Preoptic and hypothalamic regulation of multi-tiered, chronologically arranged female rat sexual behavior.

As in many mammalian behaviors, sexual behavior exhibits structure. Each modular components of the structure, that are linked together over time, occur in probabilistic manner. Endocrine milieu, in particular sex hormones, define the probability to synchronize the behavior with the production of gametes. Developmental experience and environmental cues affect the hormonal milieu of the brain. This is especially true in female mammals, in which ova mature with certain intervals along with ovarian secretion of sex hormones. Estrogens secreted by mature ovarian follicles support both affiliative and executive components of female sexual behavior. In the absence of the ovarian steroids, females avoid males when possible, or antagonize and reject males when put together. Female sexual behavior is intimately linked with the estrous cycle in many species such that females are only receptive for a brief period at the estrus stage surrounding ovulation. Thus, in the rat, females strongly influence the outcome of mating encounter with a male. Affiliative or solicitatory behavior shown by females in estrus leads to the female adapting the lordosis posture, which is characterized by hindleg postural rigidity and lordotic dorsiflexion of the spine, in response to touch-pressure somatosensory stimuli on the skin of the flanks, rump-tail base, perineum region given by male partner. The posture facilitates intromission and consequently fertilization. Although dependence on estrogens is the most important feature of female rat sexual behavior, cervical probing combined with palpation of the hindquarter skin acts as a supranormal stimulus to elicit lordosis. Thus, lordosis behavior is a hub of multi-tiered, chronologically arranged set of behaviors and estrogen appear to alter excitability of neural network for lordosis.

Oxytocin is indispensable for conspecific-odor preference and controls the initiation of female, but not male, sexual behavior in mice.

Oxytocin (OT) has been demonstrated to be involved in various social behaviors in mammals. However, OT gene knockout (OTKO) mice can conceive and deliver successfully, though females cannot rear their pups because of lack of lactation. Here, we investigated the sociosexual behavior of both sexes in two experimental setups: olfactory preference for sexual partner's odor and direct social interaction in an enriched condition. In the preference test, mice were given a choice of two airborne odors derived from intact male and receptive female mice, or from intact or castrated male mice. Wild-type (WT) mice significantly preferred opposite-sex odors, whereas OTKO mice showed vigorous but equivalent exploration to all stimuli. In social interactions in the enriched condition, no difference in sexual behavior was found between WT and OTKO males. In contrast, WT female initiated sexual behavior at the second week test, while OTKO females required 4 weeks to receive successful mounts. Neuronal activation by odor stimulation was compared between WT and OTKO mice. The numbers of cFos-immunoreactive cells increased in the medial amygdala and the preoptic area after exposure to opposite-sex odors in WT mice, whereas the increase was suppressed in OTKO mice. We conclude that OT plays an important role in the regulation of olfactory-related social behavior in both male and female mice. The influence of OT was greater in female mice, especially during social interactions involving the acquisition of sexual experience.

Identification of an Intra- and Inter-specific Tear Protein Signal in Rodents.

Rodents use the vomeronasal olfactory system to acquire both inter- and intra-specific information from the external environment and take appropriate actions. For example, urinary proteins from predator species elicit avoidance in mice, while those from male mice attract female mice. In addition to urinary proteins, recent studies have highlighted the importance of lacrimal proteins for intra-specific communications in mice. However, whether the tear fluid of other species also mediates social signals remains unknown. Here, we show that a lacrimal protein in rats (predators of mice), called cystatin-related protein 1 (ratCRP1), activates the vomeronasal system of mice. This protein is specifically produced by adult male rats in a steroid hormone-dependent manner, activates the vomeronasal system of female rats, and enhances stopping behavior. When detected by mice, ratCRP1 activates the medial hypothalamic defensive circuit, resulting in decreased locomotion coupled with lowered body temperature and heart rate. Notably, ratCRP1 is recognized by multiple murine type 2 vomeronasal receptors, including Vmn2r28. CRISPR/Cas9-mediated deletion of vmn2r28 impaired both ratCRP1-induced neural activation of the hypothalamic center and decrease of locomotor activity in mice. Taken together, these data reveal the neural and molecular basis by which a tear fluid compound in rats affects the behavior of mice. Furthermore, our study reveals a case in which a single compound that mediates an intra-specific signal in a predator species also functions as an inter-specific signal in the prey species.

