DISTRIBUTION OF CALRETININ-CONTAINING NEURONSRELATIVE TO OTHER NEUROCHEMICALLY IDENTIFIED

CELL TYPES IN THE MEDIAL SEPTUM OF THE RAT

J. KISS,*¶ Z. MAGLOuCZKY,† J. SOMOGYI‡ and T. F. FREUND†*Neuroendocrine Research Laboratory, Department of Human Morphology, Semmelweis University

of Medicine, Budapest, Hungary

†Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary

‡Neurobiology Research Laboratory, Department of Anatomy, Semmelweis University of Medicine,Budapest, Hungary

Abstract––The topographic distribution of calretinin-immunoreactive neurons was studied in the medialseptum–diagonal band of Broca complex of the rat, in relation to the localization of other neurochemi-cally identified cell groups containing choline acetyltransferase, parvalbumin or calbindin D28k. Double-labelling experiments revealed that these four antigen-containing cells formed distinct dorsoventrallyrunning lamellae overlayed on top of each other similar to onion leaves. There was only a slightoverlapping of the various cell groups. None of the four antigens were co-localized in the same cells. Thelamella occupied by calretinin-positive neurons is situated at the border of the medial septum and theintermediolateral septal nucleus, and shows some overlap with the area occupied by cholinergic neurons.Retrograde transport of horseradish peroxidase from the hippocampus combined with immunostainingfor calretinin revealed that calretinin-containing neurons do not participate in the septohippocampalprojection. The lack of projection to the amygdala was also confirmed.Thus, calretinin-containing neurons represent a distinct cell group in the medial septal region, which

either projects to subcortical areas, or may function as interneurons relaying hippocampal feedback to themedial septal projection neurons. ? 1997 IBRO. Published by Elsevier Science Ltd.

Key word: Septal calretinin-containing neurons.

As a relay station the septal region plays an import-ant role interconnecting the limbic telencephalonwith the hypothalamus and brainstem.6–8,18,39 Exten-sive reciprocal connections are present between themedial septum–diagonal band complex (MSDB) andthe hippocampus3–5,10,11,16,23 while the lateral septalareas are related to several basal diencephalic, mes-encephalic and rhombencephalic regions and also thehippocampus.14,17 A large proportion of cells in theMSDB complex are cholinergic or parvalbumin-containing GABAergic22,25–28,30,36 which give riseto the septohippocampal pathway,3–5,8,10,23,32,34,38

although a considerable number of cells containneuropeptides, including enkephalin,24 luteinizinghormone-releasing hormone,15,21 calcitonin gene-related peptide, vasoactive intestinal peptide,dynorphin-B, substance-P, cholecystokinin, somato-

statin,27 neuropeptide Y27 and neurotensin.27 Ac-tivity in the septohippocampal pathway can beeffectively modified by hypothalamic inputs, eventhough axonal connections that originate in thehypothalamus and terminate on cholinergic orGABAergic neurons localized in the MSDB complexare not exactly known. Apart from the medial septalregion the intensive neuronal diversity in the septalcomplex results in considerable differences among thevarious septal nuclei with regard to cytoarchitecture,cell size, afferent and efferent connections and neuro-chemical characteristics.2,7,20 A large proportion ofcells residing in various areas of the lateral septum isknown to contain GABA as neurotransmitter,35,36 aswell as the vitamin D-dependent calcium bindingprotein (CaBP),40 a representative member of thecalmodulin superfamily. Different populations ofthese lateral septal cells are heavily innervated byvarious extraseptal afferents, including fibres contain-ing catecholamines, enkephalin, substance P, neuro-peptide Y, vasopressin, somatostatin or excitatoryamino acids.16,17

Calretinin (CR), another member of the calmodu-lin superfamily42 was found to be present in separatesets of neurons40 in various brain regions of differentspecies, including rat.43,44 A well defined group ofinterneurons immunopositive for CR has been shown

¶To whom correspondence should be addressed.Abbreviations: ABC, avidin–biotinylated horseradish per-oxidase complex; CaBP, calbindin protein; ChAT,choline acetyltransferase; CR, calretinin; DAB, 3,3*-diaminobenzidine; HRP, horseradish peroxidase; IGSS,immunogold–silver staining; MSDB, medial septum–diagonal band complex; NGS, normal goat serum; PAP,peroxidase anti-peroxidase method; PB, phosphatebuffer; PV, parvalbumin; WGA–gold, wheatgermagglutinin–colloidal gold conjugate.

