Brain Research 877 (2000) 262–270

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Research report

GFRa-1 is expressed in parvalbumin GABAergic neurons in the

hippocampus
a a b a,
Arezou Sarabi , Barry J. Hoffer , Lars Olson , Marisela Morales *
a
National Institute on Drug Abuse, Cellular Neurophysiology, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA
b
Karolinska Institute, Department of Neuroscience, S-17177 Stockholm, Sweden

Accepted 5 July 2000

Abstract

Glial cell line derived neurotrophic factor (GDNF) is a potent survival factor for several types of neurons. GDNF binds with high
affinity to GDNF-family receptor a-1 (GFRa-1). This receptor is expressed in different areas of the brain, including the hippocampus and
dentate gyrus. By using in situ hybridization and immunohistochemistry, we found that 19% to 37% of glutamic acid decarboxylase
(GAD) expressing neurons co-expressed GFRa-1 in the hippocampus. GFRa-1 / GAD co-expression was found mainly in the stratum (s)
pyramidale (29–37%) and s. oriens (20–25%). Further characterization of GFRa-1 expressing interneurons, based on their calcium-
binding protein immunoreactivity, demonstrated that many parvalbumin (PV) immunoreactive neurons express GFRa-1 in the s.
pyramidale of CA1 (72%), CA2 (70%) and CA3 (70%) subfields of the hippocampus. GFRa-1 / PV double labeled neurons were also
detected in the s. oriens of CA1 (52%), CA2 (27%) and CA3 (36%) subfields. The expression of GFRa-1 in principal neurons and in a
specific sub-population of GABAergic neurons (PV-containing neurons) suggest that GDNF might modulate, in a selective manner,
functions of the entire adult hippocampus.  2000 Elsevier Science B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters and receptors

Topic: Signal transduction: gene expression

Keywords: GDNF; Interneurons; Calcium-binding protein

1. Introduction there is also a Ret independent signaling pathway that

GDNF promotes survival and differentiation of several
distinct populations of central and peripheral neurons
[4,5,12,22,23,26,38]. Moreover, GDNF is critical for the
development of the kidney and enteric nervous system
[24,29,36]. Responses to GDNF are mediated through a
receptor complex consisting of the transmembrane Ret
receptor tyrosine kinase [7,41] and a ligand binding
glycosyl-phosphatidyl inositol linked protein, GFRa-1
[15,40]. GDNF binding to GFRa-1 induces tyrosine
phosphorylation [7,40,41] which in turn triggers various
intracellular signaling pathways [6,30,43,44], including
phosphatidylinositol 3-kinase [6,30,43,44] and Ras / ERK
pathways [43]. However, recent evidence indicate that
*Corresponding author. Tel.: 11-410-550-1409; fax: 11-410-550-
1645.
E-mail address: mmorales@intra.nida.nih.gov (M. Morales).
involves activation of Src kinases [31,43].
Following the cloning of GFRa-1 [15,40], in situ
hybridization studies were performed to determine the
pattern of expression of GFRa-1 as well as its anatomical
relationship with its signaling component, Ret [27,42].
Cellular comparison of expression of GFRa-1 and Ret
demonstrated co-existence of these transcripts in several
neurons, among them, dopaminergic cells of the ventral
tegmental area, substantia nigra and a-motoneurons
[27,42], thus supporting the idea that in these cells, GDNF
mediates its responses through Ret receptor tyrosine kinase
[7,15,40,41]. However, GFRa-1 is also expressed in cells
that are responsive to GDNF but lack Ret [27,42], such as
cochlear neurons [45]. These cells might utilize a Ret
independent GDNF-signaling pathway that involves activa-
tion of Src kinases [31,43].
In situ hybridization studies have demonstrated that
within the brain, GFRa-1 expression is not restricted to

