In situ patch-clamp recordings from Merkel cells in rat whisker hair follicles, an experimental protocol for studying tactile transduction in tactile-end organs
© Ikeda et al.; licensee BioMed Central. 2015
Received: 23 March 2015
Accepted: 13 April 2015
Published: 25 April 2015
Mammals use tactile end-organs to perform sensory tasks such as environmental exploration, social interaction, and tactile discrimination. However, cellular and molecular mechanisms underlying tactile transduction in tactile end-organs remain poorly understood. The patch-clamp recording technique may be the most valuable approach for detecting and studying tactile transduction in tactile end-organs, but it is technically challenging because tactile transduction elements in an end-organ are normally inaccessible by patch-clamp recording electrodes. Here we describe an in situ patch-clamp recording protocol for the study of tactile transduction in Merkel cells of rat whisker hair follicles, one of the most sensitive tactile end-organs in mammals. This technique offers an opportunity to explore the identities and properties of ion channels that are involved in tactile transduction in whisker hair follicles, and it may also lend a useful tool for researchers to study other tactile end-organs. The experimental protocol describes procedures for 1) tissue dissection and whisker hair follicle preparation, 2) device setup and steps for performing patch-clamp recordings from Merkel cells in a whisker hair follicle, 3) methods of delivering mechanical stimuli, and 4) intra-follicle microinjection for receptor knockdown in whisker hair follicles. The main procedures in this protocol, from tissue preparation to whole-cell patch-clamp recordings, can be completed in a few hours.
KeywordsPiezo2 channel Touch Mechanotransduction Tactile discrimination Merkel cells Whisker hair follicles
The sense of touch by mammalian tactile end-organs is indispensable for environmental exploration, social interaction, tactile discrimination and other tasks in life. Four major tactile end-organs have been identified so far, including Merkel discs, Ruffini’s corpuscles, Pacinian corpuscles, and Meissner’s corpuscles . In addition, lanceolate nerve endings and some free nerve endings also are involved in tactile transduction . The identification of these tactile end-organs was initially based on anatomical evidence showing the presence of these specialized structures at tactile sensitive spots in mammals. For example, Merkel discs were found in the touch domes throughout the body and in finger tips as well as whisker hair follicles [2-4]. Later the application of nerve fiber recordings led to the detection of afferent nerve impulses following mechanical stimulation at tactile sensitive spots that had the tactile end-organs [5,6]. Previous studies using this classical recording technique revealed a number of important properties of the tactile end-organs. These properties include the tactile sub-modality, receptive field and response dynamic range of each tactile end-organ . Merkel discs were found to be most sensitive to sustained indentation of the skin with very small receptive field, and the sustained indentation generated slowly adapting type I nerve impulses (SAI) [5,7]. The SAI responses of Merkel discs are believed to be sensory encodings essential for tactile discrimination for the texture and shape of an object [8-10]. While the recordings from afferent nerve fibers have revealed many functional aspects of the tactile end-organs, this classical electrophysiological approach is not powerful in revealing cellular and molecular insights of tactile transduction in the tactile end-organs. A recent advance in the field of tactile sensory physiology is the use of developmental biological and genetic approaches to delineate the formation and neuronal connectivity of different tactile end-organs [11,12]. However, a critical question that remains to be answered is how tactile stimuli are transduced into electric signals in the tactile end-organs.
Patch-clamp recording technique may be the most direct way of detecting and studying mechanotransduction in a cell. McCarter et al. have first described mechanically activated currents (MA) in dissociated rat sensory neuron somata by using the whole-cell patch-clamp recording method . The MA currents in sensory neuron somata have been characterized in more details by a number of subsequent studies using the patch-clamp recording technique [14-18]. By combining patch-clamp recordings and molecular biology approaches, it has recently been discovered that MA currents in most sensory neuron somata are mediated by Piezo2 protein, an ion channel that opens in response to mechanical displacement of cell membranes . However, it remains unclear as to what extent the mechanotransduction delineated in sensory neuron somata represents tactile transduction in tactile end-organs. In addition, MA currents detected in most previous studies were evoked by directly displacing sensory neuron membranes with a glass probe. It is unknown whether the mechanical stimulation with a glass probe well mimics tactile stimulation.
