Ayuka Ehara

Application of Fluoro-Jade C in Acute and Chronic Neurodegeneration Models: Utilities and Staining Differences

Recent neuropathological studies have shown that Fluoro-Jade C (FJC), an anionic fluorescent dye, is a good marker of degenerating neurons. However, those studies have mostly examined acute rather than chronic models of neurodegeneration. We therefore compared FJC staining using the intrastriatal 6-hydroxydopamine (6-OHDA)-injected rat as an acute model and the zitter rat as a chronic model, as both show dopaminergic (DA) neurodegeneration. In the 6-OHDA-injected rat, FJC-positive neurons were found in the substantia nigra pars compacta (SNc) before the loss of tyrosine hydroxylase (TH)-positive DA neurons. In the zitter rat, FJC-labeled fibers were first detected at 1 month old (1M) and were considerably increased in the striatum at 4M, whereas FJC-labeled cell bodies were found at 4M, but not at 1M in the SNc. Furthermore, FJC-labeled neurons of the zitter rat showed TH-immunoreactivity in fibers, but little in cell bodies, while those from the 6-OHDA-injected rat showed TH-immunoreactivity even in the cell bodies. These results demonstrate that FJC is a useful tool for detecting chronically degenerating neurons, and suggest that intracellular substances bound to FJC may accumulate in the cell bodies from fibers at a slower rate in the chronic model than in the acute model.

Neuroprotective effects of melatonin on the nigrostriatal dopamine system in the zitter rat

Melatonin has ubiquitous actions, both as a direct free-radical scavenger and as an indirect anti-oxidant. The present study examined in vivo neuroprotective effects of melatonin on the nigrostriatal dopaminergic system in zitter (zi/zi) rat, which displays abnormal metabolism of superoxide leading to age-related degeneration of the dopaminergic system. For up to 9 months after weaning, zi/zi rats had ad libitum access to drinking water containing melatonin. Chronic treatment with melatonin attenuated the decreases of dopamine and its metabolite in zi/zi rat caudate-putamen (CPU). Immunohistochemistry for tyrosine hydroxylase (TH) was consistent with neurochemical data in the CPU and demonstrated substantial sparing of the reduction of TH-immunoreactive neurons in the substantia nigra pars compacta. Quantitative polymerase chain reaction (qPCR) was performed to analyze mRNA expressions of pro-inflammatory cytokines (IL-1β and TNF-α) and the anti-oxidant enzymes (catalase (CAT), superoxide dismutase (SOD) 1 and 2, and glutathione peroxidase (GPx1)) in the striatum and midbrain in order to examine the neuroprotective effect of melatonin. IL-1β and TNF-α mRNA expressions were significantly increased in both areas of 3-month-old zi/zi rats, whereas there was a significant decrease in CAT mRNA expression in the striatum of 6-month-old zi/zi rat as compared to age-matched controls. With the exception of the high TNF-α mRNA expression in 3-month-old zi/zi midbrains, chronic treatment of melatonin attenuated expressional changes of IL-1β, CAT, SOD1, and GPx1. These results suggest that besides its direct scavenger effects, chronic melatonin treatment provides a neuroprotective effect against dopaminergic degeneration by suppressing pro-inflammatory cytokines and up-regulating anti-oxidant enzyme expression.

Effects of environmental enrichment on the activity of the amygdala in micrencephalic rats exposed to a novel open field: Fos activation in micrencephalic rats

Environmental enrichment (EE) mediates recovery from sensory, motor, and cognitive deficits and emotional abnormalities. In the present study, we examined the effects of EE on locomotor activity and neuronal activity in the amygdala in control and methylazoxymethanol acetate (MAM)‐induced micrencephalic rats after challenge in a novel open field. Control rats housed in EE (CR) showed reduced locomotor activity compared to rats housed in a conventional cage (CC), whereas hyperactivity was seen in MAM rats housed in a conventional cage (MC) and in MAM rats housed in EE (MR).

