Gadolinium inhibits group III but not group IV muscle afferent responses to dynamic exercise

Abstract

Dynamic exercise has been shown to stimulate rapidly both group III and IV muscle afferents. The often rapid (i.e. 2 s) onset latencies of the group IV afferents is particularly surprising because these unmyelinated afferents are thought to respond to the gradual accumulation of metabolites signalling a mismatch between blood/oxygen demand and supply in exercising muscles. One explanation for the rapid onset to exercise by group IV afferents is that they are mechanosensitive, a concept that has been supported by the finding that these afferents were stimulated by vasodilatation induced by injection of vasoactive drugs. We therefore examined in decerebrated cats the effect of gadolinium, a blocker of mechanogated channels, on the responses of group III and IV muscle afferents to dynamic exercise induced by electrical stimulation of the mesencephalic locomotor region. We found that gadolinium (10 mm; 1 ml) injected into the abdominal aorta had no significant effect (P > 0.05) on the responses of 11 group IV afferents to dynamic exercise. In contrast, gadolinium markedly attenuated the responses of 11 group III afferents to exercise (P < 0.05). Our findings suggest that group IV afferents are not responding to a mechanical stimulus during exercise. Instead their rapid response to dynamic exercise might be caused by a chemical substance whose concentration is directly proportional to blood flow, which increases in the skeletal muscles when they are dynamically exercising.

The exercise pressor reflex is believed to be evoked by both mechanical and metabolic stimuli arising in statically contracting muscles (Mitchell et al. 1983). These stimuli, in turn, activate group III and IV muscle afferents to elicit increases in sympathetic outflow, mean arterial pressure, heart rate and contractility (Coote et al. 1971; Coote & Pérez-González, 1970; McCloskey & Mitchell, 1972). Many of the discharge properties of group III and IV muscle afferents have been described (Kaufman & Forster, 1996). One property that seems to distinguish group III from group IV afferents is their responsiveness to mechanical stimuli. For example, group III afferents, whose axons are thinly myelinated, usually respond briskly within a second or two at the onset of static contraction. Moreover they respond to stretch as well as to non-noxious probing of their receptive fields (Kaufman et al. 1983). In contrast, group IV afferents, whose axons are unmyelinated, respond occasionally with an impulse at the onset of static contraction, but usually discharge most of their impulses 5–30 s latter. Moreover, group IV afferents do not respond to either stretch or non-noxious probing of their receptive fields; they usually require noxious pinching or squeezing of their receptive fields to be activated by mechanical stimulation (Paintal, 1960; Kaufman et al. 1983).

The difference in mechanical sensitivity between group III and IV muscle afferents became apparent when decerebrated cats were made to exercise on a treadmill. Although both types responded to dynamic exercise, group III afferents often discharged in synchrony with the contraction phase of the step cycle, whereas group IV afferents rarely did so (Pickar et al. 1994; Adreani et al. 1997; Hayes et al. 2006). The synchronous discharge by group III afferents occurred even when oxygen consumption of the exercising triceps surae muscles was increased by only two and a half fold, a level which is about 25% of maximum (Adreani et al. 1997). These findings raised the possibility that mechanical stimuli arising from low levels of muscle contraction were sufficient to evoke the exercise pressor reflex. Such a finding would be surprising because the reflex was commonly thought to serve solely as a mechanism correcting a mismatch between blood/oxygen supply and demand in exercising muscles. Typically, a mismatch of this nature would be expected to occur at high levels of tension development or when the arterial supply to the muscles is occluded.

