Microwave oscillator using piezoelectric thin-film resonator aiming for ultraminiaturization of atomic clock.

We developed a microwave oscillator and a micro electromechanical systems-based rubidium cell for the miniaturization of atomic clocks. A thin-film bulk acoustic resonator (FBAR) having a resonant frequency of the fundamental mode in the 3.5 GHz band was employed instead of a crystal resonator. It delivers a clock transition frequency of Rb atoms of 3.417 GHz without the need for a complicated frequency multiplication using a phase-locked loop. This topology considerably reduces the system scale and power consumption. For downsizing the atomic clock system toward the chip level as well as mass production, a microfabricated gas cell containing Rb and N2 gases was also developed. These microcomponents were incorporated into an atomic clock test bench, resulting in a clock operation with a short-term frequency instability of 2.1 × 10-11 at 1 s. To the best of our knowledge, this is the first report of a coherent population trapping clock operation using an FBAR-based microwave oscillator as well as a microfabricated gas cell.

An intraoperative irrigation device using disposable syringes and an extension tube: technical note.

BACKGROUND: The authors present a simple irrigation device used in a microsurgical setting. METHODS: The system consists of a disposable i.v. catheter, an extension tube, a three-way stopcock and two disposable syringes, capable of assembly during a surgical procedure. RESULTS: The length of the handpiece of this device is comparable to other microsurgical tools, which allow both the assistant and the surgeon to grab and irrigate in a coordinated fashion. Clots, tumor contents, and tissue debris are effectively washed away. This tool is particularly useful for lavage of subarachnoid clots during surgery for ruptured intracranial aneurysms, allowing precise anatomic orientation. This device is also practical for cooling tissues adjacent to drilling sites. CONCLUSION: This system is as efficacious as other available self-irrigating microneurosurgical instruments.

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.

Serotonin modulates expression of VIP and GRP mRNA via the 5-HT(1B) receptor in the suprachiasmatic nucleus of the rat.

The expression of vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) in the suprachiasmatic nucleus (SCN) changes depending on light. VIP mRNA increases and GRP mRNA decreases in the light phase, while they do not show change without light. In the present study we investigated the involvement of serotonin (5-HT) in the expression of VIP and GRP messenger RNA in the SCN of the rat. The decrease in VIP mRNA and the increase in GRP mRNA in the light phase were amplified by 5-HT depletion using 5,6-dihydroxytryptamine injected into the lateral ventricle. These enhancements due to 5-HT depletion were reversed to control levels by applying 5-HT(1B) agonists TFMPP and CGS12066A, but not a 5-HT(1A)/5-HT(7) agonist, 8-OH-DPAT. The 5-HT(1B) receptor is known to exist on the terminals of the retinohypothalamic tract (RHT). Therefore, next we investigated the morphological relationship of RHT and 5-HT terminals by double-labeling immunocytochemistry and demonstrated that 5-HT-immunoreactive fibers and cholera toxin B subunit-labeled RHT terminals were intermingled in the ventrolateral SCN, and 5-HT axon processes had close contact with RHT terminals. Collectively, these pharmacological and morphological results suggest that 5-HT afferents from raphe nuclei modulate VIP and GRP expression in neurons of the ventrolateral SCN by activating the 5-HT(1B) receptor in the RHT.

A novel function of prolactin-releasing peptide in the control of growth hormone via secretion of somatostatin from the hypothalamus.

The present study examined a novel function of PRL-releasing peptide (PrRP) on the neuroendocrine. PrRP-immunoreactive nerve fibers and nerve terminals were located in the vicinity of the somatostatin (SOM)-neurons in the hypothalamic periventricular nucleus (PerVN). Immuno-electron microscopy revealed that PrRP-immunoreactive nerve terminals made synaptic contacts with nonimmunoreactive neuronal elements in the PerVN. Intracerebroventricular (icv) administration of PrRP induced immediate early gene, NGFI-A, in SOM-neurons in the PerVN. Double-labeling in situ hybridization showed that some parts of SOM-neurons in the PerVN expressed PrRP receptor messenger RNA. Therefore, some parts of SOM-neurons in the PerVN are considered to be directly innervated by PrRP via PrRP receptor. In addition to the above morphological characteristics, icv administration of PrRP decreased plasma GH levels. Such inhibitory effects of PrRP on the secretion of GH from the anterior pituitary were diminished by depletion or neutralization of SOM. From these findings it was strongly suggested that SOM-neurons respond to PrRP and secrete SOM into the portal vessels and thus inhibit GH secretion from the anterior pituitary.

