Brain-derived neurotrophic factor promotes the survival of neurons ...

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 210-214, January 1995 Neurobiology

Brain-derived neurotrophic factor promotes the survival of neurons arising from the adult rat forebrain subependymal zone (neurogenesis/neurotrophin/stem cells/ventricular zone)

BARRY KIRSCHENBAUM AND STEVEN A. GOLDMAN* Department of Neurology and Neuroscience, Cornell University Medical College, New York, NY 10021

Communicated by Dominick P. Purpura, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY, September22, 1994

neurogenic in selected regions but more generally becomes vestigial (14). On this basis, we reasoned that in those areas that do not exhibit neurogenesis, its absence might in part reflect a lack of postmitotic trophic support. We therefore sought to identify those humoral agents capable of promoting the differentiation and/or survival of neurons generated from adult precursor cells. We also sought to define more thoroughly those areas of the SZ harboring neuronal progenitors by identifying those segments of the adult rat SZ capable of giving rise- to new neurons in vitro. Members of the neurotrophin family, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor (NT)-3, and NT-4/5 (15), have been reported to permit or enhance the survival of postmitotic neurons in a variety of central systems (16). Among these, however, only BDNF is expressed to a significant extent in the adult neocortex (17). We therefore asked whether BDNF might affect the differentiation or survival of newly generated neurons in the adult rat brain. To this end, we compared neuronal outgrowth and survival between BDNF-treated SZ explants and their untreated controls, as well as to matched plates treated with NGF or NT-3. We found that BDNF, alone among the agents tested, substantially accentuated the survival of these new neurons, rescuing >40% from otherwise predictable death by 3 weeks in vitro. These neurons emigrated from SZ explants taken from a wide area of the adult lateral ventricles (LVs), reaching >6 mm back from the rostral tip of the lateral ventricle, and roughly corresponding to the anterior two-thirds of the ventricular system. This region included, but was not limited to, that area of the SZ that gives rise to olfactory bulb neurons in adulthood (9-11). Over this entire expanse, BDNF acted as a permissive factor for the recruitment and survival of new, SZ precursor-derived neurons.

Neuronal precursor cells persist in the adult ABSTRACT forebrain ependymal/subependymal zone (SZ) and have been found to produce neurons in cultures derived from birds, rodents, and humans. We postulated that the survival of neurons generated from these cells might be constrained in adulthood by the local absence of trophic support. To test this hypothesis, we established explant cultures of adult rat forebrain SZ and assessed the effect of defined neurotrophins on the survival of new neurons arising from these explants. We found that microtubule-associated protein 2+ neurons arose from explants derived from a wide area of the SZ, spanning the rostral 6 mm of the ventricular system. In cultures exposed to brain-derived neurotrophic factor (BDNF), >35% of new neurons survived at 22 days in vitro (DIV), and >25% survived at 42 DIV, concurrent with the virtually complete loss of neurons in unsupplemented controls. The surviving cells expressed trkB, the high-affinity receptor for BDNF. In contrast, neither nerve growth factor nor neurotrophic factor 3 enhanced neuronal survival. Thus, BDNF supports the survival of neurons produced by the adult rat forebrain and may act as a permissive factor for neuronal recruitment in adulthood. In the adult mammalian brain, neurogenesis appears largely limited to the olfactory bulb and hippocampal dentate gyrus (1, 2). We have found that the avian forebrain, in contrast, continues to generate neurons throughout adulthood from mitotic subependymal zone (SZ) precursor cells (3). In explant cultures of adult songbird brain, these SZ cells continued to generate neurons (4), particularly under reduced serum conditions (5). However, higher serum levels in these cultures were associated with more net neuronal outgrowth (5), suggesting that serum-borne agents promoted the differentiation and/or maintenance of new neurons either directly or indirectly through their effects on cocultured glial and ependymal cells. Neuronal precursor cells have been identified in cultures derived from diverse areas of the postnatal rodent brain (6, 7) and likely also derive from the SZ (8). In vivo, the rostral 2 mm of the rodent SZ exhibits ongoing neurogenesis, with neuronal migration to the olfactory bulbs (9-11). In vitro, explants of this region generate an outgrowth of neurons, which can be prelabeled with [3H]thymidine given in the days before sacrifice (8). In primates, although the postnatal forebrain SZ largely ceases neurogenesis in vivo (12), it too retains the capacity for neuronal production in vitro, as noted in explants of adult human temporal lobe SZ (13). In all of these cultures, including those derived from canary (4), mouse (8), and human (13), striatal and cortical explants that did not harbor SZ failed to yield neuronal outgrowth. Taken together, these studies suggested the persistence into adulthood of a relatively widespread SZ progenitor cell population, which remains

