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The circling mouse serves as a hearing loss model. It has spontaneous tmie gene mutations that cause hair cell and cochlear degeneration. However, little is known about the role of the tmie gene in superior olivary complex (SOC) regions, in which sound information from the two ears is integrated and primarily relayed to the nuclei of the lateral lemniscus and inferior colliculus. Several studies have reported that abnormal calcium (Ca2+) homeostasis is associated with the pathology of hearing loss. This study investigated the distribution of Ca2+-binding proteins (CaBPs), such as calbindin D28k, parvalbumin, and calretinin, in the SOC of the circling mouse on postnatal day 16. A comparison of wild-type (+/+), heterozygous (+/cir), and homozygous (cir/cir) mice showed that CaBP immunoreactivity was significantly decreased in the auditory nucleus of the SOC of homozygous (cir/cir) mice. A decline in the CaBPs level in the SOC may be the result of hearing loss through hair cell and cochlear degeneration following tmie gene mutation.

Circling mice is a mutant model of spontaneous deafness exhibiting degenerated spiral ganglion cells in the cochlea and loss of organ of Corti. The balance between glycinergic inhibition and glutamatergic excitation in the lateral superior olive (LSO) is essential for the detection of inte-raural level differences. Long term weakening of glycinergic synaptic inhibition in the LSO may lead to the downregulation of synaptic release of glycine in dorsal cochlear nucleus and downregulation of postsynaptic glycine receptor (GlyR) activity in the LSO, which may contribute to hearing loss. The present study utilized an immunohistochemical method to assess changes in GlyR immunoreactivity (IR) and the cell number in the superior olivary complex (SOC) of heterozygote (+/cir) and homozygote (cir/cir) circling mice. A significant decrease in the IR was observed in all nuclei of the SOC of homozygous mice. Loss of GlyR immunoreactive cells and a decrement in cell size was also observed in the homozygotes. A decrease in the GlyR IR in the neurons and neuropils, cell number and size of the cir/cir, may lead to profound changes in inhibitory transmission and the functional properties in the SOC nuclei. Therefore, the functional loss of inhibitory neurotransmitters in the brainstem may result in deafness of adult cir/cir mice.

Raising health concerns about the biological effects from radiofrequency exposure, even with conflicting results, has prompted calls for formulation of a guideline of the biological safety level. Given the close proximity between a mobile phone and the ear, it has been suggested that the central auditory system may be detrimentally influenced by radiofrequency exposure. In the auditory system, neurotrophins are important in the regulation of neuron survival, especially mammalian cochlear neurons. Neurotrophic factors like brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) present in the auditory system are responsible for the maintenance of auditory neurons. BDNF and GDNF may protect against acoustic trauma and prevent from hearing defect. The present study applied radiofrequency at a specific absorption rate (SAR) of 1.6W/kg (E1.6) or 0W/kg group to determine the distribution of BDNF and GDNF in the nuclei of superior olivary complex (SOC). In the E1.6 group, significant decrements of BDNF immunoreactivity (IR) were noted in the lateral superior olive, medial superior olive, superior paraolivary nucleus and medial nucleus of the trapezoid body. GDNF IR was also significantly decreased (p

Calcineurin (CaN)-mediated excitotoxicity impairs γ-aminobutyric acid (GABA) transmission and induces neuronal apoptosis. Ca(2+)-dependent K(+)-Cl(-) cotransporter 2 (KCC2) participates in GABAergic inhibitory transmission. However, the mechanism by which CaN mediates GABA receptor-mediated KCC2 in seizures is not fully understood. In the present study, we investigated the altered expression of KCC2 and the effects of the CaN inhibitor FK506 on KCC2 expression in the mouse hippocampus following kainic acid (KA) treatment. FK506 was injected twice 24 h and 30 min before KA treatment and then mice were treated with KA and killed 2 days later. FK506 had anticonvulsant effect on KA-induced seizure activities. CaN cleavage was evident in the hippocampus 24 h after KA treatment. FK506 pretreatment blocked the truncation of CaN in the KA-treated hippocampus. Cresyl violet and TUNEL staining showed that FK506 prevented KA-induced hippocampal cell death. In particular, Western blot analysis showed that KCC2 expression was time dependent, with a peak at 6 h and a return to decreased levels at 48 h, whereas FK506 pretreatment inhibited the KA-induced decrease in KCC2 expression in the hippocampus. Immunofluorescence showed that FK506 pretreatment protected the loss of inhibitory GABAergic KCC2-expressing neurons following KA treatment. Taken together, these results provide evidence that altered KCC2 expression may be associated with Ca(2+)-mediated seizure activity and indicate that neuron-specific KCC2 may be involved in neuroprotection after seizures.

