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Acidic fibroblast growth factor mRNA is expressed by basal forebrain and striatal cholinergic neurons

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THE JOURNAL OF COMPARATIVE NEUROLOGY 366379-389 (1996)
Acidic Fibroblast Growth Factor mRNA
is Expressed by Basal Forebrain
and Striatal Cholinergic Neurons
JENNIFER L. BIZON, JULIE C. LAUTERBORN, PAUL J. ISACKSON,
AND CHRISTINE M. GALL
Departments of Psychobiology (J.L.B., C.M.G.) and Anatomy and Neurobiology
(J.C.L., C.M.G), University of California at Irvine, Irvine, California 92717;
Department of Biochemistry and Molecular Biology, The Mayo Clinic-Jacksonville,
Jacksonville, Florida 32224 (P.J.I.)
ABSTRACT
Evidence for the importance of the basal forebrain cholinergic system in the maintenance
of cognitive function has stimulated efforts to identify trophic mechanisms that protect this cell
population from atrophy and dysfunction associated with aging and disease. Acidic fibroblast
growth factor (aFGF) has been reported to support cholinergic neuronal survival and has been
localized in basal forebrain with the use of immunohistochemical techniques. Although these
data indicate that aFGF is present in regions containing cholinergic cell bodies, the actual site of
synthesis of this factor has yet to be determined. In the present study, in situ hybridization
techniques were used to evaluate the distribution and possible colocalization of mRNAs for
aFGF and the cholinergic neuron marker choline acetyltransferase (ChAT) in basal forebrain
and striatum. In single-labeling preparations, aFGF mRNA-containing neurons were found to
be codistributed with ChAT mRNA+ cells throughout all fields of basal forebrain, including the
medial septumidiagonal band complex and striatum. By using a double-labeling (colormetric
and isotopic) technique, high levels of colocalization (over 85%) of aFGF and ChAT mRNAs
were observed in the medial septum, the diagonal bands of Broca, the magnocellular preoptic
area, and the nucleus basalis of Meynert. The degree of colocalization was lower in the striatum,
with 64% of the cholinergic cells in the caudate and 33% in the ventral striatum and olfactory
tubercle labeled by the aFGF cRNA. These data demonstrate substantial regionally specific
patterns of colocalization and support the hypothesis that, via a n autocrine mechanism, aFGF
provides local trophic support for cholinergic neurons in the basal forebrain and the
striatum.
1996 Wilcy-Liss, Inc.
Indexing terms: in situ hybridization, choline acetyltransferase, neurotrophic factor, caudate,
Alzheimer’s disease
Basal forebrain cholinergic neurons are believed to be
critical for the maintenance of cognitive abilities in humans
and in rodents, and they are particularly vulnerable to
dysfunction and degeneration with age and disease (Fischer
et al., 1989, 1991; Gallagher et al., 1990; Stroessor-Johnson
et al., 1992; Armstrong et al., 1993). A large body of
literature has correlated the atrophy of these cells and the
concomitant decline in cholinergic indices with memory and
attentional deficits associated with such disorders as Alzheimer’s disease (Whitehouse et al., 1982; Coyle et al., 1983;
Collerton, 1986). Because of this age-related pathology, a
great deal of effort has been made to identify endogenous
factors that sustain cholinergic neuronal viability. Whereas
past research in this area has primarily focused on distant
target-derived neurotrophic factors, recent findings suggest
1996 WILEY-LISS. INC.
that there are important trophic interactions within the
basal forebrain itself.
The role of target-derived nerve growth factor (NGF) in
the survival of basal forebrain cholinergic cells is well
documented. It has been demonstrated that NGF is synthesized in the hippocampus (Gall and Isackson, 1989; Ernfors
et al., 1990; Phillips et al., 19901, sequestered by cholinergic
afferent axons, and retrogradely transported back to the
cell bodies located in the medial septum (Schwab et al.,
1979). Following fimbriaifornix transection, which severs
cholinergic afferents to the hippocampus and, thereby,
Accepted September 14, 1995.
Address reprint requests to Christine M. Gall, Department of Anatomy
and Neurobioloa, University of California at Irvine, Irvine, CA 92717.
E-mail:cgall~~r
uci.edu
380
blocks retrograde transport, the majority of the medial
septal neurons undergo changes in phenotype, including
marked decreases in choline acetyltransferase (ChAT; Gage
et al., 1986; Armstronget al., 1987; Sofroniew et al., 1987).
In these fields, the number of ChAT-immunoreactive neurons is reportedly reduced by as much as 75% a t 3 weeks following transection (Sofroniew et al., 1987; Peterson et al.,
1990). However, the cholinergic cells can be rescued by the
infusion of various trophic factors, including not only NGF
(Kromer, 1987; Koliatsos et al., 1990; Venero et al., 1994)
but also brain-derived neurotrophic factor (BDNF; Morse
et al., 1993; Koliatsos et al., 1994; Venero et al., 1994) and
fibroblast growth factor (FGF; Anderson et al., 1988; Otto
et al., 1989; Gomez-Pinilla et al., 19921, all ofwhich are synthesized normally in the hippocampus (Emoto et al., 1989;
Ernfors et al., 1990: Phillips et al., 1990; Gall et al., 1994).
