Choong Ik Cha

Distribution of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide receptors (VPAC1, VPAC2, and PAC1 receptor) in the rat brain.

To examine the distributions of VIP/PACAP receptors (VPAC1, VPAC2, and PAC1 receptors) in the brain and to identify the cell types that express these receptors, we performed immunohistochemistry and double immunofluorescence in the rat brain with specific antibodies. The immunohistochemistry revealed that the receptors had distinctive, complementary, and overlapping distribution patterns. High levels of the VPAC1 receptor were expressed in the cerebral cortex, hippocampal formation, deep cerebellar nuclei, thalamus, hypothalamus, and brainstem. The VPAC2 receptors were concentrated in the cerebral cortex, hippocampal formation, amygdalar regions, cerebellar cortex, deep cerebellar nuclei, hypothalamus, and brainstem. On the other hand, the PAC1 receptors had a more restricted distribution pattern in the brain, and high levels of the PAC1 receptors were confined to the cerebellar cortex, deep cerebellar nuclei, epithalamus, hypothalamus, brainstem, and white matter of many brain regions. Also, many fibers expressing the PAC1 receptors were observed in various areas, i.e., the thalamus, hypothalamus, and brainstem. The double immunofluorescence showed that the VIP/PACAP receptors were confined to the neuroglia as well as the neurons. All three types of the VIP/PACAP receptors were expressed in the astrocytes, and the PAC1 receptors were also expressed in the oligodendrocytes. These findings indicate that VIP and PACAP exert their functions through their receptors in specific locations in different combinations. We hope that this first demonstration of the distributions of the VIP/PACAP receptors provides data useful in the investigation of the mechanisms of the many functions of VIP and PACAP in the brain, which require further elucidation. J. Comp. Neurol. 476:388–413, 2004.

Reactive astrocytes express nitric oxide synthase in the spinal cord of transgenic mice expressing a human Cu/Zn SOD mutation

THE distribution of the neuronal isoform of nitric oxide synthase (nNOS) in the spinal cord of transgenic mice expressing a mutated human copper/zinc super-oxide dismutase gene was enhanced when investigated by immunocytochemistry. Immunocytochemistry showed intensely stained NOS-immunoreactive (IR) glial cells with the appearance of astrocytes in the spinal cord and brain stem of transgenic mice, but none were observed at these sites in control mice. Using antisera directed against GFAP, the specific marker for astrocyte, the glial cells were confirmed by immunocytochemistry to be astrocytes. This immunocytochemical evidence suggests that nitric oxide may mediate glutamate neurotoxicity, and this study provides the firs in vivo evidence that nitric oxide may be implicated in the pathologic process of human familial amyotrophic lateral sclerosis.

Immunocytochemical study on the distribution of NOS-immunoreactive neurons in the cerebral cortex of aged rats

NITRIC oxide (NO) involvement has been demonstrated in mechanisms of synaptic plasticity, particularly in hippocampal long-term potentiation, a mechanism that underlies certain forms of learning and memory. Several findings suggest that NO production may be decreased in the aged rats. Changes in the nNOS-containing neurons with ageing were demonstrated by immunocytochemistry. NOS-immunoreactive (IR) cells in aged rats were present in all cortical areas and the hippocampus, and the pattern of distribution was similar to that of the control group. The number of NOS-IR cells in the cerebral cortex was significantly decreased in the aged rats, but the extent of changes was variable in each area, and ranged from mild decrease (< 30 %) to severe decrease (> 50%). Severely decreased areas were the cingulate cortex, parietal cortex area 1, temporal cortex area 1, 2, 3, medial part of occipital cortex area 2, monocular and binocular part of occipital cortex area 1, entorhinal cortex, hippocampus proper, dentate gyrus and subiculum. Morphologically, the number of dendritic branches seemed to be decreased in aged group and the length of dendrites of NOS-IR neurons showed a tendency to shorten. These results indicate the involvement of neuronal system containing NOS in the ageing brain, and provide the first morphological evidence for the loss of NOS neurons in the cerebral cortex of the aged rats by immunocytochemistry.

Caveolin-1 upregulation in senescent neurons alters amyloid precursor protein processing.

