GABAergic neurons in the rat dentate gyrus are innervated by subcortical calretinin-containing afferents

THE JOURNAL OF COMPARATIVE NEUROLOGY 364:425-438 (1996)

GABAergic Neurons in the Rat Dentate Gyrus Are Innervated by Subcortical

Calretinin-Containing Afferents

ROBERT NITSCH AND CSABA LERANTH Institute of Anatomy, Humboldt University Clinic (Charite), D- 10098 Berlin, Federal Republic of Germany (R.N.); Department of Obstetrics and Gynecology and Section of

Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06520-8063 (C.L.)

ABSTRACT Fibers of supramammillary origin establish putatively excitatory asymmetric synaptic

connections with dentate granule cells. The present study was designed to determine whether hippocampal y-aminobutyric acid (GABAI-ergic nonprincipal cells are also targets of these calretinin (CR)-containing subcortical afferents. Light and electron microscopic double immunostaining for CR and parvalbumin (PA) or calbindin (CB) were performed in the rat dentate gyrus ipsilateral and contralateral to a unilateral fimbria-fornix transection. GABA- postembedding immunostaining was performed on ultrathin sections of this double-labeled material. Contralateral to the transection, CR-immunoreactive fibers formed multiple large boutons in the inner molecular layer. These fibers also impinged on PA-containing basket cells located adjacent to the granular layer and on CB-immunoreactive hilar neurons. Ipsilateral to the transection, CR-containing fibers in the inner molecular layer and boutons impinging on PA-containing or CB-immunoreactive neurons were absent. Parent cell bodies of extrinsic CR-containing afferents were traced using wheat germ agglutinin-conjugated horseradish peroxidase. Additional CR immunostaining of the subcortical region unveiled retrogradely labeled neurons that were also immunostained for CR only in the supramammillary area and the nucleus reuniens. The latter projection, however, terminates in CA1 and not in the dentate gyrus. Subcortical afferents impinging on dentate nonprincipal cells formed exclusively asymmetric synapses. Postembedding immunostaining demonstrated that CB-containing cells contain GABA, whereas CR-positive axon terminals forming asymmetric synapses are devoid of this labeling. These data indicate that dentate inhibitory neurons receive a putative excitatory input originating from the supramammillary nucleus. Thus, the supramamillo-hippocampal pathway may exert a powerful feed-forward inhibitory control of the signal flow in the rat dentate gyrus. 1996 Wiley-Liss, Inc.

Indexing terms: supramammillary nucleus, calcium-binding protein, feed-forward inhibition, calbindin, parvalbumin

Recent studies have suggested that calretinin (CR)- immunoreactive axon terminals in the inner molecular layer of the rat dentate gyrus originate in the supramammillary nucleus (SMN; Gulyas et al., 1992; Magloczky et al., 1994). These authors have shown that these fibers establish exclusively asymmetric synapses with cell bodies and proximal dendrites of dentate granule cells, which might indicate an excitatory nature of this hypothalamo-hippocampal pathway. In fact, in the monkey, colocalization studies have provided evidence that all of the large supramammillary projection neurons contain both CR and substance P (SP) but lack y-aminobutyric acid (GABA) as an inhibitory transmitter (Nitsch and Leranth, 1993; Nitsch and Ler- anth, 1994). Furthermore, it has been shown that SP-

containing projection systems exert an excitatory action (Nicoll et al., 1980). Conversely, in the rat, the functional properties of the hypothalamo-hippocampal afferent system have been reported to be at least partially inhibitory (Segal, 1979). Hence, the question arises as to how the physiological action on hippotampal principal neurons of a putatively excitatory pathway becomes inhibitory, which was shown by Segal (1979). A possible explanation for this is that hypothalamic afferents heavily innervate hippocampal short axon

Accepted July 10, 1995 Address reprint requests to Robert Nitsch, M.D., Institute of Anatomy,

Humboldt University Clinic (Charitel, 10098 Berlin, Federal Republic of Germany.

,$, 1996 WILEY-LISS. INC.

426 R. NITSCH AND C. LERANTH

neurons, i.e., local circuit GABAergic cells, which, although they represent only a small population of hippocampal neurons, are known to control the discharge of a large number of hippocampal principal neurons (Lubbers and Frotscher, 1987; Halasy and Somogyi, 1993; Han et al., 1993). Innervation of GABAergic inhibitory neurons by supramammillary afferents has recently been demonstrated in the monkey hippocampus. The peculiar innervation pattern of this afferent system provides a morphological basis for the capacity of supramammillary fibers to filter the signal flow in the monkey hippocampal formation (Leranth and Nitsch, 1994).

The aim of the present study is 1) to determine whether nonprincipal neurons are targets of subcortical afferents to the dentate gyrus by applying double-immunostaining techniques for CR and parvalbumin (PA) or calbindin (CB) ipsilateral and contralateral to a fimbria-fornix transection; 2) to reveal the cells of origin of the CR-containing subcortical afferents using combined wheat germ agglutinin- conjugated horseradish peroxidase (WGA-HRP) tracing and CR immunostaining; and 3) to demonstrate the type of synaptic contacts established by these afferent fibers on dentate nongranule cells. If these PA- or CB-containing cells are synaptic targets of extrinsic CR-containing fibers, then the question arises whether these calcium-binding, protein-containing neurons are in fact GABAergic inhibitory cells. Because all of the hippocampal PA-containing neurons are known to be GABAergic (Kosaka et al., 1987; Katsumaru et al., 1988; Nitsch et al., 19901, we only performed postembedding immunostaining for GABA on the CB-positive population, which is contacted by hypothalamic fibers.

