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Keywords:

  • Rat striatum;
  • Quinolinate;
  • Grafting;
  • Survival

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

Abstract: Intrastriatal injection of quinolinate has been proven to be a very useful animal model to study the pathogenesis and treatment of Huntington's disease. To determine whether growth factors of the neurotrophin family are able to prevent the degeneration of striatal projection neurons, cell lines expressing brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4/5 (NT-4/5) were grafted in the adult rat striatum before quinolinate injection. Three days after lesioning, ongoing cell death was assessed by in situ detection of DNA fragmentation. In animals grafted with the control cell line, quinolinate injection induced a gradual cell loss that was differentially prevented by intrastriatal grafting of BDNF-, NT-3-, or NT-4/5-secreting cells. Seven days after lesioning, we characterized striatal projection neurons that were protected by neurotrophins. Quinolinate injection, alone or in combination with the control cell line, induced a selective loss of striatal projection neurons. Grafting of a BDNF-secreting cell line prevented the loss of all types of striatal projection neurons analyzed. Glutamic acid decarboxylase 67-, preproenkephalin-, and preprotachykinin A- but not prodynorphin-expressing neurons were protected by grafting of NT-3- or NT-4/5-secreting cells but with less efficiency than the BDNF-secreting cells. Our findings show that neurotrophins are able to promote the survival of striatal projection neurons in vivo and suggest that BDNF might be beneficial for the treatment of striatonigral degenerative disorders, including Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded polyglutamine repeat in the huntingtin gene (MacDonald and Gusella, 1996; Mangiarini et al., 1996). Its predominant pathological feature is a massive and progressive degeneration of striatal output neurons without substantial loss of striatal interneurons and afferents (for review, see DiFiglia, 1990). Intrastriatal injection of quinolinate (QUIN), an NMDA receptor agonist, replicates many neurochemical, histological, and behavioral features of HD (Beal et al., 1986; DiFiglia, 1990). Striatal projection neurons containing enkephalin are affected to a greater extent than substance P-containing neurons, and those neurons surviving the lesion express reduced levels of their mRNAs both in HD (Reiner et al., 1988; Richfield et al., 1995) and after QUIN injections (Pérez-Navarro et al., 1999a,b). Excitotoxicity and apoptosis have been suggested to be involved in the degeneration of neurons in HD (Thomas et al., 1995; Petersén et al., 1999) and after QUIN injection (Ferrer et al., 1995; Portera-Cailliau et al., 1995; Hughes et al., 1996). Furthermore, intrastriatal QUIN injections have been found to induce huntingtin mRNA (Carlock et al., 1995) and protein (Tatter et al., 1995), providing a possible link between the QUIN model and HD.

Members of the neurotrophin family have been suggested as therapeutic candidates for neurodegenerative disorders because they promote neuronal survival in different lesion models (for review, see Hefti, 1994; Connor and Dragunow, 1998). Neurotrophins show high-affinity interactions with the tyrosine kinase receptors TrkA, TrkB, and TrkC. These receptors mediate neurotrophin signaling, a process that involves tyrosine phosphorylation. Nerve growth factor binds to TrkA, and brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) bind to TrkB, whereas neurotrophin-3 (NT-3) binds preferentially to TrkC and to a lesser extent to TrkA and TrkB (for review, see Barbacid, 1994). In addition, the p75 neurotrophin receptor is able to bind all neurotrophins and could represent an important switch between life and death signaling (Carter and Lewin, 1997). During development, both p75 and Trk receptors are expressed in the striatum (Ernfors et al., 1992), and adult striatal projection neurons express both TrkB and TrkC mRNAs (Merlio et al., 1992). In agreement with the presence of neurotrophin receptors on striatal projection neurons, BDNF, NT-3, and NT-4/5 have been found to promote the survival of striatal projection neurons in vitro (Ardelt et al., 1994; Widmer and Hefti, 1994; Nakao et al., 1995a, b; Ventimiglia et al., 1995). In contrast, the effects of these neurotrophins on striatal projection neurons in vivo remain unclear. Although some reports did not find any neuroprotective effect of BDNF or NT-3 (Frim et al., 1993; Anderson et al., 1996), other studies have found that BDNF and NT-4/5 partially protect striatal projection neurons from excitotoxic lesions (Martínez-Serrano and Björklund, 1996; Alexi et al., 1997) and that BDNF, NT-3, and NT-4/5 differentially regulate the phenotype of striatal projection neurons (Pérez-Navarro et al., 1999a).

In the present study, we have examined whether BDNF, NT-3, and/or NT-4/5 may prevent the death and promote the survival of different populations of striatal projection neurons in a QUIN model of HD. Stable cell lines secreting high levels of recombinant BDNF, NT-3, or NT-4/5 were implanted in adult rat striatum before QUIN injection. We show that the three neurotrophins differentially prevent the death of different subsets of striatal projection neurons in vivo and that BDNF is the most efficient survival factor.

