RESUMO
Nociception, a somatic discriminative aspect of pain, is, like touch, represented in the primary somatosensory cortex (S1), but the separation and interaction of the two modalities within S1 remain unclear. Here, we show spatially distinct tactile and nociceptive processing in the granular barrel field (BF) and adjacent dysgranular region (Dys) in mouse S1. Simultaneous recordings of the multiunit activity across subregions revealed that Dys neurons are more responsive to noxious input, whereas BF neurons prefer tactile input. At the single neuron level, nociceptive information is represented separately from the tactile information in Dys layer 2/3. In contrast, both modalities seem to converge on individual layer 5 neurons of each region, but to a different extent. Overall, these findings show layer-specific processing of nociceptive and tactile information between Dys and BF. We further demonstrated that Dys activity, but not BF activity, is critically involved in pain-like behavior. These findings provide new insights into the role of pain processing in S1.
Assuntos
Nociceptividade , Percepção do Tato , Animais , Mapeamento Encefálico/métodos , Camundongos , Nociceptividade/fisiologia , Dor , Córtex Somatossensorial/fisiologia , Percepção do Tato/fisiologiaRESUMO
Although there are few well-validated antibodies against ESR2 proteins, a recent validation assessment identified a specific monoclonal antibody against human ESR2 proteins (PPZ0506). Furthermore, our previous study confirmed its cross-reactivity and specificity against rodent ESR2 proteins, enabling the determination of true ESR2 distribution profiles in rodents. Therefore, we aimed to determine optimal conditions for ESR2 detection by PPZ0506 immunostaining and analyze ESR2 distribution in rats. We evaluated several staining conditions using paraffin-embedded and frozen ovary sections. Immunohistochemical staining with PPZ0506 antibody required strong antigen retrieval and appropriate antibody dilution. Subsequent immunohistochemical analysis in multiple tissues under optimized conditions revealed that rat ESR2 proteins are expressed in a more localized manner than previously assumed. Our results suggest that previous immunohistochemical studies using inadequately validated antibodies against ESR2 proteins overestimated their distribution profiles. We expect that optimized immunohistochemical detection with PPZ0506 antibody can help researchers solve several conflicting problems in ESR2 research.
Assuntos
Anticorpos Monoclonais/metabolismo , Receptor beta de Estrogênio/metabolismo , Animais , Antígenos/metabolismo , Receptor beta de Estrogênio/genética , Feminino , Secções Congeladas , Temperatura Alta , Imuno-Histoquímica , Especificidade de Órgãos , Ovário/metabolismo , Inclusão em Parafina , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Reprodutibilidade dos TestesRESUMO
RF-amide peptides, a family of peptides characterized by a common carboxy-terminal Arg-Phe-NH2 motif, play various physiological roles in the brain including the modulation of neuroendocrine signaling. Neuropeptide FF (NPFF) receptors exhibit a high affinity for all RF-amide peptides, which suggests that the neurons expressing these NPFF receptors may have multiple functions in the brain. However, the distribution of the neurons expressing NPFF receptors in the rat brain remains poorly understood. This study aimed to determine the detailed histological distribution of mRNA that encodes the neuropeptide FF receptors (Npffr1 and Npffr2) in the rat brain using in situ hybridization. Neurons with strong Npffr1 expression were observed in the lateral septal nucleus and several hypothalamic areas related to neuroendocrine functions, including the paraventricular nucleus (PVN) and arcuate nucleus, whereas Npffr2-expressing neurons were observed mainly in brain regions involved in somatosensory pathways, such as several subnuclei of the thalamus. Npffr1 expression was observed in 70% of corticotropin-releasing hormone neurons, but in only a small population of oxytocin and vasopressin neurons in the PVN. Npffr1 expression was also observed in the dopaminergic neurons in the periventricular nucleus and the dorsal arcuate nucleus, and in the kisspeptin neurons in the anteroventral periventricular nucleus. These results suggest that NPFFR1-mediated signaling may be involved in neuroendocrine functions, such as in reproduction and stress response. In conjunction with a detailed histological map of NPFFRs, this study provides useful data for future neuroendocrine research.
