Comparison of murine behavioural and physiological responses after forced exercise by electrical shock versus manual prodding.

NEW FINDINGS: What is the central question of this study? Forced treadmill exercise using electrical shock is the most common technique in rodent exercise studies. Here, we examined how the use of electrical shock during forced treadmill exercise affects behavioural and physiological responses in comparison to a novel non-electrical shock technique. What is the main finding and its importance? In comparison to mice that underwent traditional treadmill running induced by electrical shock, mice that underwent forced running using a novel technique involving gentle prodding to induce running showed: (i) higher locomotor activity; (ii) less anxiety-like behaviour; and (iii) altered exercise-induced muscle pain immediately after exercise. ABSTRACT: Animal models of exercise have been useful to understand underlying cellular and molecular mechanisms. Many studies have used methods of exercise that are unduly stressful (e.g., electrical shock to force running), potentially skewing results. Here, we compared physiological and behavioural responses of mice after exercise induced using a prodding technique that avoids electrical shock versus a traditional protocol using electrical shock. We found that exercise performance was similar for both techniques; however, the shock group demonstrated significantly lower locomotor activity and higher anxiety-like behaviour. We also observed divergent effects on muscle pain; the prodding group showed hyperalgesia immediately after exercise, whereas the shock group showed hypoalgesia. Corticosterone concentrations were elevated to a similar extent for both groups. In conclusion, mice that were exercised without shock generated similar maximal exercise performance, but postexercise these mice showed an increase in locomotor activity, less anxiety-like behaviour and altered muscle pain in comparison to mice that exercised with shock. Our data suggest that running of mice without the use of electrical shock is potentially less stressful and might be a better technique to study the physiological and behavioural responses to exercise.

ASICs are required for immediate exercise-induced muscle pain and are downregulated in sensory neurons by exercise training.

Exercise training is an effective therapy for many pain-related conditions, and trained athletes have lower pain perception compared with unconditioned people. Some painful conditions, including strenuous exercise, are associated with elevated levels of protons, metabolites, and inflammatory factors, which may activate receptors and/or ion channels, including acid-sensing ion channels (ASICs), on nociceptive sensory neurons. We hypothesized that ASICs are required for immediate exercise-induced muscle pain (IEIP) and that exercise training diminishes IEIP by modulating ASICs within muscle afferents. We found high-intensity interval training (HIIT) reduced IEIP in C57BL/6 mice and diminished ASIC mRNA levels in lumber dorsal root ganglia, and this downregulation of ASICs correlated with improved exercise capacity. Additionally, we found that ASIC3 -/- mice did not develop IEIP; however, the exercise capacity of ASIC3 -/- was similar to wild-type mice. These results suggest that ASICs are required for IEIP and that diminishment of IEIP after exercise training correlates with downregulation of ASICs in sensory neurons.NEW & NOTEWORTHY Exercise performance can be limited by the sensations of muscle fatigue and pain transmitted by muscle afferents. It has been proposed that exercise training abrogates these negative feedback signals. We found that acid-sensing ion channels (ASICs) are required for immediate exercise-induced muscle pain (IEIP). Moreover, exercise training prevented IEIP and was correlated with downregulation of ASICs in sensory neurons.

ASIC2 subunits facilitate expression at the cell surface and confer regulation by PSD-95.

Acid-sensing ion channels (ASICs) are Na+ channels activated by changes in pH within the peripheral and central nervous systems. Several different isoforms of ASICs combine to form trimeric channels, and their properties are determined by their subunit composition. ASIC2 subunits are widely expressed throughout the brain, where they heteromultimerize with their partnering subunit, ASIC1a. However, ASIC2 contributes little to the pH sensitivity of the channels, and so its function is not well understood. We found that ASIC2 increased cell surface levels of the channel when it is coexpressed with ASIC1a, and genetic deletion of ASIC2 reduced acid-evoked current amplitude in mouse hippocampal neurons. Additionally, ASIC2a interacted with the neuronal synaptic scaffolding protein PSD-95, and PSD-95 reduced cell surface expression and current amplitude in ASICs that contain ASIC2a. Overexpression of PSD-95 also reduced acid-evoked current amplitude in hippocampal neurons. This result was dependent upon ASIC2 since the effect of PSD-95 was abolished in ASIC2-/- neurons. These results lend support to an emerging role of ASIC2 in the targeting of ASICs to surface membranes, and allows for interaction with PSD-95 to regulate these processes.

Acid-sensing ion channels (ASICs) are differentially modulated by anions dependent on their subunit composition.

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. ASIC1a channels possess intersubunit Cl(-)-binding sites in the extracellular domain, which are highly conserved between ASIC subunits. We previously found that anions modulate ASIC1a gating via these sites. Here we investigated the effect of anion substitution on native ASICs in rat sensory neurons and heterologously expressed ASIC2a and ASIC3 channels by whole cell patch clamp. Similar to ASIC1a, anions modulated the kinetics of desensitization of other ASIC channels. However, unlike ASIC1a, anions also modulated the pH dependence of activation. Moreover, the order of efficacy of different anions to modulate ASIC2a and -3 was very different from that of ASIC1a. More surprising, mutations of conserved residues that form an intersubunit Cl(-)-binding site in ASIC1a only partially abrogated the effects of anion modulation of ASIC2a and had no effect on anion modulation of ASIC3. The effects of anions on native ASICs in rat dorsal root ganglion neurons mimicked those in heterologously expressed ASIC1a/3 heteromeric channels. Our data show that anions modulate a variety of ASIC properties and are dependent on the subunit composition, and the mechanism of modulation for ASIC2a and -3 is distinct from that of ASIC1a. We speculate that modulation of ASIC gating by Cl(-) is a novel mechanism to sense shifts in extracellular fluid composition.

