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Volume 17, Number 20,
Issue of October 15, 1997
pp. 7644-7654
Copyright ©1997 Society for Neuroscience
A Role for TrkA during Maturation of Striatal and Basal Forebrain
Cholinergic Neurons In Vivo
Anne M. Fagan1,
Melinda Garber2,
Mariano Barbacid2,
Inmaculada Silos-Santiago2, and
David M. Holtzman1
1 Departments of Neurology, Molecular Biology, and
Pharmacology, and the Center for the Study of Nervous System Injury,
Washington University School of Medicine, St. Louis, Missouri 63110, and 2 Department of Molecular Oncology, Bristol-Myers
Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Nerve growth factor (NGF), acting via the TrkA receptor, has been
shown to regulate the survival and maturation of specific neurons of
the peripheral nervous system. Furthermore, exogenous NGF has potent
actions on TrkA-expressing cholinergic neurons of the basal forebrain
(BFCNs) and striatum. However, initial analysis of mice lacking NGF or
TrkA revealed that forebrain cholinergic neurons were present in these
animals through the fourth postnatal week. Because of the potential
effects of NGF/TrkA interactions on these developing neurons, we have
analyzed quantitatively the striatal and basal forebrain cholinergic
neurons in trkA knock-out mice. By postnatal day (P)
7/8, forebrain cholinergic neurons are smaller in trkA
( /) mice than those in wild-type littermate controls. However,
cholinergic neuron number and fiber density in the hippocampus, a
target region of BFCNs, are grossly intact. Interestingly, by P20-P25
trkA knock-outs contain significantly fewer (20-36%)
and smaller cholinergic neurons in both the striatum and septal
regions, as compared with controls. Cholinergic fiber density within
the hippocampus also is depleted in knock-outs by the end of the second
postnatal week. Contrary to some predictions, despite expression of
p75NTR in the absence of trkA in
BFCNs of these knock-out mice, many cells, although smaller, are still
alive at P25. Our data suggest that, in the absence of NGF/TrkA
signaling, striatal cholinergic neurons and BFCNs do not mature fully
and that BFCNs begin to atrophy and/or die surrounding the time of
target innervation.
Key words:
TrkA;
nerve growth factor;
neurotrophin;
knock-out;
development;
p75NTR
INTRODUCTION
The neurotrophins are a family of
neurotrophic factors, which include nerve growth factor (NGF),
brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and
neurotrophin 4/5 (NT-4/5) (for review, see Thoenen, 1991 ; Chao, 1992).
All neurotrophins bind to the low-affinity receptor
p75NTR (Chao et al., 1986; Radeke et al., 1987;
Rodriguez-Tebar et al., 1990, 1992), the function of which remains
controversial. Recently, however, p75NTR has been
implicated in processes leading to cell death (Rabizadeh et al., 1993;
Rabizadeh and Bredesen, 1994; Carter and Lewin, 1997). Neurotrophins
also bind members of the Trk family of receptor tyrosine kinases (for
review, see Barbacid, 1994), and these interactions have been shown to
be key mediators of neurotrophin actions in the peripheral nervous
system (PNS) (Snider, 1994). NGF binds the TrkA receptor (Kaplan et
al., 1991; Klein et al., 1991; Meakin and Shooter, 1991), and several
studies have shown that NGF/TrkA interactions are critical for the
survival and maintenance of many dorsal root ganglion neurons and most,
if not all, neurons in the sympathetic nervous system (Johnson and
Gorin, 1980; Levi-Montalcini, 1987; Crowley et al., 1994; Smeyne et
al., 1994; Silos-Santiago et al., 1995; Fagan et al., 1996). Although
the role of NGF and TrkA in the development of the PNS has been well
defined now, their role in CNS development is less clear.
Two of the main populations of NGF-responsive CNS neurons that have
been characterized extensively are the cholinergic neurons of the basal
forebrain and striatum (Gnahn et al., 1983; Martinez et al., 1985;
Mobley et al., 1985, 1986; Vantini et al., 1989; Longo et al., 1992).
Basal forebrain cholinergic neurons (BFCNs) are projection neurons, the
axons of which extend throughout the hippocampus and neocortex and are
important for attention, learning, and memory functions (Coyle et al.,
1983; Richardson and DeLong, 1988; Olton et al., 1991). Striatal
cholinergic neurons are large interneurons involved in the control of
movement (Schwarz et al., 1986). Studies have shown that both
populations of neurons express TrkA (Holtzman et al., 1992, 1995;
Steininger et al., 1993; Sobreviela et al., 1994; Li et al., 1995) and
that NGF is expressed in the target regions of these cells (Large et
al., 1986; Whittemore et al., 1986; Lu et al., 1989; Mobley et al.,
1989). Furthermore, the expression levels of NGF are low at birth in
the target of both populations and then increase substantially in the
postnatal period (Large et al., 1986; Whittemore et al., 1986; Lu et
al., 1989; Mobley et al., 1989), correlating with the maturation of both striatal cholinergic neurons and BFCNs. Evidence suggesting that
endogenous NGF directly participates in BFCN development includes
studies in which intracerebroventricular injections of NGF antibodies
were shown to attenuate neurochemical maturation of BFCNs during
development (Vantini et al., 1989; Li et al., 1995). These studies did
not demonstrate the effects of these antibodies on striatal cholinergic
neurons.
