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Immunoreactivity to p-tyramine, one of the natural trace amines, was studied in the rat brain by an anti-p-tyramine antibody. Immunoreactivity to this amine is very weak in the nigrostriatal dopaminergic neurons and terminals, and weak in the locus coeruleus noradrenergic ones. It was intensified in these structures after monoamine oxidase inhibition. On the other hand, this amine was highly concentrated in the median eminence of the mediobasal hypothalamus, in which its physiological function on prolactin release has been demonstrated.

Accumulating evidence indicates that somatostatin (SS) is a key substance for the circadian rhythm of rodents. In the present study, we investigated whether SS mRNA coexists with arginine-vasopressin (AVP) mRNA, vasoactive intestinal peptide/peptide histidine isoleucine amide (VIP/PHI) mRNA and glutamate decarboxylase (GAD) mRNA in neurons of the rat suprachiasmatic nucleus (SCN) by double labeling in situ hybridization technique. SS mRNA-positive neurons were scattered in the whole region of rostral SCN, in the intermediate region between dorsomedial and ventrolateral region at the middle level, and in the mid to lateral region at the caudal level. These neurons were located in the close vicinities of the dorsomedial AVP and ventrolateral VIP/PHI mRNA-positive cell clusters. They rarely coexpressed AVP mRNA or VIP/PHI mRNA, but mostly coexpressed GAD mRNA. Thus, SS-synthesizing neurons are GABAergic and form a distinct cell group different from AVP or VIP/PHI cell groups.

Nitrergic ganglionic cells located in the canine intrinsic laryngeal muscle were studied by NADPH-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. Cells intensely stained by NADPH-d histochemistry were found between the striated muscle fibers of the intrinsic laryngeal muscle. Most of these cells were bipolar or pseudounipolar in form. Some NADPH-d negative cells were observed to be enveloped in a mesh by varicose NADPH-d positive nerve fibers. The findings obtained by nNOS immunohistochemistry corresponded well with those obtained by NADPH-d histochemistry, indicating that NADPH-d activity in the ganglion in the intrinsic laryngeal muscle is nNOS. The present findings clearly indicate that some of the ganglion cells located in the canine intrinsic laryngeal muscle are nitrergic, and that the ganglionic cells synapse together with the participation of nitric oxide in integrating ganglionic cells.

The influence of serotonergic inputs on the levels of vasoactive intestinal peptide (VIP) mRNA in the rat suprachiasmatic nucleus (SCN) was examined by in situ hybridization combined with morphometrical analysis. Depletion of serotonin by p-chlorophenylalanine methyl ester (300 mg/kg i.p. daily for 3 days) caused a marked decrease in VIP mRNA signal levels in the ventrolateral part of the SCN. This finding suggests that serotonergic input controls VIP production at the mRNA level.

Norepinephrine (NE) contents in the suprachiasmatic nucleus (SCN) in rats kept under light-dark (LD) or constant dark (DD) conditions displayed significant variations over one day with a peak during the subjective day and a trough during the subjective night. These results indicate that rhythmic variation of NE in the SCN is driven by an endogenous pacemaker and independent of external light. In agreement with these observations, immunocytochemical study localized tyrosine hydroxylase immunopositive fibers in the medial part of the SCN. Unlike serotonin, histamine and neuropeptide Y projections to the ventrolateral SCN, NE innervation may represent the general activity level of the brainstem, from where these fibers arise.

Nitric oxide (NO), which was firstly identified as an endothelium-derived relaxing factor, has recently been demonstrated to be a neurotransmitter in the central and peripheral nervous systems. In the hypothalamus, abundant nitric oxide synthase (NOS) immunoreactivity and its histochemical marker, NADPH-diaphorase activity, have been demonstrated in the hypothalamo-neurohypophyseal system. In the present study, we examined whether NOS is coexpressed with posterior pituitary hormones in the rat hypothalamus by combination of oxytocin and vasopressin immunofluorescence and NADPH-diaphorase histochemistry. Most oxytocin-immunoreactive neurons in the paraventricular and supraoptic nuclei expressed NADPH-diaphorase activity, but virtually no vasopressin-immunoreactive neurons contained NADPH-diaphorase activity. This suggests that oxytocin neurons are the main source of NO production in the hypothalamic-pituitary system.

