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Functional properties of mechanoreceptors in mouse whisker hair follicles determined by the pressure-clamped single-fiber recording technique
Neuroscience Letters 707 (2019) 134321
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Functional properties of mechanoreceptors in mouse whisker hair follicles determined by the pressure-clamped single-fiber recording technique
T
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Mayumi Sonekatsu, Jianguo G. Gu
Department of Anesthesiology and Perioperative Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35294, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Mechanoreceptors Whisker hair follicles Tactile Impulses Touch
Several types of mechanoreceptors have been identified anatomically in rodent whisker hair follicles, but their functional properties have not been fully studied. Here we developed a pressure-clamped single-fiber recording technique to record impulses on mouse whisker hair follicle afferent nerves following displacements of whisker hair follicles. On the basis of the patterns of impulses evoked by the mechanical stimulation, three functional types of mechanoreceptors were identified, including rapidly adapting (RA), slowly adapting type 1 (SA1), and slowly adapting type 2 (SA2) mechanoreceptors. Impulses of all these mechanoreceptors were almost completely abolished by 30 nM TTX, and were largely suppressed by cooling temperatures at 15°C. Tested at different displacement distances as different stimulation intensity, RA mechanoreceptors showed a limited capacity for stimulation intensity encoding, but both SA1 and SA2 mechanoreceptors displayed linear increases of impulse numbers with increased stimulation intensity. Tested with different ramp speed of displacements, RA impulses were only evoked by rapid ramp stimulation but SA1 and SA2 impulses could be evoked by both rapid and slow ramp stimulation. Tested with different stimulation frequency, all three types of mechanoreceptors well followed the stimulation at 10–100 Hz. Taken together, this study revealed some important functional properties of RA, SA1 and SA2 mechanoreceptors, which helps better understand the encoding of tactile information by different types of low-threshold mechanoreceptors.
1. Introduction
to study mechanoreceptors [4] for their functions and mechanisms underlying mechanical transduction [5,6]. Previous studies have identified several morphologically distinct types of primary afferent endings which are believed to be low-threshold mechanoreceptors in rodent whisker hair follicles [7,8]. For example, Merkel discs are found to be mostly located underneath the ring sinus region in front of the ringwulst of whisker hair follicles [8]. Lanceolate endings are located in front of the ringwulst and Ruffini-like endings in rear of the ringwulst [8]. Using in vivo electrophysiological recordings from primary afferent nerves innervating whisker hair follicles, several functional types of mechanoreceptors have been identified in whisker hair follicles [4] but the detailed functional properties of these mechanoreceptors were not characterized. Using isolated whisker hair follicles, we have recently uncovered mechanical transduction via Piezo2 [5] and mechanical transmission via 5-HT [6] in Merkel discs. However, our previous studies recorded afferent impulses from nerve bundles of whisker hair follicle afferent fibers which may contain impulses of multiple types of mechanoreceptors. Here we have developed the pressure-clamped single-fiber recording technique to characterize functional properties of each type of mechanoreceptor in whisker hair follicles.
Tactile sensory tasks such as environmental exploration, social interaction and tactile discrimination are essential in life and rely on mechanoreceptors in the body [1,2]. Different types of mechanoreceptors, based on morphological features, have been identified in tactile sensitive spots in the body, including Meissner’s corpuscles, Merkel discs, Pacinian corpuscles and Ruffini endings [2]. These mechanoreceptors detect distinct forms of mechanical stimuli such as a briefly gentle touch and a sustained pressure on the skin [2,3]. Mechanoreceptors are highly enriched in fingertips of humans and whisker hair follicles of non-primate mammals [3,4]. In fact, whisker hair follicles in non-primate mammals are functionally equivalent to human fingertips. Mechanoreceptors in human hands have been classified into four general types based on encoding properties for mechanical stimulation. They are rapidly adapting type 1 mechanoreceptors (RA1), slowly adapting type 1 mechanoreceptors (SA1), rapidly adapting type 2 mechanoreceptors (RA2), and slowly adapting type 2 mechanoreceptors (SA2) [3]. Whisker hair follicles of rodents have been used as a model system
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Corresponding author. E-mail address: [email protected] (J.G. Gu).
https://doi.org/10.1016/j.neulet.2019.134321 Received 1 April 2019; Received in revised form 4 June 2019; Accepted 6 June 2019 Available online 07 June 2019 0304-3940/ © 2019 Published by Elsevier B.V.
Neuroscience Letters 707 (2019) 134321
M. Sonekatsu and J.G. Gu
2. Materials and methods
2.4. Tests of functional properties of mechanoreceptors
2.1. Whisker hair follicle preparations
To test the effects of low concentrations of tetrodotoxin (TTX), 30 nM TTX was bath applied to whisker hair follicles for 10 min. Mechanical responses were elicited by the standard stimulation protocol before (control) and following the application of TTX. Mechanical responses were recorded from whisker hair follicle afferent nerves using the pressure-clamped single-fiber recording. To determine temperature effects on mechanical responses, whisker hair follicles were mechanically stimulated using our standard protocol. The experiments were performed with the Krebs solution at temperatures of 29, 24, and 15 °C. The temperatures of the Krebs solution were controlled by a dual in-line heater/cooler system. To determine effects of stimulation intensity on mechanical responses, several forward movement steps were applied with probe displacement at 2, 5, 10, 20 and 38 μm. The time was 100 ms for the ramp of each step and 2500-ms at each holding step. To determine effects of ramp-speed on mechanical response, in addition to the standard mechanical stimulation protocol (38 μm, 100-ms ramp and 2500ms holding), mechanical stimulation was also applied at slower ramps of 200, 500, 1000, or 1500 ms and other parameters remain the same as the standard mechanical stimulation protocol. To determine effects of stimulation frequency on mechanical responses, mechanical stimuli were applied with probe displacements each at 38 μm and duration at 10 ms, and stimulation was applied for a total time of 1 s at the frequency of 10, 50, and 100 Hz.
Male C57BL/6 mice aged 8–10 weeks. Animal care and use conformed to NIH guidelines for care and use of experimental animals. Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Alabama at Birmingham. In brief, animals were anesthetized with 5% isoflurane and then sacrificed by decapitation. Whisker pads were dissected out and placed in a 35-mm petri dish that contained 5 ml ice cold L-15 medium. Each whisker hair follicle together with its afferent bundle and hair shaft was then gently pulled out. The capsule of each whisker hair follicle was cut open to two small holes at the enlargement and end parts of the capsule to facilitate solution exchange. The whisker hair follicle preparations were affixed into a recording chamber by a tissue anchor and submerged in a Krebs solution. The Krebs solution contained (in mM): 117NaCl, 3.5 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2NaH2PO4, 25NaHCO3 and 11 glucose (pH 7.3 and osmolarity 325 mOsm), and was saturated with 95% O2 and 5% CO2.
2.2. The pressure-clamped single-fiber recording technique The recording chamber was mounted on the stage of the Olympus BX50WI microscope. The whisker hair follicle preparations were then exposed to a mixture of 0.05% dispase II plus 0.05% collagenase for 3–5 min, and the enzymes were washed off with the Krebs solution. Recording electrodes were made by thin walled borosilicate glass tubing without filament (inner diameter 1.12 mm, outer diameter 1.5 mm). They were fabricated using P-97 Flaming/Brown Micropipette Puller and fire polished to make tip diameter of 5–10 μm. The recording electrode was filled with the Krebs solution, mounted onto a holder which was connected to a high-speed pressure-clamp device (ALA Scientific Instruments, Farmingdale, NY). Under a 40x objective, the cutting end of a single whisker afferent nerve fiber was separated out by a low positive pressure (˜10 mmHg) from the recording electrode. The nerve end was then aspirated into the recording electrode by a negative pressure at about -10 mmHg. Once the nerve end reached ˜10 μm in length within the electrode, the electrode pressure was readjusted to -5 to -1 mmHg and maintained throughout the experiment. Nerve impulses were recorded using a Multiclamp 700A amplifier and signals sampled at 20 KHz with low pass filter set at 1 KHz. Unless otherwise indicated, all experiments were performed at 24 °C.
