Papel de los canales iónicos de calcio dependientes de voltaje en la modulación de las señales nociceptivas en la médula espinal

Abstract

The principal aims of the present Thesis were (1) to develop new procedures to study the transmission of nociceptive information across spinal circuits using a mouse model of the spinal cord in vitro, and (2) to use this new technology to study the role of voltage dependent calcium channels (VDCC) on spinal nociceptive transmission. On the basis of existing models of the hemisected rat spinal cord in vitro, we have established a viable preparation of the entire mouse spinal cord in vitro. This model allowed for the simultaneous recording of activity in motor and sensory transmission pathways in response to the electrical stimulation of a lumbar dorsal root. We have recorded ascending sensory information from single axons in the anterolateral quadrant of the cord (contralateral to the site of stimulation) which is the main projection area for spinothalamic pathways. In the first experiments we were able to characterize and classify ascending axons in terms of their responses to dorsal root stimulation. We also obtained recordings from the ipsilateral dorsal columns, showing the potential of the model used. In order to test whether or not motor and sensory pathways could be independently modulated, we studied the effects of a halogenated anaesthetic agent (sevoflurane) and an amine (noradrenalin) in simultaneous recording experiments. Results showed that sevoflurane depressed transmission across both pathways, whereas noradrenalin depressed sensory transmission but potentiated motor transmission. This demonstrated that both pathways can be modulated by different mechanisms. Once established the reliability of the technique, we proceeded to study the effects of agonists and antagonists of VDCC on motor and sensory responses to afferent stimulation. We used verapamil, diltiazem and nimodipine, each representing one of the three main families of VDCC antagonists of the L-type currents. These antagonists tended to show marked depressant effects on motor pathway but little effects on sensory pathways. Nimodipine appeared to be the most selective antagonist, whereas verapamil and diltiazem could activate different targets from the L-type VDCC when applied at high concentrations. Finally, we studied the action of the agonist of the L-type VDCC Bay K8644 on spinal transmission. This agonist produced a long-lasting increase of the responses in motor pathways. The long duration of the effect appeared to be due to a strong interaction of the agonist with the channel, excluding an established process of increased responses started by calcium entry, since perfusion of diltiazem abolished the effects produced by Bay K8644. This agonist did not show any effect on the responses to afferent stimulation in the sensory pathway but increased spontaneous activity. In summary, we have established a new model of the in vitro mice spinal cord that allowed for simultaneous recordings in sensory and motor pathways. The use of this new model has allowed us to conclude that L-type calcium currents are involved in the immediate codification of persistent nociceptive signals across somato-motor pathways without intervening in the initiation of a process of spinal sensitization. We also confirmed that L-type currents do not play a fundamental role in modulating nociceptive sensory transmission in the present study conditions.