the sections. The immunoreactive profiles at the light microscopic level were identified by their colour difference (i.e. somatostatinpositive profiles were black, the second markers were brown). Some of the somatostatin-immunoreactive boutons in close contact with dendrites and somata of the neurons immunostained by the second marker were photographed and re-embedded for further ultrathin sectioning. At the electron microscopic level, the same profiles were identified and examined whether they form synapses. Postembedding immunogold staining for GABA Areas with dense somatostatin-immunoreactive axonal staining from animals perfused with fixative B were selected both in the ventral and dorsal part of stratum lacunosummoleculare of the CA1 and CA3 subfields and stratum moleculare of the dentate gyrus and were re-embedded. Ultrathin sections were cut on a Reichert ultramicrotome and adjacent sections were mounted on copper and nickel grids. Postembedding GABA immunostaining was carried out on the nickel grids according to the protocol of Somogyi and Hodgson,66 using a well-characterized antiserum against GABA.33 Incubations were performed on droplets of solutions in humid Petri dishes. Briefly: 2% periodic acid (H5IO6) for 10 min; washed in distilled water; 2% sodium metaperiodate (NaIO4) for 10 min; washed in distilled water; three times 2 min in TBS (pH 7.4); 30 min in 1% ovalbumin dissolved in TBS; three times in TBS containing normal goat serum (NGS); 1.5 h in rabbit anti-GABA antiserum (1:3000 in NGS/TBS); two times 10 min TBS; 10 min in Tris buffer containing 1% BSA, 0.05% Tween 20; 2 h goat anti-rabbit IgG-coated colloidal gold (1 nm or 15 nm, Amersham, 1:20 in NGS/TBS) in the same solution as before; two times 5 min washed in distilled water; 30 min saturated uranyl acetate; washed in distilled water; stained with lead citrate; washed in distilled water. In the case of 1 nm gold particles, additional postfixation with 1%

Fig. 2. Camera lucida drawings of somatostatin-immunoreactive neurons of the CA1 subfield, reconstructed from 10 consecutive sections (60 µm each). (A) The ‘‘Classic’’ type of somatostatin-positive neurons showing typical morphology of O-LM cells. Namely, the somata and dendrites are confined to stratum oriens, while the main axons arborize predominantly in stratum lacunosum-moleculare. Note that the axons in each case emerge from proximal dendrites, which is a typical feature of O-LM cells. Cell 1 is also shown in Fig. 1D. (B) A type of somatostatin-positive neuron showing striking differences from those in A. The soma is located in the alveus, whereas some dendrites extend to stratum pyramidale. The axon emerges from the cell body and bifurcates several times close to the axon hillock. One of the main axons disappears in the alveus (arrowheads), and the others arborize profusely in stratum pyramidale. Another main axon crosses the hippocampal fissure, but it was impossible to follow further due to the dense axonal meshwork in stratum moleculare (see Fig. 1A–C.). The axon carried several drumstick-like boutons (see Fig. 1G), some of them are indicated by arrows. It should be noted that none of the axon collaterals could be followed to their natural end, they were either cut on the section surface, or lost in dense terminal networks. Thus, each reconstruction is very partial.

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glutaraldehyde was performed, and silver intensification (Amersham) was carried out for 4 min to increase the size of the label. There was no difference in the specificity and sensitivity between the two methods. Somatostatin-immunoreactive boutons were searched for in sections on copper grids, and when they formed a synapse, the GABA content of the postsynaptic element was examined in the adjacent, GABA-immunostained sections on nickel grids. 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. Profiles containing the same or lower numbers of gold particles as asymmetrical terminals (background level) were considered GABA-negative, whereas postsynaptic elements with GABA-immunoreactivity between these two levels were regarded as unidentified. The electron micrographs were taken on a Hitachi 7100 electron microscope. The studies were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals. RESULTS

