THE JOURNAL OF COMPARATIVE NEUROLOGY 378:320–336 (1997)

Immunostaining for Substance P

Receptor Labels GABAergic Cells With
Distinct Termination Patterns in the
Hippocampus
L. ACSÁDY,1 I. KATONA,1 A.I. GULYÁS, 1 R. SHIGEMOTO,2 AND T.F. FREUND1*
1Institute of Experimental Medicine, Hungarian Academy of Sciences,

Budapest, H-1450, Hungary

2Department of Morphological Brain Science, Faculty of Medicine,

Kyoto University, Kyoto 606-01, Japan

ABSTRACT
A specific antiserum against substance P receptor (SPR) labels nonprincipal neurons in
the cerebral cortex of the rat (T. Kaneko et al. [1994], Neuroscience 60:199–211; Y. Nakaya et
al. [1994], J. Comp. Neurol. 347:249–274). In the present study, we aimed to identify the types
of SPR-immunoreactive neurons in the hippocampus according to their content of neurochemi-
cal markers, which label interneuron populations with distinct termination patterns. Markers
for perisomatic inhibitory cells, parvalbumin and cholecystokinin (CCK), colocalized with SPR
in pyramidallike basket cells in the dentate gyrus and in large multipolar or bitufted cells
within all hippocampal subfields respectively. A dense meshwork of SPR-immunoreactive
spiny dendrites in the hilus and stratum lucidum of the CA3 region belonged largely to
inhibitory cells terminating in the distal dendritic region of granule cells, as indicated by the
somatostatin and neuropeptide Y (NPY) content. In addition, SPR and NPY were colocalized
in numerous multipolar interneurons with dendrites branching close to the soma. Twenty-five
percent of the SPR-immunoreactive cells overlapped with calretinin-positive neurons in all
hippocampal subfields, showing that interneurons specialized to contact other gamma-
aminobutyric acid-ergic cells may also contain SPR. On the basis of the known termination
pattern of the colocalized markers, we conclude that SPR-positive interneurons are function-
ally heterogeneous and participate in different inhibitory processes: (1) perisomatic inhibition
of principal cells (CCK-containing cells, and parvalbumin-positive cells in the dentate gyrus),
(2) feedback dendritic inhibition in the entorhinal termination zone (somatostatin and
NPY-containing cells), and (3) innervation of other interneurons (calretinin-containing cells).
J. Comp. Neurol. 378:320–336, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: tachykinins; neuropeptides; receptors; colocalization; interneurons

Combined morphological and electrophysiological stud-
ies have revealed that interneurons with different termina-
tion patterns are responsible for diverse inhibitory pro-
cesses in the hippocampus (Lacaille et al., 1987; Lacaille
and Schwartzkroin, 1988a,b; Buzsáki et al., 1992; Gulyás
et al., 1993a,b; Soltész and Deschenes, 1993; Buhl et al.,
1994; McBain et al., 1994; Bragin et al., 1995; Buckmaster
and Schwartzkroin, 1995; Cobb et al., 1995; Sı́k et al.,
1995; Ylinen et al., 1995; Miles et al., 1996). The functional
classification of the interneuron populations became in-
creasingly difficult because morphologically identified types
and immunocytochemically characterized populations were
often not or poorly correlated. The relationship to physi-
ological characteristics was even more difficult to estab-
lish, but there have been encouraging attempts (Sı́k et al.,
1995). Recent studies on the termination pattern of hippo-
campal interneurons showed that on the basis of their role
in the inhibitory networks interneurons can be classified
into three basic categories, each consisting of several
subtypes. The first two kinds of interneurons contact
principal cells in two different membrane domains, i.e., in
the perisomatic region (basket and chandelier cells) and in
Contract grant sponsor: Human Frontier Science Program Organization;
Contract grant sponsor: The Howard Hughes Medical Institute; Contract
grant sponsor: OTKA; Contract grant number: T 16942.
*Correspondence to: Tamás F. Freund, Institute of Experimental Medi-
cine, Hungarian Academy of Sciences, Budapest, P.O. Box 67, H-1450,
Hungary. E-mail: freund@.koki.hu
Received 11 June 1996; Revised 18 September 1996; Accepted 27
September 1996

r 1997 WILEY-LISS, INC.