3. Results We developed the pressure-clamped single-fiber recording technique (Fig. 1A–D). In this new recording technique, a single whisker afferent fiber was gently aspirated into a recording electrode and pressure-clamped to tightly fit in the recording electrode (Fig. 1C&D). The pressure-clamp of a single fiber was achieved by a pressure-clamp device which can fine adjust the intra-electrode pressure. In our experiments, following a gentle aspiration of a single fiber into a recording electrode, we normally kept a negative pressure of -5 mmHg to -1 mmHg in the recording electrode to maintain stable recordings that could last for more than 2 h. Using this recording technique, three types of mechanical responses were recorded from individual whisker afferent nerves (Fig. 1E–G). On the basis of impulse firing patterns, mechanical responses could be classified as rapidly adapting (RA) response (Fig. 1E), slowly adapting type 1 (SA1) response (Fig. 1F), and slowly adapting type 2 (SA2) response (Fig. 1G). Accordingly, we termed the respective whisker afferent endings as RA, SA1, and SA2 mechanoreceptors. For RA mechanoreceptors, impulses fired only during the ramp phase (38 μm in 100 ms), i.e., the dynamic phase, of mechanical displacement (Fig. 1E). In the step holding phase, i.e., static phase, there was no impulse. It was noted that in some RA mechanoreceptors impulses appeared during both forward ramp phase and reverse ramp phase (Fig. 1E) and in other RA mechanoreceptors impulses appeared only during forward ramp phase. SA1 mechanoreceptors displayed impulse firing during both dynamic and static phases of mechanical displacements and the impulses were fired irregularly (Fig. 1F). SA2 mechanoreceptors displayed impulse firing during both dynamic and static phases of mechanical displacements but the impulses were fired regularly (Fig. 1G). Using the coefficient of variance (CoV) of inter-spike intervals, a measure of impulse regularity, CoV values were 0.82 ± 0.05 (n = 24) for SA1 responses and 0.30 ± 0.02 (n = 24) for SA2 responses, and they were significantly different (Fig. 1H, p < 0.001). We examined receptive fields of mechanoreceptors, i.e., the most sensitive spots where displacements evoked impulses. The receptive fields for both RA and SA1 responses were mainly located in front of the ringwulst (Fig. 1I). In contrast, the receptive fields of SA2 were mainly located in the rear of the ringwulst (Fig. 1I).
2.3. Mechanical stimulation Mechanical stimulation was applied to the body of a whisker hair follicle using a 20 gauge needle as a probe. The needle was mounted on a holder and attached to a piezo device. The tip of the needle was positioned at an angle of 45 degrees to the surface of the hair follicle. The piezo device with the mechanical probe was mounted on a micromanipulator. The piezo device was computer-programmable with the pCLAMP10 software to deliver forward stepwise mechanical stimulation. In each experiment, a receptive field was first probed manually with the mechanical probe controlled by the micromanipulator. Once identified, the vertical position of the probe tip was adjusted such that no nerve impulses were evoked at this position but a 1-μm forward movement of the probe would evoke nerve impulses. Unless otherwise indicated, the stepwise forward movement of the probe consisted of a 100-ms ramp to 38-μm step (dynamic phase) followed by a 2500-ms holding position at the 38-μm step (static phase) and then a 100-ms ramp back to baseline. This is our standard protocol of mechanical stimulation.
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Neuroscience Letters 707 (2019) 134321
M. Sonekatsu and J.G. Gu
Fig. 1. Identification of three types of mechanoreceptors in whisker hair follicles by the pressure-clamped single-fiber recording technique. A) The setting of the pressure-clamped single-fiber recording technique. B) Image shows a fresh whisker hair follicle preparation anchored in a recording chamber. The cutting end of the whisker afferent nerve is shown in a box. C) Image shows a cutting end of a single nerve (arrow indicated) isolated with a positive electrode pressure of 10 mmHg. D) Image shows the nerve end was aspirated into the recording electrode by a negative electrode pressure of -5 mmHg. E) Top panel, sample trace shows RA impulses evoked by a 38-μm displacement step. Inset below shows RA impulses at an expanded time scale. Bottom panel, plot of instantaneous frequency of RA impulses. Inset in the graph shows individual RA impulses aligned at their peaks. F) Similar to E except an example of SA1 impulses are shown. G) Similar to E except an example of SA2 impulses are shown. H) Summary data of coefficient of variation (CoV) of SA1 (n = 24) and SA2 (n = 24) responses. I) Receptive fields on whisker hair follicles where RA, SA1, and SA2 mechanoreceptors were identified. Each receptive field location is in reference to the ringwulst. Data represent Mean ± SEM, ***p < 0.001, student’s t-test.
mechanoreceptors, impulse numbers were progressively decreased from 80.1 ± 7.8 at 29°C to 49.2 ± 8.4 at 24°C (n = 9, p < 0.01) and to 15.8 ± 5.2 at 15 °C (n = 9, p < 0.01, Fig. 3B). Similar to SA1, impulses of SA2 mechanoreceptors also were progressively reduced from 293.8 ± 26.0 at 29 °C to 241.4 ± 22.4 at 24 °C (n = 10, p < 0.01) and to 107.0 ± 12.8 at 15 °C (n = 10, p < 0.001, Fig. 3C). We determined how stimulation intensities are encoded by the three types of whisker follicle mechanoreceptors. In this set of experiments, mechanical responses of these whisker follicle mechanoreceptors were tested by stimuli at displacements of 2, 5, 10, 20, and 38 μm. For RA mechanoreceptors, impulse numbers had limited increases with
We determined whether impulses of these mechanoreceptors are sensitive to TTX. As shown in Fig. 2, application of 30 nM TTX for 10 min almost completely abolished impulses of all three types of mechanoreceptors (Fig. 2A–C). We examined effects of temperatures on whisker hair follicle mechanoreceptors by determining mechanical responses at temperatures of 29, 24 and 15 °C. For RA responses, there was no significant differences in impulse numbers when temperatures dropped from 29 °C to 24 °C (n = 12) but the impulse numbers were significantly reduced when temperatures were further decreased to 15 °C (Fig. 3A). The impulse numbers were 3.0 ± 0.6 (n = 12) at 24 °C and reduced to 1.2 ± 0.2 (n = 12, p < 0.05) at 15°C. For SA1
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Neuroscience Letters 707 (2019) 134321
M. Sonekatsu and J.G. Gu
Fig. 2. Effects of low concentrations of TTX on mechanical responses of RA, SA1 and SA2 mechanoreceptors. A) Left, sample traces of RA responses before (control, top) and following the application of 30 nM TTX10 min (bottom). Right, summary data of RA impulse numbers in control (n = 7) and following the application of 30 nM TTX for 10 min (n = 7). B) Left, sample traces of SA1 responses before (control, top) and following the application of 30 nM TTX for 10 min (bottom). Right, summary data of SA1 impulse number before (control, n = 6) and following the application of 30 nM TTX for 10 min (n = 6). C) Left, sample traces of SA2 responses before (control, top) and following the application of 30 nM TTX for 10 min (bottom). Right, summary data of SA2 impulse numbers before (control, n = 7) and following the application of 30 nM TTX for 10 min (n = 7). Data represent Mean ± SEM, * p < 0.05, ***p < 0.001, paired t-test.
