RESUMO
Estrogens regulate synaptic properties and influence hippocampus-related learning and memory via estrogen receptors, which include the G-protein-coupled estrogen receptor 1 (GPER1). Studying mice, in which the GPER1 gene is dysfunctional (GPER1-KO), we here provide evidence for sex-specific roles of GPER1 in these processes. GPER1-KO males showed reduced anxiety in the elevated plus maze, whereas the fear response ('freezing') was specifically increased in GPER1-KO females in a contextual fear conditioning paradigm. In the Morris water maze, spatial learning and memory consolidation was impaired by GPER1 deficiency in both sexes. Notably, in the females, spatial learning deficits and the fear response were more pronounced if mice were in a stage of the estrous cycle, in which E2 serum levels are high (proestrus) or rising (diestrus). On the physiological level, excitability at Schaffer collateral synapses in CA1 increased in GPER1-deficient males and in proestrus/diestrus ('E2 high') females, concordant with an increased hippocampal expression of the AMPA-receptor subunit GluA1 in GPER1-KO males and females as compared to wildtype males. Further changes included an augmented early long-term potentiation (E-LTP) maintenance specifically in GPER1-KO females and an increased hippocampal expression of spinophilin in metestrus/estrus ('E2 low') GPER1-KO females. Our findings suggest modulatory and sex-specific functions of GPER1 in the hippocampal network, which reduce rather than increase neuronal excitability. Dysregulation of these functions may underlie sex-specific cognitive deficits or mood disorders.
Assuntos
Hipocampo , Receptores de Estrogênio , Masculino , Feminino , Camundongos , Animais , Receptores de Estrogênio/genética , Potenciação de Longa Duração/genética , Sinapses/fisiologia , Cognição , Plasticidade Neuronal/genéticaRESUMO
G-protein-coupled-estrogen-receptor 1 (GPER1) is a membrane-bound receptor that mediates estrogen signaling via intracellular signaling cascades. We recently showed that GPER1 promotes the distal dendritic enrichment of hyperpolarization activated and cyclic nucleotide-gated (HCN)1 channels in CA1 stratum lacunosum-moleculare (SLM), suggesting a role of GPER1-mediated signaling in neuronal plasticity. Here we studied whether this role involves processes of structural plasticity, such as the regulation of spine and synapse density in SLM. In organotypic entorhino-hippocampal cultures from mice expressing eGFP, we analyzed spine densities in SLM after treatment with GPER1 agonist G1 (20 nM). G1 significantly increased the density of "non-stubby" spines (maturing spines with a spine head and a neck), but did so only in cultures from female mice. In support of this finding, the expression of synaptic proteins was sex-specifically altered in the cultures: G1 increased the protein (but not mRNA) expression of PSD95 and reduced the p-/n-cofilin ratio only in cultures from females. Application of E2 (2 nM) reproduced the sex-specific effect on spine density in SLM, but only partially on the expression of synaptic proteins. Spine synapse density was, however, not altered after G1-treatment, suggesting that the increased spine density did not translate into an increased spine synapse density in the culture model. Taken together, our results support a role of GPER1 in mediating structural plasticity in CA1 SLM, but suggest that in developing hippocampus, this role is sex-specific.
Assuntos
Hipocampo , Receptores de Estrogênio , Animais , Espinhas Dendríticas/metabolismo , Feminino , Proteínas de Ligação ao GTP , Hipocampo/metabolismo , Masculino , Camundongos , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G , Sinapses/metabolismoRESUMO
Clinical and animal studies have revealed sex-specific differences in histopathological and neurological outcome after traumatic brain injury (TBI). The impact of perioperative administration of sex steroid inhibitors on TBI is still elusive. Here, we subjected male and female C57Bl/6N mice to the controlled cortical impact (CCI) model of TBI and applied pharmacological inhibitors of steroid hormone synthesis, that is, letrozole (LET, inhibiting estradiol synthesis by aromatase) and finasteride (FIN, inhibiting dihydrotestosterone synthesis by 5α-reductase), respectively, starting 72 h prior CCI, and continuing for a further 48 h after CCI. Initial gene expression analyses showed that androgen (Ar) and estrogen receptors (Esr1) were sex-specifically altered 72 h after CCI. When examining brain lesion size, we found larger lesions in male than in female mice, but did not observe effects of FIN or LET treatment. However, LET treatment exacerbated neurological deficits 24 and 72 h after CCI. On the molecular level, FIN administration reduced calpain-dependent spectrin breakdown products, a proxy of excitotoxicity and disturbed Ca2+ homeostasis, specifically in males, whereas LET increased the reactive astrocyte marker glial fibrillary acid protein specifically in females. Examination of neurotrophins (brain-derived neurotrophic factor, neuronal growth factor, NT-3) and their receptors (p75NTR , TrkA, TrkB, TrkC) revealed CCI-induced down-regulation of TrkB and TrkC protein expression, which was reduced by LET in both sexes. Interestingly, FIN decreased neuronal growth factor mRNA expression and protein levels of its receptor TrkA only in males. Taken together, our data suggest a sex-specific impact on pathogenic processes in the injured brain after TBI. Sex hormones may thus modulate pathogenic processes in experimental TBI.
Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Encéfalo/efeitos dos fármacos , Di-Hidrotestosterona/antagonistas & inibidores , Estradiol/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Antagonistas de Estrogênios/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/efeitos dos fármacos , Caracteres SexuaisRESUMO
HCN1 compartmentalization in CA1 pyramidal cells, essential for hippocampal information processing, is believed to be controlled by the extracellular matrix protein Reelin. Expression of Reelin, in turn, is stimulated by 17ß-estradiol (E2). In this study, we therefore tested whether E2 regulates the compartmentalization of HCN1 in CA1 via Reelin. In organotypic entorhino-hippocampal cultures, we found that E2 promotes HCN1 distal dendritic enrichment via the G protein-coupled estrogen receptor GPER1, but apparently independent of Reelin, because GST-RAP, known to reduce Reelin signaling, did not prevent E2-induced HCN1 enrichment in distal CA1. We therefore re-examined the role of Reelin for the regulation of HCN1 compartmentalization and could not detect effects of reduced Reelin signaling on HCN1 distribution in CA1, either in the (developmental) slice culture model or in tamoxifen-inducible conditional reelin knockout mice during adulthood. We conclude that for HCN1 channel compartmentalization in CA1 pyramidal cells, Reelin is not as essential as previously proposed, and E2 effects on HCN1 distribution in CA1 are mediated by mechanisms that do not involve Reelin. Because HCN1 localization was not altered at different phases of the estrous cycle, gonadally derived estradiol is unlikely to regulate HCN1 channel compartmentalization, while the pattern of immunoreactivity of aromatase, the final enzyme of estradiol synthesis, argues for a role of local hippocampal E2 synthesis.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Dendritos/efeitos dos fármacos , Estrogênios/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Hipocampo/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Animais , Dendritos/metabolismo , Estradiol/metabolismo , Estradiol/farmacologia , Estrogênios/metabolismo , Hipocampo/metabolismo , Neurônios/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Células Piramidais/metabolismo , Ratos Wistar , Proteína ReelinaRESUMO
The basolateral amygdala (BLA) integrates sensory input from cortical and subcortical regions, a function that requires marked synaptic plasticity. Here we provide evidence that cytochrome P450 aromatase (AROM), the enzyme converting testosterone to 17ß-estradiol (E2), contributes to the regulation of this plasticity in a sex-specific manner. We show that AROM is expressed in the BLA, particularly in the basolateral nucleus (BL), in male and female rodents. Systemic administration of the AROM inhibitor letrozole reduced spine synapse density in the BL of adult female mice but not in the BL of male mice. Similarly, in organotypic corticoamygdalar slice cultures from immature rats, treatment with letrozole significantly reduced spine synapses in the BL only in cultures derived from females. In addition, letrozole sex-specifically altered synaptic properties in the BL: in acute slices from juvenile (prepubertal) female rats, wash-in of letrozole virtually abolished long-term potentiation (LTP), whereas it did not prevent the generation of LTP in the slices from males. Together, these data indicate that neuron-derived E2 modulates synaptic plasticity in rodent BLA sex-dependently. As protein expression levels of AROM, estrogen and androgen receptors did not differ between males and females and were not sex-specifically altered by letrozole, the findings suggest sex-specific mechanisms of E2 signaling.SIGNIFICANCE STATEMENT The basolateral amygdala (BLA) is a key structure of the fear circuit. This research reveals a sexually dimorphic regulation of synaptic plasticity in the BLA involving neuronal aromatase, which produces the neurosteroid 17ß-estradiol (E2). As male and female neurons in rodent BLA responded differently to aromatase inhibition both in vivo and in vitro, our findings suggest that E2 signaling in BLA neurons is regulated sex-dependently, presumably via mechanisms that have been established during sexual determination. These findings could be relevant for the understanding of sex differences in mood disorders and of the side effects of cytochrome P450 aromatase inhibitors, which are frequently used for breast cancer therapy.
