neurocalcin-immunoreactive cells in the rat hippocampus ... · neurocalcin-immunoreactive cells in...

Neurocalcin-Immunoreactive Cells in the RatHippocampus are GABAergic Interneurons

Francisco J. Martı́nez-Guijarro,1* Jesús G. Briñón,2

José M. Blasco-Ibáñez,1 Katsuo Okazaki,3

Hiroyoshi Hidaka,3 and José R. Alonso2

1Department of Cellular Biology, Faculty of BiologicalSciences, University of Valencia, Valencia, Spain2Department of Cellular Biology and Pathology, Facultyof Biology, University of Salamanca, Salamanca, Spain3Department of Pharmacology, Nagoya University Schoolof Medicine, Nagoya, Japan

ABSTRACT Neurocalcin (NC) is a recently described calcium-bindingprotein isolated and characterized from bovine brain. NC belongs to theneural calcium-sensor proteins defined by the photoreceptor cell-specificprotein recoverin that have been proposed to be involved in the regulationof calcium-dependent phosphorylation in signal transduction pathways.We analyzed the distribution and morphology of the NC-immunoreactive(IR) neurons in the rat dorsal hippocampus and the coexistence of NC withGABA and different neurochemical markers which label perisomaticinhibitory cells [parvalbumin (PV) and cholecystokinin (CCK)], mid-proximal dendritic inhibitory cells [calbindin D28k (CB)], distal dendriticinhibitory cells [somatostatin (SOM) and neuropeptide Y (NPY)], andinterneurons specialized to innervate other interneurons [calretinin (CR)and vasoactive intestinal polypeptide (VIP)]. NC-IR cells were present inall layers of the dentate gyrus and hippocampal fields. In the dentate gyrus,NC-IR cells were concentrated in the granule cell layer, especially in thehilar border, whereas in the CA fields they were most frequently found inthe stratum radiatum. NC-IR cells were morphologically heterogeneousand exhibited distinctive features of non-principal cells. In the dentategyrus, pyramidal-like, multipolar and fusiform (horizontal and vertical)cells were found. In the CA3 region most NC-IR cells were multipolar, butvertical and horizontal fusiform cells also appeared. In the CA1 region,where NC-IR cells showed most frequently vertically arranged dendrites,multipolar, bitufted and fusiform (vertical and horizontal) cells could bedistinguished. All the NC-IR cells were found to be GABA-IR in allhippocampal layers and regions, and they represented about 19% of theGABA-positive cells. NC/CB, NC/CR and NC/VIP double-labeled cellswere found in all hippocampal regions, and represented 29%, 24% and18% of the NC-IR cells, respectively. NC and CCK did not coexist in thedentate gyrus; however, 9% of the NC-IR cells in the CA fields alsocontained CCK. No coexistence of NC with PV, SOM or NPY was found inany hippocampal region.

We conclude that NC is exclusively expressed by interneurons in the rathippocampus. NC-IR cells are a morphologically and neurochemically

heterogeneous subset of GABAergic non-principal cells,which, on the basis of the known termination pattern ofthe colocalizing markers, are also functionally heteroge-neous and are mainly involved in feed-forward den-dritic inhibition in the commissural-associational andSchaffer collateral termination zones (CB containingcells), in innervation of other interneurons (CR- andVIP-containing cells), and in perisomatic inhibition(CCK-containing cells). NC is never present in periso-matic inhibitory PV-containing cells, or in feed-backdistal dendritic inhibitory SOM/NPY-containing cells.Hippocampus 1998;8:2–23. r 1998 Wiley-Liss, Inc.

KEY WORDS: calcium binding proteins; neuropep-tides; colocalization; non-pyramidal cells; inhibition

INTRODUCTION

The hippocampus is composed of two major classes ofneurons: excitatory principal cells and GABAergic inhibi-tory non-principal cells (interneurons). Principal cells ineach hippocampal region, i.e., granule cells in thedentate gyrus and pyramidal cells in the Ammon’s horn,are morphologically and functionally homogeneous neu-ronal populations. In contrast, inhibitory interneuronsinclude a wide variety of cells with distinct somatoden-dritic and axonal termination patterns (Freund andBuzsáki, 1996).

Combined morphological and electrophysiologicalstudies have shown that interneurons with differenttermination patterns are responsible for diverse inhibi-tory processes in the hippocampus (Lacaille et al., 1987;Lacaille and Schwartzkroin, 1988a,b; Buzsáki et al.,1992; Gulyás et al., 1993a,b; Soltész and Deschenes,1993; Buhl et al., 1994; McBain et al., 1994; Bragin et

Grant sponsor: Spanish FIS; Grant number: 95/0689; Grant sponsor:DGICYT; Grant number: PB 94-1388; Grant sponsor: ‘‘Junta de Castilla yLeón.’’*Correspondence to: Dr. F.J. Martı́nez-Guijarro, Departmento de Biologı́aCelular, Facultad de Ciencias Biológicas, Universidad de Valencia, C/ Dr.Moliner, 50, E-46100 Burjasot, Spain. E-mail: [email protected] for publication 22 October 1997

HIPPOCAMPUS 8:2–23 (1998)

r 1998 WILEY-LISS, INC.

al., 1995; Buckmaster and Schwartzkroin, 1995; Cobb et al.,1995; Sik et al., 1995; Miles et al., 1996). Some calcium bindingproteins, as well as some neuropeptides, have been shown to beneurochemical markers differentially expressed by subsets ofGABAergic hippocampal interneurons with distinct terminationpatterns. Thus, the immunocytochemical detection of certaincalcium-binding proteins and neuropeptides is a powerful tool forclassification of hippocampal interneurons (Freund and Buzsáki,1996). Hippocampal interneurons fall into three basic categorieswith regard to their termination pattern. The first type includesinterneurons which contact principal cells in the perisomaticregion (basket and axo-axonic cells)(Somogyi et al., 1983, 1985;Soriano and Frotscher, 1989; Soriano et al., 1990; Gulyás et al.,1993a; Buhl et al., 1994), and have effect on the timing andrepetitive firing of sodium-dependent action potentials of pyrami-dal cells (Cobb et al., 1995; Miles et al., 1996). The calciumbinding protein parvalbumin (PV) and the neuropeptide cholecys-tokinin (CCK) are present in two non-overlapping subsets ofperisomatic inhibitory cells, the former including both basket andaxo-axonic cells (Kosaka et al., 1987; Katsumaru et al., 1988;Nitsch et al., 1990; Soriano et al., 1990), and the latter consistingexclusively of basket cells (Gulyás et al., 1991; Acsády et al.,1996a). Interneurons included in the second type contact thedendritic domain of principal cells in conjunction with differentexcitatory afferents (Gulyás et al., 1993a,b; Han et al., 1993; Buhlet al., 1994; Sı́k et al., 1995; Gulyás and Freund, 1996) and maybe involved in regulation of calcium-dependent dendritic electro-genesis (Miles et al., 1996). Within the interneurons belonging tothe second type, the calcium binding protein calbindin D28k(CB) is present in a subset of interneurons innervating themid-proximal dendrites of pyramidal cells, whereas the neuropep-tides Y (NPY) and somatostatin (SOM) have been found in cellsinnervating the distal dendritic portion of granule and pyramidalcells (Köhler et al., 1986; Deller and Léránth, 1990; Léránth et al.,1990; Buckmaster et al., 1994; Sı́k et al., 1995, 1997; Gulyás andFreund, 1996; Katona et al., 1996). The third group is specializedto innervate other interneurons, and the majority of vasoactiveintestinal polypeptide (VIP)- and calretinin (CR)-containing cellsbelong to this group (Acsády et al., 1996b; Gulyás et al., 1996;Hájos et al., 1996). They may be involved in the high-frequencypopulation oscillations and/or disinhibition of principal cells(Müller and Misgeld, 1990; Michelson and Wong, 1991; Buzsákiand Chrobak, 1995; Whittington et al., 1995).

Neurocalcin (NC)(molecular weight 23-24 kDa, pI 5.3-55) is arecently described calcium-binding protein with three EF-handdomains (Terasawa et al., 1992; Nakano et al., 1992), whichbelongs to the neural calcium-sensor proteins defined by thephotoreceptor cell-specific protein, recoverin. NC has beenisolated and characterized from bovine brain, where four isoformsof NC have been identified (Terasawa et al., 1992). However, itsprecise function is poorly understood and its localization has notbeen studied extensively. Immunoblot analysis demonstrated thatNC is present in olfactory bulb, cerebrum, cerebellum, brainstem,spinal cord, retina, pituitary and adrenal glands (Bastianelli et al.,1993; Nakano et al., 1992,1993; Hidaka and Okazaki, 1993).Immunohistochemical studies have shown NC-immunoreactivity

in amacrine and ganglion cells of the bovine retina (Nakano et al.,1992), in tufted, periglomerular, Van Gehuchten’s and Cajal’s cellsof the rat olfactory bulb (Bastianelli et al., 1993; Porteros et al.,1996), in neurons of the posterior horn of the spinal cord anddorsal root ganglia (Okazaki et al., 1994), in receptor cells of theolfactory epithelium and vomeronasal organ (Bastianelli et al.,1995; Iino et al., 1995a), in neurons of the spiral and vestibularganglia, and in sensory epithelial cells of the vestibular end organs(Iino et al., 1995b).

Some recoverin-like neural calcium-sensor proteins have beenfound in hippocampal principal cells; e.g., hippocalcin andNCS-1 have been found in hippocampal pyramidal cells (Kobaya-shi et al., 1992; Schaad et al., 1996), and visinin-like protein(VILIP) and neural visinin-like Ca (21)-binding protein 2(NVP2) have been found in dentate granule cells and hippocam-pal pyramidal cells (Saitoh et al., 1994; Lenz et al., 1996).

