Prokr2-deficient mice display vascular dysmorphology of the fetal testes: potential implications for Kallmann syndrome aetiology.

Kallmann syndrome is a form of hypogonadotropic hypogonadism also associated with the loss of smell. It is a phenotypically and genetically heterogeneous disorder, with mutations in several known causative genes now accounting for approximately 30% of cases. The prevalence for the disease is also much higher in males than in females, a phenomenon that remains to be fully explained. Here, we show that loss of Prokr2, which is linked to autosomal recessive Kallmann syndrome type 3 (KAL3; OMIM 244200), affects fetal testis differentiation in mice. We find that Prokr2 is specifically expressed in the XY gonads during sex determination and fetal sexual differentiation, and knockout mice display a variable degree of compromised vasculature in the fetal testes. This phenotype offers potential insight into the clinical heterogeneity observed within familial cases, and may contribute to the gender bias in Kallmann syndrome patients.

In situ identification of hepatitis C virus RNA in muscle.

The authors examined skeletal muscle specimens from four patients with myositis and hepatitis C virus (HCV) infection. PCR analysis identified HCV RNA in muscle homogenates from two patients. In situ hybridization signals for HCV RNA were detected within muscle fibers as well as in infiltrating lymphocytes from the same patients. The results may relate to the pathomechanism of myositis in patients with HCV infection.

Constitutive expression and delayed light response of casein kinase Iepsilon and Idelta mRNAs in the mouse suprachiasmatic nucleus.

Casein kinase Iepsilon (CKIepsilon) and casein kinase Idelta (CKIdelta) phosphorylate clock oscillating mPER proteins, and play a key role in the transcription (post)translation feedback loop that generates circadian rhythm. In the present study, the expression profiles of CKIepsilon and CKIdelta mRNAs were examined in the mice clock center, suprachiasmatic nucleus (SCN). Moderate levels of CKIepsilon and CKIdelta mRNAs were constantly expressed in the SCN in both light:dark and constant dark conditions. This finding supports the hypothesis that CKI may form a constant threshold to the nuclear entry of mPER proteins as in the Drosophila homologue, double-time. Further, we demonstrated that the light exposure at subjective night induced a delayed increase in CKIepsilon and CKIdelta mRNAs in the SCN. CKIepsilon and CKIdelta proteins may play a role on light-induced phase-shift.

Different developmental profiles of the expression of preprosomatostatin and preprotachykinin-A mRNAs in rat SCN neurons.

The suprachiasmatic nucleus (SCN), a central circadian oscillator of mammals, contains various peptides arranged in the compartment specific manner. In the present study, we examined a distinct population of neurons in the central part of the SCN. In situ hybridization histochemistry has demonstrated that these neurons coexpressed both preprosomatostatin (PPSS) and preprotachykinin A (PPT-A) mRNAs, but the developmental expression profiles were different among two. PPSS mRNA first appeared in the SCN at postnatal day 1(P1). The intensity and number of PPSS mRNA signals increased and peaked at P7-P14 and gradually decreased as to adult age (P56). However, PPT-A mRNA-positive appeared late at P7, and gradually increased up to P56. These findings suggest that neurons encoding both the PPSS and PPTA genes first express PPSS and then express PPT-A at a later stage of maturation.

Expression of the Per1 gene in the hamster: brain atlas and circadian characteristics in the suprachiasmatic nucleus.

