Mechanosensory encoding in ex vivo muscle-nerve preparations

Abstract

Our objective was to evaluate an ex vivo muscle-nerve preparation used to study mechanosensory signalling by low threshold mechanosensory receptors (LTMRs). Specifically, we aimed to assess how well the ex vivo preparation represents in vivo firing behaviours of the three major LTMR subtypes of muscle primary sensory afferents, namely type Ia and II muscle spindle (MS) afferents and type Ib tendon organ afferents. Using published procedures for ex vivo study of LTMRs in mouse hindlimb muscles, we replicated earlier reports on afferent firing in response to conventional stretch paradigms applied to non-contracting, that is passive, muscle. Relative to in vivo studies, stretch-evoked firing for confirmed MS afferents in the ex vivo preparation was markedly reduced in firing rate and deficient in encoding dynamic features of muscle stretch. These deficiencies precluded conventional means of discriminating type Ia and II afferents. Muscle afferents, including confirmed Ib afferents were often indistinguishable based on their similar firing responses to the same physiologically relevant stretch paradigms. These observations raise uncertainty about conclusions drawn from earlier ex vivo studies that either attribute findings to specific afferent types or suggest an absence of treatment effects on dynamic firing. However, we found that replacing the recording solution with bicarbonate buffer resulted in afferent firing rates and profiles more like those seen in vivo. Improving representation of the distinctive sensory encoding properties in ex vivo muscle-nerve preparations will promote accuracy in assigning molecular markers and mechanisms to heterogeneous types of muscle mechanosensory neurons.

Keywords: electrophysiology; in vitro; mechanoreceptor; mouse; muscle spindle; proprioceptor; sensory; tendon organ.

FIGURE 1

Recording paradigm and firing behaviours of a confirmed muscle spindle afferent. (a) Experimental features of ex vivo muscle–nerve preparation (see Methods for details). Muscle fixed and submersed in fluid‐filled chamber (HEPES‐buffer solution in this case). Suction electrode attached to nerve recorded spikes evoked by servo‐motor controlled stretch or muscle contraction elicited by bipolar stimulating electrode. Muscle spindle and tendon organ receptors (objects shown within the muscle) are indicated in locations taken from Sonner et al. (2017) for mouse soleus muscles. (b,c) Representative spiking activity (blue vertical lines) produced by one MS afferent in response to mechanical stimulation of EDL muscle at resting muscle length L _o. (b) Electrical stimulation (bottom black trace indicating timing of two suprathreshold 50 μs pulses delivered at 100 spikes/s (sps)) delivered by stimulating electrode to induce isometric twitch (top black trace). Periodic spiking (blue vertical lines) produced by stretching muscle to >L _o; arrowhead indicates expected time of occurrence of the next periodic spike that was instead delayed to the end of muscle twitch contraction (superimposed black trace). The afferent was classified MS by the pause in firing. Stimulus artifacts were removed from afferent firing record for clarity. (c) Ramp–hold–release muscle stretch (bottom trace) from L _o to 7.5% L _o at constant ramp and release velocities 60% L _o/s temporally aligned with corresponding afferent spiking (blue middle trace) and instantaneous firing rate (IFR in spikes per second, sps; black dots), with peak and static firing rates defined. (d,e) Muscle length vibrations (bottom black traces) at 10 Hz (d) designated the maximum entrainment frequency by evoking a spike (top blue traces) with each vibration cycle in contrast with faster vibration frequencies, for example 25 Hz (e) that did not evoke cycle‐to‐cycle entrainment.

FIGURE 2

Ambiguity in distinguishing spike encoding of muscle stretch between MS and TO afferents in HEPES‐buffer solution. (a) Representative case of spiking activity recorded simultaneously from one muscle spindle (MS: blue) and one tendon organ (TO: green) afferent in response to ramp–hold–release stretch (bottom trace). Inset (b) shows four trials of spiking during isometric twitch contraction that consistently interrupted or initiated spiking, respectively, to confirm MS and TO afferent identity. Note that pauses in slow background firing rates for the MS afferent were sometimes difficult to identify and required multiple trials for confirmation. (c,d) Coloured circles plot instantaneous firing rates generated by the MS (c) and TO (d) afferents in response to ramp–hold–release stretch. (e) Encoding parameters: static firing rate, dynamic index and peak firing rate, for individual afferents (filled circles) averaged from four trials of ramp–hold–release (7.5% L _o at constant velocity 60% L _o/s) plotted for all colour‐coded TO and MS afferents together with corresponding box and whisker plots (markers for median, quartiles and range). Grey rectangles document the minimum and maximal values estimated from data reported in Wilkinson et al. (2012) to show similarity of firing responses recorded under identical conditions in this study.

FIGURE 3

Muscle spindle subtypes classified by conventional measures in Bicarb‐buffer solution. Firing patterns observed in two confirmed MS afferents. (a,c) Simultaneous records of ramp–hold changes in muscle length (bottom length traces), evoked spikes (middle traces) and instantaneous firing rates (top traces). Presence versus absence of high frequency initial burst firing at stretch onset, respectively, distinguish type Ia versus II afferents (see also Figure 4a, b). (b,d) Simultaneous records of muscle length during vibration (bottom traces) and evoked spikes at maximum entrainment frequency (top traces). (b) High (100 Hz) versus low (25 Hz) frequency entrainment, respectively, typify type Ia versus II afferents. (e) Values of maximum entrainment frequency plotted for each afferent (circles) sampled in Bicarb‐buffer solution at 24°C; filled versus open circles, respectively, represent presence or absence of initial burst firing determined during ramp–hold–release stretch (data not shown). High frequency entrainment coupled with initial burst designated subtype Ia afferents; the converse of these two properties designated subtype II afferents. (f) dynamic index for afferents represented by circles as described in (e). Shift toward higher values of dynamic index expected for subtype Ia versus II afferents. (g) Plot same as (e) for a separate set of afferents sampled in HEPES‐buffer solution.

FIGURE 4

Spike encoding properties of the three main muscle LTMR subtypes in Bicarb‐buffer solution. Spike encoding parameters (static firing average, dynamic index and peak firing rate) measured ex vivo for two separate afferent samples studied in Bicarb‐buffer solution at 24°C (a) or 34°C (b). Values for individual afferents plotted as circles together with corresponding box and whisker plots (markers for median, quartiles and range). Overlain traces to the right side of each panel show instantaneous firing rate profiles generated by single afferents of colour‐coded identity in response to ramp–hold–release stretch ( 7.5% L _o at constant velocity 60% L _o/s). Note the high frequency initial burst firing at stretch onset for type Ia afferents. For comparison, dashed‐line rectangular boxes in (a) outline ranges for various firing properties found for MS and TO afferents sampled in the present study in HEPES‐buffer solution at 24°C (Figure 2).