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Volume 17, Number 10,
Issue of May 15, 1997
pp. 3554-3567
Copyright ©1997 Society for Neuroscience
Complementary and Overlapping Expression of Glial Cell
Line-Derived Neurotrophic Factor (GDNF), c-ret Proto-Oncogene, and GDNF
Receptor- Indicates Multiple Mechanisms of Trophic Actions in the
Adult Rat CNS
Miles Trupp,
Natale Belluardo,
Hiroshi Funakoshi, and
Carlos F. Ibáñez
Division of Molecular Neurobiology, Department of Neuroscience,
Karolinska Institute, 171 77 Stockholm, Sweden
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Glial cell line-derived neurotrophic factor (GDNF), the most potent
trophic factor yet described for both dopaminergic neurons of the
substantia nigra and spinal motorneurons, has recently been shown to
signal through a multireceptor complex composed of a novel
glycosylphosphatidylinositol-anchored GDNF receptor- (GDNFR-) and
the receptor tyrosine kinase product of the c-ret proto-oncogene (RET).
Despite its importance, the individual expression patterns and the
relationships between domains of expression of the different components
of this trophic system are not understood. We show here by in
situ hybridization that GDNF mRNA is expressed in the normal
adult rat brain in several targets of substantia nigra neurons,
including striatum, nucleus accumbens, thalamic nuclei, olfactory
tubercle, hippocampus, cerebellum, and cingulate cortex as well as in
the internal granular cell layer of the olfactory bulb. Within the
basal ganglia we observe a pronounced segregation of regions expressing
GDNF from those expressing GDNF receptors, suggesting that within these
structures GDNF is functioning in its anticipated role as a
target-derived trophic factor. In addition, the expression of GDNF and
both GDNF receptors within the cerebellum, hippocampus, and olfactory
bulb may indicate a paracrine mode of action. Importantly, we also see
expression of RET mRNA in cellular populations within the cerebellum
and the glomerular layer of the olfactory bulb, as well as in the
subthalamic nucleus, which lack GDNFR- expression, indicating that
RET functions either independently of GDNFR- or with GDNFR-
presented in trans. Conversely, GDNFR- is widely
expressed in many regions in which RET expression is absent, suggesting
that GDNFR- may associate with additional signaling receptors.
Finally, RET and GDNFR- show distinct patterns of regulated
expression in the brain after kainic acid stimulation and in the
sciatic nerve after nerve transection. Taken together these findings
indicate that GDNF, RET, and GDNFR- utilize multiple mechanisms to
comprise physiologically relevant trophic circuits for different
neuronal populations.
Key words:
in situ hybridization;
GDNF;
ret;
GDNFR-;
mRNA regulation;
cerebellum;
olfactory;
kainate-induced activation;
sciatic nerve transection;
motorneuron regeneration
INTRODUCTION
Although the initial purification and cloning of
glial cell line-derived neurotrophic factor (GDNF) generated a great
deal of interest centered around its pronounced effects on cultured dopaminergic neurons of the substantia nigra (Lin et al., 1993 ), subsequent studies have shown that GDNF has similarly potent effects on
a wide range of neuronal populations. First characterized were the
survival-promoting activities of GDNF on primary cultures of spinal
motorneurons (Henderson et al., 1994) and lesioned motorneurons in vivo (Li et al., 1995; Oppenheim et al., 1995; Yan et
al., 1995). Additionally, GDNF was seen to have pronounced effects on
cultures of dissociated neurons from various chick peripheral ganglia sympathetic, sensory, and enteroceptive (Bujbello et al., 1995; Ebendal et al., 1995; Trupp et al., 1995). Further in
vitro studies identified Purkinje cells as a population that
responds to GDNF in primary cultures (Mount et al., 1995), and several in vivo studies have indicated that noradrenergic neurons of
the locus coeruleus (Arenas et al., 1995) and cholinergic neurons of
the basal forebrain (Williams et al., 1996), as well as thalamic and
hippocampal neurons (Martin et al., 1995), all benefit from treatment
with GDNF prior or subsequent to experimental lesioning. While GDNF has
widespread effects in the adult CNS, its critical role as a morphogen
in the developing excretory and enteric nervous systems is highlighted
by the phenotype of GDNF null-mutated mice, which die shortly after
birth (Moore et al., 1996; Pichel et al., 1996; Sanchez et al.,
1996).
The diverse actions of GDNF may be better understood in light of the
finding that GDNF signaling is mediated by the receptor tyrosine kinase
encoded by the c-ret proto-oncogene (RET) (Durbec et al., 1996; Trupp
et al., 1996). Indeed, although very little is known about the
expression of RET mRNA in the adult mammalian nervous system, several
of the populations responsive to GDNF express RET. These include spinal
motorneurons (Pachnis et al., 1993; Tsuzuki et al., 1995), dopaminergic
neurons of the adult substantia nigra (Trupp et al., 1996), and various
subpopulations of peripheral gangliasympathetic, nodose, enteric, and
some sensory (Pachnis et al., 1993; Tsuzuki et al., 1995). Strikingly,
the RET protein is known to be expressed in the ureteric buds of the metanephric kidney, where it is absolutely required for ureteric branching induced by GDNF from the adjacent mesoderm (Schuchardt et
al., 1994; Durbec et al., 1996).
Although it is clear that reciprocal RET/GDNF interactions are
necessary for normal development, a newly cloned
glycosylphosphatidylinositol (GPI)-anchored GDNF binding protein, GDNF
receptor- (GDNFR-), is believed to enhance the affinity of GDNF
for its signaling receptor (Jing et al., 1996; Treanor et al., 1996).
These studies also revealed that GDNFR- can assist in GDNF binding
to RET even when administered in a soluble form. Preliminary studies
indicate some overlap between GDNFR and RET expression patterns such
that both transcripts appear in embryonic rat motor neurons, ureteric buds of developing nephrons, and adult substantia nigra neurons (Treanor et al., 1996).
