RESUMO
Focal cortical dysplasias (FCDs) are malformations of cortical development that often result in medically refractory epilepsy, with a greater incidence in the pediatric population. The relationship between the disturbed cortical morphology and epileptogenic activity of FCDs remains unclear. We used the BCNU (carmustine 1-3-bis-chloroethyl-nitrosourea) animal model of cortical dysplasia to evaluate neuronal and laminar alterations and how these result in altered activity of intracortical networks in early life. We corroborated the previously reported morphological anomalies characteristic of the BCNU model, comprising slightly larger and rounder neurons and abnormal cortical lamination. Next, the neuronal activity of live cortical slices was evaluated through large field-of-view calcium imaging as well as the neuronal response to a stimulus that leads to cortical hyperexcitability (pilocarpine). Examination of the joint activity of neuronal calcium time series allowed us to identify intracortical communication patterns and their response to pilocarpine. The baseline power density distribution of neurons in the cortex of BCNU-treated animals was different from that of control animals, with the former showing no modulation after stimulus. Moreover, the intracortical communication pattern differed between the two groups, with cortexes from BCNU-treated animals displaying decreased inter-layer connectivity as compared to control animals. Our results indicate that the altered anatomical organization of the cortex of BCNU-treated rats translates into altered functional networks that respond abnormally to a hyperexcitable stimulus and highlight the role of network dysfunction in the pathophysiology of cortical dysplasia.
Assuntos
Carmustina , Malformações do Desenvolvimento Cortical , Ratos , Animais , Criança , Humanos , Cálcio , Pilocarpina , NeurôniosRESUMO
We recently reported that rodent anterior pituitary (AP) cells (with the exception of corticotrophs and melanotrophs) express neuronal markers, including 68-kDa neurofilaments (NF68) in an oestrogen-dependent manner. The functional significance of neurofilament (NF) expression in the AP is unknown, but recent data in myelinated nerve fibres from NF-null mice suggest that NFs can regulate ion channel function. Because Ca(2+) influx through voltage-gated Ca(2+) channels is required for hormone secretion in AP cells, and oestrogen regulates the expression of Ca(2+) channels in AP cells, the present study examined the expression of alpha1 subunits of voltage gated Ca(2+) channels in relation to that of NF68. Using quantitative immunofluorescence, we demonstrate that alpha 1C and alpha 1D subunits are abundantly expressed in female AP cells, alpha 1A subunits are moderately expressed, and alpha 1G and alpha 1B subunits are expressed at the lowest levels. Double-immunostaining showed that NF68 expression is not correlated with that of alpha 1C, alpha 1D or alpha 1B. Expression of alpha 1G and NF68 appear to be mutually exclusive from each other. Moreover, alpha 1A subunit and NF68 expression are significantly correlated and alpha 1A immunoreactivity is sexually dimorphic (i.e. low in males and high in females) and its levels of expression vary during the oestrous cycle, similar to NF68. Finally, omega-agatoxin IVA, a specific blocker of P/Q type Ca(2+) currents that are a result of the activity of alpha 1A subunits, inhibited to a greater extent spontaneous [Ca(2+)](i) fluctuations in AP cells from females in oestrous and dioestrous, whereas cells from females in pro-oestrous and males were less affected by this toxin. These results suggest a preferential participation of P/Q-type Ca(2+) channels and hence alpha 1A subunits, in regulating spontaneous Ca(2+) transients in AP cells under conditions where the proportion of NF68-expressing cells is high. It remains to be determined whether the expression of NF68 affects that of alpha 1A Ca(2+) channel subunits or vice versa.
Assuntos
Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/metabolismo , Imuno-Histoquímica , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Neurofilamentos/metabolismo , Subunidades Proteicas/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo N , Canais de Cálcio Tipo P/genética , Canais de Cálcio Tipo Q/genética , Ciclo Estral/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Subunidades Proteicas/genéticaRESUMO
The plant alkaloid ryanodine (Ry) is a high-affinity modulator of ryanodine receptor (RyR) Ca(2+) release channels. Although these channels are present in a variety of cell types, their functional role in nerve cells is still puzzling. Here, a monosubstituted fluorescent Ry analogue, B-FL-X Ry, was used to reveal the distribution of RyRs in cultured rat sympathetic neurons. B-FL-X Ry competitively inhibited the binding of [3H]Ry to rabbit skeletal muscle SR membranes, with an IC(50) of 150 nM, compared to 7 nM of unlabeled Ry. Binding of B-FL-X Ry to the cytoplasm of sympathetic neurons is saturable, reversible and of high affinity. The pharmacology of B-FL-X Ry showed marked differences with unlabeled Ry, which are partially explained by its lower affinity: (1) use-dependent reversible inhibition of caffeine-induced intracellular Ca(2+) release; (2) diminished voltage-gated Ca(2+) influx, due to a positive shift in the activation of voltage gated Ca(2+) currents. B-FL-X Ry-stained sympathetic neurons, viewed under confocal microscopy, showed conspicuous labeling of crescent-shaped structures pertaining to the Golgi complex, a conclusion supported by experiments showing co-localization with Golgi-specific fluorescent probes and the breaking up of crescent-shaped staining after treatment with drugs that disassemble Golgi complex. The presence of RyRs to the Golgi could be confirmed with specific anti-RyR(2) antibodies, but evidence of caffeine-induced Ca(2+) release from this organelle could not be obtained using fast confocal microscopy. Rather, an apparent decrease of the cytosolic Ca(2+) signal was detected close to this organelle. In spite of that, short-term incubation with brefeldin A (BFA) suppressed the fast component of caffeine-induced Ca(2+) release, and the Ca(2+) release process lasted longer and appeared less organized. These observations, which suggest a possible role of the Golgi complex in Ca(2+) homeostasis and signaling in nerve cells, could be relevant to reports involving derangement of the Golgi complex as a probable cause of some forms of progressive neuronal degeneration, such as Alzheimer's disease and amyotrophic lateral sclerosis.