Cell grafting and QUIN lesion

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

BDNF- (F3N-BDNF), NT-3- (F3A-NT3), or NT-4/5- (F3A-NT4/5) transfected Fischer-344 rat 3T3 fibroblasts have been described previously and shown to produce >100 ng of each neurotrophin/106 cells per day, in vitro (Arenas and Persson, 1994; Neveu and Arenas, 1996). Mock-transfected Fischer-344 rat 3T3 fibroblasts (F3A-MT) were used as a control (Arenas and Persson, 1994). All cell lines were grown in Dulbecco's modified essential medium supplemented with 10% fetal calf serum, 1 mg/ml penicillin-streptomycin, 1 mg/ml glutamine, and 200 μg/ml G-418 (37°C, 5% CO2). For grafting, cells in active growth phase were washed and collected in serum-free medium at a concentration of 2.5 × 105 cells/μl as described (Arenas and Persson, 1994). A microinjection cannula was implanted into the left striatum, and 3 μl containing 7.5 × 105 cells was injected (1 μl/min) at the following coordinates: 1.8 mm cranial to bregma, 3.2 mm lateral to the midline. Two microinjections of 34 nmol of QUIN each were performed 24 h later, as described previously (Pérez-Navarro et al., 1996, 1999a,b), at the following coordinates: (a) 2.2 mm cranial to bregma, 2.9 mm lateral to the midline; and (b) 0.8 mm cranial to bregma, 3.5 mm lateral to the midline. In all cases, the injection was performed at 5.2 mm under the dural surface with the incisor bar at 5 mm above the interaural line.

BDNF and NT-3 ELISA

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

Seven days after QUIN injection in F3A-MT-, F3N-BDNF-, or F3A-NT3-grafted striata, rats were killed by decapitation. Brains were removed, and neostriata were quickly dissected out, frozen on dry ice, and stored at -80°C. Samples were analyzed using a BDNF or NT-3 ELISA kit according to the recommendations of the manufacturer (Promega). In brief, striata were lysed in 1 ml of lysis buffer [137 mM NaCl, 20 mM Tris, 1% (octylphenoxy)polyethoxyethanol (IGEPAL), 10% glycerol, 10 mM NAF, 2 mM sodium vanadate, and proteinase inhibitor cocktail tablets]. A volume of 25 μl of the sample was diluted in 75 μl of sample buffer and incubated in a plate coated with a BDNF or NT-3 antibody. Two dilutions (1:100 and 1:10,000) of each sample were analyzed. Standard curves of pure BDNF or NT-3 protein, provided by the kit, were used to quantify the production of BDNF or NT-3.

Immunohistochemistry

Seven days after QUIN injection in F3A-MT- or F3N-BDNF-grafted striata, animals were killed by decapitation. Brains were removed, frozen on dry ice, and stored at -70°C. Cryostat-cut horizontal sections (14 μm) through the whole striatum were serially collected on silane-coated slides. Sections were incubated overnight at 4°C with a polyclonal antibody against phosphorylated Trk (pY-490; 1:10; a generous gift from Dr. Rosalind A. Segal) alone or in the presence of 1 μM competing phosphopeptide (686) in Tris-buffered saline with 1 mM sodium orthovanadate, 5% goat serum, and 0.5% NP-40. After washing, the slides were incubated for 1 h at room temperature with a secondary tetramethylrhodamine B isothiocyanate-labeled goat anti-rabbit antibody (1:50, in Tris-buffered saline with 1 mM sodium orthovanadate), washed, and mounted in Tris-buffered saline with glycerol containing p-phenylenediamine. Optical density was quantified using a Quantimed 570 Image processing and Analysis System linked to a Microphot-FXA microscope (Nikon, Garden City, NY, U.S.A.) by an interfacing CCD videocamera. Measurements were performed in horizontal sections through the maximal diameter of the graft. Readings of optical density were obtained from the border of the graft, to the end of the striatum, in a sagittal and caudal direction. Measures of background staining, obtained from sections preincubated with the competing phosphopeptide, were subtracted from the values obtained for the noncompeted pY-490 staining in adjacent sections and expressed as net optical units. Three sections per animal, in three animals per condition, were analyzed.