Characterization of rodent constitutively active estrogen receptor α variants and their constitutive transactivation mechanisms.

Estrogen receptor α (ERα) mRNAs exhibit remarkable heterogeneity owing to complicated alternative splicing. Some encode C-terminally-truncated ERα proteins, which display ligand-independent transactivation or dominant-negative activity. We previously characterized C-terminally-truncated ERα mRNA variants with cryptic sequences in humans and mice, and demonstrated that helices in the ligand-binding domains (LBDs) of ERα variants contribute to ligand-independent transcriptional activity. However, existence of non-conventional coding exons and generation of constitutively active ERα variants have remained to be examined in rats. To comparatively analyze modular organization and splicing profiles of the ERα genes, the range of C-terminally-truncated ERα variants was explored in rats and mice using rapid amplification of cDNA ends and RT-PCR cloning. Furthermore, their functions were determined in transiently transfected cells using expression constructs and luciferase reporter assays. Multiple cryptic exons and C-terminally-truncated ERα variant mRNAs were identified in rats and mice. Naturally occurring and artificially truncated variants/constructs lacking helix 5 potentially exhibited gain-of-function in transfected cells. Although cryptic exons and splicing profiles were poorly conserved among humans, mice, and rats, constitutively active variants were generated from the ERα genes. Moreover, the primary mechanism of ligand-independent activation by C-terminally-truncated ERα variants is C-terminus to helix 5 deletion in the LBD. This comparative study documented the complexity of ERα genes, mRNAs, and proteins, and further determined the underlying structural basis of ligand-independent activation by C-terminally-truncated ERα variants.

Vomeronasal signal deficiency enhances parental behavior in socially isolated male mice.

We previously reported that social isolation promotes parental care in sexually naïve male mice. This effect was blocked by exposure to chemosensory and auditory social signals derived from males in an adjacent compartment. In the present study, we examined whether the chemosensory signals detected in the vomeronasal organ (VNO) are involved in parental behavior by using mice deficient for a VNO-specific ion channel (Trpc2-/-) and thus impaired in VNO-input signaling. We housed virgin homozygous Trpc2-/- and heterozygous Trpc2± males for 3weeks during puberty (5-8weeks old) alone or in groups of 3-5 males. At 8weeks of age, the mice were placed with three pups in an observation cage and tested for parental behavior. The Trpc2-/- males housed under isolated conditions spent significantly longer in the vicinity of pups than did the Trpc2-/- males than had been group housed, whereas no isolation effect was observed in heterozygous Trpc2± males. Both Trpc2 knockout and isolation housing significantly increased the time males spent licking pups and crouching (arched back posture over pups to enable nursing), whereas only isolation housing increased the incidence of retrieval behavior. These results demonstrated that social signals transmitted not only through the VNO but also from other modalities, independent of each other, suppress the expression of parental behavior during puberty in sexually naïve males.

Oxytocin mediates copulation-induced hypoalgesia of male rats.

