Increased neurogenesis and the ectopic granule cells after intrahippocampal BDNF infusion in adult rats
Introduction
BDNF is a member of the neurotrophin family, and is highly expressed in hippocampus. In the dentate gyrus, BDNF protein is strongly expressed in granule cells, where it appears to be anterogradely transported to the axons of the granule cells, called the mossy fibers (Conner et al., 1997, Yan et al., 1997). Functionally, BDNF appears to have several effects in the dentate gyrus. BDNF influences the growth and survival of granule cells in primary culture (Holtzman and Lowenstein, 1995, Patel and McNamara, 1995), and the morphology of adult granule cells (Danzer et al., 2002). It has the potential to regulate the morphology and activity of inhibitory cells within the dentate gyrus as well (Marty, 2000, Olofsdotter et al., 2000). Exposure to BDNF increases excitatory transmission, and this has been shown not only in area CA1 (see Lu, 2004 for review) and CA3 (Scharfman, 1997), but also in the dentate gyrus (Asztely et al., 2000, Messaoudi et al., 1998). It also regulates excitability in other ways (Blum et al., 2002, Wardle and Poo, 2003).
Because of the extensive literature showing that BDNF is important to the growth and development of the CNS (Eisch, 2002, Gould et al., 1999, Lu, 2004) and the evidence that granule cells of the hippocampus undergo neurogenesis throughout life, several studies have investigated whether BDNF might influence neurogenesis. For example, Benraiss et al. (2001) showed that increasing BDNF in the adult subventricular zone using an adenovirus approach increased the number of new neurons in several brain areas outside the hippocampus. Pencea et al. (2001) showed that i.c.v. BDNF led to increased numbers of new neurons in several areas adjacent to the ventricles, such as the striatum, septum, and thalamus. In the dentate gyrus, Lee et al. (2000) showed that dietary restriction increased BDNF, and that there was an increase in dentate gyrus neurogenesis as well. They and others (Linnarsson et al., 2000) subsequently showed that the number of new cells in the dentate gyrus was decreased in heterozygote BDNF knockout mice. Katoh-Semba et al. (2002) reported that riluzole, a drug currently approved for amyotrophic lateral sclerosis, increased BDNF in hippocampus, and there was increased proliferation of granule cell precursors. Consistent with a role of BDNF, they found that infusion of an antibody to BDNF into the ventricles blocked the increase in proliferation.
Interestingly, the results of other studies of BDNF, in the context of ischemia, have not led to similar conclusions. In these studies, BDNF after ischemia appeared to block the increase in neurogenesis that ischemia produces. Thus, using an adeno-viral vector approach, increased hippocampal BDNF after ischemia led to a decrease in neurogenesis (Larrsson et al., 2002). Scavenging BDNF with trkB-IgG after ischemia promoted neurogenesis (Gustafsson et al., 2003). These studies led us to question what infusion of BDNF itself would do to neurogenesis in the normal adult rat. We also questioned whether BDNF administration to the hippocampus directly, that is, direct to the subgranular zone, would lead to different effects than the studies based on transgenic mice, studies of remote administration, or use of viral vectors. Our question was based on the fact that transgenic mice may have developmental compensatory changes, remote administration is limited by the inability of BDNF to reach the hippocampus, and the viral vector approach may not lead to large changes in BDNF concentration in the subgranular zone, where presumably it exerts its actions on neurogenesis.
We also had a secondary purpose for our experiments. We questioned whether BDNF infusion would lead to new cells that would develop in abnormal, “ectopic” locations, as occurs following severe, continuous seizures (status epilepticus; Schmidt-Kastner et al., 1996). Such cells may be important to understand because thus far it has been assumed that any increase in neurogenesis is beneficial, but inappropriate migration and development of new cells may disrupt hippocampal function. One factor that may be involved in the development of ectopic cells is BDNF because the development of ectopic cells after status occurs when BDNF protein levels are elevated (Scharfman et al., 2000, Scharfman et al., 2002b). One way to address this possibility is to directly infuse the dentate gyrus with BDNF in the absence of status epilepticus.
Thus, BDNF infusion allowed us to address two issues. To use this approach, the effects of BDNF infusion were compared to the effects of vehicle infusion (phosphate-buffered saline; PBS), and infusion of bovine serum albumin (BSA), which is a large protein (like BDNF) but does not bind to the trk or p75 receptors that mediate neurotrophin effects.
Section snippets
Materials and methods
The animals used were adult male Sprague–Dawley rats (300–350 g; Taconic Farms, Germantown, NY), housed in individual cages, exposed to stable temperature 68–72°C, a 12-h light/dark schedule (lights on at 7:00 a.m.), and fed ad libitum. All animal care and use met the guidelines of the National Institutes of Health and New York State Department of Health. All chemicals were purchased from Sigma Co. (St. Louis, MO) unless stated otherwise.
BrdU/NeuN double-labeling
The number of cells labeled with a BrdU-stained nucleus and NeuN-labeled cytoplasm (BrdU+/NeuN+) in the granule cell layer were quantified in the dorsal hippocampus. Both the animals infused with a low dose of BDNF and a high dose showed higher values relative to controls (Table 1; statistics are described in the table legends). There was no statistical difference in the average number of cells/section between the low dose and high dose groups (Table 1). Fig. 1 shows a representative example of
Summary
The results demonstrate that BDNF infusion increases neurogenesis in the dentate gyrus. Neurogenesis increased both ipsilateral and contralateral to the infusion site, and most new neurons appeared to become granule cells. BDNF infusion also led to the formation of ectopic granule cells located in the hilar region.
Acknowledgments
We thank Ning Cai, Annmarie Curcio, and Qing Zhang for technical and secretarial assistance. This study was supported by NS 39562 and NS 38285 to H.E.S.
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