Although these findings suggest the importance of targetderived neurotrophic factors in the survival of cholinergic
neurons following insult, a growing body of evidence indicates that factors from distant targets may not be an
essential determinant of viability under normal circumstances. First, although the cholinergic neurons are particularly susceptible to pathology that is associated with the
aging process, a n age-related decline of NGF levels in the
hippocampus has not been demonstrated in rat (Hellweg et
al., 1990; Crutcher and Weingartner, 1991) nor have postmortem examinations of cholinergic cortical targets in
Alzheimer's patients revealed deficits in NGF mRNA (Jette
et al., 1994)or protein (Allen et al., 1991).Second, although
the medial septal cholinergic cells atrophy following transection of their axonal projections to the hippocampus, these
cells survive the removal of all distant trophic support by
excitotoxic lesion of hippocampal neurons, which spares the
cholinergic axons themselves (Sofroniew et al., 1990, 1993;
Kordower et al., 1992). Moreover, transgenic mice that do
not express NGF (Crowley et al., 1994) or its high-affinity
receptor TrkA (Smeyne et al., 1994) retain cholinergic
perikarya in basal forebrain, although the TrkA mutants
reportedly fail to maintain cholinergic basal forebrain
afferents to the hippocampus (Smeyne et al., 1994). Survival of the cholinergic neurons despite the absence of their
target cells, NGF, or the NGF receptor suggests that the
cholinergic cells may receive trophic support 1)other than
NGF and 2 ) from outside their principal cortical targets.
Because very few of the neurons in the medial septum
project both to the hippocampus and to another region
(Amaral and Kurz, 1985), the idea of local trophic support
for these cells becomes compelling.
FGFs were noted originally for their ability to promote
mitosis in fibroblasts (for review, see Burgess and Maciag,
1989). These broadly acting factors are now known to
promote the development and survival of cultured neurons
from virtually all brain regions tested (Morrison et al.,
1986; Walicke, 1988; Ferrari et al., 1989; Grothe et al.,
1989; Garcia-Rill et al., 1991; Kushima et al., 1992).In vivo,
they can rescue neurons in basal forebrain (Anderson et al.,
1988; Otto et al., 1989; Gomez-Pinilla et al., 1992) and
entorhinal cortex (Cummings et al., 1992) from the degenerative effects of axotomy, and they support hippocampal
CA1 neurons following cerebral ischemia (Nakata et al.,
1993).Acidic and basic FGF (aFGF and bFGF, respectively)
act via the same receptors (Asai et al., 1993; Yazaki et al.,
19941, and, generally, both are effective in the support of
neuronal survival in vitro and in vivo. However, with regard
to the viability of cholinergic basal forebrain cells, the
.J.L. BIZON ET AL.
involvement of aFGF (a.k.a. FGF1) is of particular interest.
Recently, immunoreactivity for this FGF family member
has been localized in neuronal perikarya within regions of
the cholinergic basal forebrain, including the septum and
the nucleus basalis of Meynert (Stock et al., 1992),suggesting that this factor may be synthesized locally. However,
the distribution of protein immunoreactivity does not settle
the issue of the site of synthesis. The possibility exists that
aFGF, like other neurotrophic factors that are associated
with the cholinergic system, is produced elsewhere and
then is transported to the basal forebrain.
The goal of the present study was to determine the
distribution of those cells that synthesize aFGF mRNA in
relation to forebrain neurons expressing the mRNA for
ChAT, which is a cholinergic cell marker. In addition, the
possibility that aFGF acts as an autocrine neurotrophic
factor for the cholinergic forebrain neurons was investigated using an in situ hybridization technique that allowed
for the simultaneous localization of mRNAs for ChAT and
aFGF. The results demonstrate substantial but regionspecific patterns of colocalization and are consistent with
the hypothesis that aFGF acts as an autocrine neurotrophic
factor for cholinergic neurons of basal forebrain and striatum.
MATERIALS AND METHODS
Adult male Sprague Dawley rats ( n = 14) were killed with
an overdose of sodium pentobarbital (100 mgikg) and were
perfused with 4% paraformaldehyde in 0.1 M phosphate
buffer, pH 7.4. The brains were removed from the crania,
postfixed in perfusate for 24 hours, cryoprotected in 20%
sucrose and 4% paraformaldehyde for 48 hours, and then
sectioned on a freezing microtome (25 Fm, coronal plane)
through the rostra1 forebrain, beginning just caudal to the
olfactory bulbs and ending at the midrostral hippocampus.
Adjacent series of every ninth section were collected into
cold 4%,paraformaldehyde.
Free-floating tissue sections were processed either for
single-labeling or for double-labeling in situ hybridization
as previously described (Lauterborn et al., 1993a,b). For
single-labeling experiments, adjacent sections from one
group of animals (n = 3 ) were processed for the localization
of either ChAT mRNA or aFGF mRNA by using:35SS-labeled
cRNA probes. For double-labeling experiments, sections
from another group of animals ( n = 11)were processed for
the simultaneous detection of ChAT mRNA and aFGF
mRNA within individual tissue sections by using a digoxigenin (DIG)-labeledChAT cRNA ("'"ChAT) and :%labeled
aFGF cRNA. The ChAT riboprobe was transcribed from a
431 base pair fragment of rat C U T cDNA (Lauterborn et
al., 1993b). For the sense and antisense ChAT cRNAs, the
constructs were digested with Pvu 11, and transcripts were
generated in the presence of 5'-a-I % lthiotriphosphate by
using T3 and T7 RNA polymerase. The aFGF :W-labeled
riboprobe was transcribed from a 510 base pair fragment
(corresponding to nucleotides 245-755 in Goodrich et al.,
1989) of rat aFGF cDNA. The aFGF cDNA was obtained by
the polymerase chain reaction (PCR) from rat neocortical
cDNA 24 hours after the induction of limbic seizures. The
oligonucleotide primers (20 bases in length) used for PCR
were synthesized based on the published sequence of rat
heparin-binding growth factor 1 (Goodrich et al., 1989).