MATERIALS AND METHODS Animals and surgery

Thirty adult Sprague-Dawley (250-280 g) rats of both sexes were used in this study. Animals were kept under standard laboratory conditions. Eighteen deeply anesthe- tized rats (Nembutal; 50 mgikg b.w., i.p.) were fixed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA), and, from a dorsal penetration, after sucking off overlying cortical areas and corpus callosum, the right fimbria fornix was transected under visual control. Ten days later, animals were killed under deep Nembutal anesthesia by transcardial perfusion of 50 ml heparinized saline followed by a fixative containing 4% paraformaldehyde, 0.1% glutaraldehyde, and 15% saturated picric acid in 0.1 M phosphate buffer (PB) at pH 7.35. Two rats were fixed with another fixative containing 4% paraformaldehyde and 0.9% glutaraldehyde in PB for triple immunostaining with CR, CB, and GABA antibodies. The hippocampi ipsilateral and contralateral to the fimbria-fornix transection were dissected out, divided into three pieces along their septo- temporal axis, and postfixed for 2 hours in the same, but glutaraldehyde-free, fixative. Under deep Nembutal anesthesia, ten animals stereotactically received multiple unilateral injections of 0.1 pl 2.5% WGA-HRP into the hippocampus (+2.9 mm to +6.2 mm from interaural line; +4.8 mm to +2.0 mm from midline; +8 mm to +3.5 mm from skull surface; Paxinos and Watson, 1986; see Fig. 2a). Seventy-two hours following WGA-HRP injection, the animals were transcar- &ally perfused with 50 ml of heparinized saline followed by a fixative containing 1% paraformaldehyde and 1% glutaraldehyde in PB. The brains were rapidly removed from the skull

and postfixed in 1% paraformaldehyde in PB for 2 hours at 4°C. After postfixation, the entire brains of the WGA-HRP- injected animals were frontally cut on a Vibratome. These 60-80 pm thick sections were immersed in a solution containing 15 mg diaminobenzidine (DAB), 0.12 mgglucose oxidase, 12 mg ammonium chloride, and 60 mg P-D-glucose in 30 ml PB for 30 minutes. Subsequent immunostaining for CR was performed as described in detail below, except a plain DAB reaction was used to visualize the tissue-bound peroxidase.

Tissue preparation Tissue blocks of fimbria fornix-lesioned animals were

rinsed in several changes of ice-cold PB, and 60 pm Vibratome (Lancer) sections were prepared perpendicular to the longitudinal axis of the hippocampus. In order to enhance the penetration of antisera, sections for electron microscopy were transferred into vials containing 0.5 ml 10% sucrose (in PB) and were rapidly frozen by immersing the vial in liquid nitrogen, then thawed to room temperature, and washed in PB. Subsequently, sections for both light and electron microscopy were treated with 1% sodium borohydride in PB for 10-30 minutes to eliminate unbound aldehydes from the tissue (Kosaka and Hama, 1986).

Double immunostaining for CR and CB or PA Both light and electron microscopic double immunostain-

ing for CR and CB or PA was carried out according to the same protocol. However, sections for light microscopy were double immunostained by antisera containing 0.3% Triton X-100. Sections were incubated for 48 hours at 4°C in a polyclonal rabbit anti-CR (Rogers, 1989) and were then diluted 1:20,000 in PB containing 1% normal goat serum and 0.1% NaN3. After three 15 minute washes in PB, sections were further processed according to the ABC technique (Hsu et al., 1981) using the Vectastain Elite Kit (Vector Laboratories, Burlingame, CA): incubation for 2 hours at room temperature in biotinylated goat anti-rabbit immunoglobin (IgG) diluted to 1:250 in PB, three 15 minute rinses in PB, and incubation for 2 hours at room temperature in ABC-Elite reagent diluted 1:250 in PB. After several rinses in PB, imrnunoreactivity for CR was visualized by our modification (see, e.g., Horvath et al., 1992) of the dark blue-to-black Nickel(Ni)-DAB reaction: 15 mg DAB, 0.12 mg glucose oxidase, 12 mg ammonium chloride, 600 pl 0.05 M nickel ammonium sulfate, and 600 pl 10% P-D-glucose in 30 ml PB for 7-10 minutes. After several rinses in PB, sections were further immunostained for CB or PA. In this procedure, after a 48 hour incubation at 4°C in a monoclonal mouse anti-CB (Sigma, St. Louis, MO) or a monoclonal mouse anti-PA (Sigma), both diluted 1:5,000 in PB containing 1% normal goat serum and 0.1% NaN3, sections were rinsed in PB and were further incubated at room temperature in goat anti-mouse IgG diluted 1:50 in PB (Organon Technica, Durham, NC) for 2 hours, rinsed three times for 15 minutes in PB, and incubated for 2 hours at room temperature in mouse peroxidase- antiperoxidase (PAP) (Organon Technica Durham, NC) diluted 1:150 in PB. After several rinses in PB, the second tissue antigen was visualized by the brown DAB reaction (14.5 mg DAB in 25 ml PB containing 165 ~ 1 0 . 3 % H20,; 10 minutes). After several rinses in PB, sections for light microscopy were placed on gelatin-coated slides, dehydrated through increasing ethanol concentrations, and mounted with Permount.