Assuntos
Inibidores da Aromatase/farmacologia , Aromatase/fisiologia , Complexo Nuclear Basolateral da Amígdala/fisiologia , Plasticidade Neuronal/fisiologia , Caracteres Sexuais , Animais , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Letrozol , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Nitrilas/farmacologia , Técnicas de Cultura de Órgãos , Ratos , Triazóis/farmacologiaRESUMO
HCN channels are important regulators of neuronal excitability. The proper function of these channels is governed by various mechanisms, including post-translational modifications of channel subunits. Here, we provide evidence that ubiquitination via a ubiquitin ligase, neuronal precursor cell expressed developmentally downregulated (Nedd)-4-2, is involved in the regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We identified a PY motif (L/PPxY), the characteristic binding motif for Nedd4-2 in the C terminus of the HCN1 subunit, and showed that HCN1 and Nedd4-2 interacted both in vivo (rat hippocampus, neocortex, and cerebellum) and in vitro [human embryonic kidney 293 (HEK293) cells], resulting in increased HCN1 ubiquitination. Elimination of the PY motif reduced, but did not abolish, Nedd4-2 binding, which further involved a stretch of â¼100 aa downstream in the HCN1 C terminus. Coexpression of Nedd4-2 and HCN1 drastically reduced the HCN1-mediated h-current amplitude (85-92%) in Xenopus laevis oocytes and reduced surface expression (34%) of HCN1 channels in HEK293 cells, thereby opposing effects of tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b)-(1a-4), an auxiliary subunit that promotes HCN1 surface expression. Regulation may further include N-glycosylation of HCN1 channels, which is significantly enhanced by TRIP8b(1a-4), but may be reduced by Nedd4-2. Taken together, our data indicate that Nedd4-2 plays an important role in the regulation of HCN1 trafficking and may compete with TRIP8b(1a-4) in this process.
Assuntos
Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Regulação da Expressão Gênica , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Motivos de Aminoácidos , Animais , Encéfalo/metabolismo , Regulação para Baixo , Eletrofisiologia , Feminino , Glicosilação , Células HEK293 , Humanos , Ubiquitina-Proteína Ligases Nedd4 , Oócitos/citologia , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Xenopus , Xenopus laevisRESUMO
Hyperpolarization-activated, cyclic nucleotide-gated nonselective (HCN) channels modulate both membrane potential and resistance and play a significant role in synaptic plasticity. We compared the influence of HCN channels on long-term depression (LTD) at the medial perforant path-granule cell synapse in early postnatal (P9-15) and adult (P30-60) rats. LTD was elicited in P9-15 slices using low-frequency stimulation (LFS, 900 pulses, 1 Hz; 80 ± 4% of baseline). Application of the specific HCN channel blocker ZD7288 (10 µM) before LFS significantly enhanced LTD (62 ± 4%; P < 0.01), showing HCN channels restrain LTD induction. However, when ZD7288 was applied after LFS, LTD was similar to control values and significantly different from the values obtained with ZD7288 application before LFS (81 ± 5%; P < 0.01), indicating that HCN channels do not modulate LTD expression. LTD in slices from adult rats were only marginally lower compared to those in P9-15 slices (85 ± 6%), but bath application of ZD7288 prior to LFS resulted in the same amount of LTD (85 ± 5%). HCN channels in adult tissue hence lose their modulatory effect. In conclusion, we found that HCN channels at the medial perforant path-granule cell synapse compromise LFS-associated induction, but not expression of LTD in early postnatal, but not in adult, rats.