SPR-CONTAINING INTERNEURONS IN THE HIPPOCAMPUS 321

the dendritic region (Han et al., 1993; Gulyás et al., the termination pattern was hindered by the lack of axonal
1993a,b; Buhl et al., 1994; Miles et al., 1996), and the third staining. In previous experiments, combined morphologi-
kind is specialized to innervate other interneurons (Ac- cal and neurochemical studies of GABAergic cells were
sády et al., 1996b; Gulyás et al., 1996; Hájos et al., 1996). useful in separating functionally different sets of interneu-
Electrophysiological and modeling studies ascribe differ- rons labeled by the same marker (Acsády et al., 1996a,b).
ent roles for somatic and dendritic inhibition and to the Using similar methods, our aim in the present study was
local inhibitory control of gamma-aminobutyric acid to identify the morphologically heterogeneous SPR-
(GABA)-ergic cells. Basket and axo-axonic cells innervate immunoreactive neurons in the hippocampus according to
the perisomatic region of pyramidal cells (Somogyi et al., the content of neurochemical markers and to establish the
1983, 1985; Gulyás et al., 1993a; Buhl et al., 1994) and types of inhibitory circuits they are involved in.
have profound effects on the timing and repetitive firing of
sodium-dependent action potentials of pyramidal cells
(Cobb et al., 1995; Miles et al., 1996). The axons of den- METHODS
dritic inhibitory cells innervate the dendritic surface of the
Ten male Wistar rats (300–350 g, 2 months old; Charles
pyramidal cells in conjunction with various excitatory
River, Budapest, Hungary) were deeply anesthetized with
afferents (Gulyás et al., 1993a,b, 1996; Han et al., 1993;
Equithesin (chlornembutal, 0.3 ml/100 g) and perfused
Buhl et al., 1994; Sı́k et al., 1995) and may regulate calcium-
through the heart first with saline and then with phos-
dependent dendritic electrogenesis (Miles et al., 1996).
phate buffered (PB; 0.1 M) fixative containing 4% parafor-
Interneurons of the third category terminate almost exclu-
maldehyde, 0.2% picric acid, and 0.05% glutaraldehyde.
sively on other interneurons (Acsády et al., 1996b; Gulyás
Coronal sections (60 µm thick) were cut from the hippocam-
et al., 1996; Hájos et al., 1996). Specific interactions among
pus on a Vibratome, kept in sequence, washed, cryopro-
different sets of GABAergic cells may be involved in the
tected in 30% sucrose in 0.1 M PB overnight, and freeze
high-frequency population oscillations and/or in disinhibi-
thawed over liquid nitrogen. Following extensive washes,
tion (Müller and Misgeld, 1990; Michelson and Wong,
the sections were incubated in one of the following anti-
1991; Buzsáki and Chrobak, 1995; Whittington et al.,
sera: for single immunostaining, rabbit anti-SPR (1:300,
1995). The neurochemical marker content (neuropeptides
1.6 mg/ml; Shigemoto et al., 1993), rabbit anti-somatosta-
and calcium-binding proteins) of interneurons shows re-
tin (1:20,000; Lantos et al., 1995; or 1:300, Dakopatts),
markable correlation with these three categories. Parval-
rabbit anti-NPY (1:20,000; Csiffáry et al., 1990), rabbit
bumin and cholecystokinin (CCK) are present in two
anti-calretinin (1:5,000; Rogers, 1989), rabbit anti-VIP
separate types of perisomatic inhibitory cells (Nunzi et al.,
(1:10,000; Gulyás et al., 1990), rabbit anti-CCK (1:3,000;
1985; Kosaka et al., 1987; Acsády et al., 1996a). Calbindin-
Gulyás et al., 1990), or rabbit anti-GABA (1:20,000; Beau-
immunoreactive interneurons innervate the proximal den-
lieau et al., 1994) was used as primary antiserum (see
drites of pyramidal cells, and somatostatin- and neuropep-
Table 1A). The secondary antibody was biotinylated anti-
tide Y (NPY)-containing interneurons contact pyramidal
rabbit IgG made in goat (Vector; 1:200, 2 hours), followed
cells in the distal dendritic region (Köhler et al., 1986;
by avidin biotinylated-horseradish peroxidase complex
Deller and Léránth, 1990; Léránth et al., 1990; Buckmas-
(ABC; Vector; 1.5 hours, 1:150). All the washes and dilu-
ter et al., 1994; Sı́k et al., 1995, 1996; Gulyás and Freund,
tion of antisera were done in 0.05 M Tris buffered saline
1996; Katona et al., 1996). The majority of vasoactive
(TBS), pH 7.4. The immunoperoxidase reaction was devel-
intestinal polypeptide- (VIP) and calretinin-containing
oped with 3,38-diaminobenzidine (DAB) as a chromogen.
cells selectively innervate other interneurons (Acsády et
The sections were treated with 1% OsO4 in 0.1 M PB for 15
al., 1996b; Gulyás et al., 1996; Hájos et al., 1996). Thus,
minutes, dehydrated in ethanol and propylene oxide, and
neurochemical identification provides a valuable tool to
embedded in Durcupan (ACM, Fluka). The characteriza-
study large population of inhibitory cells with similar
tion of the SPR antiserum is described in Nakaya et al.
characteristics. Large numbers of nonprincipal cells have
(1994). Briefly, the antigen is a trpE-fusion protein that
been visualized in the hippocampus by an antiserum
contains rat SPR sequence (amino acid residues 349–407).
against substance P receptor (SPR; Kaneko et al., 1994;
Cross reactivity with NK2 and NK3 receptors was ex-
Nakaya et al., 1994). Whether this antigen functions as a
cluded by immonoblot and staining analyses of the recep-
receptor for tachykinins in these locations remains to be
tor transfected cell lines (Shigemoto et al., 1993). The
established because there is considerable mismatch with
distribution of SPR immunoreactivity is generally very
the distribution of the known endogenous ligands (Liu et
consistent with the autoradiography of SP-binding sites
al., 1994). However, the great detail of the morphology
(Mantyh et al., 1989). The specificity of the other primary
provided by the immunolocalization of SPR encourages its
antisera have been tested by the laboratories of origin (see
use as a marker in connectivity studies, even before the
references above). Replacing the primary antisera with
function of this protein is understood. The functional
normal sera of the animals in which the primary antisera
classification of SPR-immunoreactive neurons based on
were raised resulted in lack of immunostaining. SPR-
immunoreactive cells were reconstructed with a drawing
tube from serial 60-µm-thick sections. To study the coexist-
Abbreviations ence of SPR with calretinin, CCK, somatostatin, VIP, NPY,
DG gyrus dentatus
and GABA, the mirror technique of Kosaka et al. (1985)
CCK cholecystokinin was used. Because no obvious difference was found be-
CB calbindin D28k tween the dorsal and ventral parts of the hippocampus, the
CR calretinin analysis was carried out in the dorsal hippocampus (5–10
NPY neuropeptide Y
PV parvalbumin
sections/animal, 4–7 animals/colocalization studied). Adja-
SOM somatostatin cent sections were treated for SPR or one of the other
VIP vasoactive intestinal polypeptide antigens, and the cell bodies cut in half were identified on
322 L. ACSÁDY ET AL.