Copulatory behavior has been reported to raise the pain threshold in male rats. In this study, we examined the effect of copulatory behavior with or without ejaculation on pain threshold measured by electrical shock via an electrode attached to the tail. It was demonstrated that ejaculation is not necessary to raise the pain threshold in male rats. In addition, we examined whether oxytocin, a hypothalamic neuropeptide, was involved in copulation-induced hypoalgesia. Sexually experienced males were subjected to stereotaxic implantation of a guide cannula targeting the lateral ventricle. After the recovery period, half of the males were intracerebroventricularly treated with an oxytocin antagonist (OTA, 100ng d(CH2)51,Tyr(Me)2,Thr4, Orn8,Tyr-NH29]-vasotocin/1µL saline) and the remaining half were administered saline without anesthesia. Fifteen minutes later, half of each group were given sexual behavior with receptive females. We found no effect of OTA on sexual activity. Immediately after ejaculation, pain threshold was measured. While raised pain threshold was observed after sexual behavior in saline-treated males, no change in pain threshold was found in OTA-treated males even after copulation. The results suggest that central oxytocin mediates copulation-induced hypoalgesia in male rats.

Human C-terminally truncated ERα variants resulting from the use of alternative exons in the ligand-binding domain.

The nuclear receptor genes contain alternative internal and terminal exons, with alternative exon incorporation yielding mRNA variants that encode various receptor types, including some with C-terminal truncation that exhibit constitutive activation or dominant-negative transcriptional transactivation. However, C-terminally truncated estrogen receptor α (ERα) variants with alternative sequences have rarely been reported in humans. Therefore, we assessed human ERα genomic organization and alternative splicing profiles, and identified both alternative exons and C-terminally truncated ERα variants. These naturally occurring C-terminally truncated ERα proteins were localized in the nuclei of transfected cells. In addition, ERαi45c and ERαΔ5 variants exhibited constitutive transactivation of an estrogen responsive element-driven promoter in transfected cells. We manufactured expression vectors encoding artificially truncated ERα constructs and evaluated their transactivation abilities to establish mechanisms determining the constitutive activity and dominant-negative properties of truncated variants. Lack of the region encoded in exon 8 eliminated basal and ligand-induced transcriptional transactivation. The C-terminally truncated ERα variants/constructs containing the helices 5 in their ligand-binding domains did not exhibit constitutive transactivation. Furthermore, we demonstrated that truncation from C-termini to helices 5 in the variant ligand-binding domains was required for constitutive activation and found that the remnant regions of the ligand-binding domains and variant-specific sequences influenced transcriptional transactivation efficiency. In conclusion, we elucidated the structural and functional features of novel C-terminally truncated ERα variants and revealed the mechanisms underlying constitutive transactivation by C-terminally truncated nuclear receptor variants.

Puerperal and parental experiences alter rat preferences for pup odors via changes in the oxytocin system.

In the rat, induction of maternal behavior depends on the parity of the female. For example, nulliparous (NP) females need longer exposure to pups than multiparous (MP) or lactating (L) females to exhibit similar maternal behavior. In this study, we investigated the role of brain oxytocin in the approaching behavior of these female rats. Olfactory preferences for pup odors were examined for 8 consecutive days. Each preference test was followed by direct overnight exposure to pups. On the 8th day, MP and L, but not NP females showed robust pup-odor preferences. After the behavioral test, half of the females were exposed to pups for 2 h, whereas the other half were not. The females were then sacrificed to analyze brain oxytocin (OXT) and vasopressin (AVP) activities by cFos immunohistochemistry and to quantify their receptor mRNA expression using real-time PCR. In the paraventricular nucleus (PVN), the percentage of cFos-positive OXT neurons was significantly larger in MP and L females than in NP females after pup exposure. No significant differences were found in cFos expression in OXT neurons of the supraoptic nucleus (SON) or in AVP neurons of either the PVN or SON. Expression of OXT receptor mRNA in the medial preoptic area and amygdala of the control groups was also higher in MP females than in NP females. Finally, we demonstrated that infusion of OXT into the lateral ventricle of NP females promoted preferences for pup odors. These results indicate that puerperal and parental experiences enhance the responsiveness of OXT neurons in the PVN to pup stimuli and establish olfactory preferences for these odors in a parity-dependent manner.

Transient reversal of olfactory preference following castration in male rats: Implication for estrogen receptor involvement.

