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The micronucleus is known to be a biomarker for genomic instability, which is a hallmark of tumors and aging. Normally, micronuclei are produced by segregation errors and mechanical stresses arising from dividing or migrating cells, leading to activation of the innate immune response pathway. Although micronuclei often emerge in damaged tissues, the quantitative procedure for analyzing micronuclei accurately has been problematic. Here, we introduce a novel MATLAB-based program for quantifying micronuclei (CAMDi: calculating automatic micronuclei distinction) in vitro and in vivo. CAMDi is adaptable to various experimental imaging techniques and is useful for obtaining reproducible data. CAMDi enables us to measure the accurate size of micronuclei from the three-dimensional images. Using CAMDi, we revealed a novel link between the emergence of micronuclei and neuroinflammation. We found that inflammatory stimulation does not increase the number of micronuclei in primary neurons. On the other hand, the administration of lipopolysaccharide into mice slightly increases micronuclei formation in neurons of the hippocampus region. These findings demonstrate that neuronal micronuclei formations are induced by an inflammatory response in a non-cell-autonomous manner. We provide a novel tool, CAMDi, to quantify micronuclei and demonstrate that neuronal micronuclei are produced not only by the cell-autonomous process but also by the intercellular communication associated with neuroinflammation in vivo., (© 2021. The Author(s).)

Competing Interests: Declaration of Competing Interest None.

Various mutations have occurred during evolution among orthologs, genes in different species that diverged from a common ancestral gene by speciation. Here, we report the remarkable deterioration of a characteristic mammalian maternal behavior, pup retrieval, in nonmammalized mice, in which the transcription factor Pou3f2 was replaced with the Xenopus ortholog lacking all of the homopolymeric amino acid repeats of mammalian POU3F2. Most of the pups born to the nonmammalized mice died within days after birth, depending on the dam genotype alone. Quantitative immunohistochemical analysis revealed decreases in the rate-limiting enzymes of dopamine and serotonin synthesis in various brain structures. Similar results were obtained in knock-in mice in which all of the homopolymeric amino acid repeats of mammalian POU3F2 were removed. Pup retrieval behavior in mammals is thus strongly related to monoamine neurotransmitter levels via the acquisition of homopolymeric amino acid repeats during mammalian evolution., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

A fast method has been established for the precise measurement and quantification of the dynamics of hyperpolarized (HP) xenon-129 ((129)Xe) in the mouse brain. The key technique is based on repeatedly applying radio frequency (RF) pulses and measuring the decrease of HP (129)Xe magnetization after the brain Xe concentration has reached a steady state due to continuous HP (129)Xe ventilation. The signal decrease of the (129)Xe nuclear magnetic resonance (NMR) signal was well described by a simple theoretical model. The technique made it possible to rapidly evaluate the rate constant α, which is composed of cerebral blood flow (CBF), the partition coefficient of Xe between the tissue and blood (λ(i)), and the longitudinal relaxation time (T(1i)) of HP (129)Xe in the brain tissue, without any effect of depolarization by RF pulses and the dynamics in the lung. The technique enabled the precise determination of α as 0.103 ± 0.018  s(-1) (± SD, n = 5) on healthy mice. To investigate the potential of this method for detecting physiological changes in the brain of a kainic acid (KA) -induced mouse model of epilepsy, an attempt was made to follow the time course of α after KA injection. It was found that the α value changes characteristically with time, reflecting the change in the physiological state of the brain induced by KA injection. By measuring CBF using (1)H MRI and (129)Xe dynamics simultaneously and comparing these results, it was suggested that the reduction of T(1i), in addition to the increase of CBF due to KA-induced epilepsy, are possible causes of the change in (129)Xe dynamics. Thus, the present method would be useful to detect a pathophysiological state in the brain and provide a novel tool for future brain study., (Copyright © 2011 John Wiley & Sons, Ltd.)

The mechanism of radiation-induced diffuse brain injury was investigated in a neonatal rat hemicranial irradiation model using immunohistochemistry. Neonatal Fischer 344 rats received hemicranial irradiation with a single dose of 15 Gy, and appropriate combinations of myelin markers were used to assess the myelin damage at various stages of myelin development. Antibodies against myelin basic protein, 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin oligodendrocyte glycoprotein were used, and the density of the antibody-positive fibers was classified into five categories. Statistical analysis showed significant differences between irradiated and unirradiated hemispheres. The differences decreased and myelination approached normality by postnatal day 70. These results show that myelination in the neonatal rat can recover from the developmental delay caused by a single 15 Gy dose of hemicranial irradiation.

The effect of excercise on brain function was investigated through animal experiments. Exercise leads to increased serum calcium levels, and the calcium is transported to the brain. This in turn enhances brain dopamine synthesis through a calmodulin-dependent system, and increased dopamine levels regulate various brain functions. There are abnormally low levels of dopamine in the neostriatum and nucleus accumbens of epileptic mice (El mice strain) and spontaneously hypertensive rats (SHR). The low dopamine levels in those animals were improved following intracerebroventricular administration of calcium chloride. Dopamine levels and blood pressure in SHR were also normalized by exercise. In epileptic El mice, convulsions normalized dopamine levels and physiologic function. These findings suggest that exercise or convulsions affect brain function through calcium/calmodulin-dependent dopamine synthesis. This leads to the possibility that some symptoms of Parkinson's disease or senile dementia might be improved by exercise.

Here we describe a novel technique for comparative analysis of the distributions of various neurochemicals visualized using multiple immunohistochemistry in the same brain slice. As an example, the distributions of tyrosine hydroxylase, substance P and glutamate decarboxylase in coronal slices of rat brains were compared. Each slice was divided into approximately 220,000-300,000 microareas at 20-microm intervals, and the immunohistochemical intensities of the three substances in each microarea were analyzed independently using a brain mapping analyzer; a microphotometry system previously developed in our laboratory (Sutoo et al., J. Neurosci. Methods, 1998; 85: 161-73). No significant differences between the distribution of each substance were observed in single- and triple-labeled slices. We believe that this method will facilitate the investigation of the functions of the central nervous system and the disorders thereof in various diseases., (Copyright 2002 Elsevier Science B.V.)

