dopaminergic neurons in rat ventral midbrain express brain-derived neurotrophic factor and...

THE JOURNAL OF COMPARATIVE NEUROLOGY 342:321-334 (1994)

Dopaminergic Neurons in Rat Ventral Midbrain Express Brain-Derived

Neurotrophic Factor and Neurotrophin-3 mRNAs

KIM B. SEROOGY, KERSTIN H. LUNDGREN, TIEN M.D. TRAN, KATHLEEN M. GUTHRIE, PAUL J. ISACKSON, AND CHRISTINE M. GALL

Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky 40536 (K.B.S., K.H.L. 1, Department of Anatomy and Neurobiology, University of California, Irvine, California 92717 (T.M.D.T., K.M.G., C.M.G.), and Department of Biochemistry and

Molecular Biology, Mayo Clinic, Jacksonville, Florida 32224 (P.J.1.)

ABSTRACT Studies of the trophic activities of brain-derived neurotrophic factor and neurotrophin-3

indicate that both molecules support the survival of a number of different embryonic cell types in culture. We have shown that mRNAs for brain-derived neurotrophic factor and neurotrophin-3 are localized to specific ventral mesencephalic regions containing dopaminergic cell bodies, including the substantia nigra and ventral tegmental area. In the present study, in situ hybridization with 35S-labeled cRNA probes for the neurotrophin mRNAs was combined with neurotoxin lesions or with immunocytochemistry for the catecholamine-synthesizing enzyme tyrosine hydroxylase to determine whether the dopaminergic neurons, themselves, synthesize the neurotrophins in adult rat midbrain. Following unilateral destruction of the midbrain dopamine cells with 6-hydroxydopamine, a substantial, but incomplete, depletion of brain- derived neurotrophic factor and neurotrophin-3 mRNA-containing cells was observed in the ipsilateral substantia nigra pars compacta and ventral tegmental area. In other rats, combined in situ hybridization and tyrosine hydroxylase immunocytochemistry demonstrated that the vast majority of the neurotrophin mRNA-containing neurons in the substantia nigra and ventral tegmental area were tyrosine hydroxylase immunoreactive. Of the total population of tyrosine hydroxylase-positive cells, double-labeled neurons constituted 25-50% in the ventral tegmental area and 10-30% in the substantia nigra pars compacta, with the proportion being greater in medial pars compacta. In addition, tyrosine hydroxylaselneurotrophin mRNA coexistence was observed in neurons in other mesencephalic regions including the retrorubral field, interfascicular nucleus, rostra1 and central linear nuclei, dorsal raphe nucleus, and supramammillary region. The present results demonstrate brain-derived neurotrophic factor and neurotrophin-3 expression by adult midbrain dopamine neurons and support the sugges- tion that these neurotrophins influence dopamine neurons via autocrine or paracrine mechanisms. These data raise the additional possibility that inappropriate expression of the neurotrophins by dopaminergic neurons could contribute to the neuropathology of disease states such as Parkinson’s disease and schizophrenia. o 1994 Wiley-Liss, Inc.

Key words: tyrosine hydroxylase, in situ hybridization, substantia nigra, coexistence, Parkinson’s disease

The neurotrophins compose a family of proteins that share structural homology to nerve growth factor (NGF) and support the survival, differentiation, and maintenance of certain populations of neurons in the nervous system. The best-characterized of the neurotrophins are NGF, brain-derived neurotrophic factor (BDNF) and neuro-

Hofer et al., 1990; Hohn et al., 1990; Leibrock et al., 1990;

Maisonpierre et al., 1990; Phillips et al., 1990; Rosenthal et al., 1990; Wetmore et al., 1990; see Snider and Johnson, 1989, Thoenen, 1991, and Ebendal, 1992 for reviews). In both the Peripheral and central nervous systems, the three

Accepted October 9, 1993, trophin-3 (NT-3) (Barde et al., 1982; Ernfors et 1990; Address reprint requests to Kim B. Seroogy, Department ofAnatomy and

Neurobiology, University of Kentucky, Lexington, Kentucky 40536-0084.

O 1994 WILEY-LISS, INC.

322 K.B. SEROOGY ET AL.

factors have overlapping but also distinct specificities of action upon defined populations of sympathetic, sensory, basal forebrain, cerebellar, and ventral mesencephalic neurons (Lindsay et al., 1991; Korsching, 1993).

Degeneration of the ventral midbrain dopaminergic neurons, and subsequent depletion of striatal dopamine, marks the basic neuropathology of Parkinson’s disease (Javoy- Agid et a]., 1984). Until recently there has been no direct evidence for the existence of a neurotrophic factor for the dopaminergic cells of the ventral mesencephalon. However, recent studies of the trophic activities of BDNF and NT-3 indicate both may play a role in the survival and biosynthetic activities of midbrain dopaminergic neurons. Two groups have reported that BDNF increases dopamine uptake activity and survival of cells containing the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) in neuron-enriched cultures of embryonic rat ventral mesencephalon (Knusel et al., 1991; Hyman et al., 1991a). A neuroprotective effect of BDNF is also suggested by results showing that treatment of fetal midbrain cultures with BDNF protected the TH-positive neurons from the neurotoxicity of 6-hydroxydopamine (6-OHDA) and 1-methyl-4- phenylpyridinium ion (MPP+) (Hyman et al., 1991a; Lindsay et al., 1991; Beck et al., 1992; Spina et al., 1992). Preliminary evidence also indicates that NT-3 enhances the survival of TH-immunoreactive neurons in fetal mesencephalic cultures (Hyman et al., 1991b). The ventral midbrain dopamine cells do not, however, respond to treatment with NGF (Knusel et al., 1991; Hyman et al., 1991a; Spina et al., 1992).