experiments, a step of 38 μm displacement was applied at ramp durations of 100 to 1500 ms, i.e., the ramp speeds from 38/100 μm*ms−1 to 38/1500 μm*ms−1. RA mechanoreceptors showed to be highly sensitivity to ramp speed and required high ramp speed for activation. For example, RA mechanoreceptor impulse numbers were 4.5 ± 1.15 with 100 ms ramp, and was rapidly reduced to 1.0 ± 0.4 with 200 ms ramp (n = 6, p < 0.05). RA impulses mostly failed to generate with ramp duration of 500 ms or longer (n = 6, Fig. 5A). In contrast to RA mechanoreceptors, impulse numbers in the dynamic phase of SA1 responses were significantly increased from 16.0 ± 3.0 with 100 ms ramp to 44.8 ± 10.3 with 1500 ms ramp (n = 8, p < 0.05) (Fig. 5B). Slow ramp displacements had no significant effects on impulse numbers during static phase of SA1 responses (Fig. 5B). Impulse numbers in dynamic phase of SA2 mechanoreceptors were 15.0 ± 1.0 with 100 ms ramp and increased to 117.0 ± 9.6 with 1500 ms ramp (n = 11, p < 0.001, Fig. 5C). Impulse numbers in static phase of SA2 responses did not show significant differences between fast ramp and slow ramp stimulation protocols (Fig. 5C). We examined how stimulation frequencies were encoded by these mechanoreceptors. In this set of experiments, 38-μm displacement pulses were applied with each pulse at the duration of 10 ms and at the stimulation frequency of 10, 50, 100 Hz. RA mechanoreceptors, which mainly responded to rapid ramp stimulation, were able to follow stimulation with 100% success rate at each stimulation frequency from 10, 50 to 100 Hz (Fig. 6A, n = 12). The total impulse numbers were proportional to stimulation frequency (Fig. 6A, n = 12). Similar to RA responses, both SA1 and SA2 mechanoreceptors also well responded to
increased displacement distances (Fig. 4A). For example, the spike numbers were 1.7 ± 0.3 (n = 15) with 10 μm displacement and were increased to 3.7 ± 0.7 (n = 15) with 38 μm displacement. RA mechanoreceptor impulses remained only in dynamic phase with no impulse occurring in static phase in all displacements tested. For SA1 mechanoreceptors, impulse numbers showed nearly linear increases in total impulses with increased displacements from 2 to 38 μm. For example, the total impulse numbers were 10.5 ± 2.7 (n = 10) with 10 μm displacement and increased to 80.1 ± 14.4 (n = 10) with 38 μm displacement. The impulse numbers in dynamic phase were more in SA1 mechanoreceptors than in RA mechanoreceptors (Fig. 4A&B). For example, at the displacement of 38 μm, the dynamic phase impulse numbers were 14.5 ± 2.11 in SA1 mechanoreceptors, significantly higher than the dynamic phase impulse numbers (3.67 ± 0.66, p < 0.001) of RA mechanoreceptors. For SA2 mechanoreceptors, total impulse numbers as well as impulse numbers in both dynamic and static phases were increased linearly with increased displacements (Fig. 4C). For example, the total impulse numbers were 45.3 ± 21.5 (n = 6) with 10 μm displacement and increased to 274.2 ± 30.5 (n = 6) with 38 μm displacement. Comparing between SA1 and SA2 responses, impulse numbers were similar in dynamic phases but in static phases were much higher in SA2 responses than in SA1 responses (Figure 4B&C). For example, at 38 μm displacement, the impulse numbers in the static phases were 261.3 ± 28.9 (n = 6) in SA2 mechanoreceptors and were 65.6 ± 12.6 (n = 10, p < 0.001) in SA1 mechanoreceptors. We determined how stimulation speeds were encoded by these mechanoreceptors in whisker hair follicles (Fig. 5A–C). In this set of 4
Neuroscience Letters 707 (2019) 134321
M. Sonekatsu and J.G. Gu
Fig. 3. Effects of temperatures on mechanical responses of RA, SA1 and SA2 mechanoreceptors. A) Left panel, sample traces of RA responses at 29 °C (top), 24 °C (middle), and 15 °C (bottom). Insets are impulses at an expanded time scale. Right panel, summary data of RA impulse numbers at 29 °C (n = 12), 24 °C (n = 12), and 15 °C (n = 12). B) Left panel, sample traces of SA1 responses at 29 °C (top), 24 °C (middle), and 15 °C (bottom). Right panel, summary data of SA1 impulse numbers at 29 °C (n = 9), 24 °C (n = 9), and 15 °C (n = 9). C) Left panel, sample traces of SA2 responses at 29 °C (top), 24 °C (middle), and 15 °C (bottom). Right panel, summary data of SA2 impulse numbers at 29 °C (n = 9), 24 °C (n = 9), and 15 °C (n = 9). Data represent Mean ± DEM, ns; not significantly different, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, Bonferroni’s post hoc test.
endings, and Ruffini-like or reticular endings [7,8]. A previous study found an uncommon type of RA mechanoreceptor in rat whisker hair follicles [4], which was not encountered in the present study. Lanceolate endings were previously shown to be located in front of the ringwulst and club-like endings near the ringwulst [7,8], consistent with the distribution of RA mechanoreceptors in our study. Merkel disc endings were previously found to be located in the front part of ringwulst [7,8], consistent with the distribution of SA1 mechanoreceptors our study. Ruffini-like endings were previously found to be located in the rear part of the ringwulst [7,8], consistent with the distribution of SA2 mechanoreceptors in our study. We show that impulses of all three types of mechanoreceptors are nearly completely abolished by 30 nM TTX, suggesting these mechanoreceptors are Aβ and Aδ low-threshold mechanoreceptors [10] primarily expressing TTX-sensitive voltage-gated Na+ channels [11]. We show that cooling temperature from 29 °C to 15 °C suppressed all three mechanoreceptors. The effects were most likely due to the suppression of voltage-gated Na+ channels on the afferents of these mechanoreceptors by cooling temperatures [12,13]. Voltage-gated Na+
the stimulation at 10, 50 to 100 Hz and showed nearly 100% success rates (Fig. 6B&C, n = 6 for both SA1 and SA2). For both SA1 (Fig. 6B, n = 6) and SA2 mechanoreceptors (Fig. 6C, n = 6), the total impulse numbers were also proportional to stimulation frequency.