Exponential rise in the use of mobile communication devices has generated health concerns due to radiofrequency (RF) exposure due to its close proximity to the head. Calcium binding proteins like calretinin regulate the levels of calcium (Ca(2+)) which plays an important role in biological systems. Ginseng is known for maintaining equilibrium in the human body and may play a beneficial radioprotectant role against electromagnetic field (EMF) exposure. In the present study, we evaluated the radioprotective effects of red ginseng (RG) extract in a mouse model. Calretinin (CR) expression was measured using a free-floating immunohistochemical method in the hippocampus of mice after 835 MHz EMF exposure for 5 h/d for 5 d at specific absorption rate=1.6 W/kg for the different experimental groups. The control animals were treated with NaCl while the experimental animals received 10 mg/kg ginseng, or 30 mg/kg; EMF exposed mice were also treated with NaCl, 10 mg/kg ginseng (E10), or 30 mg/kg (E30). Decreases in CR immunoreactivity (IR) along with loss of CA1 and CA3 interneurons and infragranular cells were observed in the ENaCl group while such losses were not observed in the E10 and E30 groups. CR IR significantly increased in the RG-treated group compared to control and EMF-exposed groups treated with NaCl. The study demonstrates that RG extract can serve as a radioprotective agent that maintains Ca(2+) homeostasis and prevents neuronal loss in the brain hippocampal region caused by RF exposure.

Objectives. We tested the possibility of differential expression and function of the potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) of heterozygous (+/cir) or homozygous (cir/cir) mice.Methods. Mice pups aged from postnatal (P) day 9 to 16 were used. Tails from mice were cut for DNA typing. For Immunohistochemical analysis, rabbit polyclonal anti-KCC2 or rabbit polyclonal anti-NKCC1 was used and the density of immunolabelings was evaluated using the NTH image program. For functional analysis, whole cell voltage clamp technique was used in brain stem slices and the changes of reversal potentials were evaluated at various membrane potentials.Results. Immunohistochemical analysis revealed both KCC2 and NKCC1 immunoreactivities were more prominent in heterozygous (+/cir) than homozygous (cir/cir) mice on P day 16. In P9-P12 heterozygous (+/cir) mice, the reversal potential (E(gly)) of glycine-induced currents was shifted to a more negative potential by 50 mu M bumetanide, a known NKCC1 blocker, and the negatively shifted EA, was restored by additional application of 1 mM furosemide, a KCC2 blocker (-58.9 +/- 2.6 mV to -66.0 +/- 1.5 mV [bumetanide], -66.0 1.5 mV to -59.8 +/- 2.8 mV [furosemide+bumetanide], n=11). However, only bumetanide was weakly, but significantly effective (-60.1 +/- 2.9 mV to -62.7 +/- 2.6 mV [bumetanide], -62.7 +/- 2.6 mV to -62.1 +/- 2.5 mV [furosernide+bumetanide], n=7) in P9-P12 homozygous (cir/cir) mice.Conclusion. The less prominent immunoreactivities and weak or absent responses to bumetanide or furosemide suggest impaired function or delayed development of both transporters in homozygous (cir/cir) mice.

Worldwide expansion of mobile phones and electromagnetic field (EMF) exposure has raised question of their possible biological effects on the brain and nervous system. Radiofrequency (RF) radiation might alter intracellular signaling pathways through changes in calcium (Ca(2+)) permeability across cell membranes. Changes in the expression of calcium binding proteins (CaBP) like calbindin D28-k (CB) and calretinin (CR) could indicate impaired Ca(2+)homeostasis due to EMF exposure. CB and CR expression were measured with immunohistochemistry in the hippocampus of mice after EMF exposure at 835 MHz for different exposure times and absorption rates, 1 h/day for 5 days at a specific absorption rate (SAR)=1.6 W/kg, 1 h/day for 5 days at SAR=4.0 W/kg, 5 h/day for 1 day at SAR=1.6 W/kg, 5 h/day for 1 day at SAR=4.0 W/kg, daily exposure for 1 month at SAR=1.6 W/kg. Body weights did not change significantly. CB immunoreactivity (IR) displayed moderate staining of cells in the cornu ammonis (CA) areas and prominently stained granule cells. CR IR revealed prominently stained pyramidal cells with dendrites running perpendicularly in the CA area. Exposure for 1 month produced almost complete loss of pyramidal cells in the CA1 area. CaBP differences could cause changes in cellular Ca(2+)levels, which could have deleterious effect on normal hippocampal functions concerned with neuronal connectivity and integration.