Assuntos
Encéfalo/metabolismo , Neurônios/metabolismo , Receptores de Neuropeptídeos/análise , Animais , Feminino , Imunofluorescência , Hibridização In Situ , Masculino , Ratos , Ratos Wistar , Receptores de Neuropeptídeos/biossínteseRESUMO
Several lines of controversial evidence concerning estrogen receptor ß (ERß) remain to be solved because of the unavailability of specific antibodies against ERß. The recent validation analysis identified a monoclonal antibody (PPZ0506) with sufficient specificity against human ERß. However, the specificity and cross-reactivity of PPZ0506 antibody against ERß proteins from laboratory animals have not been confirmed. In the present study, we aimed to validate the applicability of PPZ0506 to rodent studies. The antibody exhibited specific cross-reactivity against mouse and rat ERß proteins in immunoblot and immunocytochemical experiments using transfected cells. In immunohistochemistry for rat tissue sections, PPZ0506 showed immunoreactive signals in the ovary, prostate, and brain. These immunohistochemical profiles of rat ERß proteins in rat tissues accord well with its mRNA expression patterns. Although the antibody was reported to show the moderate signals in human testis, no immunoreactive signals were observed in rat testis. Subsequent RT-PCR analysis revealed that this species difference in ERß expression resulted from different expression profiles related to the alternative promoter usage between humans and rats. In conclusion, we confirmed applicability of PPZ0506 for rodent ERß studies, and our results provide a fundamental basis for further examination of ERß functions.
Assuntos
Anticorpos Monoclonais Murinos/química , Receptor beta de Estrogênio/biossíntese , Animais , Humanos , Imuno-Histoquímica , Camundongos , Especificidade de Órgãos , Ratos , Ratos WistarRESUMO
Estrogens play an essential role in multiple physiological functions in the brain, including reproductive neuroendocrine, learning and memory, and anxiety-related behaviors. To determine these estrogen functions, many studies have tried to characterize neurons expressing estrogen receptors known as ERα and ERß. However, the characteristics of ERß-expressing neurons in the rat brain still remain poorly understood compared to that of ERα-expressing neurons. The main aim of this study is to determine the neurochemical characteristics of ERß-expressing neurons in the rat hypothalamus using RNAscope in situ hybridization (ISH) combined with immunofluorescence. Strong Esr2 signals were observed especially in the anteroventral periventricular nucleus (AVPV), bed nucleus of stria terminalis, hypothalamic paraventricular nucleus (PVN), supraoptic nucleus, and medial amygdala, as previously reported. RNAscope ISH with immunofluorescence revealed that more than half of kisspeptin neurons in female AVPV expressed Esr2, whereas few kisspeptin neurons were found to co-express Esr2 in the arcuate nucleus. In the PVN, we observed a high ratio of Esr2 co-expression in arginine-vasopressin neurons and a low ratio in oxytocin and corticotropin-releasing factor neurons. The detailed neurochemical characteristics of ERß-expressing neurons identified in the current study can be very essential to understand the estrogen signaling via ERß.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Receptor beta de Estrogênio/biossíntese , Regulação da Expressão Gênica , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/citologia , Arginina Vasopressina/biossíntese , Feminino , Imunofluorescência , Hibridização In Situ , Masculino , Neurônios/citologia , Núcleo Hipotalâmico Paraventricular/citologia , RatosRESUMO
Male rats rarely show lordosis, a female sexual behavior, because of strong inhibition of the behavior in the lateral septum. Because neonatal treatment with estradiol (E2) in female rats decreases lordosis, it is believed that the lateral septum is a target of E2 action to defeminize or masculinize the lordosis-inhibiting system. Here, we tested the hypothesis that disruption of the lateral septum before E2 treatment prevents the effect of neonatal E2 on lordosis. Female rats that underwent radiofrequency-induced septal lesions or sham operation on postnatal day 4 (PD4, day of birth = PD1) were subcutaneously injected with E2 or sesame oil vehicle alone on PD5. Vaginal opening and smears were checked. After sexual maturation, lordosis tests were performed. The effects of neonatal septal lesions on lordosis in male rats were also observed. Sham-operated and E2-treated female rats showed a reduction in lordosis and irregular estrous cycles. Conversely, septal lesioned and E2-treated females exhibited higher levels of lordosis, although their estrous cycles were irregular. These results suggest that neonatal septal lesions prevent females from being behaviorally defeminized by neonatal E2. Additionally, neonatally septal lesioned males displayed higher levels of lordosis than sham-operated males. These results suggest that E2, which is produced by the aromatization of testicular testosterone in the neonatal period, acts on the lateral septum to organize the lordosis-inhibiting system.
