Ryohachi Arai

Single Nigrostriatal Dopaminergic Neurons Form Widely Spread and Highly Dense Axonal Arborizations in the Neostriatum

The axonal arbors of single nigrostriatal dopaminergic neurons were visualized with a viral vector expressing membrane-targeted green fluorescent protein in rat brain. All eight reconstructed tyrosine hydroxylase-positive dopaminergic neurons possessed widely spread and highly dense axonal arborizations in the neostriatum. All of them emitted very little axon collateral arborization outside of the striatum except for tiny arborization in the external pallidum. The striatal axonal bush of each reconstructed dopaminergic neuron covered 0.45–5.7% (mean ± SD = 2.7 ± 1.5%) of the total volume of the neostriatum. Furthermore, all the dopaminergic neurons innervated both striosome and matrix compartments of the neostriatum, although each neurons arborization tended to favor one of these compartments. Our findings demonstrate that individual dopaminergic neurons of the substantia nigra can broadcast a dopamine signal and exert strong influence over a large number of striatal neurons. This divergent signaling should be a key to the function of the nigrostriatal system in dopamine-based learning and suggests that neurodegeneration of individual nigral neurons can affect multiple neurons in the striatum. Thus, these results would also contribute to understanding the clinicopathology of Parkinsons disease and related syndromes.

D-amino-acid oxidase is confined to the lower brain stem and cerebellum in rat brain: regional differentiation of astrocytes.

Based on enzymatic activity, the localization and the identification of D-amino-acid oxidase-containing cells in rat whole brain was systematically studied in serial fixed sections. The oxidase activity was absent or scarce in the forebrain, was confined to the brain stem (midbrain, pons and medulla oblongata) and cerebellum, and its localization was extended to the spinal cord. In the brain stem the oxidase was mainly localized in the tegmentum, particularly in the reticular formation. The intense oxidase reactions were present in the red nucleus, oculomotor nucleus, trochlear nucleus, ventral nucleus of the lateral lemniscus, dorsal and ventral cochlear nuclei, vestibular nuclei, nuclei of posterior funiculus, nucleus of the spinal tract of the trigeminal nerve, lateral reticular nucleus, inferior olivary nucleus, and hypoglossal nucleus. In the cerebellum the activity in the cortex was much more intense than that in the medulla. In all the fields described above, the oxidase-containing cells were exclusively astrocytes including Bergmann glial cells, and neither neuronal components, endothelial cells, oligodendrocytes nor ependymal cells showed oxidase activity. These results indicated that the astrocytes regionally differentiated into two distinct types, one of which expressed oxidase in the midbrain, rhombencephalon and spinal cord, and the other which did not in the forebrain. The localization of the oxidase was inversely correlated with the distribution of free D-serine in mammalian brains (Nagata, Y., Horiike, K. and Maeda, T., Brain Res., 634 (1994) 291-295). Based on the characteristic localization of the oxidase-containing astrocytes, we discussed the physiological role of the oxidase.

Topographic atlas of monoamine oxidase-containing neurons in the rat brain studied by an improved histochemical method

The distribution of monoamine oxidase-containing neuronal somata was studied in the rat brain by using an improved enzyme histochemical technique of the coupled peroxidatic oxidation method applied to fixed free-floating sections. The majority of monoamine oxidase-containing neuronal somata appeared to correspond with well-known cell groups of monoamine-containing neurons with a few exceptions. The enzyme appeared to coexist also in histamine-containing neurons in the posterior hypothalamus. Furthermore, monoamine oxidase activity was localized in apparently non-monoaminergic cells in the mesencephalon, hypothalamus, thalamus and telencephalon.

Substance P-containing axon terminals in the mucosa of the human urinary bladder: pre-embedding immunohistochemistry using cryostat sections for electron microscopy.