MATERIALS AND METHODS Culture Preparation. Adult (300-325 g) Sprague-Dawley rats (Hilltop Labs, Philadelphia) were anesthetized by brief halothane exposure and sodium pentobarbital (40 mg/kg; i.p.). Each was perfused with Ca2+/Mg2+-free Hanks' balanced salt solution (HBSS), decapitated, and its brain was removed. Coronal sections were taken at 1.0- to 1.5-mm intervals, beginning 3 mm behind the rostral tip of the forebrain ["1.7 mm anterior to bregma (18)]. The LV surface was revealed, and its SZ was dissected to a depth of 300-400 ,um. Serial Abbreviations: SZ, subependymal zone; NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; NT, neurotrophic factor; LV, lateral ventricle; DIV, days in vitro; MAP, microtubule-associated protein; GFA, glial fibrillary acidic protein. *To whom reprint requests should be addressed at: Department of Neurology and Neuroscience, Cornell University Medical College, 1300 York Avenue, New York, NY 10021.

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Neurobiology: Kirschenbaum and Goldman sections were then dissected at the stereotaxic levels noted (see Fig. 1), and their SZs were prepared as explants. Culture Conditions. All explants were prepared as described with adult songbird brain (4, 6). Explants were distributed in groups of four into 35-mm Petri dishes, each coated with laminin, and then incubated in 5% C02/95% air at 37°C in 0.65 ml of a base medium that we found permissive for adult avian neurogenesis (5). Fetal bovine serum (FBS) was added to a final concentration of either 2% or 10% for the growth factor and regional comparisons, respectively. Regional Comparison. To define those regions capable of generating neuronal outgrowth in vitro, SZ samples were taken at five noncontiguous rostrocaudal levels of the rat ventricular system, whose locations and stereotaxic coordinates are given in Fig. 1. To optimize neuronal outgrowth by this set of explants, they were maintained in 10% FBS, and half were treated with BDNF. Control explants not harboring SZ were prepared from samples of parietal cortex taken from the same rats.

Assignment to Treatment Groups. A total of 26 rats were used, of which 20 were dedicated to the evaluation of growth factors and 6 to the comparison of neuronal outgrowth among SZ regions. The 16-24 explants obtained from each sampled SZ segment of every rat were plated in groups of four, and each plate was assigned to its treatment group randomly. Growth Factor Comparisons. All cultures were first established in medium supplemented with 10% FBS to promote initial explant attachment. After 2 days in vitro (DIV), cultures included in the comparison of growth factors underwent a complete medium change and were assigned to one of four possible treatment groups. The cultures dedicated to this experiment were derived from the most anterior SZ segment (Fig. 1, segment A), since the intent here was to compare growth factors in a preparation already known to be neurogenic. The conditions tested included (i) 2% FBS, (ii) 2% FBS with human recombinant BDNF (Regeneron Pharmaceuticals, Tarrytown, NY), (iii) 2% FBS with murine 2.5S NGF (GIBCO), and (iv) 2% FBS with human recombinant NT-3 (Regeneron). NGF and BDNF were added at 20 ng/ml, and NT-3 was added at 10 ng/ml (19). Each culture was given a complete medium change twice weekly in its appropriate test medium. PI-4.2