SUBCORTICAL INNERVATION OF HIPPOCAMPAL NONPRINCIPAL CELLS 427

After the second immunostaining, sections for electron microscopic analysis were examined under the light microscope, and drawings and color pictures were obtained of putative synaptic contacts between the black CR fibers and the brown CB- or PA-containing neurons. Drawings were used to enable a reidentification of structures under the electron microscope. Subsequently, sections were postosmi- cated (1% Os04 in PB) for 30 minutes, rinsed in PB, dehydrated in increasing ethanol concentrations (using 1% uranyl acetate in 70% ethanol; 30 minutes), flat embedded in araldite between liquid release-coated (Electron Micro- scopic Sciences, Fort Washington, PA) slides and coverslips, and cured in an oven for 48 hours at 60°C. Vibratome sections of high glutaraldehyde-fixed (0.9%) material stained for CR and CB were flat embedded in Durcupan (Fluka, Switzerland). After this, the previously selected putative synaptic contacts were again photodocumented under the light microscope. Flat-embedded sections were then fixed on cylindrical araldite (or Durcupan) blocks and trimmed, and ribbons of serial ultrathin sections (Reichert-Jung Ultramicrotome) were collected on Formvar-coated, single- slot grids and were examined using a Philips CM-10 electron microscope.

Triple immunostaining for CR, CB, and GABA In order to determine whether CB-containing neurons

innervated by CR fibers are, in fact, GABAergic, ten CB-immunostained cells were postembedding immunostained for GABA. Rats fixed with high glutaraldehyde (0.9%) were used in this experiment. First, Vibratome sections were double immunostained for CR (black Ni-DAB reaction) and CB (brown DAB precipitate), and the putative synaptic contacts were documented as described above. Sections were then flat embedded in Durcupan and reembed- ded, and serial ultrathin sections were collected on Formvar- coated, single-slot nickel or gold grids (two to three sections on each). Every second grid was postembedding immunostained for GABA using a modified version of the technique of Somogyi and Hodgson (1985). All of the steps were carried out on Millipore-filtered solutions in humid cham- bers: 1) 1% periodic acid for 10 minutes; 2) a rinse in double-distilled water (DDW); 3) 2% sodium metaperiodate in DDW for 10 minutes; 4) a rinse in DDW; 5 ) three 2 minute rinses in Tris-buffered saline (TBS), pH 7.4; 6) 1% ovalbumin (in TBS) for 30 minutes; 7 ) 1% normal goat serum iNGS) in TBS three times for 10 minutes; 8) the first incubation for 1-2 hours in rabbit anti-GABA iHodgson et al., 1985; code no. 9) diluted 1:1,000 in NGSITBS; 9) two 10 minute washes in TBS and one 10 minute rinse in 0.05 M Tris buffer, pH 7.5, containing 1% bovine serum albumin (BSA) and 0.5% Tween 20; 10) a 2 hour incubation in gold-conjugated (15 nm) goat anti-rabbit IgG diluted 1 : l O in the same buffer; 11) two 5 minute washes in DDW; and 12) contrasting with saturated uranyl acetate (30 minutes) and lead citrate (20-30 seconds).

Controls Nonspecific cross reactivity between the individual immu-

noreagents was controlled by processing sections in the full immunohistochemical sequence with one or both primary antisera replaced with preimmune serum. The controls necessary for electron microscopic double immunostaining were performed as previously described (Leranth and Pickel, 1989). No unspecific immunostaining could be observed.

RESULTS Similar to the results described in the rat and the monkey

(Gulyas et al., 1992; Nitsch and Leranth, 19931, comparative light microscopic analysis of the hippocampi ipsilateral and contralateral to the fimbria-fornix transection demonstrated the existence of both an intrinsic and an extrinsic CR-containing system. The former remains unaffected by an ipsilateral fimbria-fornix transection and consists of CR-immunoreactive neurons and terminals located in various subfields of the hippocampus. These terminals formed symmetric synaptic contacts with hippocampal targets, as shown by Gulyas et al. (1992). Immunostained cell bodies, however, were concentrated in the hilus and the pyramidal cell layer (Fig. 3c). The latter system arrives at the hippocampus via the fimbria fornix (Gulyas et al., 1992; Nitsch and Leranth, 1993). Thus, in the hippocampus contralateral to the transection, a dense plexus of CR-containing fibers in the innermost portion of the dentate molecular layer gave rise to numerous large boutons establishing exclusively asymmetric synaptic contacts, mostly with the initial dendrites (Fig. la-c) and spines (Fig. ld,e) of cells exhibiting the typical localization and ultrastructural characteristics of granule cells. It appears that the vast majority of granule cells are postsynaptic targets of this fiber plexus (see also Gulyas et al., 1992). The postsynaptic membrane specialization of these contacts was characteristically dense and thick (Fig. 11, as described by Gulyas et al. (1992). Conversely, the dense CR-containing plexus was almost completely absent on the side ipsilateral to the lesion, and large boutons establishing exclusively asymmetric synaptic contacts with granule cells could not be found.