Pergamon

Neuroscience Vol. 78, No. 2, pp. 399–410, 1997Copyright ? 1997 IBRO. Published by Elsevier Science Ltd

Printed in Great Britain. All rights reserved0306–4522/97 $17.00+0.00PII: S0306-4522(96)00508-8

399

to be present among others, in the hippocampus19,33

and in addition, a prominent CR-immunoreactivefibre network was found to innervate the granule cellsof the dentate gyrus.31 In a mapping study, CR hasbeen found to be localized in a large number ofneurons also in the rat basal forebrain,19 within, oradjacent to areas known to project to the hippo-campus, the olfactory bulb and various neocorticalareas. A well defined group of cells immunopositivefor CR has been shown to extend along the lateralborder of the medial septal area,41 which is known toinclude a large number of septohippocampal cholin-ergic neurons. However, it is still unknown whetherthere is any co-localization of cholinergic markersand CR, and whether CR-containing neurons partici-pate in the septohippocampal projection. The rela-tionship to the other CaBP-containing neurons alsoremains to be established.In the present study a detailed description of the

localization and cytoarchitectonic organization ofCR-containing neurons is provided in the medialseptal region, and the data are presented in compari-son with the distribution of choline acetyltransferase(ChAT)-, and parvalbumin (PV)-immunoreactivecells. Retrograde labelling was used to determinewhether the CR-containing cells project to hippo-campal target areas. In addition, a co-localizationstudy was performed to reveal any co-localizationof CR with ChAT-, or the other calcium bindingproteins, parvalbumin and CaBP.

EXPERIMENTAL PROCEDURES

Male Sprague–Dawley rats (Crl:CD, Charles RiverHungary, 300–350 g) were used in the present study. Sixteenrats were perfused through the heart under equithesin(chlornembutal 0.3 ml/kg) anaesthesia, first with saline(0.9% NaCl, 50 ml 2–3 min) followed by 400 ml of fixativecontaining 4% paraformaldehyde, 0.1% glutaraldehyde and0.2% picric acid in 0.1 M phosphate buffer (PB, pH 7.4) for30 min and finally with 200 ml of the same fixative contain-ing no glutaraldehyde. After perfusion the brains wereremoved, the blocks of tissue containing the septum–diagonal band region were dissected and 50 µm thick coro-nal Vibratome sections were equilibrated in 15% followedby 30% sucrose diluted in PB then freeze-thawed usingliquid nitrogen to enhance penetration.

Immunocytochemical procedures

Single-label immunocytochemistry. The free-floating sec-tions were extensively washed and incubated first in 20%normal goat serum (NGS, 1 h), then treated sequentiallywith: rabbit anti-CR antiserum (1:2000, two days), bioti-nylated sheep anti-rabbit IgG F(ab)2 fragment (Boehringer–Mannheim, Germany) at a dilution of 1:200, for 4 to 6 h,then in avidin–biotinylated peroxidase complex (Elite ABC,Vector, 1:400) overnight. All incubations were carried out at4)C. The immunoperoxidase reaction was developed using0.04% 3,3*-diaminobenzidine–4HCl (DAB) and DAB con-taining 0.001% hydrogen peroxide. Tris–HCl buffer (Tris,50 nM, pH 7.4) containing 1% NGS was used for all washesand antiserum dilutions.

Double-label immunochemistry. In order to reveal CR andChAT, CR and PV or CR and CaBP antigens simul-

taneously in the same area, sections from four rats wereimmunostained using the following mixtures of antisera.Primary antisera (for two days): rabbit anti-calretinin(1:2000) plus monoclonal rat/mouse anti-ChAT (2 µg/ml,Boehringer–Mannheim, Germany), or rabbit anti-CR(1:2000) plus monoclonal mouse anti-PV (1:1000, Celio) orrabbit anti-CR plus monoclonal mouse anti-CaBP (1:1000,Celio). Second layer (for 4–6 h): biotinylated sheep anti-ratimmunoglobulin F(ab*)2 fragments (1:200, Boehringer–Mannheim) plus goat anti-rabbit IgG (1:50, Dakopatts), orbiotinylated horse anti-mouse IgG (1:200, Vector) plus goatanti-rabbit IgG (1:50, Dakopatts). As the third layer ABCElite (1:400) was used. The first immunoperoxidase reactionwas developed using DAB, intensified with ammonium–nickel sulphate, while the second reaction following rabbitperoxidase anti-peroxidase (PAP, 1:100) was developedwith DAB alone. With this double immunocytochemicallabelling, two antigens present in different cells in thesame section can be labelled simultaneously in contrastingcolours (deep blue and brown).