TABLE 1A. Antibodies Used for Preembedding Single Staining1 manner. Various types of dendritic appendages were also
Primary antibodies Source of Secondary antibody, labeled. Principal cells (granule, mossy, and pyramidal
and dilutions primary antibody source and dilution cells) were consistently devoid of SPR immunoreaction in
Rabbit-anti Substance P Shigemoto et al., 1993 Biotinylated anti-rabbit IgG the hippocampus. Ventral subiculum showed dense neuro-
receptor (1:300, 1.6 µg/ml) made in goat (1:200), Vector pil staining. SPR-positive axons were not present in the
Rabbit anti-somatostatin Lantos et al., 1995
(1:20000)
material. The morphology of SPR-containing cells showed
Rabbit anti-somatostatin (DAKOPATTS) considerable variation within and among the hippocampal
(1:300) subfields.
Rabbit anti-NPY (1:20000) Csiffáry et al., 1990
Rabbit anti-calretinin Rogers, 1989
(1:5000) Dentate gyrus
Rabbit anti-VIP (1:10000) Gulyás et al., 1990
Rabbit anti-CCK (1:3000) Gulyás et al., 1990 The most frequently encountered SPR-positive cell type
Rabbit anti-GABA (1:20000) Beaulieau et al., 1994 in the dentate gyrus belonged to the pyramidal-like cells
(Fig. 1A,D). The medium-sized pyramidal-shaped cell bod-
TABLE 1B. Antibodies Used for Double Immunofluorescence
ies of this cell type were situated mainly in the deep
Secondary Source of stratum (str.) granulosum or at the border of str. granulo-
Primary antibody Source of antibody and secondary
and its dilution primary antibody its dilution antibody
sum and the hilus. Their single, pronounced apical den-
drites crossed str. granulosum and branched profusely in
Rabbit-anti Sub- Shigemoto et al., Lissamine-rho- Jackson str. moleculare. The two or three thinner basal dendrites
stance P receptor 1993 damine sulfonyl
(1:200, 2.5 µg/ml) chloride-conju- entered the hilus. The dendrites of this cell type were
gated anti-rabbit aspiny. Besides pyramidal-like cells, large multipolar or
IgG made in
donkey (1:50) fusiform SPR-immunoreactive cells were also occasionally
Mouse anti-calbindin Celio, 1990 Fluorescein-conju- DAKOPATTS present in str. granulosum with aspiny dendrites entering
(1:2000) gated anti-mouse
IgG made in goat
str. moleculare and the hilus. Stratum moleculare con-
(1:50) tained a few stellate or fusiform SPR-positive cells, with
Mouse anti-parval- Celio, 1990 Fuorescein-conju- DAKOPATTS thin primary dendrites largely confined to this layer.
bumin (1:500) gated anti-mouse
IgG made in goat
(1:50) Hilus of the dentate gyrus
1Abbreviations:CCK: cholecystokinin, NPY: neuropeptide Y; VIP: vasoactive intestinal The most prominent SPR-immunostaining was present
polypeptide, SPR.
in the hilus (Fig. 1A). The profuse dendritic arbor of
numerous SPR-immunoreactive cells formed a dense mesh-
the common surfaces of both sections by using capillaries work filling the hilus. Close examination of this region
as landmarks. First, bisected SPR-positive cell bodies were showed that the hilar plexus consists of morphologically
identified on the surface of the sections by using 1003 different cell types. The most characteristic hilar SPR-
oil-immersion objective. Second, the corresponding halves containing cells were large, intensely stained, spiny neu-
of the cells were looked for on the matching surface of the rons (Fig. 1B–C). Two three-thick primary dendrites origi-
adjacent section. Cells were only included in the analysis if nated from the fusiform somata of these cells, running
the matching half could be unequivocally identified parallel to the granule cell layer. The dendrites of the spiny
(whether negative or positive). In the case of antibodies SPR-positive cells were restricted mainly to the hilus: they
that gave excellent dendritic staining, this method could never penetrated the granule cell layer but occasionally
be extended to examine cut main dendrites, as described entered str. radiatum of the CA3c region. The entire
previously (Acsády et al., 1996a). For fluorescent double dendritic arbor and the somata of these cells were densely
staining, the following mixtures of antisera were used (see covered with spines. The spines had thin, long, sometimes
Table 1B): rabbit anti-SPR (1:200, 2.5 mg/ml) and mouse branching, necks and small heads. Forty percent of all the
anticalbindin (1:2,000; Celio, 1990) or rabbit anti-SPR and SPR-containing cells belonged to this type in the hilus.
mouse antiparvalbumin (1:500; Celio, 1990). In all cases, Thorny excrescences, characteristic of mossy cells, were
the second layer was lissamine-rhodamine sulfonyl chlo- encountered neither on this nor on any other hilar SPR-
ride (LRSC)-conjugated anti-rabbit IgG made in donkey containing cell types. The hilus contained various smooth
(Jackson, 1:50) and fluorescein-conjugated anti-mouse IgG dendritic SPR-positive interneurons. Medium-sized multi-
made in goat (1:50; Dakopatts). The sections were mounted polar neurons with thick primary dendrites and stellate-
on glass slides in Vectashield (Vector) embedding medium. like cells with thin primary dendrites were distinguish-
The results were obtained from 9–12 sections from three able. In contrast to the spiny hilar SPR-positive cells, the
animals and evaluated with a Leitz Laborlux microscope dendrites of aspiny interneurons were radially oriented
with Ploemopak fluorescence illuminators using filters for and regularly crossed str. granulosum and branched in str.
fluorescein isothiocyanate (type I3, exciting filter band- moleculare.
pass 450–490) and LRSC (type N2, exciting filter bandpass
530–560). The CA3 region
A large number of SPR-immunoreactive interneurons
RESULTS were found in all strata of the CA3 region. The most
abundant cell type in all layers of the CA3 region were
Morphological characterization aspiny multipolar SPR-immunoreactive cells, with radial
Immunostaining for SPR labeled numerous, morphologi- thin primary dendrites (Fig. 2A). The 5–7 primary den-
cally heterogeneous nonprincipal cells in the hippocampus drites of these cells branched in close proximity to the
as reported previously (Nakaya et al., 1994). The immuno- round or irregularly shaped soma. Various larger multipo-
precipitate was membrane-bound and visualized cell bod- lar or bitufted SPR-containing interneurons were also
ies and proximal and distal dendrites in a Golgi-like observed with thick, more distally branching, primary
 Fig 1. A: Low magnification light micrograph of the dentate gyrus
immunostained for substance P receptor (SPR). Note the extremely
dense meshwork of dendrites in the hilus, which is formed mainly by
spiny dendrites of large fusiform cells (see B–C) and the pyramidal-
like cells (arrowheads in D) at the hilar border of the granule cell layer.
B,C: Spiny cells in the hilus restricted their dendrites to the hilus
(arrowheads in B) and always proved to be positive for somatostatin.
D: In contrast, dendrites of SPR-positive pyramidallike cells in str.
granulosum (s.g.) are smooth and extend into str. moleculare. Approxi-
mately half of the SPR-immunoreactive cells in str. granulosum
contained parvalbumin (see Fig. 5), demonstrating that they are
perisomatic inhibitory cells. Scale bars 5 100 µm in A,C, 30 µm in B,
20 µm in D.
324
dendrites. A characteristic subgroup of these cells confined
the majority of their dendrites to str. oriens and had a few
stubby spines. Stratum lucidum contained numerous SPR-
positive spiny dendrites restricted to this layer and run-
ning parallel to str. pyramidale. These dendrites origi-
nated from large spiny cells, with morphology similar to
hilar spiny SPR-positive neurons (Fig. 2B).
The CA1 region
The CA1 region contained multipolar cells of various
sizes (Fig. 2C). The aspiny multipolar cells looked similar
to their counterparts in the CA3 region. Their aspiny
dendrites branched near the cell bodies and arborized in
the vicinity of the soma in a stellatelike manner (Fig. 3A).
In contrast, the thick primary dendrites of large, darkly
stained, multipolar or bitufted cells (Fig. 2D) crossed
several layers and occasionally bore large numbers of
