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 neuron's 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 Parkinson's disease and related syndromes.

Isolation of specific peptides that home to dorsal root ganglion neurons in mice.

We isolated peptides that home to mouse dorsal root ganglion (DRG) from a phage library expressing random 7-mer peptides fused to a minor coat protein (pIII) of the M13 phage. An in vitro biopanning procedure yielded 113 phage plaques after five cycles of enrichment by incubation with isolated DRG neurons and two cycles of subtraction by exposure to irrelevant cell lines. Analyses of the sequences of this collection identified three peptide clones that occurred repeatedly during the biopanning procedure. Phage-antibody staining revealed that the three peptides bound to DRG neurons of different sizes. To determine if the peptides would recognize neuronal cells in vivo, we injected individual GST-peptide-fusion proteins into the subarachnoid space of mice and observed the appearance of immunoreactive GST in the cytosol of DRG neurons with a similar size distribution as that observed in vitro, indicating that the GST-peptide-fusion proteins were recognized and taken up by different DRG neurons in vivo. The identification of homing peptide sequences provides a powerful tool for future studies on DRG neuronal function in vitro and in vivo, and opens up the possibility of neuron-specific drug and gene delivery in the treatment of diseases affecting DRG neurons.

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.

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.

Fusion of proinsulin-producing bone marrow-derived cells with hepatocytes in diabetes.

We previously reported that diabetes in mice is associated with the appearance of proinsulin-producing (Proins-P) cells in the liver. It was unclear, however, whether these Proins-P bone marrow-derived cells (BMDC) merely transit through the liver or undergo fusion with hepatocytes, normally an extremely rare event. In this study, we found that, in diabetes, BMDC in the liver produce not only Proins but also TNF-alpha, suggesting that diabetes reprograms gene expression in BMDC, turning on "inappropriate" genes. Bone marrow transplantation using genetically marked donor and recipient mice showed that fusion occurs between Proins-P BMDC and hepatocytes. Cell fusion is further supported by the presence of the Y chromosome in Proins-P cells in female mice that received male bone marrow transplantation cells. Morphologically, Proins-P fusion cells are albumin-producing hepatocytes that constitute approximately 2.5% of the liver section area 5 months after diabetes induction. An extensive search failed to reveal any fusion cells in nondiabetic mice. Thus, diabetes causes fusion between Proins-P BMDC and hepatocytes in vivo, an observation that has implications for the pathophysiology of diabetes as well as the fundamental biology of heterotypic cell fusion.

Presence of monoamine oxidase type B protein but absence of associated enzyme activity in neurons within the inferior olive nucleus of the rat.

A previous study demonstrated that monoamine oxidase type B (MAOB) mRNA is located in the inferior olive complex (IO). The purpose of the present study was to examine whether neuronal cell bodies within the IO also express MAOB protein and whether they exhibit associated MAOB enzyme activity. Using immunohistochemistry and enzyme histochemistry, we demonstrated that IO neuronal cell bodies were positive for MAOB immunohistochemistry but negative for MAOB enzyme histochemistry. These findings indicate that IO neuronal cell bodies express MAOB mRNA and produce MAOB protein but curiously do not exhibit MAOB enzyme activity, as might be expected. The mechanism responsible for the failure of MAOB protein to result in enzymatic activity in IO neuronal cell bodies is clearly of significance in terms of functionality but remains to be elucidated.

Immunohistochemical localization of monoamine oxidase type B in pancreatic islets of the rat.

Monoamine oxidase (MAO) is regarded as a mitochondrial enzyme. This enzyme localizes on the outer membrane of mitochondria. There are two kinds of MAO isozymes, MAO type A (MAOA) and type B (MAOB). Previous studies have shown that MAOB activity is found in the pancreatic islets. This activity in the islets is increased by the fasting-induced decrease of plasma glucose level. Islet B cells contain monoamines in their secretory granules. These monoamines inhibit the secretion of insulin from the B cells. MAOB is active in degrading monoamines. Therefore, MAOB may influence the insulin-secretory process by regulating the stores of monoamines in the B cells. However, it has not been determined whether MAOB is localized on B cells or other cell types of the islets. In the present study, we used both double-labeling immunofluorescence histochemical and electron microscopic immunohistochemical methods to examine the subcellular localization of MAOB in rat pancreatic islets. MAOB was found in the mitochondrial outer membranes of glucagon-secreting cells (A cells), insulin-secreting cells (B cells), and some pancreatic polypeptide (PP)-secreting cells (PP cells), but no MAOB was found in somatostatin-secreting cells (D cells), nor in certain other PP cells. There were two kinds of mitochondria in pancreatic islet B cells: one contains MAOB on their outer membranes, but a substantial proportion of them lack this enzyme. Our findings indicate that pancreatic islet B cells contain MAOB on their mitochondrial outer membranes, and this enzyme may be involved in the regulation of monoamine levels and insulin secretion in the B cells.

Immunohistochemical localization of monoamine oxidase type B in the taste bud of the rat.

We have used immunohistochemistry to examine the subcellular localization of monoamine oxidase type B (MAO-B) in the taste bud of the rat circumvallate papilla. Electron microscopy showed that MAO-B was localized to the outer membranes of mitochondria in nerve terminals of afferent and efferent fibers, as well as in taste bud cells. MAO-B also existed on the mitochondrial outer membranes within myelinated and unmyelinated axons in the lamina propria beneath the taste bud. It is suggested that MAO-B-containing mitochondria are localized in peripheral branches and their terminals of sensory neurons for taste. The present study is the first to reveal the localization of MAO-B in sensory organs.

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.