The patch-clamp recording technique may be also the most useful approach to study tactile transduction in a tactile end-organ. However, it is technically challenging because tactile transduction elements in a tactile end-organ are normally inaccessible by a patch-clamp recording electrode. Nevertheless, recently we have successfully applied patch-clamp recording technique to study tactile transduction in Merkel discs of rat whisker hair follicles, and demonstrated that Merkel cells transduce tactile stimulation via Piezo2 channels . Our study, together with two other recent studies using cultured Merkel cells [21,22], have for the first time revealed cellular and molecular mechanisms underlying tactile transduction in the Merkel discs. A brief description of the methods of our study has been reported elsewhere . Here we describe the technical details about the in situ patch-clamp recordings from Merkel cells in rat whisker hair follicles. This technique offers an opportunity to further characterize the properties of tactile transduction and encoding in Merkel discs of whisker hair follicles. It may also lend a useful technical tool for studying other tactile end-organs.
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. Unless otherwise indicated, experimental animals were Sprague Dawley rats aged 10–22 days purchased from Harlan Laboratories.
The following instruments were used in our experiments for preparing whisker hair follicles, performing patch-clamp recordings, delivering mechanical stimuli, and knocking down candidate tactile transducers: Dissection microscope (Olympus); Dissection scissors and Forceps; Brown-Flaming P-97 programmable pipette puller (Sutter Instrument Company, Novato, CA); Gravity-fed bath perfusion system; Microforge (World Precision Instruments, Sarasota, FL); Computer-programmable Piezoelectric actuator (E-625 LVPZT; Physik Instrumente); Thin-walled borosilicate glass tubing (inner diameter 1.12 mm, outer diameter 1.5 mm, World Precision Instruments); 35-mm culture dishes (Thermo Scientific); Syringe (50 ml); Patch-pipette fillers with solution filter (4 mm diameter, 0.2 μm pore size, World Precision Instruments); Olympus IX50 upright microscope equipped with IR-DIC and fluorescent imaging systems (Olympus); Micromanipulators and pipette holder for patch clamping (Sutter Instrument Company); Multiclamp 700A amplifier, Digidata 1322A, and pCLAMP10 software (Molecular Devices, Sunnyvale, CA); Vibration isolation table and perimeter Faraday cage (TMC, Peabody, MA); Cool SNAP™ HQ2 CCD camera (Photometrics, Tucson, AZ); MetaFluor Imaging System software (Molecular Devices); High-speed pressure-clamp device (ALA Scientific Instruments, Farmingdale, NY 11735); Isoflurane anesthesia machine (World Precision Instruments); Microinjection system (World Precision Instruments); Digitized stereotaxic apparatus (World Precision Instruments).
Reagents and solution preparation
Reagents for making recording electrode internal solution and bath solution were obtained from Sigma-Aldrich (St. Louis, MO). For whole-cell voltage-clamp experiments, Cs+-based internal solution was used and the solution contained (in mM): 70 Cs2SO4, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 HEPES, 5 Na2ATP and 0.5 GTP-TRIS salt; the pH of the solution was adjusted to 7.3 with CsOH. For whole-cell current-clamp experiments, K+-based internal solution was used and the solution contained (in mM): 135 K-gluconate, 5 KCl, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 HEPES, 5 Na2ATP and 0.5 GTP-TRIS salt; the pH of the solution was adjusted to 7.3 with KOH. The K+-based internal solution for whole-cell current-clamp recordings could also be used for whole-cell voltage-clamp experiments. The recording electrode internal solutions were aliquoted (0.5 ml each tube) and stored at −20°C.
Normal Krebs solution was used as the bath solution for the perfusion of whisker hair follicle tissues during patch-clamp recording experiments. A 10X stock Krebs solution was first made and the stock solution contained (in mM): 1170 NaCl, 35 KCl, 25 CaCl2, 12 MgCl2, 12 NaH2PO4, 250 NaHCO3 and 110 glucose. The stock Krebs solution was diluted by 10 times with de-ionized distilled water, and the pH adjusted to 7.35 with NaOH and osmolarity adjusted to 325 mOsm with sucrose to form the final Krebs bath solution. The bath solution was saturated with 95% O2 and 5% CO2 during experiments, and the solution was only used in the same day. All patch-clamp recording experiments were performed with the temperature of Krebs bath solution at 23°C.
An enzyme solution was used to help removing tissues that covered the Merkel cell layer in a hair follicle. The enzyme solution contained 0.05% dispase II plus 0.01% collagenase. The solution was prepared freshly with Krebs bath solution and used in the same day. Quinacrine stock solution (0.3 mM) was made in de-ionized distilled water and stored at 4°C. The solution was diluted 1000 times with Krebs bath solution to the final concentration of 0.3 μM and used in the same day. Lentiviral particles that carry Piezo2 shRNAs were obtained from Santa Cruz Biotechnology, Inc.