The topographic alternation of vasoactive intestinal peptide (VIP) immunoreactivity after long-term bilateral eye enucleation (80 days) was investigated in the suprachiasmatic nucleus (SCN) and the superior colliculus of the adult rat with the peroxidase-antiperoxidase method. In the SCN, the immunostaining of VIP-immunoreactive (IR) cell bodies increased, and the intensity and density of VIP-IR fibers and terminals were markedly enhanced after eye enucleation. On the other hand, after eye enucleation, no VIP-IR cell bodies and fibers were visible in the stratum griseum superficiale of the superior colliculus, although fusiform-shaped VIP-IR neurons were distributed in the layer of unoperated control rats. The present results, therefore, have revealed that the long-term blockage of retinal input induces changes of VIP immunoreactivity in different manners, according to the target brain areas.

The expression of the brain rich orphan nuclear receptor ROR beta (retinoid-related orphan receptor beta) was investigated in the mouse brain by in situ hybridization using antisense cRNA probe. Positive ROR beta mRNA signals were detected in various parts of the brain with high expression in the suprachiasmatic nucleus (SCN). In the SCN, ROR beta mRNA signals showed a peak at early daytime (ZT/CT4) and a trough at early nighttime (ZT/CT16) in both light-dark and constant dark conditions. Light exposure at subjective night did not alter the expression level. These findings suggest that ROR beta is a new member of a transcription factor possibly related to the circadian pacemaking system.

Single brief and discrete light treatments are sufficient to reset the overt mammalian rhythms of nocturnal rodents. In the present study, we examined the phase-dependent response of the mammalian clock genes, Per1 and Per2, to a brief strong light-stimulus (1000 lux) in the circadian oscillator center, the suprachiasmatic nucleus (SCN) of rats. Light-induced elevation of Per1 mRNA was observed through the subjective night (CT16, CT20 and CT0 (=CT24)) with a marked peak at the subjective dawn (CT0). However, the light influence was very limited for the induction of Per2; only weak elevation of Per2 mRNA was detected at CT16. The effect of light-stimulus on the Per1 gene was transient, and the effect was restricted to ventrolateral SCN neurons in both CT0 and CT16 after light exposure. Since it is known that these rats show a light-induced behavioral phase-shift throughout the subjective night with being strongest at subjective dawn, the present results suggest that the transient induction of Per1 in ventrolateral SCN neurons is a critical step in the resetting of the biological clock to environmental light–dark schedule.

Using immunohistochemistry, we demonstrated that paraventricular hypothalamic neurons immunoreactive for tyrosine hydroxylase (TH) were not immunopositive for the second step catecholamine synthesizing enzyme L-amino acid decarboxylase (AADC) in the human and monkey Macaca fuscata. In the latter species, they were not immunoreactive for dopamine. It is most likely that primate paraventricular TH-containing neurons do not synthesize dopamine.

By means of double staining technique of NADPH-diaphorase (NADPH-d) histochemistry and calcitonin gene-related peptide (CGRP) immunohistochemistry, we investigated the coexistence of NADPH-d reactivity and CGRP immunoreactivity in the canine superior cervical ganglion (SCG). Most of NADPH-d reactivity and CGRP immunoreactivity were coexisted in the principal postganglionic neurons. These neurons were distributed throughout the ganglion without specific localization. The present findings suggest the intimate role of CGRP and nitric oxide in postganglionic neurons of the canine SCG.

Age-related decreases in circadian oscillating activity are speculated to be one of the causes of psychiatric symptoms. To explore the effects of aging on vasoactive intestinal peptide (VIP) synthesis in the suprachiasmatic nucleus (SCN), we investigated the changes in VIP mRNA levels in aged rats compared with young-adult rats under a light/dark cycle using in situ hybridization combined with microcomputer-based imaging analysis. In the young-adult rats, total signals of VIP mRNA in the light-phase showed a significant decrease compared with those on the dark-phase. The VIP signal level in the aged rats was markedly lower than that in young-adults in both light and dark phases. Moreover, in the aged rats, there were no significant differences in VIP mRNA level between the light and dark phases. These results suggest that gene expression of VIP neurons, a main component of the circadian oscillating system, becomes disturbed in the aged rat brain.