We have sought to further characterize possible GDNF/RET/GDNFR-
interactions in vivo by analyzing expression of these mRNA species using in situ hybridization and RNase protection
assays. We first show that some of these interactions may be
physiologically relevant in the normal adult rat brain by describing
constitutive expression of GDNF in several targets of substantia nigra
neurons. We also describe RET mRNA expression patterns that suggest
novel neuronal populations that may respond to GDNF. Finally, we see distinct patterns of regulation for the GDNF receptors after
experimental manipulations indicating novels modes of action for GDNF
that may be amenable to therapeutic manipulations.
MATERIALS AND METHODS
In situ hybridization. Fourteen micrometer
sections of spinal cord or brain were thawed onto 3-aminopropyl
ethoxysilane-coated slides for hybridization with radiolabeled probes
as follows. After fixation in 4% paraformaldehyde for 15 min, slides
were rinsed once in PBS and twice in distilled water. Tissue was
deproteinated in 0.2 M HCl for 10 min, acetylated with
0.25% acetic anhydride in 0.1 M ethanolamine for 20 min,
and dehydrated with increasing concentrations of ethanol. Slides were
incubated 16 hr in a humidified chamber at 58° with 8 × 105 cpm of probe in 300 µl of hybridization cocktail
(50% formamide, 20 mM Tris-HCl, pH 7.6, 1 mM
EDTA, pH 8.0, 0.3 M NaCl, 0.1 M dithiothreitol, 0.5 mg/ml yeast tRNA, 0.1 mg/ml polyA
RNA, 1× Denhardt's solution, and 10% dextran sulfate). Slides were
first washed at room temperature in Formamide/SSC (1:1) followed by 30 min at 65° in 1× SSC. Single-stranded RNA was digested by RNase
treatment (10 mg/ml) for 30 min at 37° in 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5, 2 mM EDTA. Tissue was washed twice with 1× SSC in 65° for
30 min before it was dehydrated in ethanol and air-dried. Slides were
either exposed to  -max x-ray film (Amersham, Buckinghamshire, UK)
for 10-20 d or dipped in NTB-2 photoemulsion diluted 1:1 in water
(Eastman Kodak, Rochester, NY) exposed at 4° for 3-5 weeks,
developed with D19 (Eastman Kodak), fixed with Al-4 (Agfa Gevaert,
Kista, Sweden), and counterstained with cresyl violet.
RNA isolation, cRNA probes, and RNase protection assays.
Total RNA was isolated from frozen samples homogenized in guanidine isothiocyanate and -mercaptoethanol by the addition of 1:10 vol of 2 M sodium acetate followed by phenol/chloroform extraction and ethanol precipitation. cRNA probes, generated from linearized plasmids using T3 or T7 RNA polymerase (Promega Biotec, Madison, WI)
and 35S-UTP or 32P-CTP (Amersham),
respectively, were used for in situ hybridization as above,
and RNase protection assays (RPA) were performed according to
manufacturer's instructions (Ambion, Austin, TX). The
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobe for RPA has
been described previously (Trupp et al., 1995). GDNF cRNA probes were
transcribed from a new fragment (nucleotides 279-700 of the originally
published rat GDNF cDNA) subcloned for use as an in situ
hybridization probe containing 422 nucleotides homologous to both
published GDNF transcripts. The rat RET riboprobe has been described
previously (Trupp et al., 1996). GDNFR- probes of 250 (RPA) and 400 nucleotides (in situ hybridization) were generated by RT-PCR
using primers designed from the previously described cDNA sequence
(Treanor et al., 1996). Control sense probes for in situ
hybridization were generated from all plasmids using the reciprocal RNA
polymerase.
Kainic acid stimulation. Adult male Wistar rats (150-170
gm) received bilateral intracerebroventricular injections of kainic acid (0.35 µg/0.5 µl; Sigma, St. Louis, MO),
because in our experience this offers the advantage of a standardized
latency of initiation and intensity of seizure activity with abrupt
termination of status epilepticus, which contributes notably to a
reduction of interanimal variability. Animals were mounted in a
stereotactic frame and injected using the following stereotaxic
coordinates: anteroposterior 0.2, lateral 1.5, ventral 4.2 from the
bregma. At the indicated time points animals were decapitated after
ether anesthesia, and the brains were removed, frozen in  40°
isopentane, stored at 70°, and cryosectioned for in situ
hybridization.
Sciatic nerve transection. Adult male Sprague Dawley rats
(200-250 gm) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) before transection of the right sciatic
nerve, distal to the obturator tendon. To minimize religation of nerve
segments, both the proximal and distal ends of the transected nerve
were reflected. At the indicated times after axotomy, the animals were sacrificed, and the gastrocnemius muscle, lumbosacral portion of the
spinal cord, and four segments (distal/distal; distal/proximal; proximal/distal; proximal/proximal) of the sciatic nerve were dissected
out. All tissues were frozen immediately on dry ice and stored at
70° before preparation of RNA.
Sciatic nerve cultures. Sciatic nerves dissected out from
decapitated early postnatal rat pups (P1-5) were incubated in PBS containing 0.2% collagenase II (Sigma) for 15 min at 37°, followed by an additional 15 min incubation after the addition of trypsin to
0.25% final concentration. Nerves were washed, triturated, and plated
in DMEM supplemented with 10% fetal calf serum (Life Technologies,
Gaithersburg, MD), glutamine, penicillin, and streptomycin. Cultures
were washed with the above media twice for 2 hr before affinity-labeling with iodinated recombinant GDNF, as described previously (Trupp et al., 1996).