General pattern of somatostatin-immunostaining in the hippocampus Somatostatin-immunoreactive neurons showed the same basic morphological features as observed in several previous studies in the rat21,34,36,38,50,64 with some additional morphological details due to the enhanced sensitivity of the present antiserum. Briefly, cell bodies were located in stratum oriens of the CA1, stratum oriens and stratum lucidum of the CA3 regions and in the hilus of the dentate gyrus (Fig. 1A). Occasionally, some cells were also found in stratum radiatum of the CA1 and CA3 subfields. The vast majority of dendritic processes were confined to the same layers as the cell bodies. At higher magnification, spines were observed on both the dendrites and perikarya (Fig. 1E–F). This is consistent with the observation that dendrites of somatostatin-positive cells visualized by immunostaining for metabotropic glutamate receptor 1á subunit (mGluR 1á)13 and SPR4 are covered by long thin spines. The most striking immunoreactive structure was the dense meshwork of axons in stratum lacunosummoleculare of the CA1 and CA3 subfields and in the outer two thirds of stratum moleculare in the dentate gyrus (Fig. 1A–B). Most of the main axons crossed the cell body and proximal dendritic layers with only occasional side-branches and turns, nevertheless roving axons were seen in all layers throughout the hippocampus. These axon trunks often carried small round varicosities resembling ‘‘en passant’’ boutons, but in some cases drumstick-like terminals were also observed mainly in the hilus, stratum oriens and stratum radiatum (Fig. 1G). Main axons crossing the hippocampal fissure were also found (Fig. 1C). Reconstruction of somatostatin-immunoreactive neurons CA1 subfield. Despite strong axonal immunostaining, it was hard to reconstruct somatostatin-positive

neurons due to the rare staining of proximal main axon segments. Thick axons were followed back from stratum lacunosum-moleculare to stratum oriens in many cases, however only four of them could be unequivocally connected to its cell body. Three cells showed typical morphology of O-LM cells (Fig. 2A).43,47,54,61 The somata and dendrites were located in stratum oriens. The axons emerged from a proximal dendrite (Figs 1D, 2A), as also seen in intracellularly filled O-LM cells,29,47 crossed stratum pyramidale and radiatum, and began to arborize with much thinner collaterals in stratum lacunosummoleculare. Because of the dense meshwork of axon collaterals in this layer, it was impossible to follow one reliably for a considerable distance. Therefore, we reconstructed only some of the primary and secondary branches of the arborization in stratum lacunosum-moleculare, which clearly indicated that indeed, somatostatin-containing O-LM neurons project to stratum lacunosum-moleculare in the CA1 subfield (Fig. 2A). The main axons also gave some collaterals in stratum oriens and stratum pyramidale. The axons of the reconstructed O-LM cells were studded by ‘‘en passant’’ boutons, whereas drumstick-like terminals were not seen on them. The fourth cell showed some striking differences from the conventional O-LM type cells (Fig. 2B). First, one of the main axons was faintly labelled and disappeared in the alveus. Second, it had a more profuse axonal arborization both in stratum oriens and stratum radiatum, whereas the main axon ran directly towards the hippocampal fissure, although it was impossible to follow through due to the very dense somatostatin-positive axon staining. Moreover, the axons bore several drumstick-like boutons (Fig. 1G) in addition to the ‘‘en passants’’ varicosities. Other two main axons with similar features have also been reconstructed, but their cell bodies could not be recovered. These main axons were followed from the stratum oriens-alveus border to the hilus across the hippocampal fissure. They arborized extensively both in the hilus and in stratum radiatum of CA1, but also gave several axon collaterals in all other layers. It is important to note that, in addition to the ‘‘en passant’’ varicosities, these axons bore large numbers of drumstick-like boutons mainly in the hilus and in stratum oriens. In spite of the lack of recovered cell bodies, these striking morphological similarities with the fourth cell and conspicuous differences from the three former cells strongly suggest that these latter axons and Cell 4 belong to a second type of somatostatin-immunoreactive neurons in the CA1 subfield of the hippocampus. CA3 subfield. The stratum lacunosum-moleculare of the CA3 subfield also showed dense somatostatinimmunoreactive axonal immunostaining. These axons probably originated from the somatostatinpositive somata of stratum oriens.26 In stratum radiatum, many roving axons bearing drumstick-like