Dissect whisker hair follicles from rat whisker pads ● TIMING ~30 min
Prepare hair follicles for in situ patch-clamp recordings from Merkel cells ● TIMING ~45 min
Label Merkel cells and pre-identify them for in situ patch-clamp recordings ● TIMING ~20 min
Although Merkel cells are mainly located in the outer root sheath in the enlargement part of a whisker hair follicle, many other cells including keratinocytes are also present in this region and intermingled with Merkel cells. When observed under microscope with a 40X objective in the bright field, Merkel cells and other cells are not distinguishable morphologically in the whisker hair follicles (Figure 2H). Therefore, one cannot assume that a recording made from a cell in this region is from a Merkel cell. To solve this problem, we labeled Merkel cells using quinacrine, a fluorescent dye that could specifically vital-stain Merkel cells . Whisker hair follicle preparations were incubated with 0.3 μM quinacrine in the Krebs bath solution for 15 min to vital-stain Merkel cells. After the staining, the preparations were continuously perfused with the Krebs bath solution at a flow rate of 1.5 ml/min. Quinacrine-labeled cells (Merkel cells) were identified (Figure 2I) using a fluorescent imaging system. The fluorescent imaging system was controlled by the MetaFluor Imaging System software to acquire images with short exposure time (~200 ms). The short exposure time is required because quinacrine fluorescent intensity in Merkel cells would drop rapidly to the undetectable level with a prolonged exposure. Observing quinacrine-labeled Merkel cells through the eye pieces of a microscope should be avoided because this would lead to the photo bleach of quinacrine fluorescence due to the prolonged UV exposure. To perform patch-clamp recordings, the acquired digital fluorescent image that had Merkel cells (Figure 2I) was first displayed on a computer monitor. In the meantime the live image for the same field was viewed under bright field to identify Merkel cells (Figure 2H), and then the identified Merkel cell was approached by a patch-clamp electrode.
Perform in situ patch-clamp recordings from Merkel cells ● TIMING 45 min
We used a P-97 Brown-Flaming Micropipette Puller to make recording electrodes for in situ patch-clamp recordings from Merkel cells. Our recording electrodes usually had a resistance of ~8 MΩ, about 2 times higher than the resistance of whole-cell patch-clamp recording electrodes used for other cells. The use of high-resistant electrodes facilitates the formation of gigaohm seal between the recording electrodes and Merkel cell membranes. We found that it became difficult to form the gigaohm seal with lower resistant electrodes. Since the input resistance of Merkel cells is normally extremely high (>2 gigaohm), the use of the high-resistant electrodes would not yield intolerable voltage-clamp errors. Recording electrodes were filled with K+-based internal solution for both whole-cell current-clamp and whole-cell voltage-clamp experiments, and filled with Cs+-based internal solution for whole-cell voltage-clamp experiments.
We found that it was difficult to establish the whole-cell configuration by manually applying a pulse of negative pressure, a commonly used method to rupture the membrane patch for whole-cell recordings. In our experiments, a constant low negative pressure of about −45 mmHg was kept in the recording electrode by using the HSPC device. In the meantime, electric pulses (200 ms each) were applied to rupture Merkel cell membranes and establish whole-cell configuration. The magnitude of electric pulses started from 200 mV with an increment of 25 mV until breaking into whole-cell configuration. For most recordings, whole-cell configuration was established with the electric pulses of 300–450 mV. An increase of capacitive transient in membrane seal tests is an indicator for the formation of the whole-cell configuration (Figure 3C left panel), but this change could be overlooked because the capacitive transient increase was small. The reason for the small change in membrane seal tests was because Merkel cells had very high membrane input resistance (>2 GΩ) and very small whole-cell membrane capacitance (~4 pf). To further confirm the formation of whole-cell configuration, we usually examined membrane responses to a series of voltage steps in the voltage-clamp mode (Figure 3D) or to a series of current steps in the current-clamp mode (Figure 3E). In the voltage-clamp mode, prominent outward currents could be evoked in response to depolarizing voltage steps if the whole-cell configuration was established. In the current-clamp mode, a resting membrane potential of −40 mV or more negative was a good indication of the formation of whole-cell configuration. Firing regenerative action potentials in response to the injection of depolarizing currents (40 pA to 80 pA) was another clear indication of the establishment of whole-cell configuration (Figure 3E). In some recordings, abortive potentials rather than regenerative action potentials were observed. The fail of showing regenerative action potentials in a Merkel cell was most likely due to an unhealthy condition of the cell. In addition, an imperfect whole-cell mode, either due to a partial break-in or partial membrane reseal, could also be a reason for the fail of observing regenerative action potentials in Merkel cells.