The aim of the present study is to analyze the distribution andmorphology of NC-containing neurons in the rat dorsal hippocam-pus. In addition, in order to characterize the chemical nature ofNC-IR cells, the coexistences of NC with GABA and differentneurochemical markers for perisomatic inhibitory cells (PV andCCK), dendritic inhibitory cells (CB, SOM, and NPY), andinterneurons specialized to innervate other interneurons (CR andVIP) were also analyzed. A preliminary account of these results hasbeen published elsewhere (Martı́nez-Guijarro et al., 1996).

MATERIALS AND METHODS

Twelve male Wistar rats (275-300 g; Harlan Interfauna Iberica,Barcelona, Spain) were deeply anaesthetized with ketamine (Keto-lar, 50 mg/kg body weight) and perfused through the heart firstwith saline and then with a phosphate-buffered (PB; 0.1 M, pH7.4) fixative containing either 4% paraformaldehyde and 0.2%picric acid for morphological study and colocalization analysis ofNC with CCK, SOM and VIP, or 3% paraformaldehyde, 0.2%picric acid, and 0.3% glutaraldehyde for morphological analysisand colocalization analysis of NC with GABA, PV, CB, CR andNPY.

Brains were removed from the skull and coronal sections (50mm thick) were cut from the dorsal hippocampus on a vibratome(VT 1000E, Leica, Spain) and kept in sequence.

Alternatively, 400 mm thick coronal slabs were obtained,dehydrated in an ascending ethanol series, and flat embedded inDurcupan (ACM, Fluka AG, Switzerland) between acetate sheets.After polymerization, slabs were reembedded and serial semithin 1mm sections were mounted alternately on gelatin-coated slides.

Immunostaining and Coexistence Analysis

Free-floating vibratome sections

Following extensive washes in PB, sections were treated with1% NaBH4 in PB for 30 minutes, and washed again thoroughly inPB. Adjacent 50 mm thick free-floating vibratome sections were

____________________________________________________ NEUROCALCIN IN THE RAT HIPPOCAMPUS 3

first immersed in 10% normal goat serum (NGS) for 1 h, andthen incubated to reveal different antigens in one of the followingantisera: rabbit anti-NC (1:10,000; Nakano et al., 1992), rabbitanti-CB (1:15,000; SWant, Bellinzona), rabbit anti-CR (1:8,000,SWant, Bellinzona; Schwaller et al., 1993), rabbit anti-CCK(1:1,000; Incstar), rabbit anti-SOM (1:20,000; Lantos et al.,1995), or rabbit anti-VIP (1:10,000; Gulyás et al., 1990).Incubation in primary antisera was carried out for 2.5 days at 47C.Then the sections were incubated in biotinylated goat anti-rabbitIgG (1:200, Vector) for 3 h at room temperature followed byavidin-biotinylated horseradish peroxidase complex (1:100, ABCstandard kit, Vector, Burlingame, CA) for 2 h at room tempera-ture. PB containing 1% NGS and 0.1% Triton X-100 was usedfor washing and antisera dilutions. For some series of sections,devoted exclusively to morphology analysis, 0.5% Triton X-100was used to enhance NC antiserum penetration. The immunoper-oxidase reaction was developed using 0.05% 3,38-diaminobenci-dine-4HCl (DAB) as a chromogen. After immunostaining thesections were treated with 0.5% osmium tetroxide for 30 min,dehydrated in ethanol, and flat-embedded in Durcupan.

The coexistence of NC and any of the other antigens in thesame cell was analyzed using the mirror technique described byKosaka et al. (1985). Immunoreactive perikarya cut in half at thesurface of the sections, as well as nearby capillaries, were drawnwith the aid of a camera lucida using a 3100 oil immersionobjective. The other half of the immunoreactive somata werelocated on the matching surface of the adjacent section (incubatedfor the other antigen) using the capillaries as landmarks. Somedouble-labeled cells were partially reconstructed from sectionpairs and used for morphological characterization of the doublelabeled cells.

Semithin plastic sections

Post-embedding immunostaining of consecutive 1 mm semi-thin plastic sections was used for colocalization studies of NC withGABA, PV, CB, CR, and NPY. The resin was removed bytreatment with sodium ethoxide, and the sections were hydrated,treated with 1% NaBH4 in PB for 15 min, and incubated at roomtemperature to reveal different antigens, first in 10% normal goatserum (NGS) for 1 h, and then in one of the following antisera:rabbit anti-NC (1:1,000; Nakano et al., 1992), rabbit anti-GABA(1:2,000; Sigma), rabbit anti-CB (1:1,000; SWant, Bellinzona),rabbit anti-CR (1:1,000, SWant, Bellinzona; Schwaller et al.,1993), or rabbit anti-NPY (1:1,000; Csiffáry et al., 1990).Incubation in primary antisera was performed overnight. Thenthe sections were incubated in biotinylated goat anti-rabbit IgG(1:200, Vector) for 1 h followed by avidin-biotinylated horserad-ish peroxidase complex (1:100, ABC standard kit,Vector) for 1 h.PB containing 1% NGS and 0.1% Triton X-100 was used forwashing and antisera dilutions. The immunoperoxidase reactionwas developed using 0.05% 3,38-diaminobencidine-4HCl (DAB)as a chromogen. After immunostaining the sections were treatedwith 0.01% osmium tetroxide for 1 min to enhance DABstaining, dehydrated in ethanol, cleared in xylene, and cover-slipped with Eukitt.

Using camera lucida drawings of the sections, all the NC-IRcell bodies were plotted and their immunoreactivity for any of thedifferent neurochemical markers was analyzed in the adjacentsection.

The specificity of the primary antisera used in this study wastested by the companies and laboratories of origin (see referencesabove). Immunocytochemical controls included the substitutionof the primary antisera by normal rabbit serum, which resulted ina complete loss of immunostaining of neuronal structures in allcases. In addition, for NC immunoreactivity control, incubationswith NC antiserum preadsorbed with native bovine neurocalcinin excess were also performed, and no immunostaining wasobserved under this condition.

Data shown in Tables 1-6 derive from direct counting ofimmunoreactive somata in semithin sections or at the cut surfacesof vibratome sections. Thus, these data have to be considered assemi-quantitative since no stereological correction was performed.

RESULTS

Laminar Distribution and Morphologyof NC-IR Cells

NC-immunoreactivity was found exclusively in neurons. Cellbodies and dendrites were immunostained and, in 1 mm semithinsections, a substantial accumulation of NC immunoreactivity wasseen associated to cell membranes (see Figs. 6,7,10). However,neither axonal arborizations nor terminal fields of immunoreac-tive boutons were stained strong enough to be clearly visualized.Moreover, the whole extent of the dendritic tree was not revealed,due to poor penetration of the antiserum (in spite of detergenttreatment and prolonged incubation time). This situation wasspecially remarkable for small cells located in the molecular layerof the dentate gyrus and in the stratum lacunosum-moleculare ofthe CA1 region, where NC-IR cell bodies frequently appeareddevoid of stained dendrites or only very short portions ofemerging dendrites were observed. Therefore, the cells weregrouped considering their major dendritic orientation and cellbody shape. NC-IR cells were diverse in size and morphology, butall of them exhibited the location and general morphologicalfeatures characteristic of non-principal cells. NC-IR perikaryawere present in all layers of the dentate gyrus and hippocampalsubfields (Fig. 1).

In the dentate gyrus (Figs. 2,3) NC-IR cells were concentratedin the granule cell layer (70.7%), especially in the hilar border,being less abundant in the hilus (18.4%) and in the molecularlayer (10.9%)(Table 1). In the granule cell layer of the dentategyrus, typical pyramidal-like neurons were found. Their medium-large pyramidal shaped cell body was located in the hilar border ofthe granule cell layer or just beneath it. They bore a single apicaldendrite which crossed the granule cell layer, but only in a fewcases could it be followed further in the molecular layer. Inaddition, they showed two to four basal dendrites which runparallel to the granule cell layer or extended into the hilus.

4 MARTÍNEZ-GUIJARRO ET AL.

Horizontal fusiform cells were found in the hilar border of thegranule cell layer. They extended horizontally running dendriteswhich usually remained in the subgranular zone, but occasionallywere seen to curve and, after crossing the granule cell layer, runinto the molecular layer. Horizontal fusiform cells were alsoobserved deeper in the hilus and also in the molecular layer nearthe hippocampal fissure. Vertical fusiform and multipolar cellswere found in the molecular layer, hilar region, and occasionally inthe granule cell layer.

In the CA3 region, NC-IR cells were scarce in the CA3cincreasing their number towards the CA3a-b. The largest numbersof NC-IR cells were found in the stratum radiatum (52%)followed by the strata pyramidale (17.7%) and lucidum (16.5%).A small proportion of NC-IR cells was found in the stratumoriens (10.1%) and they were rare in the stratum lacunosum-moleculare (3.6%)(Table 1). In the CA3 region (Figs. 2,4), mostNC-IR cells were multipolar with their cell bodies located at anylayer and their dendrites sometimes extending through differentlayers. In addition, fusiform cells with vertically oriented dendritescrossing different layers or with horizontally arranged dendritesnearly restricted to a single layer were observed in the stratalucidum, radiatum and lacunosum-moleculare.

In the CA1 region no evident clustering or differences in thedistribution of NC-IR cells were found along the transverse axis.In this area NC-IR cells were most frequently found in thestratum radiatum (41.6%) followed by the stratum pyramidale(33%), and were present at lower rates in the strata lacunosum-moleculare (18.5%) and oriens (6.9%)(Table 1).