In situ detection of DNA fragmentation

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

Three days after intrastriatal QUIN injection, animals grafted with F3A-MT, F3N-BDNF, F3A-NT3, or F3A-NT4/5 were deeply anesthetized and immediately perfused transcardially with saline, followed by 4% paraformaldehyde/phosphate-buffered saline (0.1 M, pH 7.4). Brains were removed and postfixed for 1-2 h in the same solution, cryoprotected by immersion in 30% sucrose in phosphate-buffered saline, and then frozen in dry ice-cooled isopentane. Cryostat-cut horizontal sections (14 μm) through the whole striatum were serially collected on silane-coated slides. DNA fragmentation was histologically examined using the in situ Apoptosis Detection System, Fluorescein (Promega). Sections were stained according to the manufacturer's recommendations. In brief, sections were immersed in cool ethanol/acetic acid (2:1 vol/vol) for 5 min and washed in two changes of phosphate-buffered saline at room temperature. Sections were treated with proteinase K (20 μg/ml) during 10 min and postfixed in 4% paraformaldehyde in phosphate-buffered saline for 5 min. Sections were incubated with the equilibration buffer for 5 min at room temperature and then with fluorescein-12-dUTP and terminal deoxynucleotidyl transferase (TdT). After a 1-h incubation at 37°C, sections were washed with 2× saline-sodium citrate for 15 min, and three times with phosphate-buffered saline for 10 min and mounted with Mowiol. As a negative control, adjacent sections were processed following the standard procedure, except that TdT was substituted by water.

In situ hybridization

Seven days after QUIN injection, animals were killed by decapitation. Brains were removed, frozen on dry ice, and stored at -70°C. Cryostat-cut horizontal sections (14 μm) through the whole striatum were serially collected on silane-coated slides, fixed with 4% paraformaldehyde in phosphate-buffered saline, dehydrated in graded ethanol solutions, treated with chloroform, and air-dried. Adjacent sections were processed for in situ hybridization with oligonucleotide probes for rat glutamic acid decarboxylase 67 (GAD), preproenkephalin (PPE), preprotachykinin A (PPTA), or prodynorphin (DYN) as previously described (Arenas et al., 1996; Pérez-Navarro et al., 1999b). The slides were exposed to β-Max x-ray film for 20 days, dipped in NTB-2 photoemulsion (diluted 1:1 in water) for 40 days at 4°C, developed in D-19, fixed, and lightly counterstained with cresyl violet before analysis.

Image analysis and morphometry

All morphometry and cell counting were performed in a blind coded fashion. Area of the lesion and cell counting measurements were performed using PC-Image analysis (Foster Findlay) on a computer attached to an Olympus microscope as previously described (Pérez-Navarro et al., 1999a,b).

For the lesion size estimations, consecutive sections (average of 20-22 sections per animal and probe) were visualized on a computer, and the border of the lesion was outlined. The volume of the lesion was estimated by multiplying the sum of all the sectional areas (in square micrometers) by the distance between successive sections (98 μm), as previously described (Coggeshall, 1992).

PPE-, GAD-, PPTA-, and DYN-positive neurons were counted in a region caudal to the graft, in four different fields (130 × 86 μm, each). The first field analyzed was the one where the first labeled neurons were observed. This field was located at different distances from the border of the graft depending on the in situ probe used (Pérez-Navarro et al., 1999a). The other three regions were successively located 325 μm away from the previous one and aligned with the center of the graft, as previously described (Pérez-Navarro et al., 1999a,b). Cell counts were converted to the number of cells per square millimeter by dividing by the area corresponding to each field examined (0.0112 mm2). TdT-mediated UTP nick end-labeling (TUNEL)-labeled cells were also quantified in a region caudal to the graft by direct counting of fluorescein-stained nuclei. Three adjacent fields (305 × 205 μm, each) beginning at the border of the graft and aligned with the center of the graft were counted.

All striatal neuronal populations and TUNEL-labeled cells were counted in 10 sections per animal, separated by 150 μm, and in four animals per condition (F3A-MT, F3N-BDNF, F3A-NT3, or F3A-NT4/5 grafting with or without QUIN injection).

Materials

All the reagents used for cultures were from GIBCO. QUIN and IGEPAL were obtained from Sigma Chemical Co. The ELISA kit and in situ Apoptosis Detection System, Fluorescein were from Promega. β-Max x-ray film was from Amersham. NTB-2 photoemulsion and D-19 developer were from Kodak. Tetramethylrhodamine B isothiocyanate-labeled goat anti-rabbit antibody was from Jackson, Mowiol was purchased from Calbiochem, and proteinase inhibitor cocktail tablets were from Boehringer Mannheim.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

Characterization of graft size and neurotrophin production by grafts

To control the growth rate of the different cell lines, the size of the grafts was measured 8 days after grafting, in all the sections used to count the number of striatal neurons. The mean radius (in μm) of the grafts in non-lesioned animals was similar for all the cell lines used (Fig. 1A). In QUIN-lesioned animals the size of the grafts was also similar for all the cell lines but slightly larger than in nonlesioned animals (Fig. 1A).

image

Figure 1. Characterization of graft size and neurotrophin production. A: Radius (in μm) of the grafts 8 days after grafting (nonlesioned animals) or 7 days after intrastriatal QUIN injection. Data are mean ± SEM values (n = 4 animals per condition). All grafts showed similar size but were slightly larger in QUIN-lesioned striata. B and C: Content of (B) BDNF or (C) NT-3, measured by ELISA, in striata of adult rats receiving grafts of F3A-MT (MT), F3N-BDNF (BDNF), or F3A-NT3 (NT3) cell lines and QUIN (Q) injections. *p < 0.01, **p < 0.001 compared with MT + Q-injected striata by Student's t test for unpaired data.