Somatostatin-immunoreactive interneurons in hippocampus

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Fig. 3. Camera lucida drawings of somatostatin-immunoreactive neurons in the hilus of the dentate gyrus. This somatostatin-containing cell type was most frequently encountered in the hilus and showed many characteristics of HIPP cells. The somata and dendrites are confined to the hilus, while the axons crossing stratum granulosum arborize extensively in stratum moleculare. Two of the axons emerge from proximal dendrites (Cells 1 and 2), while the third one originates from the cell body (Cell 3). The main axon of Cell 1 had a collateral penetrating the hilus.

boutons could be observed. They were often found to cross the border of the CA1 and CA3 subfields. Many somatostatin-immunoreactive cell bodies were located in stratum lucidum and their axon initial segments were only occasionally immunostained. However, these axons became usually faintly labelled beyond the axon initial segment. Some of them could be followed through stratum pyramidale, but most of them disappeared already in stratum lucidum. Dentate gyrus. Three somatostatin-immunoreactive neurons were partially reconstructed in the dentate gyrus, all of them showed typical morphological characteristics of HIPP cells7,15,32,59 (Fig. 3). The somata and the dendritic trees were located in the hilus, whereas the main axons crossed stratum granulosum and arborized in the outer two-thirds of stratum moleculare. They frequently carried ‘‘en passant’’ varicosities, while drumstick-like boutons were never found on these axons. They could be only partially reconstructed, because the axons had been lost in the dense axonal meshwork in stratum moleculare. The axons emerged from the cell body in two cases (Fig. 3, Cells 2–3) and from a proximal dendrite (Fig. 3, Cell 1) in one case. This latter cell also had an axon collateral remaining in the hilus. Several somatostatin-immunoreactive cells had a main axon that crossed stratum granulosum into stratum moleculare, coursed parallel with stratum granulosum, then returned back into the hilus.

Postsynaptic targets of somatostatin-immunoreactive boutons are mainly, but not exclusively GABAnegative In the present study we used postembedding GABA-immunostaining66 to explore the GABAergic or non-GABAergic nature of postsynaptic targets of somatostatin-immunoreactive boutons. Altogether 101 boutons were examined in GABA-immunostained sections at the electron microscopic level in stratum lacunosum-moleculare of the CA1 and CA3 subfields and in stratum moleculare of the dentate gyrus (Table 1). All of them gave single symmetric synapses. The vast majority (76%) of the postsynaptic targets were clearly GABA-negative (Figs 4, 5), while some (8%) of the profiles were GABA-positive (Fig. 6, for criteria see Experimental Procedures). The remaining elements (16%) were regarded as unidentified due to equivocal staining of the targets, or relatively high background staining. There were no striking differences in the ratio of GABA-positive/ negative targets among the three examined regions (Table 1). Regardless of the targets, somatostatinimmunoreactive boutons were always strongly positive for GABA. The postsynaptic elements were mainly dendritic shafts (72%, Figs 4B, 5A), but numerous boutons contacted spines (27%, Fig. 5D). In one case a synapse was found on a GABA-positive soma in the CA1 subfield. The precise localization of somatostatin/GABA-positive synapses was

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Fig. 4.

Somatostatin-immunoreactive interneurons in hippocampus Table 1. Postsynaptic target selection of somatostatinimmunoreactive axon terminals

GABA-negative GABA-positive Unidentified Dendritic shafts Dendritic spines Cell body

Dentate gyrus n=33

CA3 n=34

CA1 n=34

Total n=101

79% 9% 12% 61% 39% 0%

76% 6% 18% 76% 24% 0%

74% 9% 17% 79% 18% 3%

76% 8% 16% 72% 27% 1%

The postsynaptic targets of 101 randomly found somatostatin-immunoreactive boutons were identified electron microscopically using postembedding immunogold staining for GABA.