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 00 )02682-2
 A. Sarabi et al. / Brain Research 877 (2000) 262 – 270
those brain areas originally shown to be responsive to
GDNF (i.e. motoneurons and dopaminergic cells). As
GFRa-1 is widely expressed in the adult brain [27,40,42],
it has been suggested that GDNF may activate several
sub-types of cells. GFRa-1 is expressed in both embryonic
and adult rodent hippocampus [21,27,40,42]. RT-PCR
measurements demonstrate that the highest levels of
hippocampal GFRa-1 expression are reached between
postnatal days 0 and 14 [21], corresponding to crucial
periods for cellular migration and synaptogenesis, and
implying that GDNF through GFRa-1 might participate in
the development and maturation of the hippocampus.
Additional studies have demonstrated that GFRa-1 expres-
sion in the hippocampus is highly regulated by physiologi-
cal changes. For example, up-regulation of GFRa-1 ex-
pression is triggered in the adult hippocampus by kainic
acid treatment [33,42] and kindling-evoked stimulation
[19], as well as after global forebrain ischemia ([19];
unpublished observations) and cortical injury (unpublished
observations).
While hippocampal expression of GFRa-1 has been
documented [27,40,42], the phenotypic properties of hip-
pocampal cells which express GFRa-1 have not been
determined. Within the hippocampus, there are gluta-
matergic and GABAergic neurons. Moreover, hippocampal
GABAergic neurons constitute a heterogeneous population
with regard to their morphology, biochemical composition
and synaptic connections [8–10,16,20,37]. Sub-populations
of GABAergic neurons can be identified according to their
content of calcium binding protein; parvalbumin (PV),
calbindin (CB) and calretinin (CR) [3]. In the present
communication, we have used double labeling techniques
to determine the cellular phenotype of hippocampal GFRa-
1 expressing neurons to better understand GDNF target
cells in this brain structure.
2. Material and methods
2.1. Tissue preparation
Adult Sprague–Dawley male rats (200–250 g body
weight) were anesthetized with chloral hydrate (35 mg /
100 g) and perfused transcardially with a solution of 4%
paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.3.
Brains were then removed from the skull, postfixed from 2
to 15 h at 48C, rinsed with PB and sequentially transferred
to 12, 14 and 18% sucrose solutions. Brains were frozen
on dry ice and sectioned on a cryostat to obtain coronal
sections of 40 mm in thickness. All procedures were
approved by NIDA Animal Care and Use Committee.
2.2. In situ hybridization
In situ hybridization was performed as previously
described [25]. After pre-hybridization, sections were
263
hybridized at 558C for 16 h with 10 7 cpm / ml sense or
antisense [ 35 S]-labeled GFRa-1 probes (corresponding to
nucleotides 273–1741 of the rat GFRa-1 cDNA, original
GFRa-1 plasmid was kindly provided by Dr. Andreas
Tomac, NIDA). Sections were treated with 4 mg / ml RNase
A at 378C for 1 h, washed in 13SSC, 50% formamide at
558C for 2 h, and in 0.13SSC at 688C for 1 h. Sections
were mounted on coated slides, air dried, dipped in nuclear
track emulsion and exposed for several weeks prior to
development.
2.3. Double in situ hybridization
For double in situ hybridization material was processed
as described above using a [ 35 S]-labeled antisense GFRa-1
probe and digoxigenin-labeled antisense probes for the two
isoforms of rat glutamic acid decarboxylase (GAD 65 and
GAD 67 , generously provided by Dr. Allan Tobin, Uni-
versity of California Los Angeles, Los Angeles, CA). After
