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We previously reported the presence of transiently tyrosine hydroxylase (TH)-only-immunoreactive (ir), non-catecholaminergic (non-CAnergic) neurons in some brain regions of postnatal mice; anterior olfactory nucleus, medial geniculate nucleus, and spinal trigeminal nucleus, where CAnergic terminals but not cell bodies are present in the adult wild mouse. These transiently TH-ir brain regions of the postnatal wild mouse showed stable TH-immunoreactivity in the adult brain of the human TH transgenic (hTHTg) mice. TH expression was also observed in the nucleus parabigeminalis of the hTHTg mice. Using the antiserum against GTP cyclohydrolase I (GCH), first rate-limiting enzyme of the biosynthesis of tetrahydrobiopterin (BH4), the cofactor for TH, we proved that these TH-only-ir neurons in the wild mice and in the hTHTg mice were not stained with the antiserum against GCH. The results indicate that these TH-only-ir neurons which do not synthesize the BH4 cofactor do not produce dihydroxyphenylalanine, suggesting a new unknown function of TH in these neurons.

Immunoreactivity (IR) of tyrosine hydoroxylase (TH), which is the rate- limiting enzyme of catecholamine (CA) synthesis, was observed in the serotonergic neurons of the raphe nucleus (RN) of the newborn laboratory shrew from postnatal day (P) 0 to P14. Using an immunohistochemical method involving highly specific antibodies produced in our laboratory, we found that these RN neurons were TH-, GTP cyclohydrolase I-, aromatic L-amino acid decarboxylase-immunoreactive, but DOPA- and dopamine-immunonegative. In addition, they were tryptophan-, 5-hydroxytryptophan- and serotonin-immunoreactive. These results suggest that TH in serotonergic neurons of RN of laboratory shrew has no function as a CA-synthesizing enzyme but may play some role as a regulator or a subsidiary factor in the postnatal development of serotonergic neurons.

Publisher Summary Tyrosine hydroxylase (TH) has been used as a marker enzyme of catecholamine (CA) biosynthesis, such as dopamine (DA), noradrenaline, and adrenaline. GTP cyclohydrolase I (GCH) is the first enzyme for biosynthesis of tetrahydrobiopterin (BH4), the cofactor of phenylalanine hydroxylase, TH, tryptophan hydroxylase, and nitric oxide synthase. TH and GCH, aromatic L-amino acid decarboxylase (AAD), or serotonin (5HT), have been detected on the serial sections of the mouse brain by use of an avidin-biotin peroxidase complex (ABC) method. The transient appearance of GCH-immunoreactive (IR) cells in the ventral lateral geniculate (VLG) nuclear region of mice has been detected by immunocytochemistry. The transient appearance of TH-IR cells in the medial geniculate (MG) nuclear region of mice has been investigated at P7. The numbers of TH-IR cells have reached maximum at P14 to P21 and decreased at P29. These TH-IR cells have been GCH-negative, AAD-negative, DA-negative, and do not belong to the CAnergic neuron system, because they do not produce DA. The novel TH-IR/GCH-negative cells are distributed from MG to nucleus brachium inferior colliculus. Novel GCH-IR cells in the VLG are TH-negative, and novel TH-IR cells in the MG are GCH-negative, suggesting that both GCH and TH have new independent functions in the GCH-IR or TH-IR cells.

Pheochromocytoma is a catecholamine (CA)-producing tumor that is classified into two types: the norepinephrine (NE) and the mixed NE and epinephrine type (E-type) from plasma CA levels. Phenylethanolamine N-methyltransferase (PNMT) is the terminal enzyme in CA synthesis; it catalyzes the synthesis of E from NE. It is not known whether the absence of immunoreactive PNMT is paralleled by a lack of PNMT mRNA. The mRNA of tyrosine hydroxylase (TI-I) and PNMT in seven pheochromocytomas, five NE-type and two E-type tumors, were examined by Northern blot analysis and in situ hybridization (ISH) technique. TH mRNA was detected in all tumors but PNMT mRNA was limited only to the E-type tumors. In addition to our previous immunohistochemical study of 70 pheochromocytomas and paragangliomas in which all pheochromocytomas had cells immunoreactive to TH, but PNMT was expressed only in E-type, we concluded that NE-type pheochromocytoma lacks PNMT both at the mRNA and protein levels, resulting in an inability to produce E. The essential difference between NE-type and E-type pheochromocytoma is that the NE-type lacks PNMT.