remarkable as they usually contacted their target adjacent to an asymmetric, probably excitatory synapse (Fig. 4A–B). The average bouton diameter (n=101) was 0.800.30 µm (major axis) and 0.400.12 µm (minor axis). There were no differences among the subfields. Different interneuron types innervated somatostatin-immunoreactive boutons

by

Nearly 8% of the postsynaptic targets of somatostatin-immunoreactive boutons were clearly GABAergic (Fig. 6). This ratio roughly corresponds to the estimated occurrence of GABAergic and non-GABAergic elements in the hippocampus.5,71 However, recent studies described novel interneuron types, which selectively innervate other interneurons, rather than principal cells.2,24,28 Interestingly, one of the VIP-positive cell types has a dense dendritic arbor confined to stratum lacunosum-moleculare, and other VIP-immunoreactive cells also have extensive branches in this layer. Therefore, we aimed to determine whether the somatostatin-immunoreactive boutons terminate randomly on all types of interneurons in stratum lacunosum-moleculare or they show some degree of selectivity for specific subpopulations. In stratum lacunosum-moleculare and stratum moleculare nearly all of the somata and dendrites (over a hundred examined for each cell type) were contacted by somatostatinimmunoreactive boutons, belonging to all examined interneuron types visualized by parvalbumin-, cholecystokinin-, SPR-, calretinin- and VIPimmunostaining in stratum lacunosum-moleculare

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and stratum moleculare. To confirm our light microscopic observation that somatostatinimmunoreactive boutons (black, Ni–DAB) contact interneuron dendrites and somata (brown, DAB), some selected boutons were examined at the electron microscopic level as well. Five somatostatinimmunoreactive boutons were examined on parvalbumin-immunoreactive dendrites (Fig. 7) and six boutons each on calretinin- and VIP-positive dendrites and somata (Fig. 8). All of the 17 boutons formed symmetric synapses with their interneuron targets. Interestingly, in one case a somatostatinimmunoreactive bouton gave two synapses next to each other, one was found on a VIP-positive soma and another on a VIP-positive proximal dendrite probably originating from the same soma (Fig. 8D–F). DISCUSSION

On the basis of morphological characteristics, somatostatin-immunoreactive interneurons could be separated into three distinct subtypes, (i) HIPP cells of the dentate gyrus, (ii) conventional O-LM cells in stratum oriens of the CA1 and CA3 regions and (iii) neurons with dendrites similar to O-LM cells, but with a characteristically different axon. The first two subtypes have dense axonal projections to stratum lacunosum-moleculare (O-LM) or stratum moleculare (HIPP), where entorhinal afferents terminate. The postsynaptic targets of somatostatinimmunoreactive boutons in these layers showed a quasi-random termination pattern, as most of their targets were GABA-negative principal cell dendrites, but all examined GABAergic interneuron types were also innervated. Interneurons (strata oriens/lacunosum-moleculare/ hilar perforant path-associated) involved in feedback circuits contain somatostatin Two of the most characteristic interneuron types, the O-LM and HIPP cells are wired with a unique specificity to participate in feedback inhibition of principal cells. The special features indicating this role are the localization of dendritic tree in the same layer where recurrent axon collaterals of local principal cells arborize, and the overlap of their axonal arbor with the distal dendritic tree of local principal cells. Direct anatomical evidence for the recurrent activation of O-LM and HIPP cells has been

Fig. 4. Postsynaptic targets of somatostatin-immunoreactive boutons in stratum moleculare of the dentate gyrus were examined by electron microscopy of serial ultrathin sections and postembedding immunogold staining for GABA of a random sample of somatostatin-positive boutons. The somatostatin-containing boutons, always positive for GABA (b1 in A; b2 in B), form symmetrical synaptic contacts (arrows) with GABA-negative dendritic shafts (d), as indicated by the lack of immunogold particles in the postsynaptic targets (C and E are serial sections of b1, D and F are of b2). Note that somatostatin-containing boutons are often localized adjacent to asymmetrical synapses (open arrows), which probably originate from the entorhinal cortex. The stars label other GABA-negative profiles, whereas SOM-negative GABA-positive boutons are indicated by asterisks. Scale bars=0.4 µm.

