The overall goal of this procedure is to demonstrate a quick and simple method to combine live cell imagining and electrophysiological recording, in notoriously difficult to access, mechanically sensitive, neuron terminals. This method can help address key questions in mechanosensation. Such as, the role of synaptic-like vesicles or the pharmacology of candidate mechanotransducer channels in fully differentiated terminals, in situ.
The main advantage of this technique, is that it is so simple, yet gives visual access to fully differentiated terminals for physiological investigation. Although this method is focused on hair follicle afference, It can also give insight into other lower threshold mechanosensory systems, such as muscle spindles, merkel sensory terminals, and baroreceptors. The implications of this technique extend towards developing new drug targets to regulate the mechanical sensors, including therapy of abnormal blood pressure regulation.
After decapitating a young adult mouse, place the head dorsal side up, in gassed Liley's solution, in a 50 centimeter wide bottom dish, under a stereo dissection mircoscope. About every 10 minutes, thoroughly rinse the dissection area with gas silane. Now, carefully incise and separate the skin at the base of the pinna using a spring bow scissors and a number three forceps.
Expose the innervation and cartilage of the external auditory meatus. Identify the branches of the mandibular division of the trigeminal and great auricular nerves as the emerge from the skull in the cleft of the mandibular joint. These two nerve branches project through the cartilage to innervate the concave and convex aspects of pinna skin.
Then, in the grove between the jaw and the mastoid process, identify where the trigeminal nerve branches exit. Expose their lengths by gently pulling the pinna away from the skull. Once thus exposed, cut the nerves as close to the skull as possible.
Next, use spring bow scissors to remove the pinna from the head. Avoid cutting the distal stumps of the divided nerves and minimize the amount of dense hair. Transfer the pinna to a silicone rubber lined dish filled with gassed Liley's solution.
Now, open the external auditory meatus by dividing it at its narrowest point. Then, flatten the pinna with the concave side down and pin down that side along the margins with regularly spaced, very fine insect pins. Next, completely remove the exposed dorsal skin from the pinna and the underline cartilage by blunt dissection using scissors.
Do not damage the anterior skin during this step. Then, using forceps, grip a fine insect pin and gently extend the transected branches of the mandibular division that emerge posteriorly between the skin, and external auditory meatus cartilage. Secure these nerve branches to the plate with the finest possible pins.
Impaling the connective tissue, adjoining their cut ends, and not the nerve chunks themselves. Complete the tissue preparation by removing most of the surrounding connective tissue from around the nerve branches, but do not damage the nerves with excessive pulling or cutting. In order to treat the hair follicle mechanosensory endings with vital dyes or drugs, carefully peel away the foamy fibroelastic cartilage of the subdermal adipose layer along five millimeters of the pinna margin.
Thus, open a square window, exposing the dermis and base of the hair follicles. Using a young mouse minimizes the adhesion between the anterior and posterior skin layers and the difficulty of removing the underlying cartilage. Both of these with increase the likelihood of seeing successful staining.
Being by transferring the preparation to the silicon-lined base of a recording chamber. Using fine insect pins, secure the ear around the edges, placing the clean nerves of the base of the ear near two suction electrodes. One for recording and the other, an indifferent electrode, to provide the neutral signal to a differential amplifier.
For the recording electrode, carefully match the aperture and internal diameter of the opening to the thickness of the nerve selected for recording. Once done, this will usually be constant for any series of experiments. A good length of nerve needs to fit snugly inside.
To draw the nerve into the electrode, use gentle suction from a two milliliter syringe, attached via silicone tubing. In the recording electrode, ensure the nerves are straight and not folded or doubled up, by gently withdrawing and reinserting the nerve with a fine insect pin during suction. Now, suck in some surrounding connective tissue or a soft, pliable adipose tissue, to form a water tight seal in the electrode orifice.
Developing a balanced impedance between the electrodes is essential for high quality, deferential recording. Critical to this is developing a high resistance, water tight seal around the nerve. Next, fill the indifferent electrode with saline by suction, if not already filled by spontaneous capillary action.
Then, place identical silver recording wires into the internal bore of the recording and indifferent electrodes. At the other end, the wires should be connected to different cores of a two core screen cable. Ensure the wire in the recording electrode contacts the saline and is near the nerve but without disrupting the seal.