RESULTS
Physiological trophic circuits in the basal ganglia
Despite the several critical brain regions that respond to GDNF
treatment in the adult rodent brain, endogenous GDNF expression has
been verified only at very low levels using a highly sensitive RNase
protection assay (Arenas et al., 1995), and one in situ hybridization study reported the absence of GDNF mRNA expression in
adult striatum (Strömberg et al., 1993), raising the possibility that the rescue of lesioned adult neurons by exogenous GDNF may not
represent the endogenous activities of this factor. We therefore have
sought to resolve more clearly the expression of GDNF by performing
in situ hybridization using radiolabeled cRNA probes. Several regions of the adult brain are labeled with GDNF cRNA probes,
with the general pattern being moderate labeling of only scattered
cells. Most striking is the number of regions that are innervated by
the various projections of substantia nigra neurons, which have been
shown to express both GDNF receptors. Importantly, we see strong
labeling over scattered, medium-sized neurons throughout the caudate
nucleus, putamen, and internal and external segments of the globus
pallidus (Fig.
1A,B), whereas
neither GDNFR- nor RET mRNA were detected anywhere in the striatum
(Fig. 1, C and D, respectively). As with all
regions studied, no labeling was detected with radiolabeled sense
probes complementary to those used to assay mRNA expression (not
shown). In addition, slightly stronger labeling for GDNF is seen in
several thalamic nucleiventromedial, anteroventral, and
ventropostero-lateral and -medial nuclei (Fig. 1E;
and data not shown), none of which show expression of either receptor
(Fig. 1G,H; and data not shown). Conversely, the only RET
labeling of thalamic nuclei appears at high levels in the reticular
thalamic nucleus and zona incerta (Fig. 1H) and the lateral habenula (Fig. 6), all of which express GDNFR- (Fig. 1G). With the noted exception of GDNF-expressing
populations, GDNFR- mRNA is seen at moderate to high levels
throughout the thalamus, with the most pronounced labeling appearing in
cells of the zona incerta and reticular thalamic nucleus. Generally, within the basal ganglia there appears a clear-cut separation of
populations that express GDNF from those expressing mRNA encoding the
GDNF receptors (Fig. 1). These observations suggest that GDNF is
functioning as a target-derived factor to maintain neuronal circuits
within the basal ganglia. We observe GDNF expression in several targets
of efferent projections of substantia nigra, namely olfactory tubercle,
nucleus accumbens, and the second layer of the cingulate cortex (Fig.
1A), indicating that external sources of GDNF may
also have effects on neurons in the basal ganglia.
Fig. 1.
Segregated patterns of expression within the basal
ganglia. In situ hybridization of 14 µm coronal
sections through striatal (A-D) and thalamic
(E-H) levels of the basal ganglia demonstrates a
clear separation of domains of expression of mRNA encoding GDNF and its
receptors. A, Autoradiogram indicating GDNF expression in scattered cells throughout the striatum (str),
nucleus accumbens (na), and cingulate cortex
(cc), and stronger labeling throughout the olfactory
tubercle (ot). B, Bright-field
photomicrograph showing that medium-sized neurons throughout the
striatum express low levels of GDNF, whereas some scattered neurons
express considerably higher levels. C, Although no
GDNFR- is expressed in the striatum, very high levels are seen
throughout the lateral septum (ls) and in some
peripheral regions of the medial septum. Strong labeling is also seen
in the claustrum (cl), in the dorsal endopiriform nucleus (ep), and in scattered cells in all layers of
the cortex. D, Again, although no RET is expressed in
the striatum, low levels are seen throughout the medial septum
(ms). E, GDNF mRNA is detected at high
levels in ventromedial and ventrolateral thalamic nuclei (th) and at low levels throughout the hippocampus.
F, Bright-field photomicrograph of the ventrolateral
thalamic nucleus indicates that a large proportion of medium-sized
neurons express GDNF mRNA. G, Although thalamic nuclei
that produce GDNF express neither of its receptors, high levels of
GDNFR- mRNA are seen in the reticular thalamic nucleus
(rt), zona incerta (zi), and medial habenula (mh). In addition, GDNFR- labeling can be
seen throughout the amygdala (am) and hypothalamus
(ht). H, Similarly, RET mRNA is detected
in the zona incerta and reticular thalamic nucleus. Scale bar (shown in
F): B, F, 93 µm.
[View Larger Version of this Image (120K GIF file)]
Fig. 6.
Segregated domains of expression within the
olfactory bulb. A, An autoradiogram of a sagittal
section through the olfactory bulb indicates that RET mRNA is expressed
at high levels throughout the glomerular layer
(gl) and at lower levels in some regions of the internal granular layer (igl). B,
C, Conversely, GDNF mRNA is detected in the internal granular
cell layer, whereas GDNFR- is most prominent within the external
plexiform layer (epl). D, Dark-field photomicrograph showing detail of red boxed
region in A indicates that RET mRNA is expressed
in cells surrounding glomeruli. E, Detail of
green boxed region in C indicates that GDNFR- is expressed at high levels in cells throughout the external plexiform layer and at low levels surrounding some glomeruli on the
periphery of the external plexiform layer. F, G,
Bright-field photomicrograph of one glomerulus
(F) and scattered cells in the external plexiform
layer (G). H, Superimposition of
colorized autoradiograms A-C. RET labeling was colored
red, GDNF blue, and GDNFR-
green. Scale bar (shown in G): D,
E, 150 µm; F, G, 30 µm.
[View Larger Version of this Image (119K GIF file)]
RET mRNA expression indicates novel
GDNF-responsive populations
In addition to the various neuronal populations of the basal
ganglia, some of which have been shown to respond to GDNF in vivo, we see mRNA encoding receptors for GDNF in several other brain regions. We have shown previously that lesioned noradrenergic neurons of the locus coeruleus can be rescued by GDNF (Arenas et al.,
1995) and show here that both RET and GDNFR- mRNAs are expressed by
these neurons (Fig. 2A,B,D). We also
detected both RET and GDNFR- mRNA in sensory neurons of the
trigeminal mesencephalic nucleus (Fig. 2A,C). Also as
expected, brain stem motor nuclei, including facial (Fig.