The isolated aFGF cDNA was cloned into the EcoRl site of
pBS. For antisense aFGF cRNA, the construct was digested
aFGF EXPRESSION BY CHOLINERGIC NEURONS
38 1
with P w 11, and transcripts were generated with T3 RNA using sense DIG- or :35S-cRNAprobes or 2) omitting the
polymerase in the presence of "S-UTP. An internal Pvu I1 antisense probes. Cellular labeling was not detected in either
site exists in the aFGF sequence; therefore, the final result of these conditions. To determine whether the immunohistois a 350-base antisense probe. For sense aFGF cRNA, the chemical procedure produced a nonspecific autoradiographic
construct was digested with BamH1, and transcripts were signal, the double-labeling technique was performed as
generated with T7 RNA polymerase in the presence of described above, but the 35S-aFGFcRNA was omitted from
the initial hybridization incubation. No autoradiographic
:35SS-UTP.
For both single-labeling and double-labeling experi- grains were detected under these control conditions.
ments, free-floating sections were pretreated with proteinAnalysis of colocalization
ase K (1 mg/ml in 0.1 M Tris buffer, pH 8.0, with 50 mM
EDTA) for 30 minutes, then in 0.25% acetic anhydride in
By using a combination of light- and darkfield micros0.1 M triethanolamine for 10 minutes, and then rinsed in copy, tissue processed according to the double-labeling
2 x saline sodium citrate buffer (SSC; 1 x SSC = 0.15 M technique was evaluated to determine the distribution and
NaC110.015 M Na-citrate, pH 7.0). Tissue was incubated for the number of cells in specific fields of the rat basal
40-48 hours at 60°C in hybridization buffer (50% for- forebrain that were hybridized with both the D'GChAT
mamide, 10% dextran sulfate, 0.7% ficoll, 0.7% polyvinyl cRNA and the "SS-aFGF cRNA (double-labeled) or with
pyrrolidone, 0.7% bovine serum albumin, 0.15 mg/ml yeast each cRNA alone (single-labeled). The regions analyzed
transfer RNA, 0.33 mgiml denatured herring sperm DNA, included the medial septa1 nucleus, the horizontal and
and 40 mM dithiothreitol) containing either the :j5SS-labeled vertical limbs of the diagonal bands of Broca, the caudate/
ChAT cRNA or the 35s-labeled aFGF cRNA separately (for putamen, the olfactory tubercle, the magnocellular preoptic
the single-labeling studies) or containing a cocktail of the area, and the nucleus basalis of Meynert. Single- and
"I(;ChAT cRNA and the "5S-labeled aFGF cRNA (for the double-labeled cells were counted from six camera lucida
double-labeling studies). In all cases, the 35S-labeled cRNA drawings from each of four rats that corresponded to
was added to a final density of 1 x lo6 cpmilO0 ~ 1and
, the rostrocaudal levels represented in plates 12-17 of Paxinos
DIG-labeled cRNA was diluted 1:1,000 in the hybridization and Watson (Paxinos and Watson, 1986). Because of diffibuffer. After hybridization, tissue processed for single- culty in determining cytoarchitectonic boundaries in the
labeling was rinsed in 4 x SSC, treated with 20 pgiml RNase-treated tissue, the field designated as ventral striaribonuclease A in 10 mM Tris saline, pH 8.0, with 1 mM tum included the ventral one-third of neostriatum and the
EDTA for 30 minutes at 45"C, washed through descending nucleus accumbens. The percentages of DIGCUTcRNA+
concentrations of SSC to a final wash in 0.1 x SSC at 60°C, cells that were also labeled by the :35S-aFGFcRNA and
of Y3-aFGF cRNA+ cells that were also labeled by the
and then mounted onto gelatin-coated slides.
For tissue processed for double-labeling, following hybrid- DrGChATcRNA were determined for select regions.