Photic stimulus and serotonin (5-hydroxytryptamine; 5-HT) are two factors known to regulate vasoactive intestinal peptide (VIP) synthesis in the suprachiasmatic nucleus (SCN). To explore the role of 5-HT in the photic stimulus-induced change in VIP synthesis, we investigated the changes in level of VIP mRNA under a 12 h light/12 h dark cycle following depletion of 5-HT by intraperitoneal administration of p -chlorophenylalanine (PCPA) methyl ester (200 mg/kg concentration) for 3 successive days. To estimate VIP mRNA expression, we performed in situ hybridization using imaging plates combined with microcomputer-based imaging analysis. In light-phase, total signals of VIP mRNA from the PCPA-treated rats showed a signifanct decrease compared with those from the saline-treated control rats. However, in dark-phase, there were no significant decreases between the PCPA-treated rats and the saline-control rats. The present results strongly suggest that 5-HT neuronal inputs to the SCN interfere with the effect of photic stimulus on VIP synthesis at the mRNA level.

Daily profiles of somatostatin mRNA expression were investigated in the rat suprachiasmatic nucleus (SCN) by semiquantitative in situ hybridization histochemistry. Under 12 h light/12 h dark conditions, somatostatin mRNA signals were higher during the day time (Zeitgeber time (ZT) 1) than during the night time (ZT 16). This day-night difference was still maintained in constant darkness where the somatostatin mRNA was higher in the subjective day (circadian time (CT) 1) than in the subjective night (CT 16). Together with previous Northern blot hybridization studies, the present observation suggests that the level of somatostatin mRNA in SCN neurons is controlled by the circadian clock, independent of photic environment.

The expression of vgf gene, first isolated as a gene induced by nerve growth factor in PC12 cells, was investigated in neurons of the suprachiasmatic nucleus (SCN) by in situ hybridization. In the rat forebrain, the vgf mRNA was found most densely in the SCN. Neurons which express vgf mRNA were found both in the dorsomedial and ventrolateral subdivisions. Soluble-labeling of vgf in situ hybridization and peptide immunocytochemistry demonstrated that vgf mRNA was expressed in most vasopressin- and neurophysin-immunoreactive neurons in the dorsomedial part and in vasoactive intestinal peptide (VIP)- and peptide histidine isoleucine amide (PHI)-immunoreactive neurons in the ventrolateral part. These findings suggest that vgf is a highly expressed gene in both vasopressin/neurophysin neurons and VIP/PHI neurons which were speculated to be involved in the generation and entrainment of circadian rhythm.

Day-night variations of gastrin releasing peptide (GRP) in the neuronal somal area of the SCN and the nearby region on the anterior hypothalamic area (AHA) where GRP fibers project, were examined by semiquantitative immunocytochemistry, and compared with those of vasoactive intestinal peptide (VIP). Both in the SCN and AHA, GRP immunoreactivity was higher during the day than at night, although VIP immunoreactivity was higher at night than during the day. These observations suggest that, although they are produced in the same ventrolateral subdivision of the SCN and, in part, coexist in the same neurons [13], the day-night changes of GRP and VIP immunoreactivity are in the opposite direction to each other.

Pituitary adenylate cyclase-activating polypeptide-like immunoreactive (PACAP-LI) neuronal elements in the rat hypothalamus including the median eminence (ME) were investigated by light and electron microscopic immunocytochemistry. PACAP-LI neuronal perikarya with well-developed cell organelles and dense granules were distributed mainly in the magnocellualr portion of the paraventricular nucleus and throughout the entire supraoptic nucleus. In the ME, numerous PACAP-LI neuronal processes were found in the internal layer (IL), and immunoreactive terminals containing dense granules, vesicles and mitochondria were detected around portal capillaries which penetrated into the IL from the external layer. Thereafter, PACAP is released into the portal capillaries in the IL, transported to the anterior pituitary and plays a role in the stimulation of adenylate cyclase of anterior pituitary cells.