last SSC wash, sections were rinsed with TBS buffer (20
mM TRIS, 0.5 M NaCl), pH 8.2. Sections were incubated
with an alkaline phosphatase-conjugated antibody against
digoxigenin (Dig, Boehringer Mannheim; Indianapolis,
IN) overnight at 48C; alkaline phosphatase reaction was
developed with nitroblue tetrazolium and 5-bromo-4-chlo-
ro-3-indolyl phosphate (Life Technologies; Gaithersburg,
MD). Sections were dipped in nuclear track emulsion and
exposed for several weeks prior to development.
2.4. In situ hybridization and immunocytochemistry
In situ hybridization combined with immunolabeling
was performed as previously described [25]. After pre-
hybridization, sections were hybridized with [ 35 S]-labeled
antisense GFR a-1 probe at 558C for 16 h. Sections were
treated with 4 mg / ml RNase A at 378C for 1 h, washed in
13SSC, 50% formamide at 558C for 2 h, and in 0.13SSC
at 688C for 1 h. Material was rinsed with PB prior to
processing for immunocytochemistry, incubated in 4%
bovine serum albumin (BSA) supplemented with 0.3%
Triton X-100 in PB for 1 h, followed by incubation with
the corresponding primary antibody for 24 h at 48C.
Primary antibody dilutions were done in PB containing 1%
BSA and 0.3% Triton X-100. The following antibodies
were used at 1:2000 dilutions: anti-parvalbumin (Incstar,
Sillwater, MI), anti-calbindin mouse monoclonal and anti-
calretinin rabbit polyclonal antibodies (Swant, Switzer-
land). After rinsing 3310 min in PB, sections were
processed with an ABC kit (Vector Laboratories, Burling-
ame, CA). Material was incubated in a 1:200 dilution of
the biotinylated secondary antibody. After rinsing with PB,
sections were incubated with avidin-biotinylated horserad-
ish peroxidase for 2 h. Samples were rinsed and the
peroxidase reaction was developed with 0.05% 3,3-
diaminobenzidine-4 HCl (DAB) and 0.003% hydrogen
264 A. Sarabi et al. / Brain Research 877 (2000) 262 – 270
peroxide (H 2 O 2 ). Sections were mounted on coated slides,
air dried, dipped in nuclear track emulsion and exposed for
several weeks prior to development. All used antibodies
resulted in specific pattern of labeling as previously
described for PV [3,20], CR [3,11,14] and CB [2,3,39].
2.5. Data analysis
Sections processed for in situ hybridization and im-
munocytochemistry were analyzed and photographed with
bright field or polarized microscopy. Observation of the
entire hippocampus indicated lack of GFRa-1 expressing
in either CR- or CB-immunoreactive neurons, thus, quan-
titative analysis was omitted for this material. The GFRa-1
expressing cells, and neurons containing GAD mRNA or
PV-immunoreactivity were counted in random sections of
the CA1–CA3 subfields of the hippocampus and dentate
gyrus. For each region, the percentage of neurons con-
taining GFRa-1 and GAD in the total population of GAD
expressing neurons was calculated. Similarly, the per-
centage of neurons containing GFRa-1 mRNA and PV-
immunoreactivity in the total population of PV-immuno-
labeled neurons was calculated. A neuron was considered
double labeled when its soma was brown (PV-immuno-
label) or purple (GAD-expression) and contained an
aggregation of silver particles clearly above the immedi-
ately surrounding background. Typically a minimum of
15–20 silver grains represented weak label above back-
ground. Background was also evaluated from slides hy-
bridized with sense probes. Double-labeled cells were
counted in 4–6 random sections from 4 rats.
3. Results
We confirmed and extended previous reports
[21,27,40,42] showing expression of GFRa-1 in the hip-
pocampus (Fig. 1A–B). GFRa-1 transcripts were observed