Peptidylarginine deiminases (PADs) are a group of posttranslational modification enzymes that convert protein arginine residues to citrulline residues. In the rat cerebrum, the type II PAD is thought to be expressed mainly in glial cells, especially astrocytes, and to become activated early in the neurodegenerative process. To determine whether hyperplastic glial cells express PAD type II, we examined the rat brain after kainic acid-evoked neurodegeneration. Western blot and reverse transcriptase-polymerase chain reaction analyses revealed increased and type II-specific expression of PAD in the brain at 4-7 days after kainate administration. Immunocytochemically, no PAD type II immunoreactivity was observed in the glial fibrillary acidic protein-positive astrocytes, but such immunoreactivity was present coincident with a microglial marker recognized by Bandeiraea simplicifolia isolectin B4 in the damaged regions. These results clearly indicate that PAD type II is specifically and abundantly expressed in activated microglial cells and suppressed in hyperplastic astrocytes following neurodegeneration.

The immunohistochemical distributions of tyrosine hydroxylase (TH), calmodulin (CaM) and calcium/CaM-dependent protein kinase II (CaMKII) in the rat forebrain were compared quantitatively to confirm our previous finding that TH activity and dopamine synthesis in the brain are regulated by a calcium/CaM-dependent system. The same slice was triply stained and the above substances were detected immunohistochemically. Their distributions in the slice were measured using a brain mapping analyzer which is a microphotometry system for the analysis of the distribution of neurochemicals in a large tissue slice. Each coronal section was divided into approximately 250000 to 310000 microareas at 20-microm intervals, and the immunohistochemical fluorescence intensities of the three substances in these microareas were analyzed independently. Quantitative images of the distributions were reconstructed from the data, and the distribution of each substance was investigated. Immunoreactive staining of TH, CaM and CaMKII was observed in almost all areas of the brain, but the intensities varied. Markedly intense TH-, CaM- and CaMKII-like immunoreactivities were distributed in the anterior dorsolateral and posterior areas of the neostriatum, nucleus accumbens and olfactory tubercle. In the previous study, the amount of dopamine was increased by the intracerebroventricular administration of calcium chloride in the neostriatum and nucleus accumbens. Combining these results with those previously reported, it is suggested that TH activity and dopamine synthesis in these regions are regulated by calcium ions via CaM and CaMKII. This method is a powerful technique for quantitative and comparative analysis of the distributions of various neurochemicals in the same slice, and we believe that it will facilitate investigation of the functions of the central nervous system and disorders thereof in various diseases.

Myelin basic proteins (MBPs) are the major protein components of myelin. MBP isoforms are known to have different expression patterns. In order to distinguish the different expression patterns on myelination, we have developed a novel antibody reacting with the four major isoforms of MBPs with molecular masses of 21.5 kDa, 18.5 kDa, 17.0 kDa, and 14.0 kDa. These MBPs were initially separated by acid urea gel and sodium dodecyl sulfate polyacrylamide gel electrophoreses and detected with the luminol reaction. Then the antibody developed was used to determine the relative amounts of MBP isoforms. The MBPs of oligodendrocytes were detected by the enhanced luminol reaction using Renaissance (Dupont NEN, Boston, MA). From the immunological aspect, the MBP monoclonal antibody (Sires et al. [1981] Science 214:87-89) was revealed to recognize MBPs with molecular masses of 21.5 kDa and 18.5 kDa. Furthermore, we found that Ile-166 in the rat 18.5-kDa MBP isomers was replaced by methionine. The 14.0-kDa and 18.5-kDa isoforms of MBP are the most abundant MBP species and comprise more than 70% of the total MBPs in 3.5-and 24-month-old rats. MBPs are expressed during development and the compositions of MBPs in mature (3.5 months old) and aged (24 months old) rats were almost the same. The expression of the 14.0-kDa and 18.5-kDa MBPs occurred earlier in the cerebellum and the spinal cord than in the cerebrum by approximately 1 week. MBPs are also expressed upon oligodendrocyte maturation by interacting with astrocytes. The above results suggest that the regulation of MBP isoforms during development and oligodendrocyte differentiation may indicate the point of occurrence of both the unique patterns of isoform expression and the shift in intracellular localization of MBPs with the maturation of oligodendrocytes., (Copyright 2002 Wiley-Liss, Inc.)

Cognitive dysfunction and histological changes in the brain were investigated following irradiation in 20 Fischer 344 rats aged 6 months treated with whole brain irradiation (WBR) (25 Gy/single dose), and compared with the same number of sham-irradiated rats as controls. Performance of the Morris water maze task and the passive avoidance task were examined one year after WBR. Finally, histological and immunohistochemical examinations using antibodies to myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and neurofilament (NF) were performed of the rat brains. The irradiated rats continued to gain weight 7 months after WBR whereas the control rats stopped gaining weight. Cognitive functions in both the water maze task and the passive avoidance task were lower in the irradiated rats than in the control rats. Brain damage consisting of demyelination only or with necrosis was found mainly in the body of the corpus callosum and the parietal white matter near the corpus callosum in the irradiated rats. Immunohistochemical examination of the brains without necrosis found MBP-positive fibers were markedly decreased in the affected areas by irradiation; NF-positive fibers were moderately decreased and irregularly dispersed in various shapes in the affected areas; and GFAP-positive fibers were increased, with gliosis in those areas. These findings are similar to those in clinically accelerated brain aging in conditions such as Alzheimer's disease, Binswanger's disease, and multiple sclerosis.

The effect of intracerebroventricular (i.c.v.) administration of magnesium on calcium- and dopamine-dependent brain function was investigated behaviorally and biochemically. The duration of ethanol-induced sleeping time in mice was prolonged following i.c.v. administration of calcium chloride (10 micromol/kg) or dopamine (30nmol/mouse); however, it was not affected by magnesium chloride (10 or 40 micromol/kg). The ability of calcium to prolong ethanol-induced sleeping time was inhibited by the administration of magnesium chloride. The brain dopamine level in mice was significantly increased following i.c.v. administration of calcium chloride. Taking into consideration these results and those from previous studies, it is suggested that calcium enhances dopamine synthesis in the brain through a calmodulin-dependent system, and the increase in dopamine level prolongs ethanol-induced sleeping time. However, magnesium inhibits dopamine release. Therefore, magnesium may inhibit calcium-dependent brain function through dopaminergic neurons, and consequently reduce the effect of calcium on ethanol activity.

