Yasuhiko Ibata

New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals.

Only a few RFamide peptides have been identified in mammals, although they have been abundantly found in invertebrates. Here we report the identification of a human gene that encodes at least three RFamide-related peptides, hRFRP-1–3. Cells transfected with a seven-transmembrane-domain receptor, OT7T022, specifically respond to synthetic hRFRP-1 and hRFRP-3 but not to hRFRP-2. RFRP and OT7T022 mRNAs are expressed in particular regions of the rat hypothalamus, and intracerebroventricular administration of hRFRP-1 increases prolactin secretion in rats. Our results indicate that a variety of RFamide-related peptides may exist and function in mammals.

Vasoactive intestinal peptide (VIP)-like immunoreactive neurons located in the rat suprachiasmatic nucleus receive a direct retinal projection.

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.

Neurons expressing relaxin 3/INSL 7 in the nucleus incertus respond to stress

Relaxin 3/INSL 7 has recently been identified as a new member of the insulin/relaxin superfamily. Although it was reported to be dominantly expressed in the brain, its detailed distribution and function in the central nervous system are still obscure. In the present study we demonstrated that in the rat relaxin 3 was mainly expressed in neurons of the nucleus incertus (NI) of the median dorsal tegmental pons. Other relaxin 3‐expressing neurons were scattered in the pontine raphe nucleus, the periaqueductal gray and dorsal area to the substantia nigra in the midbrain reticular formation. Relaxin 3‐immunoreactive fibers projected particularly densely in the septum, hippocampus, lateral hypothalamus and intergeniculate leaflet of the thalamus. Ultrastructural examination revealed that relaxin 3 was localized in the dense‐cored vesicles in the perikarya and was also observed in the synaptic terminals of axons. As almost all relaxin 3‐containing neurons express corticotropin‐releasing factor (CRF) type 1 receptor in the NI, we examined the response of relaxin 3 neurons to intracerebroventricular administration of CRF; 65% of relaxin 3 neurons expressed c‐Fos 2 h after intracerebroventricular administration of 1 µg CRF. We then confirmed that c‐Fos was induced in 60% of relaxin 3 neurons in the NI and the expression of relaxin 3 mRNA increased significantly in the NI after water‐restraint stress. Collectively, these results suggest that relaxin 3 produced in the NI is released from nerve endings and is involved in the regulation of the stress response.

ELECTRON MICROSCOPIC DEMONSTRATION OF ZINC IN THE HIPPOCAMPAL FORMATION USING TIMM'S SULFIDE-SILVER TECHNIQUE

Ultrastructural localization of zinc in the hippocampus of rabbits and rats was investigated by the application of Timms sulfide-silver and zinc-dithizonate methods. Best results were obtained by fixation of tissue in 3% glutaraldehyde saturated with H2S, followed by treatment of small blocks of tissue with Timms solution for 1 hr. In sections cut from these blocks, silver particles were found on the synaptic vesicles in the terminal boutons of the mossy fibers. The nervous tissue of the hippocampal formation was devoid of silver particles, as were the perikarya of the granular cells of the fascia dentata and the pyramidal cells of the hippocampus. The cytochemical significance of zinc in nerve endings of the granular cells is discussed.

Branching of regenerating retinal axons and preferential selection of appropriate branches for specific neuronal connection in the newt

Abstract Exact trajectory and pattern of branching of individual regenerating retinal axons derived from different retinal quadrants was detected within the tectum by labeling selected retinal axons with horseradish peroxidase (HRP) at the 4th, 6th, and 10th weeks after transection of the optic nerve in adult newt Cynops pyrrhogaster . In the early phase of regeneration, each regenerating retinal axon sprouted several branches, in random directions within the tectum. As the regeneration proceeded, only branches directed toward the site of normal innervation were maintained, while the sproutings toward the ectopic part of the tectum were retracted or became atrophic. The surviving branches then sprouted several new branches. Sprouting of new branches and the disappearance of old branches occurred simultaneously throughout the process of regeneration. At the 10th week of regeneration, most regenerating retinal axons possessed branches only at the distal part within the site of normal innervation. These observations strongly suggest that the retinotopic central connection of regenerating retinal axons may be reestablished by means of a stepwise selection of widespread axonal branches.

Coexistence of oxytocin and NADPH-diaphorase in magnocellular neurons of the paraventricular and the supraoptic nuclei of the rat hypothalamus

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.

Vasoactive intestinal peptide immunoreactive neurons in the rat suprachiasmatic nucleus demonstrate diurnal variation

The suprachiasmatic nucleus (SCN) of mammals is considered to be a circadian oscillator and it also demonstrates circadian rhythmicity of its multiple unit activity. A number of neuropeptides have been found in the SCN. Vasoactive intestinal peptide (VIP)- and vasopressin-containing neurons comprise large populations of these cells and have a distinct distribution within the nucleus. Therefore we attempted to examine whether the VIP neurons show a diurnal alteration of their immunoreactivity by combined immunocytochemistry and color image analysis. Our results demonstrate that VIP-like immunoreactive neurons show a diurnal change in the amount of immunoreactivity. Immunoreactivity was most intense in the sections from rats maintained in the cyclic photoperiod and sacrificed at 02.00 h and weakest in the SCN from animals sacrificed at 14.00 h. We considered that VIP-like immunoreactive neurons showed diurnal variation of VIP synthesis depending strongly on the light from the retina.

l-DOPA-immunoreactive neurons in the rat hypothalamic tuberal region

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.

Localization and neuronal response of RFamide related peptides in the rat central nervous system

RFamide related peptides (RFRP)-1 and RFRP-3 are neuropeptides derived from the same preproprotein. We have examined the distribution of RFRP-1 and RFRP-3 immunoreactivities (irs) in the rat central nervous system using specific antibodies. Neuronal cell bodies containing both RFRP-1 and RFRP-3 were detected within the caudal portion of the hypothalamus, the periventricular nucleus (PerVN), and the portion around or above the ventromedial nucleus of the hypothalamus. Both immunohistochemical and in situ hybridization analyses showed that neurons containing RFRP immunoreactivity and mRNA were distinct from those of neuropeptide FF, which contains the same structure at the C-terminus, Pro-Glu-Arg-Phe-NH2, as RFRP-3. Fibers containing both RFRP-1 and RFRP-3 were widely distributed in the brain: the lateral septal nucleus in the telencephalon, the paraventricular thalamic nucleus, various hypothalamic nuclei, the periaqueductal gray in the midbrain, the parabrachial nucleus in the pons, and the nucleus tractus solitarius (NTS) in the medulla oblongata. Only RFRP-1-ir was detected within the posterior gray horn in the spinal cord. Only RFRP-3-ir was detected in several thalamic nuclei and the spinal cord, especially at the posterior intermediate sulcus and within the anterior gray horn. Intracerebroventricular administration of RFRPs induced c-Fos expression in the anterior portion of the NTS, locus coeruleus, the nucleus of incertus, supraoptic nucleus, PerVN and the arcuate nucleus of the hypothalamus. These results show that RFRP-1 and RFRP-3 are widely distributed in the rat central nervous system and might be involved in various functions such as the neuroendocrine system or pain modulation.

Coexistence of dopamine and neurotensin in hypothalamic arcuate and periventricular neurons

The coexistence of dopamine and neurotensin in the same neuronal perikarya in the arcuate nucleus of the rat hypothalamus was examined by combined fluorescence histochemistry and immunohistochemistry on the same tissue sections and we obtained the evidence of the coexistence of two substances. The functional significance of those two substances for the prolactin release from the anterior pituitary was also briefly discussed.