These in vitro studies suggest that BDNF and NT-3 may be important for the survival of mesencephalic dopaminergic neurons in vivo. Moreover, they raise the issue of the endogenous source of trophic support for the dopamine cells. Studies of NGF have given rise to the general hypothesis that neurotrophic molecules are derived from a target by the axon of a responsive neuron and retrogradely transported to the soma, where viability, growth, biosynthetic activities, etc. could be affected (Thoenen, 1991). This led to the expectation that BDNF and NT-3 would be available to the dopaminergic neurons via their projection targets. However, localization of BDNF and NT-3 has not been reported in the caudate-putamen or nucleus accumbens, the principal targets of the dopaminergic cells of the substantia nigra pars compacta and ventral tegmental area (VTA), respectively. An alternative to the “target-derived’’ theory of neurotrophic function proposes that trophic factors may be produced locally, in the immediate vicinity of the responsive neuron. Accordingly, the neurons could derive neurotrophic support via autocrine or paracrine mechanisms. Evidence from recent in situ hybridization studies supports such a concept for the neurotrophins (Ernfors et al., 1992; Schecterson and Bothwell, 1992). In particular, we have previously shown that NGF mRNA is produced by neurons distributed within basal forebrain regions of NGF-responsive cholinergic perikarya (Lauter- born et al., 1991). More recently, we demonstrated that both BDNF and NT-3 mRNAs are expressed within regions of all major dopaminergic cell groups of the ventral midbrain (i.e., substantia nigra, VTA, and retrorubral region) in the adult rat (Gall et al., 1992). These results indicate an intimate association between these two neurotrophins and the dopaminergic mesotelencephalic projection neurons and suggest that the dopamine cells may depend primarily

on local synthesis of BDNF and NT-3 for neurotrophic support.

The high correspondence between the distributions of the neurotrophin mRNAs and TH mRNA within the ventral midbrain raises the possibility that the dopaminergic neurons, themselves, synthesize the trophic factors. Since not all neurons of the substantia nigra and VTA are dopaminergic (van der Kooy et al., 1981; Swanson, 19821, the extent of neurotrophinldopamine coexistence must be verified by demonstration of the colocalization of dopaminergic markers and the neurotrophic factor mRNAs. In the present study, we used two approaches to address this issue: 1) the neurotoxin 6-OHDA was used to selectively lesion the ventral midbrain dopamine cells to examine whether the neurotrophin mRNAs in the same regions were also depleted; and 2 ) in situ hybridization for the neurotrophic factor mRNAs was combined with TH immunocytochemistry on the same midbrain tissue section to directly demonstrate coexistence within individual neurons. Both approaches revealed that the neurotrophins are indeed synthesized by mesencephalic dopamine neurons.

MATERIALS AND METHODS 6-OHDA treatment

Adult male and female Sprague-Dawley rats (200-300 g) were used for these experiments. The animals were anesthetized with 7% ( w h ) chloral hydrate (i.p.1 and received unilateral injections of 6-OHDA HC1 (Sigma) stereotaxi- cally placed into either the ventral mesencephalon (two injections, spaced 0.5 mm apart, of 10 pg in 0.5 ~1 0.9% NaCl with 0.2% ascorbic acid; n = 6) or the ascending medial forebrain bundle (one injection of 10 pg in 1.0 p1 0.9% NaCl with 0.2% ascorbic acid; n = 6). Two additional rats for each injection site received isovolumetric injections of vehicle; these control injections did not result in a reduction of hybridization for either TH or neurotrophin mRNA in the ventral midbrain. All rats received desmethy- limipramine (Research Biochemicals Inc.; 25 mg/kg; i.p.) 30 minutes prior to 6-OHDA treatments to protect noradrenergic terminals (Breese and Traylor, 1971). Following survival periods ranging from 6 days to 5 weeks, the rats were deeply anesthetized with an overdose of sodium pentobarbital and were either immediately decapitated or perfused via the ascending aorta with 2% paraformaldehyde in 0.1 M phosphate buffer (PB). In the unperfused animals, the brains were removed and rapidly frozen over dry ice. Tissue sections throughout the mesencephalon were cut in the coronal plane at 10 pm in a cryostat, thaw-mounted onto Vectabond (Vector Labs)-coated glass slides, and stored at -80°C until processing for hybridization. In the perfused animals, the brains were removed, postfixed in the perfusate for 1 hour, and placed in 10% sucrose overnight at 4°C. The brains were then cut in a cryostat as described above, and the sections were stored at - 80°C until hybridization.

In situ hybridization Adjacent sections through the ventral midbrain of rats

that had received 6-OHDA or vehicle were processed for the in situ hybridization localization of mRNAs for BDNF, NT-3, and TH. Pretreatment of the unperfused sections consisted of equilibration to room temperature, immersion in 4% paraformaldehyde for 10 minutes, washes in PB, glycine/PB, 0.25% acetic anhydride in 0.1 M triethan-