GABAergic and glutamatergic neuronal systems in adult normal human brains were shown quantitatively and in detail through the distributions of glutamate decarboxylase (GAD) and glutamate dehydrogenase (GDH), respectively. Consecutive coronal sections containing part of the striatum and the substantia nigra were obtained from the right hemisphere of three deceased persons with no history of neurological or psychiatric diseases and were stained immunohistochemically for GAD and GDH. Each stained section was divided into approximately 3 million microareas and the immunohistochemical fluorescence intensity in each area was measured by a human brain mapping analyzer, which is a microphotometry system for analysis of the distribution of neurochemicals in a large tissue slice. In the analyzed brain regions, conspicuously intense GAD-like immunoreactivity was observed in the substantia nigra, globus pallidus, and hypothalamus. GDH was widely and rather evenly distributed in the gray matter compared to GAD, although intense GDH-like immunoreactivity was observed in the lateral geniculate nucleus and substantia nigra. Within the substantia nigra, the globus pallidus, and other regions, characteristic distributions of GAD- and GDH-like immunoreactivity were found. We believe that the analysis of the human brain by this novel technique can help to understand the functional distribution of neuronal systems in the normal human brain and may be able to identify abnormal changes in the diseased human brain. It can also provide basic data to help in the interpretation of functional magnetic resonance imaging or positron emission tomography.

Several lines of evidence have implicated Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II), a multifunctional protein kinase, in the regulation of signal transduction after chronic administration of psychostimulants. CaM-Kinase II activities were decreased in discrete brain regions after a single methamphetamine (METH) injection to rats. Pretreatment with either SCH 23390 (a dopamine D1 receptor antagonist) or NMK-801 (an N-methyl-D-aspartate receptor antagonist) prevented the acute METH-induced decrease in CaM-kinase II activity in the parietal cortex, nucleus accumbens, and substantia nigra/ventral tegmental area (SN/VTA). Striatal CaM-kinase II activity was significantly lower than that of the chronic saline-treated controls after a one-week, but not a four-week, abstinence from chronic administration of METH. A METH challenge after a four-week abstinence period decreased CaM-kinase II activity in rats chronically injected with METH to a greater extent than in rats chronically injected with saline. Western blot analysis revealed that protein amount of CaM-kinase II was not altered after a single METH injection or after chronic METH injections, as compared with saline-treated controls. However, amounts of phosphorylated (Thr286) CaM-kinase II in the parietal cortex, striatum, and SN/VTA were significantly decreased at 3 h after an acute METH injection compared with saline-treated controls. It is suggested that dephosphorylation of CaM-kinase II may contribute to the decreased enzyme activities induced by acute METH administration, and that chronic treatment with METH leads to an enhanced capacity of METH to decrease CaM-kinase II activity after an extended withdrawal period.

A Ca(2+)/calmodulin-dependent signaling cascade has been implicated in the regulation of dopaminergic neurotransmission after chronic administration of amphetamine and methamphetamine (METH). We found a decrease in Ca(2+)/calmodulin-dependent protein kinase II (CaM-kinase II) activity in five regions of the rat brain (parietal cortex; frontal cortex; hippocampus; striatum; and nucleus accumbens) after a single injection of METH. Pretreatment with the selective dopamine D1 receptor antagonist SCH 23390 prevented the acute METH-induced decrease in CaM-kinase II activity in the parietal cortex, striatum, nucleus accumbens, and substantia nigra/ventral tegmental area (SN/VTA). Pretreatment with the N-methyl-D-aspartate receptor antagonist MK-801 significantly restored the acute METH-induced decrease in CaM-kinase II activity in the parietal cortex, nucleus accumbens, and SN/VTA. Striatal CaM-kinase II activity was still significantly lower than that of the chronic saline-treated controls after a 1-week, but not a 4-week, abstinence from chronic administration of METH. A METH challenge after a 4-week abstinence period induced a more pronounced decrease in CaM-kinase II activity in rats chronically injected with METH than in rats chronically injected with saline. Western blot analysis revealed that the amount of CaM-kinase II protein was not altered after a single METH injection or after chronic METH injections, compared with saline-treated controls. However, amounts of phosphorylated (Thr(286)) CaM-kinase II in the parietal cortex, striatum and SN/VTA were significantly decreased at 3 h after an acute METH injection compared with saline-treated controls. These results suggest that dephosphorylation of CaM-kinase II may contribute to the decreased enzyme activities induced by acute METH administration, and that chronic treatment with METH leads to an enhanced capacity of METH to decrease CaM-kinase II activity after an extended withdrawal period., (Copyright 2000 Wiley-Liss, Inc.)

The distribution of substance P (SP)-like immunoreactive neurons in the brains of aged normal human was analyzed quantitatively. Consecutive coronal sections in which the striatum and the substantia nigra were exposed widely, were obtained from the right hemisphere and stained immunohistochemically for SP. Each stained section was divided into approximately three million microareas and the immunohistochemical fluorescence intensity in each area was measured using a human brain mapping analyzer, which is a microphotometry system for analysis of the distribution of neurochemicals in a large tissue slice. These distributions are displayed in color and monochromatic graphics. In the analyzed brain regions, conspicuously intense SP-like immunoreactivity was observed in the substantia nigra and the internal segment of the globus pallidus. Within the substantia nigra, the SP-like immunoreactive intensity in the pars compacta was 25%, higher than that in the pars reticulata, and the distribution of melanin-containing neurons corresponded well to the distribution of the SP-containing structures. SP-like immunoreactive intensity in the internal segment of the globus pallidus, which was lower than that in the substantia nigra, was approximately twice as high as that in the external segment of the globus pallidus. Very intense immunoreactivity was localized at the most medial area of the internal segment of the globus pallidus. The SP-like immunoreactive intensity in the caudate nucleus and putamen was moderate, and the distribution was heterogeneous and observed in patches.