The Role of Attractin in Neurodegeneration Caused by Oxidative Stress

Oxidative stress is linked to dopaminergic (DA) neurodegeneration in Parkinson’s disease. Our laboratory reported slowly progressive DA neurodegeneration in the zitter (zi) rat, which is Attractin (Atrn) deficient. However, little is known about the function of Atrn in the central nervous system (CNS). Thus, we investigated whether DA neurodegeneration in the zi rat was induced by oxidative stress, and how Atrn affects oxidative stress. First, we summarize our previous in vivo data, which revealed suppression of DA neurodegeneration using antioxidants (vitamin E and melatonin) in zi rats. Second, our current ex vivo and in vitro studies are introduced. Using primary neuronal cultures of zi mesencephalon as a model of Atrn-deficient neurons or AtrnGFP-overexpressing HEK293 cells, accumulation of reactive oxygen species (ROS) in mitochondria and cell viability was examined under oxidative stress. Atrn-deficient neurons accumulated excess ROS in mitochondria, resulting in neurodegeneration, whereas Atrn-overexpressing cells showed suppression of ROS accumulation under oxidative stress. These results showed that Atrn plays a suppressive role against ROS and that the loss of Atrn function induced excess ROS accumulation and led to DA neurodegeneration. This is the first report to show that Atrn directly modulates mitochondrial ROS accumulation in the CNS.

Tyrosine hydroxylase afferents to the interstitial nucleus of the posterior limb of the anterior commissure are neurochemically distinct from those projecting to neighboring nuclei

The interstitial nucleus of the posterior limb of the anterior commissure (IPAC) is exclusively innervated by tyrosine hydroxylase-immunoreactive (TH-IR) fibers as observed in the other nuclei of the rat forebrain such as the striatum and nucleus accumbens. Distinguishing TH-IR afferents to the IPAC from those projecting to neighboring nuclei has been difficult. However, we previously showed that the TH-IR fibers projecting to the IPAC were invulnerable to neurodegeneration in zitter mutant rats, whereas almost all TH-IR afferents fibers to the dorsolateral striatum were lost, indicating that these two groups of TH-IR afferents have distinct neurochemical properties. Here, to explore this observation further, we injected Fluorogold (FG) retrograde tracers to identify neurons projecting to the IPAC or dorsal striatum. We also determined the distribution of attractin mRNA and protein, causative factors for the pathological phenotypes of zitter mutant rats, within the normal rat midbrain. In rats injected with FG into the dorsal striatum, we detected many FG-positive neurons in the ventral aspect of the substantia nigra pars compacta (SNC). In contrast, many FG-positive neurons were observed in the dorsal aspect of the SNC of rats injected with FG into the IPAC. Immunohistochemistry and in situ hybridization studies of intact animals revealed that both attractin mRNA and protein were expressed at higher levels in the ventral aspect of the SNC, whereas both attractin mRNA and protein were expressed at lower levels in the dorsal aspect of the SNC. Taken together, these results indicate that TH-IR afferents to the IPAC have distinct neurochemical properties from those to the striatum and may account for the differential vulnerability to neurodegeneration observed in zitter mutant rats.

Temporally distinct expression of vesicular glutamate transporters 1 and 2 during embryonic development of the rat olfactory system.

To study the development of glutamatergic neurons during the main olfactory bulb morphogenesis in rats, we examined the expression of vesicular glutamate transporters 1 (VGLUT1) and 2 (VGLUT2). On VGLUT1, expressions of mRNA and immunoreactivity were first detected in the mitral cell layer on embryonic day (E) 17.5 and E18.5, respectively, and persisted in the E20.5 olfactory bulb. Much earlier (on E12.5) than VGLUT1, expressions of VGLUT2 mRNA and/or immunoreactivity were found in the olfactory epithelium, migratory cells and telencephalon. On E14.5, the mRNA expression was also observed in the prospective bulbar region and vomeronasal organ, while immunoreactivity existed in migratory cells and growing fibers. Some fibers were observed in the deep telencephalic wall. From E16.5 onward, mRNA expression became gradually detectable in cells of the mitral cell layer with development. On E17.5, immunoreactivity was first found in fibers of the developing olfactory bulb and in some immature mitral cells from E18.5 to E20.5. The present study clarifies the expression of VGLUT2 precedent to VGLUT1 during olfactory bulb morphogenesis, suggesting differential contribution of the two VGLUT subtypes to glutamate-mediated embryonic events.