spines. In the CA1 region, numerous SPR-positive den-
drites ran horizontally at the border of str. radiatum and
str. lacunosum-moleculare, but relatively few entered str.
lacunosum-moleculare. In summary, four major cell types
were distinguished throughout the hippocampus on the
basis of location and dendritic morphology: (1) spiny,
horizontal, fusiform cells were confined to the hilus and
str. lucidum of the CA3 region, (2) pyramidal-shaped cells
or fusiform cells, present only in str. granulosum of the
dentate gyrus, (3) aspiny multipolar cells with thin radial,
primary dendrites branching close to the soma, and (4)
larger, more robust aspiny or sparsely spiny multipolar or
bitufted cells with thick, more distally branching den-
drites. Several SPR-positive cells showed mixed features
of types 3 and 4. These multipolar cells were present
throughout all subfields and all layers in the hippocampus.
Because SPR immunocytochemistry provides no axonal
staining to investigate the role of various SPR-positive
interneurons in the hippocampal network and to examine
the correlation of morphology and neurochemical charac-
teristics, colocalization studies were carried out with neu-
rochemical markers that label well-defined, functionally
distinct sets of GABAergic cells and with GABA itself.
Colocalization of SPR with GABA
The mirror technique of Kosaka et al. (1985; see Meth-
ods) was used to establish the colocalization of SPR and
GABA. From 103 SPR-immunoreactive interneurons, 92
(89%) were unequivocally GABA positive. The remaining
11 GABA-negative or weakly positive neurons all belonged
to the spiny fusiform cell type situated in the hilus and str.
lucidum of the CA3 region (Fig. 3B–C). This number
represented 67% of all the hilar spiny SPR-positive neu-
rons (n 5 17). The hilar region contains a large number of
neurons with distant projections in which GABA cannot be
detected by immunocytochemical methods, at least in their
soma. Thus, the GABA negativity of these cells might be
explained by their long projecting nature.
Colocalization with markers of interneurons
innervating the perisomatic region of
pyramidal cells (CCK, parvalbumin)
Parvalbumin and CCK label discrete interneuron popu-
lations with different calcium-binding protein and neuro-
peptide content and with distinct intrahippocampal and
extrahippocampal connections but with similar target
selectivity, i.e., the perisomatic region of pyramidal cells.
In the hippocampus, parvalbumin is present in basket and
L. ACSÁDY ET AL.
axo-axonic cells, whereas CCK labels another nonoverlap-
ping population of basket cells (Gulyás et al., 1991, 1996;
Acsády et al., 1996a). The CCK–SPR colocalization was
examined by the mirror technique, and fluorescent double
staining was used to establish the coexistence of parvalbu-
min and SPR. The CCK and parvalbumin staining was
similar to the results of others (Baimbridge and Miller,
1982; Nunzi et al., 1985; Kosaka et al., 1987; Celio, 1990).
Briefly, CCK labeled mainly basket cells scattered through
all layers and all regions of the hippocampus, and parval-
bumin immunofluorescence was present in basket and
axo-axonic cells of the dentate gyrus, hilus, and in str.
pyramidale and oriens of the Ammon’s horn. Nearly all
CCK-immunoreactive cells (n 5 67) showed SPR immu-
nopositivity in all regions, whcih represented 4–19% of all
SPR-containing interneurons in the various subfields (see
Table 2). The majority of the CCK1/SPR1 cells in all
regions were large, darkly stained, multipolar or bitufted
cells with thick primary dendrites, which occasionally
crossed several layers without branching (Fig. 4). The
dendrites of CCK1/SPR1 interneurons rarely penetrated
str. lacunosum-moleculare and were occasionally deco-
rated with substantial numbers of spines in the CA1
region. Some of the SPR-immunoreactive pyramidal-
shaped or fusiform cells in the dentate gyrus also con-
tained CCK. Significant colocalization between SPR and
parvalbumin was only found in the pyramidal-shaped or
fusiform cells of the dentate gyrus (Fig. 5A–B). Parvalbu-
min-containing cells in str. granulosum of the dentate
gyrus contained SPR in 90% of the cases (n 5 69), which
represented 59% of the SPR-immunoreactive cells in this
layer (n 5 78). In sharp contrast, only 5% of the SPR-
positive interneurons colocalized parvalbumin in the hilus
and the Ammon’s horn (n 5 190), which corresponds to 6%
of parvalbumin-positive cells in these regions (n 5 129).
Colocalization with markers of interneurons
innervating the dendritic region of pyramidal
cells (calbindin, somatostatin, NPY)
The colocalization of SPR-somatostatin and SPR–NPY
were studied by the mirror technique, and fluorescent
double staining was used to establish the coexistence of
SPR and calbindin. The distribution of the interneurons
labeled by the markers were similar to the earlier results.
Briefly, somatostatin-containing cells were present in the
hilus and in str. oriens of CA3 and CA1 regions (Morrison
et al., 1982; Köhler and Chan-Palay, 1983). NPY-positive
cells showed a similar distribution; however, a number of
cells were situated in str. pyramidale and radiatum of the
CA1 region and at the base of granule cell layer (Köhler et
al., 1986; Deller and Léránthh, 1990). Antisera to calbin-
din stained the granule cells of the dentate gyrus, the CA1
pyramidal cells, and scattered interneurons in the den-
dritic region of the principal cells (Baimbridge and Miller,
1982; Celio, 1990; Tóth and Freund, 1992). SPR and
somatostatin colocalization showed large regional hetero-
geneity (Table 2). In the hilus and str. lucidum of the CA3
region, all large spiny fusiform SPR-containing cells (n 5
25) were immunoreactive for somatostatin (Fig. 6A–C). In
the hilus, these cells represented 73% of all somatostatin-
positive cells (n 5 25). In contrast, in the CA1 region only
8% of the somatostatin-positive cells colocalized SPR. In
addition to spiny cells in the hilus and str. lucidum of the
CA3 region, some multipolar SPR-positive cells also colocal-
ized with somatostatin in str. oriens of CA3 region (Fig.
 Fig. 2. A: Low magnification light micrographs of the CA3 region
immunostained for SPR. Stratum lucidum is outlined by a dense
network of SPR-positive dendrites (arrowheads). The border between
CA3 and CA1 is indicated by a dashed line. CA1 contains less intensive
dendritic staining than CA3. B: The dense network of SPR-containing
dendrites in str. lucidum (s.l.) mainly contains long, tangentially
running spiny dendrites (arrowheads, inset) of large SPR-immunore-
active cells. C: Low magnification light micrograph of the CA1 region
immunostained for SPR showing the morphological heterogeneity of
positive cells. Arrows point to large multipolar cells (see Fig. 3), which
were sparsely spiny and always cholecystokinin (CCK) positive.
Arrowheads identify small smooth dendritic cells, which were either
calretinin positive or contained neuropeptide Y (NPY). D: Light
micrograph of a group of large multipolar SPR-positive cells in the
CA1 region. This type proved to be CCK-positive basket cells. s.p., str.
pyramidale. Scale bars 5 200 µm in A, 50 µm in B, 100 µm in C, 50 µm
in D.
 Fig. 3. A: Camera lucida drawings of small multipolar SPR-
immunoreactive neurons in the CA1 region. These cells have many
primary dendrites, pronounced dendritic tufts close to the cell body,
and frequently contain NPY. B–C: Colocalization of SPR and gamma-
aminobutyric acid (GABA) immunoreactivity in different neurons of
the dentate gyrus. A large spiny SPR-positive cell (s1) is only faintly
positive for GABA, whereas an SPR-containing pyramidallike cell (s2)
shows strong GABA immunoreactivity. Arrow points to a GABA-
positive SPR-negative interneuron with a similar soma shape and
location as s2. Granule cells are negative for both antigens. c1 and c2
label capillaries that help identify the cells. str. rad., str. radicum; str.
pyr., str. pyramidale; str. ori., str. oriens. Scale bars 5 20 µm.
SPR-CONTAINING INTERNEURONS IN THE HIPPOCAMPUS 327