4. Discussion In the present study we have developed the pressure-clamped single-fiber recording technique to record impulses of three types of mechanoreceptors in mouse whisker hair follicles. In this new recording technique only a single afferent nerve fiber is pressure-clamped and recorded. In contrast, the classical single-fiber recording technique commonly used with a teased nerve is not a true single fiber recording technique because the teased nerve contains many nerve fibers [9]. Therefore, our pressure-clamped single-fiber recording technique detects impulses only from a single mechanoreceptor. The three functional types of mechanoreceptors in whisker hair follicles in our study are consistent with anatomically identified mechanoreceptors including lanceolate and club-like endings, Merkel disc 5
Neuroscience Letters 707 (2019) 134321
M. Sonekatsu and J.G. Gu
Fig. 4. Encoding of different stimulation intensities by RA, SA1 and SA2 mechanoreceptors. A) Left panel, three sample traces of RA impulses in response to displacement stimuli at 10 μm (top), 20 μm (middle), and 38 μm (bottom). Insets are RA impulses at an expanded time scale. Right panel, three graphs are summary data (n = 15) of total impulse numbers (left), impulse numbers in dynamic phase (middle) and static phase (right) evoked by displacements at 2, 5, 10, 20, and 38 μm. B) Left panel, three sample traces of SA1 impulses in response to displacement stimuli at 10, 20, and 38 μm. Right panel, three graphs are summary data (n = 10) of total impulse numbers (left), impulse numbers in dynamic phase (middle) and static phase (right) evoked by displacements at 2, 5, 10, 20, and 38 μm. C) Left panel, three sample traces of SA2 impulses in response to displacement stimuli at 10, 20, and 38 μm. Right panel, three graphs are summary data (n = 6) of total impulse numbers (left), impulse numbers in dynamic phase (middle) and static phase (right) evoked by displacements at 2, 5, 10, 20, and 38 μm. Data represent Mean ± SEM.
encoding, a property similar to the function of RA1 mechanoreceptors in human fingertips [3]. In contrast to RA mechanoreceptors, SA1 and SA2 mechanoreceptors of whisker hair follicles could be activated at very slow ramp displacement. They fire impulses during both ramp step (dynamic phase) and step holding period (static phase) and impulse numbers showed nearly linear increases with increased displacement steps. These features would make them more suitable for detecting graded mechanical stimulation or encoding stimulation intensity and also for sensing prolonged (static) stimulation. We show that all three types of mechanoreceptors could equally well encode stimulation at frequencies from 10 to 100 Hz. These are the whisking frequency
channels sensitive to TTX are highly sensitive to cooling temperatures [12,13]. Thus, through the suppression of TTX-sensitive voltage-gated Na+ channels, cooling temperatures may block impulses of these mechanoreceptors. These results are consistent with poor tactile acuity at cooling temperatures. We show that RA mechanoreceptors only fire impulses during ramp step (dynamic phase), and RA impulse numbers are low and limitedly increased with increased displacement steps. RA mechanoreceptors require very rapid ramp displacement to be activated. These features of whisker RA mechanoreceptors are suitable for detecting transient mechanical stimulation without a good capacity for stimulation intensity
Fig. 5. Encoding of stimuli at different stimulation ramp speeds by RA, SA1 and SA2 mechanoreceptors. A) Left panel, two sample traces of RA responses following a 38-μm displacement at ramp duration of 100 ms (left) or 1500 ms (right). Right panel, summary data (n = 6) of experiments represented in the sample traces show RA impulse numbers with stimulation ramp duration of 100, 200, 500, 1000, and 1500 ms. B) Left panel, two sample traces of SA1 responses following a 38-μm displacement at ramp duration of 100 ms (left) or 1500 ms (right). Right panel, summary data (n = 8) of SA1 impulse numbers in dynamic (left) and static phases (right) following 38-μm displacements at ramp duration of 100 ms or 1500 ms. C) Left panel, two sample traces of SA2 responses following a 38-μm displacement at ramp duration of 100 ms (left) or 1500 ms (right). Right panel, summary data (n = 11) of SA2 impulse numbers in dynamic (left) and static phases (right) following 38-μm displacements at ramp duration of 100 ms or 1500 ms. Data represent Mean ± SEM, ns, no significant difference, *p < 0.05, ***p < 0.001, paired t-test.
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Neuroscience Letters 707 (2019) 134321
M. Sonekatsu and J.G. Gu
Fig. 6. Encoding of stimuli at different frequencies by RA, SA1 and SA2 mechanoreceptors. A) Left panel, 4 sample traces from top to bottom show RA impulses evoked by a 2.5-s displacement step at 38 μm (1st trace) and the impulses evoked by a train of brief displacements at 10 Hz (2nd trace), 50 Hz (3rd trace), and 100 Hz (4th trace) for a duration of 1 s. Each brief displacement stimulus was at 38 μm for a pulse duration of 10 ms, which were indicated by the dots above the 2nd to 4th traces. Right panel, two bar graphs are summary data of stimulation success rate (n = 12, left) and total numbers of impulses (n = 12, right) during the 1-s stimulation at 10, 50, and 100 Hz. A successful stimulus was defined as at least one impulse was generated following the stimulus. B) Left panel, similar to the sample traces in A except an SA1 mechanoreceptor (1st trace) was tested with the brief displacement stimuli at 10 Hz (2nd trace), 50 Hz (3rd trace), and 100 Hz (4th trace) for a 1-s duration. Right panel, two bar graphs are summary data of stimulation success rate (n = 6, left) and total numbers of impulses (n = 6, right) during the 1-s stimulation at 10, 50, and 100 Hz. C) Left panel, similar to the sample traces in A except an SA2 mechanoreceptor (1st trace) was tested with the brief displacement stimuli at 10 Hz (2nd trace), 50 Hz (3rd trace), and 100 Hz (4th trace) for a 1-s duration. Right panel, summary data of stimulation success rate (n = 6, left) and total numbers of impulses (n = 6, right) during the 1-s stimulation at 10, 50, and 100 Hz. Data represent Mean ± SEM, ns, not significantly different, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, Bonferroni’s post hoc test.
commonly used in rodents during environmental exploration [14]. Thus, all three types of mechanoreceptors may be engage in whisking for environmental exploring.
mechanoreceptors in sinus hair follicles of the cat, J. Physiol. 235 (1973) 287–315. [5] R. Ikeda, M. Cha, J. Ling, Z. Jia, D. Coyle, J.G. Gu, Merkel cells transduce and encode tactile stimuli to drive Abeta-afferent impulses, Cell 157 (2014) 664–675. [6] W. Chang, H. Kanda, R. Ikeda, J. Ling, J.J. DeBerry, J.G. Gu, Merkel disc is a serotonergic synapse in the epidermis for transmitting tactile signals in mammals, Proc. Natl. Acad. Sci. U. S. A. 113 (2016) E5491–5500. [7] S. Tonomura, S. Ebara, K. Bagdasarian, D. Uta, E. Ahissar, I. Meir, I. Lampl, D. Kuroda, T. Furuta, H. Furue, K. Kumamoto, Structure-function correlations of rat trigeminal primary neurons: emphasis on club-like endings, a vibrissal mechanoreceptor, Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 91 (2015) 560–576. [8] S. Ebara, K. Kumamoto, T. Matsuura, J.E. Mazurkiewicz, F.L. Rice, Similarities and differences in the innervation of mystacial vibrissal follicle-sinus complexes in the rat and cat: a confocal microscopic study, J. Comp. Neurol. 449 (2002) 103–119. [9] Y.B. Peng, M. Ringkamp, J.N. Campbell, R.A. Meyer, Electrophysiological assessment of the cutaneous arborization of Adelta-fiber nociceptors, J. Neurophysiol. 82 (1999) 1164–1177. [10] S.G. Lechner, G.R. Lewin, Hairy sensation, Physiology (Bethesda) 28 (2013) 142–150. [11] V. Pinto, V.A. Derkach, B.V. Safronov, Role of TTX-sensitive and TTX-resistant sodium channels in Adelta- and C-fiber conduction and synaptic transmission, J. Neurophysiol. 99 (2008) 617–628. [12] K. Zimmermann, A. Leffler, A. Babes, C.M. Cendan, R.W. Carr, J. Kobayashi, C. Nau, J.N. Wood, P.W. Reeh, Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures, Nature 447 (2007) 855–858. [13] I. Sarria, J. Ling, J.G. Gu, Thermal sensitivity of voltage-gated Na+ channels and Atype K+ channels contributes to somatosensory neuron excitability at cooling temperatures, J. Neurochem. 122 (2012) 1145–1154. [14] M. Szwed, K. Bagdasarian, E. Ahissar, Encoding of vibrissal active touch, Neuron 40 (2003) 621–630.