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Fig. 5. Somatostatin-immunoreactive boutons (b1 in A; b2 in D) in stratum lacunosum-moleculare of the CA3 subfield form symmetric synaptic contacts (arrows) with a GABA-negative dendritic shaft (d in A and B) and with a GABA-negative spine (s in C and D). Other GABA-negative profiles are labelled with stars, while GABA-positive profiles are labelled with asterisks. Scale bars=0.4 µm.

provided by Blasco-Ibanez and Freund14 and Acsa´dy et al.,3 respectively, whereas local principal cells as their major targets were identified in the present study. Several recent intracellular labelling studies identified the HIPP and O-LM types of interneurons in all regions of the hippocampal formation both

in vitro26,32,47 and in vivo.15,59,61 The morphological characteristics of these cells strongly resemble the first two types of reconstructed somatostatinimmunoreactive neurons with somata in the hilus and in stratum oriens of CA1, and with axons in stratum moleculare and stratum lacunosummoleculare, respectively. In addition to the present

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Fig. 6. In some cases, postsynaptic targets (d) of somatostatin-immunoreactive boutons (b1 in A and C; b2 in B) proved to be GABA-positive. These photographs were taken from stratum moleculare of the dentate gyrus. The larger size of gold particles is due to silver intensification of 1 nm gold particles used in some of the experiments (see Experimental Procedures). The arrows point to symmetrical synapses formed by somatostatin-immunoreactive boutons on GABA-positive dendritic shafts. The asterisk shows another GABA-positive profile in C, while GABA-negative profiles are labelled with stars. Scale bars=0.4 µm.

direct demonstration, some earlier indirect evidence also suggests that this type of somatostatinimmunoreactive neurons in the hippocampal formation can be identified as the O-LM or HIPP cells. Although the neurochemical marker content of these intracellularly labelled neurons is largely unknown, in one case a HIPP cell was shown to contain neuropeptide Y (NPY).22,60 Since NPY and somatostatin co-localize in many hilar interneurons,37 it indicates that HIPP cells express somatostatin as well. Electrophysiological data showing that the mGluR agonist 1S,3R-aminocyclopentane dicarboxylic acid (ACPD) generates large inward currents and current oscillations in identified O-LM neurons47 presumably by activating mGluR1á receptors, which densely cover somatostatin-immunoreactive neurons in stratum oriens,13 provide further indirect evidence that O-LM cells and somatostatin-containing cells in CA1 are identical.

Interneurons resembling back-projection neurons also contain somatostatin Most hippocampal interneuron types have an axonal and dendritic arbor limited to only one and the same subfield. However, in a recent study a specific interneuron type, which projects from stratum oriens of the CA1 subfield to the other hippocampal and dentate subfields was described.62 The present study revealed several features, which suggest that a small proportion of somatostatin-immunoreactive neurons can be classified as back-projection neurons: (i) somatostatin-immunostaining labelled several axons which crossed the hippocampal fissure or the border of the CA3 and CA1 subfields; (ii) these axons frequently bore small drumstick-like appendages, which is a typical feature of the axon of the back-projection cell; (iii) several somatostatinimmunoreactive somata were localized in the alveus.

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Fig. 7. (A) Parvalbumin-immunoreactive dendrites (d1 and d2) were frequently seen to be contacted by numerous somatostatin-positive boutons (b1 and b2) at the light microscopic level. (B) A random sample was cut for correlated electron microscopy, two of which are shown here (b1 and b2 in A and B). (C–D) All of the examined boutons formed symmetrical synapses (arrows) with their parvalbuminimmunoreactive targets as seen on these high power electron micrographs of b1 and b2. Open arrow shows an asymmetrical synapse located close to the somatostatin-positive bouton in the same manner as in the case of GABA-negative postsynaptic targets. Scale bars: (A)=7.5 µm; (B)=2 µm; (C–D)=0.2 µm.

