inhibits neuronal sprouting in a rat model of epilepsy (kindling/neurotrophic factors/hippocampus/synaptic reorganization)

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 92, pp , October 1995 Neurobiology A nerve growth factor peptide retards seizure development and inhibits neuronal sprouting in a rat model of epilepsy (kindling/neurotrophic factors/hippocampus/synaptic reorganization) KASHIF RASHID*, CATHARINA E. E. M. VAN DER ZEE*t, GREGORY M. Rosst, C. ANDREW CHAPMAN, JOLANTA STANISZ*, RicHARD J. RIOPELLEt, RONALD J. RAcINE, AND MARGARET FAHNESTOCKS *Department of Biomedical Sciences, McMaster University, Hamilton, ON Canada L8N 3Z5; tdepartment of Medicine (Neurology), Queens University, Kingston, ON Canada K7L 2V7; and Psychology Department, McMaster University, Hamilton, ON Canada L8S 4K1 Communicated by Daniel E. Koshland, Jr., University of California, Berkeley, CA, June 15, 1995 ABSTRACT Kindling, an animal model of epilepsy wherein seizures are induced by subcortical electrical stimulation, results in the upregulation of neurotrophin mrna and protein in the adult rat forebrain and causes mossy fiber sprouting in the hippocampus. Intraventricular infusion of a synthetic peptide mimic of a nerve growth factor domain that interferes with the binding of neurotrophins to their receptors resulted in significant retardation of kindling and inhibition of mossy fiber sprouting. These rmdings suggest a critical role for neurotrophins in both kindling and kindling-induced synaptic reorganization. Recent studies have demonstrated that kindling results in mossy fiber sprouting and upregulation of neurotrophins and their receptors (1-3). In kindling, subconvulsive electrical stimulation applied repeatedly to various regions of the forebrain through indwelling electrodes evokes progressively prolonged electrographic and behavioral responses that culminate in generalized clonic seizures (4-6). Sutula et al. (3) have reported that perforant path kindling induced sprouting of mossy fibers (axons of dentate gyrus granule cells) into the supragranular molecular layer of the dentate gyrus. Represa and Ben-Ari (7) have reported that amygdaloid kindling induces sprouting of mossy fibers and synaptic reorganization in the CA3 region of the hippocampus. Both studies reported sprouting in the absence of any noticeable morphological damage, although Cavazos et al. (8) recently reported progressive neuronal loss in the hilus of the dentate gyrus and area CAl with amygdaloid kindling. Electrophysiological studies have supported the possibility that sprouting mossy fibers make functional synaptic connections (7, 9), and it has been proposed that the resulting synaptic reorganization contributes to the epileptogenic process (3). Hippocampal mossy fiber sprouting is observed in patients with childhood epilepsy (1, 11) and with partial complex epilepsy (12). Neurotrophins are essential for the development, maintenance, and regulation of various classes of neurons (13, 14), and nerve growth factor (NGF) has been shown to be required for sympathohippocampal and septohippocampal sprouting in response to injury (15, 16). Neurotrophins may thus play a role in kindling-induced sprouting. In the central nervous system, the highest mrna levels of neurotrophins are found in the hippocampus (17, 18), but they exhibit different patterns of expression. NGF mrna is expressed in the pyramidal layer of the hippocampus, the stratum oriens, and in the hilar region of the dentate gyrus (19, 2); brain-derived neurotrophic factor (BDNF) mrna is found in pyramidal neurons of the CA1, CA2, and CA3 regions as well as in the hilus; and neurotrophin 3 (NT-3) mrna is expressed in the CAl and CA2 regions, the pyramidal cell layer, the hilus, and the granule cell layer of the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact dentate gyrus (19, 21). Hippocampal kindling induces the rapid and transient synthesis of NGF and BDNF mrna (1) and increases the production of NGF protein in the dentate gyrus and the parietal and piriform cortices (2). These findings, alongside studies demonstrating that