Recent progress in study on the molecular component of mammalian clocks has claimed that mammals and Drosophila share the similar fundamental clock oscillating system. In the present study, we investigated expression of Per1, the first gene of the mammalian homolog of the Drosophila clock gene period, in the hamster brain, and we also examined its circadian expression pattern in the mammalian clock center, the suprachiasmatic nucleus (SCN). In situ hybridization using isotope-labeled cRNA probes revealed a wide and region-specific distribution of Per1 in the hamster brain and spinal cord. High levels of Per1 were found in the internal granular layer of the granular cells of the olfactory bulb, anterior olfactory nuclei, tenia tecta, olfactory tubercle, piriform cortex, suprachiasmatic nucleus, and gyrus dentatus of hippocampus. Moderate levels of expression were detected in many brain regions including the granular layer of the cerebellum, anterior paraventricular thalamic nucleus, caudate-putamen, inferior colliculus, pontine nuclei, inferior olive, and nucleus of the solitary tract. We examined the circadian profile of hamster Per1 mRNA in the SCN in constant darkness and found that Per1 expression showed a peak at subjective day (circadian time [CT] 4) and formed a trough at subjective night (CT16-CT20). A brief exposure of light at CT16 could acutely induce large quantities of Per1 mRNA in the hamster SCN, except for its dorsomedial subdivision. These findings suggest that the characteristics of Per1 gene expression in the mammalian circadian center (showing a peak in the daytime and a trough in the nighttime and a rapid inducibility by light) are common among mammalian species. Lastly, in hamster brain, Per1 gene is also inducible in extra-SCN brain nuclei, since light at night also elicited Per1 mRNA in neurons of the hypothalamic paraventricular nucleus.

Per1 and Per2 gene expression in the rat suprachiasmatic nucleus: circadian profile and the compartment-specific response to light.

Expression profiles of rPer1 and rPer2 messenger RNAs, rat homologues of the Drosophila clock gene period, were examined in the rat suprachiasmatic nucleus, a main locus of circadian oscillation, with special reference to the topographical compartmentation of the suprachiasmatic nucleus. Quantitative in situ hybridization of rPer1 and rPer2 messenger RNAs showed a robust circadian rhythm in the suprachiasmatic nucleus, with a characteristic peak/trough profile in each gene: the peak of rPer1 messenger RNA was in the daytime and that of rPer2 messenger RNA was at the transition time of day to night in both light-dark and constant dark conditions. Light exposure at circadian time 16 increased both rPer1 and rPer2 messenger RNAs in the suprachiasmatic nucleus. In a detailed histological analysis, we found that light exposure at circadian time 16 induced the expression of rPer1 and rPer2 genes in neurons limited to the ventrolateral part of the suprachiasmatic nucleus, although the usual circadian rPer1 and rPer2 messenger RNA oscillation in light-dark and constant dark conditions occurred strongly in neurons in the dorsomedial part but weakly in neurons in the ventrolateral part of the suprachiasmatic nucleus. These rPer expression profiles indicate that the two major subpopulations of neurons in the suprachiasmatic nucleus play different roles in the generation of circadian rhythm: a strong autonomous expression ability with no light response in dorsomedial neurons and a strong light responsiveness with a weak autonomous expression in ventrolateral neurons.

A putative transcription factor with seven zinc-finger motifs identified in the developing suprachiasmatic nucleus by the differential display PCR method.

The suprachiasmatic nucleus (SCN) is a mammalian central circadian pacemaker. This nucleus develops in the last stage of fetal life and matures to make strong synaptic connections within 2 weeks of postnatal life to establish strong oscillation characteristics. To identify factors that initiate the circadian oscillation, we applied a differential display PCR method to developing SCN, and isolated a gene with seven zinc-finger motifs, Lot1, which encodes a gene that appeared at a very high level in the SCN during the early postnatal days. Lot1 mRNA first appeared at postnatal day 1 (P1) at a very high level, and the signal in the SCN continued to be very high until P10 and thereafter rapidly decreased until P20 and was expressed at a very faint level during adulthood. Lot1 mRNA expression was observed only in neurons of the dorsomedial SCN throughout the course of development. During the developmental stage, Lot1 mRNA expression shows a circadian rhythm with a peak in the day time and a trough at night time in both light-dark and constant dark conditions. These observations imply that Lot1 is the first identified putative transcription factor expressed only in the period of active synaptogenesis in the SCN, where Lot1 might play a role in establishing autonomous oscillation.

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.

A new mammalian period gene predominantly expressed in the suprachiasmatic nucleus.