ization, the sections were rinsed in 50% formamidei2 x
SSC a t 60"C, rinsed in 2 x SSC at room temperature (RT),
RESULTS
and treated with ribonuclease A (as above). The tissue was
Distribution of aFGF in the basal forebrain
then washed in descending concentrations of SSC to a final
wash in 0.1 x SSC a t 60"C, rinsed in 50% formamidei2 x
The distribution of labeling in tissue sections processed
SSC ( 5 minutes) at 60°C, and incubated in 0.1 x SSC for hybridization of either aFGF cRNA or ChAT cRNA alone
containing 2% normal sheep serum (NSS) and 0.05% revealed similar patterns of expression in basal ganglia and
Triton-X 100 at RT for 1 hour. Following several rinses in in other regions of basal forebrain. Figure 1A,B shows that,
Tris-buffered saline (TBS; 100 mM Tris-HCL, 150 mM for both aFGF and ChAT cRNAs, there were large numbers
NaCI, pH 7.5), the tissue was incubated with a sheep of heavily labeled cells in the medial septum and the diagonal
anti-DIG antiserum (Boehringer Mannheim) diluted 1:2,000 bands of Broca, and there were somewhat fewer densely
in TBS containing 2% NSS,0.05% Triton-X 100, and 10 labeled cells scattered across the caudateiputamen and the
mM sodium thiosulfate at 37°C for 2 hours. The tissue was olfactory tubercle. Unlike ChAT mRNA, aFGF mRNA
then washed in TBS a t RT, rinsed in 0.1 M Tris-HCLi100 expression was also detected throughout the lateral septum
mM NaClI50 mM MgC12, pH 9.5 (Tris-MgC12), and incu- (Fig. 1B) and the lateral preoptic area, although, in these
bated in Tris-MgClz containing nitroblue tetrazolium (0.34 regions, the autoradiographic grains were diffusely scatmgiml), 5-bromo-4-chloro-3-indolyl phosphate (0.18 mgi tered, and individual labeled neurons were not easily identified. Figure 1C,D shows that, in more caudal regions, cells
ml), and levamisole (0.24 mgiml) in the dark for 18-24
hours at RT. Following this colormetric reaction, tissue was containing aFGF and ChAT mRNAs were distributed simirinsed in 10 mM Tris-HCl/l mM EDTA, pH 8.0, and larly within the magnocellular preoptic area and the nucleus
basalis of Meynert. At these same levels, several other brain
mounted onto gelatin-coated slides.
The slide-mounted tissue sections were defatted through regions lacking ChAT mRNA expression contained cells
alcohol and Americlear, rehydrated, and then air dried. that were heavily labeled by the aFGF cRNA. Most particuSlides for single-labeled tissue were dipped in Kodak emul- larly, aFGF mRNA expression was high in the anterodorsal,
sion (NTBS), and slides for double-labeled tissue were paraventricular, and reuniens nuclei of the thalamus;
dipped in Ilford K5.d emulsion. All slides were stored a t 4°C labeling in the anteromedial and anteroventral thalamic
for 4-6 weeks, after which the autoradiograms were devel- nuclei was less dense. There was a notable absence of aFGF
oped with Kodak D19 (1:l with H 2 0 )and fixed with Kodak cRNA labeling in the paratenial nucleus of thalamus.
regular fixer. The slides were then rinsed through an
Colocalization of aFGF mRNA with ChAT
ascending series of alcohols (50-100%) and Americlear and
mRNA in basal forebrain and striatum
were coverslipped with Permount.
Labeling specificity was assessed by processing tissue
In tissue hybridized simultaneously with DIGChATand
according to the aforementioned protocol but either 1) 35S-aFGF probes, cells were visualized through a combina-
. J . L BIZON ET AL.
Fig. I . Darkfield photomicrogmphs showing the autoradiographic
localization of Y-choline acetyltransferase t ChAT) cRNA lA,C) and
"S-acidic. fibroblast growth factor IaFGF! cRNA IB,D) hybridization in
semiadjacent sections through control rat forebrain a t t he level of the
medial wptal nucleus tA,B; MS) and the rostra1 thalamus and nucleus
basalis of Mevnert 1C.D; nB). A,B: Both cRNAs label cells tha t a re
similarly distributed in t he medial septum, the diagonal bands of Broca
tion of light- and darkfield microscopy and were examined
for double-labeling. Examples of the pattern of cellular
labeling are shown in Figure 2, where the colormetrici
immunohistochemical label (seen as a blue reaction product
in the tissue section) indicates the localization of C U T
mRNA, and the autoradiographic silver grains mark aFGF
cRNA hybridization. Figure 2A,B shows that some neurons
were singly labeled for either aFGF mRNA or C U T
mRNA. whereas others cells were found to contain mRNAs
for both ChAT and aFGF. Plots of the distributions of cells
labeled with ChAT cRNA alone or with both cRNAs are
presented in Figure 3 .
In all of the forebrain regions analyzed, some degree of
aFGF cRNA labeling was observed in cholinergic cells,
although the density of labeling with each probe varied with
respect to anatomical location. Characteristic of the cholinergic neurons distributed throughout the basal forebrain,
the ChAT mRNA+ cells in medial septum had large round
perikarya that displayed intense color labeling. Figure 2C
and the plots of labeled cells presented in Figure 3A show
that all of the ChAT mRNA' (immunohistochemically
labeled) cells in this region were also labeled with dense
accumulations of autoradiographic grains. The r'i';ChAT
cRNA-labeled perikarya in the horizontal and vertical limbs
(NDB), the olfactory tubercle (Or,,and the caudatesputamen (<:Pi.
Labeling with the a FG F cRNA alone is seen within the lateral septum
(LS).C,D: In more caudal sections, ChAT cRNA ( C )and aFGF cRNA
(D! both label cells in the mafinocellular preoptic area tMCPAi and the
nucleus basalis. A t these levels, the a FG F cRNA also labels the
and the intralaminar fields suranterodorsal thalamic niicleus (AD!
rounding the paratenial nucleus (asterisk,. Scale bar = 750 p m .
of the diagonal bands were reminiscent of the medial septa1
cholinergic neurons, although, within peripheral aspects of
these fields, the labeled cells had somewhat smaller somata.
