Tetsuya Shirokawa

Specific uptake of Aβ1-40 in rat brain occurs in astrocyte, but not in microglia

In the brain of a patient with Alzheimers disease, beta amyloid peptide (Abeta) is thought to be taken up by glial cells such as astrocyte and microglia to be degraded. However, it is unclear whether the Abeta is absorbed by astrocyte or microglia. The purpose of our study is to determine which type of glial cell, astrocyte or microglia, can take up Abeta. Beta amyloid 1-40 (Abeta1-40) was directly infused into the frontal cortex or hippocampus for 14 days. Dual-labeling immunohistochemistry for Abeta1-40 with an astrocytic (GFAP) or microglial (CD11b) marker was performed to examine co-localization of Abeta1-40 and glial markers. In the Abeta1-40 infused site, immunoreactivity of Abeta1-40 was observed only in astrocytes, not in microglia. In addition, Abeta40-1, a reverse peptide of Abeta1-40, was not taken up by astrocytes. These results suggested that the astrocyte-specific uptake of Abeta occurred in the rat brain.

BDNF is necessary for maintenance of noradrenergic innervations in the aged rat brain.

In the axon terminals of the locus coeruleus (LC) neurons, a high level of axonal branching was occurred in the middle-aged brain, and the increased branching was maintained in the aged brain. In the present study, we hypothesized that neurotrophic support is necessary for the morphological age-related changes seen in the noradrenergic innervations from the LC to frontal cortex. Through immunohistochemical and quantitative image analyses, we examined the age-dependent effects of brain-derived neurotrophic factor (BDNF) on the noradrenergic axon terminals in the frontal cortex of F344 rats. We continuously infused BDNF into the frontal cortex of young (6-months-old), middle-aged (13-months-old), or aged (25-months-old) rats. Exogenous BDNF infusion caused a marked increase in the density of noradrenergic axons in the aged brain, but no trophic action of BDNF was observed in the young and middle-aged brain. Neutralization of endogenous BDNF with a specific function-blocking antibody to BDNF led to a reduction in noradrenergic axons in the frontal cortex of 19-month-old rats. The present results suggest that BDNF is not involved in the augmentation of noradrenergic innervations in the aging brain, but it is necessary for the maintenance of noradrenergic innervations in the aged brain.

Age-dependent changes in noradrenergic innervations of the frontal cortex in F344 rats

Noradrenergic innervations of the frontal cortex with advancing age (9, 13 and 25 months) in male F344 rats were quantified by immunohistochemistry for dopamine-beta-hydroxylase (DBH), which is a marker enzyme for noradrenergic axons. The density of DBH-positive axons, varicosities (swellings along an axon from which noradrenaline is released), and the number of varicosities per unit length of axon were measured in the frontal cortex. We found that the density of axons and varicosities significantly decreased at an earlier stage of aging (9-13 months), but not at a later stage (13-25 months). On the other hand, the number of varicosities per unit length of axon did not change with age. The result shows that the density of varicosities, which represent the synapses of noradrenergic neurons, decrease in the frontal cortex in the early aging process.

Changes in brain tryptophan metabolism elicited by ageing, social environment, and psychological stress in mice

The kynurenine (KYN) pathway, which is initiated by indoleamine 2,3-dioxygenase (IDO), is a tryptophan (TRP) metabolic pathway. It shares TRP with the serotonin (5-hydroxytryptamine, 5-HT) pathway. In major depression, activation of the KYN pathway may deplete 5-HT. In the present study we investigated the influence of various risk factors for depression, such as ageing, social isolation and psychological stress, on TRP metabolism. Male ICR mice (postnatal day, PND, 21) were divided into two housing conditions, isolation and group housing, reared for 4 weeks (young adult) or 5 months (adult) and exposed to novelty stress. We measured TRP, KYN and 5-HT contents in the prefrontal cortex, hippocampus, amygdala and dorsal raphe nuclei to investigate the balance between the KYN and 5-HT pathways. Ageing decreased TRP and KYN and increased 5-HT. Thus, ageing shifted the balance to the latter. In the younger group, social isolation decreased TRP and KYN and increased the 5-HT/TRP ratio, whereas novelty stress increased TRP and KYN and decreased the 5-HT/TRP ratio. Thus, social isolation shifted the balance to the latter, whereas novelty stress shifted it to the former. In the older group, these effects were restricted to specific brain regions. Ageing and social isolation counteracted novelty stress effects on TRP metabolism.

Involvement of neurotrophic factors in aging of noradrenergic innervations in hippocampus and frontal cortex

In the present study, we investigated the age-dependent changes in the axon terminals of the locus coeruleus (LC) neurons in the frontal cortex and hippocampus, in which a high degree of axonal branching in the middle-aged brain was suggested to occur in our previous electrophysiological study. We used 6-, 13- and 25-month-old male F344/N rats, and performed Western blot analysis of the norepinephrine transporter (NET), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). NET expression level increased in the 13-month-old hippocampus, but was not altered by aging in the frontal cortex. BDNF expression level increased in the hippocampus, but did not change with age in the frontal cortex. On the other hand, GDNF expression level was increased with age in the frontal cortex, but was not in the hippocampus. These results suggest that the LC noradrenergic innervations may be locally regulated by different neurotrophic factors that exert their trophic actions at different target sites.