3A,B), hypoglossal (Fig. 3C,D),
trigeminal (Fig. 3E,F), and oculomotor (Fig.
4A-D), are seen to express high levels of both RET and GDNFR-. Within the motor nucleus of the trigeminal, both large and small neurons were labeled with RET and
GDNFR- (Fig. 3F; data not shown). Cholinergic neurons of the basal forebrain have also been described as responsive to the
survival-promoting effects of GDNF (Williams et al., 1996); however,
although GDNFR- is seen to be expressed at high levels in neurons of
the lateral septum (Figs. 1C, 2E),
labeling was observed only on the periphery of the medial septum (Fig.
1C). Conversely, RET mRNA was detected in scattered neurons
in the medial septum (Figs. 1D, 2F)
and not at all in the lateral septum (Fig. 1D). Thus,
there appears to be some segregation of RET and GDNFR- expression
within the basal forebrain.
Fig. 2.
RET and GDNFR- coexpression correlates with
in vivo responsiveness to GDNF. Previous studies of GDNF
in vivo activities have identified noradrenergic neurons
of the locus coeruleus and cholinergic neurons of the basal forebrain
as responsive to GDNF (see introductory remarks). A,
Dark-field photomicrograph showing intense labeling of sensory neurons
of the trigeminal mesencephalic (mt) nucleus and lower,
diffuse labeling of the locus coeruleus (lc), which is
shown encircled in dashed white lines. B,
Bright-field photomicrograph of the medial-dorsal edge of the locus
coeruleus showing diffuse, specific labeling over the densely packed
neurons of the lc. C, Intense labeling
for RET mRNA is observed over large proprioceptive neurons of the
trigeminal mesencephalic nucleus. D, GDNFR- mRNA is
also observed throughout the locus coeruleus. E,
Although intense labeling for GDNFR- is observed over large neurons
of the lateral septum, less strong labeling is also seen over smaller
neurons. F, In the medial septum, RET mRNA is detected
in scattered neurons. Scale bar (shown in F):
A, 250 µm; B, C, E, F, 21 µm;
D, 27 µm.
[View Larger Version of this Image (133K GIF file)]
Fig. 3.
RET and GDNFR- are highly expressed in motor
nuclei of cranial nerves. Motor neurons of the rodent brainstem,
including facial (VII) and hypoglossal (XII), have been shown
previously to respond to GDNF survival-promoting effects.
A, Dark-field photomicrograph depicting high levels of
RET mRNA in the facial motor nucleus. B, Bright-field
image showing that the RET labeling is over the large motorneurons.
C, D, In addition, motor neurons of the
hypoglossal nucleus express mRNA encoding GDNFR-. E,
Dark-field image of the motor nucleus of the trigeminal (V) indicates
that RET is expressed widely and at varying levels within the nucleus.
F, Higher magnification bright-field image of this
section reveals that RET is expressed in both small and large neurons
within this nucleus. GDNFR- mRNA is also detected in both small and
large neurons within the motor nucleus of the trigeminal (not shown). Scale bar (shown in F): A, C, E, 230 µm; B, D, 20 µm; F, 25 µm.
[View Larger Version of this Image (153K GIF file)]
Fig. 4.
Coexpression of GDNFR- and RET suggests
novel GDNF-responsive populations. A, Dark-field
photomicrograph showing high GDNFR- mRNA levels in caudal linear
(CLi) and dorsal Raphe (DR) nuclei and
lower levels in motor nuclei of the oculomotor nerve (III). B, Conversely, hybridization of an adjacent section with
an RET cRNA probe indicates that RET is more highly expressed in
oculomotor (OM) neurons and at lower levels
in both caudal linear and dorsal Raphe. C, D,
Higher-magnification bright-field images of the dorsal Raphe indicate
that expression of GDNFR- mRNA is more widespread than that of RET
mRNA. Lower levels of transcripts encoding both receptors were also
detected in the medial Raphe nucleus (not shown). E, F,
Dark-field photomicrographs depicting GDNFR- mRNA expression in the
dorsal cochlear nucleus (E) and the lateral vestibular
nucleus (F). G, H, Bright-field
micrographs of adjacent sections hybridized with RET antisense cRNA
probes indicate that RET is expressed in neurons of both the dorsal
cochlear (G) and lateral vestibular
(H) nuclei. Although expression of both GDNFR- and RET mRNA is confined to the dorsal region of the cochlear nucleus,
both transcripts were detected in medial and superior vestibular nuclei
in addition to the lateral region (not shown). Scale bar (shown in
H): A, B, 107 µm; C,
D, 21 µm; E, F, 160 µm; G, 11 µm; H, 16 µm.
[View Larger Version of this Image (116K GIF file)]
In addition to these previously characterized GDNF-responsive
populations, we find novel neuronal populations, which, based on the
expression of both GDNF receptor components, are likely to benefit from
the neurotrophic activities of GDNF. Of considerable interest is the
finding that serotonergic neurons of the dorsal Raphe nucleus express
both GDNF receptors (Fig. 4A-D). As this population
has been shown to degenerate in some cases of Parkinson's disease,
this suggests that GDNF may be an even more efficient therapeutic agent
for this disorder. In support of this notion, preliminary results
indicate that GDNF can promote the survival of a portion of lesioned
serotonergic neurons of the dorsal Raphe (P. Åkerud and E. Arenas,
personal communication). Additional populations that express both RET
and GDNFR- mRNAs include dorsal cochlear (Fig.
4E,G), lateral vestibular
(4F,H), medial and lateral habenular (see Fig.
6J,L), and several hypothalamic nuclei, including arcuate and dorsomedial compacta (see Fig.
6H,M). We also see many populations of neurons
that express GDNFR- in the absence of RET, including all cortical
layers, lateral geniculate nucleus, and the superior colliculus (Table
1; and data not shown).
