Norio Iijima

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.

Local inflammation increases vanilloid receptor 1 expression within distinct subgroups of DRG neurons

Vanilloid receptor 1 (VR1) is essential to the development of inflammatory hyperalgesia. We investigated whether inflammation can increase in VR1 positive neuronal profiles in rat DRG neurons using histochemical methods. We also used size frequency analysis and double staining with several neuronal markers to investigate whether or not inflammation alters VR1 expression. Inflammation induced a 1.5-fold increase in percentage of VR1-like immunoreactivity (LI) positive profiles per total neuronal profiles, suggesting that the number of heat and pH sensitive neurons increase during inflammation. Area frequency histograms showed that VR1 expression increased in small and medium-sized neurons after inflammation. Double labeling of VR1 with NF200 showed that VR1 positive neurons with NF200 positive profiles significantly increased, indicating that the medium-sized VR1 positive neurons were neurons with myelinated A-fibers. Local inflammation thus increases in VR1 protein level within distinct subgroups of DRG neurons that may participate in the development and maintenance of inflammatory hyperalgesia.

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 2u2003h after intracerebroventricular administration of 1u2003µg 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.

Circadian and Light-Induced Transcription of Clock Gene Per1 Depends on Histone Acetylation and Deacetylation

ABSTRACT Circadian clock genes are regulated through a transcriptional-translational feedback loop. Alterations of the chromatin structure by histone acetyltransferases and histone deacetylases (HDACs) are commonly implicated in the regulation of gene transcription. However, little is known about the transcriptional regulation of mammalian clock genes by chromatin modification. Here, we show that the state of acetylated histones fluctuated in parallel with the rhythm of mouse Per1 (mPer1) or mPer2 expression in fibroblast cells and liver. Mouse CRY1 (mCRY1) repressed transcription with HDACs and mSin3B, which was relieved by the HDAC inhibitor trichostatin A (TSA). In turn, TSA induced endogenous mPer1 expression as well as the acetylation of histones H3 and H4, which interacted with the mPer1 promoter region in fibroblast cells. Moreover, a light pulse stimulated rapid histone acetylation associated with the promoters of mPer1 or mPer2 in the suprachiasmatic nucleus (SCN) and the binding of phospho-CREB in the CRE of mPer1. We also showed that TSA administration into the lateral ventricle induced mPer1 and mPer2 expression in the SCN. Taken together, these data indicate that the rhythmic transcription and light induction of clock genes are regulated by histone acetylation and deacetylation.

Characteristics and distribution of endogenous RFamide-related peptide-1.

We have recently identified RFamide-related peptide (RFRP) gene that would encode three peptides (i.e., RFRP-1, -2, and -3) in human and bovine, and demonstrated that synthetic RFRP-1 and -3 act as specific agonists for a G protein-coupled receptor OT7T022. However, molecular characteristics and tissue distribution of endogenous RFRPs have not been determined yet. In this study, we prepared a monoclonal antibody for the C-terminal portion of rat RFRP-1. As this antibody could recognize a consensus sequence among the C-terminal portions of rat, human, and bovine RFRP-1, we purified endogenous RFRP-1 from bovine hypothalamus on the basis of immunoreactivity to the antibody. The purified bovine endogenous RFRP-1 was found to have 35-amino-acid length that corresponds to 37-amino-acid length in human and rat. We subsequently constructed a sandwich enzyme immunoassay using the monoclonal antibody and a polyclonal antibody for the N-terminal portion of rat RFRP-1, and analyzed the tissue distribution of endogenous RFRP-1 in rats. Significant levels of RFRP-1 were detected only in the central nervous system, and the highest concentration of RFRP-1 was detected in the hypothalamus. RFRP-1-positive nerve cells were detected in the rat hypothalamus by immunohistochemical analyses using the monoclonal antibody. In culture, RFRP-1 lowered cAMP production in Chinese hamster ovary cells expressing OT7T022 and it was abolished by pre-treatment with pertussis toxin, suggesting that OT7T022 couples G(i)/G(o) in the signal transduction pathway.

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.

