Role of the Excitability Brake Potassium Current IKD in Cold Allodynia Induced by Chronic Peripheral Nerve Injury

Abstract

Cold allodynia is a common symptom of neuropathic and inflammatory pain following peripheral nerve injury. The mechanisms underlying this disabling sensory alteration are not entirely understood. In primary somatosensory neurons, cold sensitivity is mainly determined by a functional counterbalance between cold-activated TRPM8 channels and Shaker-like Kv1.1-1.2 channels underlying the excitability brake current I_KD Here we studied the role of I_KD in damage-triggered painful hypersensitivity to innocuous cold. We found that cold allodynia induced by chronic constriction injury (CCI) of the sciatic nerve in mice, was related to both an increase in the proportion of cold-sensitive neurons (CSNs) in DRGs contributing to the sciatic nerve, and a decrease in their cold temperature threshold. I_KD density was reduced in high-threshold CSNs from CCI mice compared with sham animals, with no differences in cold-induced TRPM8-dependent current density. The electrophysiological properties and neurochemical profile of CSNs revealed an increase of nociceptive-like phenotype among neurons from CCI animals compared with sham mice. These results were validated using a mathematical model of CSNs, including I_KD and TRPM8, showing that a reduction in I_KD current density shifts the thermal threshold to higher temperatures and that the reduction of this current induces cold sensitivity in former cold-insensitive neurons expressing low levels of TRPM8-like current. Together, our results suggest that cold allodynia is largely due to a functional downregulation of I_KD in both high-threshold CSNs and in a subpopulation of polymodal nociceptors expressing TRPM8, providing a general molecular and neural mechanism for this sensory alteration.SIGNIFICANCE STATEMENT This paper unveils the critical role of the brake potassium current I_KD in damage-triggered cold allodynia. Using a well-known form of nerve injury and combining behavioral analysis, calcium imaging, patch clamping, and pharmacological tools, validated by mathematical modeling, we determined that the functional expression of I_KD is reduced in sensory neurons in response to peripheral nerve damage. This downregulation not only enhances cold sensitivity of high-threshold cold thermoreceptors signaling cold discomfort, but it also transforms a subpopulation of polymodal nociceptors signaling pain into neurons activated by mild temperature drops. Our results suggest that cold allodynia is linked to a reduction of I_KD in both high-threshold cold thermoreceptors and nociceptors expressing TRPM8, providing a general model for this form of cold-induced pain.

Keywords: 4-AP; Kv1 channels; PBMC; TRPM8; thermotransduction; α-DTx.

Figure 1.

Nocifensive behavior of sham and injured mice in response to innocuous cold stimulation. A, Time course of the cold-evoked nocifensive behavior assessed by acetone response net score in mice, evaluated at 0 (basal), 1, 3, 7, and 14 d after injury (see Materials and Methods). Red line and red dots represent CCI animals. Black line and dots represent sham operated mice; n = 6 for each group. B, Bar graph of the nocifensive behavior after application of acetone to the plantar surface of the hindpaw in sham and CCI animals, before and after pharmacological suppression of I_KD by intraplantar injection of 10 mm 4-AP (n = 6). A, B, Intergroup analyses of nociceptive behavior net scores were performed by means of two-way ANOVA followed by the Bonferroni post hoc multiple-comparisons test: **p < 0.01; ***p < 0.001. B, Intragroup analyses were assessed by means of paired t test: ^##p < 0.01; ^###p < 0.001.

Figure 2.

Altered cold sensitivity of primary sensory neurons in response to chronic nerve damage. A, Ratiometric [Ca²⁺]_i response to cooling (middle) in one representative low-threshold CSNs from a sham animal, recorded simultaneously with the temperature of the bath (bottom). Top, Transmitted (a) and pseudocolor ratiometric [Ca²⁺]_i images of this neuron, that correspond with b (control) and c (cold) points in the middle panel; the fluorescence images in b and c correspond with the time points marked in magenta. B, Percentage of active population recruited during a cooling ramp in CSNs from sham versus CCI mice. C, Dot plot of temperature thresholds exhibited by CSNs from sham (n = 135) and CCI (n = 140) mice. Temperature thresholds were compared using a two-tailed unpaired Student's t test: ***p < 0.001. D, Summary bar plot of the magnitude of the cold-induced responses in CSNs from sham (black bar) and CCI (red bar) animals. Intracellular calcium increases in CSNs were compared using unpaired Student's t test: *p = 0.040. All values in this figure that include error bars indicate mean ± SEM. E, Pie plots showing the percentage of the different populations of CSNs in sham and CCI condition. CINs are cold-insensitive neurons. **p = 0.005 (F test). ***p < 0.001 (F test).