Complete trophic circuits within the cerebellum
In correlation with the recent finding that endogenous RET protein
in the cerebellum is constitutively phosphorylated on tyrosine residues
(Colucci-D'Amato et al., 1996), we see GDNF expression in scattered
cells of the granular layer of the cerebellum (Fig. 5A,D). In addition, strong labeling for both
GDNFR- and RET mRNAs is detected in a narrow band at the external
edge of the granular cell layer (Fig. 5B,C) over cells
directly adjacent to Purkinje cells (Fig. 5E,F). The
strong labeling for RET in this region dissipates gradually both toward
the granular layer and into the molecular layer, creating gradients of
RET expression (Fig. 5C). Importantly, this demonstrates a
population of cells within the granular layer, which do not appear to
be granule cells (Fig. 5G), that express RET but not
GDNFR-. Interestingly, although GDNF has been shown to have effects
on Purkinje cells in mixed cultures of embryonic cerebellum (Mount et
al., 1995), we do not detect mRNA for either of the previously
characterized GDNF receptors in Purkinje cells of adult animals (Fig.
5D,F); however, we do observe GDNFR- and RET
mRNAs as well as weak labeling for GDNF mRNA in deep cerebellar nuclei
(Fig. 5A-C,H,I). The pattern of RET mRNA expression
detected in the cerebellar cortex suggests that this receptor is
activated by GDNF produced within the structure. GDNF secreted by
granule cells may be acting on afferent mossy fibers as well. It is
interesting to note in this context the expression of GDNF receptors in
the pontine, red, cochlear, and vestibular nuclei (Table 1), all of
which extend projections to the cerebellar granular layer.
Fig. 5.
Complete trophic circuits within the cerebellum.
A-C, Autoradiograms of hybridized sagittal sections
through the cerebellum show that GDNF, RET, and GDNFR- mRNAs are all
expressed in the cerebellar cortex as well as the deep cerebellar
nuclei. D, Bright-field photomicrograph of the granular
layer reveals that scattered cells within this layer that do not seem
to be granule cells express GDNF mRNA. E, GDNFR- mRNA
is seen to be expressed most highly in the Purkinje layer in cells
directly adjacent to Purkinje cells (possibly basket cells) and
also at lower levels reaching into the molecular layer.
F, RET mRNA is also observed in cells similarly placed
next to Purkinje cells, as well as within the molecular layer.
G, High-magnification bright-field photomicrograph of
the granular layer of the cerebellum reveals that RET mRNA is highly expressed in cells that stain very lightly with cresyl violet. H, I, Neurons of deep cerebellar nuclei are seen to
express both GDNF receptors. Scale bar (shown in
I): D, 29 µm; E, F, H,
I, 20 µm; G, 14 µm.
[View Larger Version of this Image (161K GIF file)]
Segregated domains of expression of GDNF and its receptors within
the olfactory bulb
Analysis of the expression patterns of the GDNF receptors
within the olfactory bulb indicates that RET mRNA is present in cells
that express no or very low levels of GDNFR; labeling for RET mRNA
is seen at high levels within the olfactory nerve layer and the
glomerular layer of the olfactory bulb (Fig.
6A,D,F). This suggests that RET
is using another co-receptor or collaborating with GDNFR- expressed
on the surface of an adjacent cell or as a soluble protein. Indeed,
either of these latter mechanisms seems possible, because GDNFR-
expression is pronounced in the external plexiform layer of the bulb
(Fig. 6C), juxtaposed to the nerve and glomerular layers. In
this region, GDNFR- is detected at high levels in scattered cells
(Fig. 6E,G). In addition, we see GDNF mRNA expression
in the internal granular layer of the olfactory bulb (Fig.
6B), which suggests that GDNF could activate RET
expressed within the olfactory bulb but could also act on
RET-containing projections from afferent nuclei, such as the dorsal
Raphe or locus coeruleus. Superimposition of the expression patterns of the three molecules reveals unexpectedly segregated domains of expression for GDNF and its receptors within the olfactory bulb (Fig.
6H).
Kainic acid-induced regulation of GDNF and its receptors in the
adult brain
RET-expressing populations within the septum could be
physiologically activated by GDNF expressed in the normal adult
hippocampus at low levels (Figs. 1E, 7A).
We have studied the effects of kainic acid-induced activation on the
expression of GDNF and its receptors in adult hippocampus. Similar to
previous studies (Humpel et al., 1994), we see an upregulation of GDNF
in dentate gyrus after 2 hr that peaks at 4 hr (Fig.
7B,C). Twelve hours after kainic acid treatment, increased GDNF mRNA levels are observed in all regions of
the hippocampus (Fig. 7D), whereas after 24 hr, labeling
remains elevated in only the CA3 and hilar regions (Fig.
7E). In view of the present results, the previously
described kainate-induced induction of GDNF mRNA in granule cells of
the dentate gyrus should now be reconsidered as an upregulation of
endogenous GDNF mRNA levels. This increase of ligand is in close
correlation with that which we observe for the receptors. As with GDNF,
the upregulation of GDNFR- mRNA in dentate gyrus is pronounced and
rapid, already increasing after 2 hr (Fig. 7G), reaching
peak levels at 4 hr (Fig. 7H), and returning to basal
levels after 48 hr (not shown). Although this time course of
upregulation is similar in the CA3 region, GDNFR- expression is
greatly increased in CA1 only after 24 hr (Fig. 7J)
and remains high after 48 hr (data not shown). Finally, an increase in
CA4 is seen to begin at 12 hr and peak at 24 hr (Fig.