Assuntos
Estradiol/fisiologia , Postura , Núcleos Septais/fisiologia , Caracteres Sexuais , Comportamento Sexual Animal/fisiologia , Animais , Animais Recém-Nascidos , Estradiol/administração & dosagem , Ciclo Estral , Feminino , Masculino , Ratos Wistar , Núcleos Septais/efeitos dos fármacosRESUMO
Sex steroids play a major role in sexually dimorphic brain development during not only the perinatal period but also the pubertal period. We previously showed that, in male mice, the estrogen receptor-α (Esr1) and aromatase (Cyp19a1) genes are essential to the sexually dimorphic formation of the anteroventral periventricular nucleus (AVPV) and the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), but the estrogen receptor-ß (Esr2) gene is not necessary. We also showed that the androgen receptor (Ar) gene is essential to the sexually dimorphic formation of the BNSTp. These genes are expressed in the AVPV and BNSTp of perinatal mice. However, it remains unknown whether these genes are expressed in the AVPV and BNSTp during puberty, and whether the expression, if any, differs by sex, age, and brain region. Here, we dissected the AVPV and BNSTp from Nissl-stained brain sections of male and female mice on postnatal day (PD) 20 (prepuberty), PD30 (puberty onset in females), PD40 (puberty onset in males), and PD60 (young adult) using a laser microdissection system. We then examined the mRNA levels of Esr1, Esr2, Cyp19a1, and Ar in these brain regions. In the AVPV, Esr1 mRNA levels were greater in females than males during PD20-60. Esr2 and Ar mRNA expressions did not differ between sexes. Ar mRNA levels were higher at PD30 than PD20. Cyp19a1 mRNA was not detected in the AVPV at PD20-60. In the BNSTp, Esr1 and Esr2 mRNA levels were higher in females than in males during PD20-60, although the mRNA levels of Cyp19a1 and Ar did not differ between sexes. Additionally, we revealed that orchiectomy at PD20 induced a failure of normal formation of the male BNSTp and testosterone replacement in the prepubertal period rescued the effect of orchiectomy at PD20. Taken together, it is suggested that pubertal testosterone transported to the AVPV is not converted to estradiol there and does not act via ESR1 and ESR2. By contrast, the formation of the male BNSTp may be affected by testicular testosterone during puberty via AR and/or via ESR1 after conversion to estradiol by CYP19A1.