BACKGROUND: In mammals, two possible clock genes (Clock, Per1) have very recently been reported. mPer1 (the first identified mouse period gene), in particular, shows a circadian expression in suprachiasmatic nuclei (SCN), the mammalian circadian centre. However, only mPer1 and Clock as clock components may not be sufficient to understand all the events in circadian oscillation and entrainment. RESULTS: A mammalian period complementary DNA, mPer2, has been isolated from the mouse brain. The amino acid sequence of mPer2 is similar to mPer1 and Drosophila Period (dPer), indicating that mPer2 is a member of the family which contains mPer1, itself a homologue of dPer. mPer2 mRNA is predominantly expressed in SCN. A robust circadian rhythmic expression in the SCN supports the view that mPer2 is a clock gene. mPer2 is strongly expressed at the subjective afternoon in constant darkness, distinct from a morning-phase clock mPer1. Our precise quantitative in situ hybridizations have revealed that the peak expression of mPer2 transcripts is delayed by 8 h in LD (light-dark) or 4 h in DD (dark-dark) conditions when compared to mPer1. A short brief light exposure at the early subjective night, prompting a phase-shift in locomotor rhythms, induces a transient increase of mPer2 transcripts with delayed onset, as compared to mPer1 mRNA induction. Furthermore, mPer2 is co-expressed with mPer1 in single SCN cells. CONCLUSIONS: Mammalian period genes show molecular heterogeneity, each of which is composed of a different oscillator, and may serve to establish stable circadian rhythms in mammalian oscillating cells.

Circadian oscillation of a mammalian homologue of the Drosophila period gene.

Many biochemical, physiological and behavioural processes in organisms ranging from microorganisms to vertebrates exhibit circadian rhythms. In Drosophila, the gene period (per) is required for the circadian rhythms of locomotor activity and eclosion behaviour. Oscillation in the levels of per mRNA and Period (dPer) protein in the fly brain is thought to be responsible for the rhythmicity. However, no per homologues in animals other than insects have been identified. Here we identify the human and mouse genes (hPER and mPer, respectively) encoding PAS-domain (PAS, a dimerization domain present in Per, Amt and Sim)-containing polypeptides that are highly homologous to dPer. Besides this structural resemblance, mPer shows autonomous circadian oscillation in its expression in the suprachiasmaticnucleus, which is the primary circadian pacemaker in the mammalian brain. Clock, a mammalian clock gene encoding a PAS-containing polypeptide, has now been cloned: it is likely that the Per homologues dimerize with other molecule(s) such as Clock through PAS-PAS interaction in the circadian clock system.

Peptide expression in GABAergic neurons in rat suprachiasmatic nucleus in comparison with other forebrain structures: a double labeling in situ hybridization study.

We investigated the characteristics of GABAergic neurons in the rat suprachiasmatic nucleus (SCN) in normal untreated rats by examination of co-expressed peptides. We adopted double labeling in situ hybridization using a digoxigenin-labeled glutamic acid decarboxylase (GAD) riboprobe and 35S-labeled peptide riboprobes. GAD mRNA-positive neurons were distributed throughout the SCN from the rostal to the caudal pole. In the dorsomedial part of the SCN, most GAD mRNA-positive neurons co-expressed arginine vasopressin mRNA. In the ventrolateral part of the SCN, about two thirds of GAD mRNA-positive neurons co-expressed vasoactive intestinal peptide (VIP) mRNA. Co-expression of GAD and somatostatin mRNA was observed in virtually all neurons of the intermediate part of the SCN. In contrast, these peptidergic traits were poorly expressed in hypothalamic GABAergic neurons outside the SCN. Vasopressin mRNA-positive cells in the supraoptic nucleus did not express GAD mRNA, and co-expression of somatostatin mRNA and GAD mRNA was rare in the periventricular hypothalamic nucleus. Similarly, the VIP mRNA co-expression ratio of GABAergic neurons in the cerebral cortex was far lower than that in the SCN.

Co-localization of preprosomatostatin mRNA and preprotachykinin A mRNA in neurons of the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN), a circadian oscillator, contains various peptides arranged in the compartment-specific manner. Somatostatin (SS) and substance P (SP), a peptide derived from preprotachykinin A (PPT-A), are expressed in neurons in the intermediate zone, a narrow area between the major dorsomedial and ventrolateral subdivisions. In the present study, we examined the possibility of co-localization of SS and SP in the SCN by a double-labeling in situ hybridization method using 35S-labeled and digoxigenin-labeled cRNA probes. In the SCN, most of preprosomatostatin (PPSS) mRNA-containing neurons expressed PPT-A mRNA (86%) and, in turn, almost all preprotachykinin A (PPT-A) mRNA-expressing neurons expressed PPSS mRNA signals (97%). Both PPSS and PPT-A mRNAs were also detected in the cerebral cortex and the caudate-putamen, however, their co-existence was extremely rare (< 4%) in these regions. Since the pharmacological effects of SS and SP are similar to that of the light pulses exposed on animals under constant darkness, the co-release of peptides might be an important process for entraining the circadian clock in the SCN.