Figure 3.

Effect of the pharmacological suppression of I_KD on thermal threshold of CSNs from sham and CCI mice. A, Ratiometric [Ca²⁺]_i response in a representative CSN from sham group during two consecutive cooling stimuli in control solution and the presence of 100 μm 4-AP. In this neuron, the cold threshold was shifted to higher temperatures, from 26.6°C to 29.2°C (black arrowheads). B, Summary of effect of 100 μm 4-AP on temperature threshold of cold-evoked responses in 24 CSNs from sham group. C, Mean values of the temperature threshold of CSNs in sham (black bar), control + 4-AP (dashed black), CCI (red bar), and CCI in the presence of the inhibitor of I_KD. The mean threshold of CCI CSNs was unaffected by the pharmacological suppression of I_KD. D, Ratiometric [Ca²⁺]_i response in a CSN from CCI group during two consecutive cooling ramps in control solution and in the presence of 100 μm 4-AP. In this neuron, the cold threshold was unaffected by the drug (31.9°C vs 31.1°C, red arrowheads). E, Summary of the effect of 100 μm 4-AP on temperature threshold of cold-evoked responses in 25 CSNs from CCI mice. F, Mean values of the temperature threshold of CSNs in control (black bar), control + α-DTx (dashed black bar), CCI (red bar), and CCI in the presence of the inhibitor of I_KD (dashed red bar). The mean threshold of CCI CSNs was unaffected by the suppression of I_KD by this toxin. Temperature threshold shifts after treatment with 4-AP (C) or α-DTx (F) were assessed using paired Student's t test: ***p < 0.001 and **p = 0.008, respectively. C, F, Temperature thresholds exhibited by CSNs from sham and CCI mice were compared using a Student's t test: **p = 0.001; *p = 0.044. All values in this figure that include error bars indicate mean ± SEM.

Figure 4.

Reversible shift of thermal threshold induced by pharmacological suppression of I_KD in primary sensory neurons. A, Simultaneous recording of membrane potential (top) and bath temperature (bottom) during three consecutive cooling ramps in a cold-sensitive DRG neuron recorded in current-clamp mode (I_hold = 0 pA). Application of 100 μm 4-AP reversibly shifted the temperature threshold and enhanced the firing of action potentials during the cooling ramp. B, Left, Response-threshold temperatures of five CSNs during cold applications in control solution, 100 μm 4-AP and after washing. Colors of the symbols represent each neuron studied using this protocol. Right, Bar plot of mean firing frequency during the cold-induced responses measured for the neurons in B. C, Simultaneous recording of membrane potential (top) and bath temperature (bottom) during three consecutive cooling ramps in a cold-insensitive DRG neuron recorded in current-clamp mode (I_hold = 0 pA). Note the reversible firing response induced by the pharmacological suppression of I_KD by 4-AP during the cooling ramp. D, Left, Response-threshold temperatures of nine cold-insensitive neurons during cold applications in control solution supplemented with 100 μm 4-AP. Colors of the symbols represent each CIN studied using this protocol. Right, Bar plot of mean firing frequency during cold ramps measured for the neurons in D. B, D, Temperature threshold and firing frequencies were compared using paired Student's t test: **p = 0.002 and *p = 0.022, respectively. All values in this figure that include error bars indicate mean ± SEM.

Figure 5.

Biophysical properties of I_KD and differential expression of I_KD and I_TRPM8 in CSNs from sham and CCI mice. A, 4-AP-sensitive currents (top), obtained from the digital subtraction of whole-cell currents in control and 100 μm 4-AP with an activation protocol in a CSN from a sham animal. The voltage protocol is shown at the bottom. Note the slow inactivation at negative membrane potentials. Dotted line indicates the 0 current level. B, Average activation (orange) and steady-state inactivation (blue) curves obtained from five control CSNs. Note the window current around the resting membrane potential. C, Whole-cell current in a representative CSNs from sham (left) and CCI (right) mice during a bipolar voltage protocol. The holding potential was −50 mV, and a hyperpolarizing pulse to −120 mV (see Materials and Methods) was applied before to reach the subthreshold membrane potential of −40 mV to reveal I_KD. D, Mean I_KD current density in sham (n = 34) and CCI (n = 36) CSNs: p = 0.003 (t test). E, Simultaneous recording of membrane current (top) and bath temperature (bottom) during a long cooling step (20°C) combined with application of menthol (100 μm) in a representative CSN from a sham animal. Dotted line indicates the 0 current level. F, Bar plot summarizing the mean I_cold and I_cold+menthol (I_TRPM8) current density (measured at the pick of both currents) in 26 CSNs from sham and 20 CSNs from CCI animals (V_hold = −50 mV): p = 0.477 and p = 0.267 (t test), respectively. G, Dot plot of I_KD and I_cold+menthol current density in sham and CCI CSNs separated into HT- and LT-CSNs. The abscissa represents the thermal threshold, measured using [Ca²⁺]_i imaging. *p < 0.05 (t test). **p < 0.01 (t test). Same as in C, I_KD was measured at −40 mV 1 s after the return from a holding potential of −120 mV, in the presence of 0.5 μm TTx.