Assuntos
Cardiotônicos/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/fisiologia , Via Perfurante/efeitos dos fármacos , Pirimidinas/farmacologia , Sinapses/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Técnicas In Vitro , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
Inhibitors of aromatase, the final enzyme of estradiol synthesis, are suspected of inducing memory deficits in women. In previous experiments, we found hippocampal spine synapse loss in female mice that had been treated with letrozole, a potent aromatase inhibitor. In this study, we therefore focused on the effects of letrozole on long-term potentiation (LTP), which is an electrophysiological parameter of memory and is known to induce spines, and on phosphorylation of cofilin, which stabilizes the spine cytoskeleton and is required for LTP in mice. In acute slices of letrozole-treated female mice with reduced estradiol serum concentrations, impairment of LTP started as early as after 6 h of treatment and progressed further, together with dephosphorylation of cofilin in the same slices. Theta-burst stimulation failed to induce LTP after 1 week of treatment. Impairment of LTP was followed by spine and spine synapse loss. The effects were confirmed in vitro by using hippocampal slice cultures of female mice. The sequence of effects in response to letrozole were similar in ovariectomized female and male mice, with, however, differences as to the degree of downregulation. Our data strongly suggest that impairment of LTP, followed by loss of mushroom spines and spine synapses in females, may have implications for memory deficits in women treated with letrozole.
Assuntos
Inibidores da Aromatase/farmacologia , Aromatase/fisiologia , Potenciação de Longa Duração/fisiologia , Nitrilas/farmacologia , Caracteres Sexuais , Triazóis/farmacologia , Animais , Células Cultivadas , Cofilina 1/metabolismo , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/ultraestrutura , Estradiol/sangue , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Letrozol , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Sinapses/ultraestruturaRESUMO
The functions of HCN channels in neurons depend critically on their subcellular localization, requiring fine-tuned machinery that regulates subcellular channel trafficking. Here we provide evidence that regulatory mechanisms governing axonal HCN channel trafficking involve association of the channels with specific isoforms of the auxiliary subunit TRIP8b. In the medial perforant path, which normally contains HCN1 channels in axon terminals in immature but not in adult rodents, we found axonal HCN1 significantly increased in adult mice lacking TRIP8b (TRIP8b(-/-)). Interestingly, adult mice harboring a mutation that results in expression of only the two most abundant TRIP8b isoforms (TRIP8b[1b/2](-/-)) exhibited an HCN1 expression pattern similar to wildtype mice, suggesting that presence of one or both of these isoforms (TRIP8b(1a), TRIP8b(1a-4)) prevents HCN1 from being transported to medial perforant path axons in adult mice. Concordantly, expression analyses demonstrated a strong increase of expression of both TRIP8b isoforms in rat entorhinal cortex with age. However, when overexpressed in cultured entorhinal neurons of rats, TRIP8b(1a), but not TRIP8b(1a-4), altered substantially the subcellular distribution of HCN1 by promoting somatodendritic and reducing axonal expression of the channels. Taken together, we conclude that TRIP8b isoforms are important regulators of HCN1 trafficking in entorhinal neurons and that the alternatively-spliced isoform TRIP8b(1a) could be responsible for the age-dependent redistribution of HCN channels out of perforant path axon terminals.
Assuntos
Axônios/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Proteínas de Membrana/metabolismo , Via Perfurante/metabolismo , Canais de Potássio/metabolismo , Animais , Células Cultivadas , Giro Denteado/citologia , Giro Denteado/metabolismo , Giro Denteado/ultraestrutura , Córtex Entorrinal/citologia , Córtex Entorrinal/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Via Perfurante/citologia , Peroxinas , Isoformas de Proteínas/metabolismo , Transporte Proteico , Ratos , Ratos Wistar , Frações Subcelulares/metabolismo , Inclusão do Tecido , TransfecçãoRESUMO
Presynaptic metabotropic glutamate receptors (mGluRs) at glutamatergic synapses play a major role in governing release probability. Previous reports indicated a downregulation of group III mGluRs at the lateral perforant path-granule cell synapse in the chronically epileptic hippocampus. Here, we investigated the mGluR-dependent presynaptic inhibition at the medial perforant path-granule cell synapse in the pilocarpine-treated chronically epileptic rat. The specific group II mGluR agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV, 10µM) significantly depressed medial perforant path-evoked responses in control slices, but significantly more so in epileptic tissue. This depression was accompanied by a significant increase of the paired-pulse ratio in both animal groups indicating a presynaptic mechanism. Moreover, we also found that this significantly enhanced DCG-IV effect in the medial perforant path recorded in slices from pilocarpine-treated rats was due to a significant increase of mGluR2, but not mGluR3 transcripts in the entorhinal cortex using quantitative real-time reverse transcriptase-PCR. Immunohistochemistry confirmed the increased expression of group II mGluRs in the epileptic medial molecular layer. These results demonstrate that chronic epilepsy not only causes downregulation of mGluRs in the hippocampus, but may also lead to enhanced expression of these receptors - at least in the medial perforant path.