Double-label immunocytochemistry using immunogoldstaining procedure. In order to examine the co-existence oftwo antigens in the same cell, immunogold labelling29

was combined with CR or CaBP-immunocytochemistry on‘‘Vibratome’’ sections (50 µm thick) cut from the septal–diagonal band region of four rats. The sections were firstincubated in 5% NGS for 1 h, then in ChAT (monoclonalrat/mouse anti-ChAT, Boehringer–Mannheim, 2 µg/ml) orin PV (monoclonal mouse anti-PV, 1:2000) antiserum fortwo days. This was followed by treatment with gold-conjugated goat anti-rat IgG (GARaGo, AuroProbe EMGARaG10, Amersham, 1:5, diluted in 0.1 M Tris–HCl,pH 8.2 at 4)C overnight) for visualizing ChAT, or withgold-conjugated goat anti-mouse IgG (GAMGo, Auro-Probe EM GAMG10, Amersham, 1:5 diluted in 0.1 MTris–HCl, pH 8.2, at 4)C overnight) to reveal PV antigens.After treatment with the gold-conjugated immunoglobulins,sections were intensified with a silver intensification kit,IntenSEM. Sections showing positive reaction for ChAT orPV were processed further for CR immunocytochemistryusing DAB as chromogen (as described above), andmounted on gelatine-coated slides for light microscopicexamination.

Comparative analysis of the distribution of calretinin-,choline acetyltransferase-, parvalbumin-, and calcium bindingprotein-immunoreactive cells. The distribution of CR- andChAT-immunoreactive cells in the MSDB region was evalu-ated in sections from six rostrocaudal levels of the MSDBcomplex. The sections double-stained for ChAT (DAB–Nickel) and CR (DAB alone) were evaluated by plottingimmunopositive cells with the aid of the Neurolucida imageanalysis setup (MicroBrightField, Inc., Colchester, VT)connected to a Zeiss Axioscope microscope. The data fromthe same levels were printed separately. At level 400 µmanterior to bregma, two alternate sections, one doublestained for ChAT and CR, the other double stainedfor PV and CaBP were plotted separately then super-imposed in a way that the image was imitating a quadrupleimmunostaining.

Retrograde labelling of septohippocampal neurons com-bined with calretinin-immunocytochemistry. In a separateexperiment, horseradish peroxidase (HRP, SIGMA, 20%dissolved in saline, four rats) or wheatgerm agglutinin–colloidal gold conjugate (WGA–gold, 5 nm, SIGMA, threerats) was injected under Equitesin (chlornembutal, 0.3 ml/100 g body weight) anaesthesia into the dorsal hippocampusat the coordinates of 3.6 mm posterior to bregma, 1.9 mmlateral to the superior sagittal sinus and two to threeinjection sites between 3.4 and 2.5 mm vertically below thepial surface. Injections (0.2 µl each) were made with a

400 J. Kiss et al.

stereotactic apparatus, equipped with a glass micropipetteconnected to an air pressure system, over 20 min, and themicropipette was left in place for an additional 10 minbefore being slowly withdrawn. Two days later, the ratswere anaesthesized again and killed by transcardiacperfusion-fixation as described above. The region contain-ing the entire medial septum–diagonal band complex wasdissected and serial ‘‘Vibratome’’ sections of 50 µm werecut. The retrogradely-transported HRP or WGA–gold wasthen visualized either by using DAB intensified withammonium–nickel sulphate, giving a black granular reac-tion endproduct, or the gold particles conjugated to theretrogradely transported WGA were silver-intensified byusing the IntenSEM silver enhancement kit (Amersham)resulting in a high contrast black granular signal. The samesections were then immunostained for CR, as describedabove, using DAB as a chromogen giving the cells anhomogeneous brown staining.