intraventricular administration of antibodies to NGF delay kindling (22, 23) and inhibit kindling-induced mossy fiber sprouting (23), suggest that neurotrophins may mediate synaptic reorganization within the hippocampus and thereby contribute to the development of an epileptogenic state. Neurotrophins bind to members of the trk family of receptor tyrosine kinases and to the low-affinity nerve growth factor receptor (p75ngfr) (24). Although the receptor-binding domains of the neurotrophin molecules have yet to be fully elucidated, a variety of data including a 2.3-A x-ray crystal solution of NGF (25) implicate the variable regions within the molecule in receptor binding (26-29). Although intraventricularly administered antibodies against NGF have already been shown to inhibit amygdaloid kindling (22, 23) and kindlinginduced mossy fiber sprouting (23), it has not yet been demonstrated directly that these in vivo effects are receptor mediated. The intraventricular infusion of synthetic peptides designed to prevent neurotrophin binding to their receptors is one approach to examining the mechanism of kindling and of neurotrophin-induced neuronal sprouting in the central nervous system. MATERIALS AND METHODS Kindling Surgery and Stimulation. Three-month-old male Long-Evans hooded rats, 3-4 g, were housed individually, maintained on an ad lib feeding schedule, and kept on a 12-hr-on/12-hr-off light cycle. The rats were anesthetized with a mixture of ketamine hydrochloride (.9 ml/1 g) and xylazine hydrochloride (.4 ml/1 g) and placed in a stereotaxic frame. Teflon-coated, stainless steel bipolar electrodes with an exposed tip (diameter, 19,um) were implanted into the right basolateral amygdala. The stereotaxic coordinates (3) were 2.8 mm posterior, 4.8 mm lateral, and 8.6 mm ventral to bregma. Immediately after electrode implantation, an Alzet brain infusion cannula (Alza) was implanted into the lateral ventricle with the following coordinates:.6 mm posterior, 1.3 mm lateral, and 5 mm ventral to bregma. The electrode and cannula were firmly attached to the skull with dental cement and anchored with three stainless steel screws. A fourth screw placed above the contralateral parietal cortex served as a ground electrode. Lyophilized peptide was dis- Abbreviations: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin 3. tpresent address: Department of Anatomy and Neurobiology, Dalhousie University, Halifax, NS Canada B3H 4H7. tto whom reprint requests should be addressed at: Department of Biomedical Sciences, McMaster University, 12 Main Street West, Hamilton, ON Canada L8N 3Z5.

2 9496 Neurobiology: Rashid et al. Proc. Natl. Acad. Sci. USA 92 (1995) solved in.9% physiological saline to a final concentration of 45.3,uM to deliver 64.8,ug of peptide per ml. The control group received intraventricular saline. A flow-regulated miniosmotic pump (Alzet model 22; flow speed,.5,li/hr; effective up to 14 days; Alza) was filled, connected to the cannula with polyethylene tubing (Intramedic nonradiopaque 7415; Clay Adams), and placed subcutaneously in the dorsal neck area. Pump implantation was performed "blind," and the code was broken only after all stimulations were complete. The animals were given 3 days for recovery after the surgery, before the twice daily kindling stimulations were initiated. Each stimulation consisted of a 1-s 6-Hz train of 1.-msec duration biphasic pulses at an intensity that ranged from 2 to 5 p.&a. This low-intensity stimulation was sufficient to trigger epileptiform afterdischarges following each stimulation. Animals were stimulated for a period of 11 days, and the duration and magnitude of the afterdischarges were recorded electrographically. The behavioral progression of kindling was also monitored by rating the seizures on a scale of 1 to 5 according to Racine's classification (5): stage 1, mouth and facial twitches; stage 2, clonic head movements; stage 3, unilateral forelimb clonus followed by contralateral clonus; stage 4, clonic rearing; stage 5, loss of postural control. Animals were considered fully kindled when they exhibited three