Lipid rafts provide a platform for regulating cellular functions and participate in the pathogenesis of several diseases. However, the role of caveolin-1 in this process has not been elucidated definitely in neuron. Thus, this study was performed to examine whether caveolin-1 can regulate amyloid precursor protein (APP) processing in neuronal cells and to identify the molecular mechanisms involved in this regulation. Caveolin-1 is up-regulated in all parts of old rat brain, namely hippocampus, cerebral cortex and in elderly human cerebral cortex. Moreover, detergent-insoluble glycolipid (DIG) fractions indicated that caveolin-1 was co-localized with APP in caveolae-like structures. In DIG fractions, bAPP secretion was up-regulated by caveolin-1 over-expression, which was modulated via protein kinase C (PKC) in neuroblastoma cells. From these results we conclude that caveolin-1 is selectively expressed in senescent neurons and that it induces the processing of APP by β-secretase via PKC downregulation.

Replacement of microglial cells using Clodronate liposome and bone marrow transplantation in the central nervous system of SOD1G93A transgenic mice as an in vivo model of amyotrophic lateral sclerosis

Disease progression of amyotrophic lateral sclerosis (ALS) is partially mediated by the toxic microenvironment established by microglia. In the present study, we used SOD1G93A transgenic mice as an in vivo ALS model and replaced microglia expressing mutant SOD1 (mSOD1) with microglia expressing wild-type SOD1 (w/tSOD1) to modulate the toxic microenvironment. Stereotactic injection of Clodronate liposome, a selective toxin against the monocyte/macrophage system, into the fourth ventricle of the brains of 12-week-old asymptomatic ALS mice reduced the number of microglia effectively in the central nervous system. Subsequent bone marrow transplantation (BMT) with bone marrow cells (BMCs) expressing w/tSOD1 and GFP leads to replacement of the endogenous microglia of the ALS mice with microglia expressing w/tSOD1 and GFP. The expression of mSOD1 in the other neural cells was not influenced by the replacement procedures, and immunological side effects were not observed. The replacement of microglia significantly slowed disease progression and prolonged survival of the ALS mice compared with the ALS mice treated by stereotactic injection of PBS-liposome and BMT with BMCs expressing mSOD1 or w/tSOD1. These results suggest that replacement of microglia would improve the neural cell microenvironment, thereby slowing disease progression. The mechanisms and functional implications of this replacement require further elucidation.

Age-related changes in glycogen synthase kinase 3β (GSK3β) immunoreactivity in the central nervous system of rats

Although glycogen synthase kinase 3beta (GSK3beta) is emerging as a prominent drug target in the treatment of neurodegenerative diseases such as Alzheimers disease (AD) and stroke, very little is known about age-related changes in GSK3beta expression and GSK3beta phosphorylation. Therefore, we examined age-related changes in immunoreactivities for GSK3beta and phosphorylated GSK3beta (pGSK3beta) in the central nervous system. In aged rats, there were significant increases in GSK3beta immunoreactivity in the cell bodies and processes of pyramidal cells in most cortical regions. GSK3beta immunoreactivity was also significantly increased in the pyramidal layer of CA1-3 regions, and the granule cell layer of dentate gyrus. Age-related increases were prominent in lateral septal nuclei, compared to the medial septal nuclei. Interestingly, both GSK3beta and pGSK3beta was increased in the prefrontal cortex, while GSK3beta and pGSK3beta was differentially localized in the cerebellar cortex. The first demonstration of age-related alterations in immunoreactivities for GSK3beta and pGSK3beta in the basal forebrain area and cholinergic projection targets may provide useful data for investigating the pathogenesis of age-related neurodegenerative diseases including AD.

Reactive astrocytes express p53 in the spinal cord of transgenic mice expressing a human Cu/ Zn SOD mutation

In a previous study, we reported increased NOS expression in the astrocytes in the spinal cord of SOD mutant transgenic mice that are used as ALS animal model. Recently, Messmer and Brune suggested that nitric oxide-induced apoptosis is intimately related with p53-dependent signaling pathway, and de la Monte et al. reported increased p53-immunoreactivity in the spinal cord of ALS patients. In the present study, we performed immunocytochemical studies to investigate the changes of p53-immunoreactivity in the brains of the mutant transgenic mice expressing a human Cu/Zn SOD mutation. Immunocytochemistry showed intensely stained p53-IR glial cells with the appearance of astrocytes in all levels of the spinal cord of the mutant transgenic mice, but no p53-IR glial cells were observed in the spinal cord of the control mice. P53-IR astrocytes were also detected in the brain stem of the mutant transgenic mice. In the medulla, they were observed in the medullary reticular formation, hypoglossal nucleus, vestibular nucleus, dorsal motor nucleus of the vagus and nucleus ambiguus. In the pons, their presences were noted in the pontine reticular formation, and trigeminal and facial nuclei. In the midbrain, astrocytes were detected in the mesencephalic reticular formation, red nucleus and periaqueductal gray matter. In the cerebellum, intensely stained p53-IR astrocytes were detected in the intracerebellar nuclei. In contrast to the mutant transgenic mice, no p53-IR astrocytes were detected in the brain stem and spinal cord of the control mice. Further multidisciplinary investigations involving p53-mediated cellular damage and pathogenesis of ALS are needed to clarify the importance of these results.

