Increased neurogenesis and the ectopic granule cells after intrahippocampal BDNF infusion in adult rats

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Abstract

There is evidence that BDNF influences the birth of granule cells in the dentate gyrus, which is one of the few areas of the brain that demonstrates neurogenesis throughout life. However, studies to date have not examined this issue directly. To do so, we compared the effects of BDNF, phosphate-buffered saline (PBS), or bovine serum albumin (BSA) on neurogenesis after infusion into the hippocampus of the normal adult rat, using osmotic pumps that were implanted unilaterally in the dorsal hilus.

BDNF, PBS, and BSA were infused for 2 weeks. The mitotic marker bromodeoxyuridine (BrdU) was administered twice daily during the 2-week infusion period. At least 1 month after infusion ended, brains were processed immunocytochemically using antibodies to BrdU, a neuronal nuclear protein (NeuN), or calbindin D28K (CaBP), which labels mature granule cells. Stereology was used to quantify BrdU-labeled cells in the dorsal hippocampus that were double-labeled with NeuN or CaBP. There was a statistically significant increase in BrdU+/NeuN+ double-labeled cells in the granule cell layer after BDNF infusion relative to controls. The values for BrdU+/NeuN+ cells were similar to BrdU+/CaBP+ cells, indicating that most new neurons were likely to be granule cells. In addition, BrdU+/NeuN+-labeled cells developed in the hilar region after BDNF infusion, which have previously only been identified after severe continuous seizures (status epilepticus) and associated pathological changes. Remarkably, neurogenesis was also increased contralaterally, but BDNF did not appear to spread to the opposite hemisphere. Thus, infusion of BDNF to a local area can have widespread effects on hippocampal neurogenesis.

The results demonstrate that BDNF administration to the dentate gyrus leads to increased neurogenesis of granule cells. They also show that ectopic granule cells develop after BDNF infusion, which suggests that ectopic migration is not necessarily confined to pathological conditions. These results are discussed in light of the evidence that BDNF increases neuronal activity in hippocampus. Thus, the mechanisms underlying neurogenesis following BDNF infusion could be due to altered activity as well as direct effects of BDNF itself, and this is relevant to studies of other growth factors because many of them have effects on neuronal excitability that are often not considered.

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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