Apply mechanical stimulation and record mechanically activated currents ● TIMING 60 min
Hair movement is a natural tactile stimulus. We applied this natural tactile stimulus to evoke MA currents from Merkel cells in our hair follicle preparation. In preparing the setup for delivering stepwise hair movement, a 26-gauge injection needle was bent into an “L” shape and was used as a holder of whisker hair. The needle was stably attached to the piezo device and a whisker hair shaft was then inserted into the tip of the needle. The hair shaft was tightly against the inside wall of the needle so that no dead space was present between hair shaft and the wall of the needle. Hair shaft was deflected by stepwise needle movements driven by the piezo device (Figure 4E). As shown in Figure 4F, hair movement elicits rapidly adapting MA currents from Merkel cells as well.
Perform intra-follicle microinjection for knockdown of tactile transducers TIMING ~45 min
The protocol presented here has been successfully used by us in a recent study that demonstrates Piezo2 channel-mediated tactile transduction in Merkel cells of rat whisker hair follicles .This protocol can also be used for future studies to further understand 1) the transduction and encoding of tactile signals at Merkel discs, 2) the transmission of tactile signals from Merkel cells to afferent nerves, and 3) the regulation of tactile transduction, encoding and transmission at Merkel discs.
There are several advantages for the use of our in situ patch-clamp recording technique to study tactile transduction in whisker hair follicles. First, we used freshly harvested whisker hair follicles rather than dissociated Merkel cells. Merkel cells in our whisker hair follicle preparations remain intact and are in an in vivo-like condition. This avoids a potential change of tactile transduction. It has been known that receptor expression in a cell often becomes altered after the cell is dissociated and grown in culture. Previous studies have shown that dissociated Merkel cells lose their processes , the sites where mechanical transducers may reside in . Indeed, dissociated Merkel cells failed to respond to mechanical stimulation in previous studies . Interestingly, dissociated Merkel cells grown in a culture condition were reported to be mechanically sensitive in two recent studies [21,22]. Second, by using our whisker hair follicle preparations, we are able to apply several different types of mechanical stimuli to study mechanical transduction in Merkel discs of whisker hair follicles. These three stimuli are direct and indirect mechanical stimulation as well as hair movement. Importantly, since hair movement is a natural tactile stimulus, the MA currents following hair movement shown in our study thus directly illustrates tactile transduction. In dissociated Merkel cells, mechanical stimulation was achieved by direct membrane displacement with a glass probe. This stimulation paradigm does not allow one to define MA currents in Merkel cells as tactile transduction currents. Third, in our in situ patch-clamp recordings from Merkel cells in whisker hair follicles, we found that Merkel cells were able to fire regenerative Ca2+-action potentials in a slowly adapting manner . On the other hand, regenerative action potentials were not demonstrated in dissociated Merkel cells [21,22,28]. The discrepancy may suggest that dissociation process and/or culture conditions alter the expression of some voltage-gated ion channels that are essential for firing regenerative action potentials in Merkel cells. The firing of Ca2+-action potentials in Merkel cells in response to tactile stimulation provides some insights into initial tactile encoding downstream to tactile transduction. More needs to be done using our in situ patch-clamp recordings to further study tactile transduction, encoding and transmission in whisker hair follicles.
In the present protocol we also describe in details the procedures for intra-follicle microinjection of Piezo2 shRNA lentiviral particles to knockdown Piezo2 channel expression in Merkel cells. As reported in our recent paper, the approach effectively knocked down Piezo2 in Merkel cells to result in a significant decrease of Merkel cell MA currents. The technical advantage of this approach is that Piezo2 shRNA lentiviral particles microinjected into a whisker hair follicle is restricted in the injected whisker hair follicle due to the insulation of its capsule. This allows highly efficient and tissue specific knockdown. This approach may also be used to knockdown other proteins of interest in Merkel cells (e.g. TRP channels) to study their potential roles in tactile transduction, encoding, and transmission in whisker hair follicles.
In addition to Merkel discs, other tactile end-organs such as Ruffini’s corpuscles, Pacinian corpuscles, and Meissner’s corpusles are involved in tactile transduction. The transduction mechanisms in these tactile organs remain to be elucidated. Our success in performing patch-clamp recordings from Merkel discs may also give a hope for the use of this technique (with modification) to study tactile transduction in these tactile end-organs.
This study was supported by NIH grants DE018661 and DE023090 to J.G.G.
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