In the CA1 area (Figs. 2,5), NC-IR cells showed mostfrequently vertically arranged dendrites. Nevertheless, neuronswith horizontally running dendrites could be observed in all

layers. The most frequent NC-IR cells in the CA1 were multipolarneurons. They occurred in all layers, but were most abundant inthe stratum radiatum with the cell body located at any level insidethe layer. In the strata radiatum, pyramidale and oriens, multipo-lar NC-IR cells showed a predominant radial dendritic orienta-tion, independently of the initial disposition of the proximaldendrites. Multipolar cells located in the stratum lacunosum-moleculare were scarce, and usually showed their dendrites mostlyrestricted to this layer. Bitufted cells were found in the stratapyramidale and radiatum, with radially running dendrites, some-times crossing several layers and entering the stratum lacunosum-moleculare. Fusiform cells with horizontal or vertical polarizationwere observed. Vertical fusiform cells had their cell bodies locatedin the strata radiatum, pyramidale, or occasionally oriens, andextended radially oriented dendrites which were sometimes seento enter the stratum lacunosum-moleculare. Horizontal fusiformcells were most frequently found in the stratum radiatum/lacunosum-moleculare border and in the stratum lacunosum-moleculare proper, but they were also occasionally detected in thestratum oriens.

Colocalization Studies

Due to the lack of consistent axonal staining with NCantiserum, in order to establish the possible termination pattern ofNC-containing cells, colocalization studies were performed withneurochemical markers that label functionally and morphologi-cally distinct subsets of GABAergic interneurons and with GABAitself. Immunostaining of consecutive 1 mm semithin plasticsections and/or the mirror technique were used to determine thedegree of coexistence and the morphology (when possible) of the

FIGURE 1. Camera lucida drawing from a coronal section of therat dorsal hippocampus, showing the distribution of NC-IR cells.Dots represent cell bodies cut in half at both surfaces of a singlevibratome section. Dashed lines mark the borders of the different

layers on each hippocampal subfield. DG, dentate gyrus; CA1, CA1region; CA3, CA3 region; g, granule cell layer; h, hilus; l, stratumlucidum; lm, stratum lacunosum-moleculare; o, stratum oriens; p,stratum pyramidale; r, stratum radiatum. Scale bar: 200 mm.


double-labeled cells. The distribution of cell bodies immunoreac-tive for GABA and the different neurochemical markers used inthis study were similar to those previously described in the rathippocampus and dentate gyrus (Freund and Buzsáki, 1996).

Coexistence of NC with GABA

The distribution and morphology of the NC-IR cells stronglysuggested that they were GABAergic neurons. To address thispossibility, consecutive 1 mm semithin plastic sections wereimmunostained for NC and GABA (Fig. 6). The analysisdemonstrated that all the NC-IR cells analyzed (n 5 454) werealso GABA-IR in all hippocampal layers and regions, and theyshowed medium-strong GABA-immunoreactivity. They repre-sented about 19% of GABA-positive cells in the same areas (n 52,382), with little regional variation (Table 2).

Colocalization of NC with markers of perisomaticinhibitory cells (PV, CCK)

PV and CCK are markers for distinct subsets of perisomaticinhibitory cells. To analyze the degree of coexistence of NC withPV (Fig. 7) and CCK (Fig. 8), immunostaining of consecutive 1mm semithin plastic sections and the mirror technique were usedrespectively. The coexistence analysis showed that NC-IR cellsnever contain PV (100 NC-IR cells analyzed per region), and 9%

of the NC-IR cells in the CA3-1 regions colocalize CCK (Table3). In the DG, in spite of the highly similar morphology of theNC- and CCK-IR cells, many of which showed characteristicshape of pyramidal-like cells, no colocalization was observed.However, about 13% and 5% of the NC-IR cells were CCK-IR inthe CA3 and CA1 regions respectively, and they were multipolarin all cases. In the CA3 area, double-labeled cells were found in thestrata radiatum (70%) and oriens (30%), whereas in the CA1 area,double-labeled cells were found exclusively in the stratum radia-tum.

Colocalization of NC with markers of dendriticinhibitory cells (SOM, NPY, CB)

SOM, NPY, and CB label inhibitory interneurons whichinnervate the dendritic domain of principal cells. However,whereas SOM and NPY cells innervate the distalmost dendriticsegments, CB cells project to the mid-proximal dendrites ofprincipal cells (Gulyás and Freund, 1996). Colocalization analysis(75 NC-IR cells per region) showed no coexistence at all of NCwith SOM or NPY at any hippocampal region (Fig. 9). Incontrast, about 29% of all the NC-IR cells in the hippocampuswere shown to be positive for CB, with a higher proportion in theCA3 area (40.6%) compared to the DG (30.1%) or the CA1(22.1%) regions (Table 4) (Fig. 10). In the DG, most double-

FIGURE 2. Composite camera lucida drawing of NC-IR cells inthe different regions of the hippocampus. Multipolar (1), horizontalfusiform (2), and vertical fusiform (3) cells were found in allhippocampal regions. In addition, pyramidal-like (4) and bitufted(5) cells were found in the dentate gyrus (DG) and CA1 regionrespectively. The cells are drawn in the same location where theyappeared in the immunostained sections. The composition represents

morphology and position, but not the density or frequency ofappearance of the NC-IR cell types. Stippled lines represent theborders of the different layers on each hippocampal subfield. Dashedline marks the border between CA1 and CA3 regions. g, granule celllayer; h, hilus; l, stratum lucidum; lm, stratum lacunosum-moleculare; o, stratum oriens; p, stratum pyramidale; r, stratumradiatum. Scale bar: 200 mm.


labeled cells were located in the granule cell layer (89.2%),preferentially on its hilar border, and only a few of them werefound in the hilus (10.8%), whereas no double-labeled cells weredetected in the stratum moleculare. The CB immunoreactivityexhibited by the granule cells and the mossy fibers made difficultthe morphological characterization of the double-labeled cellsusing the mirror technique in thick sections. However, in thosecases in which the analysis was possible, horizontal bipolar NC-IRcells located in the hilus and hilar border of the granule cell layer,and, most frequently, pyramidal-like cells were also CB-IR.Double-labeled NC/CB pyramidal-like cells represented about 45%(nine of 20) of the NC-IR pyramidal-like cells in the dentate gyrus.

In the CA3 region, NC/CB double-labeled cells usuallycorresponded to multipolar cells. They were most frequently

found in the stratum radiatum (77.8%), and at a lower proportionin the stratum lucidum (16.7%), being rare in the stratapyramidale (3.7%) and lacunosum-moleculare (1.8%). No double-labeled cells were found in the stratum oriens.

In the CA1 region, most of the double-labeled cells weredetected in the stratum radiatum (64.7%). They usually corre-sponded to multipolar cells, and were preferentially located in theupper half of the layer, with dendrites extending in the stratumradiatum. In addition, some bitufted and vertical fusiform cellsalso showed immunoreactivity for both NC and CB. All thesecells usually had dendrites coursing in the stratum radiatum oreven reaching the stratum oriens, but they usually did not extendinto the stratum lacunosum-moleculare. About 23.5% of theNC/CB double-labeled cells were found in the stratum lacunosum-

FIGURE 3. NC-IR cells in the dentate gyrus (DG). g, granulecell layer; h, hilus; m, molecular layer. A: Microphotograph showingNC-IR cells in the granule cell layer hilar border and hilus. Note thepresence of a typical pyramidal-like cell (arrow). Scale bar: 25 mm. B:Survey of the dentate gyrus showing several NC-IR cells (arrows)

scattered throughout the molecular layer. Several cells can also beseen in the granule cell layer hilar border (arrowheads). Scale bar: 25mm. C,D: Microphotograph of a multipolar (C) and a verticalfusiform (D) NC-IR cell of the molecular layer. Scale bar: 25 mm.


moleculare. Many of them had their cell body located close to theborder with the stratum radiatum. Using the mirror technique,these cells were shown to have horizontal fusiform cell bodies andextend their dendrites in the strata lacunosum-moleculare andradiatum. Double-labeled cells were found at a lower proportionin the stratum pyramidale (9.8%), corresponding to multipolar orbitufted cells, and were rare in the stratum oriens (2%) where theyshowed multipolar morphology.

Colocalization of NC with markers of interneuronsspecialized to innervate other interneurons(CR, VIP)

Most CR- and VIP-IR cells have been recently shown toselectively innervate other GABAergic interneurons in the dentategyrus and hippocampus (Acsády et al., 1996b; Gulyás et al., 1996;Hájos et al., 1996).

The population of NC-IR cells which also contained CR (Fig.10) represented 24.2% of all the NC-positive cells, with a slightlyhigher proportion in the DG (25.6%) and CA1 (26.2%) areasthan in the CA3 region (19.1%)(Table 5). In the DG, double-labeled cells were most frequently found in the hilar region(44.1%), followed by the granule cell layer (35.3%) and themolecular layer (20.6%). In the hilus, NC/CR double-labeledcells corresponded to fusiform (both horizontal and vertical) ormultipolar cells, with dendrites that sometimes were seen to crossthe granule cell layer and enter the stratum moleculare. In thegranule cell layer, double-labeled cells could be found within thelayer, corresponding to small fusiform cells, or most frequently in

the hilar border where they usually corresponded to horizontalbipolar cells, but occasionally they showed morphology ofpyramidal-like cells. Nevertheless, the latter represented a lowproportion of the NC-containing pyramidal-like cells (two of 21).In the molecular layer, multipolar and vertical fusiform double-labeled cells were found.

In the CA3 area, double-labeled cells usually were multipolar.They were most frequently found in the stratum pyramidale(59.1%), but they also appeared in the strata lucidum (22.7%)and radiatum (13.6%), being rare in the stratum lacunosum-moleculare (4.5%). No double-labeled cells were detected in thestratum oriens.

In the CA1 area, NC/CR double-labeled cells were mostabundant in the strata pyramidale (51.9%) and radiatum (37%),where they showed vertical fusiform, bitufted or multipolarmorphologies, with dendrites running radially and sometimesreaching the stratum lacunosum-moleculare and the stratumoriens. Double-labeled cells were occasionally found in thestratum lacunosum- moleculare (5.5%) or in the stratum oriens(5.5%). Using the mirror technique, some CR-IR cells alsocontaining NC were sometimes found to stablish dendro-dendritic contacts, a typical characteristic of the CR-IR cells(Gulyás et al., 1992, 1996).

