Upregulation of Ih expressed in IB4-negative Aδ nociceptive DRG neurons contributes to mechanical hypersensitivity associated with cervical radiculopathic pain

Abstract

Cervical radiculopathy represents aberrant mechanical hypersensitivity. Primary sensory neuron's ability to sense mechanical force forms mechanotransduction. However, whether this property undergoes activity-dependent plastic changes and underlies mechanical hypersensitivity associated with cervical radiculopathic pain (CRP) is not clear. Here we show a new CRP model producing stable mechanical compression of dorsal root ganglion (DRG), which induces dramatic behavioral mechanical hypersensitivity. Amongst nociceptive DRG neurons, a mechanically sensitive neuron, isolectin B4 negative Aδ-type (IB4(-) Aδ) DRG neuron displays spontaneous activity with hyperexcitability after chronic compression of cervical DRGs. Focal mechanical stimulation on somata of IB4(-) Aδ neuron induces abnormal hypersensitivity. Upregulated HCN1 and HCN3 channels and increased Ih current on this subset of primary nociceptors underlies the spontaneous activity together with neuronal mechanical hypersensitivity, which further contributes to the behavioral mechanical hypersensitivity associated with CRP. This study sheds new light on the functional plasticity of a specific subset of nociceptive DRG neurons to mechanical stimulation and reveals a novel mechanism that could underlie the mechanical hypersensitivity associated with cervical radiculopathy.

Figure 1. Setup of cervical radiculopathic pain (CRP) model following chronic compression of C7/C8 DRGs.

(a) Schematic illustration of the method for producing a chronic compression of the C7/C8 DRGs in rat. Position and direction of a stainless steel rod inserted into the intervertebral foramen are shown. (b) Magnitude and time course of mechanical hypersensitivity to plantar von Frey hair application are shown in CRP and sham control rats. Note that paw withdrawal mechanical threshold decreased significantly from the 1st day after chronic compression and remained so up to the latest time point tested as compared to controls. (c) Showing the magnitude and time course of thermal hyperalgesia to radiant heat in CRP rats. Note that paw withdrawal thermal latency was significantly reduced till 3 days after operation, as compared to controls. (d) Spontaneous pain developed in CRP rats, as compared to controls. All data are expressed as mean ± S.E.M. *P < 0.05 as compared to their basal levels using repeated measures one-way analysis of variance (ANOVA).

Figure 2. Induction of c-Fos expression and ERK1/2 phosphorylation in the DRG and spinal dorsal horn of the pain pathway following chronic compression of C7/C8 DRGs.

(a) Typical examples of immunoreactivity for c-Fos in the DRG (upper panels) and spinal dorsal horn (lower panels) from control rats and CRP rats at 12 h, 24 h and 48 h post operation. (b) Quantification of the number of Fos-positive small cells in the DRG derived from control and CRP rats at different time points (n = 3 rats per group). (c) Quantification of the number of Fos-positive cells in the superficial and deep layer of spinal dorsal horn from control and CRP rats at different time points (n = 3 rats per group). (d) Typical examples of immunoreactivity for phosphorylated ERK1/2 in the DRG (upper panels, white solid triangle) and spinal dorsal horn (lower panels, white solid triangle) derived from control rats and CRP rats at 12 h post operation. (e) Quantification of phosphorylated ERK1/2 in small DRG neurons from control and CRP rats (n = 3 rats per group). (f) Quantification of phosphorylated ERK1/2 in the superficial layer of dorsal horn from control and CRP rats (n = 3 rats per group). All data are expressed as mean ± S.E.M. *P < 0.05 indicates statistically significant differences between control and CRP rats (ANOVA). Scale bars represent 100 μm in (a) and upper panels in (d), 50 μm in the lower panels in (d).

Figure 3. Membrane property and cell excitability of different subsets of nociceptive DRG neurons in control and CRP rats.

(a) A representative action potential of whole-cell configuration evoked by a depolarizing current injection from control (in black) and from CRP (in red) group. (b) Classification of all small DRG neurons recorded. Note that three subtypes were determined by IB4 staining, shape of action potential (left panels) and type of the afferent. A single electrical stimulation of dorsal root 3 mm away from DRG was applied to distinguish C type afferent from Aδ type. (c) Showing resting membrane potential (RMP), action potential (AP) properties, such as amplitude, half-width, rheobase and threshold as well as repetitive firing in response to current injection in IB4⁻ Aδ-, IB4⁻ C- and IB4⁺ C-type DRG neurons from control and CRP rats. All data are expressed as mean ± S.E.M. *P < 0.05 indicates statistically significant differences between control and CRP rats (ANOVA).

Figure 4. Occurrence of spontaneous activity (SA) in different subsets of nociceptive DRG neurons in CRP rats.

(a) The proportion of cells expressing SA in IB4⁻ Aδ-, IB4⁻ C- or IB4⁺ C-type DRG neurons in control and CRP rats. (b) Typical example of a spontaneous firing of an IB4⁻ Aδ-type neuron from CRP rats at 3d after surgery. (c) Quantitative analysis of SA frequency in IB4⁻ Aδ-type neurons in control and CRP rats. (d) The time course of a typical example of spontaneous firing observed in IB4⁻ Aδ-type neuron from CRP rats showing frequency (grey column), peak amplitude (filled red circle), RMP (filled black rectangle) and duration of SA. (e) Three firing patterns of SA in IB4⁻ Aδ-type neuron from CRP rats are shown: bursting activity (upper panel), periodic discharges (middle panel) and irregular firing (lower panel). *P < 0.05 indicates statistically significant differences between control and CRP rats (ANOVA).