We examined the effects of the sex steroid milieu on sexual odor preference of sexually-experienced male rats using an alternate choice paradigm after endocrine manipulations. Gonadally intact (GI) males showed a male typical preference, i.e. spent longer time sniffing estrous females than males or ovariectomized females. At 1-2 weeks after orchidectomy (ORx), the males exhibited a transient preference for sexually vigorous males, a female typical preference pattern, followed by a total loss of preference after 4 weeks. Subcutaneous implantation of a Silastic capsule containing formestane (4-OHA), an aromatase inhibitor, had no effect on the preference of gonadally intact rats, but successfully prevented the emergence of the female typical preference after ORx. Capsules containing testosterone (T), dihydrotestosterone (DHT), or estradiol benzoate (EB), but not those with cholesterol (CH), restored masculine typical preference in ORx males at 2 weeks after the placement. The feminine preference for males was observed at 2-3 weeks after removal of T or EB capsules, but not by the removal of DHT and CH capsules. The results suggest that either exogenous androgen or estrogen maintains the masculine typical odor preference. Estrogen itself or produced through aromatization of circulating T, induces a transient feminine typical preference at a certain decreased titer during its disappearance from the circulation. Estrogen at different titers might determine appearance of masculine or feminine typical olfactory preference in adult ORx rats.

Characterization of the fundamental properties of the N-terminal truncation (Δ exon 1) variant of estrogen receptor α in the rat.

The estrogen receptor α (ERα) directs transactivation of target genes, and splice variants have been shown to exhibit altered activation properties. We previously documented the complicated alternative promoter usage and splicing patterns of the rat ERα gene; however, the information was restricted to a few specific organs. Therefore, we re-examined the rat mRNA profiles of ERα, including the generation of the exon 1-skipping, ERα46 transcript in a wider variety of rat organs and further characterized the fundamental functional properties of rat ERα46 variants. With the use of RT-PCR, we discovered unique distribution and splicing patterns for promoter-specific ERα isoforms, as well as the extensive expression of the Δ exon 1 variant in the rat. Similar to wild-type ERα, an immunocytochemical analysis showed a predominant localization of ERα46 proteins in the nuclei of transfected cells. Luciferase reporter assays revealed that ERα46 variants stimulated the transcriptional activity of an estrogen response element-driven promoter in response to estrogen. In addition, the variants exhibited distinct transactivation and reactivity to 4-hydroxytamoxifen in different cell types. Although the alternative splicing patterns are species-specific, the profiles of the alternative use of promoters, and the fundamental properties of the rat ERα46 variant are similar to those of human and mouse homologs. Therefore, the present study provides fundamental and useful information for further research into the regulation and functions of ERα gene variants.

Social isolation prompts maternal behavior in sexually naïve male ddN mice.

Maternal behavior in mice is considered to be sexually dimorphic; that is, females show maternal care for their offspring, whereas this behavior is rarely shown in males. Here, we examined how social isolation affects the interaction of adult male mice with pups. Three weeks of isolation during puberty (5-8 weeks old) induced retrieving and crouching when exposed to pups, while males with 1 week isolation (7-8 weeks old) also showed such maternal care, but were less responsive to pups. We also examined the effect of isolation during young adulthood (8-11 weeks old), and found an induction of maternal behavior comparable to that in younger male mice. This effect was blocked by exposure to chemosensory and auditory social signals derived from males in an attached compartment separated by doubled opaque barriers. These results demonstrate that social isolation in both puberty and postpuberty facilitates male maternal behavior in sexually naïve mice. The results also indicate that air-borne chemicals and/or sounds of male conspecifics, including ultrasonic vocalization and noise by their movement may be sufficient to interfere with the isolation effect on induction of maternal behavior in male mice.

Social evolution. Oxytocin-gaze positive loop and the coevolution of human-dog bonds.