About 18% of the NC-IR cells contained VIP (Fig. 11), with ahigher proportion in the DG (20%) and CA1 (21%) than in theCA3 region (11.5%)(Table 6). In the DG, the number ofNC/VIP double-labeled cells was slightly higher in the granulecell layer (35.3%) and hilus (35.3%) than in the molecular layer(29.4%). Double-labeled cells in the molecular layer showedvertical fusiform morphology. In the hilus, double-labeled cellswere multipolar or vertical fusiform with dendrites sometimescrossing the granule cell layer. Double-labeled cells in the granulecell layer corresponded to pyramidal-like cells or horizontalfusiform cells with dendrites running in the subgranular zone,sometimes turning and crossing the granule cell layer, andreaching the molecular layer.

In the CA3 region, NC/VIP double-labeled cells were scarceand most of them were found in the strata radiatum (44.4%) andlucidum (44.4%) and only a reduced proportion was located inthe stratum pyramidale (11.1%). No double-labeled cells werefound in the strata oriens or lacunosum-moleculare. In those casesin which the morphology of the double-labeled cells could beanalysed, they were identified as multipolar or vertical fusiformcells.

In the CA1 region, NC/VIP double-labeled cells were found inall layers, although they were less frequently found in the stratumoriens (9%) than in the strata lacunosum-moleculare (27.3%),radiatum (31.8%), or pyramidale (31.8%). Double-labeled cellslocated in the stratum lacunosum-moleculare usually had theirdendrites restricted to that layer, whereas cells in the strataradiatum or pyramidale, typified as vertical fusiform or bituftedcells, had occasionally very long dendrites extending throughoutall layers.

TABLE 1. _____________________________________________Laminar Distribution of NC-IR Cells

N of NC1 cells % of NC cells

Dentate gyrusMolecular layer 28 10.9Granule cell layer 181 70.7Hilus 47 18.4Total 256

DISCUSSION

The major finding of this study is that in the dorsal hippocam-pus of the rat, NC is expressed by a morphologically heterogenouspopulation of interneurons, which are located in all hippocampalfields and layers and are all of them GABAergic. The NC-IR cellsare also neurochemically heterogeneous, and they partially colocal-ize CB, CR, VIP, and CCK, but never PV, SOM, or NPY.

NC Is Present in GABAergic Interneurons

The distribution of the NC-IR cells, their morphologicalfeatures, and the direct evidence of their GABAergic nature,provided by the colocalization analysis of the present study, clearlydemonstrate that they are GABAergic non-principal cells. More-over, the consistent GABA immunoreactivity exhibited by theNC-positive cells suggests that they are most likely short-axonneurons, since GABAergic cells with distant projections havesomatic GABA-levels usually below the immunocytochemical

FIGURE 4. NC-IR cells in the CA3 region. l, stratum lucidum; o,stratum oriens; p, stratum pyramidale; r, stratum radiatum. Scale bar:25 mm. A,B,C: Microphotographs showing NC-IR cells in differentlayers of the CA3 region. IR cells are multipolar or fusiform. In C,

two NC-IR cells located in stratum lucidum can be seen. One of them(large arrow) shows horizontal dendrites in stratum lucidum (smallarrows). The other cell (arrowhead) shows vertically arranged den-drites (small arrows).


detection threshold and show weak GABA immunoreactivity(Miettinen et al., 1992; Tóth et al., 1993).

The GABA-containing cell population in the hippocampus hasbeen shown to include different subsets of cells characterized bytheir content of calcium binding proteins or neuropeptides

(Freund and Buzsáki, 1996). In this study we have analyzed thecoexistence of NC and different neurochemical markers whichlabel perisomatic inhibitory cells (PV, CCK), dendritic inhibitorycells (SOM, NPY, CB), and interneurons specialised to contactother interneurons (CR, VIP). These markers (with the exception

FIGURE 5. NC-IR cells in the CA1 region. l, stratum lucidum;lm, stratum lacunosum-moleculare; o, stratum oriens; p, stratumpyramidale; r, stratum radiatum. Scale bar: 25 mm. A: Microphoto-graph showing NC-IR cells (arrows) in different layers of the CA1region. B: Microphotograph of two horizontal fusiform NC-IR cells(arrows) located in stratum lacunosum-moleculare. C,D: Micropho-tographs of large multipolar NC-IR cells (arrow) located in stratum

radiatum of the CA1 region. E: Large fusiform cell with verticallyrunning dendrites located in stratum radiatum. A descending den-drite can be seen to penetrate stratum pyramidale (small arrows). Twosmaller vertical fusiform NC-IR cells can be seen in stratumpyramidale (arrowheads). F: Bitufted NC-IR cell (arrow) located inthe pyramidal cell layer, with dendrites in stratum radiatum andoriens.


of VIP), as previously established, have been found in non-overlapping GABAergic cell subpopulations (Kosaka et al., 1985;Köhler et al., 1986; Sloviter and Nilaver, 1987; Gulyás et al.,1991; Miettinen et al., 1992; Freund and Buzsáki, 1996). Thus,on the basis of the known termination pattern of the colocalizingmarkers, NC-IR cells may be further characterized.

NC Is Present in Perisomatic InhibitoryCCK-Containing Cells but Neverin PV-Containing Cells

PV and CCK are markers for perisomatic inhibitory cells(Harris et al., 1985; Nunzi et al., 1985; Kosaka et al., 1987;Katsumaru et al., 1988; Soriano et al., 1990; Acsády et al.,1996a,b; Freund and Buzsáki, 1996). NC-IR cells never colocalizePV at any hippocampal region. No coexistence of NC with CCKhas been found in the DG; however, 9% of the NC-IR cells in theCA3 fields were CCK-IR. On the basis of the distribution ofCCK-IR boutons, which are mostly restricted to the principal celllayers, all CCK-containing neurons have been considered to bebasket cells regardless of their location in the different hippocam-pal laminae (Harris et al., 1985; Nunzi et al., 1985; Freund and

Buzsáki, 1996). NC-IR cells colocalizing CCK should be thusconsidered as basket cells. PV and CCK have been shown to bemarkers for two distinct subpopulations of perisomatic inhibitorycells in the hippocampus (Acsády et al., 1996a), which, apart fromdifferential content of these markers, show additional differenceson their subcortical raphe input (Freund et al., 1990), localcontrol by other interneuron types (Acsády et al., 1996b), andsubstance P receptor content (Acsády et al., 1997). NC immuno-reactivity shown by some CCK-IR cells in the CA fields addsfurther differential characteristics between both subsets of basketcells, which argues for functional differences between them.

NC Is Present in Mid-Proximal DendriticInhibitory CB-Containing Cells but AbsentFrom Distal Dendritic Inhibitory CellsContaining SOM or NPY

SOM, NPY and CB are neurochemical markers for dendriticinhibitory interneurons. Both SOM and NPY label inhibitoryneurons which innervate the distal dendritic segments of principalcells (Deller and Léránth, 1990) in conjunction with entorhinalafferents, whereas CB has been found in GABAergic interneurons

FIGURE 6. Colocalization of NC with GABA. Microphoto-graphic pairs of consecutive semithin (1 mm) sections immuno-stained for neurocalcin (NC)(A,C,E) and GABA (B,D,F), correspond-ing to the dentate gyrus (DG)(A-B), CA3 region (C-D), and CA1region (E-F). Arrows point to cells positive for both NC and GABA.

Note that, in all areas, all the NC-IR cells are always GABA positive,whereas there are GABA-IR cells which do not contain neurocalcin(arrowheads). Note also the accumulation of reaction productassociated with cell membranes in NC-positive cells. Scale bar:25 mm.

___________________________________________________ NEUROCALCIN IN THE RAT HIPPOCAMPUS 11

innervating preferentially the mid-proximal dendritic segments ofpyramidal cells (Gulyás and Freund, 1996), in combination withcommisural-associational afferents and Schaffer collaterals. NC-IRcells did not colocalize SOM or NPY while, on the contrary, asignificant coexistence was found with CB in all hippocampalregions. In general, the distribution and morphology of NC/CB

double-labeled neurons in the CA3 and CA1 areas was similar tothat found for CB alone, with the difference that NC/CB cellswere almost absent from the stratum oriens. Nevertheless, mostCB cells in the stratum oriens have been found to containsomatostatin (Katona et al., 1996), which, as we have found,never colocalizes with NC. Double-labeled NC/CB cells were

FIGURE 7. Colocalization of NC with parvalbumin (PV). Micro-photographic pairs of consecutive semithin (1 mm) sections immuno-stained for neurocalcin (NC)(A,C) and PV (B,D), corresponding to

the dentate gyrus (DG)(A-B) and CA1 region (C-D). NC-positivecells (arrows) are always negative for PV, and PV-containing cells(arrowheads) never show NC-immunoreactivity. Scale bars: 25 mm.

TABLE 2. ___________________________________________________________________________Coexistence of NC and GABA

N ofNC cells

N ofGABA cells

N of NC andGABA cells

% of NC cellsalso GABA

% of GABAcells also NC

Dentate gyrusMolecular layer 15 100 15 100 15.0Granule cell layer 90 453 90 100 19.9Hilus 22 157 22 100 14.0Total 127 710 127 100 17.9

CA3 RegionStr. lac.-mol. 5 34 5 100 14.7Str. radiatum 39 230 39 100 17.0Str. lucidum 20 75 20 100 26.7Str. pyramidale 20 142 20 100 14.1Str. oriens 12 114 12 100 10.5Total 106 595 106 100 17.8

CA1 RegionStr. lac.-mol. 52 207 52 100 25.1Str. radiatum 89 291 89 100 30.6Str. pyramidale 69 363 69 100 19.0Str. oriens 11 216 11 100 5.1Total 221 1077 221 100 20.5

Data taken from 12 pairs of semithin sections belonging to four animals.


most frequently found in the stratum radiatum and correspondedto multipolar, bitufted and vertical fusiform cells with radiallyrunning dendrites. A similar morphology was shown by double-labeled cells located in the stratum pyramidale. In addition, somehorizontal fusiform cells were found in the stratum lacunosum-moleculare of the CA1 region. Thus, the somatodendritic patternof double-labeled NC/CB cells suggests that they may includebistratified cells, which innervate pyramidal cell dendrites in thestrata radiatum and oriens (Buhl et al., 1994a; Sik et al., 1995;Miles et al., 1996), neurons with axon and dendrites in thestratum radiatum, which innervate mid-proximal apical pyrami-dal cell dendrites (Kawaguchi and Hama, 1988; Gulyás et al.,1993a; Miles et al., 1996), and interneurons in the stratumlacunosum-moleculare, which have an axon arborizing predomi-nantly in the stratum lacunosum-moleculare or distal stratumradiatum (Kunkel et al., 1988). All these three types of cells aredendritic inhibitory cells which are mostly driven in a feed-forward manner by their excitatory afferent inputs; bistratifiedcells and CA3 neurons with dendrites and axon in the stratumradiatum may be also activated by local pyramidal cell recurrentcollaterals in a feed-back manner (Freund and Buzsáki, 1996).