The effect of exercise on blood pressure in spontaneously hypertensive rats (SHR) was investigated assuming a mechanism involving calcium-dependent dopamine synthesis in the brain. Male SHR (13 weeks of age) were forced to run for 1 h at a speed of 10 m/min using a programmed motor-driven wheel cage. Systolic blood pressure was reduced after running, and this effect of exercise was decreased by prior intracerebroventricular administration of EDTA (1 nmol/rat), alpha-methyltyrosine (inhibitor of tyrosine hydroxylase, 1 mg/rat), sulpiride (D2 receptor antagonist, 50 microg/rat) or eticlopride (D2 receptor antagonist, 100 microg/rat), but was not changed by administration of SCH 23390 (D1 receptor antagonist, 30 microg/rat). Also, the calcium levels in the serum and brain were increased by exercise. Combining these results with our previous reports, it is suggested that exercise leads to an increase in the serum calcium level and subsequently an increase in the brain calcium level. This, in turn, leads to increased brain dopamine synthesis through a calmodulin-dependent system, with the increased dopamine levels inhibiting sympathetic nerve activity via the dopamine D2 receptor in the brain and causing a reduction in blood pressure., (Copyright 1999 Elsevier Science B.V.)

The activator protein-1 (AP-1) binding activities induced by a separate challenge with SKF38393 and quinpirole after 1 weeks' abstinence from chronic methamphetamine (4 mg/kg/day, 14 days) were increased significantly in the striatum, nucleus accumbens and cingulate cortex compared with the saline-treated controls. Quinpirole-, but not SKF38393-induced AP-1 binding activities were still significantly higher after a 4-week abstinence period in the chronic methamphetamine group than in the chronic saline control group. Downward sniffing, which occurred following a quinpirole-challenge, was significantly intensified after both a 1 and 4 weeks' abstinence from chronic methamphetamine. These results indicate that chronic administration of methamphetamine induces alterations of the interactions of dopamine D1 and D2 receptors which are reflected as enhanced AP-1 binding activities.

We developed a human brain mapping analyzer to determine the quantitative distribution of specific molecules, such as neurotransmitters or neuromodulators, based on a fluorescence microphotometry system that we had previously developed. The immunohistochemical fluorescence emitted from each microarea of a brain slice is collected into a photomultiplier tube through the pinhole and objective lens of a microscope. The brain slice is moved in the x- or y-direction by a motorized scanning stage under the objective lens, and the fluorescence intensities are measured quantitatively. The scanning speed is approximately 100 microareas/s, the maximum stage motion is 150 x 150 mm, and an unlimited amount of data can be gathered continuously by transfer to external memory devices. In this paper, this analyzer is characterized in detail, and the methods used for the preparation and analysis of human brain slices are described. As an example, the cholinergic distribution in hemispheric coronal slices of the adult human brain is analyzed. Each slice, immunohistochemically stained for choline acetyltransferase, was divided into approximately 3 million microareas (one area is 50 microm in diameter), and the distribution of the cholinergic neurons is shown.

The effect of acute and chronic administration of methamphetamine (METH) on the levels of activity-regulated cytoskeleton-associated protein (arc), an effector-immediate early gene, mRNA has been investigated in rat brain using in situ hybridization. Levels of arc mRNAs in the brain regions examined increased significantly from 0.5-1 h after an acute METH (4 mg/kg) administration compared with basal levels. The increase in arc mRNA continued by 3 h, and then subsided to basal levels by 6 h. The degree of increase in arc mRNA and the peak time after METH administration varied according to brain area. Arc mRNA in cerebral cortices showed robust increase 1 h after METH administration. In the striatum and hippocampus, it showed earlier and later increase, respectively, and its degree of both was less than in the cortices. Microscopic examination revealed that the METH-induced arc mRNAs in the parietal cortex were enriched in layers IV and VI, and those in the striatum existed mainly in the medium-sized neuron. Pretreatment with either 0.5 mg/kg SCH23390 or 0.25 mg/kg MK-801 almost completely blocked the enhanced striatal arc mRNA levels induced by acute METH administration, whereas such pretreatments only partially reduced the effect of METH in the cerebral cortical regions. In the chronic treatment experiment, the arc mRNA levels of the group that received chronic treatment with METH followed by a METH challenge showed an increase like seen after acute METH administration. Since previous studies proposed that arc is one of cytoskeleton-associated proteins and is selectively localized in neural dendrites, the results of the present study suggested that arc may play an important role in the synaptic plasticity underlying METH-induced adaptational changes including behavioral sensitization.

Stress ulceration of the stomach in mice was investigated from the aspect of the calcium/calmodulin-dependent dopamine synthesizing system in the brain. Cold stress was induced in mice by restraining them at 4 degrees C. Serum and brain calcium levels were increased by cold stress, and an increased brain calcium level was found to enhance dopamine synthesis and a successively increased brain dopamine level induced gastric ulcer formation. Development of gastric ulcers elicited by cold stress was significantly decreased by i.p. pretreatment with EDTA (1 micromol/mouse, 1 h before restraint) or alpha-methyltyrosine (a tyrosine hydroxylase inhibitor, 100 mg/kg, 24 h before restraint), and was further significantly increased by pretreatment with CaCl2 (40 micromol/kg, 1 h before restraint). These findings suggest that the development of gastric ulcers in cold-stressed mice may be linked with the enhancement of calcium/calmodulin-dependent catecholamine synthesis in the brain.

The effects of calcium on blood pressure regulation remain controversial. Although the mechanism by which calcium increases blood pressure when it is given intravenously and acutely has been elucidated, that by which calcium reduces blood pressure when it is supplemented chronically and slightly through daily diet is unclear. From a number of animal experiments concerning the effects of calcium on blood pressure, we believe that calcium ions have two separate roles in the regulation of blood pressure through both central and peripheral systems: (1) calcium ions reduce blood pressure through a central, calcium/calmodulin-dependent dopamine-synthesizing system and (2) calcium ions increase blood pressure through an intracellular, calcium-dependent mechanism in the peripheral vasculature. These concepts were applied to elucidate the mechanisms underlying hypertension in spontaneously hypertensive rats (SHR) and changes in blood pressure in other experimental animals, and the following conclusions were reached. The decrease of the serum calcium level in spontaneously hypertensive rats (SHR) causes a decrease in calcium/calmodulin-dependent dopamine synthesis in the brain. The subsequent low level of brain dopamine induces hypertension. The increase in susceptibility to epileptic convulsions and the occurrence of hypertension in epileptic mice (El mice) may be linked through a lowering of calcium-dependent dopamine synthesis in the brain, and epilepsy and hypertension may be associated. Exercise leads to increases in calcium-dependent dopamine synthesis in the brain, and the increased dopamine levels induce physiological changes, including a decrease in blood pressure. Cadmium which is not distinguished from calcium by calmodulin, activates calmodulin-dependent functions in the brain, and increased dopamine levels may decrease blood pressure. In this report, our studies are considered in light of reports from many other laboratories.