Figure 6.

Thermal modulation of cold-insensitive neurons by suppression of I_KD. A, Time course of the [Ca²⁺]_i in a CIN from a sham animal transformed in CSN in response to the pharmacological suppression of I_KD by 4-AP. Inset, Histogram representing the mean value of the Ca²⁺ response of the population of recruited neurons from sham mice: ***p < 0.001 (paired Student's t test). B, I_KD current density in recruited (n = 23) and CSNs (n = 21), recorded in the same conditions: **p = 0.003 (unpaired Student's t test). C, Stacked bar graph representing the percentage of CSNs, CINs, and 4-AP-recruited neurons in sham and CCI conditions. Note the reduction of the population of 4-AP-recruited neurons in response to injury: *p = 0.042 (F test). D, E, Simultaneous recording of membrane potential (top) and bath temperature (bottom) during two consecutive cooling ramps in two control cold-insensitive DRG neurons recorded under current-clamp (I_hold = 0 pA; V_rest= −50 and −49 mV, respectively), transformed into cold-sensitive ones by pharmacological suppression of I_KD by 4-AP. D, E, Insets, First action potential in both neurons in response to a depolarizing current pulse at 34°C. Note the hump in the repolarizing phase in the action potential from the nociceptor-like neuron in D and its lower firing frequency compared with the cold thermoreceptor-like neuron in E.

Figure 7.

Electrophysiological properties of CSNs from sham and injured mice. A, B, Voltage responses to 500 ms hyperpolarizing and depolarizing current (I_ext) pulses in two CSNs from injured mice, showing the phenotype of a canonical cold thermoreceptor (A) or broad action potentials similar to nociceptive neurons (B). Note the hump in the repolarizing phase and the lower firing frequency in the nociceptor-like CSN; both characters are often found in CSNs from injured animals. Also note the rebound firing of the neuron in A (orange trace). Insets, First action potential of neurons in A and B (black and blue arrowheads). C, Plot of the firing frequency versus depolarizing current in neurons of both phenotypes in sham and CCI mice. Left, Cold thermoreceptor-like (CT-like) neurons. Black circles represent control CT-like neurons (n = 20). Red circles represent CCI CT-like neurons (n = 7). Right, Nociceptor-like (N-like) neurons. Black circles represent control N-like neurons (n = 2). Red circles represent CCI N-like neurons (n = 8).

Figure 8.

Evaluation of the responses to chemical agonist of thermo-TRP channels of cold-sensitive neurons from sham and injured mice. A, Protocol in calcium imaging used to evaluate the responses to cold, menthol (100 μm), cold plus menthol, AITC (100 μm; TRPA1 agonist), and capsaicin (200 nm; TRPV1 agonist) in cold-sensitive neurons from sham and CCI animals. A, The CSN corresponds to a LT control neuron, which is insensitive to AITC but sensitive to capsaicin. B, Pie plots summarizing the percentage of menthol-, AITC-, and capsaicin-sensitive CSNs from sham and CCI animals. Menthol responses included an increase in the [Ca²⁺]_i at 34°C or a shift (>1°C) in the cold threshold toward higher temperatures. C, Bar histograms represent the maximal response to menthol at 34°C and/or cold plus menthol (left panel), AITC at 34°C (central panel), and capsaicin at 34°C (right panel) in CSNs responding to these agonists, isolated from sham (black bars) and CCI animals (red bars). *p = 0.045 (F test).

Figure 9.