consecutive stage-5 seizures. Timm Staining of Hippocampal Mossy Fibers. The Timm method (3, 31) was used to stain the Zn2+-containing mossy fibers. At 15 days postsurgery, rats were anesthetized with sodium pentobarbitol (65 mg/kg) and perfused transcardially with 5 ml of sodium sulfide perfusion medium (8.9 g of Na2S 9H2O, 1.9 g of sucrose, and 1.19 g of Na2PO4 H2O per 1 ml), with the descending aorta clamped. The brains were removed, covered with tissue-tek embedding medium (Miles), and immediately frozen on dry ice. Horizontal 36-gm sections of the hippocampal area at mm ventral to bregma were cut on a cryostat and mounted on chromium potassium sulfate (chrom alum)-coated slides. The sections were developed in the dark for 6-8 min in gum arabic (5 g/1 ml)/ hydroquinone (5.67 g/1 ml)/citric acid (6.375 g/25 ml)- sodium citrate (5.875 g/25 ml) buffer (6:3:1, vol/vol);.5 ml of a silver nitrate solution (1.7 g AgNO3/1 ml) was added per 1 ml of developing mixture. Determining Sprouting Density of Mossy Fibers. Mossy fiber sprouting was evaluated by comparing the density of Timm granules in the stratum oriens of kindled rats receiving intraventricular saline or peptide infusions with the values obtained from the nonkindled control animals. The group of control animals consisted of seven nonkindled rats without an implanted electrode and three nonkindled rats with an implanted electrode. Density values of seven nonkindled control rats did not differ from values obtained from three nonkindled rats with an implanted electrode, demonstrating that implantation of an electrode in the amygdala does not detectably influence CA3 Timm staining (ANOVA with supplemental Student's t test; P >.5) (23). Neither control group had an implanted cannula. Digitized images of the horizontal sections from the dorsal dentate gyrus were examined at x 1 magnification by an image analysis system (Micro Computer Imaging Device, Brock University, St. Catherines, ON Canada). The density of Timm granules was measured at 2 adjacent Table 1. Neurite outgrowth inhibition by synthetic NGF peptides No. of neurons, % inhibition of Type of neurons Growth factor Inhibitor mean ± SEM neurite outgrowth SCG NGF ±.48 Linear 16 ± Monocyclic Bicyclic 8 ±.41 6 Anti-NGF ±. 1 DRG NGF 21.5 ± 2.22 Linear 13.5 ± Monocyclic 1.25 ± Bicyclic 7.5 ±.5 65 Anti-NGF 3.5 ± DRG BDNF ±.85 Linear ± Monocyclic 14.5 ±.65 8 Bicyclic ±.85 1 Anti-NGF ±.48 3 Nodose BDNF - 16 ±.91 Linear ± Monocyclic ± Bicyclic ± Anti-NGF ± DRG NT-3-19 ±.58 Linear 15.5 ± Monocyclic ±.48 2 Bicyclic ± Anti-NGF 3 ± Nodose NT ± 1.1 Linear ± Monocyclic 11.5 ± Bicyclic 7.5 ± Anti-NGF 1.25 ± Neurite outgrowth of dissociated dorsal root ganglion (DRG), superior cervical ganglion (SCG), and nodose ganglion neurons from newborn mice in response to recombinant NGF, BDNF, and NT-3 was inhibited by the addition of anti-ngf IgG (control) or by linear, monocyclic, or depsibicyclic R-11 peptides (see Materials and Methods). The depsibicyclic peptide blocked neurite outgrowth in the bioassay to a greater extent than the depsimonocyclic or the linear peptides.

3 Neurobiology: Rashid et al. 1.3-cm2 positions along the extent of the stratum oriens. Background values were provided by readings at 1 cursor placements in the stratum radiatum of the CA3. A regression line was drawn using 8 separate points drawn from control sections with various background values (23). For each kindled rat, the density was expressed as a percentage increase over nonkindled controls with the same background density. Peptide Activity in in Vitro Bioassay. Recombinant NGF, BDNF, and NT-3 were affinity-purified from baculovirusinfected insect cell cultures (M.F., unpublished data) and were used at a concentration just sufficient to evoke maximal neurite outgrowth. Two hundred fifty micromolar peptide, or anti-ngf IgG (1,ug/ml) as a control, was added to dissociated dorsal root ganglion, superior cervical ganglion, or nodose ganglion neurons from newborn mice (day 1) in individual wells of a terasaki plate. After 48 hr, the number of neurons exhibiting neurites with lengths greater than twice