The effect of acute and chronic administration of methamphetamine (METH) on the levels of calmodulin (CaM) and its mRNAs has been investigated in rat brain using antisense oligonucleotides to three distinct rat CaM genes (CaM I, CaM II, CaM III). CaM I mRNA was reduced in the striatum and nucleus accumbens within 2 h of acute administration of 4 mg/kg METH, but returned to the control level by 6 h. The CaM content in both the cytosolic and membrane fractions of the striatum was reduced 0.5, 2, and 6 h after acute administration of METH. In the chronic experiments, rats were treated with either 4 mg/kg METH or saline once daily for 14 days. This was followed by a withdrawal period of 28 days, and thereafter, the animals were challenged with either METH (4 mg/kg, i.p.) or saline. All the animals were decapitated 6 h after this injection. There were four treatment groups: METH-METH (MM); METH-saline (MS); saline-METH (SM); and saline-saline (SS). There was a significant decrease in the mRNA for CaM I and CaM II in the striatum, and CaM II and CaM III in the nucleus accumbens in the MS group and the MS and MM groups, respectively, when compared to the SS group. The CaM content in the striatal membrane fraction decreased in both the SM and MS groups but not in the MM group. In contrast, the CaM content in the membrane fraction of the mesolimbic area showed a significant increase in the MM group. The CaM content in the cytosolic fraction of these brain areas decreased in both the SM and MM groups. The total CaM decreased significantly in the SM and MM groups of the striatum, but increased significantly in the MM group of the mesolimbic area. The mRNA for CaM I and CaM III decreased significantly in the MM group, and in the SM and MM groups, in the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA), respectively. The CaM content in both the cytosolic and membrane fractions and total CaM content of the SN/VTA decreased significantly in the SM, MS and MM group as compared with the SS group. In the medial prefrontal cortex and hippocampus the significant increase of CaM content in the membrane fraction of the MM group was also found, but neither the CaM content in the cytosol fraction nor total CaM content changed. These results suggest that chronic METH administration leads to a translocation of CaM from the cytosolic to membrane fractions; these may underlie METH-induced behavioral sensitization.

Several neuron-specific cDNAs were identified using large-scale collection of 3'-directed partial cDNA sequences from a human neuroblastoma cell line. One of such cDNAs encoded a protein homologous to the mouse NeuroD. The mouse NeuroD, a basic helix-loop-helix (bHLH) protein seems to function as a differentiation factor for neurogenesis, as the gene is transiently expressed in postmitoic differentiating neurons. The human counterpart has a bHLH domain completely identical to that of the mouse NeuroD. But its expression was not only in the fetal brain but also in the adult cerebellum. The result of cross-species in situ hybridization also showed the transcripts were detected in the granule cell layer of the adult mouse cerebellum. The results suggest that the human as well as the mouse neuroD may play some important functions not only in the fetal brain but also in the fetal brain but also in the matured neurons of the cerebellum.

The effect of exercise on central nervous system function was investigated in relation to the mechanism of calcium-calmodulin-dependent dopamine synthesis in the brain. It is shown here through animal experiments that exercise leads to an increase in the calcium level in the brain. This in turn enhances brain dopamine synthesis, and through this increased dopamine modifies and/or affects brain function, which might induce physiological, behavioral, and psychological changes.

Using K+ phosphate buffer with 25 nM spiperone, [3H]YM-09151-2 binding showed a high affinity for sigma receptors but no affinity for D2 dopamine or 5-HT1A receptors in rat brain. The order of pKi values of various sigma compounds at [3H]YM-09151-2 binding sites and stereoisomer selectivity were consistent with previous studies using other sigma ligands such as (+)-[3H]SKF-10047, [3H]DTG and (+)-[3H]3-PPP. Although Scatchard analysis fitted a one-site model, competition between [3H]YM-09151-2 and (+)-pentazocine revealed two sites, sigma 1 and sigma 2 receptors, at which the Ki values of YM-09151-2 were 8.4 nM and 9.6nM, respectively. Autoradiography using [3H]YM-09151-2 also showed a characteristic distribution of sigma receptors in rat brain. [3H]YM-09151-2 is, therefore, a potent and useful radioligand for sigma 1/sigma 2 receptor subtypes.

Expression of the mammalian major apurinic/apyrimidinic (AP) endonuclease (designated as APEX nuclease, or HAP1, APE or Ref-1 gene product) during mouse brain development was investigated by in situ and northern blot hybridizations. The enzyme is known to be a redox factor (Ref-1) stimulating DNA binding activity of AP-1 binding proteins such as Fos and Jun as well as a multifunctional DNA repair enzyme having 5' AP endonuclease, DNA 3' repair diesterase, 3'-5' exonuclease and DNA 3'-phosphatase activities. In the embryonic and postnatal development, APEX mRNA was expressed at high levels in the proliferative zone of various brain regions, with showing temporal and spatial changes. Its expression decreased in association with brain development to the basal expression level which was observed even in adulthood, with the exception of its expression in the hippocampal formation. The growth-dependent expression of APEX gene suggests that it has some roles on cell proliferation and/or differentiation in developmental brain. Its expression on the hippocampal formation became significant from postnatal day 7 and then increased. The pyramidal and granule cell layers expressed it at a higher level than most other brain regions at postnatal day 21. The developmental change of APEX gene expression was not necessarily associated with the changes of expression of c-fos and c-jun genes measured by northern blot hybridization. However, the present results suggested that APEX/Ref-1 gene product can interact with AP-1 binding proteins in brain, especially in the hippocampal formation, to regulate some brain functions by redox-activation.