In the DG, most NC/CB double-labeled interneurons corre-sponded to pyramidal-like cells located in the deep granule celllayer and hilar border. In addition, some horizontal fusiformNC/CB cells located in the hilar border of the granule cell layerwere also found. In the DG, neurons with pyramidal-likemorphology have been found among the cells labeled by most ofthe analyzed neurochemical markers. Pyramidal-like cells havebeen shown to include at least two neurochemically differentgroups innervating the perisomatic membrane domains of granulecells, one immunoreactive for PV (Kosaka et al., 1987), and theother positive for CCK (Kosaka et al., 1985; Acsády et al., 1996a).CCK-IR cells also innervate hilar mossy cells (Léránth andFrotscher, 1986). In addition, CR- and NPY-IR cells withmorphological features of pyramidal-like cells have also beendescribed and their target field is most likely the hilar region(Gulyás et al., 1992; Freund and Buzsáki, 1996). Many NC-IRcells in the DG show morphology of pyramidal-like cells.However, they are always negative for PV, CCK, or NPY, and only10% of them are CR-IR. In contrast, we have found that about45% of the pyramidal-like NC-IR cells in the DG also containCB. These findings suggest that there are pyramidal-like CB-containing cells, and that about 45% of the NC-IR pyramidal-likecells do not contain any of the other analyzed markers. Thus,pyramidal-like cells appear as a highly neurochemically heteroge-neous neuronal population, which can express CB and/or NC inaddition to other previously described markers. The dendritic

FIGURE 8. Colocalization of NC with cholecystokinin (CCK).Photomicrographs of paired surfaces of consecutive vibratome sec-tions immunostained for NC (A,C,E) and CCK (B,D,F), correspond-ing to the dentate gyrus (DG)(A-B), CA3 region (C-D), and CA1region (E-F). Large arrows point to NC-positive cells found to benegative (A-B) or positive (C-D,E-F) for CCK. Arrowheads point toCCK-positive cells shown to be negative for NC. Small arrows markcapillaries used as landmarks. Scale bars: 25 mm.


morphology of the pyramidal-like cells suggests that they may beactivated in both a feed-forward manner by entorhinal cortex andthe commissural-associational pathway or even CA3 pyramidalcells and in a feed-back manner by mossy fiber collaterals (Ribakand Peterson, 1991; Kneisler and Dingledine, 1995a,b). Thetarget domain of the NC/CB pyramidal-like cells is unknownsince axons of CB-IR interneurons in the dentate gyrus have notbeen reconstructed yet. Nevertheless, interneurons with pyramidal-shaped cell bodies have been shown to innervate, apart from theperisomatic region, dendritic domains of the granule cells (Ama-ral, 1978; Scharfman, 1995). CB-containing interneurons havebeen shown to be specialized in dendritic innervation of principalcells both in the cerebral cortex and hippocampus (DeFelipe et al.,1989; del Rio and DeFelipe, 1995; Gulyás and Freund,1996).Thus, NC/CB-containing pyramidal-like cells in the DGare likely to be involved in dendritic inhibition, which seems to bea common characteristic shared by CB-containing cells in diversecortical areas.

NC Is Present in Different Types of InterneuronsSpecialized to Innervate Other Interneurons(Interneuron Selective Cells)

CR-containing cells have been demonstrated to include spinyand aspiny cells. Spiny cells were found in the hilus and in theCA3 stratum lucidum, in association with mossy fibers (Gulyás etal., 1992). We have found NC/CR double-labeled cells in thesame location as spiny CR-IR cells; however, different evidenceindicates that the NC/CR double-labeled cells correspond toaspiny CR-IR neurons probably located between spiny neurons.Thus, the strong GABA immunoreactivity shown by the NC-containing cells argues for their aspiny nature, since CR-IR spiny

cells lack GABA immunoreactivity (Miettinen et al., 1992). Inaddition, CR-IR spiny cells have been recently demonstrated tocontain SOM (Katona et al., 1996), which never colocalizes withNC. Thus, NC/CR double-labeled cells most likely correspond toCR-IR aspiny cells.

Most CR- and VIP-IR cells have been recently shown toselectively innervate other GABAergic interneurons in the dentategyrus and hippocampus and they have been named interneuron-selective (IS) cells. This class of interneurons has been demon-strated to include at least three types of cells (IS-1, IS-2, and IS-3)according to their connectivity and neurochemical characteristics(Freund and Buzsáki, 1996). IS-1 cells are characterized by theirCR immunoreactivity and extensive dendritic and axonic intercon-nection (Gulyás et al., 1992, 1996). In the DG, they are located inthe hilus and granule cell layer and extend dendrites in all layers.In the hippocampus they correspond to bitufted, vertical fusiformand multipolar cells located in the stratum radiatum, pyramidaleand oriens. Their axons innervate dendrites and cell bodies ofother interneurons, mainly CB-IR cells, located in the hilus and inthe stratum radiatum. They may be mainly activated by commis-sural-associational fibers and Schaffer collaterals, but they may bealso innervated by afferents from the entorhinal cortex. IS-2 cellshave been characterized in the hippocampus alone, and theyextend the axon in the stratum radiatum. They comprise twosubsets of cells, of which one corresponds to VIP-IR CR-negativecells with dendrites restricted to the stratum lacunosum-moleculare, whereas the other includes bitufted or verticalfusiform VIP-IR cells located in the stratum radiatum that mayalso contain CR. Their major input is considered to be ofentorhinal origin, and their major targets are CB-IR interneuronsinvolved in mid-proximal dendritic inhibition in the Schaffercollateral and commissural-associational termination zone (Ac-sády et al., 1996a,b). IS-3 cells are VIP-IR, CR-positive neurons,located in the molecular layer or granule cell layer of the dentate

TABLE 3. _____________________________________________________________________________Coexistence of NC and CCK

N ofNC cells

N ofCCK cells

N of NCand CCK cells

% of NCcells also CCK

Laminar distributionof NC/CCK cells

CA3 RegionStr. lac.-mol. 3 2 0 0 0Str. radiatum 47 28 7 14.9 70.0Str. lucidum 9 16 0 0 0Str. pyramidale 11 12 0 0 0Str. oriens 5 9 3 60.00 30.0Total 75 67 10 13.3

gyrus, and in the strata radiatum or pyramidale of the hippocam-pus. They are bipolar or bitufted cells with radially runningdendrites that, in the hippocampus, reach the stratum lacunosum-moleculare. They appear to be driven mainly by entorhinal

afferents. Their axons form a plexus in the oriens-alveus borderand in the hilus, and innervate interneurons involved in distaldendritic inhibition in the entorhinal termination zone (Acsády etal., 1996a,b).

FIGURE 9. Colocalization of NC with somatostatin (SOM) andneuropeptide Y (NPY). A-D: Photomicrographs of paired surfaces ofconsecutive vibratome sections immunostained for NC (A,C) andSOM (B,D), corresponding to the dentate gyrus (DG)(A-B) and CA3region (C-D). Large arrows point to NC-positive cells which werefound to be negative for SOM. Arrowheads point to SOM-positive

cells always negative for NC. Small arrows mark capillaries used aslandmarks. Scale bars: 25 mm. E,F: Microphotographic pair ofconsecutive semithin (1 mm) sections immunostained for NC (K) andNPY (L), corresponding to the dentate gyrus (DG). An NC-positivecell (arrow) is shown to be negative for NPY, and all the NPY-positivecells (arrowheads) are negative for NC. Scale bar: 25 mm.


FIGURE 10. Colocalization of NC with calbindin (CB) andcalretinin (CR). Microphotographic pairs of two consecutive semi-thin (1 mm) sections immunostained for neurocalcin (NC)(A,C,E)and calbindin (CB)(B,D,F) or neurocalcin (NC)(G,I,K) and calreti-

nin (CR)(H,J,L), corresponding to the dentate gyrus (DG)(A-B,G-H), CA3 region (C-D, I-J) and CA1 region (E-F,K-L) Arrows markNC-IR cells also containing CB or CR. Arrowheads point to NC-IRcells which are negative for CB or CR. Scale bars: 25 mm.


FIGURE 11. Colocalization of NC with vasoactive intestinalpolypeptide (VIP). Photomicrographs of paired surfaces of consecu-tive vibratome sections immunostained for NC (A,C,E,G,I,K) andVIP (B,D,F,H,J,L), corresponding to the dentate gyrus (DG)(A-B,C-D), CA3 region (E-F), and CA1 region (G-H,I-J,K-L). Large arrows

point to NC-positive cells, found to be positive for VIP. Arrowheadsin K-L mark cut dendrites immunoreactive for both markers, seen tocontinue in the matching section. Small arrows mark capillaries usedas landmarks. Scale bar: 25 mm.


