entorhinal cortical innervation of parvalbumin-containing neurons (basket and chandelier cells) in...

HIPPOCAMPUS 6239-246 (1996)

Entorhinal Cortical Innervation of Parvalbumin- Containing Neurons (Basket and Chandelier Cells) in the Rat Ammon’s Horn Jozsef Kiss,l Gyorgy Buzsaki,2 Jon S. M o r r o ~ , ~ Susan B. G l a n t ~ , ~ and Csaba Leranth3

lJoint Research Organization of the Hungarian Academy of Sciences and Semmelweis Medical University, Neuroendocrine Unit, Budapest, Hungary; 2Center for Molecular and Behavioral Neuroscience, Rutgers University, Newark, New Jersey; 3Depa~tments of Pathology and Obstetrics and Gynecology and Section of Neurobiology, Yale University School of Medicine, New Haven, Connecticut

ABSTRACT: Physiological data suggest that in the CA1-CA3 hippocampal areas of rats, entorhinal cortical efferents directly influence the activity of interneurons, in addition to pyramidal cells. To verify this hy- pothesis, the following experiments were performed: 1) light microscopic double-immunostaining for parvalbumin and the anterograde tracer Phaseolus vulgaris-leucoagglutinin injected into the entorhinal cortex; 2) light and electron microscopic analysis of cleaved spectrin-immunostained (i-e., degenerating axons and boutons) hippocampal sections following entorhinal cortex lesion; and 3) an electron microscopic study of parvalbumin-immunostained hippocampal sections after entorhinal cortex lesion. The results demonstrate that in the stratum lacunosum-molec- dare of the CAI and CA3 regions, entorhinal cortical axons form asymmetric synaptic contacts on parvalbumin-containing dendritic shafts. In the stratum lacunosum-moleculare, parvalbumin-immunoreactive dendrites represent processes of GABAergic, inhibitory basket and chandelier cells; these interneurons innervate the perisomatic area and axon initial segments of pyramidal cells, respectively. A feed-forward activation of these neurons by the entorhinal input may explain the strong, short- latency inhibition of pyramidal cells. 0 1996 Wiley-Liss, inc.

KEY WORDS: cleaved spectrin, PHA-1, anterograde tracing, degeneration, feed-forward inhibition, theta activity

A major hippocampal input, the perforant path to the CA1 region, is critical for sustaining physiological patterns, such as hippocampal theta and concurrent gamma activity (Buzsaki et al., 1983, 1986; Leung, 1984; Lopes da Silva et al., 1990; Brankack et al., 1993; Bragin et al., 1995; Charpak

~~~~~~~

Accepted for publication April 11, 1996. Address correspondence and reprint requests to Csaba Leranth, M.D., Ph.D., Department of Obstetrics and Gynecology, Yale University, School of Medicine, 339 Farnam Memorial Building, P.O. Box 208063, New Haven, CT 06520-8063.

et al., 1995). The excitatory nature of this input is il- lustrated by the presence of currents sink in the stratum lacunosum-moleculare in response to acoustic stimulation. Such natural stimuli produce a simultaneous and short-latency activation of the dendrites of CAI pyramidal cells and granule cells, with a stronger sink in the lacunosum-moleculare than in the molecular layer of the dentate gyrus (Buzsaki et al., 1995). Electrical activation of the perforant path can discharge CAI pyramidal neurons under certain circumstances, but the typical response to electrical stimulation is inhibition, rather than discharge of the pyramidal cells (Bragin and Otmakhov, 1979; Doller and Weight, 1982; Yeckel and Berger, 1990; Empson and Heinemann, 1995; Pare and Llinas, 1995; Soltesz, 1995; Buzsaki et al., 1995). It has been suggested repeatedly that the relative dominance of feed- forward inhibition is responsible for curtailing excitation of the distal apical dendrites of CAI pyramidal cells, although the exact circuitry and interneurons involved have not yet been revealed.

Previous anatomical studies have demonstrated that entorhinal cortex neurons projecting to the dentate gyrus and Ammon’s horn differ in their location. Caudomedial portions of the entorhinal cortex distribute fibers mainly to the dentate gyrus (DG). O n the other hand, pro- gressively more rostra1 and lateral parts of the entorhinal cortex project more strongly to the CA1 region, with fewer fibers terminating in the DG. The rostrolateral part of the entorhinal cortex, adjacent to the olfactory cortex and amygdaloid complex, projects almost exclusively to the CA1 region (Witter et al., 1988, 1992; Amaral and Witter, 1995). The source of the CAI projection is the pyramidal cells of layer 111, whereas stellate cells of layer I1 project to the DG and the CA3 region (Tamamaki and Nojyo, 1993). Electron

0 1996 WLEY-LISS, INC.