7I,J). Although moderate levels of GDNFR- are seen
in all regions of the normal adult hippocampus, RET mRNA is expressed
only at very low levels (Fig. 7K). Also, distinct patterns of expression for these receptors are observed after kainic
acid treatment: GDNFR- mRNA is observed in all cells, whereas RET
mRNA is seen to be strongly expressed in only a subset of neurons
scattered throughout the pyramidal layer (Fig. 7N). In addition, RET mRNA is seen to be upregulated after 12 and 24 hr in
scattered extrapyramidal cells (Fig. 7N,O). Furthermore, upregulation of transcripts encoding both RET and GDNFR- is observed to begin in the piriform cortex after 12 hr (Fig.
7I,N), peak at 24 hr (Fig. 7J,O), and
subside after 48 hr (not shown).
Fig. 7.
Kainate-induced activation upregulates GDNF, RET,
and GDNFR- mRNAs. GDNF mRNA upregulation is detected in the dentate
gyrus (DG), CA1, CA2, CA3, and hilar
(Hil) regions of the hippocampus. In addition to
an increase in all hippocampal regions, GDNFR- mRNA levels also
increase in the piriform cortex. RET mRNA increases are most pronounced
in DG after 4 hr and in CA3 and piriform
cortex after 12 hr. Also, extrapyramidal cells of the hippocampus
increase RET mRNA expression after 12 and 24 hr of induced activation. In contrast to GDNFR- and GDNF patterns, RET mRNA appears to be
expressed by only a subset of neurons scattered within and outside of
the pyramidal layers. Arcuate hypothalamic nucleus (Arc), dorsal-medial hypothalamic nucleus, compacta
(DMC), lateral habenular nucleus (LHb),
subthalamic nucleus (STh).
[View Larger Version of this Image (141K GIF file)]
A role for endogenous GDNF and GDNFR- in the repair of
lesioned motorneurons
We have shown previously that GDNF mRNA is upregulated
dramatically in distal segments of the adult rat sciatic nerve after mechanical transection (Trupp et al., 1995). We have now analyzed the
expression of GDNFR- in similarly treated nerve segments by RNase
protection assay. We observe a very dramatic upregulation of GDNFR-
mRNA appearing at 3 d, with a maximum at 7 d (Fig. 8A). Importantly,
this regulation is most pronounced in the more distal segments of the
sciatic nerve (Fig. 8B), resulting in a distally
increasing gradient of GDNFR- mRNA expression. Although no RET mRNA
was detected anywhere in the sciatic nerve (data not shown), expression
is detected in the sacrolumbar spinal motorneurons that contribute
axons to this nerve (Fig. 9A). We observe an
upregulation of RET mRNA expression in spinal motorneurons after
transection of the sciatic nerve similar to that shown previously in
chick spinal motorneurons after axotomy (Nakamura et al., 1996).
Likewise, levels of GDNFR- mRNA were seen to increase in primary
motorneurons at the L5 level after axotomy (Fig. 9A). In
addition, we observe an increase of GDNF mRNA expression in axotomized
muscle 3 d after nerve transection (Fig. 9B), as well
as a distally increasing gradient of GDNF mRNA expression in cells of
the lesioned sciatic nerve (data not shown) similar to that seen for
GDNFR- mRNA. Taken together, these expression patterns suggest a
novel mechanism of trophic support for degenerating motorneurons.
Fig. 8.
Upregulation of GDNFR- mRNA after sciatic nerve
transection. A, Autoradiogram of an RPA revealing a
pronounced increase in GDNFR- mRNA in the distal segment of
transected sciatic nerve 3 and 7 d after surgery.
B, A distally increasing gradient of GDNFR- mRNA is
demonstrated in an autoradiogram of an RPA of sciatic nerve samples
collected 3 d after nerve transection. C, Photomicrographs of GDNFR- in situ hybridization of
nerve segments from animals sacrificed 3 d after nerve
transection. The GDNFR- mRNA-expressing cells are clearly present in the Schwann cell layer, whereas the epineurium
(ep) is devoid of labeling. For analysis, the transected
nerve was dissected into four parts: moving laterally from the cord,
the proximal segment of the proximal stump
(prox-prox), distal segment of the proximal stump
(dist-prox), proximal segment of the distal stump
(prox-dist), and distal segment of the distal
stump (dist-dist). Scale bar (shown in
C): dark field, 134 µm; bright field, 18 µm.
[View Larger Version of this Image (32K GIF file)]
Fig. 9.
Regulation of GDNF mRNA in muscle and its
receptors in spinal motorneurons. A, Dark-field
micrographs of transverse sections through L5 spinal motorneurons
reveal that GDNFR- mRNA (top panels) and RET mRNA
(bottom panels) are upregulated in spinal motorneurons 3 d after nerve transection. B, Autoradiogram of an
RPA showing that sciatic nerve transection induces GDNF mRNA expression
in gastrocnemius muscle. Note, this cRNA probe protects two mRNA fragments of slightly different lengths that are present in both previously characterized GDNF transcripts. Scale bar: A,
134 µm in top panels and 107 µm in bottom
panels.
[View Larger Version of this Image (56K GIF file)]
GDNFR- produced by spinal motorneurons is likely to function as a
co-receptor with RET in a GDNF binding complex; however, the GDNFR-
expressed in the sciatic nerve in the absence of RET expression must be
either acting as a co-receptor for another signaling receptor distinct
from RET or alternatively used in trans to cooperate with
RET expressed by motorneurons. To address this question, we have
investigated GDNF receptor complexes in primary cultures of dissociated
sciatic nerve cells by affinity-labeling followed by chemical
cross-linking. Although we see pronounced binding of GDNF to a protein
of the molecular weight of GDNFR-, we do not find affinity-labeling
of any other proteins (Fig. 10), suggesting that
GDNFR- may be acting in these cells to present GDNF to injured motor
neurons.
Fig. 10.