Authors’ contributions MS designed and performed the experiments and then analyzed the data. JGG wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by NIH/NIDCR grants DE018661 and DE023090 to J.G.G. References [1] K.O. Johnson, The roles and functions of cutaneous mechanoreceptors, Curr. Opin. Neurobiol. 11 (2001) 455–461. [2] A. Zimmerman, L. Bai, D.D. Ginty, The gentle touch receptors of mammalian skin, Science 346 (2014) 950–954. [3] R.S. Johansson, J.R. Flanagan, Coding and use of tactile signals from the fingertips in object manipulation tasks, Nat. Rev. Neurosci. 10 (2009) 345–359. [4] K.M. Gottschaldt, A. Iggo, D.W. Young, Functional characteristics of
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Functional properties of mechanoreceptors in mouse whisker hair follicles determined by the pressure-clamped single-fiber recording technique
T
⁎
Mayumi Sonekatsu, Jianguo G. Gu
Department of Anesthesiology and Perioperative Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35294, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Mechanoreceptors Whisker hair follicles Tactile Impulses Touch
Several types of mechanoreceptors have been identified anatomically in rodent whisker hair follicles, but their functional properties have not been fully studied. Here we developed a pressure-clamped single-fiber recording technique to record impulses on mouse whisker hair follicle afferent nerves following displacements of whisker hair follicles. On the basis of the patterns of impulses evoked by the mechanical stimulation, three functional types of mechanoreceptors were identified, including rapidly adapting (RA), slowly adapting type 1 (SA1), and slowly adapting type 2 (SA2) mechanoreceptors. Impulses of all these mechanoreceptors were almost completely abolished by 30 nM TTX, and were largely suppressed by cooling temperatures at 15°C. Tested at different displacement distances as different stimulation intensity, RA mechanoreceptors showed a limited capacity for stimulation intensity encoding, but both SA1 and SA2 mechanoreceptors displayed linear increases of impulse numbers with increased stimulation intensity. Tested with different ramp speed of displacements, RA impulses were only evoked by rapid ramp stimulation but SA1 and SA2 impulses could be evoked by both rapid and slow ramp stimulation. Tested with different stimulation frequency, all three types of mechanoreceptors well followed the stimulation at 10–100 Hz. Taken together, this study revealed some important functional properties of RA, SA1 and SA2 mechanoreceptors, which helps better understand the encoding of tactile information by different types of low-threshold mechanoreceptors.
1. Introduction
to study mechanoreceptors [4] for their functions and mechanisms underlying mechanical transduction [5,6]. Previous studies have identified several morphologically distinct types of primary afferent endings which are believed to be low-threshold mechanoreceptors in rodent whisker hair follicles [7,8]. For example, Merkel discs are found to be mostly located underneath the ring sinus region in front of the ringwulst of whisker hair follicles [8]. Lanceolate endings are located in front of the ringwulst and Ruffini-like endings in rear of the ringwulst [8]. Using in vivo electrophysiological recordings from primary afferent nerves innervating whisker hair follicles, several functional types of mechanoreceptors have been identified in whisker hair follicles [4] but the detailed functional properties of these mechanoreceptors were not characterized. Using isolated whisker hair follicles, we have recently uncovered mechanical transduction via Piezo2 [5] and mechanical transmission via 5-HT [6] in Merkel discs. However, our previous studies recorded afferent impulses from nerve bundles of whisker hair follicle afferent fibers which may contain impulses of multiple types of mechanoreceptors. Here we have developed the pressure-clamped single-fiber recording technique to characterize functional properties of each type of mechanoreceptor in whisker hair follicles.
Tactile sensory tasks such as environmental exploration, social interaction and tactile discrimination are essential in life and rely on mechanoreceptors in the body [1,2]. Different types of mechanoreceptors, based on morphological features, have been identified in tactile sensitive spots in the body, including Meissner’s corpuscles, Merkel discs, Pacinian corpuscles and Ruffini endings [2]. These mechanoreceptors detect distinct forms of mechanical stimuli such as a briefly gentle touch and a sustained pressure on the skin [2,3]. Mechanoreceptors are highly enriched in fingertips of humans and whisker hair follicles of non-primate mammals [3,4]. In fact, whisker hair follicles in non-primate mammals are functionally equivalent to human fingertips. Mechanoreceptors in human hands have been classified into four general types based on encoding properties for mechanical stimulation. They are rapidly adapting type 1 mechanoreceptors (RA1), slowly adapting type 1 mechanoreceptors (SA1), rapidly adapting type 2 mechanoreceptors (RA2), and slowly adapting type 2 mechanoreceptors (SA2) [3]. Whisker hair follicles of rodents have been used as a model system
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Corresponding author. E-mail address: [email protected] (J.G. Gu).
https://doi.org/10.1016/j.neulet.2019.134321 Received 1 April 2019; Received in revised form 4 June 2019; Accepted 6 June 2019 Available online 07 June 2019 0304-3940/ © 2019 Published by Elsevier B.V.
Neuroscience Letters 707 (2019) 134321
M. Sonekatsu and J.G. Gu
2. Materials and methods
2.4. Tests of functional properties of mechanoreceptors
2.1. Whisker hair follicle preparations
To test the effects of low concentrations of tetrodotoxin (TTX), 30 nM TTX was bath applied to whisker hair follicles for 10 min. Mechanical responses were elicited by the standard stimulation protocol before (control) and following the application of TTX. Mechanical responses were recorded from whisker hair follicle afferent nerves using the pressure-clamped single-fiber recording. To determine temperature effects on mechanical responses, whisker hair follicles were mechanically stimulated using our standard protocol. The experiments were performed with the Krebs solution at temperatures of 29, 24, and 15 °C. The temperatures of the Krebs solution were controlled by a dual in-line heater/cooler system. To determine effects of stimulation intensity on mechanical responses, several forward movement steps were applied with probe displacement at 2, 5, 10, 20 and 38 μm. The time was 100 ms for the ramp of each step and 2500-ms at each holding step. To determine effects of ramp-speed on mechanical response, in addition to the standard mechanical stimulation protocol (38 μm, 100-ms ramp and 2500ms holding), mechanical stimulation was also applied at slower ramps of 200, 500, 1000, or 1500 ms and other parameters remain the same as the standard mechanical stimulation protocol. To determine effects of stimulation frequency on mechanical responses, mechanical stimuli were applied with probe displacements each at 38 μm and duration at 10 ms, and stimulation was applied for a total time of 1 s at the frequency of 10, 50, and 100 Hz.