RESUMO
Kisspeptin (KISS1; encoded by Kiss1) neurons in the arcuate nucleus (ARC) coexpress tachykinin 3 (TAC3; also known as neurokinin B) and dynorphin A (PDYN). Accordingly, they are termed KNDy neurons and considered to be crucial in generating pulsatile release of gonadotropin-releasing hormone. Accumulating evidence suggests that Kiss1 and Tac3 are negatively regulated by estrogen. However, it has not been fully determined whether and how estrogen modulates Pdyn and PDYN. Here, we examined the expression of Pdyn mRNA and PDYN by in situ hybridization and immunohistochemistry, respectively, in the ARC of female rats after ovariectomy (OVX) and OVX plus low- or high-dose beta-estradiol (E2) replacement. We also investigated the effect of E2 on expression of Kiss1, KISS1, Tac3, and TAC3. Furthermore, colocalization of PDYN and estrogen receptor alpha (ESR1) was determined. Subsequently, we found that low-dose E2 treatment had no effect on Pdyn mRNA-expressing cells, but increased PDYN-immunoreactive (ir) cell numbers. In contrast, high-dose E2 treatment resulted in prominent reductions in both Pdyn mRNA-expressing and PDYN-ir cell numbers. Changes induced by low or high doses of E2 were similarly observed in the expression of Kiss1, KISS1, Tac3, and TAC3. The majority of PDYN-ir neurons coexpressed ESR1 in all groups. Our results indicate that E2 regulates the expression of PDYN, as well as KISS1 and TAC3, with regulation by E2 differing according to its levels.
Assuntos
Núcleo Arqueado do Hipotálamo/citologia , Dinorfinas/metabolismo , Estradiol/farmacologia , Neurônios/metabolismo , Animais , Dinorfinas/genética , Estradiol/administração & dosagem , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Hibridização In Situ , Kisspeptinas/genética , Kisspeptinas/metabolismo , Neurocinina B/genética , Neurocinina B/metabolismo , Neurônios/efeitos dos fármacos , Ovariectomia , RNA Mensageiro/metabolismo , RatosRESUMO
Sex steroid action is critical to form sexually dimorphic nuclei, although it is not fully understood. We previously reported that masculinization of the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), which is larger and has more neurons in males than in females, involves aromatized testosterone that acts via estrogen receptor-α (ERα), but not estrogen receptor-ß (ERß). Here, we examined sex steroid action on the formation of the anteroventral periventricular nucleus (AVPV) that is larger and has more neurons in females. Morphometrical analysis of transgenic mice lacking aromatase, ERα, or ERß genes revealed that the volume and neuron number of the male AVPV were significantly increased by deletion of aromatase and ERα genes, but not the ERß gene. We further examined the AVPV and BNSTp of androgen receptor knockout (ARKO) mice. The volume and neuron number of the male BNSTp were smaller in ARKO mice than those in wild-type mice, while no significant effect of ARKO was found on the AVPV and female BNSTp. We also examined aromatase, ERα, and AR mRNA levels in the AVPV and BNSTp of wild-type and ARKO mice on embryonic day (ED) 18 and postnatal day (PD) 4. AR mRNA in the BNSTp and AVPV of wild-type mice was not expressed on ED18 and emerged on PD4. In the AVPV, the aromatase mRNA level was higher on ED18, although the ERα mRNA level was higher on PD4 without any effect of AR gene deletion. Aromatase and ERα mRNA levels in the male BNSTp were significantly increased on PD4 by AR gene deletion. These results suggest that estradiol signaling via ERα during the perinatal period and testosterone signaling via AR during the postnatal period are required for masculinization of the BNSTp, whereas the former is sufficient to defeminize the AVPV.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Hipotálamo Anterior/embriologia , Hipotálamo Anterior/metabolismo , Núcleos Septais/embriologia , Núcleos Septais/metabolismo , Caracteres Sexuais , Testosterona/metabolismo , Análise de Variância , Animais , Aromatase/metabolismo , Pesos e Medidas Corporais , Primers do DNA/genética , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Receptores Androgênicos/genéticaRESUMO
Female sexual behavior in rodents, termed lordosis, is controlled by facilitatory and inhibitory systems in the brain. It has been well demonstrated that a neural pathway from the ventromedial hypothalamic nucleus (VMN) to the midbrain central gray (MCG) is essential for facilitatory regulation of lordosis. The neural pathway from the arcuate nucleus to the VMN, via the medial preoptic nucleus, in female rats mediates transient suppression of lordosis, until female sexual receptivity is induced. In addition to this pathway, other regions are involved in inhibitory regulation of lordosis in female rats. The lordosis-inhibiting systems exist not only in the female brain but also in the male brain. The systems contribute to suppression of heterotypical sexual behavior in male rats, although they have the potential ability to display lordosis. The lateral septum (LS) exerts an inhibitory influence on lordosis in both female and male rats. This review focuses on the neuroanatomy and sex differences of the lordosis-inhibiting system in the LS. The LS functionally and anatomically links to the MCG to exert suppression of lordosis. Neurons of the intermediate part of the LS (LSi) serve as lordosis-inhibiting neurons and project axons to the MCG. The LSi-MCG neural connection is sexually dimorphic, and formation of the male-like LSi-MCG neural connection is affected by aromatized testosterone originating from the testes in the postnatal period. The sexually dimorphic LSi-MCG neural connection may reflect the morphological basis of sex differences in the inhibitory regulation of lordosis in rats.