240 KlSS ET AL.

microscopic studies reveal that perforant path fibers in the stratum lacunosum-molecular contact predominantly dendritic spines of presumed pyramidal cells, but a small portion of the terminals innervate dendritic shafts (Desmond et al., 1994). In the present study, we provide evidence that at least some of these shaft contacts correspond to synaptic innervation of parvalbumin- immunoreactive basket or chandelier cells.

Sixteen adult Sprague-Dawley (250-280 g B.W.) rats of both sexes were used in this study. Animals were kept in standard laboratory conditions, with tap water and regular rat chow ad Libi- tum, in a 12-h light, 12-h dark cycle. Ten rats were used for anterograde tracing experiments combined with parvalbumin (PA) immunocytochemistry and six for a degeneration study combined with light and electron microscopic immunohistochemistry for either cleaved spectrin or PA.

Surgical Procedures

Antevogvade tracing experiments

Entorhinal cortical fibers projecting to the hippocampus were visualized by anterogradely transported Pbareolus vulgavis-leucoagglutinin (PHA-L; 2.5% in saline, Vector Laboratories, Burlingame, CA). The tracer was iontophoretically applied (using a 5-kA, 5-s on 5-s off, positive current for 15 min) into the entorhinal cortex of rats positioned in a Kopf stereotaxic instru- ment (David Kopf Instruments, Tujunga, CA). The injections into the lateral entorhinal area were made with a pipette (1 5 p m inner diameter) angled 10 degrees to the vertical plane at the following coordinates: 5.6 mm posterior to the bregma, 7.4 mm lateral to the midline, and 7.8 mm or 8.6 mm below the pial sur- face. Nine to 11 days after the PHA-L injection, the animals were anesthetized again with 0.5 ml of 7% chloralhydrate and perfused through the heart with 100 ml saline followed by a fixative containing 4% paraformaldehyde, 0.1% glutaraldehyde, and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.35 (PB). The brains were removed, and blocks of the injection site and the ipsilateral hippocampus were dissected out and postfixed in the same, but glutaraldehyde-free, solution for an additional 2 h.

Entovhinal lesion

Deeply anesthetized rats were fixed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA), and from a dorsal penetration, under visual control, layers I-V of the entorhinal cortex were aspirated. Two days later, animals were killed under ether anesthesia by transcardial perfusion of 50 ml heparinized saline, followed by a fixative containing 4% paraformaldehyde, 0.1 Yo glutaraldehyde, and 15% saturated picric acid in PB. Ipsi- and contralateral hippocampal formations were dissected out, divided into three pieces along their septo-temporal axis, and postfixed for 2 h in the same, but glutaraldehyde-free, fixative.

Tissue Preparation and Immunostaining

Fifty-micron-thick coronal sections were cut from all tissue blocks on a Vibratome (Lancer, St. Louis, MO) and rinsed in several changes of ice-cold PB. 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 rapidly frozen by immersing the vial in liquid nitrogen, thawed to room tem- perature, and washed in PB. Subsequently, sections for both light and electron microscopy were treated with 1% sodium borohy- dride in PB for 10-30 min to eliminate unbound aldehydes from the tissue (Kosaka et al., 1986).

Following extensive washing in PB, sections for the PHA-I. study were incubated first in 10% normal goat serum containing 1% Triton X-100 in PB, and then in a mixture of primary antisera directed against PHA-L (biotinylated goat anti-1'HA-L, 1:200, Vector Labs., Burlingame, CA) and PA (mouse anti-PA, 1:5,000, Sigma, St. Louis, MO) for 48 h at 4°C. Sections were then placed in a mixture containing avidin biotinylated-horse- radish peroxidase complex (ABC Elite, 1 :200, Vector Labs., Burlingame, CA) and horse anti-mouse IgG (DAKO, 1:50 Carpinteria, CA) for 6 h at 20°C. The first immunoperoxidase (for PHA-L) was developed using a dark-blue to black, nickel-in- tensified diaminobenzidine (Ni-DAB) reaction (1 5 mg DAB, 0.12 mg glucose oxidase, 12 mg ammonium chloride, 12 mg nickel ammonium sulfate, and 60 mg P-u-glucose in 30 ml PB for 10-15 min). Then, the sections were further incubated in a mouse peroxidase-antiperoxidase complex (mouse PAP, 1 : 100, DAKO), and the tissue-bound peroxidase (for PA) was visualized using a brown diaminobenzidine (DAB) reaction (15 mg DAB, 165 pl 0.3% Hz02 in 30 ml PB). Secrions were thoroughly washed in PB between each incubation step. All antibody dilu- tions were made in PB containing 1 Yo normal goat serum, 0.3% Triton X-1 00, and 0.1 % sodium azide.