PBMC blocks TRPM8-mediated responses in TRPM8(+)-HEK293 cells and suppress generator potentials in CSNs from sham and injured mice. A, Ratiometric [Ca²⁺]_i response in a TRPM8(+)-HEK 293 cell during three consecutive cooling ramps to 18°C, in control solution, in the presence of PBMC at 1 μm, and after 5 min washing this TRPM8 blocker. B, Percentage of inhibition of cold-evoked calcium signals in TRPM8(+)-HEK293 cells by different concentrations of PBMC. Gray curve indicates the dose–response fit to the Hill equation, with an IC₅₀ of 92.7 nm and a Hill coefficient of 1.3 ± 0.2 (n > 20 cells for each concentration). C, D, Representative simultaneous recordings of [Ca²⁺]_i (top) and bath temperature (bottom), during two consecutive cooling drops from 34°C to 20°C, in a CSN isolated from DRG of a sham (C, black open circles) and an injured mouse (D, red open circles). PBMC at 1 μm produced a large reduction in the cold-evoked response, with only minor effects on the response induced by a 30 mm elevation in extracellular K⁺. E, F, Normalized [Ca²⁺]_i responses to cold and to elevated K⁺ in control extracellular solution (Cold and KCl), in extracellular solution supplemented with 1 μm PBMC (Cold + PBMC), and with 30 mm KCl (KCl + PBMC) for both sham (C, black filled and stripped bars) and injured mice (D, red filled and stripped bars) (n = 6 CSNs neurons for each condition. **p = 0.003 (paired t test). ***p < 0.001 (paired t test).

Figure 10.

Mathematical model of CSNs with different densities of I_KD and I_TRPM8. A, Left, Voltage-clamp simulation of the I_KD current inserted into the model. Voltage protocol is shown at the bottom. The maximum density (g_KD) is 0.1 mS/cm². Middle, Activation (m∞) and inactivation (h∞) curves. Right, Activation time constants at different voltages were obtained with the equation described in Materials and Methods. B–D, Firing rate (left) and voltage trace (right) of model CSNs with different densities of I_KD current and I_TRPM8 in response to the cooling stimulus depicted at the bottom. The Kv1.1–1.2 and TRPM8 maximum conductance configurations are indicated at the left (Kv1.1–1.2) and top (TRPM8) in mS/cm². Note the shift of the thermal threshold to higher temperatures induced by a reduction of I_KD density (C) and the response to cold induced in a cold-insensitive neuron when I_KD density is reduced, even with a very low density of I_TRPM8 (D).

Figure 11.

Systematic exploration of g_KD and g_TRPM8 parameters. A, Basal firing (Hz), thermal threshold (°C), and maximum firing rate (Hz) at different densities of g_TRPM8 and g_KD. Top and bottom rows represent parameter sets 1 and 2, respectively, from Figure 10 and Table 3. The points indicated as a, b1, b2, c1, and c2 are the parameter combinations used in Figure 10. Black represents complete absence of action potentials. B, Average thermal threshold and maximum firing rate of the model with the g_KD and g_TRPM8 combinations that were used in Figure 10. These data are the mean value of five combinations of the remaining free parameters of the model (Fig. 10; Table 3). p values were obtained using a t test for related samples. ***p < 0.001 in both cases.

Figure 12.

Hypothetical model of cold detection mechanisms and thermal sensitivity of primary sensory neurons in sham and CCI conditions. Left, Schematic representation of cold detection mechanisms in primary sensory neurons under physiological conditions. Boxes in nerve endings represent the ion channels that are involved in the detection of cold stimuli by the functional subtypes of peripheral sensory neurons. The temperature ranges in which these nerve endings would be excited by cold are shown by the colored thermometers at left of each box. These neurons may have excitatory (+) or inhibitory (−) (indirect) actions on higher-order neurons of central sensory pathways, leading ultimately to distinct cold and cold-induced pain sensations. The size of labels reflects the relative density of these channels in the different subclasses of neurons. Right, Schematic representation of these mechanisms in injured mice, emphasizing the functional variations in response to peripheral nerve damage. The relative change of the size of the labels reflects the expected changes in functional expression levels for the different ion channels after injury. After axonal damage, the reduction in the functional expression of I_KD would recruit polymodal nociceptors normally activated by noxious cold temperatures inducing pain that will respond to mild cold temperatures in CCI animals (red arrows indicate this transition). HT-CSNs will respond to innocuous cold temperatures signaling cold discomfort, represented by the cyan neuron (cyan arrows indicate this transition). LT-CSNs expressing low levels of I_KD would remain unaffected. Thus, after injury, the reduction in the functional expression of I_KD increases the cold sensitivity of HT-CSNs signaling cold discomfort and recruits nociceptors normally activated by extremely cold temperatures that cause pain.