Changes in cortical noradrenergic axon terminals of locus coeruleus neurons in aged F344 rats

The noradrenergic innervations and noradrenaline contents of the frontal cortex in two age groups (9 and 25 months) of male F344 rats have been quantified by electrophysiological and biochemical methods. In the electrophysiological study, the percentage of locus coeruleus (LC) neurons activated antidromically from the frontal cortex decreased with age. In contrast, the percentage of LC neurons showing multiple antidromic latencies, which suggests axonal branching of individual LC neurons, increased markedly between 9 and 25 months in the frontal cortex. In the biochemical study, we found no significant difference in noradrenaline levels in the cortical terminal fields of LC neurons during aging. These results suggest that LC neurons give rise to axonal branches to retain noradrenaline levels in their target fields in the aged brain. Our findings show that LC neurons preserve a strong capability for remodeling their axon terminals even in the aged brain.

Shifting the balance of brain tryptophan metabolism elicited by isolation housing and systemic administration of lipopolysaccharide in mice.

The kynurenine (KYN) pathway, which is initiated by indoleamine 2,3-dioxygenase, is a key tryptophan (TRP) metabolic pathway. It shares TRP mainly with the serotonin (5-HT) pathway. Activation of the KYN pathway by stimulation of the inflammatory response system (IRS) is known to induce depressive symptoms. Thus, we considered that shifting the balance between the KYN and 5-HT systems in the brain to the KYN pathway closely relate to the etiology of depression. In the present study, we investigated the influence of environmental risk factors for depression, such as social isolation and activation of the IRS, on brain TRP metabolism. Male ICR mice (postnatal day 21) were divided into two housing conditions, isolation and group housing, reared for 4 weeks, and then given an intraperitoneal injection of lipopolysaccharide (LPS). We measured the TRP, KYN, and 5-HT levels in the prefrontal cortex, hippocampus, amygdala, and dorsal raphe nuclei. Isolation housing decreased the KYN/5-HT ratio in the amygdala and dorsal raphe nuclei. LPS increased the KYN/5-HT ratio in all regions except the dorsal raphe nuclei. Thus, isolation housing shifted the balance between the KYN and 5-HT pathways to the 5-HT pathway, whereas systemic administration of LPS shifted it to the KYN pathway.

Age-related changes in the release and uptake activity of presynaptic axon terminals of rat locus coeruleus neurons

Age-related changes in the release and uptake activity of presynaptic axon terminals of rat locus coeruleus (LC) noradrenergic neurons were studied in the frontal cortex using an extracellular single unit recording technique in vivo. Clonidine, a selective alpha(2) adrenergic agonist, and nisoxetine, a selective noradrenaline uptake inhibitor, were infused locally into the frontal cortex to examine the effects of these drugs on release and uptake activities of the axon terminals of LC neurons. Although the infusion of clonidine produced a marked suppression of release, the effect did not change with age. Infusion of nisoxetine caused an inhibition of uptake, but the effect was attenuated in aged rats. These results suggest that the release activity mediated by the presynaptic autoreceptor did not change with age, but the uptake activity mediated by the NA transporter declined with age in the axon terminals of LC neurons.

Effects of BDNF infusion on the axon terminals of locus coeruleus neurons of aging rats

Using in vivo electrophysiological techniques and continuous local infusion methods, we examined the effects of brain-derived neurotrophic factor (BDNF) and its specific antibody (anti-BDNF) on the noradrenergic axon terminals of the locus coeruleus (LC) neurons in the frontal cortex of aging rats. Recently, we observed that LC neurons with multiple-threshold antidromic responses (multi-threshold LC neurons) increased critically between 15 and 17 months of age. To examine whether the BDNF is involved in this change occurred in the aging brain, we continuously infused BDNF into the frontal cortex for 14 days. Exogenous BDNF produced a marked increase in the multi-threshold LC neurons in the 13-month-old brain, accompanied with a decrease in threshold current. However, no morphological change in the noradrenergic axons was observed in the BDNF-infused cortex. In contrast, infusion of anti-BDNF led to a dose-dependent reduction of the multi-threshold LC neurons in the 19-month-old brain, accompanied with an increase in threshold current. These findings suggest that BDNF may contribute to functional changes in the presynaptic axon terminals of LC neurons in the aging brain.

Changes in electrophysiological properties of axon terminals of locus coeruleus neurons with age in F344 rat

Age-dependent changes in electrophysiological properties of locus coeruleus (LC) neurons were studied in urethane anesthetized male F344 rats aged 8, 17 and 22 months. These properties, such as spontaneous firing rate, conduction time, and threshold currents were measured for individual LC neurons antidromically activated from terminals in the frontal cortex. We found no change in the electrophysiological properties with age except for an increased number of low-threshold LC neurons in 22-month-old rats compared to that of 8-month-old rats. These results suggested that the electrophysiological properties of cortical axon terminals of LC neurons changed with age.