RESUMO
We have previously reported that pigment dispersing factor (PDF) neurons, which are essential in the control of rest-activity cycles in Drosophila, undergo circadian remodeling of their axonal projections, a phenomenon called circadian structural plasticity. Axonal arborizations display higher complexity during the day and become simpler at night, and this remodeling involves changes in the degree of connectivity. This phenomenon depends on the clock present within the ventrolateral neurons (LNvs) as well as in glia. In this work, we characterize in detail the contribution of the PDF neuropeptide to structural plasticity at different times across the day. Using diverse genetic strategies to temporally restrict its downregulation, we demonstrate that even subtle alterations to PDF cycling at the dorsal protocerebrum correlate with impaired remodeling, underscoring its relevance for the characteristic morning spread; PDF released from the small LNvs (sLNvs) and the large LNvs (lLNvs) contribute to the process. Moreover, forced depolarization recruits activity-dependent mechanisms to mediate growth only at night, overcoming the restriction imposed by the clock on membrane excitability. Interestingly, the active process of terminal remodeling requires PDF receptor (PDFR) signaling acting locally through the cyclic-nucleotide-gated channel ion channel subunit A (CNGA). Thus, clock-dependent PDF signaling shapes the connectivity of these essential clock neurons on daily basis.
Assuntos
Relógios Circadianos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Plasticidade Neuronal , Neurônios/fisiologia , Neuropeptídeos/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Ritmo Circadiano , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Atividade Motora , Neurônios/citologia , Neuropeptídeos/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated pore loop channels. In mammals, this family consists of four different subunits (HCN1-4) and their ion channels activity have been proposed to play an essential role in regulating the membrane potential of excitable cells. Here, we describe the expression and relative abundances of HCN channels in cerebellum and primary cultures of cerebellar granule neurons (CGN). Quantitative determination of mRNA expression levels demonstrated the existence of an accumulation pattern of transcripts in cerebellum that encode HCN2 > HCN3 = HCN4 > HCN1 subunits. Immunolocalization analyses of HCN channels in cerebella revealed positive staining in Purkinje and granule cell layers. The presence of the HCN subunits in the cerebellar granule cell layer was then confirmed in primary cultures of CGN by quantitative real-time PCR (qPCR), as well as western blot and immunofluorescence analysis, demonstrating the presence of all four channel proteins.
Assuntos
Cerebelo/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Grânulos Citoplasmáticos/metabolismo , Neurônios/metabolismo , Animais , Western Blotting , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Imunofluorescência , Imuno-Histoquímica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Odor transduction, occurring in the chemosensory cilia of vertebrate olfactory sensory neurons, is triggered by guanosine triphosphate-coupled odor receptors and mediated by a cyclic adenosine monophosphate (cAMP) signaling cascade, where cAMP opens cationic non-selective cyclic nucleotide-gated (CNG) channels. Calcium enters through CNG gates Ca(2+)-activated Cl(-) channels, allowing a Cl(-) inward current that enhances the depolarization initiated by the CNG-dependent inward current. The anoctamin channel 2, ANO2, is considered the main Ca(2+)-activated Cl(-) channel of olfactory transduction. Although Ca(2+)-activated Cl(-) channel-dependent currents in olfactory sensory neurons were reported to be suppressed in ANO2-knockout mice, field potentials from their olfactory epithelium were only modestly diminished and their smell-dependent behavior was unaffected, suggesting the participation of additional Ca(2+)-activated Cl(-) channel types. The Bestrophin channel 2, Best2, was also detected in mouse olfactory cilia and ClCa4l, belonging to the ClCa family of Ca(2+)-activated Cl(-) channels, were found in rat cilia. Best2 knock-out mice present no electrophysiological or behavioral impairment, while the ClCa channels have not been functionally studied; therefore, the overall participation of all these channels in olfactory transduction remains unresolved. RESULTS: We explored the presence of detectable Ca(2+)-activated Cl(-) channels in toad olfactory cilia by recording from inside-out membrane patches excised from individual cilia and detected unitary Cl(-) current events with a pronounced Ca(2+) dependence, corresponding to 12 and 24 pS conductances, over tenfold higher than the aforementioned channels, and a approx. fivefold higher Ca(2+) affinity (K0.5 = 0.38 µM). Remarkably, we observed immunoreactivity to anti-ClCa and anti-ANO2 antibodies in the olfactory cilia, suggesting a possible cooperative function of both channel type in chemotransduction. CONCLUSIONS: These results are consistent with a novel olfactory cilia channel, which might play a role in odor transduction.
Assuntos
Proteínas de Anfíbios/metabolismo , Canais de Cloreto/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Animais , Anuros , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Cílios/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Potenciais da Membrana/fisiologia , Mucosa Olfatória/metabolismo , Técnicas de Patch-ClampRESUMO
Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K(+) channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K(+) selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K(+) channel and the rhodopsin-guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K(+) channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus.