Gadolinium, a trivalent lanthanide, has proven to be a useful tool with which to block the discharge of group III mechanoreceptors. When gadolinium was injected into the arterial supply of the triceps surae muscles of either cats or rats it was found to attenuate the pressor reflex response to static contraction by about half (Hayes & Kaufman, 2001; Matsukawa et al. 2006). In addition, injection of gadolinium into the arterial supply of the triceps surae muscles of either cats (Hayes & Kaufman, 2001; Matsukawa et al. 2006) or rats (Smith et al. 2005) has been shown to attenuate the pressor responses to tendon stretch, a purely mechanical stimulus which selectively activates group III afferents but has no effect on the discharge of group IV afferents (Kaufman et al. 1983). In contrast, gadolinium had no effect on the pressor reflex evoked by injection of capsaicin into the arterial supply of the triceps surae muscles (Hayes & Kaufman, 2001). Capsaicin is a TRPV1 agonist that stimulated about 75% of the group IV muscle afferents and 25% of the group III afferents tested (Kaufman et al. 1982, 1983). In electrophysiological experiments, gadolinium was found to markedly attenuate the responses of group III afferents to static contraction and tendon stretch, but did not attenuate the responses to capsaicin of the few group III afferents stimulated by this agent (Hayes & Kaufman, 2001). In addition, gadolinium had no effect on the responses of group IV afferents to static contraction or to capsaicin (Hayes & Kaufman, 2001).

The effect of gadolinium on the responses of group III and IV muscles to dynamic exercise is not known. To shed light on this issue, we have recorded the impulse activity of group III and IV afferents innervating the triceps surae muscles while decerebrated cats dynamically exercised on a treadmill. Exercise was initiated by electrical stimulation of the mesencephalic locomotor region. We tested the hypothesis that gadolinium would attenuate the responses of group III but not group IV afferents to dynamic exercise.

Methods

General

The Institutional Animal Care and Use Committee of the Penn State College of Medicine approved all procedures. Thirty-two adult cats, weighing between 2.5 and 4.5 kg, were anaesthetized by inhalation of a mixture of isofluorane (5%) and oxygen. The trachea was cannulated, and the lungs were ventilated with the anaesthetic gas mixture. A common carotid artery and an external jugular vein were cannulated for monitoring blood pressure and administering fluids, respectively. Arterial blood pressure was measured by connecting the carotid arterial cannula to a Statham P23 XL transducer. Arterial , and pH were measured periodically (model ABL 700 Series, Radiometer) and were maintained within normal limits either by adjusting ventilation or by administering sodium bicarbonate (8.5% IV). Before the decerebration procedure, dexamethasone (4 mg) was injected intravenously to reduce swelling of the brainstem. The cat was then placed in a Kopf stereotaxic frame and spinal unit that were located over a treadmill. A precollicular-postmamillary decerebration was performed. The gaseous anaesthetic was gradually discontinued, and the lungs were ventilated with room air.

A lumbosacral laminectomy was performed to expose the L₆ to S₂ spinal roots. The left hindlimb was fixed in place at the ankle and knee by clamps, and the left triceps surae muscles, calcaneal tendon and sciatic nerve were exposed. The tendon was severed from the calcaneal bone, attached to a force transducer (model FT-10, Grass Technologies/Astro-Med Inc., West Warwick, RI, USA), and stretched with a rack and pinion so that it developed a resting tension of 150 g. The left peroneal, sural, gluteal, femoral, and obturator nerves, as well as the muscular branch of the sciatic nerve, were cut. A small incision was made in the skin overlying the right lateral gastrocnemius muscle and two right-angled electromyogram (EMG) electrodes (Grass Technologies) were implanted and sutured into the muscle itself. The EMG activity of the right gastrocnemius muscle was amplified (model P511, Grass Technologies), filtered (0.1–1.0 kHz), and recorded with Spike2 (Cambridge Electronic Design, Cambridge, UK) data acquisition system.

The following procedure allowed us to inject gadolinium into the arterial supply of the left triceps surae muscles (Fig. 1). First the sacral artery which perfuses the tail was ligated. A catheter with its tip pointing towards the heart was passed into the right femoral artery. A snare was placed around the abdominal aorta, which when tightened allowed us to inject fluid into the left femoral artery from the right femoral artery. This was checked in every cat by injecting saline into the catheter in the right femoral artery and seeing blood exit the left femoral artery, leaving it clear. The volume of saline needed to clear the left femoral artery, which was usually 0.15–0.2 ml, was used to flush the gadolinium injectate.

Figure 1. A catheter was inserted into the right femoral artery, advanced to the abdominal aorta and secured in place with sutures.

The sacral artery was ligated and a snare was placed around the abdominal aorta leaving the left femoral artery as the only route for the gadolinium injectate (as indicated by the arrows).