RESUMO
A widely-used plasticizer di(2-ethylhexyl) phthalate (DEHP) is known to induce apoptosis in neurons, although the mechanisms responsible for DEHP-induced apoptosis is not well explored yet. We recently showed that exposure to DEHP increases the expression of hemeoxygenase (HO)-1, an oxidative stress related enzyme, in the mice brain. In this study, we investigated whether HO-1 is involved in DEHP-induced apoptosis using a mouse neuroblastoma cell line Neuro-2a, which forcibly express SCAT3, a fluorescent indicator of caspase-3 activity. The doses of DEHP at 1, 10 or 100 µM were used in the present study to mimic the level of human exposure to DEHP. Live image analysis of SCAT3-expressing Neuro-2a cells revealed that caspase-3 activity in the cells was significantly increased by DEHP at 100 µM but not 1 or 10 µM. We measured HO-1 mRNA level in Neuro-2a cells exposed to DEHP and found significant increase in HO-1 mRNA level by DEHP at 100 µM but not 1 or 10 µM. Live image analysis of SCAT3-expresisng Neuro-2a cells was further performed to determine the effects of HO-1 siRNA in DEHP-induced apoptosis via caspase-3 activation. We found that knockdown of HO-1 gene nullifies the effects of DEHP to activate caspase-3. These results suggest that HO-1 is involved in DEHP-induced apoptosis. Moreover, this study demonstrates that high-dose DEHP exposure induces caspase-3-dependent apoptosis, which is at least partially mediated by the up-regulation of HO-1 gene, in Neuro-2a cells.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Dietilexilftalato/toxicidade , Heme Oxigenase-1/fisiologia , Neuroblastoma/patologia , Plastificantes/toxicidade , Animais , Caspase 3/metabolismo , Caspase 3/fisiologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Camundongos , Imagem Molecular , Neuroblastoma/enzimologia , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
Sexual differentiation of brain function is regulated by estrogen in the perinatal period of rodents. However, the role of the estrogen receptor subtypes ERα and ERß is still in question. Accordingly, the effects of neonatal treatment with the ERα agonist propyl pyrazole triol (PPT) or the ERß agonist diarylpropionitrile (DPN) on female reproductive functions were investigated in rats. Female rats were injected subcutaneously with 100-500 µg/10 g body weight (b.w.) PPT or DPN, 100 µg/10 g b.w. estradiol (E(2)), or saline at day 5 (birth day = day 1), and then vaginal opening and vaginal smears were examined. On day 60, their ovaries were removed and lordosis behavior was observed after subcutaneous implantation of a silicon tube containing E(2). As a result, in most PPT and all E(2) rats, vaginal opening was advanced and an irregular estrous cycle was observed. In contrast, in most rats of the DPN groups, vaginal opening was comparable to that of the control and there was a regular estrous cycle. Lordosis tests revealed that the mean lordosis quotients (LQs) in the 250- and 500-µg PPT groups was lower than in the saline group, but higher than in the E(2) group. Mean LQs in all DPN groups were comparable to those in the saline group. These results suggest that ERα plays a major role in masculinization of the system regulating the estrous cycle in the rat brain. In behavioral defeminization of the lordosis-regulation system, ERα was also found to be the main target of estrogen.