Fig. 3. Brighttield photomicrographs showing the cellular localization of ,Y%IFGF cRNA (seen as black autoradiographic g-ainsi and
digoxigenin ( DIG,-labelcd ChAT cRNA t"'%hAT cRNA; immunohistochemical color-reaction product, hybridization in basal forebrain and
striatum. Labeled cells a re shown in th e olfactory tubercle ( A , . the
, medial septa1 nucleus tC1. the vertical ( D !
caudate/putamcn ( B J the
and horizontal tE! limbs of the diagonal bands of Broca. the magnocellular preoptic area IF,, and the nucleus basalis of Meynert ( G I .Arrnwheads indicate cells labeled with the a FG F cKNA alone, open arrows indicate cclls labeled with the ChAT cRNA alone, and solid arrows
indicate double-labeled neurons. In both the olfactory tubercle (A1 and
the caudatc/putanien (HI, S O ~ Ccells a re single-labeled with aFGF
cRNA or with ChAT cRNA, whereas other similar-sized neurons are
double?-labeled. I n contrast, in the medial septum tC), all labeled cells
were large and wvre densely double-labeled by both markers. In the
vertical (D! and horizontal t E ) limbs o f t h e diagonal bands of Broca, the
&Teat majority of labeled cells were double-labeled; however. as shown
here, a few singlc,-lebeled cells were seen a t the lateral edge of each field
(in D, the lateral aspect o f t h e field appears on the right). Finally. the
large cholinergic colls of the magx)cellular preoptic area IF) and the
nucleus basalis tG! were virtually all double-labeled as in the fields
shown. Scale bar := 30 ym in A-C,F,G. 35 pn in D.E.
aFGF EXPRESSION BY CHOLINERGIC NEURONS
383
Figure 2
.J.L. BIZON ET AL.
A
c
I
i
Fig. 3 . A-D: Schematic illustrations and bar graphs showing t h e
proportion of cholinergic (i.e., ChAT mRNA' 1 cells in basal forebrain
and the striatum t h a t also contain a F G F mRNA. A: Drawings show t h e
distributions of neurons that express both a F G F mRNA and ChAT
mRNA (solid triangles) and those t h a t express ChAT mRNA alone
[open circles) in basal forebrain and basal ganglia (small symbols
indicate one cell; large symbols indicate four to six cells). Labeling in
lateral scptum, cortex, diencephalon, and bed nucleus of stria terminalis IBS'I') was not plotted, nor were cells t h a t were labeled with a F G F
cKNA alone. The three schematics, from top to bottom, show plots of
t h e rostral. middle, and caudal basal forebrain, respectively. It can be
seen here that double-labeled cells were most numerous in medial
regions, including t h e medial septum (MS)
and t h e diagonal bands of
Broca ( N D R I followed by the magnocellular preoptic area IMCPA) and
t h e nucleus basalis i nB). Double-labeled cells were fairly evcnly interspersed with single-labelcd cells in t h e caudateiputamen ( C P )and were
somewhat less frequently encountered in the olfactory tubercle ( O T ) .
B-D: Bar graphs showing counts of single- and double-labeled ChAT
mHNA* cells within t h e different cytoarchitectonic fields found in
individual tissue sections through thcsc same rostral IB), middle tC).
and caudal (D)planes. For each area, the black portion of the bar
indicates the number of cells labeled with both a F G F and ChAT cRNAs,
whereas t h e stippled portion ( n o t including black s p a n ) shows the
number labeled with ChA'T cRNA alone. AAA, anterior amygdaloid
area; AcB, nucleus accumbens; aco, anterior commissure: En, endopiriform nucleus; GP, globus pallidus; LHA, lateral hypothalamic area: LS,
lateral scptum: opt, optic tract; PIR, piriform cortex; 1'0, preoptic area;
S F , septofimbrial nucleus; VP. ventral pallidum; VS, v m t r a l striatum.
aFGF EXPRESSION BY CHOLINERGIC NEURONS
Moreover, in these cells, the color labeling within the
cytoplasm could be seen frequently surrounding a n unlabeled nuclear region. Virtually all of the DIGChATcRNA+
cells in the diagonal bands were double-labeled (Fig. 2D,E),
although the density of aFGF cRNA labeling was less than
that observed in the medial septum (Fig. 2C).
In addition to those cells in the basal forebrain cholinergic system that give rise to cortical efferents, cholinergic
cells are distributed across the caudateiputamen, the ventral striatum, and the olfactory tubercle, and previous
studies have demonstrated that the latter cells are primarily interneurons (Woolf and Butcher, 1981). In the present
material, large DIGChAT-labeledperikarya were found in
each of these regions and, as shown in Figure 2A,B and in
the plots in Figure 3, a portion of these cells also contained
aFGF mRNA. For double-labeled perikarya in the striatum
and the olfactory tubercle, the density of aFGF cRNA
labeling was less than that observed in other forebrain
areas (e.g., the medial septum). Moreover, among these
fields, both the number of double-labeled cells (Fig. 3) and
the density of W - a F G F cRNA labeling (Fig. 2) appeared
greatest in the dorsolateral caudate and decreased toward
the more medial aspects of the caudate and toward the
ventral striatum and the olfactory tubercle.
