山田 敬喜

A morphological and immunohistochemical study of the ultimobranchial body of reptiles Japanese lizard and snake was carried out. The ultimobranchial body of the Japanese lizard was located adjacent to the left arch of the aorta between the trachea and esophagus. It was found as a cluster or group of cells with no capsule. Grimelius' silver impregnation and lead-hematoxylin staining produced positive reactions in some of the clustered cells and follicular cells. The same reaction pattern was observed with anti-calcitonin using the PAP method. The PAP reactions were positive to antiserum against pig calcitonin, but negative to antiserum against synthesized human calcitonin. Furthermore, the PAP reactions were negative to antiserum against tyrosine hydroxylase. The immunofluorescent study of the snake ultimobranchial body revealed that most of the clustered cells and some of the follicular cells were calcitonin-immunoreactive but none was tyrosine hydroxylase-immunoreactive. Certain histological similarities exist between the Japanese lizard ultimobranchial body and snake ultimobranchial body, but the distribution of calcitonin-positive cells were slightly different. In the Japanese lizard, the positive cells were scattered between the folicles and the number was small. However, most of the cells which formed the cluster in the ultimobranchial body of snake were positive. The findings suggest that the configuration of amino acid in the Japanese lizard calcitonin and snake calcitonin are similar to that of pig calcitonin, and the reptile and the birds is a boundary of the tyrosine hydroxylase existence.

A morphological and immunohistochemical study of the ultimobranchial body of reptiles Japanese lizard and snake was carried out. The ultimobranchial body of the Japanese lizard was located adjacent to the left arch of the aorta between the trachea and esophagus. It was found as a cluster or group of cells with no capsule. Grimelius' silver impregnation and lead-hematoxylin staining produced positive reactions in some of the clustered cells and follicular cells. The same reaction pattern was observed with anti-calcitonin using the PAP method. The PAP reactions were positive to antiserum against pig calcitonin, but negative to antiserum against synthesized human calcitonin. Furthermore, the PAP reactions were negative to antiserum against tyrosine hydroxylase. The immunofluorescent study of the snake ultimobranchial body revealed that most of the clustered cells and some of the follicular cells were calcitonin-immunoreactive but none was tyrosine hydroxylase-immunoreactive. Certain histological similarities exist between the Japanese lizard ultimobranchial body and snake ultimobranchial body, but the distribution of calcitonin-positive cells were slightly different. In the Japanese lizard, the positive cells were scattered between the folicles and the number was small. However, most of the cells which formed the cluster in the ultimobranchial body of snake were positive. The findings suggest that the configuration of amino acid in the Japanese lizard calcitonin and snake calcitonin are similar to that of pig calcitonin, and the reptile and the birds is a boundary of the tyrosine hydroxylase existence.

Changes in the parafollicular cells in the thyroid gland of orariectomized rats were investigated to clarify the relationship between the secretory function of sex hormones and that of parafollicular cells. Compared with control rats, the ovariectomized rats exhibited decreases in 1) the number of parafollicular cells in the thyroid glands, 2) the number of secretory granules in the parafollicular cells, and 3) the area occupied by the Golgi complex. These results suggest that the lack of estrogen caused by ovariectomy reduces the synthesis and release of calcitonin in parafollicular cells, which may be one of the causes of osteoporosis.

The distribution of monoamine oxidase (MAO)-containing neuronal somata was studied in the grass parakeet (Melopsittacus undulatus) brain by using a histochemical technique involving the coupled peroxidatic oxidation method, and compared with the distribution of the tyrosine hydroxylase-, aromatic L-amino acid decarboxylase-, serotonin-immunoreactive cells in the grass parakeet brain. MAO-containing neurons were located in the nucleus locus coeruleus, around the nucleus n. facialis (nVII), lateral region to the nucleus olivaris inferior and nucleus tractus solitarius, where noradrenaline neurons exist. Another group was located in the ventral region of nervi oculomotorii, caudal region of nucleus n. oculomotorii (nIII), the nucleus raphe and ventral to the nucleus olivaris superior, where serotonin neurons exist. A third group was the non-monoaminergic neurons which were located in the lobus parolfactorius, pareostriatum augmentatum and ventrolateral part of formatio reticularis mesencephali.