The aim of the present study is to examine by immunohistochemistry whether dopamine produced from L-DOPA in serotonin neurons of the rat brain is degraded by endogenous monoamine oxidase (MAO). In rats that received intraperitoneally L-DOPA plus a peripheral decarboxylase inhibitor, carbidopa, a cluster of dopamine-immunoreactive neurons was found in the dorsal raphe nucleus (DR). In L-DOPA/carbidopa-injected rats that were pretreated with an intraperitoneal injection of a MAO inhibitor, pargyline, when compared with the L-DOPA/carbidopa-injected rats without the pargyline pretreatment, neurons of the cluster of the DR became much darker in dopamine staining. The distribution of dopamine-stained neurons in the DR of these rats corresponded very closely to the previously reported distribution of serotonin-immunoreactive neurons of normal rats. In normal or only pargyline-injected rats, dopamine-stained neurons were scarcely observed in the DR. We previously showed that serotonin neurons of the rat DR were induced to contain dopamine by the injection of L-DOPA plus carbidopa. These findings suggest that the newly produced dopamine from L-DOPA in serotonin neurons of the rat DR is degraded by endogenous MAO.

Immunohistochemistry of dopamine-beta-hydroxylase in the human hind brain indicates that neuronal cell bodies containing the antigen form prominent populations in the nucleus tractus solitarius and nearby medial and dorsal edge of the medial vestibular nucleus. They are frequent in and around the periphery of the dorsal motor nucleus of the vagus and in an oblique band extending from that region to the ventrolateral aspect of the reticular formation, where they are most numerous at the mid medullary levels. Dopamine-beta-hydroxylase immunoreactive neurons are also closely packed in the nuclei coeruleus and subcoeruleus. Concomitant immunohistochemistry for tyrosine hydroxylase demonstrates small numbers of neuronal cell bodies that are reactive only for this antigen, and which do not contain detectable dopamine-beta-hydroxylase. Such neurons are present in the nucleus tractus solitarius, the pontine lateral parabrachial nucleus and within the core of the rostral pontine reticular formation. Some medullary and pontine axon bundles similarly stain for tyrosine hydroxylase but not for dopamine-beta-hydroxylase. These differential staining patterns suggest, among other possibilities, that in humans some neurons of the caudal brainstem are dopamine (if they contain the second step catecholamine synthesizing enzyme, aromatic L-aminoacid decarboxylase) rather than noradrenaline or adrenaline containing catecholamine neurotransmitters.

Tyrosine 3-hydroxylase (TH, EC 1.14.16.2) catalyzes the first and rate-limiting step of the catecholamine biosynthetic pathway in the nervous and endocrine systems. The TH locus was disrupted in mouse embryonic stem cells by homologous recombination. Mice heterozygous for the TH mutation were apparently normal. In these mice, TH activity in the embryos and adult tissues was less than 50% of the wild-type values, but the catecholamine level was decreased only moderately in the developing animals and was maintained normally at adulthood, suggesting the presence of a regulatory mechanism for ensuring the proper catecholamine level during animal development. In contrast, the homozygous mutant mice died at a late stage of embryonic development or shortly after birth. Both TH mRNA and enzyme activity were lacking in the homozygous mutants, which thus explained the severe depletion of catecholamines. These changes, however, did not affect gross morphological development of the cells that normally express high catecholamine levels. Analysis of electrocardiograms of surviving newborn mutants showed bradycardia, suggesting an alteration of cardiac functions in the homozygous mice that may lead to the lethality of this mutation. In addition, transfer of a human TH transgene into the homozygous mice corrected the mutant phenotype, showing recovery of TH activity by expression of the human enzyme. These results indicate that TH is essential for survival of the animals during the late gestational development and after birth.

The aim of the present study is to examine whether serotonergic terminals of the rat brain are induced to contain dopamine by L-DOPA administration. In rats that received intraperitoneally L-DOPA plus a peripheral decarboxylase inhibitor, we showed by use of a double-labeling immunofluorescence method that dopamine was localized in serotonergic fibers of the striatum and cerebral cortex as well as in serotonergic cell bodies of the midbrain raphe nuclei. In normal rats, no dopamine was detected in the serotonergic fibers or cell bodies. The finding suggests that a part of the administered L-DOPA may enter the serotonergic terminals and be converted to dopamine in the rat brain.

The colocalization of serotonin and aromatic L-amino acid decarboxylase (AADC) in the avian pancreatic polypeptide-containing PP-cells and glucagon-storing A-cells of the chicken endocrine pancreas was investigated using combined pre-embedding immuno-peroxidase and post-embedding immunogold electron microscopic immunocytochemistry. The avian pancreatic polypeptide-immunoreactive cells manifested by the labeling of immunogold particles on secretory granules were also immunoreactive with antisera directed against serotonin and AADC, an enzyme involved in the synthesis of serotonin. In PP-cells immunoreactivity against the anti-serotonin serum was stronger in secretory granules than in the cytoplasmic matrix, whereas immunoreaction with the anti-AADC serum was observed to be more intense in the cytoplasmic matrix. Immunoreactions with the serotonin and AADC antisera were also found in secretory granules of glucagon-storing A-cells. These results indicate that serotonin is co-stored within secretory granules of both A- and PP-cells, and that AADC is localized within secretory granules of A-cells, and may be present in the cytoplasmic matrix of PP-cells. It is probable that serotonin is synthesized and released simultaneously with secretory granules from both A- and PP-cells of the chicken endocrine pancreas.