The ultrastructure of substance P (SP)-containing axon terminals in the mucosa of the human urinary bladder was studied. Numerous SP-immunoreactive varicose nerve fibers were seen in the lamina propria, and most of them ran freely in the connective tissue. Many SP-immunoreactive nerve fibers were observed beneath the epithelium, and perivascular SP-immunoreactive nerves were also found in the submucosal layer. We observed a total of 305 SP-immunoreactive (IR) axon terminals, of which most (89.6%) were free nerve endings at the ultrastructural level; the rest of the SR-IR axon terminale were seen in the vicinity of the epithelium and blood vessels in the lamina propria. Varicose regions of SP-IR axon terminals contained large granular and small agranular synaptic vesicles, and most of them partially lacked a Schwann cell sheath. In some SP-IR varicosities, synaptic vesicles were concentrated in the region without any Schwann cell sheath. Long storage (for more than 1 month) of fixed-tissue pieces in sucrose before freezing has improved the ultrastructure of cryostat sections in pre-embedding immunohistochemistry. Trypsin digestion for the purpose of exposing antigenic sites was also employed before applying the first antiserum.

Immunohistochemical detection of L-DOPA-derived dopamine within serotonergic fibers in the striatum and the substantia nigra pars reticulata in Parkinsonian model rats.

On the basis of our previous studies in the normal rat [Arai, R., Karasawa, N., Geffard, M., Nagatsu, I., 1995. L-DOPA is converted to dopamine in serotonergic fibers of the striatum of the rat: a double-labeling immunofluorescence study. Neurosci. Lett. 195, 195-198; Arai, R., Karasawa, N., Nagatsu, I., 1996a. Aromatic L-amino acid decarboxylase is present in serotonergic fibers of the striatum of the rat. A double-labeling immunofluorescence study. Brain Res. 706, 177-179; Arai, R., Karasawa, N., Nagatsu, I., 1996b. Dopamine produced from L-DOPA is degraded by endogenous monoamine oxidase in neurons of the dorsal raphe nucleus of the rat: an immunohistochemical study. Brain Res. 722, 181-184] we have assumed that exogenously administered L-dihydroxyphenylalanine (L-DOPA) is converted into dopamine (DA) in serotonergic (5-HT) fibers within the striatum (ST) and the substantia nigra pars reticulata (SNR). In the present study, an attempt was made to confirm the assumptions in Parkinsonian rats, which were produced by unilateral injections of 6-hydroxydopamine (6-OHDA) into the substantia nigra pars compacta (SNC). The rats exhibiting more than 150 total controversial circles were regarded as satisfactory models of Parkinson disease (PD). Using a dual immunofluorescence histochemistry, we examined DA-immunoreactivity in the 5-HT fibers within the ST and the SNR of the PD model rats after L-DOPA was injected intraperitoneally. In experimental cases with the L-DOPA administration, DA-immunoreactivity was detected in 5-HT fibers in both the ST and the SNR on the 6-OHDA injection side; no DA-immunoreactivity was found in 5-HT fibers in the ST or the SNR in control cases without the L-DOPA administration. The results support the assumption that exogenously administered L-DOPA may be converted into DA within the 5-HT fibers in the ST and SNR of the PD model rats.

Localization of D-amino acid oxidase in Bergmann glial cells and astrocytes of rat cerebellum.

The localization of D-amino acid oxidase in rat cerebellum was systematically studied in serial fixed sections at the levels of both light and electron microscopy using a coupled peroxidation method based on the intensifying effect of nickel ions. Deposits were only seen in astrocytes and Bergmann glial cells, and not in neuronal components, endothelial cells or ependymal cells. In the molecular layer, heavy deposits were present in the profiles of Bergmann glial processes around the complexes of synapses where the parallel fiber varicosities form synapses with the thorns emerging from the spiny branchlets of Purkinje cell dendrites. In the Purkinje cell layer, the oxidase-containing processes of Bergmann glial cells enveloped basket cell axons, their terminals, the terminals of the recurrent collaterals of Purkinje cell axons and Purkinje cell bodies. In the granular layer, the cerebellar glomeruli were enveloped by the heavily stained processes of astrocytes. Based on this characteristic localization of the oxidase, we discussed the physiological role of the oxidase in connection with the function of glial cells.

Demonstration of monoamine oxidase type B in serotonergic and type A in noradrenergic neurons in the cat dorsal pontine tegmentum by an improved histochemical technique

Application of a diaminobenzidine-coupled peroxidation method to monoamine oxidase (MAO) histochemistry demonstrates a detailed morphology of MAO-containing perikarya, dendrites, axons and fibers in the dorsal pontine tegmentum of the cat, sectioned with a vibratome. Further, utilization of specific inhibitors, deprenyl and clorgyline, also provides evidence that serotonergic neurons contain exclusively MAO-B and catecholaminergic ones contain MAO-A.