Tracing studies Multiple injections of WGA-HRP resulted in an intense

labeling of almost the entire hippocampus, including the dentate gyrus and Ammon’s horn (Fig. 2a). Frontal sections through the entire subcortical region revealed neurons double labeled for WGA-HRP and for CR in only the supramammillary nucleus (Fig. 2b) and the nucleus reuniens near the midline (Fig. 2c). The vast majority of these neurons could be found ipsilateral to the injected hippocampus. The characteristic WGA-HRP grains were located in typical pyramidal-shaped neurons of the supramammillary nucleus (Fig. 2d) and in small ovoid cells in the nucleus reuniens (Fig. 2e).

Double immunostaining Light microscopy. Double immunostaining revealed CR-

immunostained axons (Ni-DABidark blue chromogen) that emerged from a plexus located in the inner molecular layer (Fig. 3a) forming multiple boutons on the proximal dendrites (Fig. 3a,b) and cell bodies (Fig. 3a) of several typical PA-immunolabeled neurons (DABIbrown chromogen). These basket cell-like neurons were located at the lower border of the granule cell layer (Fig. 3a,b) or just underneath it. Some CB-immunoreactive neurons in the hilus (DABibrown chromogen; Fig. 3d) and in CA3 (Fig. 3e,f) were closely apposed by CR-immunostained axons (Ni-DAB/ dark blue chromogen). The average diameter of CB- immunopositive hilar neurons contacted by CR-containing axons was about 20-25 pm, and they exhibited dendrites extending into the deep hilus. The majority of CR- immunoreactive boutons seemed to contact the soma of the


Fig. 1. a-e: Electron micrographs of calretinin (CRI-immunostained axon terminals in the innermost portion of the rat dentate molecular layer in the hippocampus contralateral to the fimbria-fornix transection CR-immunoreactive terminals form exclusively asymmet-

ric synaptic membrane specializations (arrowheads) with the soma (a), the dendritic shaft (b,c). and dendritic spines (arrows in c-e) of granule cells. Note the large and dense postsynaptic membrane specialization established by these contacts. Scale bars = 1 Fm.


Fig. 2. Light micrographs of frontal sections demonstrating wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) tracing combined with CR immunostaining. a: The right hippocampus (short arrows) is almost entirely stained with the diaminobenzidine ( DABIiperoxidase reaction product following multiple injections of WGA-HRP. Long arrows point to needle track. b: Numerous labeled neurons outline (arrows) the supramammillary nucleus (smnj ipsilat- era1 to injected hippocampus. c: A large number of labeled neurons outline (arrows) the nucleus reuniens (nr). The mammillothalamic tract imt), the fornix (f), and the median eminence (ME) serve as

landmarks. Note that the third ventricle is dilated following the tracer injection. d High-power magnification of large pyramidal-shaped neurons (arrows) in the supramammillary nucleus (smn) that contain both the retrogradely transported WGA-HRP grains and the diffuse DAB reaction product signifying CR immunostaining. e: High-power magnification of small ovoid neurons (arrows) in the nucleus reuniens near the midline (nr ) that contain both the retrogradely transported WGA- HRP grains and the diffuse DAB reaction product indicating CR immunostaining. Scale bars = 1 mm in a, 200 km in b,c, 20 km in d,e.

CB-immunolabeled cells, but they also formed boutons on the initial dendritic segments of the CB-immunostained neurons (Fig. 3d,f). In the hippocampus ipsilateral to the fimbria-fornix transection, we were not able to detect any

nonprincipal cell immunoreactive for either PA or CB that was contacted by CR-containing axons.

Electron microscopy. Correlated electron microscopic analysis was performed on five PA-containing neurons


Figure 3


taken from different animals. These cells were located in the hippocampus contralateral to the lesion and were found to be contacted by multiple large, CR-immunoreactive boutons under the light microscope. Basket cell-like, PA- containing neurons could be identified easily due to their location within or just underneath the granule cell layer. Moreover, they exhibited the characteristic ultrastructural features, such as large amounts of perinuclear cytoplasm and deep infoldings of their nuclear membrane (Nitsch et al., 1990). The light, diffuse DAB chromogen signifying PA immunostaining was clearly distinguishable from labeling of axon terminals, which contained the heavy, dense Ni-DAB reaction product that indicates CR immunoreactivity (Fig. 4). Analysis of a minimum of 50 proximate ultrathin sections (each approximately 50-80 nm thick) through the five selected cells revealed that, contralateral to the fimbria-fornix transection, CR-immunolabeled axons formed terminals (Fig. 4a) and en passant boutons (Fig. 4c) that established exclusively asymmetric synaptic contacts with the cell bodies and the initial dendrites (Fig. 4b) of PA-containing neurons. The postsynaptic membrane specialization of these contacts was characteristically dense and thick (Fig. 4), similar to those synapses established between CR-immunostained terminals and granule cells in the control hippocampus shown in Figure 1. No CR- immunostained axon terminals that formed asymmetric postsynaptic membrane specializations with PA-containing neurons were observed to lack these characteristics.