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The levels of BDNF or NT-3 secreted by the cell lines were determined by ELISA, 7 days after QUIN injection. Our results show that grafting of the F3N-BDNF or the F3A-NT3 cell lines increased the levels of the corresponding neurotrophin in the striatum by 17 and 10 times, respectively, compared with striata receiving the control graft plus QUIN injection (Fig. 1B and C). The actual dose of neurotrophins delivered by the cells cannot be calculated per unit of time from our measures in vivo because the kinetics of internalization and metabolism of neurotrophins have not been accounted for. However, our results clearly show that for a certain given time, 7 days after QUIN injection, both cell lines were able to increase by at least one order of magnitude the levels of protein available in the striatum.

Grafting of BDNF-secreting cell line induces TrkB phosphorylation

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

We first set out to determine the range and the extent to which neurotrophins secreted by the cell lines were able to activate their receptors within the striatum. For that purpose, we examined TrkB phosphorylation in response to intrastriatal grafting of BDNF-secreting cell line because BDNF has been reported to be the neurotrophin with the lowest intraparenchymal diffusion (for review, see Mufson et al., 1999). Seven days after QUIN injection in F3A-MT- or F3N-BDNF-grafted striata, sections were processed by immunohistochemistry with an antibody against phosphorylated TrkB [pY-490 (Segal et al., 1996)]. Optical densities were measured at different distances from the border of the graft. As shown in Fig. 2A, we observed low levels of TrkB phosphorylation in the vicinity of the lesion and a gradual increase in the staining of phospho-TrkB at increasing distances from the border of the grafts. In animals grafted with F3A-MT cells and injected with QUIN, phospho-TrkB levels were very low from the border of the graft up to a distance of 300 μm, where neurons were not observed by in situ hybridization (Pérez-Navarro et al., 1999a). In this condition, phospho-TrkB levels reached levels comparable to those found in the intact striatum at a distance of 450 μm from the border of the graft and then mildly increased to reach a plateau at 600 μm. In contrast, in the F3N-BDNF-grafted striata, phospho-TrkB levels progressively increased and reached maximal levels at 900-1,050 μm from the border of the graft (Fig. 2A). Beyond 1,200 μm from the border of the graft, phospho-TrkB levels declined in both BDNF- and control-grafted striata. However, BDNF grafts maintained higher levels of phospho-TrkB than control grafts 300 μm to up to 1,500 μm from the border of the graft (Fig. 2A). Interestingly we found both strongly immunolabeled neuronal somas in the striatum (Fig. 2B) and sporadic immunoreactive fibers around the F3N-BDNF graft (Fig. 2C), indicating that TrkB phosphorylation could result from direct activation of TrkB receptors on neuronal somas and/or retrograde transport of phosphorylated TrkB to the cell bodies.

image

Figure 2. Intrastriatal grafting of the BDNF-secreting cell line induced TrkB phosphorylation. A: Optical density was measured at different distances from the border of the graft. Data are mean ± SEM (bars) values (n = 4 animals per condition). Clear higher increases in phospho-TrkB levels were detected from a distance of 300 μm in lesioned animals grafted with the BDNF cell line (•) compared with lesioned animals grafted with the control cells (MT; ○). *p < 0.05, **p < 0.01 compared with MT plus QUIN-injected striata by Student's t test for unpaired data. B: Photomicrograph shows neurons immunostained with an anti-phospho-TrkB antibody at 600 μm from the border of the graft in animals grafted with F3N-BDNF and injected with QUIN. C: Neuronal fibers positive for anti-phospho-TrkB were sporadically observed in the vicinity of the graft. Bar = 20 μm.

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Neurotrophins prevent striatal cell death induced by QUIN

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

Striatal cell death was studied 3 days after QUIN injection. We chose this time point because apoptotic profiles reach maximal levels at 48-72 h after intrastriatal QUIN injection (Hughes et al., 1996). DNA fragmentation was histologically examined using the TUNEL technique. In the absence of TdT, no nuclei were stained, confirming the specificity of the labeling. Labeled nuclei were only observed in the QUIN-injected striata, and no signal was detected in other brain areas.