Development of vasoactive intestinal peptide mRNA rhythm in the rat suprachiasmatic nucleus.

Development of the daily rhythm of vasoactive intestinal peptide (VIP) mRNA in the rat suprachiasmatic nucleus (SCN), a main locus of circadian oscillation, was investigated by in situ hybridization. The phenotypic expression of VIP neurons occurred in two developmental stages in the ventrolateral portion of the SCN (VLSCN): the first was found before birth in the rostral part, and the second occurred in the main part between postnatal day (P) 10 and P20. The latter period coincided with the time that the massive VIP-efferent fibers project to the subparaventricular zone. In the adult and P20, the VIP mRNA signals of the SCN showed a clear diurnal rhythm with a trough in the light phase and a peak in the dark phase under light/dark (LD) conditions, but under constant dark (DD) conditions, no VIP mRNA fluctuations were observed. At P10, however, it was found that SCN VIP mRNA showed a peak at the transition from night to day and a trough at early dark period in LD conditions, in sharp contrast to the night peak in the adult rhythm. In DD conditions, a light-phase peak and a dark-phase trough were also observed at P10, contrasting the arrhythmic feature at adult stage. The present findings suggest that daily VIP rhythm was first generated in the early developed clock-controlled rostral SCN neurons, and later regulated by light-dependent main VLSCN neurons.

Lesion-induced neuronal nitric oxide synthase in Purkinje cells of the rat cerebellar cortex: histochemical and in situ hybridization study.

Lesion-induced induction of neuronal nitric oxide synthase (nNOS) was examined in the rat cerebellum. The stab-lesioned cerebellar cortex was examined with NADPH-diaphorase (NADPH-d) histochemistry and in situ hybridization using nNOS cRNA probe at 1, 3, 7, 14, 35 days post-lesion. NADPH-d- and nNOS mRNA-positive Purkinje cells appeared adjacent to the lesion by 3 days after the lesion. The area of distribution expanded and the number of positive cells increased at 7 days after the lesion, and at 14 days post-lesion, shrunken NADPH-d-positive Purkinje cells with irregular surface appeared. NADPH-d activity and nNOS mRNA signal could not be detected in Purkinje cells after 35 days post-lesion. Combined NADPH-d histochemistry and in situ hybridization using glutamic acid decarboxylase (GAD) cRNA probe revealed that nNOS-expressing Purkinje cells showed fewer GAD mRNA signals than those in normal Purkinje cells. The atrophic contour and the lower expression of GAD mRNA signals in NADPH-d positive Purkinje cells suggest that nNOS is expressed under a degenerating process.

Cloning of human and mouse cDNAs encoding novel zinc finger proteins expressed in cerebellum and hippocampus.

We identified a novel gene, kf-1, highly expressed in the normal cerebellum but not in the cerebral cortex, the expression of which could have been augmented in the cerebral cortex of a sporadic Alzheimer's disease patient. We cloned human and mouse entire kf-1 cDNAs encoding conserved 79 kDa proteins containing a zinc-binding RING-H2 finger motif at the carboxy-terminus as found in acetylcholine receptor-associated protein (RAPsyn). The 3'-untranslated regions are highly conserved between human and mouse as to constitute a common mRNA secondary structure. In situ hybridization analysis of mouse brain sections revealed strong kf-1 expression in the cerebellum and hippocampus. We propose that KF-1 is involved in membranous protein-sorting apparatus similarly to RAPsyn. We mapped the human kf-1 gene to 2p11.2.

Light-induced resetting of a mammalian circadian clock is associated with rapid induction of the mPer1 transcript.