Light and electron microscopic single immunostaining for cleaved spectrin and electron microscopic single immunostaining for PA were performed on sections cut from the ipsilateral hip- pocampi of the entorhinal cortex-lesioned animals using the avidin-biotin-peroxidase (ABC; Hsu et al., 1981) method. Briefly, sections were incubated overnight at 20°C (48 h at 4°C for electron microscopy) in rabbit anti-cleaved spectrin (see below; affinity purified #7579A, 1:1500 in PB containing 1% normal goat serum, 0.3% Triton X-100 for light microscopy, and 0.1% sodium azide). Other sections were incubated under the same conditions in a monoclonal mouse anti-PA (Sigma, St. Louis, MO; 1:25,000 in PB containing 1Yn normal horse serum and 0.1% sodium azide). This was followed by incubation of the sections in the secondary antibody (biotinylated goat anti-rabbit IgG; Vector Labs., Burlingame, CA, for cleaved spectrin, or biotinylated horse anti-mouse IgG; Vector Labs., Burlingame, CA 1:250 in PB), and then, in ABC Elite (1:250 in PB, Vector Labs., Burlingame, CA), each for 2 h at 20°C. For light microscopy, the tissue-bound immunoperoxidase was visualized by a dark-blue to black Ni-DAB reaction, whereas for electron microscopy the DAB reaction was used. Light microscopic sections were placed on gelatin-coated slides, dehydrated, and mounted in Permount.

CAZ-3 PARVALBUMIN CELL ENTORHINAL AFFERENTS 241

Following immunostaining, sections for electron microscopy were postosmicated (1% Os04 in PB, 30 min), dehydrated through increasing ethanol concentrations (the 70% ethanol con- tained 1% uranyl acetate, 30 min), and embedded in Araldite. Ribbons of ultrathin sections were examined under a Philips CM- 10 electron microscope.

Preparation of Cleavage-Specific All-Spectrin Antibodies

Synthetic hexapeptides representing the novel amino- and COOH-termini created by the action of calpain I on all spectrin (fodrin) (Harris et al., 1988) were prepared by solid phase syn- thesis at the Keck Protein Chemistry Laboratory (Yale University). Each peptide incorporated five residues of native sequence and a terminal cysteine to facilitate conjugation to hemocyanin (KLH, Sigma, St. Louis, MO). The sequence of peptide I, representing all spectrin breakdown product I (a-bdp I), was C-Q-Q-E-V-Y; peptide 11, representing the COOH terminal breakdown product of all spectrin (a-bdp II), was G-M-M-P-R-C. After conjugation to KLH, antibodies were prepared by standard methods to each peptide using New Zealand white rabbits and affinity purified on Sepharose columns containing the synthetic peptide alone (Affigel = AA, Pharmacia, Piscataway, NJ). Cross-reactivity with intact all spectrin was absorbed by reaction with purified bovine brain spectrin (Harris et al., 1988). The resulting antibodies were highly specific for all spectrin that had been cleaved after residue 1176 by calpain I (de Lacoste et al., 1992; Glantz and Morrow, 1995), as detected both by Western blot and by immunocytochemistry. In the present study, the anti-a-bdp I antibody has been used exclusively. According to our unpublished observation, this antibody specifically labels degenerating boutons.

The entorhinal innervation of the different types of fascia dentate neurons (Hjorth-Simonsen and Jeune, 1972; Steward, 1976; Steward and Scoville, 1976; Wyss, 1981) and pyramidal cells of the CA1-CA3 areas (Witter et al., 1988, 1992; Desmond et al., 1994) is well known. Therefore, this study focuses on the entorhinal innvervation of PA-containing neurons located in the Ammon’s horn.

PHA-L Plus PA Double Immunostaining Neurons at the site of the PHA-L injection (in the lateral en-

torhinal area) and anterogradely labeled fibers and boutons in- vading the stratum lacunosum-moleculare of the hippocampus proper and upper third of the dentate molecular layer were immunostained dark-blue to black. The distribution pattern of the brown DAB-labeled PA-immunoreactive neurons corresponds to earlier descriptions (Nitsch et al., 1990). Briefly, in the Ammon’s horn, the majority of the large PA-containing cell bodies were located in or adjacent to the stratum pyramidale, and their fine dendritic processes extended to the stratum lacunosum-moleculare.