Stained nuclei were counted in three adjacent fields, beginning at the border of the graft and located in the region caudal to the graft. In animals grafted with the control cell line, QUIN injection induced a gradual loss of striatal neurons, as shown by the decrease in the number of labeled nuclei (Fig. 3E). Intrastriatal grafting of F3N-BDNF, F3A-NT3, or F3A-NT4/5 cell lines differentially prevented QUIN-induced cell death in all the striatal regions examined (Fig. 3E). Data obtained from these animals indicated that most of the damage induced by QUIN was limited to the area between the graft and 1 mm away. To evaluate the total effect of neurotrophins, the average density of TUNEL-labeled nuclei was calculated in the first millimeter surrounding the graft. We found that the F3N-BDNF cell line, which reduced the number of TUNEL-labeled nuclei by 57%, was the most efficient at preventing cell death. F3A-NT3 and F3A-NT4/5 grafting showed lower efficiency and prevented striatal cell death by 37 and 31%, respectively.

image

Figure 3. Neurotrophins prevent DNA fragmentation induced by intrastriatal QUIN injection. Striata were examined 3 days after QUIN injection in animals grafted with control or BDNF-, NT-3-, or NT-4/5-secreting cell lines. Images show confocal laser scanning photomicrographs of TUNEL-stained nuclei at the level of the first region (located at the border of the graft). Grafting of (B) BDNF-, (C) NT-3-, or (D) NT-4/5-secreting cells decreased the number of labeled nuclei compared with the control cell line (A). Bar in A = 40 μm. E: Mean ± SEM (bars) number of labeled nuclei (n = 4 animals per condition). The number of nuclei in every region and condition was standardized to the values obtained for the first region in animals grafted with the control cell line (values in this region were considered as 100%, as this region was the most affected by QUIN injection). MT + QUIN, mock-transfected control cell line-grafted striata plus QUIN injection (▪); BDNF + QUIN, striata grafted with BDNF-secreting cell line plus QUIN injection ([UNK]); NT-3 + QUIN, striata grafted with NT-3-secreting cell line plus QUIN injection (♦); NT-4/5 + QUIN, striata grafted with NT-4/5-secreting cell line plus QUIN injection (▴). *p < 0.05, **p < 0.01, ***p < 0.001 compared with MT + QUIN-injected striata by Kruskal-Wallis ANOVA followed by Mann-Whitney U test.

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Effect of neurotrophins on lesion size induced by QUIN

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

As neurotrophins prevented QUIN-induced cell death, we next examined which population of striatal projection neurons was protected by these factors. In situ hybridization for different markers (GAD, PPE, PPTA, and DYN), characteristic of striatal projection neurons, was performed. Intrastriatal injection of QUIN alone induced a lesion with a size (striatal volume without in situ labeled neurons) that was not modified by grafting of the control cell line (F3A-MT; Fig. 4). In agreement with previous results (Pérez-Navarro et al., 1999a, b), GAD-and PPE-positive neurons were more affected than PPTA- and DYN-positive cells (Fig. 4). Grafting of neurotrophin-secreting cell lines reduced the lesion size of neurons positive for GAD (Fig. 4A), PPE (Fig. 4B), and PPTA (Fig. 4C), showing that BDNF and NT-3 were more efficient than NT-4/5. In contrast, in the same animals, none of the grafts reduced the volume of the lesion for DYN-positive neurons (Fig. 4D).

image

Figure 4. Neurotrophins differentially reduced the size of the lesion induced by QUIN injections. The volume of the lesion was measured for each striatal neuronal population in sections hybridized with (A) GAD, (B) PPE, (C) PPTA, and (D) DYN antisense probes. Data are mean ± SEM (bars) values (n = 4 animals per condition). Grafting of the control cell line (MT+Q) did not modify the volume of the lesion induced by QUIN injection alone. In contrast, intrastriatal grafting of F3N-BDNF (BDNF+Q), F3A-NT3 (NT-3+Q), or F3A-NT4/5 (NT-4/5+Q) cell lines differentially reduced the volume of the lesion for (A) GAD-, (B) PPE-, and (C) PPTA- but not for (D) DYN-positive neurons. *p < 0.05, **p < 0.01 compared with MT+Q-injected striata by one-way ANOVA followed by Scheffé post hoc test.

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Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

The number of surviving neurons was examined by in situ hybridization after implantation of F3A-MT, F3N-BDNF, F3A-NT3, or F3A-NT4/5 in lesioned and nonlesioned animals. Positive neurons were counted in four different fields located in the region caudal to the graft (see Materials and Methods). In the nonlesioned striatum, grafting of the different cell lines did not produce changes in the total number of striatal projection neurons compared with the contralateral, noninjected side (data not shown). QUIN injection alone induced a gradual loss of neurons and resulted in a specific pattern of surviving striatal projection neurons. Moreover, this pattern was not modified by F3A-MT grafting (Table 1), indicating that the control cell line had no survival-promoting effect per se.

Table 1. Grafting of the control cell line did not modify the loss of striatal projection neurons induced by QUIN
 PPEGADPPTADYN
 QUINMT + QUINQUINMT + QUINQUINMT + QUINQUINMT + QUIN
  1. Data are mean ± SEM cell density (in cells/mm2; n = 4 animals per condition) of identified neurons found in every condition. Region 1 was defined as the first area where positive neurons were found, with other regions progressively scored at 325μm increments (see Materials and Methods). QUIN, QUIN injection alone; MT + QUIN, F3A-MT + QUIN-injected striatum. Results were statistically analyzed by Kruskal-Wallis ANOVA.