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Immunocytochemistry. To validate that the neuron-like cells of the outgrowths were neurons, selected SZ cultures derived from segment A were incubated for 7-18 days, fixed, and then double-stained with two antibodies, one directed against neuronal microtubule-associated protein 2 [MAP-2 (20); rabbit anti-MAP-2 courtesy of I. Fisher (Medical College of Pennsylvania) and S. Halpain (University of Virginia)] and the other against either of two glial antigens, glial fibrillary acidic protein (GFA), or the oligodendrocytic antigen 04 [mouse 04 IgM courtesy of R. Bansal and S. Pfeiffer (University of Connecticut) (21)]. All cultures were fixed for 10 min in 4% paraformaldehyde, stained for each antigen using immunofluorescence as described (4, 21), and visualized with an Olympus IMT2 microscope. Neurons were defined by their multipolar morphology (4) and immunoreactivity for MAP-2 but not for GFA or 04. Control cultures prepared to assess nonspecific staining were uniformly unlabeled; these were exposed to 1% rabbit serum or mouse IgG (10 mg/ml) followed by appropriate secondary antibodies. TrkB, the high-affinity receptor for BDNF (22), was localized in four cultures by immunostaining after 21 DIV, using rabbit anti-trkB gp145 (1:100; Santa Cruz, no. sc-12). This antibody was raised against amino acids 794-808 of the full-length trkB gp145 and did not recognize either the truncated, tyrosine kinase-negative (tk-) form of trkB (23) or trkA or trkC. Detection of the bound antibody with avidin-biotin complex amplification and development with diaminobenzidine/H202 were as described (24). [3H]Thymidine Labeling. The uptake of [3H]thymidine by antigenically defined neurons was used as an index of antecedent precursor cell mitosis in vitro. [3H]Thymidine (0.2 ,tCi per plate; 5 Ci/mmol; 1 Ci = 37 GBq; Amersham) was added to selected cultures after 6 hr in vitro. Cultures were exposed to [3H]thymidine for 6 DIV before complete medium exchange, fixed after at least 7 DIV, stained for MAP-2, and autoradiographed. Autoradiography was performed and labeling was evaluated as described (5). Scoring. Once an explant displayed outgrowth, its neurons were identified morphologically (4) and counted on alternate days from 2 to 22 DIV. Selected cultures were allowed to survive as long as 42 DIV with weekly observation. Other samples were subjected to cell-type identification after 8 and 18 DIV; in these, the numbers of MAP-2+ neurons and 04+ oligodendrocytes were counted for each explant. Statistical Analysis. From each treatment group, the mean number of neurons per explant was calculated for each day of observation. Each unique combination of treatment group and time point included data from at least five explants (range, 5-30; Table 1). The mean number of neurons per explant was calculated for each treatment at each time point, as were the SD and SE. For both the growth factor and regional comparisons, group means were compared by two-way ANOVA, and post hoc comparisons were made with Boneferroni adjustments for multiple comparisons. ANOVA was limited to data obtained on or after 7 DIV, the earliest point at which maximal neuronal numbers were achieved in any group.

RESULTS A

FIG. 1. Regions of rat forebrain SZ sampled. Five rostrocaudally defined levels of the adult forebrain SZ were sampled (shaded areas); segments A-D were taken from the LV surface as follows: A, anterior (A) +1.7 to +0.2; B, posterior (P) -0.3 to -1.8; C, P -2.8 to -4.2; D, P -4.2 to -5.2. Segment E was taken from the surface of the third ventricle, P -4.2 to -5.2. Stereotaxic levels are according to ref. 18. Ctx, cortex; EC, external capsule; Hpc, hippocampus; Fim, fimbria; Amg, amygdala; CP, caudate putamen; IC, internal capsule; AC, anterior commisure.