Another more direct way to determine the importance of endogenous NGF
and TrkA in the development of forebrain cholinergic neurons is to
assess the CNS of NGF and trkA knock-out mice. It was
reported initially that cholinergic neurons were present in both lines
of mice. BFCN projections to hippocampus and neocortex appeared
qualitatively normal in NGF (/) mice by the fourth postnatal week
(Crowley et al., 1994) but were decreased at the same time in
trkA-deficient mice (Smeyne et al., 1994). Abnormalities of
striatal cholinergic neurons were not reported in either knock-out strain. Because of the marked effects of NGF/TrkA interactions in the
developing PNS and their potential activities in the developing CNS, it
seems both interesting and important to determine precisely whether
endogenous NGF/TrkA signaling is essential for the normal development
of striatal and basal forebrain cholinergic neurons. Because these
neurons mature between the first and fourth postnatal weeks (Mobley et
al., 1989), we analyzed the number, size, appearance, and axonal
projection patterns of these cells from postnatal day (P) P7-P25 in
trkA knock-out mice. Our findings suggest that TrkA signaling is required for the normal maturation and possibly the survival of both basal forebrain and striatal cholinergic neurons. In
addition, they suggest that many neurons can survive for prolonged periods despite continued expression of p75NTR in
the absence of trkA.
MATERIALS AND METHODS
Tissue. Mice homozygous for the trkA
deletion [trkA (/), n = 10];
trkA heterozygous [trkA (+/),
n = 3]; and wild-type [trkA (+/+),
n = 9] littermate controls were analyzed at P7/8
(n = 8) and P20-P25 (n = 14). All pups
were generated from matings of trkA (+/) heterozygous
mice. Before fixation, tail biopsies were taken for genotypic analysis
as described (Smeyne et al., 1994). Animals were perfused
transcardially with 0.1 M PBS, followed by 4%
paraformaldehyde in PBS. Brains were removed and post-fixed in this
same fixative overnight at 4°C and then cryoprotected in 30% sucrose
in PBS. Tissue sections were cut in the coronal plane at 40 µm on a
freezing sliding microtome, and adjacent series through the striatum,
medial septal nucleus (MS), and hippocampus were processed for
immunocytochemistry and/or histochemistry. Additional sets of animals
at P7 [wild-type, n = 2; trkA (+/), n = 1; and trkA (/), n = 3] and P13 [trkA (+/), n = 2;
trkA (/), n = 2] were perfused
transcardially with PBS and frozen on dry ice. Coronal tissue sections
through the septal region were cut at 20 µm on a cryostat and
processed with the terminal deoxynucleotidyl transferase (TdT)-mediated
dUTP-biotin nick end labeling (TUNEL) method to identify cells with
fragmented DNA.
Histology. Free-floating tissue sections through the
striatum and MS were processed for peroxidase immunocytochemistry
(ICC), using antibodies to choline acetyltransferase (ChAT) (1:200,
Chemicon, Temecula, CA) as described previously (Li et al., 1995) to
visualize cholinergic neuronal cell bodies. Adjacent sections were
processed for NADPH-diaphorase (NADPH-d) histochemistry, as described
(Holtzman et al., 1994), to identify noncholinergic, nitric oxide
synthase (NOS)-containing neurons of the striatum. Cholinergic fibers
in the hippocampus were visualized with an antibody to
p75NTR (REX, 1:2000, gift of G. Weskamp and L. Reichardt, University of California San Francisco) (Weskamp and
Reichardt, 1991), as well as with acetylcholinesterase (AChE)
histochemistry, as described (Fagan et al., 1997). Cryostat sections
through the septal region of P7 and P13 animals were stained with the
TUNEL method (fluorescent Apoptag Plus kit, Oncor, Gaithersburg, MD) to
identify fragmented DNA characteristic of apoptotic nuclei.
TUNEL-positive nuclei were identified by FITC fluorescence. Tissue from
knock-out and control animals of each age was processed identically and
simultaneously for each of the histological stains to permit comparison
among animals of different genotypes and ages. In addition, staining was performed multiple times to assess reliability.