Region 1127 ± 5148 ± 23250 ± 22279 ± 4150 ± 22145 ± 10188 ± 13206 ± 29
Region 2221 ± 15258 ± 32350 ± 22349 ± 13330 ± 20314 ± 26260 ± 29257 ± 14
Region 3389 ± 6399 ± 10458 ± 20462 ± 17417 ± 17397 ± 27333 ± 17352 ± 19
Region 4522 ± 36549 ± 10542 ± 20551 ± 13517 ± 17503 ± 24467 ± 21458 ± 14

GAD-expressing cells are mainly protected by BDNF

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

QUIN injection in animals grafted with the control cell line induced a 56, 44, and 22% loss of GAD-positive neurons in regions 1-3, respectively (Fig. 5F). In agreement with the decrease in the lesion size by neurotrophins, intrastriatal grafting of neurotrophin-secreting cell lines prevented, with different efficiency, the decrease in the number of GAD-positive neurons induced by QUIN. Whereas intrastriatal grafting of F3N-BDNF or F3A-NT3 prevented GAD-positive cell loss in all the regions examined, F3A-NT4/5 only protected those neurons in the first region (Fig. 5). The total effect of neurotrophins was also evaluated in the first millimeter from the graft, the area where cell loss takes place. BDNF was the most efficient factor and protected 68% of GAD-positive neurons affected by QUIN. In contrast, NT-3 and NT-4/5 protected GAD-positive neurons by only 34 and 20%, respectively.

image

Figure 5. BDNF was the most efficient neurotrophin at preventing the degeneration of GAD-positive neurons in the QUIN (Q)-lesioned striata. All photomicrographs show GAD-positive neurons in the first region analyzed, next to the graft and lesion sites. A: F3A-MT (MT) control. Bar=20 μm. B: Grafting of the MT cell line in Q-injected striata did not prevent loss of GAD-positive neurons. In contrast, grafting of the F3N-BDNF (BDNF) cell line (C) and, to a lesser extent, the F3A-NT3 (NT-3; D) or F3A-NT4/5 (NT-4; E) cell line, prevented the loss of neurons expressing GAD mRNA. F: Mean ± SEM (bars) cell density (n = 4 animals per condition). *p < 0.01 compared with MT; +p < 0.05, ++p < 0.01 compared with MT+Q by Kruskal-Wallis ANOVA followed by Mann-Whitney U test.

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PPE-expressing cells are differentially protected by neurotrophins

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

PPE mRNA-positive neurons were the most severely affected by QUIN-induced excitotoxicity. A 72% cell loss was observed in the first field examined (region 1; compare Fig. 6A with B and see Fig. 6F). Cell loss in regions 2 and 3 was ∼54 and 30%, respectively (Fig. 6F). As observed for GAD-positive neurons, implantation of BDNF- or NT-3-secreting cells significantly reduced the loss of PPE-positive neurons in all the regions examined, whereas NT-4/5 only protected these neurons in region 1 (Fig. 6F). In the first millimeter, F3A-BDNF or F3A-NT3 grafting prevented the loss of PPE-positive neurons by 70 and 64%, respectively. In contrast, NT-4/5 only prevented the loss of 27% of PPE-positive neurons.

image

Figure 6. BDNF, NT-3, and to a lesser extent NT-4/5 prevent the degeneration of PPE-positive neurons in the QUIN (Q)-lesioned striata. All photomicrographs show the first region next to the lesioned area, where the first labeled neurons were found. A: F3A-MT (MT) control. Bar=20 μm. B: Grafting of the MT cell line did not prevent PPE-neuron loss induced by Q. Grafting of the F3N-BDNF (BDNF; C), F3A-NT3 (NT-3; D), or F3A-NT4/5 (NT-4; E) cell line differentially prevented the Q-induced degeneration of neurons expressing PPE mRNA. F: Mean ± SEM (bars) cell density (n = 4 animals per condition) of PPE-positive neurons. *p < 0.01 compared with MT; +p < 0.05, ++p < 0.01 compared with MT+Q by Kruskal-Wallis ANOVA followed by Mann-Whitney U test.