Human-like modes of communication, including mutual gaze, in dogs may have been acquired during domestication with humans. We show that gazing behavior from dogs, but not wolves, increased urinary oxytocin concentrations in owners, which consequently facilitated owners' affiliation and increased oxytocin concentration in dogs. Further, nasally administered oxytocin increased gazing behavior in dogs, which in turn increased urinary oxytocin concentrations in owners. These findings support the existence of an interspecies oxytocin-mediated positive loop facilitated and modulated by gazing, which may have supported the coevolution of human-dog bonding by engaging common modes of communicating social attachment.

Estradiol-sensitive projection neurons in the female rat preoptic area.

Electrical stimulation of the preoptic area (POA) interrupts the lordosis reflex, a combined contraction of back muscles, in response to male mounts and the major receptive component of sexual behavior in female rat in estrus, without interfering with the proceptive component of this behavior or solicitation. Axon-sparing POA lesions with an excitotoxin, on the other hand, enhance lordosis and diminish proceptivity. The POA effect on the reflex is mediated by its estrogen-sensitive projection to the ventral tegmental area (VTA) as shown by the behavioral effect of VTA stimulation as well as by the demonstration of an increased threshold for antidromic activation of POA neurons from the VTA in ovariectomized females treated with estradiol benzoate (EB). EB administration increases the antidromic activation threshold in ovariectomized females and neonatally castrated males, but not in neonatally androgenized females; the EB effect is limited to those that show lordosis in the presence of EB. EB causes behavioral disinhibition of lordosis through an inhibition of POA neurons with axons to the VTA, which eventually innervate medullospinal neurons innervating spinal motoneurons of the back muscle. The EB-induced change in the threshold or the axonal excitability may be a result of EB-dependent induction of BK channels. Recordings from freely moving female rats engaging in sexual interactions revealed separate subpopulations of POA neurons for the receptive and proceptive behaviors. Those POA neurons engaging in the control of proceptivity are EB-sensitive and project to the midbrain locomotor region (MLR). EB thus enhances lordosis by reducing excitatory neural impulses from the POA to the VTA. An augmentation of the POA effect to the MLR may culminate in an increased locomotion that embodies behavioral estrus in the female rat.

Efferent and afferent connections of the ventromedial hypothalamic nucleus determined by neural tracer analysis: implications for lordosis regulation in female rats.

Neural connections of the ventromedial hypothalamic nucleus (VMN) to and from forebrain and midbrain structures, which are involved in the neuroendocrine regulation of reproduction, were investigated. A retrograde (fluoro-gold [FG]) or an anterograde neural tracer (phaseolus vulgaris-leucoagglutinin [PHA-L]) was injected into the left side of the VMN in ovariectomized rats. Six days after injection with FG or 11 days after injection with PHA-L, brains were fixed and sectioned. After immunohistochemistry, digital images of FG-labeled neural cell bodies (FG-cells) or PHA-L-labeled fibers (PHA-L-fibers) were analyzed. Injection sites of FG and PHA-L were mainly in the ventrolateral VMN. Considerable numbers of FG-cells and PHA-L-fibers were present in the left side of the medial amygdala, ventral lateral septum, preoptic area, bed nucleus of stria terminalis, dorsomedial hypothalamic nucleus, arcuate nucleus, periventricular nucleus of thalamus, and midbrain central gray. The lateral dorsal raphe nuclei contained many PHA-L-fibers but few FG-cells. By contrast, both sides of the median raphe nucleus contained many FG-cells but few PHA-L-fibers. Reciprocal direct neural connection between the right and left side of the VMN were observed. The present results provide an anatomical basis for functional relationships between the VMN and these nuclei.

An enriched rearing environment calms adult male rat sexual activity: implication for distinct serotonergic and hormonal responses to females.