Male C57BL/6 mice aged 8–10 weeks. Animal care and use conformed to NIH guidelines for care and use of experimental animals. Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Alabama at Birmingham. In brief, animals were anesthetized with 5% isoflurane and then sacrificed by decapitation. Whisker pads were dissected out and placed in a 35-mm petri dish that contained 5 ml ice cold L-15 medium. Each whisker hair follicle together with its afferent bundle and hair shaft was then gently pulled out. The capsule of each whisker hair follicle was cut open to two small holes at the enlargement and end parts of the capsule to facilitate solution exchange. The whisker hair follicle preparations were affixed into a recording chamber by a tissue anchor and submerged in a Krebs solution. The Krebs solution contained (in mM): 117NaCl, 3.5 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2NaH2PO4, 25NaHCO3 and 11 glucose (pH 7.3 and osmolarity 325 mOsm), and was saturated with 95% O2 and 5% CO2.
2.2. The pressure-clamped single-fiber recording technique The recording chamber was mounted on the stage of the Olympus BX50WI microscope. The whisker hair follicle preparations were then exposed to a mixture of 0.05% dispase II plus 0.05% collagenase for 3–5 min, and the enzymes were washed off with the Krebs solution. Recording electrodes were made by thin walled borosilicate glass tubing without filament (inner diameter 1.12 mm, outer diameter 1.5 mm). They were fabricated using P-97 Flaming/Brown Micropipette Puller and fire polished to make tip diameter of 5–10 μm. The recording electrode was filled with the Krebs solution, mounted onto a holder which was connected to a high-speed pressure-clamp device (ALA Scientific Instruments, Farmingdale, NY). Under a 40x objective, the cutting end of a single whisker afferent nerve fiber was separated out by a low positive pressure (˜10 mmHg) from the recording electrode. The nerve end was then aspirated into the recording electrode by a negative pressure at about -10 mmHg. Once the nerve end reached ˜10 μm in length within the electrode, the electrode pressure was readjusted to -5 to -1 mmHg and maintained throughout the experiment. Nerve impulses were recorded using a Multiclamp 700A amplifier and signals sampled at 20 KHz with low pass filter set at 1 KHz. Unless otherwise indicated, all experiments were performed at 24 °C.
3. Results We developed the pressure-clamped single-fiber recording technique (Fig. 1A–D). In this new recording technique, a single whisker afferent fiber was gently aspirated into a recording electrode and pressure-clamped to tightly fit in the recording electrode (Fig. 1C&D). The pressure-clamp of a single fiber was achieved by a pressure-clamp device which can fine adjust the intra-electrode pressure. In our experiments, following a gentle aspiration of a single fiber into a recording electrode, we normally kept a negative pressure of -5 mmHg to -1 mmHg in the recording electrode to maintain stable recordings that could last for more than 2 h. Using this recording technique, three types of mechanical responses were recorded from individual whisker afferent nerves (Fig. 1E–G). On the basis of impulse firing patterns, mechanical responses could be classified as rapidly adapting (RA) response (Fig. 1E), slowly adapting type 1 (SA1) response (Fig. 1F), and slowly adapting type 2 (SA2) response (Fig. 1G). Accordingly, we termed the respective whisker afferent endings as RA, SA1, and SA2 mechanoreceptors. For RA mechanoreceptors, impulses fired only during the ramp phase (38 μm in 100 ms), i.e., the dynamic phase, of mechanical displacement (Fig. 1E). In the step holding phase, i.e., static phase, there was no impulse. It was noted that in some RA mechanoreceptors impulses appeared during both forward ramp phase and reverse ramp phase (Fig. 1E) and in other RA mechanoreceptors impulses appeared only during forward ramp phase. SA1 mechanoreceptors displayed impulse firing during both dynamic and static phases of mechanical displacements and the impulses were fired irregularly (Fig. 1F). SA2 mechanoreceptors displayed impulse firing during both dynamic and static phases of mechanical displacements but the impulses were fired regularly (Fig. 1G). Using the coefficient of variance (CoV) of inter-spike intervals, a measure of impulse regularity, CoV values were 0.82 ± 0.05 (n = 24) for SA1 responses and 0.30 ± 0.02 (n = 24) for SA2 responses, and they were significantly different (Fig. 1H, p < 0.001). We examined receptive fields of mechanoreceptors, i.e., the most sensitive spots where displacements evoked impulses. The receptive fields for both RA and SA1 responses were mainly located in front of the ringwulst (Fig. 1I). In contrast, the receptive fields of SA2 were mainly located in the rear of the ringwulst (Fig. 1I).
2.3. Mechanical stimulation Mechanical stimulation was applied to the body of a whisker hair follicle using a 20 gauge needle as a probe. The needle was mounted on a holder and attached to a piezo device. The tip of the needle was positioned at an angle of 45 degrees to the surface of the hair follicle. The piezo device with the mechanical probe was mounted on a micromanipulator. The piezo device was computer-programmable with the pCLAMP10 software to deliver forward stepwise mechanical stimulation. In each experiment, a receptive field was first probed manually with the mechanical probe controlled by the micromanipulator. Once identified, the vertical position of the probe tip was adjusted such that no nerve impulses were evoked at this position but a 1-μm forward movement of the probe would evoke nerve impulses. Unless otherwise indicated, the stepwise forward movement of the probe consisted of a 100-ms ramp to 38-μm step (dynamic phase) followed by a 2500-ms holding position at the 38-μm step (static phase) and then a 100-ms ramp back to baseline. This is our standard protocol of mechanical stimulation.
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Neuroscience Letters 707 (2019) 134321
M. Sonekatsu and J.G. Gu
Fig. 1. Identification of three types of mechanoreceptors in whisker hair follicles by the pressure-clamped single-fiber recording technique. A) The setting of the pressure-clamped single-fiber recording technique. B) Image shows a fresh whisker hair follicle preparation anchored in a recording chamber. The cutting end of the whisker afferent nerve is shown in a box. C) Image shows a cutting end of a single nerve (arrow indicated) isolated with a positive electrode pressure of 10 mmHg. D) Image shows the nerve end was aspirated into the recording electrode by a negative electrode pressure of -5 mmHg. E) Top panel, sample trace shows RA impulses evoked by a 38-μm displacement step. Inset below shows RA impulses at an expanded time scale. Bottom panel, plot of instantaneous frequency of RA impulses. Inset in the graph shows individual RA impulses aligned at their peaks. F) Similar to E except an example of SA1 impulses are shown. G) Similar to E except an example of SA2 impulses are shown. H) Summary data of coefficient of variation (CoV) of SA1 (n = 24) and SA2 (n = 24) responses. I) Receptive fields on whisker hair follicles where RA, SA1, and SA2 mechanoreceptors were identified. Each receptive field location is in reference to the ringwulst. Data represent Mean ± SEM, ***p < 0.001, student’s t-test.
