Acid-sensing ion channel subtype 3 function and immunolabelling increases in skeletal muscle sensory neurons following femoral artery occlusion

Abstract

Sympathetic nerve activity and arterial blood pressure responses to static hindlimb muscle contractions are greater in rats with femoral arteries that were previously ligated (24-72 h earlier) than in control rats. Studies further demonstrate that acid-sensing ion channel subtype 3 (ASIC(3)) in thin-fibre muscle afferents contributes to the amplified reflex muscle responses observed in occluded rats, probably due to enhanced ASIC(3) expression in muscle sensory neurons. The purpose of this study was to characterize acid-induced current with activation of ASIC(3) in dorsal root ganglion (DRG) neurons of control rats and rats with 24 h of femoral occlusion using whole-cell patch clamp methods. Also, immunohistochemistry was employed to examine existence of ASIC(3) expression in DRG neurons of thin-fibre afferents. DRG neurons from 4- to 6-week-old rats were labelled by injecting the fluorescence tracer DiI into the hindlimb muscles 4-5 days prior to the recording experiments. The results of this study show that ∼90% of current responses evoked by pH 6.7 in DRG neurons innervating the hindlimb muscles are ASIC(3)-like. The peak current amplitude to pH 6.7 is significantly attenuated with application of rAPETx2, a specific ASIC(3) antagonist. In addition, ASIC(3)-like current responses to pH 6.7 are observed in small, medium and large DRG neurons, and size distribution of DRG neurons is similar in control and occluded animals. However, the peak current amplitude of DRG neuron response induced by ASIC(3) stimulation is larger in occluded rats than that in control rats. Moreover, the percentage of DRG neurons with ASIC(3)-like currents is greater after arterial occlusion compared with control. Furthermore, results from double immunofluorescence experiments show that femoral artery occlusion mainly augments ASIC(3) expression within DRG neurons projecting C-fibre afferents. Taken together, these data suggest that (1) the majority of current responses to pH 6.7 are ASIC(3)-like in DRG neurons with nerve endings in the hindlimb muscles, (2) a greater acid-induced current responding to pH 6.7 develops when hindlimb arterial blood supply is deficient under ischaemic conditions, and (3) increased ASIC(3) expression is largely observed in thin C-fibres of DRG neurons after hindlimb ischaemia.

Figure 1. Whole-cell patch clamp methods were employed to study acid-induced currents to pH 6.7 in DRG neurons of control rats and rats with 24 h of femoral artery ligation

Two different ASIC currents were distinguished on the basis of their inactivation kinetics. One type of current exhibited a slow inactivating rate (larger τ value) and was only expressed in small DRG neurons with a cell capacitance of <20 pF. This group of currents represents typically an ASIC_1a response to pH 6.7. Another type of current had rapid inactivation kinetics (smaller τ value), and the currents were observed in small, medium and large DRG neurons with cell capacitance ranging from 10 to 60 pF. Those currents are typically observed as recombinant ASIC₃ channels are activated. Note that there were no significant differences in inactivating rate and cell capacitance for DRG neurons of control rats and occluded rats.

Figure 2. Effects of blocking ASIC_1a and ASIC₃ homomeric channels on acid-evoked currents in DRG neurons innervating the hindlimb muscles

Toxins PcTx1 and rAPETx2 were used as specific antagonists to ASIC_1a and to ASIC₃, respectively. A, original traces of ASIC_1a currents response to pH 6.7 with prior application of rAPETx2 and PcTx1. B, averaged data showing peak amplitudes of ASIC_1a currents. Toxin PcTx1 (20 nm) significantly inhibited current responses induced by pH 6.7 in DRG neurons exhibiting ASIC1a currents. *P < 0.05 vs. pH 6.7 alone. However, rAPETx2 had no significant effects on this type of current in DiI- labelled neurons. C, typical ASIC₃-like current response to pH 6.7 with prior exposure to rAPETx2 and PcTx1. D, averaged data showing that a prior application of rAPETx2 (1 μm) significantly attenuated peak amplitudes of currents evoked by pH 6.7 in DRG neurons that displayed ASIC₃-like currents. PcTx1 had negligible effects on this type of currents. *P < 0.05 vs. pH 6.7 alone. Note that the inhibitory effects of PcTx1 on ASIC_1a currents and rAPETx2 on ASIC₃-like currents were both reversible.

Figure 3. Size distribution of DRG neurons responding to pH 6.7

Acid-evoked current responses with activation of ASIC₃ were recorded in DiI-labelled DRG neurons of control rats and rats with 24 h of femoral artery occlusion. No differences between both experimental groups were observed for the average size of DRG neurons that responded to pH 6.7.

Figure 4. Effects of femoral arterial occlusion on ASIC₃-like currents response to pH 6.7

A, original traces of ASIC₃-like current responsive to pH 6.7 recorded in DRG neurons innervating hindlimb muscles in control and after 24 h arterial occlusion. B, averaged data show mean current density of ASIC₃-like currents activated by pH 6.7. Twenty-four hours of arterial occlusion induced a larger current density compared with control. *P < 0.05 vs. control. C, percentage of DRG neurons innervating muscle with ASIC₃-like currents responsive to pH 6.7. A larger percentage of DRG neurons with ASIC₃-like currents were observed in 24 h of arterial occlusion than in control. *P < 0.05 compared with control.

Figure 5. Current clamp recordings were performed on DiI-labelled neurons exhibiting ASIC₃-like currents

A, original traces. Top: spontaneous action potentials (AP). Middle: slight acidification using a pH 6.7 solution sufficiently depolarized the membrane to trigger firing. Action potentials are magnified in the inset. Bottom: the presence of rAPETx2 inhibited pH 6.7-evoked depolarization and depressed the AP threshold. B, percentage of recorded DRG neurons with spontaneous APs and APs triggered by pH drops to 6.7 in control and femoral artery occlusion. Averaged data showing that the percentage of neurons with spontaneous APs was similar in the control group compared with the femoral artery ligation group. However, femoral artery ligation increased the number of neurons in which APs could be triggered by a pH drop. *P < 0.05 vs. control.

Figure 6. Immunofluorescence was employed to examine double-labelling for ASIC₃ and peripherin

Peripherin was used to label DRG neurons that project thin C-fibres. Representative photomicrographs show ASIC₃ and peripherin staining in DRG neurons of a control rat (top panel) and an occluded rat (bottom panel). Arrows indicate representative cells positive for both ASIC₃ and peripherin after they were merged. The number of double-labelled DRG neurons is greater in occluded rats than in control rats. Scale bar, 50 μm.

Figure 7. Immunofluorescence was employed to examine double-labelling for ASIC₃ and NF200

Note that NF200 was used to identify A-fibres DRG neurons. Photomicrographs are representative to illustrate staining of ASIC₃ and NF200 in DRG neurons of a control rat (top panel) and an occluded rat (bottom panel). Arrows indicate examples for merged ASIC₃- and NF200-positive cells. No differences in the number of double-stained ASIC₃ and NF200 were observed in DRG neurons of the control and occluded groups. Scale bar, 50 μm.