the cell body diameter was counted in each well (13, 32). The percent inhibition is one minus the number of neurite-bearing neurons in the wells with peptide divided by the number of neuritebearing neurons in the wells without the peptide. The number of neurons was taken from the average of four wells. RESULTS In these experiments, we used a conformationally constrained peptide that mimics both the conformation and sequence of the variable L3 region of NGF (26), which is thought to be part of a trka-binding domain of the NGF molecule (29). The peptide R-1 1 was designed from two noncontiguous elements of NGF making up the loop region L3 (CRASNPVESGC, residues 58-68) and part of the cysteine knot region (CVC, residues 18-11) of NGF. Three forms of this peptide were infused intraventricularly in our studies: a depsibicyclic, conformationally constrained peptide (two disulfide bonds), a depsimonocyclic, partially constrained peptide (one disulfide bond), and a linear, unconstrained peptide. The depsibicyclic R-11 peptide prevented NGF binding to both p75ngfr and trka in crosslinking studies to a much greater extent than the depsimonocyclic or linear peptides (G.M.R., M. I. Dory, D. F. Weaver, and R. J. Riopelle, unpublished data). This peptide also blocked NGF-mediated neurite outgrowth (IC5 = 1,uM) by dorsal root ganglion neurons. At high concentrations, the depsibicyclic peptide blocked neurite outgrowth in an in vitro bioassay to a greater extent than the depsimonocyclic or linear peptides, and it blocked NGF- and NT-3-induced neurite outgrowth to a much greater extent than it blocked BDNF-induced neurite outgrowth (Table 1). We examined the effects of intraventricular infusion of the R-11 peptide on both kindling and kindling-induced mossy fiber sprouting. Intraventricular infusion of depsibicyclic R-11, but not the depsimonocyclic or linear forms of the peptide, resulted in a significant retardation of amygdaloid kindling at all 5 stages of behavioral seizure activity [ANOVA with repeated measures with a supplemental t test, F (1, 8) = 18.47; P <.3] (Fig. 1A). Animals that received the depsibicyclic peptide required a significantly greater number of stimulations to reach stage-5 seizures than the rats that received the linear peptide or saline (P <.5 and P <.1, respectively; one-way ANOVA with supplemental t test) (Fig. 1B). The average number of stimulations required to reach stage-5 seizures was ±.86 for depsibicyclic R-11-treated rats, as compared to 1.8 ±.4 for rats treated with linear R-11 or ± 1.36 for rats receiving saline. The animals treated with the depsimonocyclic R-11 required an intermediate number, 12.8 ± 1.5 stimulations, to reach stage-5 seizures. This was not statistically significant when compared to either the linear, the depsibicyclic R-11, or the saline group. Similarly, intraventricular infusion of depsibicyclic R-11, but not the depsimonocyclic or linear forms of the peptide, A 5. Linear R-1 1 Bicyclic R co 3. N cd B 22 2 LO, 18 c" a o E 8 o 6 6 z 4 2 Proc. Nati. Acad. Sci. USA 92 (1995) Number of stimulations Saline Linear Monocyclic Bicyclic Treatment FIG. 1. Number of stimulations required for kindling. Rats treated with intraventricular infusions of linear, depsimonocyclic, or depsibicyclic R-11 peptide were stimulated twice daily in the amygdala for a period of 11 days. The animals were considered fully kindled when they exhibited three consecutive stage-s seizures. (A) The depsibicyclic peptide (), but not the linear peptide (), inhibits kindling at all five stages. (B) The average number of stimulations required for the animals to reach the fully kindled state is significantly greater in the depsibicyclic peptide-treated animals than in the linear peptidetreated or saline-treated animals. Statistical significance: *, P <.5; **, P <.1 (ANOVA with supplemental t test). The error bars indicate SEM. reduced kindling-induced mossy fiber sprouting (Fig. 2). Timm granules were visibly increased in the stratum oriens of the linear R-11-treated kindled rats (Fig. 2B) when compared to the nonkindled control rats (Fig. 2A). However, Timm