Hyperpolarization-activated current (I(h)) contributes to excitability of primary sensory neurons in rats

Abstract

In various excitable tissues, the hyperpolarization-activated, cyclic nucleotide-gated current (I(h)) contributes to burst firing by depolarizing the membrane after a period of hyperpolarization. Alternatively, conductance through open channels I(h) channels of the resting membrane may impede excitability. Since primary sensory neurons of the dorsal root ganglion show both loss of I(h) and elevated excitability after peripheral axonal injury, we examined the contribution of I(h) to excitability of these neurons. We used a sharp electrode intracellular technique to record from neurons in nondissociated ganglia to avoid potential artefacts due to tissue dissociation and cytosolic dialysis. Neurons were categorized by conduction velocity. I(h) induced by hyperpolarizing voltage steps was completely blocked by ZD7288 (approximately 10 microM), which concurrently eliminated the depolarizing sag of transmembrane potential during hyperpolarizing current injection. I(h) was most prominent in rapidly conducting Aalpha/beta neurons, in which ZD7288 produced resting membrane hyperpolarization, slowed conduction velocity, prolonged action potential (AP) duration, and elevated input resistance. The rheobase current necessary to trigger an AP was elevated and repetitive firing was inhibited by ZD7288, indicating an excitatory influence of I(h). Less I(h) was evident in more slowly conducting Adelta neurons, resulting in diminished effects of ZD7288 on AP parameters. Repetitive firing in these neurons was also inhibited by ZD7288, and the peak frequency of AP transmission during tetanic bursts was diminished by ZD7288. Slowly conducting C-type neurons showed minimal I(h), and no effect of ZD7288 on excitability was seen. After spinal nerve ligation, axotomized neurons had less I(h) compared to control neurons and showed minimal effects of ZD7288 application. We conclude that I(h) supports sensory neuron excitability, and loss of I(h) is not a factor contributing to increased neuronal excitability after peripheral axonal injury.

Figure 1

H-current (I_h) in sensory neurons. A. In discontinuous single electrode voltage-clamp mode, hyperpolarizing voltage commands (top panel, recorded actual voltage) produce a immediate inward current and an additional slowly activating component that is sensitive to ZD7288 (middle panel), shown as the digitally subtracted difference current (bottom panel), in an Aα/β neuron. B. The current-voltage (I–V) relationship for I_h in this neuron. C. In the same neuron, discontinuous current clamp mode, hyperpolarizing current injections through the recording electrode (top panel) elicit time-dependent and voltage-dependent inward rectification (“sag”, middle panel), that is eliminated by ZD7288 (approximately 10μM, bottom panel). D. The I–V relationship shows the loss of sag during ZD7288 administration (open squares) compared to baseline conditions (filled circles).

Figure 2

Measured action potential (AP) parameters. RMP, resting membrane potential; APamp, amplitude of AP; AP95%, duration of AP after 95% repolarization; t, latency following axonal stimulation; AHPamp, amplitude of afterhyperpolarization; AHP80%, duration of afterhyperpolarization until 80% recovery to baseline; AHParea, area of the AHP.

Figure 3

Effect of ZD7288 (approximately 10μM) on firing pattern of an Aα/β neuron during injection of depolarizing current through the recording electrode using discontinuous current clamp, during baseline conditions (A.) and during administration of ZD7288 to the same neuron (B.). In both cases, the upper panel shows voltage traces in response to the current commands shown in the lower panel. Traces in the two conditions are selected to show events at comparable degrees of membrane depolarization. As demonstrated by this neuron, ZD7288 hyperpolarized the resting membrane potential, increased the input resistance of the neuron from 22.5MΩ to 35.7MΩ, increased the voltage necessary to initiate an action potential, and eliminated this neuron’s ability to fire multiple action potentials in response to sustained depolarization.

Figure 4

Effect of ZD7288 (approximately 10μM) on the ability of an A∂ neuron to conduct action potentials into the soma during tetanic stimulation. Under baseline conditions (A.), the neuronal soma can follow at 10Hz (left panel) and maintain successful firing at 50Hz (right panel), but the same neuron during ZD7288 (B.) drops action potentials at 50Hz (right panel). Note that the time scale is not the same in the right and left panels.