Quantitative analysis. The striatum (defined as the area of
striatal tissue dorsal to the anterior commissure and extending from
its incipience rostrally to the end of the fornix caudally) and the
medial septum were analyzed in their entirety. Every other section (for
P7/8) or every third section (for P20-P25) through these structures
was processed for ChAT ICC (as well as NADPH-d histochemistry for
P20-P25 animals) and visualized with a Nikon FXL microscope linked to
a computer via a Dage CCD-72 camera. ChAT-immunoreactive (ChAT-IR) and
NADPH-d-positive cell numbers were counted in an unbiased manner as
described previously (Holtzman et al., 1996), using the optical
dissector method (Gundersen, 1986; West, 1993) in combination with the
Cavalieri method for estimating reference volume (Cavalieri, 1966). A
total of 7-10 sections per structure was analyzed. Cells were sampled
with a dissector frame taped to the monitor screen. Cells were counted if they contained a nucleus that fell within the dissector frame under
a 100× objective. The cross-sectional area of counted cellular profiles also was measured with the National Institutes of Health Image
analysis system (version 1.57), as previously described (Holtzman et
al., 1996). All quantification was done blind to animal genotype.
Between-group (i.e., genotype) analysis of mean neuron number and mean
cross-sectional neuronal profile area of age-matched samples was
performed by Student's t test. Statistical significance was
defined as p < 0.05.
Quantification of TUNEL-positive nuclei in the medial septum was
performed on an additional set of P7 and P13 animals. The total number
of fluorescent profiles in the septal region in four representative,
equally spaced sections from each animal was obtained manually with a
40× objective, and comparisons were made between groups by Student's
t test. Statistical significance was defined as
p < 0.05.
RESULTS
TrkA is required for the normal maturation of striatal cholinergic
neurons during development
To investigate whether TrkA signaling is required for the normal
maturation of striatal cholinergic neurons, we analyzed
ChAT-immunostained tissue sections from postnatal trkA
(/) animals and wild-type littermate controls. Phenotypic changes
in striatal cholinergic neurons were observed in knock-out animals by
P7/8. Qualitatively, many neurons appeared smaller in knock-outs, and
ChAT immunoreactivity was often less intense (Fig.
1A,B), suggesting that
less ChAT protein was present in the absence of trkA. To
quantitate neuronal changes, we counted in an unbiased manner the total
number of ChAT-IR cells in the striatum at P7/8 [wild-type,
n = 4; trkA (/), n = 4]
via the optical dissector method (Gundersen, 1986; West, 1993) in
combination with the Cavalieri method for estimating reference volume
(Cavalieri, 1966). Although the mean number of striatal ChAT-IR neurons
in knock-out and control animals was not significantly different at
P7/8 (Fig. 2A), the
mean cross-sectional area of cholinergic neuronal profiles in the
knock-out striatum was significantly less (10-20%) than controls
(Fig. 2B).
Fig. 1.
Striatal neurons of wild-type and
trkA (/) mice stained with antibodies to ChAT or
NADPH-d histochemistry. A, ChAT immunoreactivity identifies cholinergic neurons in the striatum of wild-type mice at P7.
B, trkA (/) mice exhibit
ChAT-positive cells in the striatum at P7, but many cells are smaller
(see insets in A and B),
and immunolabeling intensity in both cell bodies and neuropil is
generally less than that observed in wild-type mice of the same age.
C, By P22, cholinergic neurons in the wild-type striatum
are large and stain darkly with antibodies to ChAT. A plexus of
immunoreactive fibers also can be observed throughout the striatal
neuropil. D, ChAT-positive cells in the
trkA (/) striatum at P22 are smaller (inset in D) than those in wild-type
animals (inset in C) and often exhibit
reduced ChAT immunoreactivity. Cholinergic fiber staining in the
striatal neuropil also is reduced in knock-outs at this age.
E, Noncholinergic neurons of the striatum, which stain
for NADPH-d, are observed throughout the wild-type striatum at P22.
F, Neurons stained for NADPH-d in P22
trkA (/) mice appear indistinguishable in size and
staining intensity from those of wild-type animals. Scale bar in
A, 100 µm.
[View Larger Version of this Image (123K GIF file)]
Fig. 2.
Quantitative analysis of the number and size
of neurons in the striatum of wild-type and trkA (/)
mice during development. A, Unbiased counting methods
were used to determine the total number of striatal neurons stained
with antibodies to choline acetyltransferase
(ChAT) and NADPH-diaphorase
(NADPH-d) at P7/8 and P20-P25. Although the mean number
of ChAT-positive cells in wild-type (n = 4) and
trkA knock-out (n = 4) mice was not
different at P7/8, trkA (/) mice
(n = 6) at P20-P25 exhibited fewer
ChAT-immunoreactive neurons than wild-type controls
(n = 5; ¥, p = 0.059). The
number of striatal neurons stained for NADPH-d did not differ between the groups at P20-P25. B, The mean cross-sectional area
of striatal profiles stained for ChAT and NADPH-d was determined.
ChAT-immunoreactive neuronal profiles in the striatum of
trkA (/) mice were significantly smaller than those
of wild-type animals at P7/8 and P20-P25. However, cells stained for
NADPH-d were of similar size in the two groups of animals at P20-P25.