NADPH oxidase is multi-component enzyme, which comprises the cytosolic proteins p40Phox, p47Phox, and p67Phox and the two membrane proteins, gp91Phox and p22Phox, and which is well characterized in phagocytic cells. NADPH oxidase is a primary source of reactive oxygen species (ROS), and recent studies indicate that free radicals and ROS might be causative factors of several brain degenerative diseases and dysfunctions. However, though previous studies have shown the presence of NADPH oxidase subunits in cell culture and mouse brain, they have not provided detailed high power resolution data. Therefore, we investigated the distributions of the p47Phox and gp91Phox subunits in rat brain using immunohistochemical approach. Cortex, hippocampus, and Purkinje cells of cerebellum were prominently stained by p47Phox and gp91Phox antibodies. As compared with the distributions of p47Phox, gp91Phox in mouse, some differences in the rat brain were observed in the hippocampus, thalamus, amygdala, reticular nucleus, and basal ganglia. Additionally, at the cellular level, most p47Phox immunoreactivity was largely confined to cell bodies and proximal portions of the dendritic tree. Taken together, the widespread observed distributions of p47Phox and gp91Phox subunits indicate that they are probably needed to maintain normal brain function.

In the present study, we performed immunohistochemical techniques to investigate the changes in ubiquitin expression in the central nervous system of the transgenic mice expressing a human superoxide dismutase 1 mutation (SOD1)G93A. Sections of brains from control mice showed virtually no immunostaining for ubiquitin, whereas sections from SOD1G93A transgenic mice contained numerous granular or linear deposits of ubiquitin. A high density of the processes containing ubiquitin was detected all around the gray matter of the spinal cord of the mutant transgenic mice. Ubiquitin immunoreactivity was also detected in the cerebellum, brainstem and midbrain of transgenic mice. The first demonstration of the distribution of ubiquitin in the whole brains of the transgenic mice may provide clues for understanding the neuronal degeneration mechanism in amyotrophic lateral sclerosis and other neurodegenerative diseases.

We used the SOD1(G93A) transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate whether alpha-synuclein is involved in the pathogenesis of ALS. In the spinal cord of transgenic mice, immunohistochemistry showed intense staining of alpha-synuclein mainly in the anterior horn. In the hippocampus of transgenic mice, differential increases in the staining density of alpha-synuclein were observed. In the cerebellar cortex of transgenic mice, the prominent immunostaining of alpha-synuclein was found in the molecular and granular layers. The present study provides the first in vivo evidence that alpha-synuclein immunoreactivity was increased in the central nervous system of SOD(G93A) transgenic mice, suggesting that alpha-synuclein might play an important role in the pathogenesis of ALS. However, the functional implications of these increases require elucidation.

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+ (Na(v)) channel in rat cerebellum by immunohistochemistry. In the cerebellar cortex of aged rats, Na(v)1.1 immunoreactivity in Purkinje cell bodies was highly increased, whereas granule cells showed lower staining intensity. In the cerebellar nuclei of aged rats, Na(v)1.1 and Na(v)1.2 expression was specifically increased in the cerebellar output neurons, which was confirmed by image analysis. The first demonstration of age-related changes in Na(v) channel expression contributes to our understanding of the mechanisms responsible for alteration in synaptic transmission during aging.

Although the role of cAMP-response-element-binding protein (CREB) binding protein (CBP) in the neuroprotective mechanisms has been the focus of many studies, very little is known about the expression or function of CBP in aged brains. We have therefore examined age-related changes in CBP expression in the cerebral cortex and hippocampus with an immunohistochemical technique. In the cerebral cortex, the distribution patterns were not different between adult and aged groups, but the staining intensity of CBP was significantly decreased in aged rats. In the hippocampus, a distinct immunoreactivity pattern was observed in the CA1-3 areas and dentate gyrus. CBP immunoreactivity was significantly deceased in the pyramidal layer of CA1-3 regions in aged hippocampus. In the dentate gyrus of aged rats, significant decreases were also found in the granule cell layer and polymorphic layer. The first demonstration of age-related decreases in CBP expression in the cerebral cortex and hippocampus may provide useful data for investigating the pathogenesis of age-related neurodegenerative diseases and depression.