Developmental expression of prolactin releasing peptide in the rat brain: localization of messenger ribonucleic acid and immunoreactive neurons.

Prolactin releasing peptide (PrRP) was recently identified as the stimulator of prolactin release from the anterior pituitary. PrRP mRNA is expressed in the medulla oblongata and the hypothalamus in the rat brain. The fibers containing PrRP are widely distributed in the brain, therefore, it was postulated that PrRP may act as a neurotransmitter or neuromodulator as well as an endocrine substance. To clarify the developmental changes in the expression of PrRP during brain development, we examined PrRP in rat fetuses and neonates using in situ hybridization and immunohistochemistry. The PrRP mRNA was expressed in the nucleus of the solitary tract (NTS) at embryonic day 18 (E18) and in the ventral and lateral reticular nucleus (VLRN) of the caudal medulla oblongata at E20. The PrRP mRNA in the hypothalamus was first expressed at postnatal day 13 (P13). Reverse transcription-polymerase chain reaction analysis (RT-PCR) for PrRP revealed that PCR product, a 268 bp band, was detected from either E18 in the medulla or P13 in the hypothalamus. Immunodetection with monoclonal antibody against prepro-PrRP revealed intensive staining of cells in the NTS at E18, in the VLRN at E20 or in the dorsomedial hypothalamus at P13. Immunohistochemistry using monoclonal antibody against mature PrRP at P6 showed PrRP fibers to be distributed in the paraventricular hypothalamic nucleus, periventricular hypothalamic nucleus, medial preoptic area, basolateral amygdaloid nucleus, dorsomedial hypothalamus, ventromedial hypothalamus, periventricular nucleus of the thalamus and bed nucleus of the stria terminalis as previously shown in the adult rat. PrRP fibers were also found in the optic chiasm, dorsal endopiriform nucleus, cingulum, intermediate reticular nucleus, and caudal ventrolateral reticular nucleus at P6 and P9. However, PrRP fibers were never found in the above regions in the adult animal. These findings suggest that PrRP fibers originating in the medulla oblongata have been widely distributed in the rat brain during the early postnatal day and PrRP may play various roles in the brain development.

Hypothalamo-pituitary-adrenal axis sensitization after chronic salt loading.

Hypothalamic parvocellular vasopressin (VP) and corticotropin-releasing hormone (CRH) in the paraventricular nucleus (PVN) are major secretagogues of corticotropin (ACTH), and central plasticity including their alteration is closely related to hypothalamic-pituitary-adrenal (HPA) axis modulation. Chronic hyperosmotic stress caused by 2% salt loading has been known to alter VP and CRH expression. We recently reported that rehydration, a recovery stage from salt loading, induced a prolonged increase in parvocellular VP mRNA expression and suggested that rehydration can modulate HPA axis function without obvious external stress. In the present study, we examined hypothalamic VP and CRH mRNA expression and their responsiveness to acute immobilization stress in control, salt-loaded and rehydrated animals, in order to clarify the precise mechanism of HPA axis regulation during rehydration. The results were further compared with plasma corticosterone and ACTH levels. Plasma corticosterone decreased during salt loading, whereas it increased during rehydration at 1 week. Basal ACTH concentration increased in 1-week-rehydrated animals, with enhanced responsiveness to the acute immobilization stress. In the hypothalamic parvocellular PVN, basal CRH mRNA levels also decreased during salt loading and increased during rehydration. Basal VP mRNA was up-regulated during both salt loading and rehydration. VP mRNA responded to additional acute stress during salt loading and rehydration, but CRH mRNA did not. These results indicate that the HPA axis activity of parvocellular neurons is still altered at 1 week of rehydration and that VP plays a dominant role in regulating ACTH release in response to acute stress. This rehydration stage may thus be a good model for analysis of post-stress sensitization of the HPA axis.

Gonadal regulation of PrRP mRNA expression in the nucleus tractus solitarius and ventral and lateral reticular nuclei of the rat.