We examined by immunohistochemistry the effect of salt loading on the expression of tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and GTP cyclohydrolase I (GCH) in Purkinje cells of the mouse cerebellum. In control mice, only a few Purkinje cells were positive for TH or AADC. No Purkinje cells were stained for GCH. Drinking 2% sodium chloride for 2 weeks resulted in an increase in the number of TH- or AADC-positive Purkinje cells in the caudal vermis, paraflocculus and flocculus of the cerebellum. In contrast, no Purkinje cells were immunoreactive to GCH or L-DOPA after the salt loading. The present findings suggest that the salt loading differentially affects the expression of TH, AADC and GCH in Purkinje cells of the mouse cerebellum.

We immunocytochemically examined the effect of chronic salt loading on the content of calretinin, a calcium-binding protein, in both the supraoptic nucleus and the magnocellular parts of the hypothalamic paraventricular nucleus. In control rats that were given water for drinking, the supraoptic nucleus contained a cluster of calretinin-stained cells. Drinking 2% sodium chloride solution for 7 days resulted in an increase of the staining intensity of calretinin in cells of the supraoptic nucleus. In both the control and salt-loaded rats, the magnocellular parts of the paraventricular nucleus were almost devoid of calretinin-labeled cells. It is suggested that expression of calretinin in cells of the supraoptic nucleus is up regulated by chronic salt loading.

We have developed a transgenic approach, termed immunotoxin-mediated cell targeting (IMCT), to ablate conditionally selective neurons in the brain with the cytotoxic activity of immunotoxins. Transgenic mice were created that express the human interleukin 2 receptor alpha subunit (IL-2R alpha) under the control of the dopamine beta-hydroxylase (DBH) gene promoter. The animals were treated intracerebroventricularly with a recombinant immunotoxin, anti-Tac(Fv)-PE40, which selectively kills animal cells bearing human IL-2R alpha. The immunotoxin caused a characteristic behavioral abnormality only in the transgenic mice. This was accompanied by a dramatic loss of DBH-containing neurons and a significant decrease in DBH activity and norepinephrine levels in various regions of the brain. IMCT should provide a general technique to create animal models of human neurodegenerative disorders by targeting neurons or other cell types.

The aim of the present study is to examine by immunohistochemistry whether exogenous l -5-hydroxytryptophan ( l -5HTP) is decarboxylated in neurons of the substantia nigra pars compacta (SNC) and locus coeruleus (LC) of the rat. In normal rats, neurons of the SNC and LC stained intensely for aromatic l -amino acid decarboxylase (AADC). No serotonin (5HT)-positive cells were found in the two regions of the normal rats. In rats that were intraperitoneally injected with l -5HTP alone, the SNC neurons stained deeply for 5HT, but the LC neurons showed only a faint staining for 5HT. In rats that intraperitoneally received both a monoamine oxidase (MAO) inhibitor and l -5HTP, when compared with the l -5HTP-injected rats, the LC neurons became much darker in 5HT staining, but the SNC neurons showed only a slight increase in 5HT staining. The present findings suggest that (i) AADC in dopaminergic neurons of the SNC and in noradrenergic neurons of the LC can catalyze the in vivo decarboxylation of exogenous l -5HTP to produce 5HT, and (ii) most of the newly produced 5HT in the LC neurons is rapidly degraded by endogenous MAO.

Since 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to produce symptoms similar to Parkinson’s disease (Langston et al., 1983), toxins with structure similar to MPTP have been hypothesized to result in cell death of mesencephalic dopamine neurons. 1,2,3,4-Tetrahydroisoquinoline (TIQ) has a structure similar to MPTP, and it, and its derivatives exist as endogenous amines in human brain (Ohta et al., 1987; Niwa et al., 1987; Niwa et al., 1989). Toxic properties of TIQ on mitochondria are similar to those of MPP+ (the oxidative product of MPTP) (Suzuki et al., 1988). N-methyltetrahydroisoquinoline (NMTIQ) is formed from TIQ by N-methyltransferase (Naoi et al., 1989a). NMTIQ is oxidized by both type A and B monoamine oxidases, and the N-methylisoquinolinium ion (NMIQ+), the oxidative product, inhibits activities of enzymes related to catecholamine metabolism (Naoi et al., 1989b). NMTIQ and NMIQ+ inhibits complex I activity, similarly to TIQ, and this inhibitory potency of NMIQ+ on complex I activity is stronger than that of MPP+ (Suzuki et al., 1992). Recently, we administered TIQ and its derivatives (NMTIQ and NMIQ+) to monkeys and observed mesencephalic dopamine neurons immunohistochemically. Using light and electron microscopy we found that NMTIQ produces changes in mesencephalic dopamine neurons.

