Kazunori Yabe

Quantitative analysis of immunohistochemical distributions of cholinergic and catecholaminergic systems in the human brain

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)

Quantitative mapping analyzer for determining the distribution of neurochemicals in the human brain

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.

Multiple analysis of tyrosine hydroxylase and calmodulin distributions in the forebrain of the rat using a microphotometry system

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.

Comparison analysis of distributions of tyrosine hydroxylase, calmodulin and calcium/calmodulin-dependent protein kinase II in a triple stained slice of rat brain.

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.

Quantitative imaging of tyrosine hydroxylase and calmodulin in the human brain

The distributions of tyrosine hydroxylase and calmodulin in adult normal postmortem human brain were analyzed quantitatively. Consecutive coronal sections were obtained from the anterior area of the right hemisphere and were stained immunohistochemically for tyrosine hydroxylase and calmodulin. Stained sections were divided into approximately 3 million microareas at 50 μm intervals, 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. Immunoreactive staining of tyrosine hydroxylase and calmodulin was observed in almost all brain regions, but its intensity varied. Relatively high levels of calmodulin were observed in brain regions with high levels of tyrosine hydroxylase, though high levels of tyrosine hydroxylase were not always observed in brain regions where high levels of calmodulin were distributed. In particular, high levels of both of tyrosine hydroxylase and calmodulin were distributed in the caudate nucleus and putamen. Previously it was shown that tyrosine hydroxylase was activated and dopamine synthesis was enhanced in the neostriatum region in mice and rats by the intracerebroventricular administration of calcium through a calmodulin‐dependent system. The present results combined with these previous findings suggest that the activity of tyrosine hydroxylase in the caudate nucleus and putamen of humans may also be regulated by a calcium/calmodulin‐dependent system. J. Neurosci. Res. 63:369–376, 2001.

Quantitative maps of GAbAergic and glutamatergic neuronal systems in the human brain.

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. Hum. Brain Mapping 11:93–103, 2000.

Quantitative imaging of substance P in the human brain using a brain mapping analyzer.

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.

Quantitative immunohistochemical distribution of choline acetyltransferase in the rostral forebrain of the rat.

The immunohistochemical distribution of choline acetyltransferase (CAT) in the rat rostral forebrain was analyzed quantitatively and minutely by means of a microphotometry system. The CAT concentrations varied greatly depending on the brain region. Within the neostriatum, CAT tended to be distributed with a lateral (high) to medial (low) gradient of approximately 1.2:1 and a caudal (high) to rostral (low) gradient of approximately 1.4:1, with the highest level in the medius lateralis. In the cortex cerebri, the CAT concentration in the area cinguli was high, while those in the area frontalis, area parietalis and area pyriformis were relatively low. High levels of CAT were also localized in other regions: e.g., hippocampus pars posterior, nucleus preopticus, nucleus anterior hypothalami, nucleus interstitialis striae terminalis, nucleus suprachiasmaticus and nucleus accumbens septi. The quantitative data obtained from the present microphotometric examination can be useful for analysis of a dynamic aspect of neurochemical substances under physiological as well as pathological conditions of the brain.

Effect of Zena F-III, a liquid nutritive and tonic drug, on the neurochemical changes elicited by physical fatigue in mice.

The effects of a liquid nutritive and tonic drug (NTD) on the neurochemical changes elicited by physical fatigue in mice were investigated in terms of the calcium-dependent dopamine synthesizing function of the brain. In this study, Zena F-III (Taisho Pharmaceutical Co., Ltd., Japan), one of the most popular NTDs in Japan, containing 15 crude drug extracts together with taurine, caffeine, and vitamins, and formulated based on the precepts of traditional Chinese medicine, was used. Male mice were forced to walk for 0-6 h at a speed of 3 m/min using a programmed motor-driven wheel cage. The serum and brain calcium levels in the mice were significantly increased following forced walking. The increase in brain calcium level began later and was more gradual than that in the serum calcium level, and reached its maximum value following forced walking for 3 h. The neostriatal dopamine level was also significantly increased, and locomotor activity significantly decreased following forced walking for 3 h. Prior oral administration of F-III (10 ml/kg) attenuated the increases in the serum and brain calcium levels, the increase in the brain dopamine levels, and the decrease in locomotor activity induced by forced walking. Taking into consideration these findings with our previous reports, it is suggested that physical fatigue leads to an increase in dopamine synthesis in the brain through a calcium/calmodulin-dependent system, thereby inducing behavioral changes, and that F-III inhibits this pathway and may alleviate overwork-induced physical fatigue.

A novel technique for quantitative immunohistochemical imaging of various neurochemicals in a multiple-stained brain slice

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.