The ChAT mRNA+ cells in the magnocellular preoptic
area were similar to neurons in the horizontal limb of the
diagonal band with regard to soma1 size, shape, and incidence of "S-aFGF cRNA labeling. However, in the magnocellular preoptic area (Fig. 2F), the density of the autoradiographic label over individual cells was somewhat lower than
in medial septum. The DrGChATcRNA-labeled perikarya in
the nucleus basalis of Meynert were very large and elongated. Figure 2G shows that all of the labeled cells in this
region were double-labeled; however, the 35SS-aFGF
cRNAlabeling was much more diffuse than in other forebrain
areas analyzed.
Quantification of ChAT/aFGF colocalization
The schematic illustrations and bar graphs in Figure 3
show that the number of double-labeled somata varied
substantially with respect to anatomical location. There
were very high levels of colocalization in regions traditionally associated with the cholinergic basal forebrain. All
DIGChATcRNA-labeled neurons in the medial septum (Fig.
3B) and the nucleus basalis of Meynert (Fig. 3D) exhibited
dense %-aFGF cRNA labeling. In the horizontal limb of
the diagonal band (Fig. 3B) and in the more caudal magnocellular preoptic area (Fig. 3C), over 85% of the ChAT
mRNA' cells were double-labeled.
Fewer of the probable cholinergic interneurons identified
in the regions of the dorsal caudate, the ventral striatum,
and the olfactory tubercle contained aFGF mRNA. Figure
3B,C and Table 1show that, among these fields, the highest
level of colocalization was detected in the caudate, where
64% of the ChAT mRNA+ cells also contained aFGF
mRNA. Fewer than 32% of the DIGChATcRNA+ neurons in
the ventral striatum and the olfactory tubercle were also
"S-aFGF cRNA+. In these regions, the total number of
aFGF mRNA-containing somata were also counted and
examined for the presence of ChAT mRNA. In general,
there were fewer cells labeled with 3sS-aFGF cRNA alone
than there were double-labeled cells. Of the aFGF mRNAcontaining cells, the highest incidence of colocalization was
found within the dorsal caudate (770/0), whereas less than
385
TABLE 1. Cell Counts and Percentages of Single- and Double-Labeled Cells
in Basal Ganglia and Olfactory Tubercle'
Single-labeled cells
Region
Dorsd caudate
Ventralstriaturn
Olfactory tubercle
Double-labeled cells
ChAT
cRNA
(no.)
aFGF
cRNA
(no.)
aFGFiChAT
cRNA
(no.)
ChAT
cRNA
(%I
(%1
23 i 9.6
11 ? 3.4
10 i 2.2
12 2 4.7
38 ? 6.2
10 i 3.4
9 I 2.5
64 2 9.8
32 2 11.7
25 I 6.2
77 i 3.3
43 i 11.6
44 2 9.2
22 t 5.9
27 2 4.9
'Cell counts and pen.entd#?s represent average per section
i
aFGF
cKNA
S.D.: analysis of t o u r rats
totdl.
43% of the cells in the ventral striatum and the olfactory
tubercle were double-labeled.
DISCUSSION
The present results demonstrate that aFGF mRNA is
distributed in all fields of the cholinergic basal forebrain
and is colocalized with ChAT mRNA in each, although the
extent of colocalization varies with respect to anatomical
region. The distribution of aFGF mRNA alone was in good
agreement with the immunohistochemical localization of
aFGF protein reported by Stock et al. (1992). In each of
these studies, labeling was dense in the diagonal bands of
Broca, the nucleus basalis of Meynert, and the anterodorsal
and paraventricular thalamic nuclei; was moderately dense
in the lateral septum and preoptic areas; and was associated
with scattered cells in the caudateiputamen and the olfactory tubercle. Together with data on the distributions of
aFGF immunoreactivity and mRNA in brainstem and
spinal cord (Bean et al., 1991; Elde et al., 1991), these
results indicate that aFGF protein generally accumulates
within, and serves as a good marker for, cells that produce
this factor. Moreover, these data reinforce the conclusion
that, in brain, aFGF mRNA is expressed by restricted
populations of neurons (Elde et al., 1991) unlike bFGF,
which has been shown to be expressed by some neuronal
groups in addition to diffusely distributed glial cells (Emoto
et al., 1989; Gall et al., 1994). Results ofthe double-labeling
analysis demonstrate that aFGF mRNA is expressed by
virtually all cholinergic neurons in the medial septum and
the nucleus basalis of Meynert. In contrast, in the horizontal limb of the diagonal bands, magnocellular preoptic area,
and striatum, there were substantial numbers of doublelabeled neurons codistributed with cells producing aFGF
mRNA or ChAT mRNA alone, suggesting a heterogeneity
of cell types within both the aFGF+ and the ChAT+
populations.