Dynamic exercise

Locomotion was evoked by electrical stimulation (40 Hz; 0.5 ms; 40-110 μA) of the mesencephalic locomotor region with a monopolar stainless steel electrode (SNEX-300, Rhodes), which was stereotaxically positioned 5 mm lateral to the midline of the brain, 2 mm caudal to the sulcus between the superior and inferior colliculi, and 2 mm below the surface of the midbrain. While the treadmill was hand driven at a speed of 0.40 m s⁻¹ (24 m min⁻¹), the stimulating electrode was lowered in 0.5 mm increments until locomotion arising from the four limbs was observed. Locomotion was monitored by measuring the tension developed by the left triceps surae muscles and the EMG from the right triceps surae muscles.

Recording single-unit activity from groups III and IV afferents

We recorded the impulse activity of individual group III and IV triceps surae muscle afferents from the distal cut end of the left L7 or S1 dorsal roots. The neural signals were passed through a high-impedance probe (Grass HIP511), amplified (Grass P511), and filtered (0.1–3 kHz band pass). The action potentials were displayed on a monitor as well as on a storage oscilloscope (Hewlett-Packard). The receptive field of an afferent was identified as being in the triceps surae muscles if a burst of impulses were discharged in response to either noxious or non-noxious probing of this muscle group. Noxious probing consisted of vigorously pinching the muscles with the fingers, whereas non-noxious probing consisted of either gently stroking the triceps surae with a blunt rod or gently squeezing the muscles with the fingers.

We classified afferents as either group III or group IV by their conduction velocities. Afferents with conduction velocities between 2.5 and 30 m s⁻¹ were classified as group III, and afferents with conduction velocities of < 2.5 m s⁻¹ were classified as group IV. We calculated conduction velocity by measuring the conduction time (Fig. 2) and distance from a stimulating electrode placed under the tibial nerve close to its exit from the triceps surae muscles and the recording electrode placed under the dorsal root filament. The criterion for a response to dynamic exercise by a group III or IV afferent was an increase 0.2 impulses (imp) s⁻¹.

Figure 2. Measurement of conduction time from the stimulating electrode placed under the tibial nerve and the recording electrode placed under the L₇ dorsal root.

Conduction time was 17 ms and the conduction distance was 139 mm, resulting in a calculated conduction velocity of 8.2 m s⁻¹, which was in the group III range.

Protocols

Once we identified a group III or IV afferent with its receptive field in the triceps surae muscles and established its resting level of activity, we recorded the response of the afferent to dynamic exercise before and after injecting gadolinium (10 mm; 1 ml) into the popliteal artery. We recorded the afferent activity for 1 min before exercise, during the exercise bout, and for 1 min immediately after the exercise bout. The snare placed around the abdominal aorta was first tightened and then gadolinium (10 mm; 1 ml) was injected into the right femoral artery. The snare around the aorta was released after a 15 min period and the left hindlimb was allowed to be perfused for another 45 min before the afferents’ response to dynamic exercise was determined. Consequently, we waited 60 min for gadolinium to exert its effect on each afferent's responses to exercise.

In a separate group of five cats, we measured interstitial concentrations of ATP before and after administering gadolinium (10 ml; 0.2 ml). Interstitial fluid was collected by microdialysis. We manufactured microdialysis probes by gluing both ends of a 4 cm length of capillary microdialysis membrane (0.20 mm in diameter, with a 13KD molecular cutoff) into nylon tubing. The nylon tubing was attached to a Luer tip adapter stub that connected the probe and the perfusate-filled syringe. Each cat had four microdialysis fibres placed in its triceps surae muscles; the fibres were separated by approximately 0.5–1 cm. The probes were inserted into the muscles via a 20-gauge cannula inserted parallel to muscle fibre orientation. The insertion and exit points were approximately 6 cm apart. The microdialysis probe was threaded through the internal lumen of the needle. The needle was withdrawn, leaving the membrane in place. The Luer tip adapter stub was attached to a syringe for administration of saline through a perfusion pump (model 402, CMA, Microdialysis, Solna, Sweden) at 5 μl min⁻¹. We inserted the microdialysis probes and then waited 2 h for equilibration. ATP concentrations in dialysate fluid were measured by chemiluminescence using a luciferin–luciferase assay. At the conclusion of the experiment, the cat was humanely killed with an overdose of pentobarbital followed by an injection of saturated KCl solution.

