Abstract
The neurosteroid allopregnanolone (ALLO) causes unconsciousness by allosteric modulation of γ-aminobutyric acid type A (GABAA) receptors, but its actions on the spinal motor networks are unknown. We are therefore testing the hypothesis that ALLO attenuates the action potential firing of spinal interneurons and motoneurons predominantly via enhancing tonic, but not synaptic GABAergic inhibition. We used video microscopy to assess motoneuron-evoked muscle activity in organotypic slice cultures prepared from the spinal cord and muscle tissue. Furthermore, we monitored GABAA receptor-mediated currents by performing whole-cell voltage-clamp recordings. We found that ALLO (100 nM) reduced the action potential firing of spinal interneurons by 27% and that of α-motoneurons by 33%. The inhibitory effects of the combination of propofol (1 µM) and ALLO on motoneuron-induced muscle contractions were additive. Moreover, ALLO evoked a tonic, GABAA receptor-mediated current (amplitude: 41 pA), without increasing phasic GABAergic transmission. Since we previously showed that at a clinically relevant concentration of 1 µM propofol enhanced phasic, but not tonic GABAergic inhibition, we conclude that ALLO and propofol target distinct subpopulations of GABAA receptors. These findings provide first evidence that the combined application of ALLO and propofol may help to reduce intraoperative movements and undesired side effects that are frequently observed under total intravenous anesthesia.
Highlights
Considerable research efforts are focusing on neuroactive steroids as adjuncts or even substitutes for commonly used intravenous anesthetics [1,2], which have a long list of adverse side effects [3]
Since we previously showed that at a clinically relevant concentration of 1 μM propofol enhanced phasic, but not tonic GABAergic inhibition, we conclude that ALLO and propofol target distinct subpopulations of GABAA receptors
The present study shows, for the first time, that ALLO induces a tonic, GABAA-R-mediated current in spinal interneurons of the ventral horn, which is accompanied by a decrease in motor output
Summary
Considerable research efforts are focusing on neuroactive steroids as adjuncts or even substitutes for commonly used intravenous anesthetics [1,2], which have a long list of adverse side effects [3]. We utilize organotypic co-cultures of the spinal cord and muscle tissue to explore the effects of ALLO on spinal motor networks This preparation’s morphological features and functional properties were described in greater detail in the landmark papers published by Spenger et al as well as by Streit et al These authors provided evidence that spinal interneurons and motoneurons, neuromuscular junctions, and stratified muscle fibers ex vivo largely resemble their counterparts in vivo [17]. These researchers identified motoneurons by intracellular filling and retrograde axonal staining, using horseradish peroxidase crystals applied to the muscle tissue. Our group makes use of this preparation to study the effects of anesthetic agents (e.g., propofol, etomidate, thiopental, and sevoflurane) [20], muscle relaxants [21], and neurotoxins [22,23,24] to further elucidate the drugs’ mechanisms of action and to develop cell-based ex vivo assays in order to reduce the need for animal testing [23,24]
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