Asterisk indicates statistical significance, p < 0.05. Error bars, SEM.
[View Larger Version of this Image (30K GIF file)]
Analysis of the striatum from animals at P20-P25 [wild-type,
n = 5; trkA (/), n = 6]
revealed additional defects in knock-out animals. The cholinergic
neuronal atrophy observed in the trkA (/) striatum at
P7/8 was still apparent at P20-P25 (Figs. 1C,D, 2B). In addition, knock-outs exhibited fewer numbers
(20%) of ChAT-IR cells than controls at this later time point,
although the difference in neuron number did not quite reach
statistical significance (p = 0.059) (Fig.
2A). It was also of note that the absolute number of
detectable ChAT-IR neurons was greater in both wild-type and knock-out
animals at P20-P25, as compared with P7/8. This is likely to be
attributable in part to the increase in ChAT expression that occurs
between these ages (Mobley et al., 1989) (discussed below). The
observed defect in the number of detectable striatal cholinergic
neurons in knock-out animals at P20-P25 cannot be attributed to
smaller brain size because, despite the fact that postnatal
trkA (/) mice that survive to this age are typically smaller (in body and total brain weight) than their wild-type littermates (Smeyne et al., 1994), striatal volume did not differ between the groups at either time point [wild-type = 1.83 mm3 ± 0.15; trkA (/) = 1.73 mm3± 0.14, p > 0.05].
Interestingly, striatal cholinergic neurons in trkA (+/)
heterozygotes appeared normal at P22 and P25 and did not differ in cell
size from wild-type littermate controls [wild-type = 204.35 µm2 ± 8.17; trkA (+/) = 194.35 µm2 ± 13.13; p > 0.05].
To assess whether the phenotypic differences observed in the striatum
were specific for the cholinergic system or merely reflected generalized neuronal death and/or atrophy in trkA-deficient
animals, we also analyzed a noncholinergic population of neurons in the striatum. Noncholinergic striatal neurons that contain nitric oxide
synthase (NOS) do not express TrkA and can be visualized with a
histochemical stain for NADPH-d (Holtzman et al., 1994). In contrast to
the differences observed in cholinergic striatal neurons by P20-P25,
we found no difference between groups in the number or size of
NADPH-d-positive cells of the striatum (Figs. 1E,F,
2A,B). Together, these results suggest that TrkA
signaling is specifically required for the normal maturation of
cholinergic neurons in the striatum.
TrkA is required for the normal maturation of basal forebrain
cholinergic neurons during development
We observed phenotypic defects in developing basal forebrain
cholinergic neurons of trkA knock-out mice that were similar in many ways to those seen in the striatum. As observed in the striatum, the intensity of ChAT immunoreactivity in septal neurons and
their fibers in knock-out animals often appeared qualitatively lighter
than in wild-type cells (Fig. 3). Also,
although cholinergic neuron number in the medial septum was not
different between knock-outs and controls at P7/8 (Fig.
4A), knock-out neurons
were significantly smaller than those in wild-type animals at this
early time point (Figs. 3A,B, 4B). By
P20-P25, trkA (/) animals exhibited significantly fewer
(36%) numbers of ChAT-IR cells in the medial septum than control
animals (Fig. 4A), and those neurons remaining were
atrophic (Figs. 3C,D, 4B). Qualitatively,
atrophic changes observed in the medial septal region also were noted
in cholinergic neurons in the vertical and horizontal limbs of the
diagonal band and in the nucleus basalis (data not shown). Cholinergic
cell size in the medial septum of trkA (+/) heterozygotes
at P22 and P25 was not different from wild-type controls
[wild-type = 179.94 µm2 ± 11.13;
trkA (+/) = 186.40 µm2 ± 13.5, p > 0.05].
Fig. 3.
Neurons in the medial septum of P7 and P22
wild-type and trkA (/) mice stained with antibodies
to ChAT. A, ChAT-immunoreactive neurons in the medial
septum of P7 wild-type mice appear larger (compare
insets in A and B) and
stain more darkly with the ChAT antibody than those in
trkA (/) mice (B) of the same
age. By P22, the difference in medial septal cell size and ChAT
immunostaining intensity between wild-type (C)
and trkA knock-out (D) mice is even clearer than at the earlier time point. ChAT-immunoreactive processes also appear more complex in the wild-type, as compared with
the knock-out mice (see insets in C and
D). In addition, there appear to be reduced numbers of
ChAT-positive neurons in the medial septum of knock-out animals at this
age. Scale bar in A, 100 µm.
[View Larger Version of this Image (90K GIF file)]
Fig. 4.
Quantitative analysis of the number and size of
cholinergic neurons in the medial septum of wild-type and
trkA (/) mice during development. A,
Unbiased counting methods were used to determine the total number of
septal neurons stained with antibodies to ChAT at P7/8 and P20-P25.