Data analysis

Afferent activity is expressed as impulses per second and was analysed using Spike2 software. The impulse activity of no more than two afferents was analysed from any single filament. When activity appeared to arise from more than two afferents, the filament was either split or discarded. The tension–time index (Perez-Gonzalez, 1981) was calculated step by step by integrating the area between the tension trace and the baseline level (Spike2). All values are expressed as means ±s.e.m. Two-by-two repeated-measures ANOVA followed by Tukey's post hoc tests were used to determine statistical significance. The criterion for statistical significance was set at P < 0.05.

Results

Group III afferents

We recorded the impulse activity of 18 group III afferents with receptive fields in the left triceps surae muscles (conduction velocity: 12.1 ± 1.6 m s⁻¹; range: 2.8–22.0 m s⁻¹). Each of the 18 responded to either non-noxious probing or stretch of the triceps surae muscles.

Dynamic exercise before Gd³⁺

Eleven of the 18 group III afferents (conduction velocity: 11.4 ± 2.2 m s⁻¹; range: 2.8–22.0 m s⁻¹) responded to dynamic exercise (Figs 3, Figs 4 and 5). The seven afferents that did not respond to dynamic exercise were discarded. Each of the 11 afferents responding to exercise increased their discharge within the first 2 s of exercise and discharged at a rate higher than baseline for the entire exercise period (Fig. 3). The mean onset latency, which was calculated from the onset of tension development by the left triceps surae muscles, was 0.31 ± 0.09 s (n= 11). On average, group III afferents responding to dynamic exercise discharged 70 ± 3% (n= 11) of their impulses during the contraction phase of the step cycle, a value that was significantly greater than the percentage of impulses discharged during the relaxation phase of the step cycle (P < 0.05). Two of the 11 group III afferents were silent before the start of exercise, whereas the remaining nine discharged at 0.6 imp s⁻¹ or less. The tension–time index (TTI) for dynamic exercise before gadolinium averaged 25.7 kg s (n= 11).

Figure 3. Gadolinium (10 mm; 1 ml) prevented the response of a group III afferent to dynamic exercise.

T, tension developed by the exercising triceps surae muscles.

Figure 4. Mean impulse activity in impulses s⁻¹ of group III and group IV muscle afferents before and 60 min after 1.0 ml of 10 mm gadolinium.

The first filled bar represents 1 min of baseline activity, the open bar represents dynamic exercise and the second filled bar represents the 1 min after exercise. The horizontal bracket represents a significant difference between the response to dynamic exercise before gadolinium and the response 60 min after gadolinium injection. Asterisks represents a significant difference from baseline and post exercise activity (P < 0.05, n= 11).

Figure 5. Histogram of a single group III (top) and single group IV (bottom) muscle afferent before and 60 min after gadolinium injection.

The vertical bars represent afferent activity in 2 s bins. The horizontal bar represents the period of dynamic exercise in seconds. A–D in bottom histogram refer to original tracings shown in Figure 6.

Dynamic exercise after Gd³⁺

We injected gadolinium (Gd³⁺) into the abdominal aorta, waited 60 min and then re-examined the effects of dynamic exercise on the discharge of the 11 group III afferents previously responsive to exercise. Gd³⁺ attenuated the responses of each of the group III afferents to exercise (Figs 2 and 3), an effect that was highly significant (P < 0.001) on average (n= 11). The percentage of impulses discharged during the contraction phase of the step cycle (66 ± 3%) after Gd³⁺ was not different from the percentage of impulses discharged during the contraction phase of the step cycle before Gd³⁺ was given (P > 0.05). Likewise, the TTI after Gd³⁺ (28.2 kg seconds) was not different from the TTI before Gd³⁺ (P > 0.05).