mainly in the s. oriens and s. pyramidale of CA1–CA3
sub-fields of the hippocampus (Fig. 1A–B). The signal for
GFRa-1 expression was uniformly moderate in the s.
pyramidale (Fig. 1A–B), indicating for the presence of
GFRa-1 in principal glutamatergic neurons. However, we
frequently observed single cells with strong label in the s.
oriens (Fig. 1A–B) and s. pyramidale (Fig. 1A–B),
suggesting that GFRa-1 might be expressed not only in
principal glutamatergic neurons but also in interneurons.
Thus, we sought to determine the phenotype of these
putative interneurons expressing GFRa-1 mRNA.
3.1. Expression of GFRa -1 in hippocampal interneurons
We used double in situ hybridization histochemistry to
investigate the phenotype of GFRa-1 expressing neurons
in the hippocampus. In this procedure, GFRa-1 transcripts
were detected with a radioactive antisense riboprobe while
the identity of interneurons was established by their
expression of GAD mRNA. Co-expression of GFRa-1 and
GAD transcripts were found mainly in s. oriens and s.
pyramidale (Fig. 2A–B). Semi-quantitative analysis (Table
1) indicated that 29%62 (sem, n5554) of the total
population of GAD expressing neurons co-express GFRa-
1 in s. pyramidale of CA1. Co-expression was also seen in
s. pyramidale of CA2 (27%62, SEM, n5194) and CA3
(37%62.9, SEM, n5592). Lower co-expression was
found in s. oriens of CA1 (20%62.2 SEM, n5389), CA2
(25%63.3; SEM, n544) and CA3 (19%62.0, SEM, n5
250) subfields of the hippocampus.
3.2. Expression of GFRa -1 in PV-immunoreactive
interneurons of the hippocampus and dentate gyrus
We sought to determine if any of the Ca 21 binding
proteins known to be present in GABAergic neurons were
detected in GFRa-1 expressing interneurons. While no CB
either CR immunoreactivity was observed in GFRa-1
expressing interneurons, GFRa-1 / PV double-labeled neu-
rons were frequently found in the s. pyramidale of the
CA1–CA3 sub-fields (Fig. 3). GFRa-1 / PV double-labeled
neurons were also found in the s. oriens of CA1–CA3
(Fig. 3) subfields but were not detected in either the s.
radiatum or s. lacunosum moleculare. Within the dentate
gyrus, GFRa-1 / PV double-labeled neurons were present in
the s. granulosum (Fig. 4). A semiquantitative analysis of
GFRa-1 / PV double-labeled cells (Table 2) indicated that
most of these cells are concentrated in the s. pyramidale of
CA1 (72%62.3, SEM, n5508), CA2 (70%62.9, SEM,
n5148) and CA3 (70%62.4, SEM, n5358) sub-fields of
the hippocampus. Lower co-localization of PV-immuno-
reactivity and GFRa-1 transcripts was observed in the s.
oriens of CA1 (52%62.7, SEM, n5152), CA2 (27%63.2,
SEM, n526) and CA3 (36%64.3, SEM, n529) sub-fields
of the hippocampus and s. granulosum (25%61.3, SEM,
n5130) of the dentate gyrus. Within the s. pyramidale, PV
immunoreactive neurons clearly showed higher concen-
trations of silver grains than the surrounding pyramidal
neurons; while most CA1 pyramidal neurons contained
between 5–18 silver grains, adjacent PV-immunoreactive
neurons had more than 50 silver grains.
4. Discussion
By using double labeling techniques, we have demon-
strated that GFRa-1 is expressed in both pyramidal and
GABAergic neurons in the hippocampus. Hippocampal
GFRa-1 expressing interneurons constitute 19–37% of the
total population of GABAergic neurons in the s.
pyramidale and s. oriens of CA1–CA3 subfields of the
hippocampus. Consistently, the levels of GFRa-1 expres-
sion were lower in pyramidal neurons when compared with
interneurons. While these results suggest that under normal
 A. Sarabi et al. / Brain Research 877 (2000) 262 – 270 265