Although the mean number of ChAT-positive cells in wild-type
(n = 4) and trkA knock-out
(n = 4) mice was not different at P7/8,
trkA (/) mice (n = 6) at
P20-P25 exhibited significantly fewer immunoreactive neurons than
wild-type controls (n = 5). B, The
mean cross-sectional area of ChAT-positive profiles in the medial
septum of trkA knock-out mice and wild-type controls was
determined. At P7, ChAT-positive neuronal profiles in knock-out animals
were smaller than those in wild-type mice (§, p = 0.053). By P20-P25, immunoreactive cells in the knock-out septum were significantly atrophic, as compared with wild-type controls.
Asterisk indicates statistical significance,
p < 0.05. Error bars, SEM.
[View Larger Version of this Image (29K GIF file)]
Two possibilities exist to explain the differences in striatal and
septal cholinergic neuron number observed between wild-type and
trkA knock-out animals at P20-P25. First, it must be noted that both wild-type and knock-out animals have greater numbers of
detectable ChAT-positive cells at P20-P25, as compared with P7/8. The
fact that there are fewer ChAT-immunoreactive neurons in
trkA (/) animals at P20-P25, as compared with controls
at this age, could reflect the lack of the normal developmental
increase in ChAT expression, which occurs in these cells between P7 and P25 (Mobley et al., 1989). Thus, if there is less of an increase in
ChAT protein in trkA knock-outs, fewer cells are detected by the ICC method and counted. A second, but not mutually exclusive, possibility is that there is an increase in developmental cell death
taking place in trkA (/) animals before P20-P25. To
address the question of cell death, we processed a sampling of sections through the septal region of P7 and P13 knock-out and control animals
with the TUNEL method to identify nuclear fragmented DNA indicative of
apoptosis. Whereas very few TUNEL-positive nuclei were detected in the
septum of either knock-out or control mice at P13, trkA
(/) animals exhibited a significantly greater number of
TUNEL-positive nuclei at P7 than was observed in controls (Fig. 5). The time-dependent pattern of TUNEL
labeling observed in control animals suggests that most naturally
occurring cell death in this region takes place before P13, in
agreement with a recent report (Van der Zee et al., 1996). Although we
cannot be certain that TUNEL labeling was present specifically in
cholinergic neurons, these results also suggest that, in addition to
decreased ChAT expression in knock-out neurons, developmental cell
death in the septal region is exacerbated in trkA-deficient
animals.
Fig. 5.
Counts of TUNEL-positive profiles in four tissue
sections through the septal region of trkA (/) mice
and littermate controls. TrkA (/) mice exhibited
significantly greater numbers of TUNEL-positive nuclei in the septal
region at P7 than were observed in littermate controls. This indicates
that at this time point there is likely to be an increase over baseline
in the amount of naturally occurring cell death in trkA
knock-out animals. By P13, both groups of mice displayed few
TUNEL-stained profiles in the septal region. Asterisk indicates statistical significance, p < 0.05. Error bars, SEM.
[View Larger Version of this Image (23K GIF file)]
TrkA is required for development of the mature pattern of
cholinergic innervation of the hippocampus
Our observation of a defect in the number and size of septal
cholinergic neurons in knock-out animals at P20-P25, together with a
greater number of TUNEL-positive nuclei in this same region at an
earlier time point, suggests that TrkA signaling is required for the
normal maturation and possibly the survival of this NGF-sensitive neuronal population. Because the neurotrophic hypothesis posits that
trophic factor-dependent neurons obtain trophic support from their
target regions during development (for review, see Thoenen, 1991), we
assessed the timing and extent of cholinergic fiber innervation of the
hippocampus, the target region of septal cholinergic neurons. Sections
through the hippocampus of trkA knock-out and control
animals were stained with antibodies to p75NTR as
well as with AChE histochemistry to identify cholinergic fibers. p75NTR-IR colocalizes with ChAT-IR in BFCNs (Hefti
et al., 1986; Batchelor et al., 1989) and in our experience proves to
be better for visualizing basal forebrain cholinergic fibers than does
the cytosolic ChAT marker. Similar to what has been shown in rats
(Milner et al., 1983), we observed cholinergic
(p75NTR-IR) fibers within the stratum oriens and
stratum radiatum of the wild-type hippocampus at P7/8 (Fig.
6). Individual fibers also could be
discerned in the molecular layer of the dentate gyrus at this time
(Fig. 6G). Interestingly, we observed a similar pattern of
p75NTR-IR in the hippocampus of trkA
(/) animals at P7/8 (Fig. 6). Although the level of innervation of
the dentate molecular layer varied among individual knock-out animals,
it was generally similar to that seen in wild-type controls.
Fig. 6.