Correlated light and electron microscopic analysis was performed in the hippocampus contralateral to the fimbria- fornix lesion on six CB-containing neurons taken from different animals. These cells were located in the hilus and were contacted by multiple large, CR-immunoreactive axons, which are demonstrated on black-and-white micrographs (Figs. 5a,b, 6a). Light microscopic analysis allowed for the unequivocal distinction between the brown DAB (CB), and black Ni-DAB (CR) reaction products. Documentation of these sections by drawings enabled a definite verification of structures in the correlated electron micrograph. The large CB-immunolabeled hilar neurons frequently contained a nucleus with infoldings of its membrane (Fig. 54 . CR- immunolabeled boutons containing the heavy, dark Ni- DAB reaction product that were determined under the light microscope to putatively impinge on CB-immunoreactive

Fig. 3. Color light micrographs taken from double-immunostained hippocampus contralateral to thc fimbria-fornix transection. CR- containing axons are highlighted by the dark blue to black nickel (NiI-DAB labeling, whereas parvalbumin (PA; a,b) and calbindin (CB) immunostaining (c-f, is signified by brown DAB reaction product. a,b: CR-inimunostained axons emergmg from a plexus in the inner dentate molecular layer (long arrows) form multiple boutons (arrows) on PA-containing neurons located a t the lower border of the granule cell layer (g r i . c: CR-immunostained neurons are concentrated mostly in the pyramidal cell layer (CA3) and in the hilus (arrows). CB immunostaining highlights dentate granule cells and their axons, the mossy fibers terminating in CA3, as well as nonprincipal neurons outside the principal cell layers. d CB-containing neuron in the deep hilus (h) contacted by numerous CR-immunostained boutons (arrows). e: CB- immunoreactive neuron (black-and-white arrow) located in CA3 in close vicinity to two CR-immunostained cells (white arrows). This neuron is shown at higher magnification in f. f: Higher magnification of CB-immunostained neuron (black-and-white arrow; also shown in e ) demonstrates the presence of multiple CR-containing boutons impinging on this nonprincipal cell. White arrows point to corresponding CR-immunostained neurons as shown at lower magnification in e. Scale bars = 30 p n in a,b,d,f, 400 Fm in c,e.

cells could be identified in the electron microscope. The light, diffuse DAB chromogen indicative of CB immunostaining was clearly distinguishable from CR-immunolabelled axon terminals, which contained the heavy, dense Ni-DAB reaction product (Figs. 5, 6). Analysis of a minimum of 50 proximate ultrathin sections (each approximately 50-80 nm thick) through the six selected CB-immunoreactive neurons revealed that, contralateral to the fimbria-fornix transection, all of the identified axosomatic (Figs. 5c, 6b) and axodendritic (Fig. 5c) synaptic contacts formed by CR-immunoreactive axon terminals exhibited an asymmetric membrane specialization (Figs. 5d, 6c). Like the synapses between CR-immunolabeled axons and PA-containing neurons, the postsynaptic membrane specialization of these cells was characteristically dense and thick (Figs. 5d, 6c). No CR-immunostained axon terminals that formed asymmetric postsynaptic membrane specializations with CB-immunostained neurons lacking these characteristics were observed.

Electron microscopy of triple-immunostained sections

Postembedding GABA immunostaining was performed on alternate serial ultrathin sections of ten identified CB-immunoreactive hilar neurons that were contacted by CR-immunolabeled boutons (Fig. 7). The large CB-immunoreactive cells showed a large rim of perinuclear cytoplasm (Fig. 7a,b) and infoldings of the nuclear membrane (Fig. 7b). Light microscopic analysis, as stated above, allowed for the unequivocal distinction of the DAB and Ni-DAB reaction products. Documentation of these sections by drawings enabled an unmistakable confirmation of structures in the correlated electron micrograph. CR-immunolabeled boutons containing the dense Ni-DAB reaction product that impinged on a CB-containing neuron are demonstrated in black-and-white photomicrographs (Fig. 7a). Due to the characteristic location of several of these boutons that contacted the CB-immunoreactive cell, they could be identified on serial ultrathin sections (Fig. 7b). GABA-postembedding immunostaining resulted in a dense and specific accumulation of gold particles in the CB-immunostained hilar neurons, which were identified by their light and diffuse DAB reaction product (Fig. 7b,c). Conversely, the CR-immunolabeled terminals were always devoid of gold particles, indicating the absence of GABA in these axons (Fig. 7c). The CR-immunoreactive axon terminals established exclusively asymmetric synapses with the CB- containing, GABA-immunolabeled neurons.