Fig. 1. Distribution of GFRa-1 expressing cells in CA1 (A) and CA3 (B) subfields of the hippocampus. (A–B). Moderate levels of GFRa-1 are seen in the
s. pyramidale of the CA1 and CA3 subfields. Note additional highly expressing cells scattered in s. pyramidale (large arrows) and s. oriens (small arrows).
SP, s. pyramidale. SO, s. oriens. SR, s. radiatum. Scale bar5100 mm.
266 A. Sarabi et al. / Brain Research 877 (2000) 262 – 270

Fig. 2. Simultaneous detection of GFRa-1 and GAD transcripts in the hippocampus. Under polarized light, GFRa-1 / GAD double-labeled cells appeared
as dark neurons (GAD expression) covered with green grains (GFRa-1 expression). (A). CA1. GFRa-1 / GAD double-labeled cells are found in the SP (big
arrows) or in its proximity (small arrow). Single GAD expressing cells (arrow heads) are seen in SO and SP. (B). CA3. GFRa-1 / GAD double-labeled cells
are present in the SP (big arrow) and SO (small arrows). Note a single GAD expressing cell (arrow head) in the SO. Scale bar525 mm for A and 32 mm for
B.

conditions, GABAergic interneurons might be the principal
sites of GFRa-1 expression, previous studies have shown
that GFRa-1 expression can be up-regulated in principal
neurons following neuronal activation [19,33,41]. For
instance, local [42] or systemic applications [33] of kainic
acid increase expression of GFRa-1 in principal neurons of
CA1, CA3 and dentate gyrus. Similar increase in expres-
sion was obtained after kindling [19], global forebrain
ischemia ([19]; unpublished observations) and cortical
injury (unpublished observations).
Hippocampal GABAergic interneurons constitute a
heterogeneous population with regard to their morphology,
biochemical composition and synaptic connections
[8,10,16,20,37]. Sub-populations of GABAergic neurons
can be identified according to their content of calcium
binding protein (CB, CR and PV). We found that many of
the GFRa-1 expressing interneurons contain parvalbumin,
these GFRa-1 / PV double-labeled cells were preferentially
distributed in the s. pyramidale, where about 70% of
PV-immunoreactive neurons expressed GFRa-1. Less co-
localization was found in the s. oriens of CA1 (52%) and
s. granulosum (25%). Our data are consistent with previ-
 A. Sarabi et al. / Brain Research 877 (2000) 262 – 270 267

Table 1 PV-containing neurons are fast spiking cells [17,18] that

Percentage of neurons expressing GFRa-1 and GAD mRNA in the exert an inhibitory effect on principal hippocampal neurons
hippocampus
[16,34]. As indicated above, hippocampal PV-interneurons
Region Percentage of double have a higher expression of GFRa-1 than the surrounding
labeled neurons a
principal neurons; the strong expression of GFRa-1 might
Mean6SEM
be related to the rapid electrical activity reported for
CA1 PV-containing neurons [17,18]. Consistent with this sug-
s. oriens 2062.2 (n5389)
s. pyramidale 2962.0 (n5554) gestion, prior studies have shown that GFRa-1 expression
is up-regulated in principal neurons in response to induced
CA2
excitation [19,33,42].
s. oriens 2563.3 (n544)
s. pyramidale 2762.2 (n5194) Recent reports have demonstrated that most of the
hippocampal PV-containing neurons (82%) express high
CA3
s. oriens 1962.0 (n5250) levels of nerve growth factor (NGF) mRNA [28,35].
s. pyramidale 3762.9 (n5592) Indeed, hippocampal NGF mRNA in PV-immunopositive
a cells corresponds to 71% of all NGF-positive cells in the
Total number of GAD-expressing cells (n) were counted in 4–6 sections
each from 4 experiments and the percentage of neurons expressing hippocampus [28,35]. Tracing studies have demonstrated
GFRa-1 and GAD was calculated. that PV/ NFG interneurons are preferential targets of the
GABAergic septohippocampal pathway [35]. This ob-
ous reports indicating that PV-immunoreactive cell bodies servation had led to the suggestion that, in addition to the
are located mainly in the s. pyramidale (40–50%) and to well established effects of NGF on cholinergic septohip-
lesser extent in the s. oriens and in the dentate granule cell pocampal neurons, NGF might participate in the formation
layer (20–30%) [16,20]. and maintenance of the GABAergic septohippocampal
Previous anatomical studies have demonstrated that all pathway [35]. GABAergic neurons in the lateral septum
hippocampal PV-containing neurons are GABAergic and express GFRa-1 and are trophically stimulated by GDNF
represent 20% of the total population of GABAergic [32]. The preferential concentration of transcripts for
neurons [20]. Hippocampal PV neurons might be basket or GFRa-1 and NGF in PV neurons raises the interesting
axo-axonic (chandelier) neurons [16], which form possibility of functional interactions between these two
symmetrical synapses on cell bodies, axon initial segments different neurotrophin systems (GDNF and NGF).
and proximal dendrites of the pyramidal cells [16,34]. While we established that under normal conditions,