DOPAMINE/NEUROTROPHIN COEXISTENCE IN MIDBRAIN 323

oleamine, dehydration through ethanols, delipidation in chloroform, and air-drying. The perfused sections were similarly processed except that the paraformaldehyde immersion step was omitted. For verification of the extent of the 6-OHDA lesions, sections were processed for the in situ hybridization localization of TH mRNA by using a 48 base synthetic oligodeoxyribonucleotide probe complementary to nucleotides 1441-1488 of rat TH (Grima et al., 1985) as described elsewhere (Seroogy et al., 1989b, 1994). For the detection of the neurotrophin mRNAs, sections were hybridized for 18-24 hours at 60°C in hybridization buffer consist- ing of 50% formamide, 10% dextran sulfate, 20 mM Tris- HC1, 1 mM EDTA, l x Denhardt's solution, 0.33 mgiml denatured salmon sperm DNA, 0.15 mgiml yeast tRNA, 40 mM dithiothreitol, and the 35S-labeled cRNA probes at a density of 1.0 x lo6 cpmi0.05 mlislide. The cRNA probes were prepared by transcription from cDNA constructs with RNA polymerase in the presence of 35S-UTP. The 550-base rat NT-3 cRNA is complementary to 392 bases of the mature rat NT-3 coding region, whereas the 540-base BDNF cRNA includes 384 bases complementary to the rat BDNF mRNA coding region (Isackson et al., 1991; Gall et al., 1992). Posthybridization treatment consisted of washes in 4 x SSC (1 x SSC = 0.15 M NaC1, 0.015 M sodium citrate, pH 7.01, incubation in ribonuclease A for 30 minutes at 45"C, and washes through descending concentrations of SSC to a final wash of 0.1 x SSC at 60°C for 1 hour. The sections were air-dried and then exposed to p-Max Hyperfilm (Amersham) for 3-8 days at room temperature for generation of film autoradiograms.

After film development, the sections were dipped in NTBB nuclear track emulsion (Kodak; 1:l in H20), air- dried, and exposed in light-tight boxes at 4°C for 2-4 weeks. The emulsion was developed in D19 (Kodak), fixed in Kodafix, counterstained with cresyl violet, coverslipped in Permount, and analyzed with a Nikon Optiphot-2 micro- scope equipped with brightfield and darkfield optics. Cells were considered labeled if the density of reduced silver grains overlying the perikarya was at least tenfold greater than background. Pseudocolor densitometric images of film autoradiograms were generated by the Microcomputer Imaging Device (Imaging Research, Inc.) with hybridization densities calibrated relative to I4C-labeled standards (American Radiolabeled Chemicals, Inc.) apposed along with the tissue to each sheet of film.

Combined in situ hybridization/ immunocytochemistry

Tissue sections through the caudal diencephalon, mesencephalon, and pons were processed for the combined immunocytochemical localization of TH and in situ hybridization localization of mRNAs for NT-3 or BDNF. Untreated male Sprague-Dawley rats (25-35 days of age; n = 6) were killed with an overdose of sodium pentobarbital and intracardi- ally perfused with isotonic saline followed by 4% paraformaldehyde in 0.1 M PB (pH 7.2). The brains were removed from the cranium, postfixed in the perfusate for 24 hours at PC, transferred to 4% paraformaldehyde in 20% sucrose/PB for 24 hours at 4"C, and then sectioned frozen in the coronal plane at a thickness of 20 or 25 pm. The tissue sections were collected into cold PB and immediately processed for in situ hybridization as free-floating sections [described in detail elsewhere (Gall et al., 1991, 199411 with 393-labeled rat NT-3 cRNA (n = 3) or 35S-labeled rat BDNF cRNA (n = 3) (Gall et al., 1992). Following hybridization

and posthybridization rinses in descending concentrations of SSC buffer, the tissue sections were transferred to PB containing 10 mM sodium thiosulfate (PB/S) and then processed for the immunocytochemical localization of TH with affinity purified polyclonal anti-TH (Pel Freeze; 1:400 for 24 hours at 4°C). The avidin-biotin technique was used with kit reagents (Vector Laboratories) and with diaminobenzidine as chromagen. Throughout these procedures, sterile PB/S was used for rinses and as diluent. Following the diaminobenzidine reaction, the tissue was mounted onto glass slides, air-dried, defatted through ethanols and chloroform, and processed for NTBB emulsion autoradiography (as above) with exposures of 4-8 weeks. Following development of the emulsion autoradiograms, the tissue was very lightly counterstained with cresyl violet and coverslipped with Permount.

To obtain a quantitative measure of neurotrophin mRNA/ dopamine coexistence, single- and double-labeled cells were counted in the VTA and substantia nigra pars compacta in ten evenly spaced sections through the rostrocaudal extent of the ventral mesencephalon. Comprehensive counts were made in one representative animal that had been processed for combined BDNF cRNA hybridizationiTH immunocytochemistry and in one processed for NT-3 cRNA hybridization/TH immunocytochemistry. Cell counts were also obtained from additional representative sections from other animals for each mesencephalic subfield that corroborated the more detailed analysis. The approximate rostral and caudal borders corresponded to levels -4.8 mm and -6.3 mm, respectively, relative to bregma, according to the rat brain atlas of Paxinos and Watson (1986). Ventral midbrain nuclear boundaries were defined according to German and Manaye (1993) and Paxinos and Watson (1986). Double- labeled neurons were defined as perikarya containing brown reaction product (indicative of TH immunostaining) with overlying clusters of silver grains (indicative of neurotrophin cRNA hybridization). The single- and double- labeled cells were identified and plotted with the aid of a Nikon drawing tube at 200 x magnification with brightfield illumination. Only cells with a discernible nucleus were plotted; counts were subsequently made from the plots.

As controls for the specificity of hybridization, additional tissue sections were either treated with ribonuclease A at 45°C for 30 minutes prior to hybridization with the anti- sense probes or processed for hybridization with 35S-labeled sense RNA sequences (transcribed from the same cDNA templates using different RNA polymerases). No cellular labeling was observed in tissue processed under these conditions. To control for the possible diminished sensitiv- ity of either the TH immunocytochemistry or the neurotrophin in situ hybridization in the combined technique, some semiadjacent tissue sections were processed in paral- lel for immunocytochemistry alone or for in situ hybridization alone. No significant reduction in TH immunostaining or in hybridization signal intensity was observed with the combined procedure. Finally, to verify that there was no interaction between the TH immunostaining and the autoradiographic emulsion, tissue sections processed for TH immunocytochemistry, but not in situ hybridization, were processed for emulsion autoradiography; in this material there were no aggregations of silver grains over immunostained cells.