Fig. 8. (A–C) Correlated light (A) and electron micrographs (B–C) of a calretinin-immunoreactive neuron (S) receiving multiple synaptic contacts from somatostatin-immunoreactive boutons (b1 and b2). The thin arrow in B shows the invaginated nucleus of the calretinin-positive neuron, typical of interneurons (also shown in F in the case of a VIP-positive neuron). (C) High power electron micrograph of somatostatinpositive boutons forming symmetrical synapses with their calretinin-immunoreactive target (SCR) as indicated by thick arrows. (D–F) somatostatin-immunoreactive boutons also heavily innervate VIPpositive cells. Bouton b1 was found to contact (thick arrows) two VIP-positive profiles, a cell body (SVIP) and a proximal dendrite (d) in the same section. Scale bars: (A, D)=7.5 µm; (B)=2 µm; (E)=1 µm; (C, F)=0.2 µm.

Somatostatin-immunoreactive interneurons in hippocampus

Fig. 8.

49

50

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Fig. 9.

Somatostatin-immunoreactive interneurons in hippocampus

One of these neurons could be partially reconstructed, and the laminar and regional distribution of these axon arborization fragments resembled that of back-projection neurons. Some of the main axons extensively arborized in stratum oriens and stratum pyramidale, while one main axon disappeared in the alveus after myelination. It was recently shown that immunostaining for muscarinic type 2 receptor (M2) labels horizontal cells at the oriens/alveus border with axons, which occasionally cross the hippocampal fissure.30 Co-localization of M2 receptors and somatostatin was found in a small number of interneurons in stratum oriens of the CA1 subfield,30 which further suggests the existence of a somatostatin-positive back-projection cell population. Moreover, in this study some neurons expressing M2 receptor were retrogradely labelled from the medial septum. Since all nonpyramidal hippocamposeptal projection neurons contain somatostatin (Katona I., Acsa´dy L., Freund T.F., unpublished observation), we suggest that these back-projection neurons, in addition to their extensive arborization in the entire hippocampus, may project to the medial septum. The postsynaptic target distribution of somatostatincontaining strata oriens/lacunosum-moleculare and hilar perforant path-associated cells Precise knowledge of connectivity, convergence and divergence properties is necessary for any predictions to be made about the functional roles of interneurons (or of any other cell types). Therefore we determined the postsynaptic targets of somatostatin-immunoreactive neurons in those layers, where they have the most dense axonal arborization. The intrinsic origin of these fibres was demonstrated by our cell reconstructions and also by earlier data demonstrating the lack of extrinsic somatostatin-containing projections to the hippocampus.12,57 Previous studies assumed that targets of these axons are the principal cells and showed that somatostatin-immunoreactive neurons contact

51

dendritic spines and sometimes shafts both in stratum moleculare and in stratum lacunosummoleculare.10,42,49 However, recent results revealed that in addition to the principal cell dendrites, several interneuron types may possess spines.4,13,25 Moreover, the cells of origin of postsynaptic dendritic shafts can rarely be identified by electron microscopy alone. On the other hand, our present results revealed that dendritic shafts occurred more frequently among the targets than spines, which correlates well with earlier data on postsynaptic targets of intracellularly labeled HIPP neurons31 and O-LM cells26 and recently described perforant path-associated neurons located in stratum radiatum.69 To provide a direct answer, we used postembedding immunogold labelling for GABA in sections containing somatostatinimmunoreactive boutons, which allowed in most cases to identify the postsynaptic elements. The majority of the postsynaptic targets were clearly GABA-negative and the somatostatin-positive synapses were usually located adjacent to asymmetric synapses. This suggests that the major mode of termination of somatostatin-containing interneurons is on distal dendrites of principal cells in close association with entorhinal excitatory afferents. However, many GABA-positive profiles were also among the targets, but not more than what could be expected for a quasi-random termination pattern. Thus, the relatively higher incidence of shaft contacts made by somatostatin-positive boutons cannot be explained by a preference for interneuron targets. Recent anatomical data have shown that parvalbumin-immunoreactive neurons are innervated by HIPP cells.59 Here we demonstrate morphologically that, in addition to principal and basket cells, basically all interneuron types examined may be among the targets. Electrophysiological data are also available to support an O-LM cell input to stratum lacunosum-moleculare interneurons, but the types of cells were not identified: synchronized spontaneous GABAergic inputs that could be modulated by ACPD were shared among lacunosum-moleculare