A few PA neurons could be observed in the stratum oriens. However, none could be detected in the stratum radiatum and lacunosum-moleculare (Fig. 1B). Through the entire stratum lacunosum-moleculate, the PHA-L-labeled entorhinal fibers run- ning parallel to the hippocampal fissure formed several putative synaptic contacts with the PA-immunoreactive, aspiny dendrites (Fig. 1C-G).

Degeneration Studies Light microscopic immunostaining for cleaved spectrin

Even multiple PHA-L injections do not label all of the entorhinal cortical efferent axons. Therefore, to gain some idea as to the density of the entorhinal cortical innervation of the CA, and since according to our recent knowledge, no trespassing hip- pocampopetal axons arrive at the hippocampus through the entorhinal cortex, an entorhinal cortical lesion was combined with light microscopic immunostaining for cleaved spectrin. Two days following entorhinal cortex lesion, this immunostaining revealed a continuous dense band of degenerating perforant pathway axons which occupied the stratum lacunosum-moleculare of all CA areas and the distal third of the dentate molecular layer (Fig. 2A).

Electron microscopic immunostaining for cleaved spectrin and PA

In the electron microscopic material immunostained for cleaved spectrin, a dense population of immunoreactive boutons and axons were observed in the stratum lacunosum-moleculare. The overwhelming majority of immunoreactive boutons formed asymmetric synaptic contacts with spines (Fig. 2BJ; however, oc- casionally, axodendtitic synapses could also be observed (Fig. 2B2). It has to be noted that in addition to the cleaved spectrin- immunopositive, degenerating axons, a few small, immunonegative, degenerating boutons were found (Fig. 2B2). These boutons exhibited a very electron-dense homogenous axoplasm and were usually surrounded by glial processes. The lack of immunoreactivity for cleaved spectrin in these axon terminals is probably due to the absence of tissue antigen because of the advanced stage of degeneration.

Forty-eight hours following entorhinal cortex lesion, electron microscopic analysis of the stratum lacunosum-moleculare of PA- immunostained hippocampal sections showed a very large number of degenerating axons and boutons throughout the stratum lacunosum-moleculare of all CA areas. These degenerating boutons, in addition to forming asymmetric synaptic membrane specializations with dendritic spines, established numerous, asymmetric synaptic contacts with the shafts of PA-immunoreactive dendrites (Fig. 2C-E). In order to characterize the magnitude of the entorhinal cortical innervation of PA neurons, it is important to note that all of the PA-positive dendritic segments, the length of which exceeded 1 pm, were contacted by degenerated axon terminals. PA-immunoreactive spines could not be detected. This observation confirms the light microscopic aspiny description of PA dendrites (Nitsch et al., 1990).

CA1-3 PARVALBUMIN CELL ENTORHINAL AFFERENTS 243

FIGURE 1. Color light micrographs demonstrate the result of a double immunostaining for PA and PHA-L injected into the rostrolateral part (A) of the entorhinal cortex (EC). In A, EC PHAL in- dicates the PHA-L deposit, the direction of the injection is labeled by a black bar, and arrowheads indicate PHA-L-labeled fibers in the straum lacunosum moleculare (slrn). B is an overview of a segment of the CAI area and dentate gyrus. The PAP-labeled, brown immunoperoxidase product represents PA-immunoreactive somata located in and adjacent to the stratum pyramidale (sp) and oriens (so) of the CAI and dentate granule cell layer (g), their axonal plexus heavily innervating the sp and g. In addition, long PA-containing dendritic processes can be seen, which can be followed from their parent neurons to the slm in the CAI and to the upper third of the stratum moleculare (sm) of the dentate gyrus. The dark-blue to black Ni-DAB-labeled perforant pathway axons (pp) occupy CAI and dentate gyrus areas separated by the hippocampal fissure (Hif), the slm, and the upper third of the sm, respectively. C-G are high-power magnifications of the putative synaptic contacts (arrows) between the Ni-DAB-labeled pp axon terminals and brown DAB-labeled PA- containing dendritic shafts (arrowheads) in the slm of the CA3 (C) and CAI (D-G). Original magnification of A: X32; B: X250; and C-G: X1,250.

In this study, we provide direct morphological evidence that entorhinal afferents form asymmetric synaptic contacts with PA- immunoreactive dendrites of basket cells and/or chandelier cells in the CA1-CA3 regions of the hippocampus. In both rodent and primate hippocampus, immunoreactivity for PA is associated exclusively with non-principal cells. The somata of these PA neurons are mostly located in the vicinity of principal cells. They represent two subpopulations of GABAergic interneurons: basket and chandelier cells, with target specificity for cell body regions, in- cluding the soma, proximal dendrites, and axon initial segments. The long, practically aspiny dendrites of both cell types extend to the stratum lacunosum-moleculare (Kosaka et al. 1987; Sloviter, 1989; Nitsch et al., 1990; Braak et al., 1991; Seress et al., 1991; Leranth and Ribak, 1991; Ribak et al., 1993; Gulyas et al., 1993). Chandelier cells emit very few dendrites in the stratum radiatum but have a tuft of thin dendrites in the stratum lacunosum-moleculare (Li et a]., 1992).

