Ion channels and intrinsic membrane properties of locomotor network neurons in the lamprey spinal cord

Abstract

NMDA-receptor dependent membrane potential oscillations and postinhibitory rebound are examples of intrinsic membrane properties of many neurons of rhythm-generating networks, and are considered to play major roles in the operation of the spinal locomotor network of the lamprey vertebrate model. A significant feature of many lamprey spinal cord neurons is their ability to generate pacemaker-like membrane potential oscillations in the presence of the glutamate agonist NMDA. These plateau potentials are voltage-dependent, resistant to sodium channel blockade by tetrodotoxin (TTX), and persist in the absence of action potentials and synaptic interaction, thus representing an intrinsic membrane property of the individual neuron. NMDA-receptor dependent, TTX-resistant plateau potentials are found both in network interneurons and motoneurons, and are believed to be important for the maintenance of a slow and stable locomotor activity. Postinhibitory rebound (PIR) can play a significant role for producing stable rhythmic motor patterns, like locomotion, by contributing to burst initiation following the phase ofinhibition. Like other membrane properties, PIR may also be a target for modulatory systems acting on the network. For both the plateau oscillation and the PIR property, the calcium influx into different parts of the neuron during activity is of key importance. The goal of this thesis has been to elucidate (I) the involvement of novel ion channel subtypes like calcium activated non-selective cation (CAN) channels and low voltage activated (LVA) calcium channels in NMDA oscillations; (II) the relation between the level of NMDA-receptor activation and membrane potential oscillation frequency, by combining experimental and mathematical modelling methodologies; (III) the spatial and temporal characteristics of calcium influx during NMDA-induced membrane potential oscillations; (IV) The PIR in identified neurons of the spinal locomotor network in lamprey, - the ionic bases of the rebound response as well as the modulatory actions exerted on the PIR property by 5-HT and dopamine. To explore the putative involvement of an ICAN current in lamprey spinal neurons, the commonly used blocker flufenamic acid (FFA) was utilized to observe the effect on fictive locomotion and on NMDAinduced oscillations. FFA deteriorated both fictive locomotion and NMDA-induced oscillations. However, several non-specific effects of FFA were found, like an influence on calcium channels and on NMDA receptors. These effects may account for the observed influence of FFA on fictive locomotionand on NMDA-induced oscillations. Thus, unlike in reticulospinal neurons of the lamprey brainstem, CAN channels do not appear to play any significant role in lamprey spinal neurons. By combining experimental and computational strategies, the relation between the level of NMDAreceptor activation and the frequency of NMDA-induced membrane potential oscillations was analyzed. The frequency increased with higher concentrations of NMDA in both experiments and model simulations, to reach a maximal frequency at even higher levels. This concentration dependence is similar to that of the network rhythm during fictive locomotion. Electrophysiological recordings and confocal laser scanning microscopy were combined to investigate intracellular calcium fluctuations in lamprey spinal neurons during NMDA-induced oscillations. Calcium fluorescence fluctuations were detected in different compartments of the neuron, in the soma as well as in proximal and distal dendrites. The calcium fluctuations were more prominent in distal dendrites than in proximal dendrites and the soma. During the oscillation cycle, an abrupt rise in calcium is initiated at the onset of depolarization and the calcium level then increases during the depolarized plateau, to reach its maximum by the end of the plateau. The timing of the calcium peak could differ between different distal dendrites. This may indicate that different regions of the dendritic tree could act partly independently and contribute to different aspects of the oscillatory trajectory. In pharmaocological experiments, LVA calcium channels of the L-type (Cav 1.3) were found to contribute to the generation of NMDA-induced membrane potential oscillations. Calcium imaging experiments corroborated the involvement of this subclass of calcium channels, which appear located primarly in distal dendritic regions in a non-uniform manner. Together with calcium entering via e.g. NMDA-channels, calcium entry via Cav 1.3 channels will activate calcium-dependent potassium (KCa) channels during the oscillation plateau and contribute to its termination and the subsequent repolarization. The calcium current via Cav 1.3 channels may also contribute to the depolarizing phase of NMDAinduced oscillations. Using injection of hyperpolarizing prepulses, postinhibitory rebound responses were demonstrated in both motoneurons and commissural interneurons in the lamprey spinal cord. The amplitude of the PIR response depends on the membrane potential level, as well as on the amplitude and duration of the hyperpolarizing prepulse. PIR responses were more prominent in commissural interneurons, suggesting that this property may be of particular significance for the reciprocal inhibition generated by these interneurons, resulting in distinct alternating activity between the hemisegments of the lamprey locomotor network. Also for the PIR property, an involvement of LVA Cav 1.3 L-type calcium channels was established. Together with T-type (Cav 3) LVA calcium channels, Cav 1.3 L-type channels may account for the PIR response, while there appears to be no significant contribution from a hyperpolarization-activated (Ih) current to the PIR response in lamprey spinal neurons. Both the 5-HT and the dopamine modulatory systems will reduce the PIR response, and this action is mediated via 5-HT1A and dopamine D2 receptors respectively. The modulatory effect of 5-HT and dopamine is due to a depressing action mainly on Cav 1.3 L-type calcium channels. The PIR effects complements previously established modulatory actions of 5-HT and dopamine on the operation of the spinal network for locomotion