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 anti-serotonin (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.

The intracellular localization of tyrosine hydroxylase (TH), which is the rate limiting enzyme in catecholamine (CA) biosynthesis, and its activity in various adrenal and other neuroendocrine tumors was studied. TH was strongly localized in adrenal medulla, pheochromocytoma, and paraganglioma, but was scatteredly expressed in neuroblastoma. TH was not detected in adrenocortical tumors, ganglioneuroma, and other neuroendocrine tumors. Neuron specific enolase (NSE) was found in all neuroendocrine tumors, but Grimelius staining showed only the secreting granules of the tumor cells. TH activity was significantly high in pheochromocytoma and paraganglioma as compared with that in normal adrenal gland, whereas TH activity was low in a neuroblastoma and was undetectable in other tumors. These findings indicate that TH correlates well with the biosynthetic function of CA in the tumor cell and, thus, both the immunostaining of TH and the measurement of its activity in adreno-medullary and related tumors may provide some information about the process of cell differentiation in these tumors.

We examined whether or not the number of noradrenergic and adrenergic neurons changes with age, using peroxidase antiperoxidase (PAP) immunohistochemistry with specific antisera against tyrosine hydroxylase (TH) phenylethanolamine- N -methyltransferase (PNMT). TH- and PNMT-immunoreactive neurons were counted in every noradrenergic and adrenergic neuron group of young (3-month-old) and old (24-month-old) rats. The differences in the counted number of TH- and PNMT-immunoreactive neurons did not reach statistical significance between the young and old rats.

We demonstrated the expression of tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) in cerebellar Purkinje cells of human and mouse brains by immunohistochemistry. At the age of 8 months, vermal Purkinje cells in the human cerebellum were immunoreactive with anti-TH or anti-human specific PNMT antiserum. We also found TH-positive Purkinje cells in the mouse brain. TH immunoreactivity first appeared in the Purkinje cells in the caudal vermis from postnatal day 8 (P8), and enlarged numbers between P13 and P15. After gradual reduction to P19, the TH-positive Purkinje cells again multiplied the number from the age of 4 weeks, and maintained in a large number up to old stage. At the age of 11 months, the other groups of TH-positive Purkinje cells appeared in the flocculus and paraflocculus of the mouse cerebellum.

The midline of the rostral ventral medulla (RVM) is the portion in which many serotonin (5-HT) neurons of the nucleus raphe magnus and the rostral nucleus raphe pallidus are located and where dense catecholaminergic (CA) fibers are distributed. In this study, we investigated the connection between spinally projecting 5-HT neurons and CA fibers in the rat RVM by light and electron microscopic immunocytochemistry. First, light microscopic immunocytochemistry using a triple labeling method revealed that the 5-HT-immunoreactive (IR) neuron containing retrograde tracer from the cervical cord was intimately surrounded by tyrosine hydroxylase (TH)-IR fibers. Second, silver-gold intensified TH-IR axon terminals were found to make synaptic contacts with 5-HT-IR neuronal perikarya and dendrites by double labeling immunoelectron microscopy. These morphological findings suggest that spinally projecting 5-HT neurons, presumed to be involved in pain modulation or sympathetic autonomic control, are directly regulated by CA neurons at the level of the RVM.

The aim of this study was to examine whether protein products of a transgene are localized within axon terminals in transgenic mice. We have previously created transgenic mice containing a chimeric gene composed of the human dopamine β-hydroxylase gene promoter and the human phenylethanolamine N-methyltransferase (PNMT) cDNA. In the present study, we used an antiserum that detects specifically human PNMT but not mouse PNMT, and examined immunocytochemically the hippocampal formation of the transgenic mice. At a light microscopic level, immunoreactivity of human PNMT was found in fiber plexuses in the outer molecular layer of the dentate gyrus, and in cell bodies of layer 2 of the entorhinal cortex. At an electron microscopic level, in the outer molecular layer of the dentate gyrus, human PNMT immunoreactivity was observed in axon terminals that formed synapses with dendritic spines. The present study provides the evidence for localization of the transgene's protein products in axon terminals, suggesting axonal transport of the products.

