Upregulation of the hyperpolarization-activated cation current after chronic compression of the dorsal root ganglion

Abstract

A chronic compression of the DRG (CCD) produces cutaneous hyperalgesia and an enhanced excitability of neuronal somata in the compressed ganglion. The hyperpolarization-activated current (I(h)), present in the somata and axons of DRG neurons, acts to induce a depolarization after a hyperpolarizing event and, if upregulated after CCD, may contribute to enhanced neuronal excitability. Whole-cell patch-clamp recordings were obtained from acutely dissociated, retrogradely labeled, cutaneous, adult rat DRG neurons of medium size. Neurons were dissociated from L4 and L5 control DRGs or DRGs that had each been compressed for 5-7 d by L-shaped rods inserted into the intervertebral foramina. I(h), consisting of a slowly activating inward current during a step hyperpolarization, was recorded from every labeled, medium-sized neuron and was blocked by 1 mm cesium or 15 microm ZD7288. Compared with control, CCD increased the current density and rate of activation significantly without changing its reversal potential, voltage dependence of activation, or rate of deactivation. Because I(h) activation provides a depolarizing current to the neuron, thus enhancing neuronal excitability, our results are consistent with the hypothesis that I(h) contributes to hyperalgesia after CCD-induced nerve injury.

Fig. 1.

The effects of CCD on the density ofI_h current. A, Current responses evoked by hyperpolarizing voltage steps from a holding potential of −50 mV in a control (left) and a CCD (right) DRG neuron. The voltage protocol is shown at thebottom. I_h current amplitude was calculated from the difference between the steady-state and the instantaneous currents (i.e., the difference between the twoarrows shown as an example at the last trace of the left top traces). In both panels, families of current responses on the top were recorded in the control bath solution. Middle traces, Cs⁺ at 1 mm.Bottom traces, After 2 min of washout. Voltage protocol is shown as aninset at bottom. B,I_h current density versus membrane potential, V_m. Squares andcircles represent the data summarized from 34 control and 51 CCD neurons, respectively. Error bars indicate SEM. * Indicates that means at the same membrane voltage are significantly different.

Fig. 2.

Effects of CCD on the conductance and reversal potential of I_h current. A, In a control (top) and a CCD (bottom) neuron, I_h was fully activated by a hyperpolarizing prepulse to −100 mV for 1 sec, followed by a depolarization to test potentials of −90 to −50 mV (inset). Tail currents were measured at the start of the test potentials (arrows). B, Leak-subtracted tail current versus test potential.Squares and circles represent control and CCD neurons, respectively. The solid lines are the linear regressions fitted to the data points for each group. The slopes of the linear regressions were 0.15 and 0.23 nS/pF for control and CCD, respectively. The reversal potentials were −21 and −23 mV for control and CCD, respectively.

Fig. 3.

Effects of CCD on the voltage dependence ofI_h activation. A,I_h was activated from a holding potential of −50 mV to prepulse potentials ranging from −40 to −120 mV, followed by a step to a test potential of −120 mV (voltage protocol as shown atbottom). The magnitude of the tail current at the start of the test potential was used as an index ofI_h activation (arrows ininsets a and b). The tail currents of a control (top) and a CCD (middle) neuron are shown. B, Activation curves obtained by fitting the data of a control (squares) and a CCD (circles) neuron (as shown in A,top and middle, respectively) with single Boltzmann functions. The fitting yielded midpoint potentials,V_0.5, and slope factors,k, of −76.2 and 5.3 mV for control and −77.4 and 6.5 mV for CCD neurons, respectively.

Fig. 4.

Effect of CCD on the activation kinetics ofI_h current. A, Current responses of a control (top) and a CCD (bottom) neuron each evoked by a series of hyperpolarizing voltage steps of −60 to −120 mV from a holding potential of −50 mV. Step duration was varied from 2150 to 1150 msec (inset at bottom right). Eachsolid line is a fitting of the sum of two exponentials to the data points (open circle) obtained at each hyperpolarizing voltage. Note the faster activation ofI_h currents at each voltage in the CCD neuron. B, The mean ratio ofA_f toA_f +A_s versus membrane potential for control (squares) and CCD (circles) neurons. C, D, The mean time constants τ_f (C) and τ_s (D) versus membrane potential for control (squares) and CCD (circles). * Indicates that means are significantly different.

Fig. 5.

Effects of CCD on the deactivation ofI_h current. A, Current response (top) of a control neuron evoked by a triple-pulse voltage protocol (bottom). A hyperpolarizing potential of −110 mV was followed by different durations of a depolarization to −30 mV to deactivateI_h current and a hyperpolarization to −90 mV to reactivate I_h (the number of traces was reduced for clarity). The amplitude ofI_h current (shown as an example in theright trace) activated by the third pulse was used to calculate the deactivation time constant. B, Current responses of a control cell (same cell as in A) to the triple protocol but with deactivating potentials of −30, −40, −50, and −60 mV (a–d, respectively). Current traces were truncated at the end of b–d to keep the same time scale as that in a. The number of data points in each current trace in both A and B were reduced to $\frac{1}{10}$. C, The effect of potential on the time course of I_h current deactivation. Data points are fit by a single exponential. D, Mean time constant of deactivation of I_h current versus membrane potential for control and CCD neurons (squares and circles, respectively). The single exponential functions fitted to the data points for each group are not significantly different for CCD and control neurons.

Fig. 6.

The effects of I_h on the resting membrane potential. Current-clamp recordings of the resting membrane potential (V_rest) of a control (A) and a CCD (B) neuron. The respective initial values ofV_rest for the control and CCD neurons were −53 and −56 mV. Current pulses of −0.6 nA, 200 msec, were injected into each cell every 3 sec to elicit a hyperpolarization and sag in voltage response. The horizontal bars indicate the durations of application of the I_h blockers Cs⁺ (1 mm) and ZD7288 (15 μm). A, B, Top trace, Voltage responses to the current injection;bottom trace, enlarged voltage traces to show the reduction in sag produced by the blocker. Note that, in both control and CCD neurons, both 1 mm Cs⁺ and 15 μm ZD7288 did not alter resting potential but had similar effects in abolishing the sag.