Extracellular chloride modulates the desensitization kinetics of acid-sensing ion channel 1a (ASIC1a).

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. The recent crystallization of ASIC1a identified potential binding sites for Cl(-) in the extracellular domain that are highly conserved between ASIC isoforms. However, the significance of Cl(-) binding is unknown. We investigated the effect of Cl(-) substitution on heterologously expressed ASIC1a current and H(+)-gated currents from hippocampal neurons recorded by whole-cell patch clamp. Replacement of extracellular Cl(-) with the impermeable and inert anion methanesulfonate (MeSO(3)(-)) caused ASIC1a currents to desensitize at a faster rate and attenuated tachyphylaxis. However, peak current amplitude, pH sensitivity, and selectivity were unchanged. Other anions, including Br(-), I(-), and thiocyanate, also altered the kinetics of desensitization and tachyphylaxis. Mutation of the residues that form the Cl(-)-binding site in ASIC1a abolished the modulatory effects of anions. The results of anion substitution on native ASIC channels in hippocampal neurons mirrored those in heterologously expressed ASIC1a and altered acid-induced neuronal death. Anion modulation of ASICs provides new insight into channel gating and may prove important in pathological brain conditions associated with changes in pH and Cl(-).

ASIC2 subunits target acid-sensing ion channels to the synapse via an association with PSD-95.

Acid-sensing ion channel-1a (ASIC1a) mediates H(+)-gated current to influence normal brain physiology and impact several models of disease. Although ASIC2 subunits are widely expressed in brain and modulate ASIC1a current, their function remains poorly understood. We identified ASIC2a in dendrites, dendritic spines, and brain synaptosomes. This localization largely relied on ASIC2a binding to PSD-95 and matched that of ASIC1a, which does not coimmunoprecipitate with PSD-95. We found that ASIC2 and ASIC1a associated in brain, and through its interaction with PSD-95, ASIC2 increased ASIC1a localization in dendritic spines. Consistent with earlier work showing that acidic pH elevated spine [Ca(2+)](i) by activating ASIC1a, loss of ASIC2 decreased the percentage of spines responding to acid. Moreover, like a reduction of ASIC1a, the number of spine synapses fell in ASIC2(-/-) neurons. These results indicate that ASIC2 facilitates ASIC1a localization and function in dendritic spines and suggest that the two subunits work in concert to regulate neuronal function.

ASIC2a and ASIC3 heteromultimerize to form pH-sensitive channels in mouse cardiac dorsal root ganglia neurons.

RATIONALE: Acid-sensing ion channels (ASICs) are Na+ channels that are activated by acidic pH. Their expression in cardiac afferents and remarkable sensitivity to small pH changes has made them leading candidates to sense cardiac ischemia. OBJECTIVE: Four genes encode six different ASIC subunits, however it is not yet clear which of the ASIC subunits contribute to the composition of ASICs in cardiac afferents. METHODS AND RESULTS: Here, we labeled cardiac afferents using a retrograde tracer dye in mice, which allowed for patch-clamp studies of murine cardiac afferents. We found that a higher percentage of cardiac sensory neurons from the dorsal root ganglia respond to acidic pH and generated larger currents compared to those from the nodose ganglia. The ASIC-like current properties of the cardiac dorsal root ganglia neurons from wild-type mice most closely matched the properties of ASIC2a/3 heteromeric channels. This was supported by studies in ASIC-null mice: acid-evoked currents from ASIC3(-/-) cardiac afferents matched the properties of ASIC2a channels, and currents from ASIC2(-/-) cardiac afferents matched the properties of ASIC3 channels. CONCLUSIONS: We conclude that ASIC2a and -3 are the major ASIC subunits in cardiac dorsal root ganglia neurons and provide potential molecular targets to attenuate chest pain and deleterious reflexes associated with cardiac disease.

Acid-sensing ion channel 3 (ASIC3) cell surface expression is modulated by PSD-95 within lipid rafts.

Acid-sensing ion channel 3 (ASIC3) is a H(+)-gated cation channel primarily found in sensory neurons, where it may function as a pH sensor in response to metabolic disturbances or painful conditions. We previously found that ASIC3 interacts with the postsynaptic density protein PSD-95 through its COOH terminus, which leads to a decrease in ASIC3 cell surface expression and H(+)-gated current. PSD-95 has been implicated in recruiting proteins to lipid rafts, which are membrane microdomains rich in cholesterol and sphingolipids that organize receptor/signaling complexes. We found ASIC3 and PSD-95 coimmunoprecipitated within detergent-resistant membrane fractions. When cells were exposed to methyl-beta-cyclodextrin to deplete membrane cholesterol and disrupt lipid rafts, PSD-95 localization to lipid raft fractions was abolished and no longer inhibited ASIC3 current. Likewise, mutation of two cysteine residues in PSD-95 that undergo palmitoylation (a lipid modification that targets PSD-95 to lipid rafts) prevented its inhibition of ASIC3 current and cell surface expression. In addition, we found that cell surface ASIC3 is enriched in the lipid raft fraction. These data suggest that PSD-95 and ASIC3 interact within lipid rafts and that this raft interaction is required for PSD-95 to modulate ASIC3.