Group IV afferents

We recorded the impulse activity of 16 group IV afferents with receptive fields in the left triceps surae muscles (conduction velocity: 1.7 ± 0.1 m s⁻¹; range: 1.2–2.4 m s⁻¹). None of the 16 group IV afferents responded to tendon stretch. Non-noxious probing of the triceps surae muscles stimulated only 1 of the 16 group IV afferents, whereas noxious probing stimulated each of them.

Dynamic exercise before Gd³⁺

Eleven of the 16 group IV afferents were stimulated by dynamic exercise (conduction velocity: 1.7 ± 0.1 m s⁻¹; range: 1.2–2.4 m s⁻¹; Fig. 4). The five group IV afferents that did not respond to dynamic exercise were discarded. Each of the 11 afferents responding to exercise increased their discharge within the first 6 s of exercise and discharged at a rate higher than baseline for the entire exercise period. The mean onset latency, which was calculated from the onset of tension development by the left triceps surae muscles, was 2.1 ± 0.4 s (n= 11), a value that was significantly longer P < 0.001) than the mean onset latency of the group III afferents responding to exercise (n= 11). On average, the 11 group IV afferents responding to dynamic exercise discharged 58 ± 2% of their impulses during the contraction phase of the step cycle, a value that was not significantly different from the percentage of impulses discharged during the relaxation phase of the cycle (P > 0.05). Two of the 11 group IV afferents were silent before the start of exercise, whereas the remaining nine discharged at 0.2 imp s⁻¹ or less. The tension–time index for dynamic exercise before gadolinium averaged 39.9 kg s (n= 11).

Dynamic exercise after Gd³⁺

We injected gadolinium (Gd³⁺) into the abdominal aorta, waited 60 min and then re-examined the effects of dynamic exercise on the discharge of the 11 group IV afferents previously responsive to exercise. Gd³⁺ had no effect on the responses to dynamic exercise of five of the group IV afferents, appeared to increase the responses of two, and slightly decreased the responses of the remaining four. On average, Gd³⁺ had no effect on the responses of the group IV afferents to dynamic exercise (P > 0.05; n= 11; Figs 4, 5 and 6). The percentage of impulses discharged during the contraction phase of the step cycle (56 ± 2%) after Gd³⁺ was not different from the percentage of impulses discharged during the contraction phase of the step cycle before Gd³⁺ was given (P > 0.05). Likewise, the TTI after Gd³⁺ (38.6 kg s) was not different from the TTI before Gd³⁺ (P > 0.05).

Figure 6. Recording of the activity of a group IV muscle afferent at rest and during dynamic exercise before (A and B) and after (C and D) gadolinium injection.

Note that A, B, C and D in this figure correspond to the time points depicted by the same letters in the previous figure. AP represents action potentials discharged by the group IV afferent; EMG represents the right leg electromyogram; T represents the left triceps surae muscle tension in kg; and BP represents the arterial blood pressure in mmHg. Note the alternating and synchronized activity of the left leg muscle tension and the right leg EMG representing rhythmical dynamic exercise; horizontal bar indicates 1 s.

Gd³⁺ and the hemodynamic responses to MLR stimulation

Gadolinium had no significant effect (P > 0.05; n= 17) on the pressor-cardioaccelerator responses to stimulation of the MLR, the technique used to induce dynamic exercise. Specifically, before gadolinium injection, MLR stimulation increased mean arterial pressure from 129 ± 8 to 167 ± 8 mmHg and heart rate from 139 ± 5 to 151 ± 5 bpm. After gadolinium MLR stimulation increased mean arterial pressure from 127 ± 8 to 161 ± 10 mmHg and heart rate from 141 ± 7 to 155 ± 7 bpm.

Effect of Gd³⁺ on interstitial concentrations of ATP

In five cats, we sampled interstitial fluid with microdialysis in order to measure ATP release during dynamic exercise both before and after Gd³⁺ injections. The effect of Gd³⁺ on interstitial ATP concentrations were complex. Specifically, Gd³⁺ significantly decreased baseline concentrations of ATP (P < 0.05; n= 5; Fig. 5); likewise it significantly decreased ATP concentrations during dynamic exercise (P < 0.05; n= 5; Fig. 7). Nevertheless, Gd³⁺ had no effect on the increase in ATP concentrations induced by dynamic exercise, averaging 0.17 ± 0.08 μm before Gd³⁺ and 0.13 ± 0.05 μm (P > 0.05) after Gd³⁺. The TTI before Gd³⁺ averaged 37.0 ± 7.7 kg s and the TTI after Gd³⁺ averaged 34.9 ± 7.5 kg s; P= 0.37).