In the present study, we used immunohistochemistry and Western blot analysis to determine region-specific changes in the distribution of voltage-gated calcium channels (VGCCs) in aged rat cerebellum. Age-dependent changes in the staining intensity of the alpha(1C) and alpha(1D) subunits were prominent in the Purkinje cells, whereas there was no change in the expression of the alpha(1A) and alpha(1B) subunits. In the aged rat, in particular, immunoreactivity for the alpha(1C) subunits were increased in the dendrites as well as in the cell bodies of Purkinje cells. On the other hand, decreases in immunoreactivity for alpha(1A) and alpha(1D) subunits were found in the molecular or granular layers. However, only alpha(1D) subunit immunoreactivity was decreased in the aged cerebellum membrane by Western blot analysis that, while not addressing regional specificity, further confirmed an age-related decrease in alpha(1D) subunit. These age-related changes in alpha(1D) subunit expression might reflect a gradual loss of regulation for L-type Ca(2+) channels in the senescent period. The first demonstration of age-related alterations in VGCC expression may provide useful data for future investigations on aging and neurodegenerative diseases.

The differential expression of specialized voltage-gated potassium (Kv) channel subtypes probably reflects the wide range of functions in the nervous system. In the present study, we investigated the distribution of six Kv1 channel subtypes in the gerbil hippocampus by immunohistochemistry. Immunoreactivities for Kv1.1, Kv1.2, Kv1.4 and Kv1.6 were observed in the pyramidal cells of the CA1-CA3 areas. In addition, many medium- to large-sized interneurons located within stratum pyramidals, stratum oriens, and stratum radiatum of CA1-CA3 were strongly immunoreactive for Kv1.4. Although there was some overlap, our results were quite different from the previous studies described in the rat and mouse hippocampus, in that we observed intense staining mainly in the cell bodies of the pyramidal cells and granule cells. As a whole, the present study has clearly shown Kv1 channel distributions in the gerbil hippocampus, which were variant between species and therefore more or less functionally significant. This study may provide useful data for the future investigations on the pathological conditions such as ischemia and epilepsy.

A role for p53-mediated modulation of neuronal viability has been suggested by the finding that p53 expression is increased in damaged neurons in models of ischemia and epilepsy. P53 gene upregulation precedes apoptosis in many cell types, and a potential role for this molecule in apoptosis of neurons has already been demonstrated in Alzheimer's disease. Recent studies suggest that p53-associated apoptosis may be a common mechanism of cell loss in several important neurodegenerative diseases. In the present study, we examined changes in p53-immunoreactive (IR) neurons in the brains of aged rats for the first time employing immunocytochemical and in situ hybridization methods. P53-IR neurons were found in the CA1 region of hippocampus, septal region and cerebellum in the aged rats, but there was no p53-IR cell in the brains of adult rats. In the hippocampus of the aged rat, p53-IR cells predominated in the stratum oriens and pyramidal layers, while the molecular layer contained relatively few p53-IR cells. The most prominent population of immunoreactive labeling in cerebellar cortex was localised within the cell bodies of Purkinje cells and dendrites in molecular layers. Upregulation of p53 in the Purkinje cells observed in this study suggests that significant loss of Purkinje cells with aging may be regulated with several apoptosis-controlling factors including p53 and oxidative stress mechanism. Further investigations are required to establish whether direct functional relations exist between p53 and the apoptotic neuronal death in normal aging or Alzheimer brains.

In the present study, we performed immunohistochemical studies to investigate the changes of Bcl-2 and Bax in the central nervous system of the transgenic mice expressing a human Cu/Zn SOD mutation. In contrast to the controls, a high density of Bcl-2-IR astrocytes were detected all around the gray matter of the spinal cord of the mutant transgenic mice. Bcl-2-IR astrocytes were also detected in the cerebellum and brainstem of transgenic mice. Specific immunoreactivity for Bax was seen in the spinal cord and brainstem of transgenic mice. Immunostaining for Bax was identified only in neurons and not in glial cells. Our present study demonstrated the distribution of Bcl-2 and Bax in detail using immunohistochemical methods through the central nervous system of the transgenic mice, for the first time.