TABLE 2. Coexistence of SPR With Calcium Binding Proteins soma (Fig. 8). Multiple dendrodendritic contacts, a charac-
and Neuropeptides1 teristic feature of some calretinin-positive cells (Gulyás et
CCK PV SOM NPY CB CR VIP al., 1992), were rarely observed. Calretinin also labels spiny
n 5 164 n 5 268 n 5 63 n 5 134 n 5 138 n 5 102 n 5 118 cells (Gulyás et al., 1992), which display anatomical features
DG 4% 59% — 28% — — 0% similar to spiny SPR-positive cells (i.e., dendrites are covered
Hilus 13% 0% 45% 60% — 25% 0% with long spines and are restricted to the hilus). However,
CA3 17% 9% 19% 12% 3% 23% 1%
CA1 19% 0% 18% 60% 8% 30% 2% from the 52 spiny SPR-immunoreactive cells, only four con-
tained calretinin. VIP-containing cells form two major morpho-
A) Percentage of SPR-positive cells immunoreactive for different markers. N indicates
number of sampled SPR-immunoreactive cells. logically and neurochemically different cell populations in the
hippocampus (Acsády et al., 1996a,b). Small bipolar or oligopo-
CCK PV SOM NPY CB CR VIP
n 5 67 n 5 198 n 5 103 n 5 102 n 5 72 n 5 77 n 5 60 lar VIP-containing cells, which innervate other GABAergic
cells, never contained SPR. However, large multipolar VIP-
DG 100% 90% — 80% — — 0%
Hilus 100% 0% 73% 84% — 38% 0% positive interneurons that innervate the perisomatic region of
CA3 81% 12% 70% 55% 14% 50% 7% pyramidal cells always showed SPR immunoreactivity. This
CA1 90% 0% 8% 51% 10% 37% 20%
observation is consistent with earlier results, because VIP-
B) Percentage of calcium binding protein- and neuropeptide-containing cells immunore- containing basket cells were reported to colocalize CCK, and
active for SPR. N indicates number of sampled calcium binding protein- and neuropep-
tide-containing cells.
nearly all CCK-positive cells contain SPR.
1Abbreviations: DG: gyrus dentatus; CCK: cholecystokinin; CB: calbindin; CR: calreti-