Primary cultures of sciatic nerve
affinity-labeled with iodinated GDNF in the absence of any known
signaling receptors. Cross-linking of iodinated GDNF to GDNFR- is
demonstrated by affinity-labeling of NIH3T3 fibroblasts
and fibroblasts stably transfected with GDNFR- cDNA
(3T3R) in a pcDNA3 plasmid (Invitrogen, San Diego, CA), left lanes. Autoradiograms of SDS-PAGE run under
reducing conditions showing iodinated GDNF affinity-labeling of
MN1 cells (a motorneuron cell line expressing RET mRNA,
which we have previously characterized as responsive to GDNF,
center lane) and primary cultures of P5 rat sciatic
nerve (1°SN, SN+cold, right
lanes) clearly indicate that although GDNF is not binding to
RET (155 kDa) in these cells, it does affinity-label a protein of
approximately the molecular weight of GDNFR- (75 kDa, including
ligand). Arrowheads indicate bands corresponding to
affinity-labeled GDNFR- or proteins of similar molecular
weight.
[View Larger Version of this Image (52K GIF file)]
DISCUSSION
We have sought to answer here the question of whether the
activities described previously for exogenous GDNF on various
populations of lesioned neurons reflect the presence of endogenous
trophic circuits of this factor within the normal adult CNS. The
studies reported here indicate that GDNF is expressed in the proper
locations to function in the maintenance of various neuronal circuits
in the normal adult rat brain. Additionally, the regulation of GDNF and
its receptors suggest that these trophic interactions may comprise
physiological responses to neurological traumas.
Endogenous target-derived GDNF trophic circuits
The initial characterization of the dopaminotrophic activities of
GDNF have generated considerable interest and effort, resulting in the
subsequent elucidation of many additional neurotrophic activities for
this factor; however, characterization of GDNF expression in the normal
adult brain has not been determined satisfactorily to date. We have
shown here that GDNF mRNA is expressed in several targets of ventral
mesencephalic dopaminergic neurons both within and outside the basal
ganglia. Within the basal ganglia, there is a striking segregation of
the pattern of expression of GDNF from that of its receptors, which
suggests that GDNF is acting as a target-derived trophic factor in the
maintenance of several neuronal circuits. As some of these neuronal
populations have been shown to respond to GDNF after mechanical or
pharmacological lesion, these trophic activities of GDNF may also be
used during neuronal degeneration.
Analysis of mRNA expression patterns for the GDNF receptors described
here and elsewhere indicates that all neuronal populations shown
previously to be responsive to GDNF in vivo express both RET
and GDNFR-. These include lesioned dopaminergic neurons of the
substantia nigra and ventral tegmental area (Beck et al., 1995; Tomac
et al., 1995), axotomized spinal and facial motorneurons, ischemic
thalamic and hippocampal neurons, 6-hydroxydopamine-treated noradrenergic neurons of the locus coeruleus, and transected
cholinergic neurons of the basal forebrain. In addition to the several
targets of ventral mesencephalic neurons that we have shown to express GDNF mRNA, we also demonstrate the presence of this mRNA in targets of
neuronal projections of thalamic nuclei, locus coeruleus, basal forebrain, and hippocampus as well as spinal motor neurons. Together, these data indicate that GDNF may be acting on many responsive populations in a target-derived fashion.
Novel GDNF-responsive populations
The strong correlation between GDNF-responsiveness and GDNF
receptor expression, combined with the biochemical studies
demonstrating GDNF activation of the RET kinase activity, suggests that
novel neuronal populations found to express GDNF receptors are also likely to benefit from the neurotrophic activities of this factor. The
Raphe nucleus, the major serotonergic nucleus in the brain, projects to
both ventral mesencephalic and striatal targets and as such is
anticipated to be the locus responsible for the increase in serotonin
levels recorded in striatum and substantia nigra after local
administration of GDNF (Beck et al., 1996). Because of the
presence of nigral afferent projections in the Raphe, these authors speculated that GDNF was influencing serotonergic neurons indirectly via direct actions on the substantia nigra. Our data suggest
that increases of serotonin levels in these paradigms could be
attributable to effects of GDNF on serotonergic neurons themselves.
This has implications for the possible therapeutic benefits of GDNF
administration, because degeneration of Raphe nucleus neurons has been
documented in some cases of Parkinson's disease (Halliday et al.,
1990).
RET and GDNFR- mRNA expression in the pontine and the red
nuclei suggests that populations of neurons that send projections to
the cerebellar granular layer are likely to respond to GDNF in
vivo. In line with the diverse roles of GDNF in establishing and
maintaining sensorimotor trophic circuits, we demonstrate that neurons
of the dorsal cochlear and lateral vestibular nuclei express mRNA
encoding both GDNF receptors. In addition, the mesencephalic nucleus of
the trigeminal is seen to express mRNA for both GDNF receptors. The
widespread expression of GDNF receptors in the various hypothalamic
nuclei could be taken as an indication of a neuroendocrine function for
GDNF. Finally, perhaps the most intense labeling for GDNFR- mRNA in
the adult brain is seen in the medial habenular nucleus, where RET mRNA
is also detected. The significance of such high levels of GDNFR-
mRNA expression is unclear, considering the high affinity of GDNFR-
for its known ligand.
Neuronal populations expressing RET but not GDNFR- mRNA
Some recent reports propose the absolute requirement of GDNFR-
for ligand binding to RET (Jing et al., 1996; Treanor et al., 1996). It
is therefore of interest to note the several populations of neurons
that we find to express RET but not GDNFR- mRNA. Cells within the
granular layer of the cerebellum that do not appear to be granule cells
express RET but no GDNFR- mRNA. Cells that are morphologically very
similar are also seen to express GDNF mRNA in the same region.
Similarly, within the olfactory bulb, strong labeling of the glomerular
layer as well as the olfactory nerve layer is observed for RET but not
GDNFR-. Furthermore, the subthalamic nucleus is also seen to contain
neurons that are positive for RET mRNA but negative for GDNFR- mRNA.