The presence of l -3,4-dihydroxyphenylalanine ( l -DOPA) immunoreactivity is reported for the first time in some neurons in the human mesencephalic region, using an immunohistochemical method with a newly raised, highly specific anti- l -DOPA antiserum. We have found many l -DOPA-positive/dopamine (DA)-positive and a few l -DOPA-positive/DA-negative cell bodies in dopaminergic regions in the midbrain. The present results suggest the existence of more than one neuronal group of l -DOPA in the human mesencephalon. l -DOPA in one group is an intermediate metabolite for decarboxylation to DA and in another group may exist as an endproduct. l -DOPA in the latter neurons could be a neuromodulator and/or neurotransmitter. Thus, we suggest that l -DOPA plays an important role besides being an intermediate of DA in the human mesencephalon.

To investigate the function of the dipeptide carnosine in the olfactory system, alterations of carnosine in the primary olfactory nerve after unilateral naris closure or olfactory bulb semilesion were studied by means of immunocytochemistry. After unilateral naris closure, carnosine staining showed no alteration, but immunoreactivity of tyrosine hydroxylase (TH), the rate-limiting enzyme for biosynthesis of dopamine in dopaminergic neurons, decreased dramatically in periglomerular neurons. This behavior of carnosine is consistent with that reported for the olfactory marker protein (OMP). On the other hand, following partial lesion of the olfactory bulb, ordinary to strong carnosine expression was seen in newly innervated glomeruli that exceeded that seen in the conventional targets. Ectopic, TH-positive, periglomerular cell-like neurons appeared around the newly formed glomeruli. These data suggest that carnosine may have some role in regulating TH expression in post-synaptic neurons.

Dopamine β-hydroxylase (DBH, EC 1.14.17.1) catalyze the conversion of dopamine to norepinephrine, the third step of catecholamine biosynthesis. We have previously created transgenic mice harboring a chimeriic gene consisting of the 4-kb DNA fragment of the human DBH gene promoter and the human phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) cDNA, to express PNMT in norepinephrine- and epinephrine-producing cells in the brain, sympathetic ganglia, and adrenal medullary chromaffin cells (Kobayashi et al., Proc. Natl. Acad. Sci. U.S.A., 89 (1992) 1631–1635). In this paper, we produced for the first time the antibody that specifically detects human PNMT, but not mouse PNMT, with the synthetic oligopeptide characteristic of the human PNMT sequence, and used this antibody to investigate the cells expressing human PNMT in transgenic mice. Immunohistochemical analysis of transgenic mice showed typical expression of human PNMT immunoreactivity in norepinephrinergic and epinephrinergic neurons in brain, as well as norepinephrine- and epinephrine-producing cells in the adrenal gland, indicating that the 4-kb 5′-flanking region is essential for the tissue-specific expression of the DBH gene. We also detected the ectopic expression in some DBH-immunonegative cells in the olfactory bulb of transgenic mice.

Copper-zinc superoxide dismutase (Cu-Zn SOD) has been localized in mouse nasal mucosa. Immunocytochemical staining using polyclonal antibody against Cu-Zn SOD revealed endogenous Cu-Zn SOD in sustentacular cells in the olfactory area, and in ciliated epithelial cells in the respiratory area of the olfactory mucosa. Since these cells are located in the superficial portion of the olfactory mucosa, Cu-Zn SOD may serve as an intracellular antioxidant.

Publisher Summary This chapter focuses on immunocytochemistry and in situ hybridization of catecholamine (CA)-synthesizing enzymes and the related neurotransmitters. It describes the methods of immunocytochemistry of neurotransmitters or neuropeptides for the CA-synthesizing enzymes, and in situ hybridization of mRNAs of the enzymes, and the results obtained by these methods. It presents a comparison of results obtained by various histochemical methods—the histofluorescence technique for monoamines, immunohistochemistry using antisera to monoamines, immunoenzyme histochemistry using antibodies against monoamine-synthesizing enzymes, and in situ hybridization of mRNA of monoamine-synthesizing enzymes. The chapter discusses the distribution of central monoamine systems of the mouse, using polyclonal antibodies against CA-synthesizing enzymes and against serotonin. It discusses the principles of immunocytochemical methods including immunofluorescence, labeled antibody, peroxidase-antiperoxidase, protein-A-gold, and avidin biotin complex procedures. The chapter also discusses the phylogenetic and ontogenetic studies of CA systems by immunocytochemistry.

The influence of antidepressant drugs on catecholaminergic neuron groups of the mouse brain was studied immunohistochemically, using a microphotometric semiquantitative method. The immunoreactive level of tyrosine hydroxylase (TH) and aromatic l -amino acid decarboxylase (AADC) in the locus coeruleus (LC) was significantly decreased after 2 weeks of antidepressant administration. Our results suggest that long-term exposure to an antidepressant drug, desipramine, significantly affects intracellular catecholamine-synthesizing enzymes. These results may suggest some significant roles of the catecholaminergic neuronal groups to the action of the tricyclic antidepressant drugs.