nin; NPY: neuropeptide Y; PV: parvalbumin; SOM: somatostatin; VIP: vasoactive

intestinal polypeptide.
6D–E). The thick, distally branching primary dendrites of
these cells were confined to str. oriens and were occasion-
ally decorated with stubby spines. Many of the spine-free
dendrites belonging to SOM1SPR1 cells became spiny
upon entering or crossing str. lucidum. The morphology of
interneurons containing both NPY and SPR differed among
the different subfields of the hippocampus. In the dentate
gyrus, pyramidallike SPR-immunoreactive cells contained
NPY in 28% of the cases (n 5 14). In the hilus and str.
lucidum of CA3, nearly all large spiny SPR-positive cells
showed NPY immunoreactivity (92%, n 5 25), also demon-
strating the colocalization of NPY and somatostatin in this
cell type (Fig. 7). From the remaining smooth dendritic
multipolar SPR-positive cells, an average of 36% contained
NPY (n 5 74) for the whole hippocampus. The highest
colocalization rate was found in the CA1 region (60%) and
the lowest in the CA3 region (12.5%). These SPR1/NPY1
multipolar cells always belonged to the aspiny multipolar
cell type, with thin primary dendrites branching close to
the soma, and showed consistently weaker immunostain-
ing for NPY than did SPR-negative NPY-containing cells.
Calbindin was present in only a very small fraction of the
SPR-immunoreactive neurons (6%, n 5 318; Fig. 5C–F).
These multipolar cells in the CA3 region emitted thick,
rarely branching, dendrites largely confined to str. oriens
and had a few stubby spines. The SPR immunoreactivity of
the hilar calbindin-positive interneurons could not be
reliably established due to the intense calbindin immuno-
fluorescence of the mossy fibers.
Colocalization with markers of interneurons
specialized to innervate other interneurons
(calretinin, VIP)
The majority of calretinin- and VIP-containing interneu-
rons selectively innervate other GABAergic cells in the
hippocampus (Acsády et al., 1996b; Gulyás et al., 1996;
Hájos et al., 1996). Their colocalization with SPR was
examined by using the mirror technique. In the whole
hippocampus, 25% of the SPR-positive cells (n 5 102)
displayed calretinin immunoreactivity, with little regional
variation, which represented 40% of the calretinin-
containing cells. The SPR1/calretinin1 cells were aspiny
multipolar cells with dendrites branching close to the
DISCUSSION
In the present study, we have shown that in the hippo-
campus SPR labels several morphologically, neurochemi-
cally, and functionally heterogeneous interneuron popula-
tions. GABAergic cells of all the three major interneuron
classes, i.e., perisomatic and dendritic inhibitory cells and
GABAergic cells specialized to contact other interneurons,
were found among the SPR-immunoreactive neurons.
Nearly all CCK-positive interneurons contained SPR,
whereas parvalbumin colocalized with SPR only in str.
granulosum of the dentate gyrus but not in other subfields.
In the hilus and str. lucidum of the CA3 region, somatosta-
tin and NPY were present in large spiny SPR-immunoreac-
tive cells. In contrast, most of the somatostatin-positive
cells were devoid of SPR in the CA1 region. Calretinin-
immunoreactive cells also showed considerable overlap
with SPR-positive neurons in all regions. Previous studies
of interneuron populations labeled by various neurochemi-
cal markers have shown that in most cases neuropeptides
and calcium-binding proteins label homogeneous popula-
tion of GABAergic cells (Baimbridge and Miller, 1982;
Somogyi et al., 1984; Roberts et al., 1984; Kosaka et al.,
1987; Sloviter and Nilaver, 1987; Celio 1990; Gulyás et al.,
1992). Moreover, the types of interneurons immunoreac-
tive for a given marker were remarkably similar among
hippocampal subfields. For example, the calcium-binding
protein parvalbumin is present in basket and axo-axonic
cells in the whole hippocampus (Kawaguchi et al., 1987),
whereas the dendritic and somatic distribution of somato-
statin-containing cells is specialized for a role in feedback
inhibition of distal principal cell dendrites in all subfields
(Baude et al., 1993; McBain et al., 1994; Blasco-Ibanez and
Freud, 1995; Sı́k et al., 1995, 1996). Occasionally, the same
proteins and peptides (e.g., VIP and calretinin) may be
present in a heterogeneous population of interneurons.
However, in these cases, correlation of morphological and
neurochemical features identify subpopulations with dis-
tinct connectivity and function (Gulyás et al., 1992; Acsády
et al., 1996a,b; Hájos et al., 1996). The high degree of
heterogeneity of SPR-immunoreactive cells found in this
study is incomparable to that of any other markers.
SPR-immunoreactive cells may contain any
of the examined markers
In the present study, all seven neurochemical markers
showed various degrees of colocalization with SPR. The
 Fig. 4. A: Camera lucida drawings of multipolar or bitufted
SPR-positive cells in the CA1 region also showing CCK immunoreactiv-
ity. Note the large cell body and the thick primary dendrites. Nearly all
CCK-positive cells also colocalized SPR. B,C: High magnification light
micrographs of an SPR-positive cell that contains CCK as demon-
strated by the mirror technique in the CA3 region. Arrow points to a
SPR-positive CCK-negative cell body. Capillaries labeled by c1–c3
serve as landmarks. D,E: Large multipolar SPR-positive cell in str.
pyramidale of the CA1 region cut in half on the surface of the section.
The same cell shows CCK immunoreactivity in the adjacent section.
The thick-cut primary dendrite (arrowhead) continues into the match-
ing section. Arrow points to a SPR-positive CCK-negative cell body.
Capillaries labeled by c1–c2 serve as landmarks. str. l.m., str. lacuno-
sum-moleculare. Scale bars 5 20 µm.
SPR-CONTAINING INTERNEURONS IN THE HIPPOCAMPUS
Fig. 5. Light micrographs of sections with double-immunofluores-
cent staining for SPR and the calcium-binding proteins parvalbumin
and calbindin. A,B: SPR-positive pyramidallike basket cells contain
parvalbumin in str. granulosum of the dentate gyrus. Parvalbumin-
positive interneurons were negative for SPR in all other subfields of
329
the hippocampus. C–D: Calbindin-immunoreactive cells showed SPR
positivity only in str. oriens of the CA3 region. The SPR-positive cell in
str. radiatum of the CA1 region (E, arrow) is negative for calbindin
(arrow in F). c, corresponding capillary. Scale bars 5 20 µm.
Figure 6
SPR-CONTAINING INTERNEURONS IN THE HIPPOCAMPUS
colocalization of these markers with each other have
largely been established. Parvalbumin does not colocalize
with any other markers, apart from a few of them that
contain somatostatin or NPY (Gulyás et al., 1991; Miet-
tinen et al., 1992; Gao and Fritschy 1994; Acsády et al.,
1996a). Calbindin-containing cells in CA1 and CA3 str.
oriens are mostly positive for somatostatin (Katona et al.,
1996) or, particularly in the CA3 region, for CCK but not
for other markers (Gulyás et al., 1991; Miettinen et al.,
1992). Calretinin colocalizes with somatostatin in spiny
calretinin-positive cells (Katona et al., 1996), but 25% of