RESULTS In agreement with our recent study (Gall et al., 19921,

neurons labeled with the NT-3 cRNA were distributed throughout the ventral mesencephalon within the VTA, substantia nigra pars compacta, and retrorubral field [AlO, A9, and A8 monoamine cell groups, respectively, according to the terminology of Dahlstrom and Fuxe (196411. The pattern of BDNF cRNA hybridization within the ventral midbrain overlapped that of the NT-3 cRNA with additional labeling of neurons found in areas including the supramammillary region, interpeduncular nuclei, periaqueductal gray matter, and the substantia nigra pars lateralis region. The distribution of the two neurotrophin mRNAs corresponded very well with the distribution of TH mRNA-containing cells, especially in the VTA and medial substantia nigra pars compacta (Fig. 1).

6-OHDA lesion studies To provide initial evidence that the ventral midbrain

dopaminergic neurons, themselves, synthesize the neurotrophins, rats were treated with 6-OHDA. Rats received unilateral injections of the neurotoxin into the ventral mesencephalon or into the ascending medial forebrain bundle; similar results were obtained for each injection site. Following a survival time ranging from 6 days to 5 weeks (results were comparable across all survival periods), a substantial or complete elimination of TH mRNA-expressing cells was found in the ipsilateral ventral midbrain, i.e., VTA and substantia nigra (Fig. 2). Adjacent sections that were processed for hybridization with the BDNF or NT-3 35S-cRNA probes showed a profound depletion of hybridization signal in the VTA and substantia nigra ipsilateral to 6-OHDA injection, whereas the contralateral side exhibited the normal pattern of hybridization (Fig. 3). Analysis of the emulsion autoradiograms revealed that, on the injected side, the vast majority, but not all, of the BDNF and NT-3 cRNA-hybridizing cells were eliminated, even when the destruction of the TH cells was complete (Fig. 4). The small numbers of labeled somata that survived the 6-OHDA treatment were situated in both the VTA and medial substantia nigra pars compacta. These data suggest substantial, but incomplete, coexistence of the neurotrophin and TH mRNAs within ventral mesencephalic neurons.

Colocalization studies: Combined in situ hybridization/immunocytochemistry

To provide the most direct evidence for the coexistence of the neurotrophin mRNAs and dopamine, sections of normal ventral mesencephalon were processed for the simulta- neous detection of BDNF or NT-3 mRNA and TH immunoreactivity.

Throughout the extent of the ventral mesencephalon, the majority of neurons in both the VTA and substantia nigra pars compacta labeled with the NT-3 cRNA probe also contained immunostaining for TH. The double-labeled cells comprised a smaller subset of the total population of TH-immunoreactive (TH-I) neurons. Labeled cells were quantified in sections at a series of rostrocaudal levels as depicted graphically in Figure 5.

In the VTA, over 90% of the cells expressing NT-3 cRNA hybridization were also TH-I, whereas a range of 2540% of the TH-I somata also contained NT-3 cRNA labeling (Fig. 5A). The double-labeled neurons were distributed throughout the entire VTA, including both the parabrachialis

NT-3 mRNAITH irnmunoreactiuity.

pigmentosus and paranigral subdivisions, and were evenly interspersed among single-labeled cells (Fig. 6B).

In the substantia nigra pars compacta, neurons exhibiting NT-3 mRNA/TH-I colocalization displayed a more restricted topography. Double-labeled perikarya were limited to medial regions of the substantia nigra pars com- pacts, and predominantly within the intermediate and dorsal sheets of the nucleus (Fig. 6A). Only infrequent examples of double-labeled somata could be found in the ventralmost tier of pars compacta neurons. At the most rostral and caudal levels of the ventral mesencephalon, less than 10% of the TH-positive cells in the substantia nigra pars compacta exhibited hybridization for the NT-3 cRNA. However, throughout the major rostrocaudal extent of the pars compacta, approximately 20% of the TH-I neurons were double labeled (Fig. 5B). An average of about 85% of the NT-3 cRNA-hybridizing cells throughout the substantia nigra pars compacta also exhibited TH immunostaining (Fig. 5B). Perikarya containing NT-3 mRNA alone were typically situated in dorsal aspects of the substantia nigra pars compacta. It should be noted that throughout the ventral mesencephalon hybridization for NT-3 mRNA was never detected in TH-I cells of the substantia nigra pars reticulata nor pars lateralis, and only extremely rarely in lateral aspects of the pars compacta.

Other regions and nuclei of the ventral mesencephalon also contained neurons exhibiting NT-3 cRNAITH-I coexistence. At caudalmost aspects of the mesencephalon, counts of three sections through the retrorubral field (A8 region) indicated that double-labeled neurons comprised about 38% (i.e., 166 of 435) of the TH-positive cells and approximately 84% (166 of 197) of the NT-3 mRNA-expressing somata. Double-labeled perikarya were prevalent in the rostral linear nucleus, interfascicular nucleus (IFN), and central linear nucleus (CLi). For example, counts from one section through the IFN indicated that approximately 33% (30 of 89) of the small TH-I somata and all of the NT-3 cRNA- labeled cells exhibited colocalization. Examples of double- labeled neurons were also found in the dorsal raphe nucleus and extended into the fountain of Sheehan. In addition, many of the TH-positive cells found adjacent to the third ventricle as well as in the central periaqueductal gray matter subjacent to the cerebral aqueduct exhibited hybridization with the NT-3 cRNA probe (Fig. 6C). Finally, it should be noted that although a few NT-3 cRNA-labeled somata were detected in the supramammillary region, none of the TH-I perikarya in this area exhibited hybridization with the neurotrophin cRNA.