RESULTS

Nomenclature of the septum–diagonal band of Brocacomplex

The terminology of the subdivisions of the septalarea used in the present study is derived largely froma description of Swanson and Cowan.46 However,the regions containing the various neurochemically-identified cell populations of this study do not exactlycoincide with any of the septal subdivisions ofSwanson. For better visual comprehension and dis-tinct characterization we use here a further parcella-tion of the medial septal area (MS), as explained inmore detail previously.22 Briefly, we have subdividedthe MS on the basis of the distribution of cholinergicand PV-containing GABAergic neurons into threemediolateral subdivisions, namely the midline part(MSm), the medial part (MSme) and the lateral part(MSl) of the medial septum. The nomenclature of thediagonal band (DB) nuclei used in this study islargely based on the description given by Zaborszkyet al.,51 and in our previous paper.22 Briefly, the DBis subdivided into dorsal and ventral parts accordingto Harkmark et al.13 These divisions are defined asthe nucleus of the vertical limb of the DB (vDB). Atmore caudal level (i.e. at the level of the decussationof the anterior commissure) the ventral division ex-tends ventrolaterally and more caudally to form thenucleus of horizontal limb of the DB (hDB) which iscontinuous caudally with cell groups in the preopticand hypothalamic areas. For delineating regions ofthe septum–diagonal band complex, the use of termi-nology outlined in the second edition of the atlas ofPaxinos and Watson37 have been maintained.

Localization and morphology of calretinin-immunoreactive neurons

In general, a large number of cells situated in themedial region of the septum and along the diagonalband complex are heavily immunostained for CR.The DAB reaction product is distributed diffuselythroughout the perikaryal cytoplasm. Proximal anddistal dendrites are also extensively stained in all

regions examined. The CR-immunoreactive cells areusually small and heterogeneous in shape and orien-tation. Elongated and bipolar-like fusiform cells,situated in the medial region of the septum, are themost common. A smaller number of cells havespheroidal or oval-shaped somata. The stained cellshave prominent nuclei which tend to fill nearly theentire cell body. Dendrites of the bipolar-like cellsrun more or less parallel in a dorsoventral direction,often for considerable distances. A lower number ofmedium-sized, multipolar cells are situated mainly atthe more lateral part of the medial septal region.Dendrites of these latter cells extend irregularly with-out showing any particular orientation, and form anirregular network. In general, the dendrites of theCR-stained cells are smooth and spine-free, exceptthe distal part of the bipolar-like fusiform cells, wherea small number of spines having long necks and smallspheroid heads, are emitted (Fig. 1).The focus of this study were septal regions impli-

cated in septohippocampal and/or hippocamposeptalconnectivity. The observations on the distribution ofCR-containing neurons were therefore restricted tothe septal region extending from 1500 µm anterior to200 µm posterior to the bregma of the adult rat (Figs2, 3). Neurons immunopositive for CR appear toform a continuous band of cells starting rostrally atthe anterior end of the septohippocampal nucleusand extending caudally as far posteriorly as theseptofimbrial nucleus. On this rostrocaudal line CR-immunopositive cells can be classified into two dis-tinct groups. One extends over the mediolateralregion of the septal complex, and the diagonal bandregion. The other population of CR-containing cellsoccupies primarily the ventrolateral areas of thelateral septum. The CR-positive neurons first ap-peared at the level of the anterior end of the septo-hippocampal nucleus (SHi), where they covered themidline and the immediate lateral areas. More ven-trally, in the medial regions of the septum corre-sponding to the rostrocaudal levels of the medialseptal nucleus (MS, where the great majority of theseptohippocampal cholinergic and PV-containingGABAergic neurons are located), a dense populationof small- and medium-sized CR-immunoreactive cellsclose to the MS form an inverted V-shape for muchof its rostrocaudal extent, like the population of thecholinergic neurons.Cells immunoreactive for CR, localized ventrally

to the medial septal area, were more scattered, andtheir number was lower, especially at more rostrallevels. In these areas, including the vDB structures,no characteristic arrangement of the cells could berecognized. The cells found here were medium-sized,mainly elongated or triangular-shaped, and arrangedin a single row lying parallel to the ventral surface ofthe brain. More caudally the number of CR-immunopositive cells was higher, in comparison withthat at rostral levels. Similar to cholinergic neurons,they merged dorsally with the mediolateral areas,

Organization of the rat medial septal region 401

whereas ventrally and laterally they fused with re-gions corresponding to the curvature of the ventralborder of the vDB. More posteriorly they extendedto the area including the hDB (Figs 2, 3). At thislevel, the CR-immunopositive cells were arranged ina circular pattern around a central immunocyto-chemically negative area (Figs 2c, 3f ), which pre-viously was observed to contain PV-positiveGABAergic neurons.