Figure 7.

Mean ATP concentration at rest (filled bars) and during dynamic exercise (open bars) Note that the absolute difference between ATP concentration at rest and during dynamic exercise was not significantly different (P > 0.05) before and 60 min after injecting gadolinium, even though the baseline concentration drops 60 min after giving gadolinium. Asterisk denotes a significant difference between baseline and dynamic exercise concentrations.

Discussion

We have found that gadolinium greatly attenuated the responses of group III afferents to dynamic exercise. In contrast, gadolinium had no overall effect on the responses of group IV afferents. In our experiments, we induced dynamic exercise by electrically stimulating the mesencephalic locomotor region (MLR) in decerebrated cats. Dynamic exercise evoked by stimulation of the MLR in decerebrated unanaesthetized cats is quite similar to dynamic exercise in intact, conscious cats. An important feature of this preparation is that activation of the MLR is believed to recruit alpha motoneurons from slow to fast (Hoffer et al. 1987a,b; Tansey & Botterman, 1996a). This order of recruitment appears to be identical to the order of recruitment of alpha motoneurons in intact conscious cats performing treadmill exercise (Hoffer et al. 1981, 1987a,b). Moreover, the discharge patterns of alpha motoneurons activated by stimulation of the MLR in decerebrated unanaesthetized cats are almost identical to the discharge patterns of these motoneurons in intact conscious cats (Tansey & Botterman, 1996b).

Our finding that gadolinium had no effect on the cardiovascular responses to MLR stimulation is not surprising. Specifically, gadolinium blocked input from group III afferents innervating only one muscle group, the triceps surae, whereas all four limbs and trunk muscles were exercising. In addition, gadolinium had no effect on the cardiovascular responses to central command, which was induced by MLR stimulation. Finally, any exercise induced afferent activity arising from cut dorsal roots innervating the triceps surae muscles could not have evoked a reflex pressor response regardless of whether it was blocked or not blocked by gadolinium.

Gadolinium probably has multiple mechanisms and sites of action on neurons, and these are likely to be concentration dependent. A previous study may shed light on the concentration that may have existed at the endings of the thin fibre muscle afferents investigated in our experiments. Specifically, Gd³⁺ at a concentration of 450 μm blocked transmission across the mouse neuromuscular junction (Molgo et al. 1991). We injected 1 ml of 10 mm Gd³⁺ into the abdominal aorta of cats. Considerable dilution must have occurred to its concentration by the time the lanthanide reached the endings of the group III and IV afferents in the triceps surae muscles. To the extent that Gd³⁺ has the same effect on the neuromuscular junction in mice as it does in cats, we can speculate that its effective concentration at the endings of group III afferents in the triceps surae muscles was less than 450 μm because the MLR stimulation still elicited locomotion after gadolinium injection. In other words, Gd³⁺ appeared to have no effect on the neuromuscular junction in our experiments.

In previous experiments, we found that group IV afferents were more responsive to injection of vasodilator agents into the arterial supply of the triceps surae muscles of cats than were group III afferents (Haouzi et al. 1999). The responsiveness of the group IV afferents to vasodilatation was attributed to mechanical stimulation by the expanding vessels within the substance of the triceps surae muscles. Electron microscopic studies have revealed that group IV afferent endings are found in the walls of small intramuscular vessels, including arterioles, venules and lymphatics, whereas group III afferent endings are found often in connective tissue (von During & Andres, 1990). Consequently, the location of the endings of group IV afferents seemed to be ideally located to signal vascular distension, induced by the increase in blood flow occurring in dynamically exercising muscles.

