Expression of TrkB subtypes in the adult monkey cerebellar cortex

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Abstract

BDNF and its specific receptor TrkB are concerned with synaptic plasticity as well as maintenance of the nervous system. TrkB has three subtypes: full-length TrkB (TK+), which has a tyrosine kinase containing intracellular domain, and two truncated TrkBs (TK−; T1 and T2), which lack tyrosine kinases. To understand the molecular interaction among these subtypes, we investigated the expression and distribution of BDNF, TK+, and T1 in the adult monkey cerebellum by single and double immunohistochemistry and Western blot analysis. We observed by single immunohistochemistry that BDNF, TK+, and T1 are distributed in almost all the somata and dendrites of Purkinje and granule cells. In the double-stained sections, three kinds of regions were observed: TK+ >T1; TK+ =T1; TK+ <T1. Moreover, three types of TrkB dimers (TK+/TK+ homodimer, TK+/TK− heterodimer, and TK−/TK− homodimer) were induced by stimulating with exogenous BDNF. These observations suggest that the functions of BDNF may be modified by interaction among subtypes of TrkB in each region of the Purkinje cells.

Introduction

BDNF and its specific receptor, TrkB, are highly expressed in the mammalian central nervous system (CNS) (Masana et al., 1993, Nawa et al., 1995, Yan et al., 1997a, Yan et al., 1997b, Ohira et al., 1999). The TrkB locus encodes at least three different subtypes: full-length TrkB (TK+) and two truncated TrkB (TK−; T1 and T2) (Klein et al., 1990a, Klein et al., 1990b). TK+ has been shown to be involved in signal transduction, and while T1 and T2 lack the tyrosine kinase domain, they exhibit various physiological functions (Beck et al., 1993, Allendoerfer et al., 1994, Biffo et al., 1995, Eide et al., 1996, Baxter et al., 1997, Fryer et al., 1997, Haapasalo et al., 1999). Both BDNF and TrkB have been shown to participate in processes of neuronal plasticity in the CNS (Bibel and Barde, 2000, Thoenen, 1995). BDNF is localized to the pre- and postsynaptic terminals (Fawsett et al., 1997, Lin et al., 1998, Aloyz et al., 1999), secreted in an activity-dependent fashion (Haubensak et al., 1998), and induces the autophosphorylation of TrkB (Aloyz et al., 1999). This BDNF-induced activation of TrkB is essential to the neuronal plasticity (Figurov et al., 1996, Akaneya et al., 1997, Kang et al., 1997, Korte et al., 1998).

Expression levels of BDNF and TrkB isoforms have been shown to be high in the cerebellum (Yan et al., 1997a, Yan et al., 1997b, Ohira et al., 1999). In the developing cerebellum, BDNF promotes survival and neurite extension of internal granule cell layers in rats (Segal et al., 1992, Gao et al., 1995). Further, BDNF knockout mice exhibit abnormal cerebellar development (Schwartz et al., 1997). Although Purkinje cells lack TrkB during embryonic development (Lindholm et al., 1997), TK+ is expressed in more mature Purkinje cells (Yan et al., 1997a, Ohira and Hayashi, 1999). In addition, BDNF mRNA levels have been reported to increase with the development of Purkinje cells (Neveu and Arenas, 1996). These results suggest that both BDNF and TrkB are of importance in the survival and maintenance of neurons in the mature cerebellum.

The cerebellum has been thought to contribute to motor skills, but recent data have led to a revision of this hypothesis regarding non-motor functions of the cerebellum (Leiner et al., 1993). Anatomically, the cerebellar efferent fibers project to thalamic neurons that innervate the dorsolateral prefrontal cortex (Middleton and Strick, 1994), which is involved in ‘spatial working memory’. Moreover, physiological and functional neuroimaging studies suggest that the cerebellum participates in the organization of higher-order functions such as concept formation and learning of paired-associates (Kim et al., 1994, Gao et al., 1996, Schmahmann and Sherman, 1998).

Taking the above into account, we consider it important to investigate the distribution of BDNF and TrkB subtypes in the non-human primate cerebellum. In this study, we immunohistochemically investigated the distribution of BDNF, TK+, and T1, and examined the interaction between TrkB subtypes in the adult monkey cerebellum by means of covalent cross-linking.

Section snippets

Experimental animals and tissue preparation

Two adult rhesus monkeys (8 and 9-years-old, Macaca mulatto) were the subjects for the cross-linking and Western blot analysis. For the immunohistochemical analysis, two adult rhesus monkeys (7 and 8-years-old) were used. All animals were pretreated with ketamine hydrochloride (10 mg/kg, intramuscularly) and deeply anesthetized with pentobarbital sodium (25 mg/kg, intraperitoneally).

For the immunohistochemical study, heparin sodium (2 ml, 1000 U/ml) was injected directly into the left

Immunoreactivity in single-stained sections

To detect and localize BDNF, TK+, and T1 proteins in the adult monkey cerebellum, we carried out single-staining immunohistochemical studies (Fig. 1). BDNF-immunoreactivity (IR) was strongly detected in cell bodies and dendrites of almost all Purkinje cells (Fig. 1D–F). Granule cells were immunoreactive. There were also abundant BDNF immunoreactive cells in the molecular layer. In addition, the entire molecular layer was weakly stained.

TK+-IR was observed in almost all Purkinje cells (Fig. 1

Distribution of BDNF in the primate cerebellum

In this study, we detected BDNF-IR in granule and Purkinje cells, whose dendrites were also immunopositive (Fig. 1). In rodent, BDNF mRNA is expressed in granule and Purkinje cells (Neveu and Arenas, 1996). In addition, the presence of BDNF protein in dendrites and cell bodies of Purkinje cells has been reported in rodents (Dugich et al., 1995). In cell-culture experiment, granule cells have been shown to synthesize BDNF protein as measured by a sensitive enzyme immunoassay (Lindholm et al.,

Acknowledgements

This work was supported partly by Grants (no. 12680772, no. 12210080) and a Grant-in-Aid for COE Research (10CE2005) from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan.

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