Assuntos
Epilepsia/metabolismo , Via Perfurante/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Regulação para Cima , Animais , Epilepsia/induzido quimicamente , Masculino , Pilocarpina , Ratos , Ratos WistarRESUMO
Output properties of neurons are greatly shaped by voltage-gated ion channels, whose biophysical properties and localization within axodendritic compartments serve to significantly transform the original input. The hyperpolarization-activated current, I(h), is mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and plays a fundamental role in influencing neuronal excitability by regulating both membrane potential and input resistance. In neurons such as cortical and hippocampal pyramidal neurons, the subcellular localization of HCN channels plays a critical functional role, yet mechanisms controlling HCN channel trafficking are not fully understood. Because ion channel function and localization are often influenced by interacting proteins, we generated a knock-out mouse lacking the HCN channel auxiliary subunit, tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Eliminating expression of TRIP8b dramatically reduced I(h) expression in hippocampal pyramidal neurons. Loss of I(h)-dependent membrane voltage properties was attributable to reduction of HCN channels on the neuronal surface, and there was a striking disruption of the normal expression pattern of HCN channels in pyramidal neuron dendrites. In heterologous cells and neurons, absence of TRIP8b increased HCN subunit targeting to and degradation by lysosomes. Mice lacking TRIP8b demonstrated motor learning deficits and enhanced resistance to multiple tasks of behavioral despair with high predictive validity for antidepressant efficacy. We observed similar resistance to behavioral despair in distinct mutant mice lacking HCN1 or HCN2. These data demonstrate that interaction with the auxiliary subunit TRIP8b is a major mechanism underlying proper expression of HCN channels and I(h) in vivo, and suggest that targeting I(h) may provide a novel approach to treatment of depression.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/deficiência , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Depressão/genética , Deleção de Genes , Hipocampo/fisiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Canais de Potássio/deficiência , Canais de Potássio/metabolismo , Subunidades Proteicas/metabolismo , Animais , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Depressão/psicologia , Depressão/terapia , Terapia Genética/métodos , Hipocampo/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxinas , Canais de Potássio/genética , Subunidades Proteicas/deficiência , Subunidades Proteicas/fisiologia , Transporte Proteico/genéticaRESUMO
Studies on the role of 17ß-estradiol (E2) in the hippocampus have mainly focused on CA1 and CA3 regions, whereas in dentate gyrus (DG), its role is largely unknown. Here, we examined potential functions of E2 in DG, particularly during development. Immunohistochemistry and in situ hybridization revealed abundance of estrogen receptor (ER)α, but not ERß, expression in DG. Similar to CA1, analysis of synapse densities revealed a reduction in spine synapse number in DG molecular layer of immature rats and adult mice after inhibition of estradiol synthesis using letrozole. Interestingly, strong expression of ERα was found in Cajal-Retzius (CR) cells, which regulate neuronal migration and synaptogenesis via the extracellular matrix protein reelin. Immunoreactivity of aromatase, the final enzyme of estradiol synthesis, was strongest in mature granule cells. In hippocampal slice cultures, exogenous application of E2 caused an increase in reelin expression in CR cells, which was abolished after blockade of ERs using ICI182,780. Vice versa, inhibition of aromatase activity by letrozole resulted in reduced reelin expression, suggesting that E2 deriving from hippocampal sources contributes to the regulation of reelin as well as to the maintenance of spine synapses in DG. E2 further regulated Notch1, a signaling protein involved in neuronal differentiation.