DISCUSSION Our results provide evidence that subcortical CR-

containing fibers form multiple, asymmetric synapses with 1) PA-positive GABAergic neurons located in or just underneath the granule cell layer and 2) CB-containing GABAer- gic cells located in the hilar area of the dentate gyrus. The morphological characteristics of these afferents suggest that they are of supramammillary origin, which was con- firmed by tracing studies. In contrast, we were unable to detect an innervation of the PA- or CB-containing neurons analyzed in our study by the intrinsic CR-containing fibers. These fibers are known to establish symmetric synaptic contacts (Gulyas et al., 1992) and to form a GABAergic inhibitory system (Miettinen et al., 1992). Thus, we will discuss our results with regard to the functional implica-


Fig. 4. a-c: Electron micrographs of sections double immunostained for CR (dark and dense reaction product) and PA (light and diffuse reaction product). A neuron immunostained for PA located at the lomer border of the dentate granule cell layer exhibits a nucleus (N) with a deep infolding (arrows in a ) and large amounts of perinuclear

cytoplasm. The cell body (in a and c ) and the proximal dendrite (in b) is contacted by CR-immunolabeled axons forming terminals (in a and b) or en passant boutons (in c) that establish asymmetric synapses (arrowheads). Scale bars = 1 +m.

Fig. 5. Demonstration of a correlated light (a,b) and electron microscopic analysis (c,d) of a CB-containing neuron that is contacted by CR-immunoreactive axon terminals. The same neuron is demonstrated in consecutive sections shown in Figure 6. a: The arrow is pointing to CB-positive neuron located in the dentate hilus (h) underneath the granule cell layer @). b Higher magnification of CB- immunoreactive neuron identified by the arrow in a. The arrow points to the bouton shown in boxed area in c, black arrowheads indicate numerous CR-positive axon terminals impinging on this cell, and black-and-white arrowheads indicate the CR-immunostained axon terminals shown in c. The star labels a capillary that is identified in c (Ca). c: CB-immunolabeled neuron shown in a and b contains a nucleus

(N) that exhibits infoldings of its membrane (arrows). Black-and-white arrowheads identify the CR-immunolabeled boutons shown in b. A capillary (Ca) is used as a landmark and is also labeled by the star in b. The boxed area is shown a t higher magnification in a consecutive ultrathin section in d. d Consecutive ultrathin section of bouton in boxed area in c demonstrates synaptic contact establishing an asymmetric synaptic membrane configuration (arrowheads) with the soma of the CB-containing cell. Note the large and dense postsynaptic membrane specialization. The capillaly identified by the star in b and by Ca in c was used as a landmark. Scale bars = 80 p n in a, 20 p n in b, 1 Km in c,d.

Fig. 6. Demonstration of a correlated light (a) and eIectron microscopic analysis (b,c) of a CB-containing neuron that is contacted by CR-immunoreactive axon terminals. The same neuron is demonstrated in a consecutive section shown in Figure 5. a: CB-positive hilar neuron is contacted by numerous CR-immunolabeled boutons. Arrowheads indicate terminals that are identified in the electron micrograph shown in b. The star labels a capillary used as landmark and is also identified by the star in b. b: The CB-containing neuron shown in a is identified by

capillary (star). Arrowheads point to boutons labeled the same way in a. The boxed area is shown a t higher magnification in c. c: Higher magnification of the bouton in the boxed area in b demonstrates synaptic contact with an asymmetric membrane configuration (arrowheads) on the soma of the CB-containing cell. Note the large and dense postsynaptic membrane specialization. N, nucleus. Scale bars = 20 pm in a, 1 Fm in b,c.

Fig. 7. Light and electron micrographs demonstrating triple immunostaining for CR, CB, and y-aminobutyric acid (GABA). a: CB- immunoreactive neuron (light reaction product) located in the dentate hilus is contacted by an axon immunostained for CR (dark reaction product), forming numerous boutons on the cell body and proximal dendrite of this hilar nonprincipal cell. The numbered arrowheads point to boutons identified under the electron microscope as shown in b and c. b: Further postembedding immunostaining for GABA of a consecutive ultrathin section of the same neuron as seen in a that resulted in specific accumulations of gold particles in the CB-

immunostained neuron. This neuron exhibits infoldings of its nuclear membrane (arrowheads). Numbered CR-immunolabeled terminals cor- respond to the boutons numbered in a. The boxed area is shown at higher magnification in c. c: Higher magnification of the boxed area in b. The numbered CR-immunoreactive axon terminal (star) is devoid of gold particles and establishes an asymmetric synapse with the cell body of the CB-immunolabeled nonprincipal neuron that contains numerous gold particles (arrowheads), signifying GABA immunoreactivity. Scale bars = 20 Fm in a, 1 pm in b,c.


tions of the termination pattern of this hypothalamic afferent system in the rat dentate gyrus.