Fig. 3. Simultaneous detection of GFRa-1 transcripts and PV immunoreactivity in the CA1 subfield of the hippocampus. Under polarized light,
GFRa-1 / PV double-labeled cells appear as brown neurons (PV-immunoreactivity) covered with green grains (GFRa-1 expression). GFRa-1 / PV
double-labeled cells are concentrated in s. pyramidale (arrows). PV single-labeled neuron is seen in s. oriens (arrow head). Scale bar525 mm.
268 A. Sarabi et al. / Brain Research 877 (2000) 262 – 270

Fig. 4. Simultaneous detection of GFRa-1 transcripts and PV immunoreactivity in the dentate gyrus. Under polarized light, GFRa-1 / PV double-labeled
cells appear as brown neurons (PV-immunoreactivity) covered with green grains (GFRa-1 expression). (A). Low magnification micrograph showing
PV-immunoreactive neurons in s. granulosum (SG). (B). At higher magnification, GFRa-1 / PV double-labeled cells with high (arrows) to low (arrow-head)
levels of GFRa-1 expression are concentrated in s. granulosum. H, hilus. Scale bar58 mm for A and 25 mm for B.

GFRa-1 transcripts are distributed in principal neurons and
interneurons in the hippocampus and dentate gyrus, it
remains to be seen whether these two kind of cells use Ret
dependent [7,15,40,41] or independent [31,43] signaling
mechanisms. Both pathways may be in place in the
hippocampus, since Ret is expressed in the hippocampus
albeit in very low levels [21,42]. In response to kainate-
induced excitation, scattered hippocampal neurons showed
increased Ret expression [42], suggesting that Ret expres-
sion is regulated by hippocampal physiological activity.
Similarly, very low levels of GDNF transcripts have been
detected in hippocampus of adult rodent brains [1,21,42].
However, increases in GDNF expression also occur as a
consequence of kainate-induced excitation [13]. The pres-
ence of GDNF transcripts within the hippocampus, suggest
that this might be a source for GFRa-1 activation but an
additional source might reside in hippocampus target
regions, such as the septum which also has low levels of
GDNF expression [1].
We concluded that GFRa-1 is expressed in specific
subsets of hippocampal neurons suggesting a role for
GDNF in the adult hippocampal function.
 A. Sarabi et al. / Brain Research 877 (2000) 262 – 270
Table 2
Percentage of PV-immunoreactive neurons expressing GFRa-1 in the
hippocampus
Region Percentage of double
labeled neurons a
Mean6SEM
CA1
s. oriens 5262.7 (n5152)
s. pyramidale 7262.3 (n5508)
CA2
s. oriens 2763.2 (n526)
s. pyramidale 7062.9 (n5148)
CA3
s. oriens 3664.3 (n529)
s. pyramidale 7062.4 (n5358)
a 198–218.
Total number of PV-immunopositive cells (n) were counted in 4–6
sections each from 4 experiments and the percentage of PV-immuno-
reactive cells expressing GFRa-1 was calculated.
Acknowledgements
The authors thank Drs. Allan Tobin and Andreas Tomac
for providing original plasmids. Supported by the In-
tramural Research Program, NIDA / NIH.
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