Distribution of calretinin-immunopositive cells incomparison with the localization of neurons immuno-reactive for choline acetyltransferase

In separate experiments the distribution of twotypes of immunocytochemically-identified neuronsin the MSDB complex was evaluated in double-immunostained sections at six rostrocaudal levels ofthe MSDB complex. The sections first stained forChAT by the ABC procedure (DAB, intensified with

ammonium nickel sulphate, deep blue to black reac-tion product) were further processed for CR withthe PAP method using DAB alone as substrate(light yellowish-brown staining). The distribution ofChAT-immunoreactive neurons in the entire rostro-caudal extent of the MSDB complex was similar tothat described earlier.22,23 CR-containing cells on thesame sections were distributed in the MSl and in theborder zone between the MS and the intermedio-lateral septum. The majority of CR-immunopositivecells was intermingled with a large number of ChAT-immunostained neurons present in the MSl. Asmaller proportion of CR-positive cells was localizedoutside the area occupied by ChAT-stained neurons(Fig. 5a). This pattern of localization of the two typesof neurons was characteristic in more ventral vDBand hDB regions as well, where large number ofCR-immunoreactive cells were found to be inter-mingled with ChAT-stained neurons. Throughoutthe MSDB complex none of the neurons contained

Fig. 1. High-power photomicrographs demonstrating the morphology of calretinin-immunoreactiveneurons in various regions of the medial septum–diagonal band complex. a) Irregular topographicorganization of immunostained cell bodies and dendrites in the border-zone between the medial- andintermediolateral septal area. b) Bipolar-shaped immunostained neurons in more lateral area of the borderzone. Dendrites of these cells run more or less parallel in a dorsoventral direction. c) Medium-sizedmultipolar cell at the intermediate septal area. Dendrites of this cell extend irregularly. d) Immunopositivedendritic portion (arrow) in the medial area of the septum. Spines are emitted in various number from the

dendritic surface. Scale bar=20 µm.

402 J. Kiss et al.

both reaction end products, suggesting that ChAT-and CR-immunoreactive neurons represent two dis-tinct populations of nerve cells (Fig. 5a). This wasalso confirmed in further experiments based onstudying co-localization of ChAT and CR in thesame cells using two clearly distinguishable reactionend products (for detail see below).These experimental procedures permitted the

analysis and comparison of CR- and ChAT-immunostained neurons on the same coronal sectionsat six rostrocaudal levels, using the Neurolucidaimage analysis system connected to a Zeiss Axioscopemicroscope. The sections were evaluated by plottingeach cell of the two distinct populations and the datafrom the two sets of cells were printed separately.As shown in Fig. 5a and d, CR- and ChAT-

immunopositive neurons form distinct neuronalpopulations which can be recognized throughout allrostrocaudal levels examined. These data clearlyshow that CR-containing cells are not confined to alateral area just outside the medial septum, buta considerable proportion of them are distributedin areas occupied by ChAT-positive neurons.More ventrally in the vDB, most of the CR-immunoreactive cells are also intermingled withChAT-positive neurons. In the region of the hDB,CR-positive cells were arranged in a circular pattern,partly intermingled with ChAT-immunoreactiveneurons around a central area (Fig. 4e, f ) that wasnegative for both CR and ChAT.In two alternate series of double-stained sections

(CR plus ChAT and PV plus CaBP; for details see

Fig. 2. Light micrographs of the MSDB complex at different rostrocaudal levels. Calretinin-containingcells are stained by single immunoperoxidase–diaminobenzidine (ABC–DAB) reaction, in order to processa comprehensive mapping study of CR-positive neurons. a) A rostral level of the septal complexcontaining the LSi and the vDB. A dense accumulation of CR-positive cells and dendritic network islocalized in the dorsal area of the vDB. The immunopositive cells in more ventral area of the vDB and inthe mediodorsal parts of the LSi are scattered (arrows). b) A mid-level area of the MSDB complexcontaining the MS, the vDB, the LSi and the ventral region of the LSv. A continuous band ofCR-immunopositive neurons and dendrites is extending dorsoventrally in the border zone between the MSand the medial part of the LSi. At the curvature of the vDB, CR-positive neurons are arranged in a rowlying parallel to the brain surface (open arrow). The LSv contains CR-positive cells immediate to the wallof the lateral ventricle and in a row bordering the dorsomedial part of the AcSh. c) A caudal area of theMSDB complex. CR-immunopositive neurons (arrows) in the area of the hDB are distributed around a

central immunocytochemically negative area. Scale bar=0.25 mm.