Fig. 9. The wiring and hypothesized activity pattern of the pyramidal cell–O-LM cell network at different phases of local intracellular theta activity (insets in the top left corners): A, near the peak, and B, at more hyperpolarized phases preceding the peak. Active neurons appear as dark symbols (blue: pyramidal cells; red: O-LM interneuron), whereas they are labelled by light shades of the same colour when inactive (in B). (A) The largest proportion of pyramidal cells are active near the positive peak of intracellular theta activity (although even then, only about 0.1% of them fire17), thus, this is the phase (time window indicated in insert) when O-LM interneurons have the highest probability of being activated during theta activity as a result of convergent input from a large number of local pyramidal cell collaterals. It is calculated that, after pyramidal cell discharge, back-propagating action potentials would arrive to stratum lacunosum-moleculare dendrites at about the same time as the disynaptic IPSP mediated by the O-LM cells (2.5–3 ms48,67). Thus, activity-dependent associative LTP of entorhinal synaptic potentials will be blocked by feedback inhibition even if the EPSPs coincide with the back-propagating action potential. (B) Place cells fire ahead of the population53,63 at more hyperpolarized phases of local theta. Within this time window only a small number of pyramidal cells fire synchronously, which is less likely to trigger an action potential in the O-LM cell.6 Thus, if perforant path synaptic potentials will coincide with activity in the place cell, potentiation of these synapses will be allowed by the lack of inhibition in stratum lacunosummoleculare. By this mechanism feedback inhibition may be able to limit synaptic plasticity both in space and time. The same connectivity and activity pattern is proposed for the granule cell-HIPP cell network as well.

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interneurons and pyramidal cells in the CA1 region.9 Functional implications The afferent connectivity of the O-LM and HIPP cells provides predictions as to under what conditions these cell types are likely to be active. Both anatomical14 and physiological44,59,61 data support the conclusion that these cells in CA1 stratum oriens and in the hilus of the dentate gyrus are driven exclusively in a feedback manner by cortical afferents, and Schaffer collateral or perforant path stimulation discharges them only if the stimulus is strong enough to evoke a population spike in CA1 or in the dentate gyrus, respectively. Thus, apparently a relatively large number of CA1 pyramidal cells or granule cells have to fire together to trigger repetitive action potential discharge in an O-LM cell or HIPP cell. The question arises, under what physiological conditions is this most likely to happen? During theta activity, principal cells have the largest probability of discharge around the positive peak of intracellular theta waves.72 Within this time window (Fig. 9A) feedback interneurons (i.e. O-LM and HIPP cells) will be activated as a result of large convergence of even distantly located principal cells68 and may prevent activity-dependent plasticity of entorhinal synapses in stratum lacunosum-moleculare and stratum moleculare. Back-propagating action potentials of pyramidal cells were suggested to underlie the Hebbian type of synaptic plasticity.45,46 On the basis of conduction velocity of backpropagating action potentials in CA1 pyramidal cells67 and the latency of disynaptic inhibition48 it may be calculated that the backpropagating action potential would arrive to the distal dendrites of CA1 pyramidal cells in approximately the same time as the inhibitory postsynaptic potential (IPSP) evoked by the same action potential via activating an O-LM cell (2.5–3 ms after the pyramidal cell action potential in vitro), giving an opportunity to feedback inhibition to veto LTP.44 Other forms of postsynaptic LTP independent of back-propagating action potentials will also be attenuated by feedback inhibition in this layer. When pyramidal cell firing carries some specific information, e.g., about space,52 then action potentials occur earlier than the positive peak, i.e. when pyramidal cells of the subfield are in more hyperpolarized phases. As the animal crosses the place field of a particular neuron, the action potentials of this place cell were shown to occur at progressively earlier phases of theta,53,63 as if its excitatory drive became stronger, overriding greater degrees of perisomatic inhibition. We propose that firing of single place cells alone, occurring earlier then that of the population, is unlikely to discharge the O-LM cells6,27 or HIPP cells, thus, the probability of recruiting feedback inhibition in this phase is much lower (see Fig. 9B).