Our morphological analysis revealed that similar to the situa- tion in the dentate molecular layer (Zipp et al., 1989), PA-immunoreactive dendrites in the stratum lacunosum-moleculare of the Ammon’s horn are synaptic targets of entorhinal cortical fibers. This observation, together with the aforementioned morphological characteristics of PA interneurons, indicate that in Ammon’s horn, the activity of PA-containing basket and axo- axonic cells can be influenced by entorhinal cortical afferents. Furthermore, although interneurons represent only a fraction (10-15%; Woodson et al., 1989) of the total number of hippocampal cells, they very effectively control large numbers of pyra- niidal cells (Li et al., 1992; Gulyas et al., 1993; Buhl et al., 1994; Sik et al., 1996).

Physiological Implications

A major challenge in the understanding of the function of the entorhinal cortex-hippocampus circuitry is to reveal the mecha- nisms which allow specific signaling in the entorhinal-hippocampal and intrahippocampal pathways (McClelland et al., 1995). The entorhinal cortex has a direct access to all subregions of the hippocampus. Layer I1 stellate cells diffusely innervate dentate granule cells and CA3 pyramidal cells. The granule cells provide a focused and convergent innervation of the CA3 subfield. The CA3 pyramids, in turn, innervate the CA1 region by their very extensive, longitudinal terminals (cf. Li et al., 1994; Amaral and Witter, 1995). In contrast, the terminal arbor of layer I11 entorhinal pyramidal cells on the distal dendrites of CAI pyramidal neurons is spatially confined (Tamamaki and Nojyo, 1992); in effect, the entorhinal cortex-mediated neocortical representa- tions are topographically mapped onto the CA1 region. A possible functional advantage of such dual, monosynaptic-multisy- naptic innervation of the CAI pyramidal cells is that the actual discharge of the pyramidal neuron would depend on the convergent excitation of the diffuse associational and the focused, direct perforant path input to the CAI region (Buzsaki et al., 1995). In the rat, electrical stimulation of the perforant path typically evokes inhibition, rather than discharge, of the pyramidal cells (Colbert and Levy, 1992; Empson and Heinemann, 1995; Pare and Llinas, 1995; Soltesz, 1995; Buzsaki et al., 1995). A quantitative electron microscopic study (Desmond et al., 1994) has shown that the majority of perforant path fibers form asymmetric contacts on dendritic spines (93%); only 6.5% of the postsynaptic targets were on dendritic shafts and none were axosomatic. Although a variety of interneurons are present in the stratum lacunosum-moleculare (Lacaille and Schwartzkroin, 1 988), their axonal targets have yet to be discovered. Some of them innervate other interneurons but not pyramidal cells (Acsady et al., 1996; Gulyas et al., 1996). O n the other hand, chandelier cells have a large tuft- like dendritic tree in stratum lacunosum-moleculare (Li et al., 1992), and some dendrites of basket cells also reach this layer (Gulyas et al., 1993; Sik et al., 1996). The present study provides direct evidence that feed-forward inhibition of CA1 and CA3 pyramidal cells by the perforant path is mediated, at least par- tially, by chandelier cells, basket cells, or both. Indeed, interneurons with their somata in the CAI pyramidal layer (putative bas- kedchandelier cells) could be discharged with very low stimulus currents and <1 ms after the population discharge of granule cells; that is they were excited and discharged monosynaptically (Buzsaki and Eidelberg, 1982). We can conclude, therefore, that it is likely that basket and/or chandelier cells are responsible for the early inhibitory postsynaptic potentials in pyramidal cells, brought about by synchronous activation of perforant path fibers.