Role of neuronal nitric oxide synthase in slowly progressive dopaminergic neurodegeneration in the Zitter rat

Neuronal nitric oxide synthase (nNOS) is involved in nigrostriatal dopaminergic (DA) neurodegeneration. However, little is known about the distribution patterns and functions of nNOS in slowly progressive DA neurodegeneration. Here we describe the spatiotemporal change in nNOS expression over the course of neurodegeneration and the effect of short- or long-term treatment with the nNOS inhibitor, 7-nitroindazole (7-NI), in zitter (zi/zi) rats. In the substantia nigra pars compacta (SNc), nNOS expression was significantly increased with progression of neurodegeneration. nNOS-immunoreactive (ir) cells were in the vicinity of tyrosine hydroxylase-ir (TH-ir) DA neurons, and some of these cells were also positive for calbindin. nNOS in the caudate-putamen (CPu) showed little difference during progression of neurodegeneration. However, immunoelectron microscopic analysis revealed that abundant TH-ir fibers in the CPu were degenerated due to compression by vacuoles that contained swollen neuronal and glial elements. Additionally, lipid peroxidation as a marker of membrane oxidation was significantly increased in zi/zi rats. Short-term 7-NI treatment attenuated the increase in lipid peroxidation and inhibited the vacuolation in the CPu. Moreover, long-term 7-NI treatment significantly protected TH-ir neurons in the SNc, and TH-ir fibers and DA contents in the CPu. These results show that nNOS exacerbates slowly progressive DA neurodegeneration, and the neuroprotective effects of 7-NI may result from suppression of membrane oxidation that causes abnormal membrane structures in zi/zi rats.

Repeated mild shaking of neonates induces transient cerebral microhemorrhages and anxiety-related behavior in adult rats

Growing evidence suggests that neonatal cerebral microhemorrhages (MHs) are implicated in neuropsychiatric diseases in adults. Although animal studies have identified the progression of the underlying mechanisms of MHs, few studies have investigated the histopathology and behavioral outcomes. In this study, we created an experimental rat model of MHs using a new experimental device for repeated mild shaking brain injury (SBI) in the neonatal period and examined temporal changes in MHs using susceptibility weighted imaging (SWI) and iron histochemistry. SWI demonstrated transient MHs in the gray matter of the cerebral cortex and hippocampus in injured rats. Iron histochemical staining demonstrated leakage of iron and iron-positive cells surrounding MHs. This staining pattern lasted for a long time and continued after disappearance of hemorrhagic signals on SWI. These data suggested the presence of iron-associated gray matter injury after MHs. In the open field test, these injured rats showed anxiety-related behavior as adults. This model may be useful for exploring the underlying mechanisms of changes that occur after MHs and the behavioral outcomes of repeated mild SBI in early development.

A rhythmic change of vesicular glutamate transporter (VGLUT) 2 expression in the rat pineal gland.

The pineal gland secretes melatonin under circadian control via nocturnal noradrenergic stimulation, and expresses vesicular glutamate transporter (VGLUT) 1, VGLUT2 and a VGLUT1 splice variant (VGLUT1v). Although we previously reported that VGLUT2 mRNA level of rat pineal gland at postnatal day 21 is higher in the nighttime than in daytime, questions remained as to the time of postnatal onset of this phenomenon and a 24-h change in the mRNA or protein level at postnatal days. The day-night difference in VGLUT2 mRNA level was evident 14 days after birth. In the adult, VGLUT2 mRNA and protein levels increased in the dark phase, with the protein level showing a 6-h delay. The nocturnal elevation in VGLUT2 mRNA level diminished under the constant light condition but persisted under the constant dark condition. The present data suggest that VGLUT2 in the rat pineal gland is involved in some nocturnal glutamatergic function.