The distribution of [Met]enkephalin-Arg 6 -Gly 7 -Leu 8 (MEAGL)-immunoreactive (-IR) neurons and its modification after enucleation have been investigated in the rat superior colliculus. In normal rats and on the ipsilateral side of monocular-enucleated rats, small sized vertically elongated fusiform-shaped weakly immunostained neurons were dispersed throughout the sublamina of the stratum griseum superficiale (SGS). In bilaterally enucleated rats and on the contralateral side of monocular-enucleated rats, many small strongly immunoreactive MEAGL-containing neurons, projecting processes horizontally or obliquely toward the surface, appeared in the deepest part of the SGS and the superficial part of the stratum opticum (SO), in contrast to the disappearance of the fusiform-shaped weakly stained neurons in the SGS. MEAGL-IR fibers increased in density throughout the sublamina of the SGS, being densest in the deep SGS, accompanying their increase in the neighboring SO. Sporadically found MEAGL-IR neurons in the deep SO and the stratum griseum intermediale did not show the detectable change of immunoreactivity. These results indicate that enkephalin biosynthesis is undergone by different type of neurons in the normal and the ocular-deprived superior colliculus, and suggest that some neurons in the adult superior colliculus have a potentiality to express the peptidergic phenotype.

The regional distribution of human leumorphin (HL)-like immunoreactivity (HL-LI) in monkey (Macaca fuscata) spinal cord and dorsal root ganglia was investigated by peroxidase-anti-peroxidase immunocytochemistry using specific antiserum. HL-LI-positive fibers and terminals were distributed densely in laminae (Rexed) I and II, and sparsely in laminae III-VII and X, but no immunoreactive elements were observed in the ventral horn, the white matter or the dorsal root ganglia. Many immunoreactive neuronal perikarya were found in laminae I and II. Intrathecal injection of colchicine also revealed the presence of immunoreactive neuronal perikarya in laminae III-VII and X. These results suggest the presence of HL-LI, which represents HL and/or its C-terminal fragment, in the neuronal elements of the monkey spinal cord.

The distribution of substance P (SP) in the rat spinal cord was investigated by peroxidase-anti-peroxidase (PAP) immunocytochemistry. A dense network of SP-immunoreactive fibers and terminals was detected in the ventral column of the rostral lumbar cord with a different density and extent from the other segmental levels. These fibers and terminals were accumulated within and around the centromedial nucleus (CM) of the L1 and L2 segments, with some bundles of immunoreactive fibers between the CM and other areas; i.e. laminae V and X and the contralateral CM. They formed a dense network, such as in arborization of immunoreactive fibers and terminals on the transverse plane and in a comb-shaped structure on the horizontal plane. The origin of the SP in this network was examined. It was determined that neither a total transection of spinal cord at a low thoracic level or mid-lumbar level, nor at an ipsilateral or bilateral section of the 3–5 dorsal roots, containing L1 and L2 roots, induced any visible changes in the SP staining pattern. An intrathecal injection of colchicine revealed the presence of SP-immunoreactive neurons in the dorsal horn and intermediate gray matter at the spinal cord including the rostral lumbar cord. The present findings suggested that the majority of SP immunoreactivities in the above network are derived from local circuit interneurons of the spinal cord.

The distribution of dopamine (DA)-immunoreactive (IR) cells is described in the rat arcuate nucleus of the hypothalamus and its adjacent areas and compared with that of tyrosine hydroxylase (TH)-IR cells. Small DA-IR cells were seen to be aggregated mainly in the dorsomedial part of the nucleus, but were hardly detectable in its ventrolateral portion and neighbouring periarcuate region which showed many larger TH-IR cells. This study reveals, for the first time, the differences in the respective topography of those neurons which actually contain detectable DA and those which contain TH, the initial synthesizing enzyme of catecholamine.