Immunoreactivity for p75NTR in
the hippocampus of wild-type and trkA (/) mice at
P7. A, p75NTR-immunoreactive fibers
are observed in the hippocampus of wild-type mice at P7.
B, A similar pattern of fiber staining is observed in
trkA knock-outs at this time. At higher magnification,
individual fibers can be seen in many regions of the wild-type
(C, E, G) and trkA (/) (D, F,
H) hippocampus, including the stratum oriens (C,
D), stratum radiatum (E, F), and
molecular layer of the dentate gyrus (G, H).
Fiber density in these regions is not markedly different in the two
groups of animals at this time. gl, Granular layer; ml, molecular layer; or, stratum oriens;
pyr, pyramidal layer; rad, stratum
radiatum. Scale bars: in A, B, 100 µm; in
C-H, 20 µm.
[View Larger Version of this Image (123K GIF file)]
By P20-P25, however, trkA-deficient animals exhibited a
profound defect in cholinergic fiber density in all regions of the hippocampus (Fig. 7), coincident with the
observed atrophy and apparent loss of cholinergic neurons in the medial
septum by this time. Similar changes also were noted in the cortex,
reflecting abnormalities in the cholinergic neurons of the nucleus
basalis. This defect in cholinergic fiber density in trkA
(/) animals was observed as early as P14 and was seen in sections
stained with p75NTR antibodies and by AChE
histochemistry (data not shown).
Fig. 7.
Immunoreactivity for p75NTR in
the hippocampus of wild-type and trkA (/) mice at
P25. A, The adult laminated pattern of cholinergic p75NTR-positive fibers in the hippocampus is
observed in wild-type mice at P25. In contrast, fiber staining is
reduced markedly in trkA (/) mice of the same age
(B). Differences in fiber staining are observed
in all regions of the hippocampus, including the stratum oriens
(C, D), stratum radiatum (E, F),
and molecular layer of the dentate gyrus (G, H).
Loss of fiber staining in the trkA knock-out mice
(B, D, F, H) likely does not merely reflect a
simple downregulation of p75NTR protein expression
because the cell bodies of these projecting neurons in the medial
septum, although clearly atrophic at this time (compare
insets in A and B), stain
darkly with antibodies to p75NTR. gl,
Granular layer; ml, molecular layer; or,
stratum oriens; pyr, pyramidal layer;
rad, stratum radiatum. Scale bars: in A, B, 100 µm; in C-H, 20 µm.
[View Larger Version of this Image (135K GIF file)]
Most, if not all, medial septal cholinergic neurons express
p75NTR (Hefti et al., 1986; Batchelor et al., 1989).
Interestingly, although there was a marked decrease in
p75NTR-positive fiber staining in the hippocampus of
trkA-null mice after the second postnatal week, cell bodies
of surviving medial septal cholinergic neurons continued to express
p75NTR (Fig. 7, insets A and
B). Thus the paucity of hippocampal fiber staining in these
animals is unlikely to be attributable simply to downregulation of
p75NTR within knock-out neurons. It could reflect,
however, an abnormality in the transport of p75NTR
to the axonal regions in knock-out animals or perhaps indicate an
actual loss of axons by this time in the absence of trkA. In support of this latter hypothesis, silver staining (Gallyas et al.,
1980) in a preliminary experiment revealed argyrophilia in regions
known to contain septal cholinergic afferents in a P16 knock-out
animal, indicating the presence of degeneration products (data not
shown). This may indicate a dying back of axons at this time. In the
same experiment argyrophilia was not observed in the wild-type
hippocampus or in knock-out animals at P7 or P22. Together, these
findings indicate that BFCNs can extend axons early in development to
innervate their target region in the absence of TrkA signaling but
require TrkA for their normal maturation and possibly their continued
survival.
DISCUSSION
Analysis of mice lacking one of the neurotrophins (Crowley et al.,
1994; Ernfors et al., 1994; Farinas et al., 1994; Jones et al., 1994;
Conover et al., 1995; Liu et al., 1995) or their signaling Trk
receptors (Klein et al., 1993, 1994; Smeyne et al., 1994; Fagan et al.,
1996) has confirmed that certain PNS neurons require neurotrophin
signaling for their survival in vivo. Recently, it has been
shown that certain CNS neurons require TrkB signaling for survival
during development (Minichiello and Klein, 1996; Alcantara et al.,
1997). What remains to be determined is whether the survival and/or
maturation of developing trkA-expressing CNS neurons is
similarly governed by trophic interactions. The present results are the
first to show that certain CNS neurons require TrkA signaling during
their normal maturation in vivo. Quantitative analysis of
developing trkA (/) mice demonstrated that
trkA is required during the normal maturation of projecting
BFCN and cholinergic interneurons of the striatum. Although there were abnormalities in cell size and ChAT staining intensity by P7/8 in both
cholinergic populations, neuron number was relatively unaffected, and
BFCN axons had reached most of their targets in the hippocampus. In
contrast, significant changes in cholinergic neuron number, as well as
BFCN projections, were apparent by P20-P25. The greater number of
TUNEL-positive cells in the septum of knock-outs at P7 suggests that
some of the decrease in BFCN cell number observed at later time points
may be attributable to cell death, not merely to downregulation of ChAT
within knock-out neurons. Interestingly, BFCNs that remained alive in
knock-out animals at P25 still expressed p75NTR,
suggesting that p75NTR expression during a 25 d
postnatal period can occur in non-trkA expressing neurons
exposed to NGF, without massive cell death ensuing.