Pkd1l1 complexes with Pkd2 on motile cilia and functions to establish the left-right axis

The internal organs of vertebrates show distinctive left-right asymmetry. Leftward extracellular fluid flow at the node (nodal flow), which is generated by the rotational movement of node cilia, is essential for left-right patterning in the mouse and other vertebrates. However, the identity of the pathways by which nodal flow is interpreted remains controversial as the molecular sensors of this process are unknown. In the current study, we show that the medaka left-right mutant abecobe (abc) is defective for left-right asymmetric expression of southpaw, lefty and charon, but not for nodal flow. We identify the abc gene as pkd1l1, the expression of which is confined to Kupffers vesicle (KV, an organ equivalent to the node). Pkd1l1 can interact and interdependently colocalize with Pkd2 at the cilia in KV. We further demonstrate that all KV cilia contain Pkd1l1 and Pkd2 and left-right dynein, and that they are motile. These results suggest that Pkd1l1 and Pkd2 form a complex that functions as the nodal flow sensor in the motile cilia of the medaka KV. We propose a new model for the role of cilia in left-right patterning in which the KV cilia have a dual function: to generate nodal flow and to interpret it through Pkd1l1-Pkd2 complexes.

Morphological survey of prolactin-releasing peptide and its receptor with special reference to their functional roles in the brain.

The gene of prolactin-releasing peptide (PrRP) was first cloned in 1998 and preproproteins encoded by cDNAs produced at least two isoforms of PrRP with different lengths; PrRP31 and PrRP20. PrRP has been shown to release prolactin from the anterior pituitary at least in vitro (Hinuma, Y.S., Habata, Y., Fuji, R., Hosoya, M., Fukusumi, S., Kitada, C., Masuo, Y., Asano, T., Matsumoto, H., Sekiguchi, M., Kurokawa, T., Nishimura, O., Onda, H., and Fujino, A., 1998. A prolactin-releasing peptide in the brain. Nature 393, 272-6). PrRP receptor has also been detected by quantitive reverse transcription polymerase chain reaction, and in situ hybridization histochernistry revealed that expression of PrRP receptor mRNA was found in the broad areas of the brain and in the anterior pituitary of the rat. This review surveys morphological studies on PrRP, PrRP mRNA and PrRP receptor mRNA in the rat brain and discusses the possible functional significance of PrRP in the brain. PrRP immunoreactive neuronal perikarya showed a similar distributional pattern to those with PrRP mRNA signals. However, distribution of nerve processes and terminals with PrRP immunoreactivity was broadly expanded in the forebrain and brainstem. They were hardly detected in the median eminence particularly in its external layer. PrRP receptor mRNA signals were distributed in the preoptic area, and the hypothalamic area, where PrRP immunoreactive nerve processes and terminals were also detected. The strongest signal of PrRP receptor mRNA was detected in the reticular nucleus of the thalamus where neither PrRP immunoreactive nerve processes nor axon terminals were distributed. From the distribution pattern of PrRP and its receptor, it is suggested that PrRP is involved in control of secretion of oxytocin, corticotropin releasing hormone and somatostatin.

Functional morphology of the suprachiasmatic nucleus.

In mammals, the biological clock (circadian oscillator) is situated in the suprachiasmatic nucleus (SCN), a small bilaterally paired structure just above the optic chiasm. Circadian rhythms of sleep-wakefulness and hormone release disappear when the SCN is destroyed, and transplantation of fetal or neonatal SCN into an arrhythmic host restores rhythmicity. There are several kinds of peptide-synthesizing neurons in the SCN, with vasoactive intestinal peptide, arginine vasopressin, and somatostatine neurons being most prominent. Those peptides and their mRNA show diurnal rhythmicity and may or may not be affected by light stimuli. Major neuronal inputs from retinal ganglion cells as well as other inputs such as those from the lateral geniculate nucleus and raphe nucleus are very important for entrainment and shift of circadian rhythms. In this review, we describe morphological and functional interactions between neurons and glial elements and their development. We also consider the expression of immediate-early genes in the SCN after light stimulation during subjective night and their role in the mechanism of signal transduction. The reciprocal interaction between the SCN and melatonin, which is synthesized in the pineal body under the influence of polysynaptic inputs from the SCN, is also considered. Finally, morphological and functional characteristics of clock genes, particularly mPers, which are considered to promote circadian rhythm, are reviewed.

Cytochemical study of prolactin-releasing peptide (PrRP) in the rat brain.

Strong positive signals for PrRP mRNA and PrRP-like immunoreactivity (PrRP-LI) were detected in the nucleus of the solitary tract and ventral and lateral reticular formation of the caudal medulla oblongata. Weak mRNA signals and immunoreactivity were seen scattered from the hypothalamic dorsomedial nucleus (DMH) to ventromedial nucleus (VMH). Nerve processes and terminals with PrRP-LI were detected from the septal region to the diencephalon. These nerve processes were also clearly visible around capillary walls and in the vicinity of the ependymal cells of the third and lateral ventricles. These observations suggested that PrRP might be secreted into the systemic circulation and cerebrospinal fluid and may play functional roles other than in the release of prolactin from the anterior pituitary.