The suggestion that group IV afferents are stimulated mechanically to respond to vascular distension is surprising because other mechanical stimuli are not effective in activating these unmyelinated afferents. For example, group IV afferents rarely respond to tendon stretch (Kaufman et al. 1983). Likewise, non-noxious probing of their receptive fields within the substance of the triceps surae muscles rarely stimulates them (Kaufman et al. 1983). We were prompted therefore to determine the effect of gadolinium on the responses of group IV afferents to dynamic exercise because it is considered to be the agent of choice to block mechanogated channels (Hamill & McBride, 1996). We found that gadolinium had no effect on the responses of the group IV afferents to dynamic exercise. In contrast, we found that gadolinium attenuated the responses of the group III afferents, which frequently display substantial mechanosensitivity.

Gadolinium has been found to block responses to mechanical stimuli in a wide variety of tissues including plants and animals (Hamill & McBride, 1996). Consequently we think that the probability is high that gadolinium would have blocked at least in part the responses of group IV afferents to dynamic exercise if these unmyelinated afferents displayed substantial mechanosensitivity. An alternative and more likely mechanism for the stimulation of group IV afferents by vasodilator agents in both our previous study (Haouzi et al. 1999) and our present one may have been the release of nitric oxide and/or cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic acid induced by the increase in blood flow through the vessels during dynamic exercise (Rotto & Kaufman, 1988; Koller et al. 1994; Gao et al. 2008).

The mechanism of action whereby gadolinium decreased the responses of group III afferents to dynamic exercise is not known. Recently, evidence was presented by Li et al. (2008) attributing this mechanism to a gadolinium-induced decrease in interstitial concentrations of ATP, which stimulates purinergic 2 receptors. Although we cannot rule out this mechanism, we think that it is unlikely to explain our finding that gadolinium attenuated the responses of group III afferents to dynamic exercise. Specifically, Group III afferents were not stimulated by purinergic 2 receptor agonists (Hanna & Kaufman, 2004) but their responses to dynamic exercise were markedly decreased by gadolinium. In contrast, group IV afferents were stimulated purinergic 2 receptor agonists (Reinöhl et al. 2003; Hanna & Kaufman, 2004), but their responses to dynamic exercise were not affected by gadolinium in our experiments. The possibility exists, nevertheless, that the decrease in interstitial ATP induced by gadolinium may have reduced the sensitivity of group III afferents to contraction (Kindig et al. 2006, 2007). However, the gadolinium-induced decrease in interstitial ATP concentrations in our experiments was about 20 times smaller than the gadolinium-induced decrease in ATP concentration in the experiments by Li et al. (2008), a difference which was probably caused by differences in the tension–time indices between the two experiments. Interstitial ATP concentrations have been shown to be directly proportional to the tension–time index (Li et al. 2003) and the tension–time index in the experiments of Li et al. (2008) were 10-fold greater than that in our experiments.

Any interpretation of our findings should be done with the following limitations in mind. Although the motoneuron input to the triceps surae muscles in our experiments was probably the same as that occurring during dynamic exercise, the muscles were fixed in place and therefore could not change their length. Consequently the muscle length–tension relationship during MLR stimulation in our experiments may not have been the same as that occurring during naturally performed exercise. Moreover, this possible change in the length–tension relationship might have had an effect on the pattern of blood flow through the muscle during exercise as well as an effect on spindle discharge.

The onset latency of the group IV afferents responding to dynamic exercise in our experiments averaged only 2.1 s and is almost identical to that reported previously by our laboratory (Adreani et al. 1997). The rapid response of a muscle afferent is attributed frequently by a process of subtraction to a mechanical stimulus in part because the brief onset latency of the afferent to exercise does not allow much time for the formation of metabolites, especially those signalling a mismatch between blood/oxygen supply and demand. The rapid response of group IV afferents to exercise as well as their responses to vasodilatation (Haouzi et al. 1999) prompted us to determine if gadolinium, an established blocker of mechanogated channels, would attenuate the responses of group IV afferents to dynamic exercise. Gadolinium had no effect on the responses of group IV afferents, raising the possibility that these unmyelinated muscle afferents were responding to a chemical released by an increase in blood flow. Although this possibility is an attractive one, we cannot at this point rule out the alternative, namely that a mechanogated channel that is not blocked by gadolinium was responsible for the rapid response of group IV afferents to dynamic exercise that was seen in our experiments.