BDNF mRNAITH immunoreactiuity. In general, throughout the ventral mesencephalon, neurons exhibiting BDNF mRNA/TH-I colocalization comprised a larger subset of the total population of TH-I cells than those containing NT-3 mRNA/TH-I (Fig. 7). Perikarya double labeled for BDNF mRNAITH-I were distributed throughout the rostrocaudal extent of the VTA (Fig. 8B,C), including the parabrachialis pigmentosus and paranigral subnuclei. Except at very rostral levels, over 80% of the BDNF cRNA-labeled somata contained immunostaining for TH, whereas about 40-50% of the TH-positive neurons were labeled with the neurotrophin cRNA (Fig. 7A). Rostrally, only 45% of the BDNF cRNA-hybridizing cells were double labeled, and only 25% of the TH-I perikarya were labeled with the BDNF cRNA probe. Neurons labeled with the BDNF cRNA alone were often situated in more dorsal aspects of the VTA, or typically immediately dorsal to the nucleus itself.


Fig. 1. Darkfield photomicrographs showing the autoradiographic localization of hybridization to neurotrophin-3 (NT-3) mRNA (top), brain-derived neurotrophic factor (BDNF) mRNA (middle), and tyrosine hydroxylase (TH) mRNA (bottom) in semiadjacent tissue sections

through ventral midbrain of a normal adult rat. Note the substantial overlap in the pattern of hybridization for all three mRNAs in the ventral tegmental area (VTA) and medial substantia nigra pars compacta (SNc). Scale bar = 260 km.


Fig. 2. Film autoradiogram of ventral midbrain showing complete depletion of hybridization to TH mRNA ipsilateral to an injection of 6-OHDA (right). Normal expression of TH mRNA is maintained in

both the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) contralateral to the lesion (left). Dashed line indicates section midline. Scale bar = 1,000 p,m.

Fig. 3. Pseudocolor densitometric image of a film autoradiogram showing the localization of NT-3 cRNA hybridization in the ventral midbrain following unilateral injection of 6-OHDA (right). Note the substantial reduction, but not complete elimination, of hybridization in the ipsilateral ventral tegmental area and substantia nigra pars com-

pacta (arrow), compared with the contralateral side (left). Results were similar in adjacent sections hybridized with the BDNF cRNA probe. Analysis of adjacent sections from this animal processed with the TH oligonucleotide probe indicated a complete depletion of TH mRNA- positive cells ipsilateral to the 6-OHDA injection (as seen in Fig. 2).

At extreme rostral levels of the substantia nigra pars compacta, about 10% of the TH-I neurons exhibited BDNF cRNA hybridization (Fig. 7B), and these cells were limited to medial aspects of the nucleus. About 70% of the BDNF cRNA-hybridizing cells were also TH positive. Slightly more caudal and extending through intermediate mesencephalic levels, the degree of BDNF mRNA/TH-I coexistence increased to 20-30% of the TH-positive perikarya and to

70-80% of the BDNF mRNA-containing somata (Fig. 7B). At caudal levels of the substantia nigra pars compacta, the incidence of colocalization declined to under 16% of the TH-I cells, In contrast to the distribution of NT-3 mRNA/ TH-I cell bodies described above, the BDNF mRNAiTH-I perikarya were found within the ventralmost sheet of pars compacta neurons, as well as in the intermediate and dorsal tiers (Fig. 8A,D-F). However, although infrequent BDNF

DOPAMINE/NEUROTROPHIN COEXISTENCE IN MIDBRAIN

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a a E Z

IPS1

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Fig. 4. High-power darkfield photomicrographs showing autoradiographic localization of NT-3 and BDNF mRNAs in the ventral mesencephalon following unilateral A-OHDA treatment. A,B: Cells labeled with the NT-3 cRNA probe in the ventral tegmental area contralateral (A) and ipsilateral (B) to 6-OHDA injection. C,D: An adjacent section through the ventral tegmental area demonstrating similar patterns of hybridization with the BDNF cRNA probe contralateral (C) and

ipsilateral (D) to the 6-OHDA injection. Arrows in A and C indicate examples of neurotrophin mRNA-containing cells found contralateral to the lesion, whereas those in B and D denote some of the few remaining labeled perikarya ipsilateral to 6-OHDA injection. CONTRA, contralateral to 6-OHDA injection; IPSI, ipsilateral to 6-OHDA injection. Scale bar = 50 pm.


VENTRAL TEGMENTAL AREA

1 2 3 4 5 6 7 8 9 10 Section number: rostral to caudal

-9- NT-3 total

SUBSTANTIA NlGRA PARS COMPACTA

Section number: rostral to caudal

Fig. 5 . Graphs showing the total number of cells in the ventral tegmental area (A) and substantia nigra pars compacta (B) containing TH immunoreactivity (open square), NT-3 cRNA hybridization (half shaded square), and double-labeling for both TH and NT-3 cRNA (circle). Counts were obtained from a series of ten evenly spaced sections from one representative animal (case #R807) spanning the rostrocaudal extent of the ventral mesencephalon [approximate levels -4.8 mm to -6.3 mm relative to bregma, according to the atlas of Paxinos and Watson (198611. Note that the double-labeled cells comprise the majority of the NT-3 cRNA-labeled neurons, but constitute a smaller subpopulation of the TH-positive neurons.

cRNA/TH-I double-labeled somata were present in lateral aspects of the substantia nigra pars compacta, the vast majority of double-labeled cells were restricted to the medial pars compacta, similar to the localization of the NT-3 mRNA-containing perikarya described above. Neu- rons labeled by hybridization of the BDNF cRNA alone were characteristically located just dorsal to the lateral substantia nigra pars compacta,

In the substantia nigra pars lateralis, somata single labeled for either BDNF mRNA or TH-I were intermingled, but no colocalization was evident (Fig. 8G). Typically, the BDNF cRNA-labeled neurons extended dorsolaterally from the pars lateralis through the peripeduncular region to the medial aspects of the medial geniculate nucleus.

