Our focus was to study the processes involved in the generation of rhythmic oscillations by a functional network made up by excitatory connections only. For this purpose we used the spontaneous rhythm generated by the in vitro neonatal rat spinal cord preparation when fast, chloride-mediated inhibition via GABAA and glycine receptors was blocked. We then addressed the issue of the role played by two distinct membrane mechanisms responsible for ionic movements across neuronal membranes, namely GABAc channels and the Na+/K+ ATPase pump. The spontaneous pattern, termed disinhibited rhythm, was stable, robust, regular and organized with intraburst oscillations. Using electrophysiological, in situ hybridisation and immunocytochemical techniques we evaluated the presence and the role of the third component of fast synaptic inhibition (chloride mediated like GABAA and glycine ones),namely the GABAc conductance. We observed that in the rat spinal cord GABAc receptor mRNA and expression was developmentally regulated. Electrophysiological results confirmed differential postnatal distribution of GABAc receptors which, however, played a minor role in rhythmogenic network activity. In fact, GABAc channel activity only modulated the disinhibited rhythm, confirming that this bursting activity was generated by a purely excitatory network. Applying a biochemical assay and electrophysiological techniques, we characterized the role of the Na+/K+ electrogenic pump in the generation and/or maintenance of rhythmic activity. After the demonstration of complete block of the sodium pump by our experimental protocol ( 4 μM strophanthidin), we studied the effect of blocking this pump on spontaneous and electrically evoked events in the spinal cord network. During a high frequency stimulus train in control solution, the pump activity normally prevented linear summation of responses and sped up network recovery. Long term strophanthidin application during disinhibited rhythm disrupted bursting which was later replaced by much longer and irregular bursts. This new activity, namely strophanthidin bursting, was very robust and organized with intraburst oscillations. Pharmacological experiments indicated that strophanthidin bursting was a network phenomenon driven by recurrent glutamatergic excitation mediated by non-NMDA and NMDA receptors. It was not possible to accelerate this pattern with conventional excitatory agents like serotonin, NMDA or high K+. Experiments based on bursts evoked by single and repeated electrical stimulation during disinhibited or strophanthidin bursting, suggested that while the former was limited by the Na+ pump activity, the latter was constrained by development of synaptic fatigue. These proposals were applied to a mathematical model developed in collaboration with Dr S. Bianchini (Istituto per le Applicazioni del Calcolo "M. Picone", Rome) who was able to provide realistic simulations of spontaneous discharges generated by the excitatory spinal cord network in the presence or in the absence ofNa+/K+ pump activity.
|Titolo:||A Study of the mechanisms regulating network rhythmicity in the neonatal rat spinal cord|
|Relatore/i esterni:||Tongiorgi, Enrico|
|Data di pubblicazione:||1-mar-2002|
|Appare nelle tipologie:||8.1 PhD thesis|