Origin of subcortical CR-containing afferents Previous studies have shown that there is a dense band of

CR-immunoreactive axons in the innermost portion of the dentate molecular layer that gives rise to remarkably large boutons in both the rat (Gulyas et al., 1992; Nitsch and Leranth, 1993; Magloczky et al., 1994) and the monkey hippocampus (Nitsch and Leranth, 1993). Following fimbria- fornix transection, this axon bundle and the large boutons disappear, whereas other CR-containing structures in the hippocampus, including small immunopositive boutons, remain unaffected (Nitsch and Leranth, 1993). These find- ings suggested the presence of two separate CR-containing systems in the hippocampus, i.e., intrinsic and extrinsic afferents, the latter arising from subcortical sources (Gul- yas et al., 1992; Nitsch and Leranth, 1993; Magloczky et al., 1994). In the rat, axons arisingfrom contralateral hippocampal neurons also terminate in the inner dentate molecular layer (Laurberg and Soerensen, 1981; Deller et al., 19951, and a conventional transection of the ipsilateral fimbria fornix results in the disruption of both subcortical and commissural pathways (Amaral and Witter, 1995; Deller and Nitsch, 1995; Deller et al., 1995). It has to be noted, however, that the morphological characteristics of CR- immunoreactive boutons in the inner molecular layer are typical for the supramammillo-hippocampal system (Pas- quier and Reinoso-Suarez, 1976, 1978; Wyss et al., 1979a,b; Riley and Moore, 1981; Stanfield and Cowan, 1984; Gulyas et al., 1992). These large boutons formed by axons located in the inner dentate molecular layer establish exclusively asymmetric synaptic contacts and exhibit a remarkably thick postsynaptic density.

Combined retrograde tracing and CR immunostaining in the monkey revealed the cells of origin of these extrinsic afferents to be in the supramammillary nucleus (Nitsch and Leranth, 1993). Recent anterograde tracing studies using Phaseolus uulgaris-leucoagglutinin (PHAL) demonstrated a supramammillary projection to the innermost portion of the dentate molecular layer in the rat (Vertes, 1992; Magloczky et al., 1994). However, the latter authors described dentate principal neurons as the major targets of supramammillary afferents and did not report a supramammillary innervation of dentate inhibitory neurons (Magloc- zky et al., 1994). I t must be kept in mind that the different technicaI approaches used in the study by Magloczky et al. (1994) and in our present study do not permit a simple comparison of the data. Anterograde tracing does not allow for a prediction of the number of PHAL-labeled neurons. In the present study, we injected WGA-HRP into the rat hippocampus, and, following immunostaining of subcortical areas for CR, only large pyramidal-shaped neurons in the supramammillary nucleus and neurons in the nucleus reuniens contained both the retrogradely transported HRP grains and the diffuse DAB reaction product indicative of CR immunostaining. Anterograde tracing using PHAL (Wouterlood et al., 1990) has revealed that the CR- containing cell population in the nucleus reuniens (Resibois and Rogers, 1992) known to project to the hippocampus terminates exclusively in the stratum lacunosum-molecu- lare of CA1. It is important to emphasize that a projection of these neurons in the nucleus reuniens to the innermost portion of the dentate molecular layer was not described in these very elaborate studies (Wouterlood et al., 1990).

Taken together, the typical mode of termination of CR- immunoreactive axons in the dentate gyrus (Gulyas et al., 1992), the characteristic dense postsynaptic membrane specialization of contacts formed by these axons (Gulyas et al., 19921, and the retrograde labeling of CR-positive neurons all suggest that the vast majority of the large CR-immunostained boutons formed by axons arising from the dense bundle in the rat dentate inner molecular layer are of supramammillary origin.

Supramammillary CR innervation of dentate gyrus PA-containing cells

CR-immunoreactive terminals in the dentate inner molecular layer are known to establish asymmetric synaptic contacts with dendritic shafts and spines of numerous granule cells (Gulyas et al., 1992). However, our data suggest that some of these supramammillary fibers also establish multiple asymmetric contacts with somata and dendrites of several PA-positive neurons located in or just underneath the granule cell layer. The location and morphological characteristics of these neurons and a large pyramidal-shaped cell body containing an infolded nucleus sur- rounded by large amounts of cytoplasmic organelles are typical of basket cells, as shown by previous studies (Seress and Ribak, 1985). It is well established that, in the rat dentate gyrus, PA-containing cells form baskets surround- ing the cell bodies and proximal dendrites of granule cells and form numerous symmetric synapses. Basket cells are common and are located rather close to each other in such a way that their axonal arborization covers an area of about 0.6-0.9 mm in which another basket cell is usually present (Cajal, 1911; Halasy and Somogyi, 1993; Han et al., 1993). We regularly found several PA-containing neurons that were evenly distributed within or just underneath the granule cell layer and that were apparently contacted by CR-immunoreactive boutons, which establish asymmetric synaptic specializations. Therefore, it can be assumed that the termination field of these PA-positive basket cells controls the discharge of all granule cells in the rat dentate gyrus. This is in contrast to the monkey dentate gyrus, where only specifically located PA-containing basket cells are postsynaptic targets of hypothalamic substance P- positive fibers (Leranth and Nitsch, 1994). Thus, in the rat, hypothalamic afferents do not exert a contrasting mechanism in the dentate gyrus, as suggested for the monkey (Leranth and Nitsch, 1994).