Organization of the rat medial septal region 403

Experimental Procedures) the superimposed data re-vealed that the four types of immunocytochemicallyidentified cell populations (CR, ChAT, PV andCaBP) are arranged around an axis of symmetry inthe MS (Fig. 4d). They are largely separated fromeach other showing a distinct laminated chemo-specific organization in the medial region of theseptum. At the border zones of these lamellaeconsiderable mixing of different cell types is visible.

Analysis of coexistence

To study the co-existence of CR and ChAT, CRand PV or CR and CaBP in the same cells, CR-immunocytochemistry was combined in the samesections with immunogold silver staining (IGSS) for

labelling ChAT, PV or CaBP. In preliminary exper-iments first the specificity of the IGSS procedureusing single silver–gold staining for ChAT or PV wastested. On sections which were processed for singleChAT labelling, a large number of neurons in theMSDB contained black granular deposit of thesilver–gold reaction end product accumulated in cellbodies and dendrites. The same labelling was ob-served when sections were stained for PV. The distri-bution of ChAT- or PV-positive neurons labelled bysilver–gold was similar to that found earlier usingother immunocytochemical procedures.22,23 In thedouble-staining experiments, sections previouslysilver-labelled for ChAT or PV by the IGSSprocedure were processed further for CR-immunostaining by the ABC–DAB technique. In

Fig. 3 (a–d). Cross-sectional maps of calretinin-immunoreactive neurons in the septal–diagonal bandcomplex at the indicated levels anterior to the bregma. The drawings of the sections in the coronal planeare arranged rostrocaudally (a–h), and they are modified from the stereotaxic atlas of Paxinos andWatson.38 Immunopositive neurons are localized in various areas of the entire septal–diagonal bandcomplex, including the regions of the Ld zone, the border zone between the MS and LSi, the vDB, thehDB and the LSv. In the rostrocaudal extent of this brain region the CR-positive neurons are distributed

in a characteristic pattern.

404 J. Kiss et al.

these sections ChAT- or PV-positive neurons con-tained the black granular silver–gold deposit,whereas the CR-immunoreactive cells were stainedyellowish-brown by DAB reaction (Fig. 5d, e).Throughout the MSDB region none of the neuronscontained both reaction end products, suggestingthat CR-positive cells represent a distinct population,containing neither ChAT nor PV (Fig. 5d, e). In thecase of CR and CaBP double-labelling the oppo-site sequence was followed, in which CR-containingcells were labelled by IGSS, followed by CaBP-immunocytochemistry with DAB staining (Fig. 5f ).The lack of co-existence of these two proteins hasbeen confirmed throughout the entire extent of theseptum and the DB complex.

Investigation of projections of calretinin-immunoreactive cells located in the medial septumand the diagonal band

HRP injections were made into the dorsal hippo-campus to study the possible participation of the

CR-containing cells, situated in the medial region ofthe septum and in the DB complex, in the septo-hippocampal projection. In the HRP-injected rats nospread of the tracer outside the hippocampal forma-tion was observed in any of the experimental animals.Much of the cross-sectional area of the dorsal hippo-campus was labelled with a dense deposition of thetracer in all layers from the stratum pyramidale up tothe hilar region of the dentate gyrus. On CR-immunostained serial sections from all levels of therostrocaudal extent of the septum and the diagonalband complex, a large number of retrogradely-labelled neurons could be observed. The vast ma-jority of the HRP-labelled cells was on the sideipsilateral to the injection, however, a few cells werealso observed in the contralateral side. Theretrogradely-labelled cells were localized mainlyalong the midline, the medial part of the MS (Fig. 5b)and in the vertical and horizontal limbs of the DB. Ingeneral, the distribution of the retrogradely-labelledneurons showed a pattern similar to that described

Fig. 3 (e–h).

Organization of the rat medial septal region 405

earlier.23 However, in the present experiments noneof the retrogradely-labelled cells were found to beimmunopositive for CR (Fig. 5b, c). A considerablenumber of CR-immunostained cells in the lateralarea of the MS were intermingled with theretrogradely-labelled neurons localized in this region.In a complementary experiment we also tested thepossibility of septal CR-positive projections to theamygdala complex. No transported HRP reaction

product was found to be present in CR-immunopositive cells in these cases either.