Agonist-induced regulation of adrenergic receptors (ARs) has an important role in controlling physiological functions in response to changes in catecholamine stimulation. We previously generated transgenic mice expressing phenylethanolamine N-methyltransferase (PNMT) under the control of a human dopamine beta-hydroxylase gene promoter to switch catecholamine specificity from the norepinephrine phenotype to the epinephrine phenotype. In the present study, we first examined changes in catecholamine metabolism in peripheral tissues innervated by sympathetic neurons of the transgenic mice, in the transgenic target tissues, a high-level expression of PNMT led to a dramatic increase in the epinephrine levels, whereas the norepinephrine levels were decreased to 48.6-87.9% of the nontransgenic control levels. Analysis of plasma catecholamines in adrenalectomized mice showed large amounts of epinephrine derived from sympathetic neurons in the transgenic mice. Subsequently, we performed radioligand binding assays with (-)-[I-125]iodocyanopindolol to determine changes in binding sites of beta-AR subtypes. In transgenic mice, the number of beta 2-AR binding sites was 56.4-74.9% of their nontransgenic values in the lung, spleen, submaxillary gland, and kidney, whereas the beta 1-AR binding sites were regulated in a different fashion among these tissues. Moreover, northern blot analysis of total RNA from the lung tissues showed that down-regulation of beta 2 binding sites was accompanied by a significant decrease in steady-state levels of the receptor mRNA. These results strongly suggest that alteration of catecholamine specificity in the transgenic sympathetic neurons leads to regulated expression of the beta-AR subtypes in their target tissues.

We attempted to compare the expression of two different reporter genes, human phenylethanolamine N-methyltransferase (PNMT) cDNA, or bacterial chloramphenicol acetyltransferase (CAT) cDNA, connected to a 5-kb fragment from the 5'-flanking region of human tyrosine. hdroxylase (TH) gene in transgenic mice. As reported previously, CAT was expressed in catecholaminergic (CAnergic) neurons. Human PNMT was also expressed in CAnergic neurons. Interestingly, however, either PNMT or CAT was expressed in dopaminergic and adrenergic neurons, but scarcely in noradrenergic neurons. The results suggest different promoter activity of the 5'-flanking region of human TH gene among different CAnergic neurons.

We investigated the localization of APUD (amine content and amine precursor uptake and decarboxylation) cells and tyrosine hydroxylase (TH)-negative, aromatic L-amino acid decarboxylase (AADC)-positive neurons (D neurons) in the hypothalamus of the bird (Melopsittacus undulatus) and the lizard (Varanus exanthematicus), and compared the differences between the two species, L-DOPA was demonstrated in the paraventricular organ (PVO) which is a characteristic region in the hypothalamus of the bird and the lizard. In the PVO of the grass parakeet, there were a small number of dopamine neurons in addition to APUD cells. However, in the PVO of the lizard, there were APUD cells but no dopamine neurons, Furthermore, TH-negative, AADC-positive neurons (D neurons) were found in the medial part of nucleus arcuatus of the bird, but not of the lizard.

Dopamine beta-hydroxylase (DBH; EC 1.14.17.1) catalyzes the production of the neurotransmitter and hormone norepinephrine in the third step of the catecholamine biosynthesis pathway. Transgenic mice were generated with multiple copies of a human DBH minigene construct containing the full-length cDNA connected downstream of the 4-kilobase upstream promoter region to achieve overexpression of DBH. Human DBH mRNA and immunoreactivity were detected tissue-specifically in the brain and adrenal gland of these transgenic mice. The transgene products were correctly processed to a glycosylated mature polypeptide with a molecular mass of 72 kDa and existed in the secretory vesicles as both soluble and membrane-bound forms. We detected a marked increase in DBH activity in various catecholamine-containing tissues of the mice that occurred as a consequence of expression of the catalytically active human DBH enzyme. However, in these transgenics the steady-state levels of norepinephrine and epinephrine were normally maintained without the acceleration of the catecholamine turnover rate, suggesting that there are some regulatory mechanisms to preserve a constant rate of norepinephrine synthesis in spite of the increased amount of DBH protein. These transgenic mice with the minigene construct provide one approach to study the mechanisms underlying biogenesis of the DBH polypeptide and regulation of norepinephrine synthesis.