The possible complication of hypertension and epilepsy was investigated through the response in epileptic El mice. The systolic blood pressure in El mice (male, 8 weeks of age) and that in normal ddY mice (the parent strain of El mice) were compared by a tail-cuff method, using a programmed sphygmomanometer. The systolic blood pressure in El mice (120.5 +/- 5.6 mm Hg) was 28% (P < 0.01) higher than that in ddY mice (93.9 +/- 5.3 mm Hg). The higher systolic blood pressure in El mice was lowered by the acute intracerebroventricular administration of CaCl2 (10 mumol/kg, 30 min before measurement) or dopamine (30 nmol/mouse, 15 min before measurement), and was also improved by the chronic oral supplementation with 1.2% calcium (Ca2+) solution. Combining these results with those in our previous reports, where it is stated that lowering of Ca(2+)-calmodulin-dependent catecholamine synthesis increases the susceptibility to epileptic convulsions, we suggest that the increase in susceptibility to epileptic convulsion and occurrence of hypertension in El mice may be linked and that the two diseases may be associated.

The ontogeny of the behavioral effects of acute cocaine administration and behavioral sensitization to cocaine in rat pups was investigated. Acute behavior stimulating effects of cocaine were observed in pups as young as 7 postnatal days (PND) old, although they needed a higher dose of cocaine than adult rats to evoke the same motor effects. An adult dose-response curve pattern of stereotypy and locomotion to acute cocaine treatment was observed at PND 21, and of rearing at PND 28. Rats aged PND 7, 14, 21, 28, and 56 received repeated injections of saline or cocaine (15 mg/kg) twice a day for 5 consecutive days. After a 3-week period of abstinence, sensitization to a challenge dose of cocaine was assessed. Cocaine-induced stereotyped behavior was enhanced significantly only in rats in which cocaine pretreatment was initiated on PND 21, 28, and 56, but not earlier on PND 7 and 14. Adult female rats given repeated cocaine injections on PND 56-60 showed significantly greater sensitization than males, but no such sex difference was observed in pups given cocaine repeatedly on PND 21-25 or 28-32. These results show clearly that cocaine-induced behavioral sensitization in rats occurred only when subchronic cocaine administration was commenced on PND 21 or later.

In situ hybridization of slide-mounted brain sections from rats subjected to acute and chronic phencyclidine treatment was carried out using synthetic oligonucleotides complementary to dopamine D2-receptor and non-N-methyl-D-aspartate (NMDA) glutamate-receptor-subunit (GluR-1) mRNAs. There was no significant difference in either the D2-receptor or the GluR-1 mRNA levels in any brain region of the acute phencyclidine (10 mg/kg)-treated and control groups. However, chronic administration of phencyclidine (10 mg/kg/day, 14 days) significantly decreased the dopamine D2-receptor mRNA level in the caudate-putamen (by 27%, P < 0.01) and significantly increased the GluR-1 mRNA level in the prefrontal cortex (by 29%, P < 0.001). These results suggest that the chronic pharmaco-behavioral effects of phencyclidine may involve expression of both dopamine- and non-NMDA glutamate-receptor mRNAs.

The distributions of the cholinergic system and catecholaminergic system in the normal human brain were analysed quantitatively by a microphotometry system. Consecutive coronal sections were obtained from the anterior area of the left hemisphere and were stained alternately with fluorescent immunohistochemical staining for choline acetyltransferase or tyrosine hydroxylase. Each stained section was divided into approximately 120,000 areas and the fluorescence intensity in each area was measured by a fluorescence microphotometry system which is a measuring microscope for distribution of fluorescence intensity in the tissue slice. Nonspecific autofluorescence was distributed in myelinated nerve fiber throughout the entire area, which was subtracted from the fluorescence intensity value in each measuring area. The obtained immunohistochemical fluorescence intensities of choline acetyltransferase and tyrosine hydroxylase were classified into eight ranks and were indicated by color graphics. Also, the intensity values of actual immunohistochemical fluorescence in the various brain regions were presented. The choline acetyltransferase and tyrosine hydroxylase concentrations varied greatly depending on the brain region. Relatively high levels of choline acetyltransferase and tyrosine hydroxylase were distributed in the putamen, caudate nucleus, claustrum, insula and some cortical regions. The immunohistochemical level of tyrosine hydroxylase was lower than that of choline acetyltransferase in a few brain regions such as the globus pallidus and amygdala. High levels of choline acetyltransferase and tyrosine hydroxylase were localized in the one area of the basal ganglia which developed from the telencephalic area, whereas middle levels of these were distributed in another, part of which developed from the diencephalic area.(ABSTRACT TRUNCATED AT 250 WORDS)

Magnetic resonance imaging (MRI) findings in 87 children with various clinical entities were used to determine the signal intensity ratio of gray/white matter in T1- and T2-weighted images using a 1.5 T MR Scanner. Signal intensity ratio changes in both T1- and T2-weighted images correlated well with advancing age (y = 0.9349-0.001575, r = -0.584, P < 0.0001 in T1-weighted images; y = 0.9798 + 0.002854, r = 0.723, P < 0.0001 in T2-weighted images), but the correlation was more linear when we included only normally developed (34) children (y = 0.9689-0.001967, r = -0.654, P < 0.0001 in T1-weighted images; y = 0.9882 + 0.002965, r = 0.747, P < 0.0001 in T2-weighted images). Abnormal ratios were observed in patients with congenital hydrocephalus, inherited metabolic diseases and cerebral palsy. Although the gray/white matter differentiation would not delineate the myelination itself, measurement of the signal intensity ratio of gray/white matter is a practical way to evaluate delayed myelination in a busy MR center.

The metabolism of calcium and brain dopamine in spontaneously hypertensive rats (SHR) after the development of hypertension was investigated as a possible model for the hypertension mechanism. Serum calcium level in SHR was lower than that in the normotensive control. Wistar Kyoto rats (WKY, the parent strain of SHR). Conversely, bone calcification of SHR was higher than that in WKY. Possible mechanisms for the lower serum calcium level seen in SHR include a decrease in the availability of calcium from bone. The immunohistochemical dopamine levels in the neostriatum and nucleus accumbens in SHR were lower than those in WKY. In these regions, the dopamine level was increased by the intraventricular administration of CaCl2 through a central, calmodulin-dependent system. This study suggests, based upon previous pharmacological studies, that the decrease of the serum calcium level in SHR causes a decrease in central, calcium-calmodulin-dependent dopamine synthesis and a subsequent low level of dopamine in the brain that produces an increase in blood pressure through functions of cerebral dopaminergic neurons and peripheral sympathetic nerves. Our results suggest that this could be one of the mechanisms of hypertension in SHR.