Differential subcellular location of mitochondria in rat serotonergic neurons depends on the presence and the absence of monoamine oxidase type B

Monoamine oxidase type A and type B are major neurotransmitter-degrading enzymes in the CNS. The type A is present on mitochondrial outer membranes in the whole extent of noradrenergic and dopaminergic neurons, including their axon terminals. The type B is present in serotonergic neurons, but its subcellular localization has not been elucidated. In the present study, we used both a double-labeling immunofluorescence method and electron microscopic immunohistochemistry to examine the subcellular localization of monoamine oxidase type B in serotonergic neurons projecting from the dorsal raphe nucleus to the suprachiasmatic nucleus in the rat brain. In the dorsal raphe nucleus, serotonin-positive neuronal cell bodies were clustered, and virtually all of these cell bodies were also positive for monoamine oxidase type B. By contrast, serotonin-negative neuronal cell bodies were mostly free of this enzyme. Within the neuronal cell bodies and dendrites that were positive for monoamine oxidase type B, most mitochondria contained this enzyme on their outer membranes, but a substantial proportion of mitochondria lacked this enzyme. In the suprachiasmatic nucleus, serotonin-positive varicosities were concentrated, but none of these varicosities exhibited monoamine oxidase type B. In this nucleus, mitochondria were found in almost all serotonin-positive axon terminals, but monoamine oxidase type B was not observed in any axon terminal that contained mitochondria. Our results show that there are two kinds of mitochondria in serotonergic neuronal cell bodies and dendrites: one containing monoamine oxidase type B on their outer membranes, and the other lacking this enzyme. In addition, mitochondria in serotonergic axon terminals do not possess monoamine oxidase type B. It is suggested in serotonergic neurons that only mitochondria lacking monoamine oxidase type B are transported by axonal flow up to axon terminals. It is also probable that mitochondria containing monoamine oxidase type B are transported along the axons, but that this enzyme undergoes a change, for example, conformational change, decomposition or removal from the membranes.

Inhibition of recombinant dipeptidyl peptidase III by synthetic hemorphin-like peptides

In order to find the most effective antagonist for dipeptidyl peptidase III degrading enkephalin, we synthesized hemorphin-like pentapeptides with aliphatic or aromatic amino acids at the N-termini, such as VVYPW, LVYPW, IVYPW, YVYPW, FVYPW and WVYPW. Among those pentapeptides, IVYPW and WVYPW showed the strongest inhibitory activity toward rDPP III. The K(i) values of IVYPW and WVYPW were 0.100+/-0.011 and 0.126+/-0.015 microM (mean+/-S.E.), respectively. The order of K(i) values was Ile> or =Trp>Phe> or =Tyr>Leu>Ala>Val>Ser>Gly. rDPP III activity is inhibited in a non-competitive manner by these peptides. The peptide VYPW did not inhibit rDPP III activity, but the sequence is essential for the expression of inhibitory activity.

Localization of L-DOPA uptake and decarboxylating neuronal structures in the cat brain using dopamine immunohistochemistry.

The present study examined dopamine-immunoreactive neuronal structures using immunohistochemistry in conjunction with an anti-dopamine antiserum, following injection of l-3,4-dihydroxyphenylalanine (L-DOPA) with or without an inhibitor of monoamine oxidase (Pargyline) in the cat brain. L-DOPA injection made it possible to detect dopamine immunoreactivity in presumptive serotonergic and noradrenergic cell bodies and axons. Weak to moderate dopamine immunoreactivity was observed in non-aminergic cells (possibly so-called D cells containing aromatic L-amino acid decarboxylase (AADC)) in several hypothalamic, midbrain, pontine and medullary nuclei. Intense dopamine immunoreactivity became visible in a large number of cells and axons (possibly containing AADC) with wide distribution in the brain following administration of L-DOPA with Pargyline. AADC is most likely active in cells and axons that take up L-DOPA, where it decarboxylates the L-DOPA to dopamine. However, newly synthesized dopamine in such cells is rapidly oxidized by monoamine oxidase.