the aspiny calretinin-immunoreactive cells contain VIP
(Acsády et al., 1996a). VIP-immunoreactive interneurons
containing calretinin innervate other GABAergic cells,
whereas VIP and CCK coexist in parvalbumin-negative
basket cells (Acsády et al., 1996a,b). The majority of
CCK-positive cells are negative for all the other markers
except VIP (Somogyi et al., 1984; Gulyás et al., 1991;
Acsády et al., 1996a). The substantial overlap between
somatostatin and NPY-immunoreactive cells was also dem-
onstrated (Köhler et al., 1987). Based on these observa-
tions and summing the proportion of SPR-immunoreactive
cells colocalizing other markers (see Table 2), we can
conclude that 80–90% of all the SPR-containing cells are
labeled by one of the markers or by a combination of them.
Although simultaneous colocalization of SPR with two
other markers was not directly demonstrated, the coexist-
ence of SPR with somatostatin and NPY in the hilar spiny
cells is highly probable. Similarly, VIP-immunoreactive
basket cells positive for colocalizing SPR are also likely
contain CCK. The large calbindin- and SPR-positive multi-
polar cells in the CA3 str. oriens may also contain somato-
statin or, occasionally, CCK.
SPR-positive perisomatic inhibitory cells
Morphologically distinct SPR-immunoreactive interneu-
rons colocalized CCK and occasionally VIP but not the
other markers. The majority of the cells in which SPR and
CCK were colocalized had few thick primary dendrites.
Previous studies have shown that CCK-positive interneu-
rons innervate the perisomatic region of pyramidal cells;
however, due to poor staining intensity, the extent of their
dendritic arbor could not be established. SPR immunostain-
ing visualized the dendritic arbor of CCK-containing cells
in a Golgilike manner. In the CA1 region, they were largely
confined to strata oriens, radiatum, and pyramidale, with
occasional thin distal dendrites penetrating str. lacunosum-
moleculare. The dendritic distribution of CCK-immunore-
active cells suggests that the principal excitatory drive to
these cells is likely to originate from ipsi- and contralateral
Fig. 6. Somatostatin-containing neurons form a distinct group of
SPR-immunoreactive cells. A: Camera lucida drawing of a large spiny
fusiform SPR-containing neuron in the hilus. The soma and the
proximal and distal dendrites are densely covered with spines. All the
cells with similar morphology examined were positive for somatosta-
tin and for NPY. The arrow indicates the axon initial segment arising
from a proximal dendrite. B,C: High magnification light micrographs
of two adjacent sections demonstrate that spiny SPR-positive cells in
the hilus also contain somatostatin. The dense meshwork of SPR-
positive dendrites in the hilus mainly consists of spiny dendrites
(arrowheads in B) of this cell type. c1 and c2 label capillaries used as
landmarks. D,E: Large aspiny or sparsely spiny SPR-immunoreactive
cells also contained somatostatin (s1, s2) in str. oriens of the CA3
region. c1–c3 labels corresponding capillaries. Scale bars 5 20 µm.
331
Schaffer collaterals. They are unlikely to participate signifi-
cantly in the perforant path-induced, feed-forward inhibi-
tion, which is pronounced in the CA1 region (Buzsáki and
Eidelberg, 1982). Thus, this effect probably originates from
parvalbumin-containing basket and chandelier cells. In
str. granulosum of the dentate gyrus, all parvalbumin-
positive (perisomatic inhibitory) cells colocalized SPR,
whereas in the hilus and Ammon’s horn, parvalbumin-
positive basket and chandelier cells did not contain SPR.
Parvalbumin-positive cells show morphological diversity
in the dentate gyrus (Baimbridge and Miller, 1982; Ribak,
1992). In the present study, however, the location of the
parvalbumin-containing cells rather than their morphol-
ogy was directly related to SPR content, i.e., in str.
granulosum all parvalbumin cells contained SPR regard-
less of their pyramidal shape or fusiform morphology,
whereas in the adjacent hilus none of the parvalbumin-
positive cell were immunoreactive for SPR. This striking
difference between parvalbumin-containing interneurons
in the hilus versus str. granulosum might be explained by
the pattern of substance P-containing innervation of this
region. In contrast to parvalbumin-containing neurons,
CCK-positive basket cells showed SPR immunoreactivity
in all hippocampal subfields, further suggesting that func-
tional differences exist between the two basket cell popula-
tions (Acsády et al., 1996a).
SPR-positive dendritic inhibitory cells and
SPR-positive interneurons innervating other
GABAergic cells
Large, fusiform, spiny cells in the hilus and str. lucidum
of the CA3 region always contained somatostatin and NPY
but were negative for the other markers. Interneurons
with identical morphology have been described in previous
Golgi studies and in vivo and in vitro intracellular labeling
experiments (Amaral, 1978; Han et al., 1993; Buckmaster
and Schwartzkroin, 1995), but their NPY content has been
directly demonstrated only for a singe cell (Sı́k et al.,
1996). In immunocytochemical studies, somatostatin/NPY-
containing cells projected to the outer two-thirds of the
molecular layer and to innervate the contralateral dentate
gyrus (Köhler et al., 1986; Deller and Léránthh, 1990;
Léránthh et al., 1990). In a recent study (Baude et al.,
1993), the dendritic distribution of somatostatin-contain-
ing cells were selectively visualized by immunostaining for
metabotropic glutamate receptor 1a (mGluR1a). Baude et
al. (1993) found only a few mGluR-positive dendrites
crossing str. granulosum; the majority were restricted to
the hilus. In the present study, the dendritic distribution of
a large number of somatostatin/NPY-containing cells could
be examined by SPR immunostaining. The dendrites of
spiny SPR-positive neurons were confined to the hilus and
occasionally penetrated str. radiatum of the CA3c region
but never crossed str. granulosum. Based on this observa-
tion, we can conclude that somatostatin/NPY-containing,
spiny SPR-positive cells receive excitatory input almost
exclusively from the mossy fiber collaterals and thus are
driven primarily in a feedback manner as their counter-
part in the CA1 region (Blasco-Ibanez and Freund, 1995).
In the hilus, spiny SPR-containing (i.e., somatostatin and
NPY-positive) cells were many times faintly labeled or
negative for GABA, whereas in a recent study all somato-
statin-containing cells contained the mRNA of GAD, the
 Fig. 7. Colocalization of SPR-immunoreactivity with NPY. A,B:
Spiny SPR cells (S) from the hilar region also proved to be positive for
NPY (S). C,D: Multipolar SPR-immunoreactive neurons (S) from CA1
str. oriens possess an extensive dendritic arbor (arrowheads) and
express low-level NPY immunoreactivity (S in D). E,F: Strongly
NPY-immunoreactive neurons on the CA1 str. oriens/alveus border
(arrow in F) never showed SPR positivity (empty cell labeled with an
arrow on E). c1–c3, capillaries serving as landmarks for alignment.
Scale bars 5 20 µm.
SPR-CONTAINING INTERNEURONS IN THE HIPPOCAMPUS 333