Assuntos
Blastocladiomycota/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Proteínas Fúngicas/metabolismo , Canais de Potássio/metabolismo , Blastocladiomycota/genética , Blastocladiomycota/fisiologia , GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Proteínas Fúngicas/genética , Potenciais da Membrana , Potássio/metabolismo , Canais de Potássio/genética , Esporos Fúngicos/metabolismoRESUMO
Properties, developmental regulation, and cAMP modulation of the hyperpolarization-activated current (I(h)) were investigated by the whole cell patch-clamp technique in vestibular ganglion neurons of the rat at two postnatal stages (P7-10 and P25-28). In addition, by RT-PCR and immunohistochemistry the identity and distribution of hyperpolarization-activated and cyclic nucleotide-gated channel (HCN) isoforms that generate I(h) were investigated. I(h) current density was larger in P25-28 than P7-10 rats, increasing 410% for small cells (<30 pF) and 200% for larger cells (>30 pF). The half-maximum activation voltage (V(1/2)) of I(h) was -102 mV in P7-10 rats and in P25-28 rats shifted 7 mV toward positive voltages. At both ages, intracellular cAMP increased I(h) current density, decreased its activation time constant (τ), and resulted in a rightward shift of V(1/2) by 9 mV. Perfusion of 8-BrcAMP increased I(h) amplitude and speed up its activation kinetics. I(h) was blocked by Cs(+), zatebradine, and ZD7288. As expected, these drugs also reduced the voltage sag caused with hyperpolarizing pulses and prevented the postpulse action potential generation without changes in the resting potential. RT-PCR analysis showed that HCN1 and HCN2 subunits were predominantly amplified in vestibular ganglia and end organs and HCN3 and HCN4 to a lesser extent. Immunohistochemistry showed that the four HCN subunits were differentially expressed (HCN1 > HCN2 > HCN3 ≥ HCN4) in ganglion slices and in cultured neurons at both P7-10 and P25-28 stages. Developmental changes shifted V(1/2) of I(h) closer to the resting membrane potential, increasing its functional role. Modulation of I(h) by cAMP-mediated signaling pathway constitutes a potentially relevant control mechanism for the modulation of afferent neuron discharge.
Assuntos
Potenciais de Ação/fisiologia , AMP Cíclico/metabolismo , Neurônios/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Subunidades Proteicas/fisiologia , Ratos , Ratos Long-Evans , Núcleos Vestibulares/citologia , Núcleos Vestibulares/crescimento & desenvolvimentoRESUMO
The steroid hormones, estrogen and progesterone, are involved mainly in the control of female reproductive functions. Among other effects, estrogen and progesterone can modulate Na(+) reabsorption along the nephron altering the body's hydroelectrolyte balance. In this work, we analyzed the expression of cyclic nucleotide-gated channel A1 (CNG-A1) and α1 Na(+)/K(+)-ATPase subunit in the renal cortex and medulla of female ovariectomized rats and female ovariectomized rats subjected to 10 days of 17ß-estradiol benzoate (2.0 µg/kg body weight) and progesterone (1.7 mg/kg body weight) replacement. Na(+)/K(+) ATPase activity was also measured. Immunofluorescence localization of CNG-A1 in the cortex and medulla was performed in control animals. We observed that CNG-A1 is localized at the basolateral membrane of proximal and distal tubules. Female ovariectomized rats showed low expression of CNG-A1 and low expression and activity of Na(+)/K(+) ATPase in the renal cortex. When female ovariectomized rats were subjected to 17ß-estradiol benzoate replacement, normalization of CNG-A1 expression and Na(+)/K(+) ATPase expression and activity was observed. The replacement of progesterone was not able to recover CNG-A1 expression and Na(+)/K(+) ATPase expression at the control level. Only the activity of Na(+)/K(+) ATPase was able to be recovered at control levels in animals subjected to progesterone replacement. No changes in expression and activity were observed in the renal medulla. The expression of CNG-A1 is higher in cortex compared to medulla. In this work, we observed that estrogen and progesterone act in renal tissues modulating CNG-A1 and Na(+)/K(+) ATPase and these effects could be important in Na(+) and water balance.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Estradiol/fisiologia , Estrogênios/fisiologia , Regulação da Expressão Gênica , Córtex Renal/metabolismo , Progesterona/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células Cultivadas , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Estradiol/farmacologia , Estrogênios/sangue , Feminino , Expressão Gênica , Taxa de Filtração Glomerular , Córtex Renal/fisiologia , Medula Renal/metabolismo , Ovariectomia , Progesterona/sangue , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
During capacitation of mammalian sperm intracellular [Ca(2+)] and cyclic nucleotides increase, suggesting that CNG channels play a role in the physiology of sperm. Here we study the effect of capacitation, 8Br-cAMP (8-bromoadenosine 3',5'-cyclic monophosphate) and 8Br-cGMP (8-bromoguanosine 3',5'-cyclic monophosphate) on the macroscopic ionic currents of mouse sperm, finding the existence of different populations of sperm, in terms of the recorded current and its response to cyclic nucleotides. Our results show that capacitation and cyclic nucleotides increase the ionic current, having a differential sensitivity to cGMP (cyclic guanosine monophosphate) and cAMP (cyclic adenosine monophosphate). Using a specific inhibitor we determine the contribution of CNG channels to macroscopic current and capacitation.
Assuntos
GMP Cíclico/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Capacitação Espermática , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , AMP Cíclico/farmacologia , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/agonistas , Canais de Cátion Regulados por Nucleotídeos Cíclicos/antagonistas & inibidores , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/fisiologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologiaRESUMO
Odour-mediated signal transduction is a complex process that occurs in the cilia of olfactory sensory neurons. To gain insight in to the molecular organization of the odour transduction machinery, we developed a procedure to purify olfactory cilia membranes by differential centrifugation of rat olfactory epithelium extracts. We tested whether known scaffolding proteins that might participate in the assembly of the complex chemotransduction apparatus are present in the purified membrane fraction. Utilizing immunoblotting and immunohistochemistry, we show that the multidomain scaffolding proteins ProSAP/Shanks and calcium/calmodulin-dependent serine protein kinase CASK are present in the olfactory cilia. Ion channels involved in chemotransduction could be reconstituted into planar lipid bilayers for electrophysiological recordings. Our procedure should allow the identification of further chemotransduction-related proteins.