Early life events induce alterations in neural function in adulthood. Although rearing in an enriched environment (EE) has a great impact on behavioral development, the effects of enriched rearing on sociosexual behavior remain unclear. In this study, we investigated the effects of rearing in an EE on male copulatory behavior and its underlying neurobiological mechanisms in Wistar-Imamichi rats. Three-week-old, recently weaned rats were continuously subjected to a standard environment (SE) or an EE comprised of a large cage with several objects, such as toys, tunnels, ladders, and a running wheel. After 6 weeks, rats reared in an EE (EE rats) showed decreased sexual activity compared with rats reared in a SE (SE rats). This included a lower number of ejaculations and longer latencies in three consecutive copulatory tests. In addition, EE rats showed decreased emotional responsiveness and less locomotor behavior in an open field. In a runway test, on the other hand, sexual motivation toward receptive females in EE males was comparable to that of SE males. Furthermore, following exposure to a female, increases in serotonin levels in the nucleus accumbens and the striatum were significantly suppressed in EE males, whereas dopaminergic responses were similar between the groups. Female-exposure-induced increases in the levels of plasma corticosterone and testosterone were also suppressed in EE rats compared to SE rats. These data suggest that rearing in an EE decreases male copulatory behavior, and serotonin and hormonal regulating systems may regulate the differences in sociosexual interactions that result from distinct rearing environments.

Estrogen-induced cell signaling in the sexually dimorphic nucleus of the rat preoptic area: potential involvement of cofilin in actin dynamics for cell migration.

Estrogen is a key factor to induce the sexually dimorphic nucleus (SDN) in the preoptic area (POA) of the rat brain. Identification of estrogen-dependent signaling pathways at SDN in POA during the critical period is a prerequisite for elucidating the mechanism. In the present study, we treated female rats with/without 17ß-estradiol (E2) at birth, designated as postnatal day 1 (P1), and prepared total RNA from brain slices containing SDN for DNA microarray analysis. Among the estrogen-responsive genes identified, protein kinase C-delta (PKC-δ) was significantly up-regulated by E2 at P5. We examined the downstream effectors of PKC-δ protein by Western blotting and found an E2-induced PKC-δ/Rac1/PAK1/LIMK1/cofilin pathway. In the pathway, E2 suppressed the phosphorylation (inactive form) of cofilin. This result was supported by immunohistochemistry, where the phosphorylation/dephosphorylation of cofilin occurred at SDN, which suggests that cell migration is a cue to create sexual dimorphism in POA.

Novel splicing events and post-transcriptional regulation of human estrogen receptor α E isoforms.

Expression of the estrogen receptor α (ERα) gene is subject to complex regulation. To elucidate the mechanisms of this regulation, the genomic organization and the physiological role of the multiple 5'-untranslated regions (5'-UTRs) must be determined. Here, we investigated the expression and splicing patterns of the human ERα E isoforms. We identified two novel untranslated exons, N1 and N2, in the 5'-region of the human ERα gene and multiple E isoform mRNA variants generated by alternative usage of non-coding internal exons. Expression of the N1-containing variants was observed only in the human breast adenocarcinoma cell line, MCF7, while the N2-containing variants were expressed in the adult liver and MCF7 cells. We examined post-transcriptional regulation of the variant mRNAs using luciferase reporter assays and quantitative PCR. The insertion of untranslated internal exons into the 5'-UTRs of the E isoforms reduced their translation efficiency, but barely influenced mRNA turnover. Our results indicate that the genomic organization of the human ERα gene and the splicing profiles of the human ERα E isoforms are more complicated than previously reported. Furthermore, the 5'-UTRs of the E isoforms post-transcriptionally control human ERα expression mainly through translational repression.

Neuronal responses in the nucleus accumbens shell during sexual behavior in male rats.