Neuronal Outgrowth Arose from Explants of the Adult Rat Forebrain SZ. Neuron-like cells began migrating from the explant borders between 2 and 5 DIV (Fig. 2). By 8 DIV, among 288 explants obtained from the most anterior portion of the SZ (segment A; n = 20 rats) that were raised in 2% FBS and pooled across treatments, 70 (24.3%) exhibited neuronal outgrowth. By this point, >80% of the morphologically identified neurons were MAP-2+ (Fig. 2). No significant differences were noted among growth factor treatment groups in their proportions of explants with neuronal outgrowth (data not shown). Each group achieved its maximal number of

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Table 1. Adult SZ-derived neuronal outgrowth and survival as a function of neurotrophin treatment % neuronal survival at 22 DIV, Neurons/SZ explant outgrowth 22 DIV [(22 DIV/8 DIV) x 100] 15 DIV 8 DIV Treatment 6 + 1.1 (n = 41) 3 ± 0.7 (n = 30) 1 ± 0.5 (n = 30) 48 ± 8.1 (n = 42) 2% FBS + NGF 3 ± 0.9 (n = 13) 1 ± 0.2 (n = 13) 6 + 1.1 (n = 26) 60 + 13.9 (n = 26) 35 ± 7.1* (n = 24) + BDNF 26 ± 6.4* (n = 24) 31 ± 5.8* (n = 33) 61 + 10.8 (n = 33) 4 ± 1.3 (n = 10) 3 ± 1.0 (n = 10) + NT-3 10 + 3.1 (n = 10) 67 + 14.8 (n = 15) Only BDNF supported the survival of neurons arising from the adult rat SZ. Relative preservation of neurons raised in BDNF first achieved statistical significance on day 11 in vitro and persisted through at least 42 DIV (see text). In contrast, no effect of either NGF or NT-3 addition was noted. For this experiment, explants were derived from segment A, the most rostral 1.5 mm of the SZ. All values given are means ± SE. Decline with time in n, the number of explants, in some groups resulted from explants being taken for immunocytochemistry. *Significant difference from control at this time point; P < 0.01.

neurons per explant between 8 and 11 DIV, after which neuronal counts fell (Table 1). Neuronal Outgrowth Did Not Arise from Cortical Explants. Previous studies have demonstrated that those neurons that migrate from adult SZ explants arise from mitotic precursors, which last divided either in the days before sacrifice or in culture (4, 8, 13). As an additional control for the possibility that neurons in our outgrowths might have included resident parenchymal neurons, we examined cortical explants prepared from the same rats. Among 48 explants of the parietal cortex, raised in either 2% (n = 16) or 10% FBS (n = 32; 16 each with and without added BDNF), no neuronal outgrowth whatsoever was observed at any time during 3 weeks of observation. This was compared to SZ explants taken from the same rats, from which neurons arose in 28% by 8 DIV (40 of 144 explants, which included 25 of 112 in 2% FBS and 15 of 32 in 10% FBS). At both serum levels, the differences in neuronal outgrowth between SZ and cortical explants were significant to P < 0.001 by x2 analysis. The restriction of neuronal outgrowth to SZ explants supported the premise that these neurons arose from SZ-derived precursors. Precursor-Derived Neurons Were Postmitotic in Vitro. To assess the possibility of mitotic neurogenesis under the conditions used in this study, 38 productive explants (16 with BDNF, 22 without BDNF) were exposed for a week to [3H]thymidine in vitro. Of 574 MAP-2+ neurons found in these outgrowths, only 9 were found to have incorporated [3H]thymidine. No difference in the frequency of [3H]thymidinelabeled neurons was noted between plates exposed to BDNF and their controls. The rarity of mitotic neurogenesis in these cultures, which had been successively exposed to 10% and 2%

FIG. 2. Neurons arise from cultured explants of the adult rat SZ. (A) Young neurons in the outgrowth of an adult rat SZ explant at 3 DIV. (B) Rapid maturation of these neurons and the associated glial substrate cell outgrowth by 6 DIV. (C) Field at 11 DIV after staining for the neuronal marker MAP-2. (D) These MAP-2+ adult neurons at higher magnification. (Bars = 50 Jxm.)

FBS, was anticipated by the finding that neuronal precursor mitosis is inhibited by serum, despite unimpaired postmitotic neuronal development (6). As a result, this preparation allowed us to examine factor effects on neuronal differentiation and survival without the interpretational difficulties posed by concurrent cellular neogenesis. Oligodendrocytes as Well as Neurons Emigrated from the Adult SZ. None of the neuron-like cells expressed GFA, even though GFA expression was common among substrate astrocytes. However, a small proportion, generally