The lack of dendritic inhibition in strata lacunosummoleculare or moleculare at this time-point would allow potentiation of those entorhinal synapses which terminate on,44 and are active synchronously with, this particular place cell (i.e. when the animal is located in that place field). This way entorhinal inputs carrying place field information will become stronger and stronger on a select population of cells, as the animal crosses repeatedly the environment. The present hypothesis suggests that the dynamic formation of place fields may begin with a quasirandom selection of cells that accidentally fire earlier than the population in a particular place field. This way they escape feedback inhibition, which allows the potentiation of their simultaneously active afferent synapses carrying spatial information. These potentiated synapses will then be responsible for phase precession of the cell’s firing upon subsequent entries of the same place field, which may lead to further enhancement of the excitatory drive and phase precession of the given place cell. This hypothesis correlates well with the notion that place fields (maps) will become more refined in time as the animal explores a new environment.70 O-LM cells are also likely to be activated6 during hippocampal sharp waves, when large populations of pyramidal cells fire synchronous bursts,18 and in turn, block LTP of perforant path synapses.44 This would ensure that, during sharp waves, only Schaffer collateral synapses will be potentiated. The diagrams in Fig. 9 are clearly oversimplified, they focus on the connectivity of this particular interneuron type and its potential role during theta activity. In future studies of entorhinal–hippocampal information transfer the functional roles of feedforward interneuron types activated by entorhinal stimulation and terminating in strata lacunosummoleculare, radiatum, or pyramidale,29,40,69 which apparently have a powerful inhibitory effect,65 should also be investigated. CONCLUSION

The present data demonstrate that somatostatincontaining GABAergic interneurons driven in a feedback manner terminate predominantly on the most distal dendritic segments of pyramidal cells in conjunction with entorhinal afferents (i.e. they are O-LM and HIPP cells). We propose that, if the role of theta oscillation is to separate signal transmission from background firing in time,19 then these feedback dendritic inhibitory cells may help confine time periods when entorhinal synapses on pyramidal cells may be effective. In addition, this feedback circuit may allow associative plasticity only for cells that carry specific information (e.g., for place cells of the given place field), and limit it to the time window when specific signal transmission takes place (i.e. during ‘‘phase precession’’). During hippocampal sharp waves, these interneurons may attenuate the

Somatostatin-immunoreactive interneurons in hippocampus

efficacy of direct entorhinal afferents, ensuring the dominance of Schaffer collateral inputs in driving CA1 pyramidal cells.44 Acknowledgements—We are grateful to Dr T. J. Go¨rcs for antisera against somatostatin, vasoactive intestinal polypeptide and cholecystokinin, to Dr K. G. Baimbridge and to Dr J. H. Rogers for antisera against parvalbumin and calretinin, respectively, and to Dr P. Somogyi for antisera against GABA. The valuable discussions with Drs A. M.

53

Thomson, Gy. Buzsa´ki, H. Markram and R. Miles concerning the activation of O-LM cells, and the time-course of back-propagating action potentials and disynaptic IPSPs are highly appreciated. The authors wish to thank for preparation of the colour figure Ms A u . L. Bodor. The excellent technical assistance of Mrs E. Boro´k, Mrs A. Zo¨ldi Szabo´ne´ and Mr Gy. Goda is also acknowledged. This work was supported by the Howard Hughes Medical Institute, the Swiss National Science Foundation, NIMH (MH54671) and OTKA (T16942) Hungary.

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