We investigated the prolactin-releasing peptide (PrRP) gene expression quantitatively in the rat brain and the involvement of estrogen and progesterone using in situ hybridization. The strongest signals were observed in the nucleus tractus solitarius (NTS), which showed approximately 70% of total PrRP mRNA in the brain. Moderate expression was observed in the ventral and lateral reticular nuclei (VLRN) of the medulla oblongata. PrRP mRNA signals in the hypothalamic ventromedial- and dorsomedial nuclei showed only 5% of total signals. The PrRP mRNA expression among female rats showing normal gonadal cycle and male rats showed that the highest levels were in female rats in proestrus. Administration of estrogen or progesterone after ovariectomy induced an increase in PrRP mRNA expression in the NTS. PrRP mRNA content in the NTS increased with the progress of the pregnancy and reached a peak on the 14th day, the mid-period of pregnancy, when plasma progesterone increases. We also observed the colocalization of PrRP and estrogen receptor alpha in the neurons distributed in the NTS by double labeling immunocytochemistry. These findings indicate that PrRP gene expression is regulated by gonadal steroid hormones in the medulla oblongata, and parts of PrRP synthesizing neurons are considered to be directly influenced by estrogen in the NTS.

Cell body size and succinate dehydrogenase activity of spinal motoneurons innervating the soleus muscle in mice, rats, and cats.

The cell body sizes and succinate dehydrogenase (SDH) activities of motoneurons in the retrodorsolateral region of the ventral horn in the spinal cord innervating the soleus muscle in mice, rats, and cats were compared using quantitative enzyme histochemistry. There was an inverse relationship between cell body size and SDH activity of motoneurons in the three species. The mean cell body sizes of both gamma and alpha motoneuron pools were in the rank order of mice < rats < cats, while the mean SDH activities of both gamma and alpha motoneuron pools were in the rank order of mice > rats > cats. It is concluded that smaller motoneurons innervating the soleus muscle have higher SDH activities than larger motoneurons, irrespective of the species, and that motoneuron pools innervating the soleus muscle in smaller animals have smaller mean cell body sizes and higher mean SDH activities than those in larger animals.

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.

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.

Development of astroglial elements in the suprachiasmatic nucleus of the rat: with special reference to the involvement of the optic nerve.

The development of astroglial cells and the effect of the retinohypothalamic tract on it were studied by vimentin and glial fibrillary acidic protein (GFAP) immunocytochemistry in the suprachiasmatic nucleus (SCN) of the rat. At the embryonic stage, vimentin-immunoreactive (VIM-IR) radial glia, precursors of astrocytes, were dominant. However, their filaments vanished in the first few postnatal days. Instead of VIM-IR glial filaments, GFAP-immunoreactive (GFAP-IR) astrocytes appeared at E20 and grew rapidly from the P3 stage. GFAP immunoreactivity in the ventrolateral portion of the SCN (VLSCN) was measured using a computer-assisted image analyzing system. In normal rats, GFAP immunoreactivity showed a stepwise pattern with two slopes at P3-P4 and P20-P25. Bilaterally eye-enucleated rats operated on the day of birth showed lower GFAP immunoreactivity than normal rats and the GFAP immunoreactivity did not increase between P20 and P25 when GFAP-IR glial processes rapidly expand. Electron microscopic investigation at P50 (adult stage) revealed that neurons in the VLSCN had often direct apposition without astroglial processes and the frequency of this finding was significantly higher in eye-enucleated rats than in the control rats. These findings strongly suggest that the postnatal development of astroglial elements, particularly the expansion of GFAP-IR processes in the SCN, is regulated by retinohypothalamic projection.

NGFI-A gene expression induced in the rat suprachiasmatic nucleus by photic stimulation: spread into hypothalamic periventricular somatostatin neurons and GABA receptor involvement.

We studied NGFI-A gene expression in response to photic stimulation in the rat suprachiasmatic nucleus (SCN) using in situ hybridization histochemistry. This gene expression spread within the SCN and extended dorsally into the anterior hypothalamus after 30 min-1 h of light exposure at circadian time (CT) CT18. It appeared first in the ventrolateral SCN where the retinohypothalamic tract (RHT) innervates, then it expanded dorsomedially in the SCN and beyond the SCN to the anterior hypothalamus. However, stimulation for 2 h light exposure decreased its expression in the SCN. NGFI-A expression in the somatostatin neurons in the periventricular nucleus increased from 8.7% to 41% with increasing exposure time from 5 to 30 min. NGFI-A mRNA expression in the SCN was suppressed by pretreatment with baclofen, the GABAB receptor agonist. The spread of photic information from the retina to the SCN was visualized at immediate early gene level not only in the SCN but also in the area beyond the SCN. Somatostatin neurons in the periventricular nucleus which project to the external layer of the median eminence and are involved in regulation of growth hormone release showed NGFI-A gene expression corresponding to the duration of photic stimulation. Photic-induced NGFI-A gene expression in the SCN was also shown to be regulated by GABAergic transmission via GABAB receptors. These NGFI-A gene-expressing cells in the SCN may be involved in the circadian entrainment by light and some of those outside the SCN may participate in the regulation of neuroendocrine function.