To investigate cis-elements responsible for catecholaminergic (CAnergic) neuron-specific expression of the tyrosine hydroxylase (TH) gene, we produced lines of transgenic mice carrying 5.0-kb, 2.5-kb and 0.2-kb fragments from the 5′-flanking region of the human TH gene fused to a reporter gene, chloramphenicol acetyltransferase (CAT), and designated them as TC 50, TC 25, and TC 02, respectively, and reporter gene expression in transgenic mice was analyzed by CAT assay by immunocytochemistry with anti-CAT antibody. High-level CAT expression was observed in the brain and adrenal gland using the 5.0-kb promoter of the TC 50 mice, but ectopic expression was consistently observed in several somatic tissues, e.g. thymus, colon, and testis. In brain, expression was achieved in CAnergic neurons with the largest construct (5.0 kb), but not with 2.5 kb or 0.2 kb of 5′ flanking sequence. However, TC 50 mice also expressed CAT immunoreactivity in non-CAnergic neurons. In the TC 25 line CAT immunoreactivity was detected only in some non-CAnergic neurons. In the TC 02 line no CAT immunoreactivity was detected in any of the tissues examined. These results indicate that the 5.0-kb DNA fragment of the TH gene upstream region contains activity to express CAT in CAnergic neurons and surprisingly, lacks some regulatory elements attenuating ectopic expression, and that the 2.5-kb and 0.2-kb fragment are not sufficient for the proper expression. We discuss the presence of the tissue-specific regulatory elements in the structure portion of the TH gene and/or 3′-flanking region.

In the rostral part of the dorsal motor nucleus of the vagus of the cat, neurons do not contain histochemically detectable catecholamines, even though many perikarya contain both intense aromatic l -amino acid decarboxylase (AADC) immunoreactivity and strong monoamine oxidase enzymatic activity. Similarly located perikarya have distinct immunoreactivities to tyrosine hydroxylase (TH) and dopamine after treatment with colchicine. Since inhibition of monoamine oxidase fails to reveal dopamine in these cells, its absence in non-colchicine-treated animals cannot be due to rapid deamination. It appears that dopamine is synthesized by TH and AADC in dorsal motor vagal cells and is then rapidly transported from the perikarya.

An antiserum was raised against l -DOPA bound to bovine serum albumin, purified by affinity chromatography, and its specificities were verified by immunoblotting and enzyme-linked immunosorbent assays. The antiserum did not cross-react with dopamine (DA), tyrosine, tyramine, octopamine, norepinephrine or epinephrine. Immunocytochemical studies using the PAP method revealed that tyrosine hydroxylase- and l -DOPA positive but aromatic l -amino acid decarboxylase- and DA-negative neurons were present in the lateral habenular nucleus of the house-shrew ( Suncus murinus ) brain. Ultrastructurally l -DOPA immunoreactive products were localized in the cytoplasmic matrix and terminals with vesicles.

Taurine (2-aminoethane sulfonic acid) and carnosine (beta-alanyl-L-histidine) are found in large quantities in the olfactory epithelium and bulb. Taurine is a structurally simple amino acid, and has been reported to have several putative roles, such as neurotransmitter, neuromodulator, neurogrowth factor and to function in membrane stabilization. Carnosine, on the other hand, has been suggested as a putative neurotransmitter in the olfactory system. We have succeeded in visualizing taurine- and carnosine-like immunoreactivities (LI) in the human olfactory mucosa, and also carnosine-LI in the human olfactory bulb. For this investigation, we collected specimens of the human olfactory bulb by autopsy and from the olfactory mucosa by biopsy, and compared localization of taurine- and carnosine-LI in several cases. By means of biopsy using Nakano's forceps, samples of olfactory mucosa were obtained from 5 cases: a 17 year old female, 23 year old male, 46 year old male, 47 year old male, and a 57 year old male. The olfactory bulb of a 1 month old male was collected at autopsy. These specimens were processed for immunohistochemical study according to the peroxidase-antiperoxidase (PAP) method. In the olfactory epithelium, taurine-LI was demonstrated in some primary olfactory neurons, and in basal cells. Carnosine-LI was observed only in primary olfactory neurons, i.e., dendrites, vesicles and axonal bundles of olfactory receptor cells, but not in basal cells. In the olfactory bulb, the olfactory nerve layer and the glomerular layer showed carnosine-LI positive reactions. Therefore, taurine and carnosine may possibly coexist in some olfactory neurons. Olfactory receptor cells are classified as sensory neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Epinephrine-producing cells are characterized by the presence of phenylethanolamine N-methyltransferase (PNMT), which catalyzes the formation of epinephrine from norepinephrine. We generated a line of transgenic mice carrying a chimeric gene containing human PNMT cDNA fused to the 4-kilobase fragment of the human dopamine beta-hydroxylase (DBH) gene promoter, to switch catecholamine phenotype in the nervous and endocrine systems. Human PNMT transcripts and immunoreactivity were mainly detected in norepinephrine neurons in brain and sympathetic ganglion as well as in norepinephrine-producing cells in adrenal medulla of transgenic mice, indicating that the human DBH gene promoter of 4 kilobases is sufficient to direct expression of the gene in norepinephrine-producing cells. Analysis of catecholamines in the various tissues showed that the expression of human PNMT in transgenic mice induced the appearance of epinephrine in sympathetic ganglion and dramatic changes in norepinephrine and epinephrine levels in brain, adrenal gland, and blood. These results indicate that the additional PNMT expression in norepinephrine-producing cells can convert these cells to the epinephrine phenotype, and suggest that norepinephrine-producing cells normally possess the basic machinery required for the synthesis of epinephrine except for PNMT. Thus it appears that the only major difference between norepinephrine- and epinephrine-producing cells is the expression of PNMT. Our transgenic animals provide an experimental model to investigate the functional differences between norepinephrine and epinephrine.