Supramammillary CR innervation of dentate gyrus CB-containing cells

Our results show that, in the dentate gyrus, subcortical CR-containing fibers, in addition to contacting proximal dendrites of the dentate granule cells and somata and dendrites of several PA-containing basket cells, impinge on a few CB-immunoreactive neurons located in the deep hilus. The few CB-containing neurons located in the hilar area that are heavily innervated by CR-containing fibers exhibit the typical morphological characteristics of hippocampal nonprincipal cells, such as their location outside the principal cell layers and an infolded nuclear membrane (Seress and Ribak, 1985). Our GABA-postembeddingimmu- nocytochemical studies revealed that, in fact, these cells are inhibitory neurons. To this day, the termination field of hilar CB-containing neurons in the rat hippocampus is not well understood. However, according to Freund et al. (1990), CB-positive boutons forming symmetric contacts


are present only in the hilar area of the dentate gyrus, and these axon terminals are more heavily stained than those establishing asymmetric membrane specializations (e.g., mossy fiber terminals). These axon terminals may well originate from the heavily stained hilar CB-containing neurons, which, in the present study, are shown to be innervated by subcortical afferents. It has to be noted, however, that these neurons may contact targets in the medial septum. Therefore, they may represent a population of hippocampal nonprincipal projecting neurons (Toth and Freund, 1992). Moreover, one cannot exclude the fact that these neurons give rise to a commissural projection to the contralateral hippocampus that is known to originate from dentate hilar neurons and that is at least partially inhibitory (Ribak et al., 1986).

Functional implications The functional properties of the hypothalamo-hippocam-

pal innervation in the rat have been reported to be at least partially inhibitory (Segal, 1979), although recent physiological data show an excitatory component within this connection. (Mizumori et al., 1989). Interestingly, recent studies have provided evidence showing that, for the most part, this connection involves substance P in various nonprimates (Gall and Selawski, 1984; Ino et al., 1988; Yanagihara and Niimi, 1988). Our previous combined WGA-HRP tracing/ substance P immunocytochemical studies have demonstrated the presence of this neuropeptide in the hypothal- amohippocampal connection of the monkey (Nitsch and Leranth, 1994). In fact, it has been shown that substance P may act as an excitatory transmitter in various systems (Nicoll et al., 1980). Furthermore, it was shown that supramammillary CR-containing cells lack GABA. Thus, it is unlikely that they exert an inhibitory action (Nitsch and Leranth, 1993). Moreover, we were able to demonstrate that CR-containing hypothalamic afferents to the hippocampus establish exclusively asymmetric synaptic contacts with both granule cells (see also Gulyas et al., 1992) and inhibitory neurons. This synaptic membrane specialization is considered to be a morphological correlate of excitatory synapses (Eccles, 1964). Taken together, these data provide strong evidence that the supramammillary pathway to the hippocampus is excitatory in nature.

In the present study, we have shown by means of double labeling that, in fact, a considerable number of GABAergic neurons in the rat dentate gyrus are targets of the presum- ably excitatory supramammillohippocampal afferents. For example, several PA-positive neurons (Kosaka et al., 1987; Katsumaru et al., 1988; Nitsch et al., 1990; Leranth and Ribak, 1991; Seress et al., 1991) are contacted by these fibers. These neurons are regarded as important compo- nents of the hippocampal inhibitory system due to their high firing rate and the termination of their axons on cell bodies and axon initial segments of principal neurons (Kawaguchi et al., 1987; Kosaka et al., 1987; Katsumaru et al., 1988; Nitsch et al., 1990; Soriano et al., 1990). Therefore, hypothalamic afferents terminating on dendrites and somata of GABAergic neurons may mediate feed-forward inhibition on the principal cells. In this way, the discharge of excitatory neurons in the hypothalamus may have an end result of inhibition in the hippocampus. It has been shown in the rat that prestimulation of supramammillary neurons significantly enhances perforant path- elicited population spikes in the fascia dentata (Mizumori et a]., 1989), an effect that could be mimicked by glutamate

injections to the lateral supramammillary area (Carre and Harley, 1991). Moreover, Dahl and Winson (1986) specu- lated about a neuronal gate-control mechanism mediated by supramammillary afferents that may facilitate the infor- mation flow in the rat dentate gyrus in a behavior- dependent manner. The pathway described in the present study may well be involved in these phenomena and may be a morphological correlate for a powerful control mechanism of hippocampal 8 activity (Crowne and Radcliffe, 1975; Wyss et al., 1979a,b; Bland, 1986; Kitt et al., 1987; Freund and Antal, 1988; Freund, 1989; Stewart and Fox, 1989a,b; Jakab and Leranth, 19951, which has been demonstrated in recent physiological studies (Kocsis and Vertes, 1994).

ACKNOWLEDGMENTS The authors thank Dr. J.H. Rogers for providing the CR

antibody. We are grateful to Marya Shanabrough and to Eva Kruschinski for excellent technical and secretarial assistance, respectively. This study was supported by grants from the Deutsche Forschungsgemeinschaft (grants Ni 34411-1, Ni 34412-1, and Ni 34415.1) to R.N. and from the National Institutes of Health (grants NS 26068 and MH 44866) to C.L.

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GABAergic neurons in the rat dentate gyrus are innervated by subcortical calretinin-containing afferents