According to their location, somatodendritic pattern, andmarker content, some NC-IR cells can be presumably ascribed tothe different IS cell types. In the DG, fusiform, multipolar, andpyramidal-like NC/CR or NC/VIP double-labeled cells located inthe hilus and granule cell layer probably belong to the IS-1 type ofinterneurons, whereas multipolar and vertical fusiform NC/CR orNC/VIP double-labeled cells located in the stratum molecularepossibly correspond to IS-3 type cells. In the hippocampus,NC/CR double-labeled multipolar cells are likely to pertain to theIS-1 type, whereas bitufted and vertical fusiform cells presumablybelong to any of the three IS types of interneurons. Bitufted andvertical fusiform NC/VIP double-labeled cells located in the strataradiatum and pyramidale may correspond to IS-2 and/or IS-3type cells, whereas NC/VIP cells located in the stratum lacunosum-moleculare of the CA1 definitely belong to the IS-2 type of cells.

IS cells, and consequently NC-IR cells which fall into thisgroup of interneurons, are likely to be activated by excitatoryinputs in a feed-forward manner, and they have been proposed toplay a role in synchronizing the activity of inhibitory cells thatconverge onto a particular group of pyramidal cells, or, alterna-tively, they may disinhibit principal cell dendrites innervated byparticular excitatory inputs (Freund and Buzsáki, 1996).

Part of the NC-IR Cells Lacks Other Markers

We have found that NC-IR cells include CB-, CR-, CCK-, andVIP-containing neurons. However, NC-IR cells colocalizing these

markers represent only part of the NC-IR neuronal population. Inthe DG, 30.1% and 25.6% of the NC-IR cells have beendemonstrated to contain CB or CR respectively, but no NC-IRcells were found to be positive for PV, CCK, SOM, or NPY. Inaddition, 20% of the NC-IR cells were found to be positive forVIP. However, since most VIP cells in the DG have been shown tocontain either CCK or CR, and no NC-IR cells were CCKpositive, NC/VIP double-labeled cells are likely to correspond toCR-IR cells. Considering that the degree of coexistence of CR andCB is very low (Miettinen et al., 1992), we can assume that about44.3% of the NC-IR cells in the DG lack other analyzed markers.

No colocalization with PV, SOM, or NPY was found in theCA3 and CA1 regions. In the CA3 region, 40.6%, 19.1%, and13.3% of the NC-IR cells were found to be CB-, CR-, andCCK-IR respectively. In the CA1 the proportions for the samemarkers were 22.1%, 26.2% and 5.5% respectively. In addition,in the CA1 at least those double-labeled NC/VIP cells located inthe stratum lacunosum-moleculare, where VIP-containing cellsdo not colocalize CR or CCK (Acsády et al., 1996a), have to beadded to the NC-IR cells also containing other markers. Conse-quently, NC-IR cells lacking other markers account for about27% and 40.5% of the NC-IR cells in the CA3 and CA1 regionsrespectively.

Thus, a substantial proportion of NC-IR cells does notcolocalize with any of the analyzed markers. This finding opensthe possibility that NC colocalizes with other markers different

TABLE 4. _____________________________________________________________________________Coexistence of NC and CB

N ofNC cells

N ofCB cells

N of NCand CB cells

% of NCcells also CB

Laminar distributionof NC/CB cells

Dentate gyrusMolecular layer 15 9 0 0 0Granule cell layer 86 —1 33 38.4 89.2Hilus 22 29 4 18.2 10.8Total 123 37 30.1

from those analyzed in the present study. From the morphologyand laminar distribution of the NC-IR cells and that of cellscontaining some other non-analyzed markers (e.g., NOS/NADPH-diaphorase or enkephalins) some indirect speculations can bedrawn. In the DG coexistence of NC and NOS/NADPH-diaphorase (Hope et al., 1991; Vincent and Kimura, 1992;Valtschanoff et al., 1993; Dun et al., 1994) is possible inmultipolar and vertical fusiform cells of the molecular layer and inpyramidal-like cells of the granule cell layer that lack PV (Freundand Búzsaki, 1996). In the CA1, coexistence may occur in verticalfusiform, bitufted, and multipolar cells distributed in strataradiatum and pyramidale of the CA1 (Freund and Búzsaki, 1996).In the CA1, NC and enkephalins are likely to coexist in verticalfusiform and multipolar cells located in strata radiatum andpyramidale (Gall et al., 1981; J.M. Blasco-Ibáñez, F.J. Martı́nez-Guijarro, and T.F. Freund, unpublished observations). Moreover,all enkephalin-IR cells in the CA1 have been found to be CR- andVIP-IR (J.M. Blasco-Ibáñez, F.J. Martı́nez-Guijarro, and T.F.Freund, unpublished observations); both markers colocalize withNC, thus arguing for a possible coexistence of enkephalinsand NC.

NC Is a Neural Calcium-Sensor Protein Presentin Hippocampal Interneurons

In general, EF-hand calcium-binding proteins have beenclassified into two major groups, ‘‘calcium-buffer proteins,’’involved in calcium buffering and transport, and ‘‘calcium-sensorproteins,’’ which have regulatory roles (da Silva and Reinach,

1991; Ikura, 1996). Neurocalcin belongs to a family of neuronal-specific EF-hand calcium-binding proteins defined by recoverin,found primarily in vertebrate brain and retina (Terasawa et al.,1992; Nakano et al., 1992; Hidaka and Okazaki, 1993), which actas calcium-sensors and have been proposed to be involved in theregulation of calcium-dependent phosphorylation in signal trans-duction pathways (De Castro et al., 1995). Moreover, NC hasbeen shown to inhibit bovine rhodopsin phosphorylation in vitro,in a calcium-dependent manner (Faurobert et al., 1996). Conse-quently, NC may play an important role in phosphorylation-dependent regulation of a variety of receptors and channels,including G-protein-coupled receptors, as well as many classes ofion channels (Jonas and Kaczmarek, 1996; Moss and Smart,1996), and thus influence firing pattern and responses to synapticinputs in NC-containing interneurons.

NC, as recoverin, has a myristoyl consensus sequence, whichmay constitute a calcium-myristoyl switch, that allows NC tobind cell membranes in a calcium-dependent manner (Zozulyaand Stryer, 1992), and may explain the substantial accumulationof NC immunostaining associated to cell membranes found insemithin immunostained sections in this study. The presence of acalcium-myristoyl switch in recoverin-like calcium-sensor pro-teins suggests that anchoring to cell membranes may be function-ally relevant, and that these proteins develop their role locally,associated to cell membrane domains, as has been shown forrecoverin in rod photoreceptor cells (Sanada et al., 1996).

NC has been found in some interneurons that also containother calcium-binding proteins of the so-called ‘‘buffer type,’’ i.e.,

TABLE 5. _____________________________________________________________________________Coexistence of NC and CR

N ofNC cells

N ofCR cells

N of NCand CR cells

% of NCcells also CR

Laminar distributionof NC/CR cells

Dentate gyrusMolecular layer 13 12 7 53.8 20.6Granule cell layer 95 18 12 12.6 35.3Hilus 25 90 15 60.0 44.1Total 133 120 34 25.6

CB and CR. The functional meaning for this coexistence in thesame cell is presently unknown, but from the variability incalcium-binding affinities as well as structural and functionaldifferences, NC and the colocalized buffer-type calcium-bindingproteins are most likely involved in different calcium-mediatedevents (Ikura, 1996).

Other neural calcium-sensor recoverin-like proteins differentfrom NC have been found in the hippocampus, includingvisinin-like protein (VILIP)(Lenz et al., 1996), neural visinin-likecalcium-binding protein 2 (NVP2)(Saitoh et al., 1994) andhippocalcin (Kobayashi et al., 1992). However, although NVP2has been shown to be transiently expressed by hippocampalinterneurons from days P7 to P28 (Saitoh et al., 1995), in adultanimals these proteins are expressed exclusively by principal cells,in contrast to NC which is found only in GABAergic interneu-rons. Thus, separate neural calcium sensor proteins seem to bepresent in principal cells and interneurons, and, although they arelikely to subserve similar general functions, the particular calcium-binding requirements of different types of cells may need theintervening of distinct proteins. Regardless of what the precisefunction of NC is, its differential expression suggests thatGABAergic interneurons containing this calcium-sensor proteinare likely to share some specific functional properties. Electrophysi-ological characterization and correlative morphological and immu-nocytochemical studies would be useful to clarify whether NC-positive cells exhibit particular functional properties which maybe related to their NC content.

In general, for a given marker, NC is present only in part of thecells expressing it, which are morphologically indistinguishablefrom other cells labeled by that marker but lacking NC (e.g., IS-2VIP cells in the stratum lacunosum-moleculare of the CA1 maycontain or lack NC). This finding suggests that for eachsubpopulation of interneurons labeled by markers which coexistwith NC, neurochemical differences exist among its cells withregard to NC content, which may imply distinct responses infront of calcium influx and /or finer tuning of calcium levels, andtherefore the existence of functionally different groups withinapparent morphologically or neurochemically homogeneous popu-lations of cells. Whether these differences are correlated withsingular functional and/or connectivity features is an openquestion.

Conclusion

NC, a calcium-sensor EF-hand protein, is present exclusively inGABAergic interneurons distributed in all layers of the dentategyrus and hippocampus. NC-containing cells are morphologicallyand neurochemically heterogeneous and, on the basis of colocaliz-ing markers, they include mid-proximal dendritic inhibitory cellscharacterized by CB immunoreactivity, interneurons specializedto innervate other interneurons, which contain CR and/or VIP,and basket cells characterized by their CCK content. On thecontrary, NC is never present in perisomatic inhibitory PV-

TABLE 6. _____________________________________________________________________________Coexistence of NC and VIP

N ofNC cells

N ofVIP cells

N of NCand VIP cells

% of NCcells also VIP

Laminar distributionof NC/VIP cells

Dentate gyrusMolecular layer 12 9 5 41.7 29.4Granule cell layer 53 15 6 11.3 35.3Hilus 20 8 6 30.0 35.3Total 85 32 17 20.0

containing cells, or in feed-back distal dendritic inhibitorySOM/NPY-containing cells.

Acknowledgments

We thank Dr. T.J. Görcs for generous gift of VIP and SOMantisera.