The differential expression of specialized voltage-gated potassium (Kv) channel subtypes in the nervous system probably reflects the wide range of functions. Although there have been previous reports in the cellular and subcellular localizations of various Kv mRNAs and proteins, the comprehensive study described here is the first in which the expression of six Kv1 channel subunits have been directly compared in the rat basal ganglia. In the present study, we have found that staining patterns of the six Kv1 channel subunits overlap in some areas of the basal ganglia, but each has a unique pattern of expression. It was noted that Kv 1.4 subunit had a strikingly high level of expression in the globus pallidus compared to the caudate–putamen. This distinct distribution formed the clear demarcations between caudate–putamen and globus pallidus. The dot-like staining pattern of Kv1 subunits was observed through the accumbens nucleus. Strong staining for Kv1.4 was observed in the cerebral peduncle, not in the subthalamic nucleus. In the substantia nigra, immunoreactivity for Kv1.4 subunit was prominent in the pars reticulata of the substantia nigra. The staining intensity for Kv1.2 was high in the pars compacta of the substantia nigra. Our immunohistochemical results may support the notion that the formation of heteromultimeric Kv channels possibly represents an important contribution to the generation of Kv channel diversity in the brain, especially in the basal ganglia.

To determine whether the vestibular nuclei are affected by inflammation of temporomandibular joint (TMJ) region, we studied vestibular nucleus neural activity using two experimental groups: (1) normal saline 0.1 cm 3 injection at right TMJ region, (2) 10% formalin 0.1 cm 3 injection at right TMJ region. Neural activity after 24 hours was assessed by immunohistochemical staining with free-floating section at the level of interaural −1.30 mm to −2.00 mm for c-Fos. In inflammation group, formalin injection produced a bilateral increase in c-Fos at vestibular nucleus with ipsilesional side higher activity. In control group, expression of c-Fos protein was also observed in the vestibular nucleus (VN), especially MVN. But stain intensity of Fos-positive neurons was much weaker and mean number of c-Fos positive cells was fewer than inflammation group. This result suggests that there is a close neural connection between TMJ and vestibular nucleus, especially in case of inflammation.

Widespread use of wireless mobile communication has raised concerns of adverse effect to the brain owing to the proximity during use due to the electromagnetic field emitted by mobile phones. Changes in calcium ion concentrations via binding proteins can disturb calcium homeostasis; however, the correlation between calcium-binding protein (CaBP) immunoreactivity (IR) and glial cells has not been determined with different SAR values. Different SAR values [1.6 (E1.6 group) and 4.0 (E4 group) W/kg] were applied to determine the distribution of calbindin D28-k (CB), calretinin (CR), and glial fibrillary acidic protein (GFAP) IR in murine hippocampus. Compared with sham control group, decreased CB and CR IRs, loss of CB and CR immunoreactive cells and increased GFAP IR exhibiting hypertrophic cytoplasmic processes were noted in both experimental groups. E4 group showed a prominent decrement in CB and CR IR than the E1.6 group due to down-regulation of CaBP proteins and neuronal loss. GFAP IR was more prominent in the E4 group than the E1.6 group. Decrement in the CaBPs can affect the calcium-buffering capacity leading to cell death, while increased GFAP IR and changes in astrocyte morphology, may mediate brain injury due to radiofrequency exposure.

Exponential interindividual handling in wireless communication system has raised possible doubts in the biological aspects of radiofrequency (RF) exposure on human brain owing to its close proximity to the mobile phone. In the nervous system, calcium (Ca(2+)) plays a critical role in releasing neurotransmitters, generating action potential and membrane integrity. Alterations in intracellular Ca(2+) concentration trigger aberrant synaptic action or cause neuronal apoptosis, which may exert an influence on the cellular pathology for learning and memory in the hippocampus. Calcium binding proteins like calbindin D28-K (CB) is responsible for the maintaining and controlling Ca(2+) homeostasis. Therefore, in the present study, we investigated the effect of RF exposure on rat hippocampus at 835 MHz with low energy (specific absorption rate: SAR=1.6 W/kg) for 3 months by using both CB and glial fibrillary acidic protein (GFAP) specific antibodies by immunohistochemical method. Decrease in CB immunoreactivity (IR) was noted in exposed (E1.6) group with loss of interneurons and pyramidal cells in CA1 area and loss of granule cells. Also, an overall increase in GFAP IR was observed in the hippocampus of E1.6. By TUNEL assay, apoptotic cells were detected in the CA1, CA3 areas and dentate gyrus of hippocampus, which reflects that chronic RF exposure may affect the cell viability. In addition, the increase of GFAP IR due to RF exposure could be well suited with the feature of reactive astrocytosis, which is an abnormal increase in the number of astrocytes due to the loss of nearby neurons. Chronic RF exposure to the rat brain suggested that the decrease of CB IR accompanying apoptosis and increase of GFAP IR might be morphological parameters in the hippocampus damages.