The coexistence of BDNF mRNA and TH-I was also noted in other mesencephalic regions. At extreme rostral levels, many BDNF cRNA-containing perikarya of the supramammillary region were also TH immunoreactive. Neurons single labeled for either TH-I or BDNF mRNA were intermingled among the double-labeled cells. In the IFN, counts from one section showed that about 30% (34 of 110) of the TH-positive somata and all of the BDNF cRNA-labeled

neurons were double labeled. BDNF cRNA-containing perikarya were distributed dorsally along the midline from the IFN to the third ventricle, but few of these cells contained TH-I. The TH-positive neurons adjacent to the third ventricle were infrequently observed to express BDNF cRNA hybridization. In the retrorubral field (A8 region), counts of several sections revealed that 28% (95 of 339) of the TH-positive cells and about 52% (95 of 184) of the BDNF cRNA-containing cells were double labeled. However, a rostrocaudal gradient of coexistence existed such that only about 20% (9 of 44) of the TH-I neurons and 23% (9 of 39) of the BDNF cRNA-expressing neurons were double labeled at caudalmost levels of the A8 region. Counts through the CLi indicated that the incidence of BDNF cRNA/TH-I colocalization comprised approximately 44% (116 of 266) of the TH-positive somata and 87% (116 of 133) of the BDNF cRNA-labeled cells. Very few of the TH-I cells of the dorsal raphe nucleus and fountain of Sheehan contained autoradiographic labeling. In addition, although many BDNF cRNA- labeled cells were localized to the periaqueductal gray matter, only a few of these were TH immunostained.

DISCUSSION The results of the present study demonstrate that dopa-

minergic neurons in adult ventral mesencephalon synthesize mRNAs for the neurotrophins BDNF and NT-3. The two different approaches used, 6-OHDA lesions and double- labeling with immunocytochemistry and in situ hybridization, both support this conclusion. The coexistence of dopamine and BDNF and of dopamine and NT-3 in normal adult midbrain neurons suggests that the neurotrophins may contribute to the maintenance of normal mesotelencephalic function in the adult, in addition to their survival and neuroprotective roles in developing midbrain cultures.

Although previous in situ hybridization studies by others have not reported the expression of neurotrophin mRNAs within the normal adult ventral midbrain, BDNF mRNA has been transiently detected within the substantia nigra of neonatal rat (Friedman et al., 1991a). In addition, Cecca- telli et al. (1991) detected BDNF mRNA within the substantia nigra of adult rat after intracerebroventricular colchicine treatment. Expression of NT-3 mRNA was not found in the ventral mesencephalon in the latter study nor in development (Friedman et al., 1991b). However, we recently demonstrated that both BDNF and NT-3 mRNAs are present within substantial numbers of neurons in normal adult ventral midbrain, including the substantia nigra and VTA (Gall et al., 1992). Our present results confirm these findings and show that the majority of these mesencephalic neurotrophin-containing neurons are also dopaminergic. Moreover, the data indicate that between 25 and 50% of dopaminergic VTA neurons and approximately 10-30% of dopaminergic substantia nigra pars compacta neurons express the neurotrophin mRNAs. Evidence is thus provided for the local synthesis of trophic molecules that are in a position to act in an autocrine or paracrine manner upon dopaminergic mesotelencephalic neurons.

The distribution of the mesencephalic dopamine neurons containing either BDNF or NT-3 is very similar in that both neurochemically defined subpopulations are localized to the VTA and medial substantia nigra pars compacta, and are generally absent from the lateral pars compacta and substantia nigra pars lateralis. Thus, the neurotrophins are expressed by dopaminergic neurons topographically situated

DOPAMINE /NEUROTROPHIN COEXISTENCE IN MIDBRAIN 329

Fig. 6. High-power (A) and lower-power (B,C) brightfield photomicrographs showing the colocalization of NT-3 cRNA hybridization and TH immunoreactivity within neurons of the medial substantia nigra pars compacta (A), VTA (B), and subjacent to the cerebral aqueduct (GI.

Solid arrows indicate examples of double-labeled cells; open arrows denote examples of somata immunostained for TH alone. Scale bar in A = 20 ym; bar in C = 27 ym for B and 20 ym for C.

to give rise to the main components of the mesotelencephalic system, including the mesostriatal, mesolimbic, and mesocortical pathways (Lindvall and Bjorklund, 1983). Of particular interest, BDNF mRNA is prominently expressed within the ventral sheet of substantia nigra pars compacta dopamine neurons, the primary origin of the nigrostriatal pathway (Fallon and Loughlin, 1985). It remains to be

determined if the expression of BDNF or NT-3 distin- guishes separate subpopulations of dopaminergic neurons or whether some of the mesencephalic dopamine cells contain both neurotrophins. In addition, the location of the neurotrophin/dopamine cells is similar to the distribution of midbrain dopamine subpopulations containing the neuro- peptides cholecystokinin and neurotensin (Seroogy et al.,


VENTRAL TEGMENTAL AREA

--- I

I.