REFERENCES

Acsády L, Arabadzisz D, Freund TF. Correlated morphological andneurochemical features indentify different subsets of VIP-immunore-active interneurons in rat hippocampus. Neuroscience 1996a;73:299–315.

Acsády L, Görcs TJ, Freund TF. Different populations of VIP-immunoreactive interneurons are specialized to control pyramidalcells or interneurons in the hippocampus. Neuroscience 1996b;73:317–334.

Acsády L, Katona I, Gulyás AI, Shigemoto R, Freund TF. Immunostain-ing for substance P receptor labels GABAergic cells with distincttermination pattern in the hippocampus. J Comp Neurol 1997;378:320–336.

Amaral DG. A Golgi study of cell types in the hilar region of thehippocampus in the rat. J Comp Neurol 1978;182:851–914.

Baimbridge KG, Miller JJ. Immunohistochemical localization of calcium-binding protein in the cerebellum, hippocampal formation andolfactory bulb of the rat. Brain Res 1982;245:223–229.

Bastianelli E, Okazaki K, Hidaka H, Pochet R. Neurocalcin immunoreac-tivity in rat olfactory bulb. Neurosci Lett 1993;581:585–588.

Bastianelli E, Polans AS, Hidaka H, Pochet R. Differential distribution ofsix calcium-binding proteins in the rat olfactory epithelium duringpostnatal development and adulthood. J Comp Neurol 1995;354:395–409.

Bragin A, Jando G, Nadasdy Z, Hetke J, Wise K, Buzsáki G. Gammafrequency (40-100 Hz) patterns in the hippocampus of the behavingrat. J Neurosci 1995;15:47–60.

Buckmaster PS, Schwartzkroin PA. Interneurons and inhibition in thedentate gyrus of the rat in vivo. J Neurosci 1995;15:774–789.

Buckmaster PS, Kunkel DD, Robbins RJ, Schwartzkroin PA. Somatosta-tin immunoreactivity in the hippocampus of mouse, rat, guinea pigand rabbit. Hippocampus 1994;4:167–180.

Buhl EH, Halasy K, Somogyi P. Diverse sources of hippocampal unitaryinhibitory postsynaptic potentials and the number of synaptic releasesites. Nature 1994;368:823–828.

Buzsáki G, Chrobak JJ. Temporal structure in spatially organizedneuronal ensembles: A role for interneuronal networks. Curr OpinNeurobiol 1995;5:504–510.

Buzsáki G, Horvath Z, Urioste R, Hetke J, Wise K. High-frequencynetwork oscillation in the hippocampus. Science 1992;256:1025–1027.

Celio MR. Calbindin D-28k and parvalbumin in the rat nervous system.Neuroscience 1990;35:375–475.

Cobb SR, Buhl EH, Halasy K, Paulsen O, Somogyi P. Synchronization ofneuronal activity in hippocampus by individual GABAergic interneu-rons. Nature 1995;378:75–98.

Csiffáry K, Görcs TJ, Palkovits M. Neuropeptide Y innervation ofACTH-immunoreactive neurons in the arcuate nucleus of the rat: Acorrelated light and electron microscopic double immunolabelingstudy. Brain Res 1990;506:215–222.

da Silva ACR, Reinach FC. Calcium binding induces conformational

changes in muscle regulatory proteins. Trends Biochem Sci 1991;16:53–57.

De Castro E, Nef S, Fiumelli H, Lenz SE, Kawamura S, Nef P. Regulationof rhodopsin phosphorylation by a family of neuronal calciumsensors. Biochem Biophys Res Commun 1995;216:133–140.

DeFelipe J, Hendry SHC, Jones, EG. Synapses of double bouquet cells inmonkey cerebral cortex visualized by calbindin immunoreactivity.Brain Res 1989;503:49–54.

del Rio MR, DeFelipe J. A light and electron microscopic study ofcalbindin D-28k immunoreactive double bouquet cells in the humantemporal cortex. Brain Res 1995;690:133–140.

Deller T, Léránth C. Synaptic connections of neuropeptide-Y (NPY)immunoreactive neurons in the hilar area of the rat hippocampus. JComp Neurol 1990;300:433–447.

Dun NJ, Dun SL, Wong RK, Forstermann U. Colocalization of nitricoxide synthase and somatostatin immunoreactivity in rat dentate hilarneurons. Proc Natl Acad Sci USA 1994;91:2955–2959.

Faurobert E, Chen CK, Hurley JB, Teng DH. Drosophila neurocalcin, afatty acylated, Ca21-binding protein that associates with membranesand inhibits in vitro phosphorylation of bovine rhodopsin. J BiolChem 1996;271:10256–10262.

Freund TF, Buzsáki G. Interneurons of the hippocampus. Hippocampus1996;6:345–471.

Freund TF, Gulyás AI, Acsády L, Görcs T, Tóth K. Serotonergic control ofthe hippocampus via local inhibitory interneurons. Proc Natl Acad SciUSA 1990;87:8501–8505.

Gall C, Brecha N, Karten HJ, Chang KJ. Localization of enkephalin-likeimmunoreactivity to identified axonal and neuronal populations ofthe rat hippocampus. J Comp Neurol 1981;198:335–350.

Gulyás AI, Freund TF. Pyramidal cell dendrites are the primary targets ofcalbindin D28k-immunoreactive interneurons in the hippocampus.Hippocampus 1996;6:525–534.

Gulyás AI, Görcs TJ, Freund TF. Innervation of different peptide-containing neurons in the hippocampus by GABAergic septal affer-ents. Neuroscience 1990;37:31–44.

Gulyás AI, Tóth K, Danos P, Freund TF. Subpopulations of GABAergicneurons containing parvalbumin, calbindin D28k, and cholecystoki-nin in the rat hippocampus. J Comp Neurol 1991;312:371–378.

Gulyás AI, Miettinen R, Jacobowitz DM, Freund TF. Calretinin ispresent in non-pyramidal cells of the rat hippocampus-I. A new typeof neuron specifically associated with the mossy fibre system.Neuroscience 1992;48:1–27.

Gulyás AI, Miles R, Hájos N, Freund TF. Precision and variability inpostsynaptic target selection of inhibitory cells in the hippocampalCA3 region. Eur J Neurosci 1993a;5:1729–1751.

Gulyás AI, Miles R, Sik A, Tóth K, Tamamaki N, Freund TF.Hippocampal pyramidal cells excite inhibitory neurons through asingle release site. Nature 1993b;366:683–687.

Gulyás AI, Hájos N, Freund TF. Interneurons containing calretinin arespecialized to control other interneurons in the rat hippocampus. JNeurosci 1996;16:3397–3411.

Hájos N, Acsády L, Freund TF. Target selectivity and neurochemicalcharacteristics of VIP-immunoreactive interneurons in the rat dentategyrus. Eur J Neurosci 1996;8:1415–1431.

Han ZS, Buhl EH, Lorinczi Z, Somogyi P. A high degree of spatialselectivity in the axonal and dendritic domains of physiologicallyidentified local-circuit neurons in the dentate gyrus of the rathippocampus. Eur J Neurosci 1993;5:395–410.

Harris KM, Marshall PE, Landis DM. Ultrastructural study of cholecys-tokinin-immunoreactive cells and processes in area CA1 of the rathippocampus. J Comp Neurol 1985;233:147–158.

Hidaka H, Okazaki K. Neurocalcin family: A novel calcium-bindingprotein abundant in bovine central nervous system. Neurosci Res1993;16:73–77.

Hope BT, Michael GJ, Knigge KM, Vincent SR. Neuronal NADPH


diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA1991;88:2811–2814.

Iino S, Kobayashi S, Okazaki K, Hidaka H. Immunohistochemicallocalization of neurocalcin in the rat inner ear. Brain Res 1995a;680:128–134.

Iino S, Kobayashi S, Okazaki K, Hidaka H. Neurocalcin-immunoreactivereceptor cells in the rat olfactory epithelium and vomeronasal organ.Neurosci Lett 1995b;191:91–94.

Ikura M. Calcium binding and conformational response in EF-handproteins. Trends Biochem Sci 1996;21:14–17.

Jonas EA, Kaczmarek LK. Regulation of potassium channels by proteinkinases. Curr Opin Neurobiol 1996;6:318–323.

Katona I, Acsády L, Gulácsi A, Freund TF. Somatostatin-containing cellsin the rat hippocampus: Connectivity and heterogeneity. Eur JNeurosci Suppl 1996;9:176.

Katsumaru H, Kosaka T, Heizmann CW, Hama K. Immunocytochemi-cal study of GABAergic neurons containing the calcium-bindingprotein parvalbumin in the rat hippocampus. Exp Brain Res 1988;72:347–362.

Kawaguchi Y, Hama K. Physiological heterogeneity of nonpyramidal cellsin rat hippocampal CA1 region. Exp Brain Res 1988;72:494–502.

Kneisler TB, Dingledine R. Spontaneous and synaptic input fromgranule cells and perforant path to dentate basket cells in rathippocampus. Hippocampus 1995a;5:151–164.

Kneisler TB, Dingledine R. Synaptic input from CA3 pyramidal cells todentate basket cells in rat hippocampus. J Physiol (Lond) 1995b;487:125–146.

Kobayashi M, Takamatsu K, Saitoh S, Miura M, Noguchi T. Molecularclonning of hippocalcin, a novel calcium-binding protein of therecoverin family exclusively expressed in hippocampus. BiochemBiophys Res Commun 1992;189:511–517.

Köhler C, Eriksson L, Davies S, Chan-Palay V. Neuropeptide Yinnervation of the hippocampal region in the rat and monkey brain. JComp Neurol 1986;244:384–400.

Kosaka T, Kosaka K, Tateishi K, Hamaoka Y, Yanaihara N, Wu JY, HamaK. GABAergic neurons containing CCK-8-like and/or VIP-likeimmunoreactivities in the rat hippocampus and dentate gyrus. JComp Neurol 1985;239:420–430.