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PPTA-positive neurons are protected by all neurotrophins to a similar extent

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

PPTA-positive neurons were the most resistant to QUIN. A significant loss of neurons was found only in regions 1 (65%) and 2 (27%) (Fig. 7F). Grafting of F3N-BDNF, F3A-NT3, or F3A-NT4/5 cells prevented the loss of PPTA-positive neurons by 30, 44, or 37%, respectively, in the first millimeter.

image

Figure 7. BDNF, NT-3, and NT-4/5 prevent with similar efficiency the loss of PPTA-positive neurons induced by QUIN (Q) injections. A: F3A-MT (MT) control. Bar = 20 μm. B: Grafting of the MT cell line did not prevent the loss of PPTA-positive neurons in the Q-injected striata. However, all of the neurotrophin cell lines—F3N-BDNF (BDNF; C), F3A-NT3 (NT-3; D), and F3A-NT4/5 (NT-4; E)—prevented the degeneration of neurons expressing PPTA mRNA. Photomicrographs show the first region where labeled neurons were counted. F: Mean ± SEM (bars) density (n = 4 animals per condition) of PPTA-positive neurons. *p < 0.01 compared with MT; +p < 0.05, ++p < 0.01 compared with MT+Q by Kruskal-Wallis ANOVA followed by Mann-Whitney U test.

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DYN-expressing cells are only protected by BDNF

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

DYN-positive neurons were also lost after QUIN injection. There was ∼50, 40, and 20% of reduction in the number of DYN-positive neurons in regions 1-3, respectively (Fig. 8F). Only F3N-BDNF grafts prevented the QUIN-induced death of DYN-positive neurons (compare Fig. 8C with B, D, and E, and see Fig. 8F). The overall response in the first millimeter was a protection of ∼52% for BDNF.

image

Figure 8. BDNF prevents the loss of DYN-positive neurons induced by QUIN (Q) excitoxicity. All photomicrographs show the first region where cell counting was performed. A: F3A-MT (MT) control. Bar = 20 μm. Grafting of MT (B), F3A-NT3 (NT-3; D), or F3A-NT4/5 (NT-4; E) cell lines did not prevent the loss of DYN-positive neurons in the Q-injected striata. Instead, grafting of the F3N-BDNF cell line (BDNF; C) prevented the loss of neurons expressing DYN mRNA. F: Mean ± SEM (bars) density (n = 4 animals per condition) of DYN-positive neurons. *p < 0.01 compared with MT; +p < 0.05, ++p < 0.01 compared with MT+Q by Kruskal-Wallis ANOVA followed by Mann-Whitney U test.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

In the present report we show that neurotrophins decrease the number of TUNEL-positive nuclei in the striatum, reduce the lesion size, and differentially prevent the loss of striatal projection neurons from intrastriatal QUIN injection. BDNF is the most efficient factor at preventing cell death and the factor with the broadest neuroprotective activity on striatal projection neurons. In situ hybridization studies show that BDNF can protect all populations of striatal projection neurons from QUIN excitotoxicity. In contrast, NT-3 and NT-4/5 do not prevent the loss of DYN-positive neurons and are less efficient than BDNF at protecting GAD-positive neurons.

Although an indirect action of neurotrophins cannot be ruled out, our results could be explained by a direct action of BDNF, NT-3, and NT-4/5 on striatal projection neurons because they express both TrkB and TrkC but not TrkA receptors (Merlio et al., 1992). In agreement with our data, in vitro studies have shown that BDNF, NT-3, and NT-4/5 promote the survival of striatal GABAergic neurons (Widmer and Hefti, 1994; Ventimiglia et al., 1995), that NT-4/5 prevents the death of substance P-containing striatal neurons (Ardelt et al., 1994), and that neurotrophins protect striatal projection neurons from different types of injury (Nakao et al., 1995a, b). With regard to in vivo studies, BDNF (Martínez-Serrano and Björklund, 1996; Bemelmans et al., 1999) and NT-4/5 (Alexi et al., 1997) have been found to prevent the degeneration of striatal projection neurons induced by intrastriatal QUIN injection. However, some other studies failed to detect any effect of BDNF (Frim et al., 1993; Anderson et al., 1996) or NT-3 (Anderson et al., 1996). In the latter cases, methodological differences, including the parameters used to assess neuronal rescue, the severity of the lesion, and/or the high doses of neurotrophins, can account for the absence of effects. In fact, it has been shown that long exposure to high doses of BDNF induces a desensitization of TrkB in vivo (Frank et al., 1996; Knüsel et al., 1996) and down-regulates TrkB mRNA or desensitizes TrkB in vitro (Carter et al., 1995; Frank et al., 1996), suggesting that treatment with high doses of BDNF or NT-3 for extended periods may reduce TrkB and TrkC responsiveness to neurotrophins. Instead, exposure to lower doses of BDNF in vivo does not down-regulate the BDNF response (Knüsel et al., 1996), TrkB expression is induced (Ferrer et al., 1998), and striatal projection neurons are protected from QUIN excitotoxicity (Martínez-Serrano and Björklund, 1996; Bemelmans et al., 1999; present study). Thus, low doses of BDNF seem to be not only sufficient, but also necessary, to activate efficiently TrkB and exploit all the therapeutic potential of this trophic factor.