The expression of aFGF within basal forebrain is particularly intriguing in light of evidence that aFGF, like the
better-characterized neurotrophins, has the ability to preserve the integrity of cholinergic forebrain neurons. Because bFGF and aFGF act at the same receptor (Asai et al.,
1993; Yazaki et al., 19941, the effects of each are indicative
of the potential functions for locally produced aFGF. Following exogenous application, both factors promote the differentiation (Burgess and Maciag, 1989; Baird and Klagsbrun,
1991) and increase the ChAT activity (Grothe et al., 1989;
Knusel et al., 1990; Kushima et al., 1992; Figueiredo et al.,
1993; Yokoyama et al., 1994) of cultured septa1 cholinergic
neurons. In vivo infusion of exogenous bFGF after a
fimbria/fornix transection (Anderson et al., 1988; Otto et
al., 1989; Gomez-Pinilla et al., 1992) or of aFGF after
neocortical lesion (Figueiredo et al., 1993) preserves ChAT
386
expression by basal forebrain neurons. Some of these
effects may involve interactions with the neurotrophins.
The FGFs are known to stimulate NGF expression and
release in vitro (On0 et al., 1991; Yoshida and Gage, 19911,
and intraventricular bFGF infusion after fimbriaifornix
transection maintains the expression of the low-affinity
NGF receptor (a.k.a. p75) by basal forebrain cholinergic
cells (Gtimez-Pinilla et al., 1992). Thus, there may be
synergistic interactions between locally produced aFGF and
target-derived neurotrophins in the support of cholinergic
neuronal viability.
It is well established that the cholinergic neurons distributed across the basal forebrain are not homogeneous but
differ with respect to both anatomical connectivity and
expression of biochemical markers. The cholinergic neurons in the medial septum and the nucleus basalis of
Meynert send axons almost exclusively to one cortical
target, the hippocampus and the neocortex, respectively
(Woolf et al., 1984; Paxinos and Butcher, 1985). Cells
distributed in lateral and ventral fields (i.e., the horizontal
limb of the diagonal bands and magnocellular preoptic
areas) make more varied and multiple projections across
telencephalic structures innervating the entorhinal and
cingulate cortices and the olfactory bulb (Woolf et al., 1984;
Paxinos and Butcher, 1985; Zaborsky et al., 1986). Within
ventral pallidum and substantia innominata, two major
subpopulations of cholinergc cells have been identified that
can be differentiated on the basis of their predominant
efferent projections to cortex or amygdala (Carlsen et al.,
1985) and on the basis of their vulnerability to either
quisqualic or ibotenic acid, respectively (Boegman et al.,
1992).
The colocalization of ChAT and aFGF mRNA, as seen
here, was greatest in the areas of least cholinergic cell
diversity. All cholinergic cells in the medial septum and
nucleus basalis and 85%of cholinergic cells in the horizontal limb of the diagonal band and magnocellular preoptic
area were double-labeled. Across ventral striatum, ventral
pallidum, and lateral substantia innominata, colocalization
was less frequently encountered, and major subpopulations
were identified that expressed either aFGF or ChAT mRNA
alone. The increased diversity of expression patterns in
fields containing greater numbers of cholinergic cell types
(differentiated by connectional and other biochemical criteria) suggests that the presence of aFGFiChAT expression
or of aFGF expression alone may be associated with specific
populations of cells. Thus, within the ventral pallidumi
substantia innominata, aFGFiChAT colocalization may be
a feature of one particular efferent population. Moreover,
differences in aFGF mRNA expression across basal forebrain areas could be indicative of distinct trophic requirements across cholinergic populations and, most particularly, could indicate differences in the degree to which these
cells depend on distant trophic support. It is well documented that the cholinergic cells of the medial septum and
the nucleus of basalis, which exhibit robust aFGF mRNA
expression, can survive the ablation of their cortical targets
and the removal of putative neurotrophic support from
these sources (Sofroniew et al., 1990, 1993; Kordower et al.,
1992).It will be interesting to determine whether cholinerg c cells lacking aFGF expression (e.g., those in the olfactory tubercle or in the lateral aspects of the diagonal bands
of Broca) are more vulnerable to degeneration following
removal of their forebrain targets and of potential distant
sources of trophic support.
.J.L. BIZON ET AL.
An additional consideration with regard to the singlelabeled cholinergic neurons in the horizontal limb of the
diagonal band and in magnocellular preoptic areas is that,
although these cells do not produce aFGF, they may receive
other local neurotrophic support. NGF-synthesizing neurons have been localized in the lateral aspects of the
horizontal limb of the diagonal band and in the magnocellular preoptic area, the olfactory tubercle, and the ventral
pallidum (Lauterborn et al., 1991; Conner et al., 1992).
These intensely NGF mRNA+ cells contain glutamic acid
decarboxylase mRNA, indicating that they are GABAergic
(Lauterborn et al., 1995). Thus, the cholinergic cells in
lateral basal forebrain may receive local trophic support
from neighboring cholinergc neurons producing aFGF as
well as from closely situated GABAergic neurons producing
NGF. In future studies, it will be of interest to determine
whether cholinergic cells in these fields have access to local
sources of both NGF and aFGF or whether separate
neuronal populations benefit from different, locally supplied trophic factors.