Sodium-dependent vitamin C transporters (SVCTs) is known to transport the reduced form of ascorbic acid into the cell, whereas the oxidized form of vitamin C (VC) is moved through a facilitative sugar transporter, such as glucose transporter (GLUT). With regard to the distribution of SVCT1 and -2 within the various organs, they were reported to be expressed in different types of cells. Especially in the central nervous system, only SVCT2 mRNA was expressed mainly in neurons and some types of neuroglial cells. However, data on the expression of SVCT proteins in the brain are scant. Therefore, we tried to develop comprehensive data on the distribution of SVCT proteins in adult rat brain by using immunohistochemical techniques for the first time. In our study, SVCT2 immunoreactivities (IRs) were intensely localized in the neurons of cerebral cortex, hippocampus, and Purkinje cells of cerebellum, and much weaker SVCT2 IRs were found in the other brain regions. Judging from double-immunohistochemical data, most of the cells expressing SVCT2 IRs were likely to be neurons or microglia, even though the cells in choroids plexus or ependymal cells around the ventricles also exhibited SVCT2 IRs. Complete mapping of the distribution of SVCT2 IRs was available by using a semiquantitative method. The subcellular localization of SVCT proteins is necessary for understanding the exact role of the protein, so the current overall mapping of SVCT IRs in the rat brain could be the basis for further studies on related subjects.

Niemann-Pick disease type C (NPC) is characterized by progressive neurodegeneration and arises from mutations in the NPC1 gene. Cholesterol has received most attention in the pathogenesis of NPC, but normalizing lipid levels in humans or mouse does not prevent neurodegeneration. In NPC mouse, neuronal degeneration in the cerebellum is the most commonly detected change, and thus previous studies have tended to focus on the cerebellum, especially Purkinje cells. Although numerous peptides have been found in the mammalian central nervous system, little data on neurotransmitters in NPC are available, and information on neurotransmitter system abnormalities could explain the complex and characteristic deficits of NPC. Thus, we performed an immunohistochemical study on NPC mouse cortices to compare cell numbers exhibiting vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and substance P (SP) immunoreactivity. In terms of VIP and NPY-immunoreactive (ir) cell numbers in the cerebral cortex, SP-ir cells were significantly reduced by about 90% in NPC (-/-) versus NPC (+/+) mouse, and were also mildly decreased in frontal and parietal NPC (+/-) versus NPC (+/+) mouse cortex. This study demonstrates for the first time, reduced number of SP-ir cells in the NPC mouse cortex.

Nitric oxide (NO) participates in synaptic plasticity, neuronal development, and apoptosis. The involvement of NO and ionic calcium in synaptic plasticity imply that NO may exert an effect on Ca2+ channels. Therefore, we investigated changes in the expressions of calcium channel subunits (Cav1.2/alpha1C, Cav1.3/alpha(1D), Cav2.1/alpha1A, and Cav2.2/alpha1B) in nNOS knock-out (-/-) (nNOS((-/-))) mouse cerebellum using an immunohistochemical approach. We found that the immunoreactivities of the Cav1.2 and Cav1.3 subunits were reduced in the cell bodies of Purkinje cells in these mice and that the signal of the Cav1.2 subunit in neurons and of the Cav1.3 subunit in the neuropils of nNOS((-/-)) mice cerebellar nuclei were significantly down-regulated. We show, for the first time, that prolonged NO deficiency in the cerebellum may affect calcium channel protein expressions, especially, of the Cav1.2 and Cav1.3 subunits.