The perforant path input to the CA1 region is essential for the maintenance of hippocampal theta activity in the behaving animal (Buzsaki et al., 1983, 1986; Lopes da Leung, 1984; Silva et al., 1990; Brankack et al., 1993; Bragin et al., 1995; Charpak et al., 1995). Distal dendritic excitation of CA1 pyramidal cells during theta activity is coupled with a powerful somatic inhibition (Fox, 1989; Leung and Yim, 1991; Soltesz and Deschenes, 1993;

FIGURE 2. Light (A) and electron micrographs (B-E) show the results of immunostaining in the hippocampus for cleaved spectrin (A,B) and parvalbumin (C-E) 48 h following entorhinal cortex lesion. A demonstrates the rich plexus of degenerating, cleaved spectrin-immunoreactive perforant pathway axons (arrows) in the entire stratum lacunosum-moleculare (slm) of the hippocampus proper and distal third of the dentate molecular layer (m). B1 and B2 show cleaved spectrin-immunoreactive axons forming asymmetric synap-

tic contacts (arrowheads) with a spine (S on B1) and dendritic shaft (D on B2). Arrow on Panel B2 points to a small, immunonegative, electron-dense, degenerated bouton. C,D demonstrate electron- dense, degenerating perforant pathway axon terminals (arrows) forming asymmetric synaptic membrane specializations (arrowheads) with PA-immunoreactive dendritic shafts in the stratum lacunosum-moleculare of the CAI (C,E) and CA3 (D) hippocampal subfields. Original magnification of A: x 32. Scale bar = 1 pm.

CAI-3 PARVALBUMIN CELL ENTORHINAL AFFERENTS 245

Ylinen et al., 1995), resulting in a net suppression of pyramidal cell firing (Buzsaki et al., 1983). An important function of theta oscillation is to keep the membrane potential of pyramidal cells close, but below the firing threshold. Perisomatic inhibition of pyramidal cells during theta derives from rhythmic discharge of basket cells and chandelier cells (Cobb et al., 1995; Ylinen et al., 1995). Theta-related activation of basket cells had been postu- lated to derive from the medial septum (Buzsaki e t al., 1983; Stewart and Fox, 1390; Ylinen et a\., 1995). I n light of the present results, an additional source of theta-related activation of basket and chandelier cells should be included: the perforant path.

During urethane anesthesia, basket cells fire on the theta phase opposite to the pyramidal cells (Buzsaki and Eidelberg, 1982; Ylinen et al., 1995), whereas in the awake rat both cell types discharge on the same phase (Buzsaki et al., 1983; Fox, 1989). This paradox may be resolved by the present anatomical observations. The excitatory theta dipole, formed by the perforant path afferents on the distal apical dendrites, is very weak during urethane anesthesia, presumably due to the drug’s suppressive effect of fast glutamate transmission (Ylinen et al., 1995). Therefore, the main source of basket and chandelier cell activation under anesthesia is the medial septa1 area. Consequently, pyramidal cells discharge when they are least inhibited, that is, out of phase with bas- kedchandelier cell activity. In the awake animal, however, the rhythmic excitatory outflow from the entorhinal cortex concur- rently excites both pyramidal cells as well as basket and chandelier cells; hence they discharge on the same phase of theta.

Acknowledgments W e thank Marya Shanabrough for her expert technical help.

This work was supported by grants from NIH (NS 26068, NS 32393 to C.L.), HFSP and the Whitehall Foundation (to G.B.), and OTKA T6372 (to J.K)

Acsady L, Gorcs T, Freund TF (1996) Different populations of VIP- immunoreactive interneurons are specialized to control pyramidal cells or interneurons in the hippocampus. Neuroscience (in press).

Arnaral DG, Witter MP (1995) The hippocampal formation. In: The rat nervous system, 2nd ed. G. Paxinos (ed), New York: Academic Press, pp 443-493.

Braak E, Strotkamp B, Braak H (199 1) Parvalbumin-immunoreactive structures in the hippocampus of the human adult. Cell Tissue Res 264:3348.

Bragin AG, Otmakhov NA (1 979) Comparison of direct influences of the perforant path on hippocampal CA1 and CA3 neurons in vitro. Neurophysiology 1 1:220-226.

Bragin A, Jando G, Nadasdy Z, Hetke J, Wise K, Buzsaki G (1995) Gamma (40-1 00 Hz) oscillation in the hippocampus of the behaving rat. J Neurosci 15:47-60.

Brankack J, Stewart M, Fox SE (1993) Current source density analysis of the hippocampal theta rhythm: associated sustained potentials and candidate synaptic generators. Brain Res. 615:310-327.

Buhl E, Halasy K, Somogyi P (1994) Hippocampal unitary inhibitory

postsynaptic potentials: diverse sources and number of synaptic re- lease sites. Nature 368:823-828.

Buzsaki G, Eidelberg E (1982) Direct afferent excitation and long-term potentiation of hippocampal interneurons. J Neurophysiol 48:597- 607.

Buzsaki G, Leung L, Vanderwolf C H (1983) Cellular bases of hippocampal EEG in the behaving rat. Brain Res Rev 6:139-171.

Buzsaki G, Czopf J, Kondakor I, Kellenyi L (1986) Laminar distribution of hippocampal rhythmic slow activity (RSA) in the behaving rat: current-source density analysis, effects of urethane and atropine. Brain Res 365:125-137.