Assuntos
Moléculas de Adesão Celular Neuronais/biossíntese , Giro Denteado/metabolismo , Estradiol/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/biossíntese , Neurogênese/fisiologia , Serina Endopeptidases/biossíntese , Animais , Western Blotting , Giro Denteado/crescimento & desenvolvimento , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Proteína Reelina , SinapsesRESUMO
HCN channels play a fundamental role in determining resting membrane potential and regulating synaptic function. Here, we investigated the involvement of HCN channels in basal synaptic transmission and long-term depression (LTD) at the Schaffer collateral-CA1 synapse. Bath application of the HCN channel blocker ZD7288 (10 microM) caused a significant increase in synaptic transmission that was due to an enhancement in AMPA receptor-mediated excitatory postsynaptic potentials. This enhancement was accompanied by a significant decrease in the paired-pulse ratio (PPR), suggesting a presynaptic mechanism. Experiments with the irreversible use-dependent NMDA receptor blocker MK-801 showed that ZD7288 led to an increase in glutamate release probability. LTD induced by brief application of (RS)-3,5-dihydroxyphenylglycine (DHPG, 100 microM, 10 min) was significantly enhanced when HCN channels were blocked by ZD7288 (10 microM) prior to DHPG application. Moreover, the concomitant increase in PPR after DHPG-induced LTD was significantly larger than without ZD7288 bath application. Conversely, ZD7288 application after DHPG washout did not alter DHPG-LTD. A significant enhancement of DHPG-LTD was also observed in HCN1-deficient mice as compared with wild types. However, LTD induced by low-frequency stimulation (LFS) remained unaltered in HCN1-deficient mice, suggesting a differential effect of HCN1 channels on synaptic plasticity constraining DHPG-LTD, but not LFS-LTD.
Assuntos
Região CA1 Hipocampal/citologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Metoxi-Hidroxifenilglicol/análogos & derivados , Canais de Potássio/fisiologia , Sinapses/efeitos dos fármacos , Animais , Biofísica , Região CA1 Hipocampal/fisiologia , Cardiotônicos/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/deficiência , Maleato de Dizocilpina/farmacologia , Estimulação Elétrica/métodos , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Técnicas In Vitro , Potenciação de Longa Duração/fisiologia , Masculino , Metoxi-Hidroxifenilglicol/farmacologia , Camundongos , Camundongos Knockout , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Técnicas de Patch-Clamp/métodos , Canais de Potássio/deficiência , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/fisiologiaRESUMO
Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are the molecular substrate of the hyperpolarization-activated inward current (I(h)). Because the developmental profile of HCN channels in the thalamus is not well understood, we combined electrophysiological, molecular, immunohistochemical, EEG recordings in vivo, and computer modeling techniques to examine HCN gene expression and I(h) properties in rat thalamocortical relay (TC) neurons in the dorsal part of the lateral geniculate nucleus and the functional consequence of this maturation. Recordings of TC neurons revealed an approximate sixfold increase in I(h) density between postnatal day 3 (P3) and P106, which was accompanied by significantly altered current kinetics, cAMP sensitivity, and steady-state activation properties. Quantification on tissue levels revealed a significant developmental decrease in cAMP. Consequently the block of basal adenylyl cyclase activity was accompanied by a hyperpolarizing shift of the I(h) activation curve in young but not adult rats. Quantitative analyses of HCN channel isoforms revealed a steady increase of mRNA and protein expression levels of HCN1, HCN2, and HCN4 with reduced relative abundance of HCN4. Computer modeling in a simplified thalamic network indicated that the occurrence of rhythmic delta activity, which was present in the EEG at P12, differentially depended on I(h) conductance and modulation by cAMP at different developmental states. These data indicate that the developmental increase in I(h) density results from increased expression of three HCN channel isoforms and that isoform composition and intracellular cAMP levels interact in determining I(h) properties to enable progressive maturation of rhythmic slow-wave sleep activity patterns.
Assuntos
Relógios Biológicos/fisiologia , Córtex Cerebral/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Canais Iônicos/biossíntese , Neurônios/metabolismo , Canais de Potássio/biossíntese , Tálamo/metabolismo , Animais , Animais Recém-Nascidos , Córtex Cerebral/crescimento & desenvolvimento , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/genética , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Neurônios/fisiologia , Canais de Potássio/genética , Isoformas de Proteínas/biossíntese , Ratos , Ratos Sprague-Dawley , Tálamo/crescimento & desenvolvimentoRESUMO
Developing neuronal networks evolve continuously, requiring that neurons modulate both their intrinsic properties and their responses to incoming synaptic signals. Emerging evidence supports roles for the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in this neuronal plasticity. HCN channels seem particularly suited for fine-tuning neuronal properties and responses because of their remarkably large and variable repertoire of functions, enabling integration of a wide range of cellular signals. Here, we discuss the involvement of HCN channels in cortical and hippocampal network maturation, and consider potential roles of developmental HCN channel dysregulation in brain disorders such as epilepsy.
