Recently, techniques of molecular biology have been widely applied to child neurology, and a new aspect of the pathogenesis of neurogenetic diseases has been revealed. In this article, recent results of molecular analysis in my laboratory were briefly reviewed on hereditary β-galactosidase deficiency. After cDNA cloning, a number of gene mutations have been identified, mainly missense mutations, such as single-base substitution, duplication, insertion, and splice site mutation. A clear phenotype-genotype correlation was established for some mutations specific to the late-onset forms of the disease. Intracellular events of mutant proteins expressed by these mutant genes were heterogeneous, and expected to be closely connected to the pathogenesis of each phenotype. On the basis of these data, a unified clinical classification was proposed for G_M1-gangliosidosis and Morquio B disease, together with a new concept of “β-galactosidosis” for the diseases with β-galactosidase gene mutations.

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In 1912, Wilson and Fleischer independently reported autopsied patients with progressive neurological disorder associated with cirrhosis of the liver, and they proposed that the syndrome could be a specific disease of toxic origin.
In 1952, Scheinberg demonstrated a deficiency of serum ceruloplasmin in Wilson's disease, and it became possible to diagnose the illness while the patient was still asymptomatic. In 1956, Walshe introduced penicillamine as the most excellent drug for treatment of the disease. These epoch- making discoveries encouraged Japanese physicians to make early diagnosis and to try prevention of the disease. This lecture was to review the changing panorama in the diagnosis, treatment and prognosis of the disease in the period of forty years focusing on the experiences in Japan.
Early detection of the patients based on hypoceruloplasminemia made it possible to investigate the onset ages of an elevation of serum GOT or GPT and the appearance of Kayser-Fleischer (KF) rings. So far, the youngest patients who exhibited high GPT level and KF rings were three years and five years old, respectively. It became popular that an unexpected elevation of serum transaminase in apparently healthy children of three years or more prompted to examine the possibility of Wilson's disease, and an increasing number of non-familial patients in late infancy have been detected. Now, the mass-screening for Wilson's disease is in progress.
Follow-up studies on the prophylaxis for more than thirty years definitely proved that the appearance of clinical symptoms was prevented with the continued penicillamine therapy. There were patients who developed symptoms such as hemolytic crisis, motor deterioration and, especially, psychotic or affective disorders after discontinuation of the therapy of several years when they reached twenty to thirty years of age. Now, the urgent problem is how to encourage all the grown-up patients to continue the lifelong therapy.

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Glutamic acid has been believed to be an excitatory transmitter in the mammalian central nervous system (CNS), and has been implicated in the pathogenesis of neuronal damage in the mammalian CNS. There are two major classes of glutamate receptors, ionotropic (iGluR) and metabotropic glutamate receptors (mGluR). Participation of iGluRs in glutamate mediated neurotoxicity has been well documented. However, much less is known about participation of mGluRs than the case for iGluRs. The physiological roles of mGluRs have been believed to regulate transmitter release and to modulate the function of iGluRs through activating various intracellular second messenger system. Recently we have discovered several potent agonists for mGluRs which would provide additional information about glutamate mediated neurotoxicity. DCGIV, one of the most potent mGluR agonists, alleviated kainate- induced limbic motor seizures in extemelv low doses in the rat, but the dose response curves showed a bell typed one. DCG-N also demonstrated severe sedative condition and markedly prolonged the sleeping time in halothane anesthesia. DCG-N depressed the duration of after-discharges and the seizures evoked by electrical stimulation in the amygdala kindling rat. DCG-Di significantly decreased in number of kainate-induced degenerated neurons in the area of hippo-campal CA1, amygdala and septum when DCG- N was continuously applied into the ventricule. In conclusion, activation of mGluRs leads the alleviation of neuron damage induced by iGluR agonists.

