Brain-derived neurotrophic factor, nerve growth factor and neurotrophin-3 in selected regions of the rat brain following kainic acid-induced seizure activity

Abstract

Changes in levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) in various regions of the rat brain following kainic acid-induced seizure activity were investigated. BDNF protein, as measured by a two-site enzyme immunoassay, increased transiently 12–24 h after the intraperitoneal administration of kainic acid to 61.6 ng/g wet weight in the hippocampus (∼10-fold increase), 19.5 ng/g in the piriform plus entorhinal cortex (∼10-fold) and 8.2 ng/g in the olfactory bulb (∼16-fold), and then rapidly decreased. Increases of 2- to 4-fold in levels of BDNF were also detected in the septum, cerebral cortex, striatum and hypothalamus, but not in the cerebellum. In contrast, levels of NGF and NT-3 decreased 24 h after the administration of kainic acid. Western and Northern blotting analyses of hippocampal tissues, respectively, revealed increase in levels of a 14-kDa protein corresponding to BDNF and its mRNA at both 4.2 and 1.4 kb. Hippocampal mRNAs for NGF and NT-3 increased and decreased, respectively, in kainic acid-treated rats. Immunohistological investigations showed that, in the hippocampus, the administration of kainic acid enhanced a homogeneous immunoreactivity of BDNF in the polymorph inner layer (the stratum radiatum of the CA3/CA4 regions and the hilar region) and in granule cells of the dentate gyrus. BDNF protein was found in neurons, but not at all in glial cells or in blood vessels, and was localized in the cytoplasm, the nucleoplasm and the primary dendrites of neurons as well as in perisynaptic extracellular spaces, but hardly in their axons. Our results show that kainic acid treatment increases levels of BDNF, but not NGF or NT-3, in various regions of the rat brain, other than the cerebellum. Also, the majority of BDNF newly synthesized by hippocampal granule neurons is secreted into the perisynaptic extracellular space in the polymorph inner layer of the dentate gyrus, supporting an autocrine-like role for the factor in synaptic functions.

Introduction

Brain-derived neurotrophic factor (BDNF) is a member of the structurally and functionally homologous neurotrophin family that includes nerve growth factor (NGF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (Leibrock et al., 1989, Ernfors et al., 1990, Hohn et al., 1990). BDNF appears to be involved in specific stages of neurogenesis, such as cell division, differentiation and axogenesis, as well as in the naturally occurring death of neuronal cells (Barde, 1989). In culture, BDNF supports the survival of several types of neuron, for example septal cholinergic, mesencephalic dopaminergic, striatal γ-aminobutyric acid (GABA)ergic and cerebellar neurons (for review, see Lindsay, 1993; also Hyman et al., 1991, Lindholm et al., 1993). In the adult rat brain, exogenously administered BDNF protects cholinergic neurons in the basal forebrain from degenerative changes after axotomy (Knüsel et al., 1992). However, excessive loss of neuronal cells has been not observed in mice with a deficiency of BDNF gene (Ernfors et al., 1994, Conover et al., 1995, Liu et al., 1995). In addition, we have shown that levels of hippocampal BDNF, but not NGF or NT-3, are higher in aged than in young adult rats (Katoh-Semba et al., 1998b) even though degenerated neurons can be clearly observed in the CA3 region of the hippocampus of aged rats (Kadar et al., 1990). Thus, there is no clear dependence of neuronal survival on BDNF.

Administration of kainic acid to rats, an analog of the excitatory amino acid glutamate (Olney et al., 1974), induces acute seizure, which resembles the sequelae of human temporal lobe epilepsy (Ben-Ari, 1985, Nadler, 1985). Systemic injection of kainic acid results in neuronal damage in the CA3 region of the hippocampus (Schwob et al., 1980). In contrast, the administration of kainic acid causes a transient increase in levels of mRNA for BDNF in the hippocampus, especially in the dentate gyrus (Zafra et al., 1990, Thoenen et al., 1991, Dugich-Djordjevic et al., 1992, Lindefors et al., 1992, Timmusk et al., 1993). Thus, kainic acid appears to be a good tool for investigation as to why BDNF is elevated in the hippocampus of aged rats and whether BDNF supports the survival of neurons in the adult brain. There have been many reports regarding the effects of kainic acid on the levels of mRNA for neurotrophins in the hippocampus (e.g. Balları́n et al., 1991, Ernfors et al., 1991, Hashimoto et al., 1992). However, protein expression, especially of BDNF, has received only limited attention (Rudge et al., 1998). We have focused on quantitative and spatial changes in levels of BDNF protein in the rat brain following kainic acid-induced seizure activity. In this article, we report the changes in levels of mRNA for BDNF and of its protein in selected regions of the brain from rats with kainic acid-induced seizure and compared them to those of total NGF and NT-3 containing a loosely and tightly bound (particle-bound) forms of NGF and NT-3 in the brain (Katoh-Semba et al., 1998a, Katoh-Semba et al., 1998b). In addition, we present immunohistochemical and immunocytochemical localization of BDNF in the brain of kainic acid-treated rats.