Assuntos
Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Neurônios/fisiologia , Animais , Modelos Biológicos , Vias Neurais/fisiologiaRESUMO
Formation of heteromeric complexes of ion channels via co-assembly of different subunit isoforms provides an important mechanism for enhanced channel diversity. We have previously demonstrated co-association of the hyperpolarization activated cyclic-nucleotide gated (HCN1/HCN2) channel isoforms that was regulated by network (seizure) activity in developing hippocampus. However, the mechanisms that underlie this augmented expression of heteromeric complexes have remained unknown. Glycosylation of the HCN channels has been implicated in the stabilization and membrane expression of heteromeric HCN1/HCN2 constructs in heterologous systems. Therefore, we used in vivo and in vitro systems to test the hypothesis that activity modifies HCN1/HCN2 heteromerization in neurons by modulating the glycosylation state of the channel molecules. Seizure-like activity (SA) increased HCN1/HCN2 heteromerization in hippocampus in vivo as well as in hippocampal organotypic slice cultures. This activity increased the abundance of glycosylated HCN1 but not HCN2-channel molecules. In addition, glycosylated HCN1 channels were preferentially co-immunoprecipitated with the HCN2 isoforms. Provoking SA in vitro in the presence of the N-linked glycosylation blocker tunicamycin abrogated the activity-dependent increase of HCN1/HCN2 heteromerization. Thus, hippocampal HCN1 molecules have a significantly higher probability of being glycosylated after SA, and this might promote a stable heteromerization with HCN2.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Ativação do Canal Iônico/fisiologia , Canais Iônicos/fisiologia , Neurônios/fisiologia , Canais de Potássio/fisiologia , Animais , Animais Recém-Nascidos , Estimulação Elétrica/métodos , Glicosilação/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Imunoprecipitação/métodos , Ativação do Canal Iônico/efeitos dos fármacos , Ácido Caínico , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp/métodos , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Tunicamicina/farmacologiaRESUMO
Epilepsy may result from abnormal function of ion channels, such as those caused by genetic mutations. Recently, pathological alterations of the expression or localization of normal channels have been implicated in epilepsy generation, and termed 'acquired channelopathies'. Altered expression levels of the HCN channels - that conduct the hyperpolarization-activated current, I(h) - have been demonstrated in hippocampus of patients with severe temporal lobe epilepsy as well as in animal models of temporal lobe and absence epilepsies. Here we probe the mechanisms for the altered expression of HCN channels which is provoked by seizures. In organotypic hippocampal slice cultures, seizure-like events selectively reduced HCN type 1 channel expression and increased HCN2 mRNA levels, as occurs in vivo. The mechanisms for HCN1 reduction involved Ca(2+)-permeable AMPA receptor-mediated Ca(2+) influx, and subsequent activation of Ca(2+)/calmodulin-dependent protein kinase II. In contrast, upregulation of HCN2 expression was independent of these processes. The data demonstrate an orchestrated program for seizure-evoked transcriptional channelopathy involving the HCN channels that may contribute to certain epilepsies.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Canais Iônicos/metabolismo , Canais de Potássio/metabolismo , Convulsões/patologia , 2-Amino-5-fosfonovalerato/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Análise de Variância , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Marcação In Situ das Extremidades Cortadas/métodos , Técnicas In Vitro , Canais Iônicos/genética , Ácido Caínico , Canais de Potássio/genética , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamenteRESUMO
Knowledge of the processes by which epilepsy is generated (epileptogenesis) is incomplete and has been a topic of major research efforts. Animal models can inform us about these processes. We focus on the distinguishing features of epileptogenesis in the developing brain and model prolonged febrile seizures (FS) that are associated with human temporal lobe epilepsy. In the animal model of FS, epileptogenesis occurs in approximately 35% of rats. Unlike the majority of acquired epileptogeneses in adults, this process early in life (in the febrile seizures model as well as in several others) does not require "damage" (cell death). Rather, epileptogenesis early in life involves molecular mechanisms including seizure-evoked, long-lasting alterations of the expression of receptors and ion channels. Whereas transient changes in gene expression programs are common after early-life seizures, enduring effects, such as found after experimental FS, are associated with epileptogenesis. The ability of FS to generate long-lasting molecular changes and epilepsy suggests that mechanisms, including cytokine activation that are intrinsic to FS generation, may play a role also in the epileptogenic consequences of these seizures.