Get the wire into the end of the indifferent electrode. Next, place the ground electrode, a silver-silver chloride pellet, into the bath. Attach this electrode to the screen of the two core cable connected to the recording electrode.
View their individual signals on a oscilloscope to check that the two channels'electrical noise levels are similar. Spontaneous action potentials in the recording electrode might not be visible until the two electrodes are balanced. To correct background noise differences, increase the impedance of one channel by sucking adipose connective tissue further into the electrode.
Once the electrodes have balanced backgrounds, switch to differential recording and look for spontaneous action potentials or invoke action potentials by stroking the hairs at the margin of the pinna with a fire polished glass rod. Next, record an electroneurogram via the computer interface. And feed the neurogram through a sound system to make the spiking audible just above the baseline noise.
This auditory feedback relay is very useful when conducting experiments. Next, confirm the innervation of the follicles near the cleared area is intact by manually stimulating the hairs. Now, fold over one to two millimeters of the margin of the ear skin at the level of the adipose cleared window to expose the underlying hairs.
Leave a saline filled gap between the opposed skin layers. While listening carefully, gently stroke the hairs protruding along the folded edge with a glass rod. Do not touch the skin.
As necessary, check the oscilloscope to evaluate the output. To make controlled recordings of stimulus-evoked action potentials, use a mechanical probe of fire-polished, ten centimeter borosilicate microelectrode glass attached to a ceramic piezo-electric actuator. Position the probe so it moves parallel to the skin-fold and touches the hairs, not the skin.
Put it about one-half to one millimeter above the skin surface. Then, manually verify the stimulation by listening for audio output when the probe moves. Now, set up the software to drive mechanical stimulation of a small area of hairs.
For example, program three second simulations at five hertz sinusoids every ten seconds. This requires a probe displacement between 200 and 2, 000 microns. Then proceed using repeated automated stimulation to check consistency of recorded responses in the nerves.
Repeatable results should be achievable. When the results are consistent, drop the repeat rate to every 30 seconds. Further instructions are provided in the text protocol.
To investigate the properties of the mechanosensory channels and label the mechanosensory terminals around the hair follicles add ten micromolar FM1-43 to the solution, and leave for 60 minutes. While continuing to stimulate the hairs with the oscillating probe. This dye blocks candidate transducer channels and sensory neurons in culture and in cochlea hair cells.
However, it does not block the firing in mature hair follicle afference in situ. The labeling of the endings shows the dye access the terminals which continue to respond to mechanical stimulation throughout dye exposure. Electroneurograms made using the described protocol typically showed ongoing action potential activity even in the absence of manual stimulation, including action potentials of the largest size.
Spontaneous activity occurred at about 10-20 spikes per second, showing no structure or signs of tonicity. The auto-correlation for the intervals between the periods of stimulation were always quite flat. During five hertz, 500 micron mechanical stimulation of the hair shafts the overall output typically rose to around 50 spikes per second.
Construction of cycle histograms revealed strong entrainment of the responses, with four to ten spikes per sinusoidal cycle. This was quite repeatable, of course, the phase of the waveform at which the responses were locked depended upon where the vibrating probe contacted the hair and its direction of movement at that point. Hair follicle lanceolate nerve ending channel pharmacology was then explored.
The channels continued to respond even when exposed to FM1-43, which can block some mechanosensory channels, such as those in sensory neurons in culture or cochlea hair cells ex vivo. Once mastered this technique up to the start of recording electroneurograms, can be done in 60 to 90 minutes. Whilst attempting this procedure, its important to develop high resistance electrical contract in the electrodes and if combining with visualizing follicles to use small mice to improve dye penetration and optical clarity.
We adapted the idea for this method from one originally developed by a close colleague and now retired professor, Clarke Slater and his MSc student Nakul Kain, when we told them of the possible role for synaptic-like vesicles in mechanosensation in the muscle spindle, and of the need for a better method of visualizing them with FM1-43. Following this procedure with the methods such as pharmacology and vital dye labeling can be performed to answer such questions as which are the mechanotransducer channels and differentiated terminals in situ and whether or not mechanical stimulation can modulate dye uptake and release by regulating synaptic-like vesicle recycling. Don't forget that FM1-43 has not been evaluated for its toxicity and should be treated as if it is hazardous.