To study the expression of tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) genes in the mouse adrenal gland and the brain, we performed in situ hybridization studies by using several types of complementary DNA probes recognizing coding regions of human TH (THc), the 3′-end of the human region of TH (TH3′), and the coding region of the human PNMT (PNMTc). THc mRNA was detected in the chromaffin cells of the mouse adrenal medulla and brain catecholaminergic neurons including the substantia nigra and ventral tegmental area. TH-immunopositive neurons were located in a similar pattern in adjacent sections. However no positive signals were detected by the TH3′ probe. Using the PNMTc probe, the majority of cells in the adrenal medulla demonstrated positive labelling. Although the mouse TH and PNMT genes have not been fully isolated and sequenced, the present study strongly suggests that the sequence of the coding regions of TH and PNMT are similar in human and mouse. The THc and PNMTc probes are particularly useful in investigating the loci of gene transcription in mouse tissues.

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.

The presence of 'tyrosine hydroxylase (TH)-only'-immunoreactive (IR) neurons is reported for the first time in the human hypothalamic region and basa ganglia, using immunohistochemical technique. 'TH-only'-IR neurons were demonstrated in the ventrolateral part of the arcuate nucleus and medial segment of globus pallidus. These neurons lacked aromatic L-amino acid decarboxylase (AADC)-, dopamine-beta-hydroxylase (DBH)-, and dopamine (DA)-immunoreactivity. The above results suggest that these 'TH-only'-IR neurons are not dopaminergic, and possibly contain L-DOPA as an end-product.

We have recently reported the production of transgenic (Tg) mice carrying the human tyrosine hydroxylase (TH) gene, and have described tissue-specific expression of the transgene in catecholaminergic (CAergic) neurons and adrenal glands. This paper describes the transgene expression in non-catecholaminergic (nCAergic) neurons in the brain of Tg mice by immunocytochemistry and in situ hybridization. In adult Tg mice, human TH was atypically expressed in the olfactory (typically, the anterior olfactory nucleus and pyriform cortex) and visual (typically, n. suprachiasmaticus and n. parabigeminalis) systems, in addition to typical CAergic neuron-rich nuclei in the brain. These results suggest the possibility that TH plays some novel roles in sensory systems.

The distribution and structural features of tyrosine hydroxylase-like immunoreactive (TH-LI) neurons were studied in the olfactory bulb of a snake, Elaphe quadrivirgata, by using pre- and post-embedding immunocytochemistry at the light microscopic level. In contrast to rodent olfactory bulbs previously reported, many TH-LI neurons were seen not only in the main olfactory bulb (MOB) but also in the accessory olfactory bulb (AOB). With regard to the TH-like immunoreactivity, there appeared no appreciable differences between MOB and AOB. As in mammalian MOB, the majority of TH-LI neurons were clustered in the periglomerular region and appeared to send their dendritic branches into glomeruli, which as a whole make an intense TH-LI band in the glomerular layer (GML). In the external plexiform/mitral cell layer (EPL/ML) of MOB and AOB as well as in the outer sublamina of the internal plexiform layer (OSL) of AOB, an appreciable number of TH-LI neurons were scattered, extending dendritic processes which appeared to make a loose meshwork. TH-LI neurons in EPL/ML (including OSL) appeared to consist of at least two morphologically different types. The first had a small perikaryon and one or two smooth dendrites which usually extended to GML and were frequently confirmed to enter into glomeruli. The second had a larger perikaryon and 2-3 dendrites which branched into several varicose processes extending in EPL/ML/OSL but appeared not to enter into glomeruli. The TH-like immunoreactivity was rarely seen in the internal plexiform layer and internal granule cell layer. The colocalization of GABA-like and TH-like immunoreactivities was further studied. Almost all TH-LI neurons in both EPL/ML/OSL and GML contained GABA-like immunoreactivity irrespectively of the type of TH-LI cells.

