Peter Placas
Marquette University
Milwaukee, WI 
Faculty Mentor: Dr. James T. Buchanan

Acetylcholine (ACh) has been demonstrated to be the neurotransmitter in both motoneurons and some classes of interneurons of the vertebrate nervous system. ACh acts on two receptor types: ionotropic nicotinic receptors and metabotropic muscarinic receptors. To test the role of ACh in CNS function, we have been using an in vitro spinal cord preparation of the lamprey, a lower vertebrate. From ventral root recordings of this preparation, motoneuron firing patterns are observed which mimic lamprey swimming (thus referred to as "fictive swimming"). Previous studies in our laboratory indicated that application of ACh directly to the spinal cord during fictive swimming decreased cycle period. The present experiments addressed the question of whether ACh is released endogenously and therefore has ongoing modulatory effects on the locomotor network.

Two properties of fictive swimming, cycle period and phase lag, were analyzed after recording motoneuron firing in the ventral roots of an in vitro spinal cord preparation. Cycle period is the measure of the time between successive ventral root bursts during fictive swimming. Phase lag is the time delay between bursts in adjacent ventral roots normalized to cycle period as the ventral root bursting propagates down the spinal cord. Swimming was induced by continuous perfusion of 0.5 mM D-glutamate so that a stable rhythm could be observed as a control. Then 25 mM eserine, an acetylcholine esterase inhibitor (AChE-I), was perfused into the bath along with the D-glutamate. In the presence of eserine, any endogenously-released ACh will accumulate due to the inhibition of the enzyme responsible for its hydrolysis. After one hour of constant eserine perfusion, two acetylcholine antagonists, scopolamine (muscarinic antagonist) (20 mM) and mecamylamine (nicotinic antagonist) (10 mM), were also perfused into the bath for the period of an hour. Cycle period and phase lag of fictive swimming were then measured from five minute intervals of the ventral root recordings of the bath perfusion experiment.

Both cycle period and phase lag were affected by the addition of the AChE-I and acetylcholine antagonists. In most cases, shortly after the beginning of AChE-I perfusion, the cycle period and phase lag decreased by an average of 33%. Conversely, whenever cholinergic blockers were perfused, both cycle period and phase lag increased by an average of 69% from the AChE-I perfusion level. In a second set of experiments, the cholinergic blockers were perfused during control fictive swimming without eserine and demonstrated similar effects of increasing phase lag and cycle period. Both blocker types contributed to the slowing of fictive swimming as shown by experiments where each blocker was perfused individually. 

These observations provide evidence for the endogenous release of ACh within the spinal cord during fictive swimming. It can be concluded that this continuous endogenous release of acetylcholine will have ongoing modulatory effects producing a decrease in cycle period and phase lag of the lamprey locomotor network.