Caracterización de los cambios en los niveles de marcadores GABAérgicos y canales de calcio, y sus implicancias funcionales, en modelos de administración de psicoestimulantes en ratón

Abstract

[ES] Nuestro laboratorio se centra en el estudio del efecto de la cocaína y el metilfenidato sobre el balance excitación/inhibición en el sistema tálamocortical (específicamente la conexión entre el núcleo reticular, el núcleo ventrobasal y la corteza somatosensorial) en modelos de ratón de la cepa SV129 (25-45días postnatales). Nuestro objetivo es dilucidar mecanismos moleculares y fisiológicos subyacentes al fenómeno de disritmia tálamocortical que nuestro grupo ha descrito previamente tras la administración aguda de cocaína. Se utilizará la técnica de western blot para estudiar los marcadores de neuronas GABAérgicas (GAD65/67) y de canales de calcio de tipo T y P/Q en tejido tálamocortical de ratones de ratones wild type y knockout para los receptores de serotonina tipo 2A (5-HT2A-/-), inyectados durante 1 día (binge de tres inyecciones separadas una hora) o 3 días (un binge por día), obteniéndose la muestra una hora o 24 horas después de la última inyección.

[EN] The thalamocortical somatosensory system is the responsible for processing information coming from the sensory nerves so as to generate a somatosensory cognitive experience, which is the same, to produce a concrete sensation from the environment. This system consists of a highly coherent and differenced neuronal circuit, which allows perception to be carried on in our daily life. The alteration of the different components of the system by psychostimulants, such as cocaine or methylphenidate, induces distinct anomalies and/or pathologies in sensory perception, as it is possible to observe in the thalamocortical dysrhythmia syndrome suffered by chronic cocaine abusers. The visualization of such alterations in the protein and electrophysiological components of the systems due to the administration of these drugs is essential to understand their effect on the thalamocortical somatosensory system. Concretely, those agents implicated in the excitation and inhibition of the components of the circuit, being the case of the T-type calcium channels and the GAD enzyme (glutamic acid decarboxylase), two proteins that, for their role in the system, are key modulators for the correct excitability and rhythm of each part of the system and, thus, to generate cognitive experiences. On the one hand, T-type calcium channels, also called low voltage activated or LVA, are responsible for the generation of the IT current, needed to trigger action potentials in the thalamic ventrobasal nucleus. On the other hand, the GAD enzyme (in its two isoforms GAD 65 and GAD 67) is responsible for the synthesis of the inhibiting neurotransmitter GABA, being important in the thalamic reticular nucleus, which modulates the excitability of the ventrobasal nucleus and the transmission of information from this part of the thalamus to the somatosensory cortex. In addition, GABA is the main inhibiting neurotransmitter in the adult nervous system, being also important in the somatosensory cortex. The alterations of these markers can be measured following two different approaches: the molecular biology approach, evaluating their protein levels by means of the Western Blot technique; or the electrophysiological approach, by the whole-cell patch-clamp technique, which allows the comparison of the densities of low (LVA) and high (high voltage activated, HVA) activation threshold calcium currents, the so called LVA/HVA ratios, and also the visualization of the GABAergic miniature currents (“minis”) caused by spontaneous GABA release.