DISCUSSION

In the present study we have provided precise andcomprehensive data on the topographic localizationof the CR-containing neurons located in the septal–diagonal band region of the rat brain, in comparison

Fig. 4. Demonstration of the Neurolucida image analysis on sections from six rostrocaudal levels of theMSDB complex, double-stained for ChAT (DAB–Nickel) and CR (DAB). The distribution of these twotypes of cells were evaluated by plotting immunopositive cells with the aid of the Neurolucida imageanalysis setup. Black dots indicate ChAT-immunopositive, red dots mean CR-immunostained neurons.Each dot represents one single cell. d) shows the organization of immunocytochemically characterizedfour different populations of neurons in the medial septal region at the rostrocaudal level 400 µm anteriorto bregma. ChAT (black dots)-, CR (red dots)-, PV (blue dots)- and CaBP (green dots)-immunoreactiveneurons were plotted separately from two alternate sections, double-stained for ChAT and CR, or for PV

and CaBP, and then superimposed by the Autocad program.

406 J. Kiss et al.

Fig. 5. Colour light micrographs of the MSDB complex. a) Immunostained neurons by double-labelimmunocytochemistry. On the same section, the ChAT-containing neurons are stained by DAB,intensified with ammonium nickel sulphate (dark bluish colour) (arrow), while the CR-positive cells arelabelled with DAB alone (light brown staining) (open arrow). Large proportion of the brown stainedCR-immunopositive cells is intermingled with bluish coloured ChAT-immunoreactive neurons. Only aminor proportion of CR-positive cells is resided in more lateral area, bordering the medial septum. (b andc) show HRP-labelled cells after tracer injection into the dorsal hippocampus, on sections immunostainedfor CR. Thin arrow in (c) points to CR-immunonegative cell that is retrogradely-labelled by HRP, thickarrow indicates CR-immunopositive neuron without HRP-labelling. (d, e and f ) ChAT- and CR-(d), PV-and CR-(e), CaBP- and CR(f )-containing neurons on the same sections are double-stained by thecombination of IGSS and immunocytochemical methods. ChAT- and PV-containing cells (in d and e) arelabelled with the dark silver–gold deposit (bluish-black granules) (thin arrows), while CR-positive cells areimmunostained with DAB (brown colour) (thick arrows). In (f ) CR-containing cell is labelled by granulardeposit of the IGSS (thick arrow), the CaBP-positive cell (open arrow) is immunostained with DAB (thickarrow). No mixing of the two markers is occurring in the same neurons. Scale bars: a=100 µm; b=200 µm;

c–f=20 µm.

Organization of the rat medial septal region 407

with neurons specifically immunolabelled for ChAT,PV or CaBP. We have shown that the CR-positivecells do not colocalize ChAT or PV, and do notparticipate in the septohippocampal projection.

Lamellar segregation of neurochemically different celltypes in the medial septum

On the basis of classical histological preparationsthe septum was considered as a non-laminatedstructure.39,45 Immunocytochemically-defined neu-rons, however, have a characteristic topographiclocalization. The best known septohippocampalneuronal populations occupy the middle area(PV-containing GABAergic neurons) and a morelateral part (cholinergic cells) of the septalcomplex.3,10,22,23,49 In the relatively separate dis-tribution of the PV-containing GABAergic9 andChAT-immunopositive cholinergic cell population,a distinct mediolateral compartmentalization hasbeen recognized.22 The distribution of CR-immunopositive cells has been recently studied inthe rat brain.19,41 The lateral mamillary andseptofimbrial nuclei, the hippocampus, variousregions of the thalamus were found to containneurons immunoreactive for CR.19 In the septum,neurons in the triangular and septofimbrial nucleiwere described which demonstrated strong immuno-reactivity for CR.41 CR-containing neurons havebeen also observed along the lateral border of themedial septal nucleus,41 without accurate identifica-tion of their exact localization. The aim of thepresent investigations was, besides the topographiclocalization of CR-positive neurons, to further char-acterize the complexity of the cytoarchitectonicsreflecting the laminated chemospecific organizationof this area of the septum. Therefore, the localiza-tion of the CR-positive cells, located between thelateral border of the medial septum and the medialarea of the intermediate lateral septal nucleus wasevaluated in comparison with the populationsof neurochemically-identified neurons, using acomputer-assisted data analysis system on paireddouble-stained sections. Our observations indicatethat all the four chemically-identified cell types(CR-, ChAT-, PV- and calbindin-immunopositive)form distinct populations (Fig. 4d). These popula-tions are arranged symmetrically, bilateral to themid-sagittal plane of the septum as the ‘‘leaves ofan onion’’ proposed earlier on the basis of devel-opmental and morphological data.20,22,23 Eventhough, double-labelled and computer-analysed re-sults showed that CR-stained and cholinergic cellswere often found to overlap with each other to aconsiderable extent, the majority of these types ofneurons were arranged largely separate from andparallel to each other.The possibility of co-localization of CR with