Synaptosomal membrane fluidity, lipid peroxide (LPO) and cytosolic superoxide dismutase (SOD) activity were examined in various brain regions (amygdala, hippocampus, striatum and frontal cortex) of amygdala-kindled rats. At 24 h after the last seizure, a significant increase of membrane fluidity was observed in all the regions examined, whereas the LPO level was significantly decreased in the four regions with enhanced activity of cytosolic SOD. At 7 days after the last seizure, membrane fluidity was decreased only in the hippocampus. At 6 weeks after the last seizure, there were no changes in membrane fluidity between control and kindled rats. These results suggest that membrane fluidity and lipid peroxidation are modulated transiently by a kindled seizure, but not at a steady state of kindling with enduring seizure susceptibility.

The effects of 2-guanidinoethanol (GEt) on the release of monoamines and on the activity of their degrading enzymes were studied in order to investigate why 3,4-dihydroxyphenylacetic acid (DO-PAC) increased to a much greater extent than homovanillic acid (HVA) after GEt injection into rat brain. In differential pulse voltammograms recorded using an electrochemically treated carbon fiber electrode, two distinct oxidation peaks, one at 130mV (DOPAC peak) and the other at 300 mV (5-hydroxyindoleacetic acid (5-HIAA) peak), were observed. In the hippocampus, the DOPAC peak increased markedly compared to the peak height recorded prior to the intracerebroventricular injection of GEt (6 mumol). Although the DOPAC peak height increased to 350% 4 hours after GEt injection, the 5-HIAA peak showed no change. In the striatum, the DOPAC peak increased to 150% 3 hours after GEt injection. Serial changes in the extracellular levels of DOPAC, HVA, and 5-HIAA were monitored in the striatum after GEt injection, using an in vivo brain micro-dialysis technique. Although the DOPAC levels strated to increase 80 minutes after GEt injection, HVA and 5-HIAA levels showed no change. On the other hand, monoamineoxidase, which metabolizes dopamine to DOPAC, was not activated and catechol-O-methyltransferase, which metabolizes DOPAC to HVA, were not inhibited by 5 mM of GEt in vitro. These data suggested that GEt increased the release of dopamine, but not of serotonin, and that GEt might restrict the DOPAC transport system.

Two molecular forms of the dopamine D2 receptor were generated by alternative RNA splicing. To investigate the relative distributions of the two mRNAs encoding the D2 receptor isoforms, D2(415) and D2(444), we performed in situ hybridization histochemistry in the rat brain with the two oligonucleotide probes. An insert probe complementary to an additional fragment of the D2 receptor mRNA cloned from the rat brain, and a spanning probe complementary to its contiguous sequence were used. These 48 base probes were 3'-end labeled with [35S]dATP. The brains were dissected from male SD rats and frozen in dry ice and acetone. Cryostat sections (16 microns) were collected on gelatin coated slides and stored at -20 degrees C. In situ hybridization studies were conducted with a probe concentration of 1 x 10(6) dpm/100 microliters of buffer per brain slice at 37 degrees C for 18-20 h in a humid chamber. The slides were washed, dried and exposed to tritium sensitive film for one week. The autoradiograph showed that both mRNA were present at high levels in the corpus striatum, accumbens nucleus and substantia nigra (pars compacta). Identical patterns of labeling were obtained in the rat brain using both the insert and spanning probes, although the optical densities detected with the insert probe were higher than those with the spanning probe in the corpus striatum. This suggests that both D2 receptor mRNAs are expressed similarly in each region of the rat brain and D2(444) expressed dominantly in the corpus striatum.

We have developed a fluorescence microphotometry system for microanalysis of the quantitative distribution of neurotransmitters and their related chemical substances in the brain slice. In the present study, the extensive distributions of cholinergic systems were analyzed quantitatively and in detail throughout the rat whole brains by this novel method through immunohistochemical staining of choline acetyltransferase (CAT). The rat whole brain was slice coronally and continuously, and 50 slices were chosen at approximately 500 microns intervals and stained immunohistochemically for CAT. Immunohistochemical fluorescence intensities were measured through a 6 microns phi (on the slice) pinhole of a microscope, the brain slice was moved along the X- or Y-axes stepwise at 40 microns intervals under the objective lens of the microscope, and the distributions of fluorescence intensities were analyzed over the entire surface of the slice. The brain was divided into approximately 5,000,000 areas, and immunohistochemical fluorescence intensities of those areas were quantitatively measured. The obtained fluorescence intensities of CAT were classified into 8 ranks and were indicated by color coding and by three-dimensional graphics. Also, the actual fluorescence intensity values in large brain regions were presented. This type of brain atlas of the neurotransmitter or its related chemical substances provides very important information on their dynamics in the brain under experimental as well as pathological conditions. Also, this quantitative and detailed analysis is useful for combining morphological data with those from neurochemical and behavioral analyses of brain function.

The contents of monoamines and their metabolites in rat brains 3 hours after the intracerebroventricular injection of 6 mumol of 2-guanidino-ethanol (GEt) were measured by HPLC. GEt which is a configurational analogue of 4-aminobutanoic acid (GABA) induced severe running fits and tonic-clonic convulsions as well as epileptic discharges. In GEt-administered rats, dopamine (DA) decreased in the cortex, hippocampus and hypothalamus. 3,4-Dihydroxyphenylacetic acid (DOPAC) increased to about the same level in all brain regions, therefore the distribution of DOPAC appeared to be homogeneous in the brain. The homovanillic acid levels also increased in the striatum and hippocampus. No significant change in the norepinephrine contents was observed in any region. The turnover ratio of DA increased significantly except in the striatum. Serotonin levels increased in the hypothalamus and midbrain by GEt administration, though 5-hydroxyindoleacetic acid levels showed no change in any of the brain regions. These data suggest that the activity of dopaminergic and serotonergic neurons are increased by GEt.