Assuntos
Encéfalo/crescimento & desenvolvimento , Modelos Animais de Doenças , Convulsões Febris/fisiopatologia , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Morte Celular/genética , Morte Celular/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Citocinas/genética , Citocinas/fisiologia , Expressão Gênica/genética , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Camundongos , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Canais de Potássio/genética , Canais de Potássio/fisiologia , Ratos , Receptores de Neurotransmissores/genética , Receptores de Neurotransmissores/fisiologia , Roedores , Convulsões Febris/genética , Convulsões Febris/metabolismoRESUMO
Increasing evidence supports roles for the current mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, I(h), in hippocampal maturation and specifically in the evolving changes of intrinsic properties as well as network responses of hippocampal neurons. Here, we describe a novel developmental plasticity of HCN channel expression in axonal and presynaptic compartments: HCN1 channels were localized to axon terminals of the perforant path (the major hippocampal afferent pathway) of immature rats, where they modulated synaptic efficacy. However, presynaptic expression and functions of the channels disappeared with maturation. This was a result of altered channel transport to the axons, because HCN1 mRNA and protein levels in entorhinal cortex neurons, where the perforant path axons originate, were stable through adulthood. Blocking action potential firing in vitro increased presynaptic expression of HCN1 channels in the perforant path, suggesting that network activity contributed to regulating this expression. These findings support a novel developmentally regulated axonal transport of functional ion channels and suggest a role for HCN1 channel-mediated presynaptic I(h) in hippocampal maturation.
Assuntos
Hipocampo/crescimento & desenvolvimento , Hipocampo/fisiologia , Plasticidade Neuronal/fisiologia , Canais de Potássio/genética , Canais de Potássio/metabolismo , Terminações Pré-Sinápticas/fisiologia , Animais , Transporte Axonal/fisiologia , Axônios/fisiologia , Axônios/ultraestrutura , Compartimento Celular/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Regulação para Baixo/fisiologia , Córtex Entorrinal/citologia , Córtex Entorrinal/crescimento & desenvolvimento , Córtex Entorrinal/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipocampo/citologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Microscopia Eletrônica , Vias Neurais , Via Perfurante/citologia , Via Perfurante/crescimento & desenvolvimento , Via Perfurante/fisiologia , Gravidez , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
The properties of the hyperpolarization-activated current (I(h)) and its roles in hippocampal network function evolve radically during development. Because I(h) is conducted by the hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels, we tested the hypothesis that understanding the quantitative developmental profiles of HCN1, HCN2, and HCN4 expression, and the isoform- and age-specific progression of their subcellular distribution, should shed light on the established modifications of the properties of I(h) throughout development. Combined quantitative in situ hybridization, regional western blots, and high-resolution, dual-label immunocytochemistry revealed striking and novel information about the expression and distribution of the HCN channel isoforms in the developing hippocampal formation. In cornus ammon 1 (CA) pyramidal cell layer, a robust increase of HCN1 mRNA and protein expression occurred with age, with reciprocal reduction of HCN4 and relatively stable HCN2 levels. These distinct expression patterns raised the contribution of HCN1 to the total HCN channel pool from 33% to 65% consonant with acceleration and reduced cyclic adenosine mono phosphate (cAMP) sensitivity of I(h) in this region with age. In CA3, strong expression of HCN1 already neonatally supports the recently established role of this conductance in neonatal, age-specific, hippocampal oscillations (giant depolarizing potentials). Notably, HCN1 channels were present and probably transported to dendritic compartments already on postnatal day (P) 2, whereas HCN2 channel protein was not evident in dendrites for the first 2 weeks of life. HCN2 mRNA and protein expression remained fairly constant subsequent to the first week of life in all hippocampal subfields examined, whereas HCN4 mRNA and protein expression declined after maximal neonatal expression, so that the contribution of this isoform to the total HCN channel pool dropped from 43% (CA1) and 34% (CA3) on P11 to 8% (CA1) and 19% (CA3) on P90. Interneuronal expression of all HCN channel isoforms in stratum pyramidale was robust in parvalbumin-but not in cholecystokinin-expressing populations and with a subunit-specific subcellular distribution. Taken together, these data suggest that early in life, HCN4 may contribute significantly to the functions of I(h) in specific hippocampal regions. In addition, these evolving, differential quantitative, and subcellular expression patterns of the HCN channel isoforms support age-specific properties and functions of I(h) within the developing hippocampal formation.