ChAT, or one or more of the other calcium-bindingproteins has been raised in the septum–diagonal band

complex because of the close topographic localiza-tion of cells immunopositive for CR, ChAT, PV orCaBP. The present co-localization study combiningimmunogold–silver staining and ABC–DAB immu-nochemistry provided direct evidence that CR-containing neurons in the medial septal–diagonalband region are not cholinergic, and that CR is notpresent in PV- or calbindin-containing cells. Thus, allfour chemically-identified cell types represent distinctgroups of neurons.

Possible role of medioseptal calretinin cells in septo-hippocampal projections

Anterograde and retrograde tracing studies re-vealed that the neuronal projections from the sep-tum to the hippocampal formation originate fromcholinergic and GABAergic neurons located exclu-sively in the medial division of the septum and thediagonal band nuclei.3–5,10,22,25,28,46 In earlierstudies Meibach and Siegel also identified cells,which were located outside the MSDB, in the re-gion of the intermediolateral septum, which termi-nated in the hippocampus and the adjacentsubicular complex.32 The lateral septum, includingalso the intermediolateral subdivision contains alarge number of GABAergic neurons as demon-strated in the rat.25,34,35,36 A large proportion ofthese GABAergic cells contains calbindin, but aconsiderable number of the lateral septal GABA-ergic cells do not contain either calbindin or PV.At present, no information is available about theGABA-content of CR-immunoreactive cells local-ized in the region investigated in the present study.The appearance and distribution of retrogradely-

labelled neurons observed in the present study corre-late well with the general location of perikaryaprojecting to the hippocampal formation from themedial septum–diagonal band complex described inprevious studies.3,4,8,23,45,47 The sections double-labelled for calretinin and the retrogradely-transported HRP injected into the hippocampalformation or amygdala demonstrated that the HRPreaction end product appeared exclusively in cellsimmunonegative for CR. Thus, it is reasonable toconclude that CR-containing cells in the MSDBregion do not participate in the septohippocampalprojections. There are, however, indications that sep-tal cells residing in the intermediolateral septumproject to the entorhinal cortex, and appear to ter-minate in layers II and IV,1 and on the other hand,these septal regions, including the lateral septum andthe diagonal band nuclei send fibres to the medialforebrain bundle and project to the supramammillaryarea.12,14,46,48 These potential projection areas re-main to be investigated. An alternative role forCR-positive cells may be to relay hippocampalfeedback to the medial septum. In this respecttheir hippocampal input and local axon collateraldistribution has to be studied.

408 J. Kiss et al.

CONCLUSION

Based on the present comparative mapping resultsit can be concluded that the medial division of theseptum is built up from chemically-different neuronalpopulations forming dorsoventrally-oriented lamel-lae. In this architecture CR cells are localized alongthe border between the medial and the intermedi-olateral septal divisions and are intermingled to someextent with cholinergic neurons. CR does not co-localize with ChAT, PV or CaBP, and CR-containingcells do not project to the hippocampus or amygdala.

Acknowledgements—We are grateful to Dr J. H. Rogers(University of Cambridge, U.K.) for a gift of antiseraagainst calretinin; to D. R. M. Celio (University ofFribourg, Suisse) for anti-parvalbumin and anti-calbindinmonoclonal antibodies and to Dr K. G. Baimbridge(University of British Columbia, Vancouver, Canada) forthe antiserum against parvalbumin. The technical assistanceof Zs. Uu jvari, E. Horvath and E. Borok is also greatlyacknowledged. This work was supported by OTKA (GrantNos T-6372 and T-16977 to J.K., T-16942 to T.F.F.,Human Frontier Science Program, and the Howard HughesMedical Institute T.F.F.).

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(Accepted 17 September 1996)

410 J. Kiss et al.