Role of TrkA in cholinergic neuronal maturation and/or survival in
the developing CNS
Our observation of cholinergic neuronal atrophy and lighter ChAT
immunostaining in the striatum and basal forebrain of trkA knock-outs by P7 suggests that these CNS neurons require
trkA even for some aspects of their normal early maturation
during development. These defects likely result from the absence of NGF signaling via TrkA, because cell size and ChAT expression can be
regulated by NGF in these developing systems (Gnahn et al., 1983;
Mobley et al., 1985, 1986; Vantini et al., 1989; Longo et al.,
1992).
What remains unclear is the extent to which TrkA is required for
neuronal survival later during development. NGF has been shown to
stimulate ChAT expression, but not neuronal survival, in BFCNs in
culture (Friedman et al., 1993). Our observations of fewer ChAT-IR
neurons in striatum and basal forebrain in knock-outs by P20-P25, as
well as a greater number of TUNEL-labeled cells in the septum at P7,
support the argument that TrkA signaling plays some role in neuronal
survival in vivo. It should be noted that, whereas some
TUNEL labeling in trkA (/) mice is likely to be
localized to cholinergic neurons, it is possible that many TUNEL-positive cells are noncholinergic. Loss of BFCNs in developing trkA (/) mice would be consistent with observations of
BFCN cell death on depletion of target-derived trophic support between the first and fourth postnatal weeks (Burke et al., 1994a,b; Cooper et
al., 1996). It is during this time (P7-P21) that NGF levels in the
target of BFCNs as well as the striatum are increasing (Large et al.,
1986; Whittemore et al., 1986; Lu et al., 1989; Mobley et al., 1989).
Thus NGF, whether derived from local sources (Lu et al., 1989;
Lauterborn et al., 1995) or from the target region, likely regulates
these cholinergic systems by activating TrkA. That decreased cell size
and ChAT immunolabeling intensity precede differences in neuron number
may reflect differences in our methodological ability to discern these
changes or may indicate an actual change in neurotrophin function
during development. For example, cell size and ChAT levels may be
regulated by local NGF interactions early in development, whereas NGF
may exert survival-promoting effects at later stages (e.g., once
targets have been reached).
Interestingly, 60-80% of identified cholinergic neurons were still
present in trkA knock-outs at P25. This contrasts markedly with the PNS, in which there is death over several days in sensory and
sympathetic neurons in the absence of NGF or trkA (Crowley et al., 1994; Smeyne et al., 1994). It is noteworthy that striatal and
basal forebrain cholinergic neurons are born prenatally and then mature
over a 4-6 week period postnatally. One might speculate that, given
the prolonged period over which these cells normally mature, all
cholinergic neurons would die in older trkA knock-outs. Unfortunately, the short lifespan of trkA (/) mice
precludes testing this hypothesis. It is also possible that, although
TrkA is required for certain aspects of neuronal development (e.g., increase in cell size and ChAT expression), the majority of cholinergic neurons can survive in its absence. Another alternate, but not mutually
exclusive, scenario is that cholinergic neuronal survival, either in
wild-type or trkA knock-outs, also may be mediated via other
trophic factor(s), such as fibroblast growth factor-2 (FGF-2), BDNF,
NT-3, or ciliary neurotrophic factor (CNTF), all known to have trophic
effects on BFCNs (Anderson et al., 1988; Otto et al., 1989; Alderson et
al., 1990; Knüsel et al., 1991; Hagg et al., 1992; Koliatsos et
al., 1994).
Role of TrkA in cholinergic process outgrowth and target
innervation during development
TrkA (/) mice at P20-P25 displayed a profound deficit in
cholinergic fiber density in the hippocampus, a defect first apparent in the second postnatal week. This paucity of fibers does not reflect a
problem in axon guidance in the absence of TrkA, because knock-outs and
controls exhibit a similar pattern of p75NTR-IR
fibers in the hippocampus at P7. Despite the fact that the level of
p75NTR can be regulated by NGF (Cavicchioli et al.,
1989; Higgins et al., 1989), it is unlikely that an absence of
detectable fibers in older knock-outs simply reflects downregulation of
p75NTR, because the cell bodies of BFCNs in these
same animals still exhibited staining for this marker (see
inset, Fig. 7B). However, we cannot rule out
completely a possible abnormality in axonal transport of
p75NTR in knock-outs.