1 i 4 4 5 6 ; a 9 l o

--P- BDNF total


SUBSTANTIA NlGRA PARS COMPACTA

OT I 1 2 3 4 5 6 7 8 9 10


Fig. 7. Graphs showing the total number of cells in the ventral tegmental area (A) and substantia nigra pars compacta (B) containing TH immunoreactivity (open square), BDNF cRNA hybridization (half shaded square), and double-labeling for both TH and BDNF cRNA (circle). Counts were obtained from a series of ten evenly spaced sections from one representative animal (case #R722) spanning the rostrocaudal extent of the ventral mesencephalon [approximate levels -4.8 mm to -6.3 mm relative to bregma, according to the atlas of Paxinos and Watson (198611. Note that the double-labeled cells comprise the majority of the BDNF cRNA-labeled neurons, but constitute a smaller subpopulation of the TH-positive neurons.

1988, 1989a,b). Future multiple-labeling studies will determine whether or not these peptides, which are known to modulate dopaminergic functions, are also present within the neurotrophin-expressing dopamine neurons.

Recently, a tremendous amount of progress has been made in the identification and characterization of functional neurotrophin receptors. There is now good evidence that members of the proto-oncogene trk family of receptor tyrosine protein kinases are essential components of the high-affinity, signal transduction receptors for the neurotrophins (Bothwell, 1991; Meakin and Shooter, 1992). In particular, BDNF appears to be the preferential ligand for the trkB receptor, whereas NT-3 is the preferred ligand for trkC (Klein et al., 1991; Lamballe et al., 1991; Squint0 et al., 1991; Ip et al., 1993). Recent studies have determined that mRNAs for these trk receptors are widely distributed throughout adult rodent brain (Klein et al., 1990; Lamballe et al., 1991; Merlio et al., 1992). In the ventral midbrain, heterogeneous labeling for both trkB and trkC mRNAs has been found in neurons of the substantia nigra and VTA (Merlio et al., 1992). These results argue for the presence of local trophic interactions in the ventral mesencephalon.

Based upon several indices, including overlap in the regional and temporal expression of neurotrophins and their receptors, as well as the particular distribution of responsive- ness to the trophic molecules, a number of investigators have postulated that the neurotrophins operate via autocrine or paracrine mechanisms in such diverse neuronal systems as sensory and sympathetic ganglia, basal forebrain, septum, hippocampus, and cerebellum (Lu et al., 1989; Lauterborn et al., 1991; Collazo et al., 1992; Ernfors et al., 1992; Roback et al., 1992; Schecterson and Bothwell, 1992; Wright et al., 1992; Korsching, 1993). The present neurotrophiniTH colocalization results suggest that within the ventral midbrain, neurotrophins produced by dopamine cells act upon the same cell (autocrine mechanism) or upon neighboring cells (paracrine mechanism) in this highly interconnected region.

Although the present data support the idea of local synthesis and action of BDNF and NT-3 in the ventral mesencephalon, it is also possible that the neurotrophins produced by ventral midbrain neurons provide trophic support for afferents to the dopaminergic cells. The most prominent projections to the substantia nigra and VTA arise from the caudate-putamen and nucleus accumbens, respectively (Fallon and Loughlin, 1985; Oades and Halli- day, 1987). These striatomesencephalic pathways utilize mainly gamma aminobutyric acid (GABA) and substance P as their neurotransmitter/neuromodulator (Graybiel and Ragsdale, 1983). Since BDNF has been shown to increase GABA uptake in fetal cultures of rat basal forebrain (Kniisel et al., 1991), it is not unlikely that striatal GABAer- gic neurons also respond to BDNF. Additional projections to the substantia nigra include afferents from the globus pallidus (GABAergic), subthalamic nucleus, prefrontal cortex, amygdala, and dorsal raphe nucleus (Fallon and Lough- lin, 1985). Additional inputs to theVTA arise from the locus coeruleus, dorsal raphe nucleus, prefrontal cortex, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, and parabrachial nucleus (Fallon and Loughlin, 1985; Oades and Halliday, 1987). All of these afferent populations are possible recipients of BDNF and/or NT-3 trophic support from their ventral mesencephalic targets. I t will be especially interesting to determine if neurotransmitter-specific afferents, such as the noradrenergic locus coeruleus neurons or the serotoninergic dorsal raphe neurons, can respond to either of the aeurotrophins in vivo. Such a possibility is supported by a recent in situ hybridization study that has demonstrated both trkB and trkC mRNAs within cells of the dorsal raphe nucleus and trkC mRNA within locus coeruleus neurons (Merlio et al., 1992). More- over, Friedman et al. (1993) recently reported that NT-3 increases the survival of TH-positive cells in embryonic cell cultures of rat locus coeruleus. Although these authors

Fig. 8. Low magnification (A) and higher magnification (B-G) brightfield photomicrographs showing the colocalization of BDNF cRNA hybridization and TH immunoreactivity within neurons of the medial substantia nigra pars compacta (A,F), VTA (B,C), middle substantia nigra pars compacta (D), and ventromedial substantia nigra pars compacta (El, and the lack of coexistence within cells of the substantia nigra pars lateralis (G). Solid arrows indicate examples of double-labeled neurons, whereas open arrows denote cells single- labeled for either TH immunoreactivity or BDNF mRNA. Note in A, E, and F that double-labeled neurons are present in the ventral tier of pars compacta cells. Scale bar = 40 pm for A; 24 km for B and F; 16 pm for C; 20 pm for D, E, and G.


postulate that the NT-3 is retrogradely transported from the hippocampus, which contains abundant NT-3 mRNA (Ernfors et al., 1990; Phillips et al., 1990; Friedman et al., 1991b; Gall, 19921, the ventral mesencephalon is an alternative source of target-derived neurotrophic support for the locus coeruleus.

