Functional Expression of T-Type Ca²⁺ Channels in Spinal Motoneurons of the Adult Turtle

<div><p>Voltage-gated Ca²⁺ (Ca_V) channels are transmembrane proteins comprising three subfamilies named Ca_V1, Ca_V2 and Ca_V3. The Ca_V3 channel subfamily groups the low-voltage activated Ca²⁺ channels (LVA or T-type) a significant role in regulating neuronal excitability. Ca_V3 channel activity may lead to the generation of complex patterns of action potential firing such as the postinhibitory rebound (PIR). In the adult spinal cord, these channels have been found in dorsal horn interneurons where they control physiological events near the resting potential and participate in determining excitability. In motoneurons, Ca_V3 channels have been found during development, but their functional expression has not yet been reported in adult animals. Here, we show evidence for the presence of Ca_V3 channel-mediated PIR in motoneurons of the adult turtle spinal cord. Our results indicate that Ni²⁺ and NNC55-0396, two antagonists of Ca_V3 channel activity, inhibited PIR in the adult turtle spinal cord. Molecular biology and biochemical assays revealed the expression of the Ca_V3.1 channel isotype and its localization in motoneurons. Together, these results provide evidence for the expression of Ca_V3.1 channels in the spinal cord of adult animals and show also that these channels may contribute to determine the excitability of motoneurons.</p></div

Tonically Active α5GABAA Receptors Reduce Motoneuron Excitability and Decrease the Monosynaptic Reflex

Motoneurons, the final common path of the Central Nervous System (CNS), are under a complex control of its excitability in order to precisely translate the interneuronal pattern of activity into skeletal muscle contraction and relaxation. To fulfill this relevant function, motoneurons are provided with a vast repertoire of receptors and channels, including the extrasynaptic GABAA receptors which have been poorly investigated. Here, we confirmed that extrasynaptic α5 subunit-containing GABAA receptors localize with choline acetyltransferase (ChAT) positive cells, suggesting that these receptors are expressed in turtle motoneurons as previously reported in rodents. In these cells, α5GABAA receptors are activated by ambient GABA, producing a tonic shunt that reduces motoneurons’ membrane resistance and affects their action potential firing properties. In addition, α5GABAA receptors shunted the synaptic excitatory inputs depressing the monosynaptic reflex (MSR) induced by activation of primary afferents. Therefore, our results suggest that α5GABAA receptors may play a relevant physiological role in motor control

Contribution of T-type channels to PIR in adult turtle motoneurons.

<p>A) Inhibition of rebound depolarization by Ni²⁺. PIR responses were elicited by hyperpolarizing pulses from a <i>V</i>_m of −58 mV (left panel). Average time courses of the normalized peak amplitude of PIR in control conditions and after Ni²⁺ application are shown in the mid panel. Arrow heads denote indicate the time at which the data were analyzed. The bar chart in the right panel shows the comparison of PIR amplitude before and after Ni²⁺ application. B) Block of rebound depolarization by NNC55-0396. PIR responses were evoked by hyperpolarizing pulses from a <i>V</i>_m of −60 mV (left panel). Average time courses of the normalized peak amplitude of PIR in control conditions and after NNC55-0396 application are shown in the mid panel. Arrow heads denote indicate the time at which the data were analyzed. The bar chart at the right shows the comparison of PIR amplitude before and after drug application. C) Sequential application of ZD7288 and NNC55-0396 significantly inhibited postinhibitory rebound, further demonstrating the contribution of HCN and T-type channels to PIR. The average time courses of the normalized peak amplitude of PIR in control conditions and after drug application are shown in the mid panel. Arrow heads denote indicate the time at which the data were analyzed.</p

Two types of postinhibitory rebound responses (PIR) in adult turtle motoneurons.

<p>A) Rebound responses evoked by hyperpolarizing current pulses with the same intensity at different <i>V</i>_m values. For clarity, in the voltage traces the PIR and the voltage sag components are indicated by arrows. The right panel shows PIR amplitudes as a function of <i>V</i>_m. PIR amplitude is larger at more negative <i>V</i>_m values which is suggestive of HCN channel activation. B) A residual PIR persists after the application of an <i>I</i>_h current blocker (ZD7288). Note that the voltage sag was eliminated by the <i>I</i>_h antagonist. C) Rebound responses evoked by hyperpolarizing current pulses at different <i>V</i>_m values. The right panel shows PIR amplitudes as a function of <i>V</i>_m. PIR amplitude is similar in the voltage range of −82 to −67 mV but is bigger at −61 mV suggesting a recruitment of T-type channels. D) PIR amplitude increases with pulse intensity. Rebound responses were evoked by hyperpolarizing current pulses of increasing amplitude (inset) from a <i>V</i>_m of −61 mV.</p

The T-type channel antagonist Ni²⁺ decreases adult turtle motoneuron excitability.

<p>A) Effect of Ni²⁺ on the AP firing rate. AP firing was elicited by applying a depolarizing current step of 1.6 nA (left panel). Note that the addition of Ni²⁺ greatly prevented AP firing (middle panel; <i>n</i> = 7). Stronger current injections restored AP bursts in the recorded cells (right panel). B) Comparison of the rheobase values in individual motoneurons before (Ctl) and after Ni²⁺ application. The bar chart summarizes the comparison of mean values in both experimental conditions. C) Comparison of the AP number as a function of current in individual (left panel) and grouped cells (middle panel) in the absence (Control) and the presence of Ni²⁺ as indicated. The bar chart compares the mean values in both conditions (right panel).</p

Ca_V3.1 channel expression in adult turtle spinal cord.

<p>A) RNA was extracted from the adult turtle spinal cord (tSC) and rat brain (rBr), used as a positive control, and subjected to RT-PCR with specific primers. Molecular weight markers are on the left, and (-) denotes negative control without RT enzyme. B) Proteins extracted from the turtle spinal cord (tSC) and rat brain (rB; used as a positive control) were subjected to Western-blot using anti-Ca_V3.1 antibodies. A ∼250 KDa band was present both in the TSc lane and the positive control. C) Representative confocal micrographs from adult turtle spinal cord slices immunostained with choline acetyltransferase (ChaT; a marker for motoneurons) shown in the left upper panel (green) and Ca_V3.1 antibodies shown in the left lower panel (red), suggesting co-localization of both proteins (right panel). Scale bar = 100 µm.</p

Electrophysiological characterization of adult turtle motoneurons.

<p>A) Measurement of the membrane input resistance (<i>R</i>_m) voltage-dependency. <i>R</i>_m was measured within ±10 mV around <i>V</i>_m. The upper panel shows typical voltage deflections of an adult turtle motoneuron. The lower panel shows <i>R</i>_m values estimated from plateau values of each voltage trace (symbols) as a function of the current pulse by calculating the slope of the linear part of the <i>I−V</i> curve. <i>R</i>_m was in a range of 9–76 MΩ. B) Spike trains elicited in an adult turtle motoneuron by current injection in the control condition. Note the spike frequency adaptation. C) Typical antidromical AP generated in a motoneuron in response to ventral root stimulation. D) Superimposed traces of PIR responses evoked by hyperpolarizing current pulses at two different <i>V</i>_m values as indicated.</p