Tissue sections stained for AChE, a marker for which the staining
intensity in vivo is not regulated by exogenous NGF (Saffran et al., 1989; Hagg et al., 1990; Holtzman and Lowenstein, 1995), revealed the same defect in hippocampal fiber density (data not shown),
suggesting an absence of fibers in the hippocampus of trkA
knock-outs by the second postnatal week. The absence of staining could
indicate a defect in axon arborization within target regions in the
absence of TrkA. Data demonstrating hyperinnervation in tissues
overexpressing NGF (Edwards et al., 1989; Albers et al., 1994; Davis et
al., 1994) are consistent with this idea. However, our observation that
cholinergic fiber density in the knock-out hippocampus at P20-P25
appeared less than what was observed initially in knock-out animals at
P7 argues for more than a defect in arborization. There may be an
actual loss of fibers with age. Such fiber loss would be expected if
BFCNs are dying or even perhaps dysfunctional.
Is the cholinergic phenotype in trkA (/) mice
attributable to an absence of NGF signaling?
Our data demonstrating impaired development of striatal and basal
forebrain cholinergic neurons in trkA knock-out mice are consistent with the known trophic effects of NGF on these neurons (for
review, see Gage et al., 1991; Longo et al., 1992) and are the first to
show that striatal cholinergic interneurons require endogenous TrkA
signaling for their normal maturation. It is noteworthy, however, that
NGF and trkA knock-outs differ in their hippocampal cholinergic phenotype. Whereas cholinergic fiber staining is markedly abnormal in the hippocampus of P20-P25 trkA (/) mice,
it is present and qualitatively normal in NGF knock-outs of the same age (Crowley et al., 1994). Whether developing NGF knock-outs exhibit
defects in BFCNs is not known. One possible explanation for the less
severe cholinergic phenotype in NGF (/) mice is that there is a
small amount of endogenous TrkA signaling in these mice, which is
ligand-independent. Alternatively, other trophic factors might signal
through TrkA in the absence of the preferred ligand of TrkA, NGF. There
is some precedence for this idea; NT-3 can signal through TrkA and TrkB
in certain cells in vitro (Lamballe et al., 1991; Soppet et
al., 1991; Squinto et al., 1991; Ip et al., 1993; Clary and Reichardt,
1994; Davies et al., 1995), albeit at a significantly higher
ED50. Whether such "crosstalk" can take place in
vivo remains to be determined.
trkA (+/) heterozygous mice did not differ from wild-type
littermates in the appearance or size of striatal and basal forebrain cholinergic neurons at P22-P25, suggesting that the level of receptor is not the limiting factor in regulating the normal maturation of these
neurons. Indeed, observations of cholinergic defects in adult NGF
(+/) heterozygotes (K. Chen and H. Phillips, personal communication)
suggest that the level of ligand is limiting.
Does p75NTR play a role in neuronal death?
In vitro data suggest that p75NTR
can mediate apoptosis in certain cell types (Rabizadeh et al., 1993;
Rabizadeh and Bredesen, 1994; Carter and Lewin, 1997). Observations of
increased numbers of BFCNs in p75NTR (/) mice
are consistent with such a role for p75NTR (Van der
Zee et al., 1996). It has been proposed that specifically those
cholinergic neurons that express p75NTR, but not
trkA, are the ones that die during postnatal development (Van der Zee et al., 1996). Our data do not support this hypothesis entirely. We observed p75NTR immunoreactivity in
BFCNs of trkA knock-out animals through P25. However,
despite the continued expression of p75NTR in the
absence of trkA during this time, most of these cells were
still present at P25. Thus, many neurons that are
p75NTR-positive and trkA-negative
encounter NGF and do not die over a 4 week period.
In sum, our data demonstrate a critical role for TrkA in the
developmental maturation of basal forebrain and striatal cholinergic neurons. TrkA signaling seems to be necessary for normal cell size and
fiber maintenance, but not initial axon outgrowth. Although our data
suggest that TrkA in the CNS also may be required for neuronal
survival, other experiments using techniques such as tissue-specific
gene targeting will be required to prove this point.
FOOTNOTES
Received May 8, 1997; revised July 30, 1997; accepted Aug. 5, 1997.
This research was supported in part by a grant from the McDonnell
Center for Cellular and Molecular Neurobiology (A.M.F.) and a Paul
Beeson Physician-Faculty Scholar Award from American Federation for
Aging Research (D.M.H.). We thank D. Xia for her excellent technical
assistance and Drs. W. Snider, W. Mobley, and E. Johnson for helpful
comments.
Correspondence should be addressed to Dr. Anne M. Fagan, Department of
Neurology, Center for the Study of Nervous System Injury, Washington
University School of Medicine, 660 South Euclid Avenue, Box 8111, St.
Louis, MO 63110.
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