Immunohistochemical distributions of tyrosine hydroxylase and calmodulin in the rat forebrain were analyzed quantitatively to confirm our previous results that the activities of central catecholamine-synthesizing enzymes are regulated by a calcium-calmodulin-dependent system. The adjacent slices of adult rat brain were stained immunohistochemically for tyrosine hydroxylase and for calmodulin, and the distributions and amounts of these proteins were measured by a fluorescence microphotometry system that was developed in our laboratory. Immunohistochemical fluorescence intensity was measured stepwise at 40 microns intervals through a 6 microns phi (on the slice) pin hole. Each stained brain slice was divided into approximately 100,000 areas, and measured for fluorescence intensity and displayed two- and three-dimensionally. Immunoreactive staining of tyrosine hydroxylase and calmodulin was observed in almost all areas of the brain, but its intensity varied. The relatively high levels of calmodulin could be observed in brain regions with high levels of tyrosine hydroxylase distribution, though high levels of tyrosine hydroxylase could not always be observed in brain regions where high levels of calmodulin were distributed. In the present study, high levels of tyrosine hydroxylase and calmodulin were distributed in the nucleus accumbens septi and the lateral part of the neostriatum regions in which the amount of dopamine was increased by the intraventricular administration of calcium. These findings suggest that the synthesis of central catecholamines is regulated by a calcium-calmodulin-dependent system.

Dopamine (DA) levels in the various brain regions of epileptic mice (El mice) were compared immunohistochemically with those in ddY mice (the mother strain of El mice) using a fluorescence microphotometry system. The fluorescence intensities of DA in the neostriatum and nucleus accumbens septi in El mice were approximately 11-15% (P less than 0.01) and 13% (P less than 0.01) lower than in ddY mice. On the other hand, the lower DA amounts in these regions of El mice were improved by intraventricular administration of CaCl2 (10 mumol/kg). The brain regions in which the amount of DA was increased by calcium were areas where high levels of calmodulin and tyrosine hydroxylase are distributed. This finding reconfirmed our previous report that the biogenic amine level disorder in El mice was related to a calcium ion level disorder through a central calcium-calmodulin-dependent biogenic amine-synthesizing mechanism, and this might increase their susceptibility to epileptic convulsions.

Intracerebral dialysis was used to study the mechanism underlying cross-behavioral sensitization between methamphetamine (MAP) and cocaine. The challenge injection of cocaine caused a significantly greater increase in striatal perfusate dopamine (DA) levels in MAP-pretreated rats than in saline-pretreated rats. Similarly, the challenge injection of MAP caused a significantly greater increase in extracellular DA levels in cocaine-pretreated rats than in control rats. These results suggest that an enhancement in striatal DA efflux may play an important role in cross-behavioral sensitization between MAP and cocaine.

The cyclic, penicillamine(beta, beta dimethylcysteine)-containing enkephalin analogs, [D-Cys2, L-Pen5]-and [D-Cys2, D-Pen5]enkephalin and the corresponding bis-penicillamine analogs, [D-Pen2, L-Pen5]-and [D-Pen2, D-Pen5]enkephalin were synthesized and evaluated for opioid activity in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays and in rat brain and neuroblastoma-glioma cell membrane binding assays. These analogs all displayed delta receptor selectivity as assessed by IC50(GPI)/IC50(MVD) ratios and by their relative potencies for displacing [3H]naloxone (NAL) vs. [3H] [D-Ala2, D-Leu5]enkephalin (DADLE) from rat brain membrane preparations. For [D-Pen2, L-Pen5]- and [D-Pen2, D-Pen5]enkephalin the observed IC50(GPI)/IC50 (MVD) ratios (1088 and 3164) and IC50NAL/IC50DADLE ratios (371 and 175) represent a vast improvement over previously reported delta receptor selective ligands.

alpha-Melanotropin (alpha-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4,D-Phe7]-alpha-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4,D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-alpha-MSH, Ac-[Nle4]-alpha-MSH4-10-NH2 and Ac-[Nle4]-alpha-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-alpha-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.

Specific binding properties of the tritium-labeled delta opiate receptor agonist [3H][2-D-penicillamine, 5-D-penicillamine]enkephalin [( 3H][D-Pen2, D-Pen5]enkephalin) were characterized in the rat brain and in a mouse neuroblastoma-rat glioma hybrid cell line (NG 108-15). Saturation isotherms of [3H][D-Pen2, D-Pen5]enkephalin binding to rat brain and NG 108-15 membranes gave apparent Kd values of 1-6 nM. These values are in good agreement with the Kd value obtained from the kinetic studies. The Bmax value in NG 108-15 membranes was 235.3 fmol/mg of protein. An apparent regional distribution of [3H][D-Pen2, D-Pen5]enkephalin binding was observed in the rat brain. A number of enkephalin analogues inhibited [3H][D-Pen2, D-Pen5]enkephalin binding with high affinity (IC50 values of 0.5-5.0 nM) in both NG 108-15 and rat brain membranes. However, putative mu receptor-selective ligands such as morphine, [D-Ala2, MePhe4, Gly5-ol]enkephalin, [MePhe3, D-Pro4]morphiceptin, and naloxone were less effective inhibitors of [3H][D-Pen2, D-Pen5]enkephalin binding in both systems tested. These data suggest that [3H][D-Pen2, D-Pen5]enkephalin is a potent and selective ligand for the delta opioid receptor.

Forty randomly selected dogs, arranged into three groups according to age, were examined light and electron microscopically. Lafora-like inclusion bodies were found in all cases of aged dogs without neurological signs. They appeared as PAS positive, round bodies measuring up to 15 mu in diameter. Typically they had central cores and radiating peripheral lines. Electronmicroscopically, the inclusions consisted of irregularly clustered, short branching filaments measuring about 80--120 A in diameter or of electron-dense homogeneous or granular central cores with filamentous peripheral areas and located both in the perikarya and neuronal processes. The inclusions were disseminated all over the brain and spinal cord. The thalamic nuclei, a circumscribed area dorsal to the aqueduct, and the molecular layer of the superior colliculus were the most severely affected. The frequency of the inclusions showed age-dependency; the inclusions were not found in dogs younger than 2 years, but were extensive in all dogs of more than 8 years of age. The occurrence of the inclusions may therefore represent an age-dependent phenomenon. The relationship between the present findings and Lafora's disease is discussed.