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.

Expanded expression of heme oxygenase-1 (HO-1) in the hypothalamic median eminence of aged as compared with young rats: an immunocytochemical study.

This study was performed to examine the differences in expression of heme oxygenase protein with age using immunocytochemistry. We compared the contents of HO-1 and HO-2 between young and aged rats using immunocytochemical methods. Stronger HO-1 expression was detected in the internal layer of the median eminence (ME) of aged than of young rats. Moreover, the cells expressing HO-1 were larger in the aged than the young animals. Electron microscopy indicated these cells with HO-1-like immunoreactivity (HO-1-LI) to be astrocytes. These findings suggested that the expression of HO-1 increased in the ME with age. The significance of this increased expression of HO-1 with age will be discussed briefly.

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.

Expression of a serine protease (motopsin PRSS12) mRNA in the mouse brain: in situ hybridization histochemical study.

Serine proteases are considered to play several important roles in the brain. In an attempt to find novel brain-specific serine proteases (BSSPs), motopsin (PRSS-12) was cloned from a mouse brain cDNA library by polymerase chain reaction (PCR). Northern blot analysis demonstrated that the postnatal 10-day mouse brain contained the most amount of motopsin mRNA. At this developmental stage, in situ hybridization histochemistry showed that motopsin mRNA was specifically expressed in the following regions: cerebral cortical layers II/III, V and VIb, endopiriform cortex and the limbic system, particularly in the CA1 region of the hippocampal formation. In addition, in the brainstem, the oculomotor nucleus, trochlear nucleus, mecencephalic and motor nuclei of trigeminal nerve (N), abducens nucleus, facial nucleus, nucleus of the raphe pontis, dorsoral motor nucleus of vagal N, hypoglossal nucleus and ambiguus nucleus showed motopsin mRNA expression. Expression was also found in the anterior horn of the spinal cord. The above findings strongly suggest that neurons in almost all motor nuclei, particularly in the brainstem and spinal cord, express motopsin mRNA, and that motopsin seems to have a close relation to the functional role of efferent neurons.

Evidence for regulation of vasopressin gene transcription by the NMDA receptor.

We investigated the effects of dizocilpine maleate (MK-801), an NMDA receptor antagonist, on arginine vasopressin heterogeneous nuclear RNA expression in the supraoptic nucleus in the rat hypothalamus. MK-801 treatment completely blocked the osmotic stimulus-induced increase in AVP hnRNA expression, but had no effect on basal AVP hnRNA expression in the SON. These observations indicate that the NMDA receptor is essential for regulation of AVP gene transcription in response to osmotic stimulation, but has no effect on steady-state AVP gene transcription.

The influence of salt loading on vasopressin gene expression in magno- and parvocellular hypothalamic neurons: an immunocytochemical and in situ hybridization analysis.

Arginine vasopressin peptide and messenger RNA expression were examined at the cellular level in the magnocellular and parvocellular neurons in the rat paraventricular nucleus after dehydration and rehydration, employing immunocytochemistry and in situ hybridization histochemistry on the same tissue sections. Most magnocellular vasopressinergic neurons of control animals expressed both vasopressin-like immunoreactivity and messenger RNA. However, neurons negative for vasopressin-like immunoreactivity but expressing messenger RNA were also detected, and their number increased during dehydration. In contrast, almost all of the parvocellular vasopressinergic neurons of dehydrated animals expressed vasopressin messenger RNA alone, with continued increase in their number after rehydration, despite return of the number of magnocellular vasopressinergic neurons to the control level. Vasopressin messenger RNA and corticotropin releasing factor-like immunoreactivity were co-localized in the same parvocellular neurons, and vasopressin-immunoreactive nerve terminals were detected in the external zone of the median eminence. These findings suggest that magno- and parvocellular vasopressinergic neurons are differentially activated during dehydration/rehydration. Osmotic stimuli activate all magnocellular vasopressinergic neurons, but the effect is not simultaneous in all of these neurons. Parvocellular vasopressinergic neurons are also activated by the stress of dehydration which effect appears to last longer than in the magnocellular system.