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Impact of the feeding eycle upon the neuronal membrane properties of rat hippocampal neurones : the involvement of voltage-gated sodium and calcium currents and the maintenance of plasma membrane organization
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Resumo(s)
Feeding behaviour and energy balance is regulated by the central nervous system, through a concerted endeavour of different brain areas. The hippocampus, historically regarded as a substrate for learning and memory processes, has also been implicated in such energy regulation. In recent years, researchers have established that hippocampal neurones form a memory of a meal and act to delay meal initiation during the postprandial period. However, more experiments are needed to identify the processes involved in such control. The present thesis starts to fill this gap, by identifying possible neuronal mechanisms by which the hippocampus processes satiety and meal termination. By assessing the functioning of ion currents/channels and the lipid composition and organization of the plasma membrane throughout the feeding cycle, this study furnishes a global perspective of the effect of post-prandial and fasting conditions upon intrinsic neuronal plasma membrane (PM) properties. The involvement of ion channels of rat hippocampal CA1 neurones in a feeding cycle context has already been studied. Indeed, the feeding cycle was found to impact the excitability of these neurones by modulating the activity of voltage-gated potassium currents. This finding has urged further investigation to evaluate the broadness of the effect of feeding cycle on the activity of other ion channels. Hence, it was critical to address the involvement of a) voltage-gated sodium (Na+) currents/channels, given their importance in the initiation and propagation of action potentials, and b) voltage-gated calcium (Ca2+) currents/channels, as they mediate the influx of this ubiquitous second messenger, with wide-ranging physiological roles, into the interior of the neurones. The influence of feeding cycle on the biophysics of Na+ and Ca2+ channels was undertaken in neurones acutely isolated from the CA1 subfield of the rat hippocampus. Two classes of neurones were used: those obtained from animals that fasted overnight (‘fasted neurones’) and those from animals that, after such period, were fed (‘fed neurones’). Voltage-gated Na+ currents were recorded by applying electrophysiological voltage clamp techniques - namely whole-cell (WC) and excised inside-out patches. Fed neurones, in comparison to fasted neurones, showed increased mean maximum macroscopic Na+ current density (1.5 ± 0.12mA.cm-2 vs. 1±0.10mA.cm-2) and a greater single-channel conductance (16.7 ± 0.76pS vs. 12.6 ± 1.30pS). Furthermore, the larger amplitude of the ‘window current’ obtained in fed neurones, derived from hyperpolarized activation curves and depolarized steady-state of inactivation curves (h∞), indicates a greater Na+ channel availability to respond to activation. Such variation is supported by a higher concentration of Nav1.2 isoform at the plasma membrane-enriched fractions of hippocampus of fed animals. Overall, the results indicate a variation in the biophysics and expression of voltage gated Na+ channels of rat hippocampal CA1 neurones, pointing out that feeding cycle changes the neuronal excitability. Voltage-gated Ca2+ currents were analysed with whole-cell recordings. It was observed heterogeneity in whole-cell Ca2+ currents, here sorted into three categories – ‘A’, ‘B’, and ‘C’ currents. The differential distribution of these currents between fed and fasted neurones determined significant alterations on the inactivation properties of Ca2+ currents. The increased values of the time-constant of inactivation - τh -, observed upon feeding, can be ascribed to a conspicuous slowly-inactivating current mainly assigned to fed neurones (current ‘A’), as oppose to the fastest kinetics of inactivation, solely seen in fasted neurones (current ‘C’). Furthermore, in fed neurones, a depolarizing shift of the most depolarized component (Vh2) of the voltage-dependence of h∞ was observed, which indicates that fasted neurones inactivate at more negative membrane potentials. Altogether, these observations point to a facilitated entry of Ca2+ into the soma of fed neurones, which, ultimately, potentiates the Ca2+-dependent intracellular events. The observed influence of feeding cycle on the biophysical and molecular expression of voltage-gated Na+ and Ca2+ channels did not have repercussions on the lipid environment of the PM. The plasma membrane-enriched fractions of rat hippocampus were labeled with molecular probes: 1,6-diphenyl 1,3,5-hexatriene (DPH) and trans-parinaric acid (t-PnA). By assessing the fluorescence properties of these probes, it was possible to study the molecular organization and lateral heterogeneity (in the membrane plane) of the lipid domains. Specifically, two types of fluorescence spectroscopy measurements were used, either in steady state (anisotropy measurements) and time-resolved domains (fluorescence intensity decay). The molecular biophysics analysis indicated that the order and rigidity of the acyl chains of the phospholipids constituents of the PM is not altered during the feeding cycle. Furthermore, the proportion of the different lipid domains at the surface of the neuronal PM is identical between conditions, which clearly indicates that the lateral heterogeneity of such domains is similar throughout the feeding cycle. This observation must be interpreted at a hydrophobic core level, where the t-Pna and DPH preferentially locate within the PM. The lipid content of the plasma membrane of rat hippocampus also did not endure any variation during the feeding cycle. The ratios calculated for the total lipid, phospholipid and cholesterol content were identical between the membranes of fed and fasted animals. The results concerning the molecular biophysics and biochemical characterization of the lipids imbedded in the neuronal plasma membrane indicate that neurones must have a shield mechanism to preserve their functional viability, regardless of the peripheral metabolic state. In summary, the greater levels of neuronal excitability and the promotion of Ca2+ entry into the neurones upon feeding may imply a subsequent increase on neuronal synaptic performance. A positive relationship between feeding and higher levels of synaptic plasticity-related phenomena (formation and consolidation of memories) is suggested, which could help to explain the role of hippocampus on the regulation of energy intake, mainly due to its role on meal-related episodic memories. This work gives new insights into the function of hippocampus on energy homeostasis, by adding new elements to the equation, namely, voltage-gated Na+ and Ca2+ channels.
Descrição
Tese de doutoramento, Bioquímica (Biofísica Molecular), Universidade de Lisboa, Faculdade de Ciências, 2018
Palavras-chave
Teses de doutoramento - 2018