In the present study, we used the superoxide dismutase 1(SODI)G93A transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of cAMP-response-element-binding protein (CREB) binding protein (CBP) in the central nervous system of transgenic mice. The distribution of CBP-immunoreactive neurons was not different between control and transgenic mice, whereas CBP-immunoreactive astrocytes were found only in transgenic mice. CBP-immunoreactive astrocytes were detected in the spinal cord, brainstem, midbrain and cerebellar nuclei of transgenic mice. The present study provides the first evidence that CBP immunoreactive astrocytes were observed in the central nervous system of transgenic mice, suggesting that reactive astrocytes may play an important role in the pathogenesis and progress of ALS.

In the present study, we examined age-related changes in 3-nitrotyrosine (NT) and neuronal nitric oxide synthase (nNOS) in rat cerebellum using immunohistochemistry. No immunoreactivity for NT was found in any layers of adult cerebellar cortex. In aged cerebellar cortex, the most prominent labeling of NT was found in the Purkinje cell layers and molecular layers. In aged cerebellar nuclei, NT immunoreactivity was observed in the surrounding neuropil. In aged rat cerebellum, nNOS immunoreactivity was significantly decreased in the molecular layer, while it was slightly increased in the granular layer. Image analysis showed no significant age-related changes in nNOS immunoreactivity in the cerebellar nuclei. In summary, this report has demonstrated that NT increases with age in the cerebellum, and suggests that NO production by the neuronal form of NOS may not be the rate limiting step in NT formation in the aged brain. Further work is needed to examine the mechanisms underlying the increased immunoreactivity for NT, and the functional implications of this increase.

In the present study, we investigated age-related changes in IGF-I receptor localization in the cerebral cortex and hippocampus of Sprague-Dawley rats using immunohistochemistry. In the cerebral cortex of adult rats, weakly stained cells were seen in layers II-III and layer V/VI in several cortical regions. In aged rats, there was a significant increase in IGF-I receptor immunoreactivity in the pyramidal cells in the same cortical regions. In the hippocampus of adult rats, several moderately stained neurons were seen in CA1-3 areas and the dentate gyrus. Levels of IGF-I receptor protein increased substantially with age in the CA3 area of the hippocampus. Our first morphological data concerning the differential regulation of IGF-I receptors in aged cerebral cortex and hippocampus may provide insights into age-related changes in trophic support as well as basic knowledge required for the study of neurodegenerative diseases such as Alzheimer's disease.

In the present study, we used immunohistochemistry to investigate the effects of transient global ischemia on pituitary adenylate cyclase activating polypeptide (PACAP) immunoreactivity in gerbil brain regions. PACAP immunoreactivity had significantly decreased in the pyramidal cells of CA1 subfield susceptible to ischemic insult at 1-4 days after transient global ischemia. On the contrary, PACAP immunoreactivity had not changed in the pyramidal cell bodies of more resistant CA3 subfield after ischemic injury. In the Purkinje cell layer, PACAP immunoreactivity had significantly decreased 1 day following transient ischemia, and had increased 2 days after ischemia. The first demonstration of the postischemic localizations of PACAP should allow us to gain a more fundamental rationale for developing methods of treating ischemic brain damage with neuroprotective peptides such as PACAP.

The present study examined the spatial localizations of voltage-gated K(+) (Kv) channels in the rat brain following transient focal ischemia, using immunohistochemistry. Increased expression of Kv1.2 was obvious in the cerebral cortex, dentate gyrus, amygdala, and hypothalamic areas at 3 days following ischemic insults. There was a significant increase in Kv1.2 immunoreactivity in several cortical regions, including cingulate cortex, infralimbic cortex, dorsal peduncular cortex and piriform cortex. On the contrary, Kv1.2 immunoreactivity had not significantly increased in the hippocampal CA1-3 regions although moderate Kv1.2 immunoreactivity was found in the cell bodies and processes of some neurons. Potentially the first demonstration of spatial changes in Kv1.2 channel expression could provide important molecular basis for altered neuronal excitability after ischemic brain injury.