granule staining in the depsibicyclic R-11-treated kindled rats resembled the staining in the nonkindled control (Fig. 2C). Image analysis and quantification of the Timm staining density (Fig. 3) demonstrated that the depsibicyclic peptide-treated kindled rats exhibited a significantly higher stratum oriens density (1% higher) compared to nonkindled control rats but significantly lower density (1% lower) when compared to the linear peptide-treated kindled rats. The Timm granule density in the depsibicyclic peptide-treated kindled rats was reduced by half compared to the linear and depsimonocyclic peptide-treated kindled rats. DISCUSSION Our ability to inhibit both kindling and kindling-induced sprouting of hippocampal mossy fibers using a conformation-

4 9498 Neurobiology: Rashid et al Proc. Natl. Acad. Sci. USA 92 (1995) A r Tf* b *. U J.9.,.'.4* -'.,X,,,,.. >.K 44 FIG. 2. Timm staining of the hippocampus. Representative examples of horizontal Timm-stained sections of the CA3 region in a nonkindled rat (A), a linear R-11 peptide-treated kindled rat (B), and a depsibicyclic R-11 peptide-treated kindled rat (C). Kindling and peptide administration were as in Materials and Methods. ally constrained peptide that perturbs both receptor binding and the in vitro biological effects of NGF, BDNF, and NT-3 implicates binding of neurotrophins to their receptors in kindling and kindling-induced mossy fiber sprouting. Preliminary data suggests that the R-11 peptide may exert its effects on NGF activity by altering NGF dimer conformation (G.M.R., M. I. Dory, D. F. Weaver, and R. J. Riopelle, unpublished data). Because the R-11 peptide influences NGF interactions with p75ngfr and trka, our in vitro data demonstrating antagonistic effects on NGF, BDNF, and NT-3 might be interpreted to suggest that the pan-neurotrophin effects are mediated not only at the level of trk family receptors but also at the level of the common neurotrophin receptor. p75ngfr is expressed in all NGF-responsive cells, including sympathetic, sensory, and basal forebrain cholinergic neurons (33). Lowaffinity and- high-affinity NGF receptors have been demonstrated in the hippocampal and dentate gyrus regions (34-36). trka mrna distribution in the adult rat brain is much more restricted than the expression of trkb and trkc mrna, however (37). trkb mrna is synthesized at high levels in the granule cell layer and throughout the pyramidal layer of the hippocampus (37, 38) and is upregulated in hippocampal pyramidal and dentate granule cells after hippocampal kindling (21). trkc mrna has been found in the granule cell layer of the dentate gyrus, throughout the pyramidal cell layer, and in the hilus (39).

5 2 Cu E co a) Cu c_ Ic Neurobiology: Rashid et al o I, v,, V'c,IAA Linear Monocyclic Bicyclic Peptide treatment FIG. 3. Mossy fiber sprouting density. Image analysis of Timm staining density in the kindled rats expressed as a percentage increase over nonkindled controls (see Materials and Methods). Kindled rats were infused with linear, monocyclic, or depsibicyclic R-11 peptide. Depsibicyclic-treated rats demonstrated a 5% reduction in mossy fiber density when compared to linear- and depsimonocyclic-treated rats. Statistical significance (*, P <.5) was determined by Student's t test. The error bars represent SEM. All density readings of the strata oriens and radiata were within the linear range of In summary, these results provide evidence that a synthetically produced domain of the NGF molecule can effectively block neurotrophin activity in vivo and that neurotrophins may play a critical role in kindling-induced synaptic reorganization and kindling epileptogenesis. Much of the experimental literature on neurotrophin effects after central nervous system insult favors their neuroprotective functions (4). Our results suggest that upregulation of neurotrophins and their receptors may in certain circumstances (e.g., epileptogenesis) be undesirable. Thus, selective inhibition of neurotrophin activity and the concommitant reduction in neuronal sprouting may be of potential therapeutic value. The question still