Cholinergic terminals and receptors in the lumbosacral spinal cord of adult and neonatal rat

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Ralcewicz, Karen Lynn
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Cholinergic input to, and cholinergic mechanisms within the lower lumbar (L6) and upper sacral (S1) spinal cord of rat may influence neuronal excitability and afferent transmission (Thor et al, 2000) and may provide the environment necessary for appropriate central nervous system control of bladder and bowel function. It is unclear, however, if cholinergic terminals and receptors are present in the L6 & S1 spinal segments of rat and when this may develop. Cholinergic mechanisms have been shown to alter sensory afferent transmission, enhance motoneuron excitability, induce plateau potentials via non-linear membrane properties in motoneurons and reveal oscillations in locomotor-related interneurons. The enhanced activity of sphincter motoneurons was attributed to non-linear properties during the continence phase of distention-evoked voiding in the decerebrate cat (Paroschy & Shefchyk, 2000). Candidate neurotransmitters inducing non-linear properties in cat sphincter motoneurons are 5-HT (Paroschy & Shefchyk, 2000) and acetylcholine via motoneuron axon collaterals (Sasaki, 1994) and other spinal sources. We have established using the antibody to the vesicular acetylcholine transporter (VAChT) that cholinergic terminals are present on ventrolateral Onuf (VLO), dorsomedial Onuf (DMO) motoneurons and parasympathetic preganglionic motoneurons (PGN) in the L6 and S1 rat spinal cord segments. Muscarinic receptor (M2), nicotinic-α4 and α7 receptor subunit immunoreactivity was also present on Onuf motoneurons and in regions dorsal to the PGN. One source of the cholinergic puncta on Onuf motoneurons may be from motoneuron axon collaterals which we observed on a postnatal day 15 VLO motoneuron. Cholinergic terminals were observed on vasoactive intestinal polypeptide-immunoreactive (VIP) afferents, interneurons in the intermediolateral (IML) region and perhaps on other afferents in the lateral and medial collateral pathway of L6 and S1 spinal segments. In the ventral horn, the cholinergic puncta and receptors appear to have a mature distribution around two weeks postnatal and the cholinergic terminals appeared to have a mature distribution in the IML region by three weeks postnatal. Using whole cell patch clamp recording techniques and thick slices of the L6 and S1 rat spinal cord, we observed excitatory responses of ventral horn neurons and motoneurons to carbachol (10-50 μM), a non-specific cholinergic agonist. Ventral horn neurons (postnatal day 8- 16) exhibited prolonged firing and prolonged depolarizations (plateau potentials) beyond the duration of the applied excitatory input from cholinergic (n=6/33) and other (n= 4/37) neurotransmitter systems. In a selection of the neurons with plateau potentials, the L-type calcium current played a role in the plateau production (n=5/5) and low frequency oscillations (n=2/2) as revealed by nifedipine. Postnatally, the voiding reflex changes from a perineal-evoked reflex, to the adult bladder-bladder reflex. Cholinergic input may be responsible in part for the bursting activity of the external urethral sphincter and the activation of the bladder, which is required for complete voiding reflexes in the adult rat. Plateau potentials and enhanced excitability due to cholinergic mechanisms could render inessential a constant excitatory drive that is required in the perineal-evoked voiding reflex in the neonatal rat and may underlie changes in the voiding reflexes that occur during postnatal development.
cholinergic, plateau potentials, motoneurons, voiding and continence, muscarinic receptors, axon collaterals, nicotinic receptors, VAChT terminals, spinal cord, electrophysiology, neonatal rat