Enhanced expression of p53 in reactive astrocytes following transient focal ischemia

Abstract The present study used immunohistochemistry to investigate p53 expression in rat brain following transient occlusion of the middle cerebral artery. In the control group, no p53-immunoreactive cells were found in any region of the central nervous system. P53 expression in reactive astrocytes was not obvious in the forebrain one day or three days following ischemic insults. Seven days following ischemic injury, increased expression of p53 was clearly detectable in reactive astrocytes in affected cortical regions, such as forelimb area, hindlimb area, and parietal cortex. At seven days of recirculation, there was also a significant increase in the number of p53-immunoreactive neurons in the cerebral cortex, striatum, and hippocampal CA1-3 regions. Although the present study has not addressed multiple mechanisms contributing to cell death following ischemic injury, the first demonstration of a significant increase in p53 expression in glial cells may prove useful for future investigations of the pathophysiology of ischemia. [Neurol Res 2002; 24: 324-328]

Postnatal development of somatostatin- andneuropeptide y-immunoreactive neurons in rat cerebralcortex : a double-labeling immunohistochemical study

The postnatal development of somatostatin (SOM)‐ and neuropeptide Y(NPY)‐immunoreactive (ir) neurons was examined in rat cerebral cortex, while considering theircoexistence in cortical neurons. Using double immunohistochemical staining for SOM and NPYwith diaminobenzidine and benzidine dihydrochloride as chromogens, we subdividedimmunoreactive cells into double‐labeled SOM/NPY‐, SOM only‐, and NPY only‐ir neurons.SOM/NPY‐ and SOM only‐ir neurons were detectable even at the day of birth, in contrast toNPY only‐ir cells which first appeared in most cortices from week two. The morphologicalfeatures of double‐labeled SOM/NPY neurons differed with those of SOM only‐ and NPYonly‐ir neurons. No apparent changes in the shape and size of single‐labeled neurons occurredwith age ; throughout their postnatal life they were round and ovoid, had a thin rim ofperinuclear cytoplasm, and short processes. However, the features of SOM/NPY‐ir neurons werenot consistent according to postnatal age ; by day P7, these neurons showed immature featuresand they began to show more advanced neuronal characteristics by week P2, when they had alarger and more intensely‐stained cytoplasm. In addition, their processes were longer, thicker andmore complex than at earlier ages. At this age, SOM/NPY‐ir somata were close to their nearmaximum size. From week P4, they became smaller and were lightly labeled. SOM/NPY‐irsomata were larger than SOM only‐ and NPY only‐ir somata at and after two weeks of age. Thepresent results, showing different postnatal maturation patterns such as time of appearance andmorphological features, raise the possibilities that double‐labeled SOM/NPY and single‐labeledimmunoreactive neurons may be different populations regulated by different mechanisms in theirdevelopment, and with different functional properties during development.

Age-related changes in the distribution of Nav1.1 and Nav1.2 in rat cerebellum

Modification of sodium channel availability and behavior is obviously a good candidate for alteration of action potential observed during aging. In the present study, we revealed age-related alterations in the expression of voltage-gated Na+ (Nav) channel in rat cerebellum by immunohistochemistry. In the cerebellar cortex of aged rats, Nav1.1 immunoreactivity in Purkinje cell bodies was highly increased, whereas granule cells showed lower staining intensity. In the cerebellar nuclei of aged rats, Nav1.1 and Nav1.2 expression was specifically increased in the cerebellar output neurons, which was confirmed by image analysis. The first demonstration of age-related changes in Nav channel expression contributes to our understanding of the mechanisms responsible for alteration in synaptic transmission during aging.