After 5-hydroxy-L-tryptophan (5-HTP) and L-3,4-dihydroxyphenylalanine (L-DOPA) were injected i.p. in the laboratory shrew Suncus murinus, immunocytochemical and immunofluorescence studies were conducted on continuous or same sections of the brain, using specific anti-tyrosine hydroxylase (TH), anti-aromatic L-amino acid decarboxylase (AADC), anti-dopamine (DA) and antiserotonin (5-HT) antisera which were produced in our laboratory. The results of double-staining by the immunofluorescence method as well as immunoelectron microscopy strongly indicate that the cells of the premammillary nucleus of the laboratory shrew brain (AADC-only-positive neurons) are capable of synthesizing DA and 5-HT simultaneously upon simultaneous administration of L-DOPA and 5-HTP.

After L-3,4-dihydroxyphenylalanine (L-DOPA) was injected intraperitoneally in the laboratory shrew (Suncus murinus), the animals were anesthetized and perfused with 5% glutaraldehyde solution. Serial sections of the brain were reacted immunohistochemically with specific anti-tyrosine hydroxylase (TH), anti-DOPA, anti-aromatic L-amino acid decarboxylase (AADC), and anti-dopamine (DA) antisera which were produced in our laboratory. We observed that all TH-negative, AADC-positive neurons (designated as D neurons in the rat brain by Jaeger) showed anti-DA immunopositive reaction. Our findings strongly suggest that D neuron system belongs to the APUD (amine precursor uptake and decarboxylation) system.

L-DOPA immunoreactivity was demonstrated in neurons of the house-shrew brain, using an immunocytochemical method in conjunction with a specific anti-L-DOPA serum. The neurons in the ventrolateral area of arcuate nuclei were tyrosine hydroxylase- and L-DOPA-immunoreactive, but aromatic L-amino acid decarboxylase- and dopamine-negative. Most of these neurons were small, round or oval in shape, and ranged from 10 to 20 mum in diameter with their long axonal processes extended within the third ventricular wall. By immunoelectron microscopy, L-DOPA-positive products were observed throughout the cytoplasmic matrix and its terminal processes which contained small (60 to 80 nm) or large (140 to 160 nm) vesicles.

We have produced transgenic (Tg) mice carrying 5.0-kb fragment from the 5'-flanking region of the human tyrosine hydroxylase (hTH) gene fused to a reporter:gene, chloramphenicol acetyltransferase (CAT) [Sasaoka et al. (1992) Mol Brain Res 16: 274-286]. In the brain of the Tg mice, CAT expression has been observed in catecholaminergic (CAnergic) neurons and also in non-CAnergic neurons. The aim of the present study is to examine in detail the cell-type specific expression of the hTH-CAT fusion gene in the brain of the Tg mice, by use of immunohistochemistry for CAT, TH, and aromatic L-amino acid decarboxylase (AADC). CAT-immunoreactive cells were found in CAnergic brain regions which contained TH-positive cells, and also in non-CAnergic brain regions which contained no TH-labeled cells. The non-CAnergic brain regions that represented CAT-stained cells were further divided into two groups: (i) regions containing AADC-labeled cells, for example, bed nucleus of the stria terminalis, nucleus suprachiasmaticus, mammillary body, nucleus raphe dorsalis, inferior colliculus, and nucleus parabrachialis, and (ii) regions containing no AADC-positive cells, for example, main olfactory bulb (except A16), accessory olfactory bulb, nucleus olfactorius anterior, caudoputamen, septum, nucleus accumbens, hippocampus, medial nucleus of the amygdala, entorhinal cortex, nucleus supraopticus, and parasubiculum. The results indicate that the 5.0-kb DNA fragment flanking the 5' end of the hTH gene may contain the element(s) specific for neuron-specific TH expression but which may be insufficient to attenuate ectopic expression.

We have previously reported the distribution of human tyrosine hydroxylase (TH) transgene expression in dopaminergic neurons (ventral tegmental area and substantia nigra), adrenal gland, and non-catecholaminergic neurons in the forebrain of transgenic (Tg) mice. In this paper, we analysed the transgene expression in catecholaminergic (CAergic) neurons in the lower brainstem of Tg mice, by in situ hybridization and immunocytochemistry at the light and electron microscopic levels. High-level hybridization signals of the human TH mRNA were observed in the locus ceruleus and nucleus tractus solitarii of the Tg brain. Intense TH immunoreactivity was expressed specifically in the Tg brainstem, as was observed in non-Tg mice. These results reveal that the human TH transgene contains the regulatory elements responsible for the expression in three kinds of CAergic (dopaminergic, noradrenergic and adrenergic) neurons of the mouse brain.