Previous behavioral studies have indicated that the nucleus accumbens (NAc) shell of a male rat is involved in its sexual behavior; however, no previous studies have investigated neuronal activities in the male rat NAc shell during sexual behavior. To investigate this issue, we recorded single unit activities in the NAc shell of male rats during sexual behavior. Of 123 NAc shell neurons studied, 53, 47, and 40 neurons exhibited significantly changed firing rates at various times during intromission, genital auto-grooming, and sniffing of females, respectively. The two types of NAc shell neurons [putative fast spiking interneurons (pFSIs) and medium spiny neurons (pMSNs)] responded differently during sexual behavior. First, more pFSIs than pMSNs exhibited inhibitory responses to thrusting with intromission and genital grooming, while pFSIs and pMSNs responded similarly to sniffing of females. Second, both pFSIs and pMSNs responded differently to thrusting with and without intromission. Furthermore, NAc shell neuronal activity was significantly different across the different phases of sexual behavior, and the number of NAc shell neurons with delta oscillation, which is related to behavioral inhibition, and high gamma oscillation, which is related to reward perception, increased after ejaculation. Together, our results suggest that the NAc shell is deeply involved in sexual behavior, and changes in NAc shell neuronal activity are related to performance of sexual behavior, encoding cues or contexts related to sexual behavior, reward-related processing, and the inhibition of sexual behavior after ejaculation.

Somatostatin inhibition of GnRH neuronal activity and the morphological relationship between GnRH and somatostatin neurons in rats.

In rodents, GnRH neurons are diffusely distributed from the medial septum through to the medial preoptic area and control gonadal functions through the pituitary. The activity of GnRH neurons is regulated by a variety of bioactive substances, including the inhibitory peptide somatostatin. In the present study, we focused on somatostatin because intracerebroventricular injection of somatostatin inhibits the LH surge in rats and reduces LH secretion in ewes. Somatostatin also decreases GnRH release from rat hypothalamic slices. In mice, somatostatin is also thought to suppress GnRH neuronal activity through contact on the soma of GnRH neurons. However, similar data are missing in rats. Moreover, rat GnRH neurons receive only a few synaptic inputs. In this study, we assessed the morphological relationship between GnRH and somatostatin neurons. Confocal microscopy on the sections from the medial septum through medial preoptic area revealed about 35 close contacts per rat between the GnRH and somatostatin neuronal fibers in the organum vasculosum of the lamina terminalis region. No contact of somatostatin fibers on the GnRH neuronal somata was observed. Multicell RT-PCR for somatostatin receptor mRNA in rat GnRH neurons was also performed, which revealed moderate expression of somatostatin receptor subtypes 1-5. In addition, patch clamp experiments were carried out in acute slice preparations. Somatostatin suppressed neuronal firing in cells recorded in a cell-attached configuration and also induced whole-cell outward currents in GnRH neurons. These findings suggest that somatostatin directly inhibits the activity of rat GnRH neurons through volume transmission in the organum vasculosum of the lamina terminalis region.

Complex organization of the 5'-untranslated region of the mouse estrogen receptor α gene: identification of numerous mRNA transcripts with distinct 5'-ends.

The 5'-untranslated region (5'-UTR) of the estrogen receptor α (ERα) gene plays an important role in determining its tissue-specific expression. We examined the 5'-UTRs of the mouse ERα mRNA variants in depth using the Basic Local Alignment Search Tool (BLAST), rapid amplification of 5'-cDNA ends (5'-RACE) and RT-PCR. We demonstrated the presence of multiple variants containing unique 5'-UTRs. We mapped the cDNA sequences onto the mouse genome, and found that both alternative splicing from four different leader exons (A, C, F1, and H) to exon 1, and combinations of 12 internal exons (X1, X2, X3, X4, F2/X5, X6, X7, X8, X9, X10, X11, and B) generate multiple ERα transcripts. Mouse exon B, that has homologies with human exon B and rat exon 0T, was used as an internal exon, not as a leader exon. RT-PCR analysis revealed distinct expression patterns of the variants, suggesting that the alternative promoter usage and alternative splicing are regulated in a tissue-specific manner. Our results indicate that the genomic organization of the mouse ERα gene is complicated as previously shown in the rat ERα gene. In addition, both alternative promoter usage and alternative splicing contribute to the remarkable mRNA diversity of the mouse ERα gene.