Regulation of the neurotensin (NT)-producing neurons by ascending catecholamine fibers in the paraventricular nucleus of the rat hypothalamus was examined using fluorescence histochemistry and immunohistochemistry after the destruction of the ascending catecholamine pathway by 6-hydroxydopamine. On the ipsilaterally operated side, the fluorescence of the catecholamine terminals decreased remarkably in the paraventricular nucleus whereas an accumulation of catecholamine fluorescence was observed in the caudal mesencephalon rostral to the locus coeruleus. In addition, the number of neural perikarya with NT-like immunoreactivity was greatly decreased in the paraventricular nucleus on the operated side compared with the intact side as determined by peroxidase-antiperoxidase immunohistochemistry. This decrease in the NT-like immunoreactive neural perikarya may be due to the disappearence of noradrenergic input to the NT-like immunoreactive neurons through axo-somatic or axo-dendritic synapses.

The ontogenic development of vasoactive intestinal polypeptide (VIP)-immunoreactive amacrine cells in the rat retina was studied using peroxidase-antiperoxidase (PAP) immunohistochemistry. In the rat retina, VIP immunoreactivity appeared entirely in postnatal stage. On the 12th postnatal day, VIP-immunoreactive amacrine cells could be first detected. However, VIP immunoreactivity was very weak and VIP-immunoreactive amacrine cell processes could not be observed at this stage. On the 21st postnatal day, VIP-immunoreactive amacrine cell bodies became more mature and their processes were distinctly observed, resembling their appearance in adult rat retinas. VIP immunoreactivity could be detected in both stratified and diffuse amacrine cells at this stage. The relationship between the first appearance of VIP-immunoreactive amacrine cells and the synaptic formation in the inner plexiform layer is briefly discussed.

The distribution of substance P (SP) in the rat spinal cord was investigated by peroxidase-anti-peroxidase immunocytochemistry combined with retrograde horseradish peroxidase (HRP) labeling via the cremaster muscle. In the male rats, a dense network of SP immunoreactive (SP-IR) fibers and terminals was detected in the ventral column of the L1 and L2 segments (Vent L1-2) with a different density and extent from the other segmental levels. These fibers and terminals were accumulated within and around the nucleus centromedialis lumbaris (CM) of the L1 and L2 segments. However, in the female rats, SP-IR fibers and terminals were sparse in the Vent L1-2 without particular segmental differences. HRP-positive motoneurons were located in the CM and surrounded by SP-IR fibers and terminals. These results indicate that the Vent L1-2 of the rat spinal cord shows sexual dimorphism with respect to the regional distribution of SP-IR fibers and terminals, and that motoneurons that innervate the cremaster muscle are innervated by dense SP-IR fibers and terminals.

The influence of ascending noradrenergic neuronal input on the neurotensin (NT)-like immunoreactive neuronal perikarya located in the dorsal part of the central nucleus of the amygdala (CNA) was examined using fluorescence histochemistry and peroxidase-antiperoxidase (PAP) immunocytochemistry. Unilateral hemitransection of the ascending noradrenergic pathway by injection of 6-hydroxydopamine into the caudal mesencephalon just rostral to the locus coeruleus caused a marked depletion of immunoreactivity in NT-like immunoreactive neuronal perikarya in the CNA. Ascending noradrenergic neuronal input, therefore, is considered to facilitate production of NT-like immunoreactive substances in neuronal perikarya and to influence on the functional role of the amygdaloid complex. In addition, we obtained evidence of unilateral direct ascending projections of NT-like immunoreactive neurons into the CNA since the disappearance of NT-like immunoreactive processes occurred mainly in the ventral part of the CNA after surgical hemitransection of the ascending neuronal pathway that interrupts the ascending NT-like immunoreactive pathway arising from the neurons in the brain stem.

The existence of GABAergic neurons in the rat suprachiasmatic nucleus (SCN) was demonstrated by three specific markers; mRNA coding for glutamic acid decarboxylase (GAD) and visualized by in situ hybridization using a 35S-labelled cDNA probe, and GAD protein and GABA were identified by immunocytochemistry using specific antisera. In situ hybridization demonstrated well labelled GAD mRNA positive cells throughout SCN, and GABA and GAD immunoreactive cells showed similar distributions. These results indicate that GABA is a transmitter of a large portion of the SCN neuronal population.