remains how the kindling-induced increase in neurotrophins and neurotrophin receptors, mossy fiber sprouting, and the development of epileptogenesis are functionally related. Neurotrophin and neurotrophin receptor induction in the forebrain after electrical stimulation may facilitate synaptic reorganization that is responsible, in turn, for the progression to or maintenance of an epileptic state. Another possibility, however, is that the sprouting is purely activation-induced. Consequently, the reduced levels of sprouting in the R-11-treated rats could be due to the reduced levels of kindling in those animals. We thank Ross Ridsdale and Beth Chick for technical assistance. This work was supported by grants from the Neuroscience Network of Centres of Excellence (R. J. Riopelle), the Natural Science and Engineering Research Council (R. J. Racine), and the National Institutes of Health (M.F.). G.M.R. was supported by a postdoctoral fellowship from the Neuroscience Network. 1. Emfors, P., Bengzon, J., Kokaia, Z., Persson, H. & Lindvall,. (1991) Neuron 7, Bengzon, J., Soderstrom, S., Kokaia, Z., Kokaia, M., Ernfors, P., Persson, H., Ebendal, T. & Lindvall,. (1992) Brain Res. 587, Proc. Natl. Acad. Sci. USA 92 (1995) Sutula, T., Xiao-Xian, H., Cavazos, J. & Scott, G. (1988) Science 239, Goddard, G. V., McIntyre, D. C. & Leech, C. K. (1969) Exp. Neurol. 25, Racine, R. J. (1972) Electroencephalogr. Clin. Neurophysiol. 32, Racine, R. J. & Burnham, W. M. (1984) in Electrophysiology of Epilepsy, eds. Schwartzkroin, P. A. & Wheal, H. (Raven, New York), pp Represa, A. & Ben-Ari, Y. (1992) Exp. Brain Res. 92, Cavazos, J. E., Das, I. & Sutula, T. P. (1994) J. Neurosci. 14, Sutula, T. P., Golarai, G. & Cavazos, J. E. (1992) Epilepsy Res. Suppl. 7, Represa, A., Le Gall La Salle, G. & Ben-Ari, Y. (1989) Neurosci. Lett. 99, Represa, A., Robain, O., Tremblay, E. & Ben-Ari, Y. (1989) Neurosci. Lett. 99, Sutula, T., Cascino, G., Cavazos, J., Parada, I. & Ramirez, L. (1989) Ann. Neurol. 26, Yamamori, T. (1992) Neurosci. Res. 12, Eide, F. F., Lowenstein, D. H. & Reichardt, L. F. (1993) Exp. Neurol. 121, Springer, J. E. & Loy, M. R. (1985) Brain Res. Bull. 15, Van der Zee, C. E. E. M., Fawcett, J. & Diamond, J. (1992) J. Comp. Neurol. 326, Ernfors, P., Whetmore, C., Olson, L. & Persson, H. (199) Neuron 5, Phillips, H. S., Hains, J. M., Laramee, G. R., Rosenthal, A. & Winslow, J. W. (199) Science 25, Ernfors, P., Ibanez, S. F., Ebendal, T., Olson L & Persson, H. (199) Proc. Natl. Acad. Sci. USA 87, Gall, C. M. & Isackson, P. J. (1989) Science 245, Merlio, J. P., Ernfors, P., Kokaia, Z., Middlemas, D. S., Bengzon, Kokaia, M., Smith, M. L., Siesko, Lindvall,. & Persson, H. (1993) Neuron 1, Funabashi, T., Sasaki, H. & Kimura, F. (1988) Brain Res. 458, Van der Zee, C. E. E. M., Rashid, K., Le, K., Moore, K. A., Stanisz, J., Diamond, J., Racine, R. J. & Fahnestock, M. (1995) J. Neurosci. 15, Chao, M. V. (1992) Neuron 9, MacDonald, N. Q., Lapatto R., Murray-Rust, J. & Blundell, T. L. (199) J. Cell. Sci. Suppl. 13, Ibanez, C. F., Ebendal, T. & Persson, H. (1991) EMBO J. 8, Longo, F. M., Vu, T. K. & Mobley, W. C. (199) Cell Regul. 1, Drinkwater, C. C., Barker, P. A., Suter, U. & Shooter, E. M. (1993) J. Biol. Chem. 31, Nanduri, J., Vroegop, S. M., Buxser, S. E., & Neet, K E. (1994) J. Neurosci. Res. 37, Paxinos, G. & Watson, C. (1982) StereotaxicAtlas ofthe Rat Brain (Academic, Sydney). 31. Danscher, G. (1981) Histochemistry 71, Coughlin, M. D. & Collins M. B. (1985) Dev. Biol. 11, Patil, N., Lacey, E. & Chao, M. V. (199) Neuron 4, Pioro, E. P. & Cuello, A. C. (199) Neuroscience 34, Altar, C. A., Burton, L. E., Bennett, G. L. & Dugich-Djordjevic, M. M. (1991) Proc. Natl. Acad. Sci. USA 88, Sobreviela, T., Clary, D. O., Reichardt, L. F., Brandabur, M. M., Kordower, J. H. & Mufson, E. J. (1994) J. Comp. Neurol. 35, Merlio, J. P., Ernfors, P., Jater, M. & Persson, H. (1992) Neuroscience 51, Klein, R., Conway, D., Parada, L. & Barbacid, M. (199) Cell 61, Lamballe, F., Klein, R. & Barbacid, M. (1991) Cell 66, Lindvall, O., Kokaia, Z., Bengzon, J., Elmer, E. & Kokaia, M. (1994) Trends Neurosci. 17,