Kosaka T, Katsumaru H, Hama K, Wu JY, Heizmann CW. GABAergicneurons containing the Ca21-binding protein parvalbumin in the rathippocampus and dentate gyrus. Brain Res 1987;419:119–130.

Kunkel DD, Lacaille JC, Schwartzkroin PA. Ultrastructure of stratumlacunosum-moleculare interneurons of hippocampal CA1 region.Synapse 1988;2:382–394.

Lacaille J-C, Schwartzkroin PA. Stratum lacunosum-moleculare interneu-rons of hippocampal CA1 region. I. Intracellular response characteris-tics, synaptic responses, and morphology. J Neurosci 1988a;8:1400–1410.

Lacaille J-C, Schwartzkroin PA. Stratum laculosum-moleculare interneu-rons of hippocampal CA1 region. II. Intrasomatic and intradendriticrecordings of local circuit synaptic interactions. J Neurosci 1988b;8:1411–1424.

Lacaille J-C, Mueller A, Kunkel DD, Schwartzkroin PA. Local circuitinteractions between oriens/alveus interneurons and CA1 pyramidalcells in hippocampal slices: Electrophysiology and morphology. JNeurosci 1987;7:1979–1993.

Lantos TA, Görcs TJ, Palkovits M. Immunohistochemical mappings ofneuropeptides in the premammillary region of the hypothalamus inrats. Brain Res Rev 1995;20:209–249

Lenz SE, Zuschratter W, Gundelfinger DE. Distribution of visinin-likeprotein (VILIP) immunoreactivity in the hippocampus of the Mongo-lian gerbil (Meriones unguiculatus). Neurosci Lett 1996;206:133–136.

Léránth C, Frotscher M. Synaptic connections of cholecystokinin-immunoreactive neurons and terminals in the rat fascia dentata: Acombined light and electron microscopic study. J Comp Neurol1986;254:51–64.

Léránth C, Malcolm AJ, Frotscher M. Afferent and efferent synaptic

connections of somatostatin-immunoreactive neurons in the rat fasciadentata. J Comp Neurol 1990;295:111–122.

Martı́nez Guijarro FJ, Briñón JG, Blasco-Ibañez JM, Okazaki K, HidakaH, Alonso JR. Neurocalcin-immunoreactive cells in the rat hippocam-pus. Eur J Neurosci Suppl 1996;9:177.

McBain CJ, DiChiara TJ, Kauer JA. Activation of metabotropic gluta-mate receptors differentially affects two classes of hippocampalinterneurons and potentiates excitatory synaptic transmission. JNeurosci 1994;14:4433–4445.

Michelson HB, Wong RKS. Excitatory synaptic responses mediated byGABA receptors in the hippocampus. Science 1991;253:1420–1423.

Miettinen R, Gulyás AI, Baimbridge KG, Jacobowitz DM, Freund TF.Calretinin is present in non-pyramidal cells of the rat hippocampus-II. Co-existence with other calcium binding proteins and GABA.Neuroscience 1992;48:29–43.

Miles R, Tóth K, Gulyás AI, Hájos N, Freund TF. Differences betweensomatic and dendritic inhibition in the hippocamus. Neuron 1996;16:815–823.

Moss SJ, Smart TG. Modulation of amino acid-gated ion channels byprotein phosphorylation. Int Rev Neurobiol 1996;39:1–52.

Müller W, Misgeld U. Inhibitory role of dentate hilus neurons in guineapig hippocampal slice. J Neurophysiol 1990;64:46–56.

Nakano A, Terasawa M, Watanabe M, Usuda N, Morita T, Hidaka H.Neurocalcin, a novel calcium binding protein with three EF-handdomains, expressed in retinal amacrine cells and ganglion cells.Biochem Biophys Res Commun 1992;186:1207–1211.

Nakano A, Terasawa M, Watanabe M, Okazaki K, Inoue S, Kato M,Nimura Y, Usuda N, Morita T, Hidaka H. Distinct regionallocalization of neurocalcin, a Ca21-binding protein, in the bovineadrenal gland. J Endocrinol 1993;138:283–290.

Nitsch R, Soriano E, Frotscher M. The parvalbumin-containing nonpyra-midal neurons in the rat hippocampus. Anat Embryol 1990;181:413–425.

Nunzi MG, Gorio A, Milan F, Freund TF, Somogyi P, Smith AD.Cholecystokinin-immunoreactive cells form symmetrical synapticcontacts with pyramidal and nonpyramidal neurons in the hippocam-pus. J Comp Neurol 1985;237:485–505.

Okazaki K, Iino S, Inoue S, Kobayashi S, Hidaka H. Differentialdistribution of neurocalcin isoforms in rat spinal cord, dorsal rootganglia and muscle spindle. Biochim Biophys Acta 1994;1223:311–317.

Porteros A, Briñón JG, Crespo C, Okazaki K, Hidaka H, Aijón J, AlonsoJR. Neurocalcin immunoreactivity in the rat accessory olfactory bulb.Brain Res 1996;729:82–89.

Ribak CE, Peterson GM. Intragranular mossy fibers in rats and gerbilsform synapses with the somata and proximal dendrites of basket cellsin the dentate gyrus. Hippocampus 1991;1:355–364.

Saitoh S, Takamatsu K, Kobayashi M, Noguchi T. Immunohistochemicallocalization of neural visinin-like Ca(21)-binding protein 2 in adultrat brain. Neurosci Lett 1994;171:155–158.

Saitoh S, Kobayashi M, Kuroki T, Noguchi, Takamatsu K. The develop-ment of neural visinin-like Ca(21)-binding protein 2 immunoreactiv-ity in the rat neocortex and hippocampus. Neurosci Res 1995;23:283–288.

Sanada K, Shimizu F, Kameyama K, Haga K, Fukada Y. Calcium-boundrecoverin targets rhodopsin kinase to membranes to inhibit rhodopsinphosphorylation. FEBS Lett 1996;384:227–230

Schaad N, De Castro E, Nef S, Hegi S, Hinrichsen R, Martone ME,Ellisman MH, Sikkink R, Rusnak F, Sygush J, Nef P. Directmodulation of calmodulin targets by neuronal calcium sensor NCS-1.Proc Natl Acad Sci USA 1996;93:9253–9258.

Scharfman HE. Electrophysiological diversity of pyramidal-shaped neu-rons at the granule cell layer/hilus border of the rat dentate gyrusrecorded in vitro. Hippocampus 1995;5:287–305.

Schwaller B, Buchwald P, Blumcke I, Celio MR, Hunziker W. Character-ization of a polyclonal antiserum against the purified human recombi-


nant calcium binding protein calretinin. Cell Calcium 1993;14:639–648.

Sik A, Penttonen M, Ylinen A, Buzsáki G. Hippocampal CA1 interneu-rons: An in vivo intracellular labeling study. J Neurosci 1995;15:6651–6665.

Sik A, Penttonen M, Buzsáki G. Interneurons in the dentate gyrus: An invivo intracellular labeling study. Eur J Neurosci 1997;9:573–588.

Sloviter RS, Nilaver G. Immunocytochemical localization of GABA-,cholecystokinin-, vasoactive intestinal polypeptide-, and somatostatin-like immunoreactivity in the area dentata and hippocampus of the rat.J Comp Neurol 1987;256:42–60.

Soltész I, Deschenes M. Low- and high-frequency membrane potentialoscillations during theta activity in CA1 and CA3 pyramidal neuronsof the rat hippocampus under ketamine-xylazine anesthesia. J Neuro-physiol 1993;70:97–116.

Somogyi P, Nunzi MG, Gorio A, Smith AD. A new type of specificinterneuron in the monkey hippocampus forming synapses exclu-sively with the axon initial segments of pyramidal cells. Brain Res1983;259:137–142.

Somogyi P, Freund TF, Hodgson AJ, Somogyi J, Beroukas D, Chubb IW.Identified axo-axonic cells are immunoreactive for GABA in thehippocampus and visual cortex of the cat. Brain Res 1985;332:143–149.

Soriano E, Frotscher M. A GABAergic axo-axonic cell in the fasciadentata controls the main excitatory hippocampal pathway. Brain Res1989;503:170–174.

Soriano E, Nitsch R, Frotscher M. Axo-axonic chandelier cells in the ratfascia dentata: Golgi-electron microscopy and immunocytochemicalstudies. J Comp Neurol 1990;293:1–25.

Terasawa M, Nakano A, Kobayashi R, Hidaka H. Neurocalcin: A novelcalcium-binding protein from bovine brain. J Biol Chem 1992;267:19596–19599.

Tóth K, Borhegyi Z, Freund TF. Postsynaptic targets of GABAergichippocampal neurons in the medial septum-diagonal band of brocacomplex. J Neurosci 1993;13:3712–3724.

Valtschanoff JG, Weinberg RJ, Kharazia VN, Nakane M, Schmidt H.Neurons in rat hippocampus that synthesize nitric oxide. J CompNeurol 1993;331:111–121.

Vincent SR, Kimura H. Histochemical mapping of nitric oxide synthasein the rat brain. Neuroscience 1992;46:755–784.

Whittington MA, Traub RD, Jefferys JG. Synchronized oscillation ininterneuron networks driven by metabotropic glutamate receptoractivation. Nature 1995;373:612–615.

Zozulya S, Stryer L. Calcium-myristoyl protein switch. Proc Natl AcadSci USA 1992;89:1569–11573.


INTRODUCTIONMATERIALS AND METHODSTABLE 1.TABLE 2.TABLE 3.TABLE 4.TABLE 5.TABLE 6.

RESULTSFIGURE 1.FIGURE 2.FIGURE 3.FIGURE 4.FIGURE 5.FIGURE 6.FIGURE 7.FIGURE 8.FIGURE 9.FIGURE 10.FIGURE 11.

DISCUSSIONREFERENCES

neurocalcin-immunoreactive cells in the rat hippocampus ... · neurocalcin-immunoreactive cells in...

Documents