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Recently medical imaging technique has greatly progressed. During these years, this technique became available not only for detection of morphological information but also for evaluation of function of variable organs. In this symposium, we focused the main topics on functional imaging techniques, and planned that the results would be presented by visually impressive methods as much as possible. This symposium consisted of 5 topics:(1) two-dimentional analysis of neural activity by intracellular calcium fluorometry, (2) photometric analysis of functional structure in the brain, (3) magnetoencephalography, (4) chemical shift imaging and functional magnetic resonance imaging, and (5) neurotransmitter and receptor mapping by positron emission tomography.

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The development of fluorescent Ca²⁺ indicators made us possible to measure the intracellular Ca²⁺ concentration easily. Furthermore the analysis of fluorescent images obtained by a fluorescence microscope became easy and popular, because of the tremendous development of computers and related hardwares. Time courses and topographical differences in Ca²⁺ concentration can be demonstrated as two dimensional color-coded images. Such images are sometimes quite persuasive. That is just “Seeing is believing”. We can detect a completely new site of biological phenomena through this method. This article will describe many different types of Ca²⁺ indicators and applications to biological image analysis. Especially the method called “macro” image analysis can demonstrate the regional difference in changes of the Ca²⁺ concentration of slice preparation during medical treatment of cerebral ischemia. Already more than ten years have passed since the first demonstration of a fluorescent Ca²⁺ indicator, quin 2, and the methods using such Ca²⁺ indicators become important in the field of experimental biology.

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To date, our understanding of the elaborate mechanism that governs the information processing of neural tissue or the brain is based mainly on data obtained from single-electrode recordings in vivo or in vitro. The refinement of electrophysiological techniques has advanced our knowledge about neural information processing, but clearly, we are still far from complete and coherent understanding of these brain functions. Although conventional approaches have provided the bulk of our knowledge about neural information processing, there is clearly a need for research tools that are better suited to investigate the detailed properties of individual neurons on the one hand and allow for studying the interactions between large numbers of neurons on the other hand.
The use of optical recording methods with voltage-sensitive dyes (extrinsic optical signal recording) seems to be one of the most promising ways to attack the above problems. This approach allows to record the neural activity at many sites simultaneously and thus provides spatio-temporal information about the flow of electrical activity in a given preparation.
The optical recording without using voltage-sensitive dyes, intrinsic optical signal recording, is also useful to detect the neural activity in the brain. The signal is thought to be originated from the metabolic change of the brain tissue which is associated with the change in neural activity. We compared the both activity mapping obtained with extrinsic signals and intrinsic signals. This kind of trial seems to be useful not only for understanding the origin of the metabolic signal, but also for treating the data obtained with PET or functional MRI.

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Electrical activities develop simultaneously with the establishment of magnetic fields following Biot-Savart's law. Excitation of cerebral neurons causes the development of electrical activities, and thereby produces magnetic fields. The term “magnetoencephalography (MEG)” refers to the detection and study of these magnetic fields. It is therefore possible to estimate accurately the origins of the magnetic fields, i. e. deep foci of electrical activities, from the outside of the scalp. This report describes the principle of magnetoencephalography, the recording method, and the estimation of intracerebral localization for rolandic discharges in benign childhood epilepsy with centrotemporal spikes.

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We have developed ¹H-chemical shift imaging (CSI) and functional magnetic resonance imaging (FMRI) methods on a clinical MRI system, in which metabolic and functional information can be obtained from the brain. ³¹P-and ¹H-CSI are in clinical use. Using ¹H-CSI, the peaks of N- acetylaspartate (NAA), choline (Cho) and creatine (Cr) are clearly detected in multiple small voxels. In normal children, the ratio of NAA/Cho increased after birth in different manners in different parts of the brain. The peak of NAA decreased in some disorders with brain damage or neuronal immaturity.
It has been shown recently that functional activation of the cortex can be visualized with MR imaging with a blood oxygen level dependent (BOLD) effect. At an activated area, the ratio of deoxyhemoglobin to oxyhemoglobin decreased in the capillary and venous beds. Therefore, with a decreaseof the effect of the T₂ susceptibility from deoxyhemoglobin, the signal intensity of the activated area increased. CSI and FMRI have a unique possibility in the field of non-invasive brain analysis.

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[in Japanese]

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