Thus, in these cases RET may be using GDNFR- delivered in
trans by other cells, or it may be associating with another
molecule related to GDNFR- for GDNF binding.
GDNF receptors exhibit distinct regulation of
expression patterns
It should be noted that although several GDNF-responsive
populations have been shown to express both previously characterized receptors, some nuclei exhibit only very low levels of receptor expression. It has been noted in previous studies that GDNF has more
pronounced biological effects on lesioned neurons than on intact
neurons. Indeed, it was shown that although normal basal forebrain
cholinergic neurons do not alter choline acetyltransferase (ChAT) mRNA
levels after stimulation with GDNF, cells that had been transected
mechanically did upregulate ChAT mRNA in response to GDNF (Williams et
al., 1996). Furthermore, we have shown previously that lesioned
noradrenergic neurons of the locus coeruleus increase tyrosine
hydroxylase mRNA levels and exhibit more pronounced sprouting after
administration of GDNF than unlesioned neurons do (Arenas et al.,
1995). It has been shown recently in the chicken that RET mRNA
expression is upregulated in both facial and spinal motorneurons after
nerve transection (Colucci-D'Amato et al., 1996; Nakamura et al.,
1996). We have shown here that GDNFR- mRNA is also upregulated in
spinal motorneurons after sciatic nerve transection and that hippocampal RET mRNA expression increases in response to excitotoxins. Because very limited regulation of GDNF itself has been detected under
various stimulations and insults, these results suggest that trophic
circuits of GDNF may be regulated dynamically at the level of the
receptors.
GDNFR- mRNA is often expressed in the absence of RET mRNA
The widespread and high levels of GDNFR- mRNA expression, in
particular in areas devoid of RET labeling, are striking. Notable among
these populations are the lateral geniculate nucleus, superior colliculus, and extensive regions of the cerebral cortex. GDNFR- may
be associating with additional signaling receptors for GDNF in these
cells. Alternatively, GDNFR- might function in these populations to
capture and concentrate diffusible GDNF from the extracellular space to
present the factor in a target-derived fashion to afferent
GDNF-responsive cells. We have found one possible example of such a
mechanism in the lesioned sciatic nerve.
A possible role for GDNF and GDNFR- in an intrinsic mechanism of
motorneuron regeneration
We have shown that GDNFR- mRNA levels are upregulated in nerve
cells in the distal segments of the lesioned sciatic nerve, which
expresses no known GDNF signaling receptors. The localization and
overall morphology of these cells resemble those of Schwann cells,
which have been shown previously to express GDNFR- mRNA by RT-PCR
analysis. We propose that lesion-induced GDNFR- may be functioning
in conjunction with GDNF to activate RET on regenerating motor axons.
In this model (Fig. 11), we envision GDNFR-
capturing and presenting soluble GDNF to function as a
membrane-anchored trophic signal for regenerating motorneurons. This
model is supported further by the observation of distally increasing
gradients of both GDNF and GDNFR- mRNAs in the lesioned sciatic
nerve as well as increased levels of GDNF mRNA in skeletal muscle.
Thus, regenerating motor axons might grow through tracts of increasing
concentration of GDNF associated with GDNFR- in a preformed complex
ready to activate the RET tyrosine kinase. Because GPI-anchored
proteins are known to be shed by the cells that produce them, a good
amount of the GDNF released by cells in the peripheral nerve may in
fact already be in a complex with GDNFR- as a co-ligand. A similar mechanism could be envisioned in regions of the brain in which GDNFR- is expressed at high levels.
Fig. 11.
GDNF/RET/GDNFR- interactions constitute a
novel mechanism of trophic support for degenerating motorneurons. Our
current data support the well characterized upregulation of RET mRNA in
spinal and facial motorneurons after axotomy (see Discussion). We have shown here that GDNFR- is also upregulated in motorneurons after transection. In addition, we see that GDNF mRNA is upregulated in the
denervated muscle as well as the axotomized distal nerve segments. Of
particular interest is the finding that GDNFR- is expressed in a
distally increasing gradient within the sciatic nerve, suggesting that
this accessory receptor acts to generate an insoluble gradient of GDNF
for presentation to regenerating motorneurons.
[View Larger Version of this Image (31K GIF file)]
Conclusions
Our expression studies indicate that GDNF is an endogenous
target-derived trophic factor in the adult brain. Importantly, most
populations that have been shown to respond to exogenous GDNF are seen
to express mRNA for both GDNF receptor components. RET mRNA expression
in the absence of GDNFR-, however, indicates that RET functions
alone, uses another co-receptor, or uses GDNFR- in trans.
Indeed, expression patterns of GDNFR- mRNA indicate that it may also
be available in trans or as a target-derived co-ligand in a
complex with GDNF. The regulation after sciatic nerve lesion of all the
components of this trophic circuit suggests an endogenous mechanism of
spinal motorneuron regeneration.
FOOTNOTES
Received Dec. 30, 1996; revised Feb. 20, 1997; accepted Feb. 25, 1997.
These studies were funded in part by The Swedish Cancer Society and The
Swedish Medical Research Council. N.B. was supported by Consiglio
Nazionale delle Ricerche. We sincerely thank Carina Raynoschek for
generating the GDNFR- expressing cell line. We gratefully
acknowledge the stimulating discussions and critical readings of Ernest
Arenas, Michael Fainzilber, and Mikael Rydén.
Correspondence should be addressed to Miles Trupp or Carlos
Ibáñez, Division of Molecular Neurobiology, Department of
Neuroscience, Karolinska Institute, Doktorsringen 12, 171 77 Stockholm,
Sweden. Miles or Carlos{at}cajal.mbb.ki.se
Dr. Belluardo's present address: Department of Physiological Sciences,
University of Catania, 95125 Catania, Italy.
Dr. Funakoshi's present address: Division of Biochemistry, Biomedical
Research Center, Osaka University, Medical School, Osaka 565, Japan.
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