DiStefano et al. 11992) recently demonstrated that la51- labeled neurotrophins (NT-3, BDNF, and NGF) are retrogradely transported by overlapping as well as distinct populations of neurons in the adult central nervous system, including neurons of the basal forebrain, cortex, thalamus, and hippocampus. The results were interpreted as provid- ing a correlation between the pattern of retrograde transport of a target-derived neurotrophic molecule and the identification of possible neuronal subpopulations selectively responsive to the particular neurotrophic factor. Unfortunately, the only CNS structure injected with labeled neurotrophin was the hippocampus. Similar studies involving ventral midbrain efferents and afferents may shed light on potential trophic lines of communication and mechanisms of neurotrophic function in the dopaminergic mesotelencephalic system.

Although BDNF and perhaps NT-3 support the survival and development of TH-positive neurons in fetal cultures, their neurotrophic role in the dopaminergic mesotelencephalic system in vivo in the adult remains controversial. Two recent reports (Knusel et al., 1992; Lapchak et al., 1993) found that intracerebroventricular or intranigral administration of BDNF failed to prevent the degeneration of ventral mesencephalic dopamine neurons following transection of the medial forebrain bundle, which contains the ascending fibers of the midbrain dopamine neurons, More- over, Lapchak et al. (1993) reported that chronic intranigral injection of BDNF in normal adult rats actually induced striatal dopaminergic hypofunction. On the other hand, Altar et al. (1992) found that supranigral infusions of BDNF in adult rats elevated dopamine turnover in the striatum and increased rotational behavior, thus augment- ing specific functional indicators of nigrostriatal dopamine neurons. In addition, the latter study provides evidence that the locus of BDNF action in the dopaminergic nigrostriatal system is presynaptic, which would be consonant with the concept of local production and action of BDNF in the ventral mesencephalon.

The present findings, showing that dopamine neurons, themselves, produce their own neurotrophic factors, raise the possibility that inappropriate expression of the endogenous trophic factors could contribute to the dysfunction of dopaminergic neurons in neurodegenerative disorders of the mesotelencephalic system, such as Parkinson’s disease. Parkinson’s disease is characterized by the degeneration of many of the dopaminergic cell bodies of the ventral mesencephalon (Javoy-Agid et al., 1984). The dopaminergic neurons are differentially susceptible to degeneration such that those situated in the medial substantia nigra pars compacta and VTA appear to be spared relative to the substantial cell death observed in more lateral parts of the substantia nigra pars compacta. I t has sometimes been suggested that the neuronal susceptibility to degeneration may be associated with the cells’ particular neurochemical phenotype. For example, dopaminergic neurons containing the calcium binding protein calbindin D Z ~ K appear to be spared in Parkinson’s disease (Lavoie and Parent, 1991; Yamada et al., 1991; but see Hirsch et al., 19921, whereas those containing melanin appear to be especially vulnerable to

degeneration (Hirsch et al., 1988). In our study, the preferential expression of both NT-3 and BDNF mRNAs by dopaminergic neurons of the VTA and medial substantia nigra pars compacta raises the possibility that the neurotrophins have a neuroprotective effect upon these more medially situated neurons. Evidence from neurotoxicity studies in cultures of embryonic midbrain suggests such a neuroprotective role for BDNF upon the TH-positive cells (Hyman et al., 1991; Lindsay et al., 1991; Beck et al., 1992; Spina et al., 1992). It will be important to determine the levels and patterns of expression of the neurotrophins by dopaminergic cells in both normal and Parkinson diseased human midbrain to explore this possibility further. I t will also be important to extend these types of analyses to other growth factors known to have atrophic role in the support of dopaminergic neurons. In particular, acidic and basic fibroblast growth factor (FGF) have both been localized to the ventral midbrain dopaminergic neurons (Bean et al., 1991; Cintra et al., 1991) and have been indirectly impli- cated in the survival of fetal dopamine cells (Ferrari et al., 1989; Engele and Bohn, 1991; Giacobini et al., 1991). Moreover, a recent study has reported a loss of basic FGF immunoreactivity in substantia nigra neurons in Parkin- son’s disease (Tooyama et al., 1993).

Beyond involvement in Parkinson’s disease, there are other disease states involving the mesotelencephalic system that could reflect activities of the neurotrophins in the ventral mesencephalon. In schizophrenia, an excess of mesotelencephalic dopaminergic transmission has been hy- pothesized (Matthysse and Kety, 1975). It is possible that overexpression of the neurotrophins within the midbrain dopamine cells could account for subsequent hyperstimula- tion of dopaminergic function via autocrine or paracrine mechanisms. Finally, in Huntington’s disease, many of the medium spiny projection neurons of the striatum degener- ate (Graveland et al., 1985; Quarrell, 1991). If the present results are considered along the traditional lines of target- derived trophic support, insufficient production of the neurotrophins in the ventral midbrain could be detrimental to the survival/maintenance of striatomesencephalic projection neurons if, in fact, striatal neurons are actually responsive to the neurotrophins. Thus, determination of the patterns of expression of the neurotrophins and their receptor proteins in the ventral mesencephalon in these disease states is warranted as well.

ACKNOWLEDGMENTS We thank Suzanne Numan for helpful comments and

assistance. This work was supported by grants from the National Parkinson Foundation, Scottish Rite Schizophre- nia Research Program, and University of Kentucky College of Medicine Research Fund to K.B.S. and Research Scien- tist Development Award MH00974 and AGO0538 to C.M.G..

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