We raised antibodies against 3 oligopeptide segments of human dopamine-beta-hydroxylase (hDBH) corresponding to the N-terminal (hDBH-N), the intermediate (hDBH-I), and the C-terminal (hDBH-C) amino acid sequences (residues 26-43, 452-468, and 582-598), respectively. We characterized the antibodies in terms of specificity by means of Western blotting and immunohistochemistry. Anti-hDBH-N antiserum recognized DBH in the brain (noradrenergic neurons in the pons and medulla oblongata) and adrenal medulla, not only of human but also of mouse, rat and house shrew. In contrast, anti-hDBH-C antiserum recognized only human DBH. These observations suggest that the antibody raised against the hDBH-C terminal peptide may specifically recognize only human DBH.

A small number of neurons in the nucleus accumbens of the rat showed tyrosine hydroxylase-like immunoreactivity (TH-LI). These TH-LI neurons had medium-sized cell bodies with several spiny dendrites. When a lesion was produced in the ventral tegmental area of the midbrain by injecting 6-hydroxydopamine, TH-LI neurons in the nucleus accumbens, as well as those in the olfactory bulb, were increased in number on the side ipsilateral to the lesion. The results indicated that expression of TH might be enhanced in some neurons deprived of dopaminergic afferent fibers.

A soluble fraction was extracted from a chemically deafferentiated striatum of adult Wistar rats after unilateral lesioning of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) injection. The soluble extract from the lesioned side enhanced the survival of cultured mesencephalic dopamine (DA) neurons of 14-day-old rat embryos as evidenced by quantitative counting of tyrosine hydroxylase-like immunoreactive cells. The neurotrophic activity of this striatal extract for DA neurons was highest 14 days after 6-OHDA injection and became negligible in 28 days. The extract showed no promoting effects on cultured γ-aminobutyric acid (GABA)-containing mesencephalic neurons. These observations indicate that the striatum of adult rats may initiate de novo synthesis of trophic substance(s) for DA neurons but not for GABA neurons when subjected to nigral dopaminergic deafferentiation.

The pineal organ of neonatal rats was transplanted to the frontal part of the cerebral cortex or the cerebral interhemispheric fissure of an isogenic adult rat to determine whether pineal differentiation and pinealopetal innervation are affected by aberrant neuronal influences. Transplants were fixed for immunohistochemistry at 1, 2 and 6 months after transplantation. When treated with an anti-serotonin antibody, cells in transplants from both locations showed intense immunoreactivity and a morphology comparable to intact pinealocytes, indicating that the transplanted pinealocytes had differentiated normally. Tyrosine hydroxylase immunohistochemistry revealed that new catecholamine fibers of central nervous origin extended only into the periphery and not into the core of transplants grafted within the cortex. However, numerous catecholamine fibers were found in transplants placed in the interhemispheric fissure. These fibers were often accompanied by blood vessels, suggesting that they derived from sympathetic ganglia. Serotonin fibers, which are densely distributed in the cerebral cortex, were seldom found to enter transplants from both locations. These observations indicate that pineal cells express their characteristic properties even when transferred to a foreign milieu and that they do not receive novel innervation from the central nerves that normally do not innervate the intact pineal body; the transplant thereby retains the property of selective pinealopetal innervation.

By means of immunohistochemical techniques it is demonstrated for the first time ever that carnosine (beta-alanyl-L-histidine)-like immunoreactivity (LI) is localized in the olfactory receptor neuron in human olfactory mucosa. Carnosine-LI was found in all regions of the receptor cells, i.e. in dendrites, olfactory vesicles, soma and axons. Supporting cells and basal cells were not immunoreactive. The present results suggest that carnosine play a major role in the human olfactory neuron and supports the concept that this peptide is a putative neurotransmitter in vertebrate olfactory systems.

The presence of phosphate-activated glutaminase in monoaminergic neurons was shown in the rat brain by a double-staining method using a mouse monoclonal anti-glutaminase antibody combined with rabbit antisera against tyrosine hydroxylase, dopamine-beta-hydroxylase, phenylethanolamine-N-methyltransferase or serotonin. Glutaminase-like immunoreactivity was detected within perikarya of many monoaminergic neurons in the substantia nigra pars compacta, locus ceruleus, raphe nuclei, etc. Possible functional significance of glutaminase in monoamine neurons was discussed.