To understand how light might entrain a mammalian circadian clock, we examined the effects of light on mPer1, a sequence homolog of Drosophila per, that exhibits robust rhythmic expression in the SCN. mPer1 is rapidly induced by short duration exposure to light at levels sufficient to reset the clock, and dose-response curves reveal that mPer1 induction shows both reciprocity and a strong correlation with phase shifting of the overt rhythm. Thus, in both the phasing of dark expression and the response to light mPer1 is most similar to the Neurospora clock gene frq. Within the SCN there appears to be localization of the induction phenomenon, consistent with the localization of both light-sensitive and light-insensitive oscillators in this circadian center.

Antidiuretic action of tachykinin NK-3 receptor in the rat paraventricular nucleus.

Studies were performed on the central antidiuretic actions via the tachykinin NK-3 receptor in the rat hypothalamic paraventricular nucleus (PVN). Microinjections of the selective tachykinin NK-3 receptor agonist senktide (2-200 pmol) into the PVN resulted in prolonged inhibition of urine output in water-loaded rats, its effect being dose-dependent. The antidiuretic action of senktide was blocked by pretreatment with the vasopressin V2 receptor antagonist OPC-31260 (1 mg/kg, i.v.), but not by microinjection of the angiotensin II AT-1 receptor antagonist losartan (1 nmol) into the PVN. NK-3 receptor mRNA was strongly detected in the magnocellular part of the PVN and the supraoptic nucleus (SON) of the hypothalamus as detected by in situ hybridization histochemistry. Moreover, [3H]senktide binding sites were also detected in the PVN and the SON by receptor autoradiography. These findings suggest that NK-3 receptors in the PVN may be involved in water regulation by stimulation of vasopressin secretion from the posterior pituitary gland, and that vasopressin caused water reabsorbtion via the kidney V2 receptor.

Somatostatin neurons form a distinct peptidergic neuronal group in the rat suprachiasmatic nucleus: a double labeling in situ hybridization study.

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.

Regional distribution of growth hormone-releasing hormone (GHRH) receptor mRNA in the rat brain.

We examined the tissue distribution of growth hormone-releasing hormone (GHRH) receptor mRNA in the rat brain because several lines of evidence have suggested that GHRH plays a functional role in the brain. GHRH receptor mRNA was detected in the hypothalamus as well as pituitary, but not in olfactory bulb, caudate putamen, cerebral cortex, hippocampus, cerebellum, or brainstem by RNase protection assay. To clarify the precise localization of GHRH receptor mRNA in the hypothalamus, reverse transcription-polymerase chain reaction (RT-PCR) was used. A PCR product of the predicted size (564 bp) was detected in the periventricular, arcuate, and ventromedial nuclei, and the anterior hypothalamic area, but not in the paraventricular nucleus in the hypothalamus. These areas where GHRH receptor mRNA was detected are possible sites of GHRH action. Another band, smaller in size than that of the predicted PCR amplification product, was detected in the anterior hypothalamic area and arcuate nucleus, respectively. Hybridization analysis with a cDNA probe for GHRH receptor demonstrated that the smaller bands as well as that of the predicted size corresponded to GHRH receptor cDNA. The role of the short form of the GHRH receptor remains unknown.

Distribution of mRNA for CCK-B receptor in the brain of Mastomys natalensis: abundant expression in telencephalic neurons.

The distribution of cholecystokinin B (CCK-B) receptors in the Mastomys brain was studied using Northern blot analysis and in situ hybridization technique. By Northern blot analysis using 32P-labeled cDNA probe, the cortex had the highest hybridization signal of CCK-B receptor mRNA in the brain. The olfactory bulb and hippocampus showed a moderate level of signals. In situ hybridization using 35S-labeled cRNA probes revealed a wide and region-specific distribution of CCK-B receptor mRNA in the telencephalon. Throughout the cerebral cortex, labeled cells were found in all layers, with higher intensities in layers II, V and VI. Pyramidal cells of the layer II of the piriform cortex showed the highest level of signals in the brain. In the hippocampus, most of the pyramidal cells of the Ammon's horn were labeled, although labeled cells were not detected in other layers. Distinct signals were also detected in the various amygdaloid nuclei, caudate-putamen, reticular thalamic nucleus, hypothalamic ventromedial nucleus and inferior colliculus. This distribution pattern may further support the prominent existence of CCK-B receptors in the brain particularly in the telencephalon.