mechanoreceptors, impulse numbers were progressively decreased from 80.1 ± 7.8 at 29°C to 49.2 ± 8.4 at 24°C (n = 9, p < 0.01) and to 15.8 ± 5.2 at 15 °C (n = 9, p < 0.01, Fig. 3B). Similar to SA1, impulses of SA2 mechanoreceptors also were progressively reduced from 293.8 ± 26.0 at 29 °C to 241.4 ± 22.4 at 24 °C (n = 10, p < 0.01) and to 107.0 ± 12.8 at 15 °C (n = 10, p < 0.001, Fig. 3C). We determined how stimulation intensities are encoded by the three types of whisker follicle mechanoreceptors. In this set of experiments, mechanical responses of these whisker follicle mechanoreceptors were tested by stimuli at displacements of 2, 5, 10, 20, and 38 μm. For RA mechanoreceptors, impulse numbers had limited increases with
We determined whether impulses of these mechanoreceptors are sensitive to TTX. As shown in Fig. 2, application of 30 nM TTX for 10 min almost completely abolished impulses of all three types of mechanoreceptors (Fig. 2A–C). We examined effects of temperatures on whisker hair follicle mechanoreceptors by determining mechanical responses at temperatures of 29, 24 and 15 °C. For RA responses, there was no significant differences in impulse numbers when temperatures dropped from 29 °C to 24 °C (n = 12) but the impulse numbers were significantly reduced when temperatures were further decreased to 15 °C (Fig. 3A). The impulse numbers were 3.0 ± 0.6 (n = 12) at 24 °C and reduced to 1.2 ± 0.2 (n = 12, p < 0.05) at 15°C. For SA1
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Neuroscience Letters 707 (2019) 134321
M. Sonekatsu and J.G. Gu
Fig. 2. Effects of low concentrations of TTX on mechanical responses of RA, SA1 and SA2 mechanoreceptors. A) Left, sample traces of RA responses before (control, top) and following the application of 30 nM TTX10 min (bottom). Right, summary data of RA impulse numbers in control (n = 7) and following the application of 30 nM TTX for 10 min (n = 7). B) Left, sample traces of SA1 responses before (control, top) and following the application of 30 nM TTX for 10 min (bottom). Right, summary data of SA1 impulse number before (control, n = 6) and following the application of 30 nM TTX for 10 min (n = 6). C) Left, sample traces of SA2 responses before (control, top) and following the application of 30 nM TTX for 10 min (bottom). Right, summary data of SA2 impulse numbers before (control, n = 7) and following the application of 30 nM TTX for 10 min (n = 7). Data represent Mean ± SEM, * p < 0.05, ***p < 0.001, paired t-test.
experiments, a step of 38 μm displacement was applied at ramp durations of 100 to 1500 ms, i.e., the ramp speeds from 38/100 μm*ms−1 to 38/1500 μm*ms−1. RA mechanoreceptors showed to be highly sensitivity to ramp speed and required high ramp speed for activation. For example, RA mechanoreceptor impulse numbers were 4.5 ± 1.15 with 100 ms ramp, and was rapidly reduced to 1.0 ± 0.4 with 200 ms ramp (n = 6, p < 0.05). RA impulses mostly failed to generate with ramp duration of 500 ms or longer (n = 6, Fig. 5A). In contrast to RA mechanoreceptors, impulse numbers in the dynamic phase of SA1 responses were significantly increased from 16.0 ± 3.0 with 100 ms ramp to 44.8 ± 10.3 with 1500 ms ramp (n = 8, p < 0.05) (Fig. 5B). Slow ramp displacements had no significant effects on impulse numbers during static phase of SA1 responses (Fig. 5B). Impulse numbers in dynamic phase of SA2 mechanoreceptors were 15.0 ± 1.0 with 100 ms ramp and increased to 117.0 ± 9.6 with 1500 ms ramp (n = 11, p < 0.001, Fig. 5C). Impulse numbers in static phase of SA2 responses did not show significant differences between fast ramp and slow ramp stimulation protocols (Fig. 5C). We examined how stimulation frequencies were encoded by these mechanoreceptors. In this set of experiments, 38-μm displacement pulses were applied with each pulse at the duration of 10 ms and at the stimulation frequency of 10, 50, 100 Hz. RA mechanoreceptors, which mainly responded to rapid ramp stimulation, were able to follow stimulation with 100% success rate at each stimulation frequency from 10, 50 to 100 Hz (Fig. 6A, n = 12). The total impulse numbers were proportional to stimulation frequency (Fig. 6A, n = 12). Similar to RA responses, both SA1 and SA2 mechanoreceptors also well responded to
increased displacement distances (Fig. 4A). For example, the spike numbers were 1.7 ± 0.3 (n = 15) with 10 μm displacement and were increased to 3.7 ± 0.7 (n = 15) with 38 μm displacement. RA mechanoreceptor impulses remained only in dynamic phase with no impulse occurring in static phase in all displacements tested. For SA1 mechanoreceptors, impulse numbers showed nearly linear increases in total impulses with increased displacements from 2 to 38 μm. For example, the total impulse numbers were 10.5 ± 2.7 (n = 10) with 10 μm displacement and increased to 80.1 ± 14.4 (n = 10) with 38 μm displacement. The impulse numbers in dynamic phase were more in SA1 mechanoreceptors than in RA mechanoreceptors (Fig. 4A&B). For example, at the displacement of 38 μm, the dynamic phase impulse numbers were 14.5 ± 2.11 in SA1 mechanoreceptors, significantly higher than the dynamic phase impulse numbers (3.67 ± 0.66, p < 0.001) of RA mechanoreceptors. For SA2 mechanoreceptors, total impulse numbers as well as impulse numbers in both dynamic and static phases were increased linearly with increased displacements (Fig. 4C). For example, the total impulse numbers were 45.3 ± 21.5 (n = 6) with 10 μm displacement and increased to 274.2 ± 30.5 (n = 6) with 38 μm displacement. Comparing between SA1 and SA2 responses, impulse numbers were similar in dynamic phases but in static phases were much higher in SA2 responses than in SA1 responses (Figure 4B&C). For example, at 38 μm displacement, the impulse numbers in the static phases were 261.3 ± 28.9 (n = 6) in SA2 mechanoreceptors and were 65.6 ± 12.6 (n = 10, p < 0.001) in SA1 mechanoreceptors. We determined how stimulation speeds were encoded by these mechanoreceptors in whisker hair follicles (Fig. 5A–C). In this set of 4
Neuroscience Letters 707 (2019) 134321
M. Sonekatsu and J.G. Gu
Fig. 3. Effects of temperatures on mechanical responses of RA, SA1 and SA2 mechanoreceptors. A) Left panel, sample traces of RA responses at 29 °C (top), 24 °C (middle), and 15 °C (bottom). Insets are impulses at an expanded time scale. Right panel, summary data of RA impulse numbers at 29 °C (n = 12), 24 °C (n = 12), and 15 °C (n = 12). B) Left panel, sample traces of SA1 responses at 29 °C (top), 24 °C (middle), and 15 °C (bottom). Right panel, summary data of SA1 impulse numbers at 29 °C (n = 9), 24 °C (n = 9), and 15 °C (n = 9). C) Left panel, sample traces of SA2 responses at 29 °C (top), 24 °C (middle), and 15 °C (bottom). Right panel, summary data of SA2 impulse numbers at 29 °C (n = 9), 24 °C (n = 9), and 15 °C (n = 9). Data represent Mean ± DEM, ns; not significantly different, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, Bonferroni’s post hoc test.
endings, and Ruffini-like or reticular endings [7,8]. A previous study found an uncommon type of RA mechanoreceptor in rat whisker hair follicles [4], which was not encountered in the present study. Lanceolate endings were previously shown to be located in front of the ringwulst and club-like endings near the ringwulst [7,8], consistent with the distribution of RA mechanoreceptors in our study. Merkel disc endings were previously found to be located in the front part of ringwulst [7,8], consistent with the distribution of SA1 mechanoreceptors our study. Ruffini-like endings were previously found to be located in the rear part of the ringwulst [7,8], consistent with the distribution of SA2 mechanoreceptors in our study. We show that impulses of all three types of mechanoreceptors are nearly completely abolished by 30 nM TTX, suggesting these mechanoreceptors are Aβ and Aδ low-threshold mechanoreceptors [10] primarily expressing TTX-sensitive voltage-gated Na+ channels [11]. We show that cooling temperature from 29 °C to 15 °C suppressed all three mechanoreceptors. The effects were most likely due to the suppression of voltage-gated Na+ channels on the afferents of these mechanoreceptors by cooling temperatures [12,13]. Voltage-gated Na+
the stimulation at 10, 50 to 100 Hz and showed nearly 100% success rates (Fig. 6B&C, n = 6 for both SA1 and SA2). For both SA1 (Fig. 6B, n = 6) and SA2 mechanoreceptors (Fig. 6C, n = 6), the total impulse numbers were also proportional to stimulation frequency.