Figure 5. Hypersensitivity to focal mechanical stimulation in IB4⁻ Aδ type neurons from CRP rats.

(a) Focal mechanical stimulation was applied to the somata of IB4⁻ Aδ neurons using a slow-moving heat-polished glass pipette (white arrow). Membrane deflection was observable by every stimulation (about 1/6 diameter of the neuron, 5–7 μm) for 1 s in duration. Response of the cell was recorded by a recording pipette (black arrow). (b) A typical example of the mechanical response in IB4⁻ Aδ neuron from control and CRP rats. Inset is a magnification of response from CRP DRG neurons. (c) Representative examples of mechanically activated property of 3 different IB4⁻ Aδ neurons from control and CRP group, respectively. Note that long-lasting afterdischarge was seen in 62.5% (5 out of 8) of DRG neurons tested in CRP group. (d) Firing frequency evoked by mechanical stimulation was much stronger in IB4⁻ Aδ neurons from CRP rats as compared to that from control rats. (e) Quantitative analysis of duration of the high-frequent discharges to mechanical stimulation in control and CRP group. The durations were determined from the onset of the high-frequent discharges until spike frequency returned to pre-stimulus level. MS: mechanical stimulation. All data are expressed as mean ± S.E.M. *P < 0.05 as compared to control rats and ^#P < 0.05 as compared to basal level (ANOVA).

Figure 6. CRP increases I_h currents in IB4⁻ Aδ-, but not in IB4⁻ C- or IB4⁺ C-type DRG neurons.

(a) I_h current was evoked by hyperpolarizing voltage steps of −50 mV to −120 mV from a holding potential at −60 mV in IB4⁻ Aδ-type DRG neurons. The voltage protocol is shown at the top. (b) I_h current recorded in IB4⁻ Aδ-type DRG neurons was blocked by bath application of Cs²⁺ (1 mM) or ZD7288 (15 μM). (c) Representative examples of I_h recorded in IB4⁻ Aδ-, IB4⁻ C- and IB4⁺ C-type DRG neurons from control (upper panels) and CRP rats (lower panels). (d) Only IB4⁻ Aδ-type neurons showed upregulated I_h currents after CRP operation, which is not seen in C-type neurons. All data are expressed as mean ± S.E.M. *P < 0.05 as compared to control rats.

Figure 7. CRP changed voltage dependence of I_h activation in IB4⁻ Aδ type neuron, but not the reversal potential.

(a) Reversal potential of I_h from control (middle panel) and CRP IB4⁻ Aδ type neurons (lower panel) were achieved by first applying a prepulse to −120 mV to fully activate I_h and then examining the tail currents after repolarization to test potentials from −110 to −50 mV (upper panel). Tail currents were plotted against test potentials. (b) No difference of reversal potential of I_h current was shown between control (black) and CRP (red) group. (c) The activation curve of I_h from control (upper panel) and CRP IB4⁻ Aδ neurons (lower panel) was constructed by measuring tail currents at −120 mV after application of prepulse potentials between −50 to −120 mV (middle inset) and fitted with a Boltzmann equation. (d) The midpoint (V_1/2) for activation of I_h in the CRP IB4⁻ Aδ neurons (red) was shifted 11 mV in the depolarizing direction compared to control neurons (black). (e) The difference of V_1/2 for activation of I_h between two groups was significant. All data are expressed as mean ± S.E.M. *P < 0.05 as compared to control rats.

Figure 8. Increased expression of HCN1 and HCN3, but not HCN2 in IB4⁻ small diameter DRG neurons in CRP rats compared to controls.

(a) Double immunofluorescence staining of DRG neurons with IB4 (red) and HCN1 (green, upper panels), HCN2 (green, middle panels), HCN3 antibody (green, lower panels) in CRP and control rats. (b,d) Quantitative summary from double immunofluorescence experiments showing that in CRP rats, HCN1 (b) and HCN3 (d) immunoreactivity in IB4⁻ small diameter DRG neurons was increased in comparison with control rats. The percentage of IB4⁻ small diameter DRG neurons expressing HCN1 immunoreactivity in all DRG neurons is shown. (c) HCN2 immunoreactivity in IB4⁻ small diameter DRG neurons did not show obvious alteration in CRP rats compared to controls. All data are represented as mean ± SEM. Scale bars represent 50 μm. *P < 0.05 as compared to control rats.

Figure 9. Spontaneous activity (SA) of IB4⁻ Aδ type neurons and its hypersensitivity to mechanical stimulation as well as behavioral mechanical hypersensitivity in CRP rats are I_h dependent.

(a) ZD7288 blocked both SA and mechanical hypersensitivity of IB4⁻ Aδ type neurons from CRP group, without obvious influence of resting membrane potential (n = 3 neurons from 3 rats). Action potential was evoked after tests to determine whether the generation of action potential was compromised. (b,c) Time course of behavioral mechanical hypersensitivity (b) and thermal hyperalgesia (c) from CRP rats before (pre) and after i.t. injection of 3, 10, 30 μg ZD7288 or saline. Note that mechanical hypersensitivity was alleviated by ZD7288 dose-dependently, while thermal hyperalgesia was unaffected (n = 9–10 rats for each group). (d) Spontaneous pain from CRP rats was largely attenuated by ZD7288 as well (n = 9–10 rats for each group). All data are expressed as mean ± S.E.M. *P < 0.05 as compared to control group.