Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicles

Samenvatting

Source: Bewick, G. et al., Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings. J. Vis. Exp. (2016).

This video demonstrates the combined recording of mechanically stimulated afferent output and nerve terminal labeling in the hair follicles of a mouse pinna. It outlines the steps involved in the mechanical stimulation of hair follicle nerve terminals before and after exposure to a fluorescent membrane dye, electrophysiology recording of the generated action potentials, and visualization of the nerve terminal labeling in the hair follicles.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Electrophysiological Recording Setup

1. Pin the mouse external ear (pinna) skin to the PDMS-lined base of a recording chamber with fine insect pins around the edges (~6-8 per ear), placing the cleaned nerves at the base of the ear near two suction electrodes – one for recording (to take the nerve) and the other an indifferent electrode (to provide the neutral signal to a differential amplifier).
2. For the recording electrode, carefully match the aperture and internal diameter of the op....

Resultaten

Figure 1: Electrophysiological Recording Arrangement and N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) Pyridinium Dibromide Labeling of Hair Follicle Afferent Lanceolate endings. (A) A pinna preparation set up for an electrophys.......

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
PDMS - Sylgard 184	Dow Corning		Flexible, inert, translucent solid silicone polymer.
No. 3 Dumont forceps	Fine Science Tools	11231-20
Austerlitz Insect pins	Fine Science Tools	26002-10	Very fine pins to attach pinna preparation securely to the PDMS with minimal damage.
AC Differential Preamplifier	Digitimer	Neurolog NL104A	Amplifying the size of the incoming afferent electroneurogram. Differential recording minimises the extraneous electrical noise and baseline drift.
High/Low-pass Filter	Digitimer	Neurolog NL125	Signal conditioning, by reducing extraneous electrical noise to ensure best signal to noise ratio.
Spike Trigger	Digitimer	Neurolog NL201	Sets the event detector threshold and displays it on the oscilloscope. This shows the action potential detection efficacy.
Audio Amplifier & speakers	Digitimer	Neurolog NL120S	Useful audio monitoring for the presencec of electrical firing of the sensory endings while adjusting the mechanical stimulation preparation down the microscope
Oscilloscope	Digitimer	PM3380A	We use this old model but any standard oscilloscope will suffice.
Piezo electroceramic wafer	Morgan Electroceramics, Southampton UK	PZT507	Electrophysiology/computer interface
Piezo electroceramic powersupply	Home made		0-200V DC output to drive the ceramic wafer displacement, with variable electronic control of output via recording/stimulation software and computer interface. We use Spike2 software and 1401micro computer interface.
Electrophysiology Software	Cambridge Electronic Design (CED)	Spike2 v7	Electrophysiology recording, stimulation and data analysis software
Laboratory interface	Cambridge Electronic Design (CED)	1401 micro	Electrophysiology interface, between the amplifier/filters and the computer. It inputs the electroneurogram and also drives the electroceramic movement.
FM1-43/Synaptogreen C4	Biotium/Cambridge Bioscience	BT70020	Fluorescent membrane probe that reversibly partitions into the outer leaflet of cell membranes. Used predominantly for monitoring vesicle membrane endo-/exocytosis.
Advasep 7	Biotium/Cambridge Bioscience	BT70029	A sulfonated b-cyclodextrin derivative that chelates FM1-43 (& other styryl pyridinium dyes) out of the exposed membranes, leaving internalised dye to be seen more clearly by lowering the background labelling/fluorescence.
Retiga Exi Fast 1394	Qimaging		Monochrome, cooled CCD camera - basic model
Volocity 3D Image Analysis Software	Perkin Elmer	Volocity 6.3	Image capture and analysis software.