Characterization of muscle spindle afferents in the adult mouse using an in vitro muscle-nerve preparation

Abstract

We utilized an in vitro adult mouse extensor digitorum longus (EDL) nerve-attached preparation to characterize the responses of muscle spindle afferents to ramp-and-hold stretch and sinusoidal vibratory stimuli. Responses were measured at both room (24°C) and muscle body temperature (34°C). Muscle spindle afferent static firing frequencies increased linearly in response to increasing stretch lengths to accurately encode the magnitude of muscle stretch (tested at 2.5%, 5% and 7.5% of resting length [Lo]). Peak firing frequency increased with ramp speeds (20% Lo/sec, 40% Lo/sec, and 60% Lo/sec). As a population, muscle spindle afferents could entrain 1:1 to sinusoidal vibrations throughout the frequency (10-100 Hz) and amplitude ranges tested (5-100 µm). Most units preferentially entrained to vibration frequencies close to their baseline steady-state firing frequencies. Cooling the muscle to 24°C decreased baseline firing frequency and units correspondingly entrained to slower frequency vibrations. The ramp component of stretch generated dynamic firing responses. These responses and related measures of dynamic sensitivity were not able to categorize units as primary (group Ia) or secondary (group II) even when tested with more extreme length changes (10% Lo). We conclude that the population of spindle afferents combines to encode stretch in a smoothly graded manner over the physiological range of lengths and speeds tested. Overall, spindle afferent response properties were comparable to those seen in other species, supporting subsequent use of the mouse genetic model system for studies on spindle function and dysfunction in an isolated muscle-nerve preparation.

Figure 1. Data analysis methods.

A. Raw waveform of 2 representative units (* and #). Spike2 software was used to identify unique waveforms based on shape. B. Response of 1 representative unit to ramp-and-hold stretch (length change of muscle shown below). The dynamic phase (D) of the stretch occurs during the ramp when the muscle is changing length. The static phase (S) of the stretch encompasses the time in which the length is held constant. C. Instantaneous frequency (Inst Fr) response to stretch of one unit. Arrows indicate time points where resting discharge (RD), dynamic peak (DP), dynamic index (DI), static response (SR), and time silenced (TS) are calculated. D-G: Afferent responses to vibration. D. No change in firing to a 25 Hz vibration. E. Entrainment at a subharmonic of 1:2 to a 25 Hz vibration. F. Multiple spikes per cycle at 10 Hz vibration. G. 1:1 entrainment at 25 Hz.

Figure 2. Effect of stretch length, speed, and temperature on spindle afferent firing.

Response of 4 ramp-and-hold stretch variables to the three levels of stretch length (x axis) and 3 speeds of stretch as indicated in the legend atop A. Stretch speeds are denoted by separate lines at 24°C (solid black) and at 34°C (dashed colored). A. Static response (SR) B. Dynamic peak (DP) C. Dynamic index (DI) D. Time silenced (TS). Error bars indicate a 95% confidence interval.

Figure 3. Spindle afferents could not be definitively classified using measures of dynamic sensitivity.

A and B. The slope of the linear regression of DI over the 3 stretch speeds tested was calculated at all 3 stretch lengths and shown for 24°C (A) and 34°C (B). No bimodal distribution was observed, though there was the largest variation in response at the largest stretch length. The response of a subset of the animals (n = 13) to a larger stretch length at 34°C (10% Lo) is shown stacked in black associated with values obtained at 7.5% Lo. No additional separation was observed at even this extreme length. The DP vs. TS responses of the 25% of the units with the highest (open circles), middle 50% (gray circles), and bottom 25% (black circles) slopes of DI are plotted at 24°C (C) and 34°C (D).

Figure 4. Frequency range of entrainment for individual units.

Frequency entrainment ranges for individual units are shown at 24°C (A) and 34°C (B). Each bar represents an individual unit and covers the frequency range that unit could entrain to. The patterns of the bar signify the number of frequencies the unit can entrain to (black  = 4, gray  = 3, hatched  = 2, white  = 1). The unit’s baseline resting discharge is shown in Hz in the bar.

Figure 5. Response to vibration of one unit at 24°C and 34°C.

Number of spikes at a given time over the vibration cycle for all 16 vibrations at 24°C (A) and 34°C (B). Bottom traces show the stage of vibration cycle. Amplitude of vibration increases down the column and frequency increases left to right. * denotes 1:1 entrainment of the unit.