Involvement of neurotrophic factors in aging of noradrenergic innervations in hippocampus and frontal cortex
Introduction
The locus coeruleus (LC) is a densely-packed cluster of noradrenergic neurons in the brain stem (Amaral and Sinnamon, 1977). These neurons innervate widely different target sites, such as the frontal cortex, cerebellum, medulla oblongata and hippocampus (Moore and Bloom, 1979, Morrison et al., 1979, Segal and Bloom, 1976, Swanson and Hartman, 1975). In particular, the LC is known as a major noradrenergic source of the hippocampus (Haring and Davis, 1985, Swanson and Hartman, 1975) and frontal cortex (Morrison et al., 1979). However, it is not clear at present how these multiple innervations of LC neurons are maintained with advancing age.
Our previous electrophysiological study suggested that the LC noradrenergic terminals are maintained during aging in the polymorphic layer of the hippocampus dentate gyrus (PoDG) (Ishida et al., 2000). In the PoDG, the densities of LC axon terminals did not decrease during aging, but the sprouting of LC axon terminals gradually increased between 17 and 25 months of age (Ishida et al., 2000). On the other hand, in the frontal cortex, the LC projections gradually decreased with age (Ishida et al., 2000). The sprouting in the frontal cortex rapidly increased after 15 months of age, and it was maintained at a high level until 25 months of age (Ishida et al., 2000). Thus, these data suggest that the hippocampus PoDG differs from the frontal cortex in the process of aging, despite both sites having the same noradrenergic source originating from the LC. Therefore, we hypothesized that the aging patterns of the LC noradrenergic innervations depend on its terminal sites. In this study, we investigated the age-dependent changes in the norepinephrine transporter (NET) expression in the hippocampus and frontal cortex. NET is located on the noradrenergic presynaptic axon terminals, and it uptakes the released norepinephrine to regulate synaptic activity (Matsuoka et al., 1997). Age-dependent changes were reported for the uptake activity of presynaptic axon terminals of LC neurons (Shirokawa et al., 2003) and for the NET expressions in the LC (Shores et al., 1999). Thus, we examined whether the age-dependent changes in NET expression occur in the terminal areas of LC noradrenergic neurons.
Neurotrophic factors have an important role for neuronal survival or in the formation of axonal branching (Arenas et al., 1995, Holm et al., 2002). Previous studies showed that neurotrophic factors are taken from axonal terminals and transported retrogradely (Leitner et al., 1999, Mufson et al., 1994, Sobreviela et al., 1996, Yan et al., 1988). If the axon terminals of LC noradrenergic neurons take up neurotrophic factors at their terminal sites, the different aging patterns of LC innervations between the hippocampus and frontal cortex may be due to the different neurotrophic factors at each terminal site. The brain-derived neurotrophic factor (BDNF) is thought to have a close relationship with the LC noradrenergic system. Previous studies showed that BDNF promotes the survival of noradrenergic neurons (Friedman et al., 1993) and the up-regulation of noradrenaline uptake (Sklair-Tavron and Nestler, 1995). In our recent study, continuous local infusion of BDNF caused an increase in the sprouting of the LC axon terminals in the frontal cortex of aged rats, but the sprouting-enhancing effect was not observed in young or middle-aged rats (Matsunaga et al., 2004). Therefore, we examined whether the glial cell line-derived neurotrophic factor (GDNF) which is known to be distributed in the LC (Choi-Lundberg and Bohn, 1995), changes during aging in the frontal cortex and hippocampus. Some previous studies showed that GDNF enhances noradrenergic innervations (Granholm et al., 2001) and protects LC neurons from 6-hydroxydopamine-induced degeneration (Arenas et al., 1995). Moreover, a relationship between GDNF expression and the aging process of LC neurons was suggested by GDNF heterozygous mice study (Zaman et al., 2003).
In the present study, we considered the NET expression as an index of the density of noradrenergic innervations in the hippocampus and frontal cortex. We first performed Western blot analysis of the hippocampus and frontal cortex to determine the age-dependent changes in NET expression. Next, Western blot analysis was also performed to examine the relationship between the aging of noradrenergic innervations and the expressions of GDNF and BDNF.
Section snippets
Animals
Six-month-old (young), 13-month-old (middle-aged) and 25-month-old (aged) male F344/N rats were used in this study. They were maintained in a 12 h light:12 h dark cycle, and had free access to food and water. All animal procedures complied with the National Institutes of Health guidelines and were approved by the Laboratory Animal Research Facilities Committee of the National Center for Geriatrics and Gerontology.
Immunohistochemistry
Rats were anesthetized with an overdose of Somnopentyl (100 mg/kg i.p.), and were
Immunohistochemistry
Fig. 1 shows the DBH-immunopositive fibers in the hippocampus and frontal cortex. In the hippocampus, dense DBH-immunopositive fibers were observed in the PoDG, but only a few DBH-immunopositive fibers were observed in other areas. The density of DBH-immunopositive fibers in the PoDG of 13-month-old rats appeared higher than that of 6- and 25-month-old rats (Fig. 1A, C and E). In the frontal cortex, in contrast, DBH-immunopositive fibers were distributed equally, and no visible differences in
Discussion
In the hippocampus, our Western blot analysis indicated that the NET expression level was significantly increased in 13-month-old rats compared with 6-month-old rats. Although the function of this transient increase in the 13-month-old hippocampus is unclear, NET is closely associated with the regulation of noradrenalin reuptake at the axon terminals (Galli et al., 1995). Our previous electrophysiological study suggested that the noradrenergic projection from LC to the hippocampus dentate gyrus
Acknowledgment
This work was supported in part by a grant from the Japan Society for the Promotion of Science, No. 1420058.
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