Single-Cell RT-PCR and Functional Characterization of Ca²⁺Channels in Motoneurons of the Rat Facial Nucleus

Abstract

Voltage-dependent Ca 2ϩ channels are a major pathway for Ca 2ϩ entry in neurons. We have studied the electrophysiological, pharmacological, and molecular properties of voltagegated Ca 2ϩ channels in motoneurons of the rat facial nucleus in slices of the brainstem. Most facial motoneurons express both low voltage-activated (LVA) and high voltage-activated (HVA) Ca 2ϩ channel currents. The HVA current is composed of a number of pharmacologically separable components, including 30% of N-type and ϳ5% of L-type. Despite the dominating role of P-type Ca 2ϩ channels in transmitter release at facial motoneuron terminals described in previous studies, these channels were not present in the cell body. Remarkably, most of the HVA current was carried through a new type of Ca 2ϩ channel that is resistant to toxin and dihydropyridine block but distinct from the R-type currents described in other neurons.Using reverse transcription followed by PCR amplification (RT-PCR) with a powerful set of primers designed to amplify all HVA subtypes of the ␣ 1 -subunit, we identified a highly heterogeneous expression pattern of Ca 2ϩ channel ␣ 1 -subunit mRNA in individual neurons consistent with the Ca 2ϩ current components found in the cell bodies and axon terminals. We detected mRNA for ␣ 1A in 86% of neurons, ␣ 1B in 59%, ␣ 1C in 18%, ␣ 1D in 18%, and ␣ 1E in 59%. Either ␣ 1A or ␣ 1B mRNAs (or both) were present in all neurons, together with various other ␣ 1 -subunit mRNAs. The most frequently occurring combination was ␣ 1A with ␣ 1B and ␣ 1E . Taken together, these results demonstrate that the Ca 2ϩ channel pattern found in facial motoneurons is highly distinct from that found in other brainstem motoneurons.

Coupling of L-type calcium channels to neurotransmitter release at mouse motor nerve terminals

“…In neonatal rats, messenger ribonucleic acid (mRNA) for the α-subunits of L-type VDCC is present in the somata of the rat facial nucleus (i.e. the brainstem nucleus innervating the levator auris longus muscle) motoneurons [36]. In addition, L-type VDCC have been implicated in the process of neurotransmitter release in immature mammalian MNT during development [38], during re-innervation [27] and during functional recovery from intoxication with botulinum toxin type-A [39].…”

Synaptic Control of Motoneuronal Excitability

“…HVA Ca 2+ current (995). In this study, single-cell RT-PCR was used to detect mRNA for the α 1A -subunit of HVA Ca 2+ channel subtypes, along with other α 1 -subunit types.…”

“…In this study, single-cell RT-PCR was used to detect mRNA for the α 1A -subunit of HVA Ca 2+ channel subtypes, along with other α 1 -subunit types. Because α 1A -subunits are thought to combine to form P/Q-type Ca 2+ channels, the absence of P-type channels in the soma suggests that P-type channels may be present elsewhere in the membrane of facial motoneurons, most likely in axon terminals (995). LVA and HVA Ca 2+ channel types are also present in spinal motoneurons (524,881).…”

Distinct Roles of Gαqand Gα11for Purkinje Cell Signaling and Motor Behavior

“…Individual Purkinje cells and material for control reactions were harvested under visual control from cerebellar slices as reported previously (Garaschuk et al, 1996;Plant et al, 1998). After solubilization in 0.5% NP-40 (Roche, Basel, Switzerland), reverse transcription (RT) was performed as described (Lambolez et al, 1992).…”

“…To test this hypothesis, we implemented a quantitative (Liss et al, 2001) real-time PCR approach (Rasmussen, 2001) that we used for total brain and single-cell RT-PCR analyses (Garaschuk et al, 1996;Plant et al, 1998). To investigate the expression of G␣ q and G␣ 11 in total brain RNA, we established external standard curves over a large range of concentrations (G␣ q , 1000 -400,000 cDNA copies; G␣ 11 , 500 -100,000 cDNA copies).…”