4. Discussion In the present study we have developed the pressure-clamped single-fiber recording technique to record impulses of three types of mechanoreceptors in mouse whisker hair follicles. In this new recording technique only a single afferent nerve fiber is pressure-clamped and recorded. In contrast, the classical single-fiber recording technique commonly used with a teased nerve is not a true single fiber recording technique because the teased nerve contains many nerve fibers [9]. Therefore, our pressure-clamped single-fiber recording technique detects impulses only from a single mechanoreceptor. The three functional types of mechanoreceptors in whisker hair follicles in our study are consistent with anatomically identified mechanoreceptors including lanceolate and club-like endings, Merkel disc 5
Neuroscience Letters 707 (2019) 134321
M. Sonekatsu and J.G. Gu
Fig. 4. Encoding of different stimulation intensities by RA, SA1 and SA2 mechanoreceptors. A) Left panel, three sample traces of RA impulses in response to displacement stimuli at 10 μm (top), 20 μm (middle), and 38 μm (bottom). Insets are RA impulses at an expanded time scale. Right panel, three graphs are summary data (n = 15) of total impulse numbers (left), impulse numbers in dynamic phase (middle) and static phase (right) evoked by displacements at 2, 5, 10, 20, and 38 μm. B) Left panel, three sample traces of SA1 impulses in response to displacement stimuli at 10, 20, and 38 μm. Right panel, three graphs are summary data (n = 10) of total impulse numbers (left), impulse numbers in dynamic phase (middle) and static phase (right) evoked by displacements at 2, 5, 10, 20, and 38 μm. C) Left panel, three sample traces of SA2 impulses in response to displacement stimuli at 10, 20, and 38 μm. Right panel, three graphs are summary data (n = 6) of total impulse numbers (left), impulse numbers in dynamic phase (middle) and static phase (right) evoked by displacements at 2, 5, 10, 20, and 38 μm. Data represent Mean ± SEM.
encoding, a property similar to the function of RA1 mechanoreceptors in human fingertips [3]. In contrast to RA mechanoreceptors, SA1 and SA2 mechanoreceptors of whisker hair follicles could be activated at very slow ramp displacement. They fire impulses during both ramp step (dynamic phase) and step holding period (static phase) and impulse numbers showed nearly linear increases with increased displacement steps. These features would make them more suitable for detecting graded mechanical stimulation or encoding stimulation intensity and also for sensing prolonged (static) stimulation. We show that all three types of mechanoreceptors could equally well encode stimulation at frequencies from 10 to 100 Hz. These are the whisking frequency
channels sensitive to TTX are highly sensitive to cooling temperatures [12,13]. Thus, through the suppression of TTX-sensitive voltage-gated Na+ channels, cooling temperatures may block impulses of these mechanoreceptors. These results are consistent with poor tactile acuity at cooling temperatures. We show that RA mechanoreceptors only fire impulses during ramp step (dynamic phase), and RA impulse numbers are low and limitedly increased with increased displacement steps. RA mechanoreceptors require very rapid ramp displacement to be activated. These features of whisker RA mechanoreceptors are suitable for detecting transient mechanical stimulation without a good capacity for stimulation intensity
Fig. 5. Encoding of stimuli at different stimulation ramp speeds by RA, SA1 and SA2 mechanoreceptors. A) Left panel, two sample traces of RA responses following a 38-μm displacement at ramp duration of 100 ms (left) or 1500 ms (right). Right panel, summary data (n = 6) of experiments represented in the sample traces show RA impulse numbers with stimulation ramp duration of 100, 200, 500, 1000, and 1500 ms. B) Left panel, two sample traces of SA1 responses following a 38-μm displacement at ramp duration of 100 ms (left) or 1500 ms (right). Right panel, summary data (n = 8) of SA1 impulse numbers in dynamic (left) and static phases (right) following 38-μm displacements at ramp duration of 100 ms or 1500 ms. C) Left panel, two sample traces of SA2 responses following a 38-μm displacement at ramp duration of 100 ms (left) or 1500 ms (right). Right panel, summary data (n = 11) of SA2 impulse numbers in dynamic (left) and static phases (right) following 38-μm displacements at ramp duration of 100 ms or 1500 ms. Data represent Mean ± SEM, ns, no significant difference, *p < 0.05, ***p < 0.001, paired t-test.
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Neuroscience Letters 707 (2019) 134321
M. Sonekatsu and J.G. Gu
Fig. 6. Encoding of stimuli at different frequencies by RA, SA1 and SA2 mechanoreceptors. A) Left panel, 4 sample traces from top to bottom show RA impulses evoked by a 2.5-s displacement step at 38 μm (1st trace) and the impulses evoked by a train of brief displacements at 10 Hz (2nd trace), 50 Hz (3rd trace), and 100 Hz (4th trace) for a duration of 1 s. Each brief displacement stimulus was at 38 μm for a pulse duration of 10 ms, which were indicated by the dots above the 2nd to 4th traces. Right panel, two bar graphs are summary data of stimulation success rate (n = 12, left) and total numbers of impulses (n = 12, right) during the 1-s stimulation at 10, 50, and 100 Hz. A successful stimulus was defined as at least one impulse was generated following the stimulus. B) Left panel, similar to the sample traces in A except an SA1 mechanoreceptor (1st trace) was tested with the brief displacement stimuli at 10 Hz (2nd trace), 50 Hz (3rd trace), and 100 Hz (4th trace) for a 1-s duration. Right panel, two bar graphs are summary data of stimulation success rate (n = 6, left) and total numbers of impulses (n = 6, right) during the 1-s stimulation at 10, 50, and 100 Hz. C) Left panel, similar to the sample traces in A except an SA2 mechanoreceptor (1st trace) was tested with the brief displacement stimuli at 10 Hz (2nd trace), 50 Hz (3rd trace), and 100 Hz (4th trace) for a 1-s duration. Right panel, summary data of stimulation success rate (n = 6, left) and total numbers of impulses (n = 6, right) during the 1-s stimulation at 10, 50, and 100 Hz. Data represent Mean ± SEM, ns, not significantly different, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, Bonferroni’s post hoc test.
commonly used in rodents during environmental exploration [14]. Thus, all three types of mechanoreceptors may be engage in whisking for environmental exploring.
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Authors’ contributions MS designed and performed the experiments and then analyzed the data. JGG wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by NIH/NIDCR grants DE018661 and DE023090 to J.G.G. References [1] K.O. Johnson, The roles and functions of cutaneous mechanoreceptors, Curr. Opin. Neurobiol. 11 (2001) 455–461. [2] A. Zimmerman, L. Bai, D.D. Ginty, The gentle touch receptors of mammalian skin, Science 346 (2014) 950–954. [3] R.S. Johansson, J.R. Flanagan, Coding and use of tactile signals from the fingertips in object manipulation tasks, Nat. Rev. Neurosci. 10 (2009) 345–359. [4] K.M. Gottschaldt, A. Iggo, D.W. Young, Functional characteristics of
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