Buzsaki G, Penttonen M, Bragin A, Nadasdy Z, Chrobak JJ (1995) Possible physiological role of the perforant path-CAl projection. Hippocampus 5: 14 1-146.

Charpak S, Pare D, Llinas RR (1995) The entorhinal cortex entrains fast CA1 hippocampal oscillations in the anesthetized guinea pig: role of the monosynaptic component of the perforant path. Eur J Neurosci 7:1548-1557.

Cobb SR, Buhl EH, Halasy K, Paulsen 0, Somogyi P (1995) Synchronization of neuronal activity in hippocampus by individual GABAergic interneurons. Nature 378:75-98.

Colbert CM, Levy WB (1 992) Electrophysiological and pharmacologi- cal characterization of perforant path synapses in CA1: mediation by glutamate receptors. J Neurophysiol 68: 1-8.

de Lacoste M-C, Lewis CE, White CL, Morrow JS (1992) Immunocy- tochemical evidence of accelerated spectrin proteolysis in hippocampus of alzheimer disease patients. SOC Neurosci Abstr 18:558.

Desmond NL, Scott CA, Jane JA, Jr, Levy WB (1994) Ultrastructural identification of entorhinal cortical synapses in CA1 stratum lacunosum-moleculare of the rat. Hippocampus 4:594-600.

Doller HJ, Weight FF (1982) Perforant path activation of hippocampal CA1 stratum pyramidale neurons: electrophysiological evidence for a direct pathway. Brain Res 237:l-13.

Empson RM, Heinemann U (1 995) Perforant path connections to area CA1 are predominantly inhibitory in the rat hippocampal-entorhinal cortex combined slice preparation. Hippocampus 5: 104-107.

Fox SE (1989) Membrane potential and impedance changes in hippocampal pyramidal cells during theta rhythm. Exp Brain Res 77: 283-294.

Glantz SB, Morrow JS (1995) The specrrin-actin cytoskeleton and membrane integrity in hypoxia. In: Tissue oxygen deprivation: develop- mental, molecular, and integrated function. (G.G. Haddad and G. Lister, eds), New York: Marcel Dekker, Inc. (in press).

Gulyas AI, Miles R, Hajos N, Freund TF (1993): Precision and vari- ability in postsynaptic target selection of Inhibitory cells in the hippocampal CA3 region. Eur J Neurosci 5:1729-1751.

Gulyas AI, Hajos N, Freund TF (1996) Interneurons containing calre- tinin are specialized to control other interneurons in the hippocampus. J Neurosci 16:3397-3411.

Harris AS, Croall D, Morrow JS (1988) The calmodulin-binding site in- fodrin is near the site of calcium-dependent protease-I cleavage. J Biol Chem 263:15754-15761.

Hjorth Simonsen A, Jeune B (1972) Origin and termination of the hippocampal perforant path in the rat studied by silver impregnation. J Comp Neurol 144:215-232.

Hsu M, Raine L, Fanger H (1981) The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (peroxidase) procedures. ] Histochem Cytochem 29:577-590.

Kosaka T, Katsumaru H, Hama K, Wu J-Y, Heizman ZW (1987) GABAergic neurons containing the Ca2+-binding protein parvalbumin in the rat hippocampus and dentate gyrus. Brain Res 41 9: 1 19- 130.

246 KISS ET AL.

Kosaka T, Nagatsul H, Wu J-Y, Hama K (1986) Use of high concen- tration of glutaraldehyde for immunocytochemistry of transmitter- synthetizing enzymes in the central nervous system. Neuroscience

Lacaille CJ. Schwartzkroin AP (1 988) Stratum lacunosiim-moleculare characteristics, synaptic responses, and morphology. Neuroscience 8: 1400-1 4 10.

Leranth C, Ribak CA (1991) Calcium-binding proteins are concentrated in the CA2 field of the monkey hippocampus: a possible key to this region’s resistance to epileptic damage. Exp Brain Res. 85:129-136.

Leung LS (1984) Model of gradual phase shift of theta rhythm in the rat. J Neurophysiol 52:1051-1065.

Leung LWS, Yim CYC (1991) Intrinsic membrane potential oscillations in hippocampal neurons in vitro. Brain Res 553:261-274.

Li X-G, Somogyi P, Tepper JM, Buzsaki G (1992) Axonal and dendritic arborization of an intracellularly labeled chandelier cell in the CAI region of the rat hippocampus. Exp Brain Res 90:519-525.

Li X-G, Somogyi P, Ylinen A, Buzsaki G (1994) The hippocampal CA3 network: an in vivo intracellular labeling study. J Comp Neurol. 339: 18 1-208.