In the present study, we have investigated the spatial and temporal distribution of voltage-gated calcium channels in the gerbil model of global cerebral ischemia using immunohistochemistry. Distinct localizations of P-type (alpha(1A)), N-type (alpha(1B)), and L-type (alpha(1C) and alpha(1D)) Ca(2+) channels were observed in the hippocampus at days 1-5 after ischemic injury. However, increased expression of N-type Ca(2+) channels was detectable in brain regions vulnerable to ischemia only at days 2 and 3 after ischemic injury. The pyramidal cell bodies of CA1-3 areas and the granule cell bodies of the dentate gyrus were intensely stained at days 2 and 3 following ischemic injury. Transient changes in N-type Ca(2+) channel expression were also observed in the affected cerebral cortex and striatum at days 2 and 3 after ischemic injury. Although the present study has not addressed the multiple mechanisms contributing to the intracellular free Ca(2+) concentration ([Ca(2+)](i)) increase in the ischemic brain, the first demonstration of the transient increase in N-type Ca(2+) channels may prove useful for future investigations.

In the present study, we have examined the expression of voltage-gated calcium channels in a rat model of transient focal ischemia using immunohistochemistry. Increased expression of class C L-type Ca2+ channels was clearly detected in reactive astrocytes in each region of the hippocampus 7 days after ischemic injury. On the contrary, class D L-type Ca2+ channels were not expressed in reactive astrocytes under these conditions. These patterns were also observed in reactive astrocytes in the affected cerebral cortex and fiber tracts. Our study showed the spatial and temporal localization of class C L-type Ca2+ channels in reactive astrocytes in ischemic rat brain, for the first time. The present studies may provide useful data for future investigations to understand the role of Ca2+ channels in reactive astrocytes following ischemia or glutamate toxicity.

We have revealed age-related changes in the expression patterns of Kv1.1 and Kv1.2 in the rat cerebellum for the first time. In the aged rat, immunoreactivity for Kv1.1 was increased in the cell bodies of Purkinje cells, while the staining intensity was significantly decreased in the granule cells. The cell bodies of cerebellar output neurons showed strong Kv1.1 immunoreactivity in the nucleus medialis, interpositus and lateralis of the aged rat. Kv1.2 immunoreactivity was found in some interneurons with their processes in this region of the aged rat. Image analysis demonstrated that immunoreactivities for Kv1.1 and Kv1.2 were increased specifically in the cell bodies of cerebellar output neurons of the aged rat. This study may provide useful data for future investigations on the channels that cause brain diseases and age-related disorders.

Voltage-gated K(+) (Kv) channels are critical for a wide variety of processes, and play an essential role in neurons. In the present study, we have demonstrated a unique pattern of expression of the six Kv1 channel subunits in the rat cerebellum, for the first time. The greatest concentration of Kv1.2 was found in the basket cell axon plexus and terminal regions around the Purkinje cells. Relatively weak immunoreactivity for Kv1.1 was also found in this area. The somatodendritic Purkinje cell areas were intensely stained with anti-Kv1.5 antibodies. In the cerebellar nuclei, the cell bodies of cerebellar output neurons showed strong Kv1.5 and Kv1.6 immunoreactivities in the nucleus medialis, interpositus and lateralis. Interestingly, Kv1.2 immunoreactivity was found in some neurons with their processes. Our immunohistochemical results may support the notion that the formation of heteromultimeric Kv channels possibly represents an important contribution to the generation of Kv channel diversity in the brain, especially in the cerebellum.

Amytrophic lateral sclerosis is characterized by relentlessly progressive degeneration of spinal cord motor neurons. During the disease, the bcl-2 family genes have been reported to alter their expression levels. To understand the meaning of these changes, the expression pattern of Bax in rat spinal cord and its changing pattern during normal aging were investigated by immunohistochemical staining. Spatial expression of the protein showed a diffuse distribution pattern with immunoreactivity more prominent in the anterior hom. With advancement of age, the cell densities of Bax-positive cells tended to increase. The increasing expression of Bax might be positively associated with amyotrophic lateral sclerosis. This work demonstrates for the first time how Bax expression changes in rat spinal cord during normal aging.

In this study, we investigated the distribution of the type I and type II Na+ channels in the gerbil cerebellum by immunohistochemistry. Strong uniform staining for type I was observed in the granular layer, whereas there was little evidence of concentrated labeling in the cell bodies and processes of Purkinje cells. The most intense staining for type II was observed in the cell bodies and dendrites of Purkinje cells, with a strong signal in the molecular layer. This localization study has shown clearly that the type I and type II Na+ channel subunits have differential distribution in the gerbil cerebellum, for the first time. The present study may provide useful data for the future investigations to understand the roles of voltage-gated sodium channels in neurological pathways.