Fig. 8. A: Camera lucida drawing of SPR-immunoreactive interneurons also containing calretinin.

Although the cells have prominent distal dendritic tufts, they have only few primary dendrites. B,C: An
SPR-positive interneuron in CA1 str. radiatum cut in half also shows calretinin immunostaining in the
adjacent section. Scale bars 5 20 µm.

synthesizing enzyme of GABA (Esclapez and Houser,
1995). This apparent discrepancy between the in situ
hybridization technique and immunocytochemistry may
be explained by the lower sensitivity of immunohistochem-
istry, which is unable to detect the low level of GABA
present in GABAergic cells with distant projection (Miet-
tinen et al., 1992; Tóth et al., 1993). In the hilus and the
CA3 region, spiny SPR-positive cells contained somatosta-
tin and NPY. In contrast, only a very small proportion of
the somatostatin-containing cells colocalized SPR in the
CA1 region. This neurochemical difference between somato-
statin-positive neurons is again surprising, because their
role (i.e., feedback inhibition in the distal dendritic region
of pricipal cells) is similar in all hippocampal subfields
(Baude et al., 1993; Han et al., 1993; McBain et al., 1994;
Blasco-Ibanez and Freund, 1995). In str. oriens of the CA3
region, large SPR-immunoreactive cells with smooth thick
primary dendrites were positive for somatostatin; more-
over, they contained calbindin, another marker for inter-
neurons responsible for dendritic inhibition. In this sub-
field, calbindin is also present in some CCK-containing
(SPR-positive) basket cells, whereas in the rest of the
hippocampus calbindin-positive cells were negative for
SPR. Multipolar SPR-immunoreactive cells with thin pri-
mary dendrites branching close to the somata were hetero-
geneous with regard to the colocalized marker. The major-
ity of these cells contained either calretinin or NPY. Recent
studies on the postsynaptic target distribution of calretinin-
positive interneurons showed that they selectively inner-
vate other interneurons (Gulyás et al., 1996), whereas
NPY-containing cells innervate the dendritic region of
pyramidal cells (Deller and Léránthh, 1990). In spite of the
heterogeneity in termination pattern, the somadendritic
morphology of SPR-immunoreactive cells containing cal-
retinin or NPY did not show consistent morphological
differences. Comparison of the SPR-positive and negative
calretinin-immunoreactive interneurons showed that the
SPR-positive cells rarely participate in dendrodendritic
contacts, which is a feature characteristic of numerous
calretinin-positive interneurons (Gulyás et al., 1992).
334
Correlation of the morphology and
neurochemical marker content of
SPR-immunoreactive interneurons
For two subpopulations of SPR-immunoreactive neu-
rons, morphological characteristics showed a direct corre-
lation with marker content. The numerous, large, spiny
SPR-positive neurons in the hilus and str. lucidum of the
CA3 region were always positive for somatostatin. Simi-
larly large, multipolar, SPR-positve cells always contained
CCK in the CA1 region. However, SPR-immunoreactive
interneurons belonging to other morphologically estab-
lished classes did not show characteristic neurochemical
marker contents. Pyramidal-like and fusiform cells in the
dentate gyrus contained parvalbumin, CCK, or NPY. These
three substances do not colocalize with each other and are
localized in cell types with different termination patterns.
Parvalbumin-positive cells innervate the perisomatic re-
gion of granule cells (Kosaka et al., 1987; Soriano et al.,
1990; Ribak et al., 1990), the majority of CCK-containing
terminals form baskets in the hilar region (Léránth and
Frotscher, 1986), and NPY axons mainly arborize in str.
moleculare (Köhler et al., 1986; Deller and Léránth, 1990).
This fact indicates that neurons with similar soma-
dendritic morphology may have different axonal distribu-
tion and target selectivity. Earlier Golgi studies have
shown that interneurons with pyramidal-like cell bodies
may innervate the perisomatic and the dendritic regions of
the granule cells (Amaral, 1978; Soriano and Frothcher,
1993), which was confirmed by a recent intracellular
labeling study (Scharfman, 1995). Here, we have demon-
strated that, in addition to morphological and electrophysi-
ological differences, pyramidal-shaped cells in the dentate
gyrus also show neurochemical heterogeneity. Similarly to
pyramidal-shaped cells, multipolar cells with thin primary
dendrites in the CA1 and CA3 regions showed no consis-
tent neurochemical characteristics because they contained
either NPY or calretinin.
Functional implications
The endogenous substrate of SPR is probably substance
P, although related tachykinins may also bind this recep-
tor. Substance P-positive axons of extra- and intrahippo-
campal origin have been described in a number of species
including guinea pig, cat, monkey, and human (Gall and
Selawski 1984; Yanagihara and Niimi, 1989; Seress and
Léránth, 1996). However, the presence and number of
substance P-containing fibers and neurons in the hippocam-
pus of the rat is still a controversial issue and seems to
depend on the fixation protocol and the antibody used
(Ljungdahl et al., 1978; Roberts et al., 1984; Davis and
Köhler, 1985; Iritani et al., 1989; Léránth and Borhegyi,
personal communication). The extrahippocampal sub-
stance P-containing fibers in the cat, guinea pig, and
monkey originate in the supramammillary nucleus (Gall
and Selawski, 1984; Ino et al., 1989; Yanagihara and
Niimi., 1989; Léránth and Nitsch, 1994) and arborize in
the supragranular layer of the dentate gyrus and in the
CA2 region. These subcortical fibers terminate almost
exclusively on principal cells in the rat (Maglóczky et al.,
1994). Recent colocalization studies have shown that the
intrahippocampal substance P-containing fibers derive
from somatostatin-positive cells and arborize in the outer
two-thirds of the molecular layer in the monkey (Seress
and Léránth, 1996), where they probably innervate gran-
L. ACSÁDY ET AL.
ule cells. Thus, there seems to be a regional and cell-type-
associated mismatch between the distribution of SPR
immunoreactivity and substance P-containing axons. SPR-
positive dendrites are abundant in regions (i.e., hilus)
where no intrahippocampal or extrahippocampal sub-
stance P-positive fibers terminate. In addition, interneu-
rons, which are not the principal targets of intrinsic or
extrinsic substance P-positive fibers, express the receptor.
The mismatch between substance P and SPR have been
directly demonstrated (Liu et al., 1994), and a nonsynaptic
mechanism was suggested as the mode of action for
substance P (Mantyh et al., 1995). Substance P diffused to
considerable distance in the spinal cord (Duggan et al.,
1990). The action of substance P is probably excitatory to
nonprincipal cells in the hippocampus (Dreifuss and
Raggenbass, 1986). Our results suggest that diverse inhibi-
tory mechanisms might be facilitated, by a diffuse release
of substance P, through SPR, which is present on interneu-
rons participating in perisomatic and dendritic inhibition
of principal cells and in the innervation of other GABAer-
gic interneurons.
ACKNOWLEDGMENTS
This sudy was supported by grants from the Human
Frontier Science Program Organisation, the Howard
Hughes Medical Institute, and OTKA (T 16942) Hungary.
We are grateful to Dr. K.G. Baimbridge and to Dr. M.R.
Celio (calbindin and parvalbumin), Dr. T. Görcs (CCK, VIP,
NPY, and somatostatin), Dr. J.H. Rogers (calretinin), and
Dr. C.G. Beaulieau (GABA) for kind gifts of antisera. The
excellent technical assistance of Mrs. E. Borók, Mrs. A.Z.
Szabó, and Mr. G. Terstyánszky is also acknowledged.
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