The influence of serotonin (5-hydroxytryptamine; 5-HT) innervation on peptide-containing neurons in the rat suprachiasmatic nucleus (SCN) was investigated by peroxidase-anti-peroxidase (PAP) immunocytochemistry. The 5-HT neuronal system was chemically severed by 5,6-dihydroxytryptamine (5,6-DHT) injection into the medial forebrain bundle bilaterally. After this treatment, a marked decrease of vasoactive intestinal peptide (VIP)-like immunoreactivity in neuronal perikarya occurred in the SCN corresponding to a decrease in number of 5-HT immunoreactive fibers and terminals. However, no alteration of arginine-vasopressin-like immunoreactivity was detected between 5,6-DHT-treated animals and the controls. It is speculated that VIP-like immunoreactive neurons play an important role in the SCN under the influence of strong 5-HT innervation.

The presence of L-DOPA-immunoreactivity is reported for the first time in the rat hypothalamic tuberal region. L-DOPA-immunoreactive neurons were demonstrated to be present in the ventrolateral part of the arcuate nucleus and periarcuate region just dorsal to the ventral surface of the brain (VLAR/PA). Weakly L-DOPA-immunostained neurons were found in the dorsomedial part of the arcuate nucleus and its neighboring periventricular nucleus (DMAR/PV). In contrast, dopamine (DA)-immunoreactive neurons were detected only in the DMAR/PV. These findings suggest that L-DOPA exists not only as a precursor of DA in neurons of the DMAR/PV, but also as an end-product in cells of the VLAR/PA.

The distribution of vasoactive intestinal peptide (VIP) and peptide histidine isoleucine amide (PHI) was investigated in the canine hypothalamus by immunocytochemistry. VIP- and PHI-like immunoreactive neurons were detected in the magnocellular supraoptic and paraventricular nucleus. These magnocellular VIP- and PHI-producing neurons coexist with vasopressin-like immunoreactivity and send axons to the median eminence and neurohypophysis. These findings may serve as an anatomical basis for studying the function of VIP and PHI on pituitary hormone secretion.

The distribution of vasoactive intestinal polypeptide (VIP) in the colon of patients with Hirschsprung's disease was investigated by the peroxidase-antiperoxidase (PAP) immunohistochemical method. Three colonic segments, ganglionic, oligoganglionic and aganglionic, were stained by the unlabeled antibody enzyme method. VIP immunoreactive nerve cell bodies, nerve fibers and nerve endings were distributed throughout the ganglionic and oligoganglionic segments. In contrast, the aganglionic segment contained no VIP nerve endings and the number of fibers was reduced. These differences are thought to be a cause of constriction of the colon in Hirschsprung's disease and VIP neurons are therefore believed to participate in the relaxation of smooth muscle.

The existence of a direct projection from retinal ganglion cells to vasoactive intestinal peptide (VIP)-like immunoreactive neuronal elements in the rat suprachiasmatic nucleus (SCN) was revealed by combining analysis of degenerating axons following enucleation and electron microscopic immunocytochemistry. Degenerating axons appeared to make synaptic contact with VIP-like immunoreactive dendrite and neuronal perikarya in the ventral part of the SCN. The possibility of neuronal input from retinal ganglion cells to axons of VIP-like immunoreactive neurons was also suspected since axo-axonic synapses were detected between degenerating axons and axons with VIP-like immunoreactivity. Thus, VIP-like immunoreactive neurons in the SCN receive several neuronal inputs, including those from the retina, and may play a significant role in circadian entrainment.

l -DOPA ( l -3,4-dihydroxyphenylalanine) immunoreactivity was demonstrated in neurons of the cat ventral midbrain and its projection areas, using an immunohistochemical method in conjunction with a newly developed highly specific anti- l -DOPA serum. l -DOPa-immunoreactive (IR) neurons were found in the substantia nigra, retrorubral area and ventral tegmental area of Tsai. l -DOPA-labeled fibers and terminals were hardly detectable in the nigrostriatal pathway and in the caudate nucleus which showed very intense dopamine-immunoreactivity. In contrast, many short labeled processes were detectable in the central amygdala and, although very few in number, in the entorhinal cortex.