Lopes da Silva RH, Witter MP, Boeijinga PH, Lohman AHM (1990) Anatomic organization and physiology of the limbic cortex. Physiol Rev 70:453-5 1 1.

McClelland JL, McNaughton BL, 0’Reilly RC (1995) Why are there complementary learning systems in the hippocampus and neocortex: insights form the successes and failures of connectionist models of learning and memory. Psycho1 Rev 102:419-457.

Nitsch R, Soriano E, Frotscher M (1990) The parvalbumin-containing nonpyramidal neurons in the rat hippocampus. Anat Embryo1 (Berl) 18 1 :4 13-425.

Pare D, Llinas RR ( I 995) Intracellular study of direct entorhinal inputs to field CAI in the isolated guinea pig brain in vitro. Hippocampus 5: 1 15-1 19.

Ribak CA, Seress L, Leranth C (1993) Electron microscopic immunocytochemical study of the distribution of parvalbumin-containing neurons and axon rerminals in the primate dentate gyrus and Ammon’s horn. J Comp Neurol 327:298-321.

Seress L, Gulyas AI, Freund TF (1991) Parvalbumin and calbindin D28k- immunoreactive neurons in the hippocampal formation of the macaque monkey. J Comp Neurol 313:162-177.

Sik A, Penttonen M, Minen A, Buzsaki G (1996) Hippocampal CAI interneurons: an in vivo intracellular labeling study. J Neurosci 15:6651-6665.

Sloviter RS (1 989) Calcium binding protein (Calbindin-Il28k) and parvalbumin immunocytochemistry: localization in the rat hippocampus

18:975-990.

with specific reference to the selective vulnerability of hippocampal neurons to seizure activity. J Comp Neurol 280:183-196.

Soltesz I (1995) Brief history of cortico-hippocampal time with a special reference to the direct entorhinal input to CA1. Hippocampus 5: 120-124.

Soltesz I, Deschenes M (1993) Low- and high-frequency membrane potential oscillations during theta activity in CA1 and CA3 pyramidal neurons of the rat hippocampus under ketamine-xylazine anesthesia. J Neurophysiol 7097-1 16.

Steward 0 (1976) Topographical organization of the projection from the entorhinal area to the hippocampal formation of the rat. J Comp Neurol 167:285-3 14.

Steward 0 , Scoville SA (1976) Cells of origin of entorhinal cortical afferents to the hippocampus and fascia dentata of the rat. J Comp Neurol 169:347-370.

Stewart M, Fox SE (1990) Do sepral neurons pace the hippocampal theta rhythm? TINS 13:163-168.

Tamamaki N, Nojyo Y (1992) Reciprocal connections between the subiculum, field CA1 and the entorhinal cortex in the rats. Soc Neurosci Abstr 18:32.

Tamamaki N, Nojyo Y (1993) Projection of the entorhinal layer I1 neurons in the rat as revealed by intracellular pressure-injection of neu- robiotin. Hippocampus 3:471-480.

Witter MP, Griffioen AW, Jorritsma-Byham B, Krijnen JLM (1988) Entorhinal projections to the hippocampal CA1 region in the rat: an underestimated pathway. Neurosci Lett 85:193-198.

Witter MP, Jorritsma Byham B, Wouterlood FG (1992) Perforant pathway projections to the ammons horn and the subiculum in the rat. An electron microscopical PHA-L study. SOC Neurosci Abstr 18:323.

Woodson W, Nitecka L, Ben-Ari Y (1989) Organization of the GABAergic system in the rat hippocampal formation: a quantitative immunocytochemical study. J Comp Neurol 280:254-271.

Wyss JM (1981) An autoradiographic study of the efferent connections of the entorhinal cortex in the rat. J Comp Neurol 199:495-512.

Yeckel MF, Berger TW (1990) Feedfonvard excitation of the hippocampus by afferents from the entorhinal cortex: redefinition of the role of the trisynaptic pathway. Proc Natl Acad Sci USA 87:5832-- 5836.

Minen A, Soltesz 1, Bragin A, Penttonen M, Sik A, Buzsaki G (1995) Intracellular correlates of hippocampal theta rhythm in identified pyramidal cells, granule cells, and basket cells. Hippocampus 5:78- 90.

Zipp F, Nitsch R, Soriano E, Frotscher M (1989) Entorhinal fibers form synaptic contacts on parvalbumin-immunoreactive neurons in the rat fascia dentata. Brain Res 495:161-166.

entorhinal cortical innervation of parvalbumin-containing neurons (basket and chandelier cells) in...

Documents