Confirming previous in situ hybridization investigations
(Ibanez et al., 1991; Lauterborn et al., 1993b1, many ChAT
mRNA+ neurons were localized throughout the basal ganglia and the olfactory tubercle in the present study. The
distribution and size of these cells indicate that they are the
well-described large, aspiny neurons thought to have primarily local projection fields (Woolf and Butcher, 1981). Only a
portion of the cholinergic striatal neurons expressed aFGF
mRNA (i.e., 64% in the dorsal and lateral aspects of the
caudate and 32% in more medial and ventral fields). In
these regions, a large proportion of the aFGF-cRNA-labeled
cells did not express ChAT mRNA (about 40% overall). The
single-labeled aFGF neurons were similar to the cholinergic
interneurons, in that they were large and were sparsely
scattered throughout the striatum. These characteristics
suggest that the single-labeled aFGF neurons are not the
medium spiny projection neurons that account for over 90%
of cells in the striatum, but they represent the second major
interneuronal group, i.e., the large aspiny GABAergic interneurons (Heimer et al., 1985).These conclusions are consistent with the immunocytochemical analysis of Stock et al.
( 1992), which suggested that aFGF-immunoreactivity was
localized in two distinct populations of interneurons in the
neostriatum. The localization of aFGF in striatal interneurons is noteworthy, in that these cells are among the least
vulnerable neurons in the striatum, which is demonstrated
by their selective survival following ischemia and excitotoxic insult (Beal et al., 1990; Chesselet et al., 1990;
Gonzales et al., 1992).This suggests that the local synthesis
and actions of aFGF may afford protection to striatal
interneurons under these circumstances.
Apart from potential autocrine actions, aFGF produced
in striatum and basal forebrain may have trophic effects on
afferents to these regions. The nigrostriatal afferents are of
particular interest in this regard. It has been reported
recently that the injection of MPTP', which destroys the
majority of the dopamine-producing cells in the substantia
nigra, increases aFGF and bFGF mRNA levels in the
striatum (Leonard et. al., 1993). Furthermore, infusion of
aFGF and bFGF into the striatum after a MPTP+ lesion
significantly increases striatal dopamine levels (Date et al.,
1990). These findings suggest that aFGF produced in the
basal ganglia and the basal forebrain may be transported
retrogradely by afferents to these regions, including the
dopaminergic somata in the substantia nigra. With regard
aFGF EXPRESSION BY CHOLINERGIC NEURONS
to this point, C U T + terminals in striatum have been
shown to lie in close proximity to tyrosine hydroxylasepositive terminals (Pickel and Chan, 1990; Dimova et al.,
19931, allowing for the possibility that FGF could be
released by cholinergic processes and then acquired and
retrogradely transported by dopaminergic afferents. The
dopaminergic neurons of the substantia nigra are known to
produce and respond to BDNF, neurotrophin-3, and FGF
(Knusel et al., 1990; Bean et al., 1991; Spina et al., 1992;
Hyman et al., 1994; Seroogy et al., 1994), suggesting that
these factors have a normal autocrine role in dopaminergic
neuronal survival. The present findings suggest that, in
addition to these locally produced trophic factors, aFGF
synthesized in the striatum may provide trophic support for
midbrain dopaminergic neurons.
For deciphering the role of aFGF in basal forebrain, it
will be important to determine which cell populations
express the receptor for aFGF and, thus, presumably
respond to this factor. Although FGF can rescue forebrain
cholinergic cells following injury, it remains unclear whether
FGF is acting directly via receptors on the cholinergic cells
or through more indirect means, such as promoting the
release of other neurotrophic factors from nearby glial cells
(Burgess and Maciag, 1989; Yoshida and Gage, 1991).
Recently, using sensitive in situ hybridization techniques,
Yoshida et al. (1994) reported partial colocalization of
mRNA for the flg FGF receptor with CUT-like immunoreactivity in cells of the diagonal bands of Broca and the
nucleus basalis but not in cells of the medial septum.
Thompson et al. (1991) have reported that the FGFR3
receptor is expressed by neurons throughout basal ganglia,
although other investigators have found that this FGF
receptor is expressed preferentially by glial cells (Yazaki et
al., 19941. An important goal for future studies will be to
determine whether these receptors are expressed by the
different populations of cholinergic basal forebrain neurons
and, thus, might mediate autocrine trophic actions.
The present findings reinforce the observation (Stock et
al., 1992) that aFGF is expressed prominently by populations of forebrain neurons that are particularly susceptible
to degeneration with age and age-related neurological disease. This correspondence gives rise to the hypothesis that
the cholinergic neurons normally benefit from aFGF action
and become vulnerable if the availability of aFGF fails
under certain pathological conditions. It is not known
whether aFGF expression is reduced with age or in association with conditions such as Alzheimer’s disease. However,
it has been demonstrated that infusion of exogenous aFGF
following cortical devascularization prevents subsequent
degeneration of nucleus basalis neurons and deficiencies on
the Morris water maze task (Figueiredo et al., 1993). This
indicates that aFGF is capable of preserving functional as
well as chemical and morphological properties of one cholinergic cell group that is particularly susceptible to atrophy in
association with pathological aging. Thus, the present data
and the available literature are consistent with the idea
that aFGF may serve as an autocrine neurotrophic factor
for basal forebrain cholinergic neurons, but future studies
are needed to determine whether changes in aFGF expression contribute to disease- and age-related neuropathology.
ACKNOWLEDGMENTS
The authors thank Julie Wong and Zhiquin Sun for their
expert technical assistance. This work was supported by
387
NIA grant AGO0538 and by NIMH Research Scientist
Development Award MH00974 to C.M.G. J.L.B. was supported by PHS grant MH14599.
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