Purinergic and cholinergic influences on hypoglossal motoneuron excitability / Matthew F. Ireland. Purinergic & cholinergic effects on XII motoneurons

Ireland, Matthew F (2005). Purinergic and cholinergic influences on hypoglossal motoneuron excitability / Matthew F. Ireland. Purinergic & cholinergic effects on XII motoneurons PhD Thesis, School of Biomedical Sciences, The University of Queensland.

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Author Ireland, Matthew F
Thesis Title Purinergic and cholinergic influences on hypoglossal motoneuron excitability / Matthew F. Ireland. Purinergic & cholinergic effects on XII motoneurons
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
Publication date 2005-01-01
Thesis type PhD Thesis
Supervisor Mark Bellingham
Total pages 156
Language eng
Subjects 06 Biological Sciences
Formatted abstract

This current thesis has focused on cholinergic and purinergic influences on hypoglossal motoneurons (HMs). During rapid eye movement sleep, a distinct phase of sleep characterized by rapid eye movements and muscle atonia, acetylcholine is the predominant neuromodulator acting on HMs, via muscarinic acetylcholine receptors (mAChR). At the present time, very little is known about muscarinic effects on HM excitability, which may contribute to respiratory disorders such as obstructive sleep apnea or sudden infant death syndrome. The primary aim of this thesis was to investigate the effects of cholinergic-muscarinic modulation of HMs. In the course of these studies, evidence emerged that the ATP-activated P2X7 receptor may be restricted to excitatory synaptic terminals in the CNS and its subsequent activation proposed to enhance excitatory transmission in the CNS. We therefore additionally investigated the role of the P2X7 receptor and its contribution to HM excitability.     

To address the first aim of this thesis, muscarinic activation was investigated in neonatal HMs (PO to P4). Whole cell current and voltage clamp recordings were made from visually identified HMs (n=60) in 200 µM thick transverse brainstem slices from mice. Bath application of muscarine (10µM) significantly depolarized the membrane potential of HMs (control 60.1±1.0 mV; muscarine 55.0±1.1 mV, n=1 0; P<0.05) and in response to current injection, muscarine significantly reduced the duration of the afterhyperpolarization (AHP) following the action potential, which resulted in a marked facilitation in steady state firing frequency (slope control 61.5 ± 9.5 Hz/nA; muscarine 111 .0 ± 15.5 Hz/nA; n=6; p<0.05). Hyperpolarizing voltage steps from a holding potential of - 30mV revealed a previously unreported slowly deactivating M-like current and also the hyperpolarization-activated inward rectifier Ih Application of oxotremorine (10)µM), a muscarine analog, suppressed both the M-like current and reduced the linear instantaneous leak K+ current, but had no significant effect on the lh current. We next examined muscarinic activation in the presence of elevated K+ (change from 3mM to 9mM). In the presence of 9mM K", we found the AHP was significantly reduced due to a decreased driving force for K+, whereas an instantaneous mixed cationic leak current was substantially increased. The net effect of which depolarized HMs and increased steady state firing frequency at lower current injections compared to values at 3mM K+. Further addition of oxotremorine in 9mM K+ ACSF had no significant effect on the AHP, M-like current or the leak current, but significantly increased firing frequency (Norm to elevated K+, 1 00pA injection, 1.4 ± 0.1 SEM, n=9).

The second aim of this thesis pharmacologically characterized the M-like current observed in neonatal HMs. In the CNS, the M-current plays a major role in regulating neuronal excitability by opposing membrane depolarization. In 23 out of 42 HMs recorded from juvenile HMs (P15 to P25), perforated patch clamp recordings revealed an M-like current that shared many pharmacological and biophysical characteristics to the native M-current. We found the M-like current was suppressed by the cognitive enhancer linopirdine (1OµM) and enhanced by the anti-convulsive retigabine (10)µM). Muscarinic activation further suppressed the M-like current, reduced a linear-cationic leak current and enhanced the hyperpolarization-activated I" current by positively shifting its voltage dependence of activation.      

For the final aim, we investigated recent reports that P2X7 receptor activation enhances glutamatergic synaptic transmission. Excitatory postsynaptic currents (EPSCs) were recorded from juvenile HMs using whole cell recording after blockade of most P2X receptors with suramin (P2X7-insensitive) and adenosine receptors with 8 phenyltheophylline (8PT). Bath application of the P2Xreceptor agonist 2'-3'-0-(4-benzoylbenzoyl)-adenosine-5'-triphosphate (BzA TP) elicited a 40.5±16.0% (mean ± S.E.M., n=8, P<0.05) increase in evoked EPSC amplitude and significantly reduced paired pulse facilitation of evoked EPSCs. Prior application of the P2X7 antagonist brilliant blue G (200nm or 2µM) completely abolished the P2X7-Iike enhancement by BzATP.     
In summary, the results of this thesis suggest muscarinic receptors acting postsynaptically play a significant role in regulating HM excitability by modulating the voltage activated M-current, the hyperpolarization-activated inward rectifier lh and a voltage insensitive "leak " cationic K+ current. Acting presynaptically we have determined activation of a P2X7like receptor enhances EPSC amplitude in an action potential dependent manner. The continuing study of the input-output properties of motoneurons is likely to contribute not only to our understanding of motor control but will also provide insights into the biophysical mechanisms of neuronal information processing elsewhere in the nervous system. 

Keyword Motor neurons
Cholinergic mechanisms
Muscarinic receptors

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
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