From a functional point of view, BDNF has been reported to be anterogradely transported to the striatum from the cerebral cortex (Altar et al., 1997), and low levels of neurotrophins are known to be expressed in the striatum and in the targets of striatal projection neurons, pallidum, and substantia nigra (Miranda et al., 1993; Seroogy and Gall, 1993; Timmusk et al., 1993). To explore whether neurotrophins could elicit functional responses on striatal projection neurons from these sites, we have previously delivered neurotrophins either in the substantia nigra (Arenas et al., 1996) or in the striatum (Pérez-Navarro et al., 1999a). Our results showed that neurotrophins are able to regulate the phenotype of striatal projection neurons both in a target-derived or in an autocrine/paracrine fashion. In the present study we now show that neurotrophins can also act within the striatum as autocrine/paracrine survival factors to prevent the degeneration of striatal projection neurons. Such a mode of action is in agreement with the presence of recurrent axon collaterals of striatal projection neurons within the striatum. Comparison of the effects of neurotrophins on the phenotype (Pérez-Navarro et al., 1999a) and survival (present study) of striatal projection neurons shows that these functions are differentially regulated by a given neurotrophin, suggesting independent mechanisms of action. For instance, NT-4/5 protected PPE- and GAD-positive neurons only in the first region examined (present study), whereas this neurotrophin also prevented the decrease in the soma area of PPE- and GAD-positive neurons induced by QUIN in all the regions examined (Pérez-Navarro et al., 1999a). Furthermore, NT-3 showed greater effects than BDNF and NT-4 at regulating the phenotype of striatal neurons (Pérez-Navarro et al., 1999a), whereas we show here that BDNF is the most efficient factor at preventing the loss of striatal neurons. Thus, our results are in agreement with the concept that binding of neurotrophins to their receptors activates different intracellular pathways to induce survival and/or differentiation (for review, see Segal and Greenberg, 1996; Springer and Kitzman, 1998).

Excitotoxicity, oxidative stress, impaired energy metabolism, and caspase activation have been proposed as mechanisms to explain neurodegeneration in HD (Goldberg et al., 1996; Petersén et al., 1999). The last possibility has recently received much attention because inhibition of caspase-1 was found to slow disease progression in a mouse model of HD (Ona et al., 1999). Furthermore, some reports have shown that neurotrophic factors can exert their protective effects by modifying caspase activity in vitro (Tamatani et al., 1998) and in vivo (Han et al., 2000). Thus, all current available data suggest that inappropriate apoptosis may take place in HD and that neurotrophins may be able to prevent cell death by activating similar molecular mechanisms as during development. In agreement with this possibility, the regulation and function of neurotrophin receptors during development and regeneration also seem to be similar. For instance, TrkB protein is developmentally regulated in the striatum (Constantini et al., 1999), and trkB-null mutant mice showed increased striatal apoptotic cell death during development (Alcántara et al., 1998). Similarly, we recently found that TrkB expression was up-regulated by intrastriatal QUIN injection (Canals et al., 1999) and that both TrkB ligands, BDNF and NT-4/5, regulate the phenotype of striatal projection neurons (Pérez-Navarro et al., 1999a) and promote their survival (present study). Thus, combined, all these data suggest an important role of neurotrophins, and in particular of TrkB ligands, both during development and regeneration of striatal neurons.

In conclusion, the present work shows that BDNF, NT-3, and NT-4/5 differentially prevent the death of identified striatal projection neurons and that BDNF is the most efficient neurotrophin in promoting survival of striatal projection neurons. Thus, our findings suggest that continuous supply of low doses of BDNF might be beneficial for the treatment of neurological disorders affecting striatal projection neurons.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Cell grafting and QUIN lesion
  5. BDNF and NT-3 ELISA
  6. In situ detection of DNA fragmentation
  7. RESULTS
  8. Grafting of BDNF-secreting cell line induces TrkB phosphorylation
  9. Neurotrophins prevent striatal cell death induced by QUIN
  10. Effect of neurotrophins on lesion size induced by QUIN
  11. Grafting of the control cell line did not prevent loss of striatal projection neurons induced by QUIN
  12. GAD-expressing cells are mainly protected by BDNF
  13. PPE-expressing cells are differentially protected by neurotrophins
  14. PPTA-positive neurons are protected by all neurotrophins to a similar extent
  15. DYN-expressing cells are only protected by BDNF
  16. DISCUSSION
  17. Acknowledgements

We are very grateful to Lotta Johanson for secretarial help, Annika Ahlsen and Maite Muñoz for technical assistance, and Anna Bosch for her support and advice in the use of confocal microscopy. Financial support was obtained from the BIOMED2 Program of the European Commission (grant BMH4-CT96-0273), CICYT (grants SAF96-0043 and SAF99-0019, Ministerio de Educación y Ciencia, Spain), La Marató de TV3 (grant 97-TV1009), the Swedish Medical Research Council (MFR, grant K98-12X-11563-03A), the Karolinska Institute, and the Jeanssonska and the Kapten Arthur Eriksson Foundations. E.P.-N. was a fellow of the European Molecular Biology Organization.

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