An ex vivo preparation of mouse pinna for recording afferent responses from guard hair follicles of hairy skin.

Banks, R.W.; Cahusac, P.M.; Watson, S.; Bewick, G.S.

Abstract

In our studies on synaptic-like vesicles (SLVs) in mechanosensory endings we have mainly used isolated nerve-muscle preparations, either of rat lumbrical or mouse soleus, both for recording muscle-spindle responses to stretch and for labelling SLVs with the fluorescent styryl dye FM1-43 (Bewick et al, 2005). Recently we extended our observations on FM1-43 labelling to the palisade endings of guard hair follicles of the external ear (pinna) of the mouse (Singh et al, 2009). We now report our development of the isolated pinna as a preparation for recording the afferent activity of palisade endings. Adult mice (C57/Bl6J or Balb/C; 20-35gm) were humanely killed by CO2 narcosis (ASPA schedule 1 method). Skin was removed near the pinnae, exposing the cartilage of the external auditory meatus (EAM) and the trigeminal (mandibular branch, MDV) and cervical (great auricular) innervation of the concave (anterior) and convex (posterior) aspects of pinna skin respectively. EAM was opened anteriorly and the pinna was flattened and pinned out, anterior skin down, onto a silicone-lined dish (Sylgard) filled with gassed (95 % O2, 5% CO2) Liley's fluid. Posterior skin and pinna cartilage were removed by blunt dissection, leaving the anterior skin. Branches (usually 2) of the MDV emerge posteriorly between the pinna and EAM cartilages. They were cleaned and inserted into a glass pipette suction electrode. Indifferent suction-electrode impedance was balanced using areolar connective tissue and the bath was earthed to the screen of the recording-electrode cable. Electrical activity was differentially amplified ( Neurolog NL104), filtered (Neurolog NL125 band-passed 0.2 - 2 kHz), and recorded using a CED 1401micro interface and Spike 2 software (Cambridge Electronic Design, UK) that also controlled a ceramic piezo-electric actuator (PZT507, Morgan Electro Ceramics, UK) fitted with a fire-polished 10 cm borosilicate microelectrode glass capillary for mechanical stimulation (3 s at 5 Hz sinusoidal every 30 s) of 1 or 2 hairs. Part of the adipose layer was removed making a window to the base of the skin to access follicles for observation or drug application. To stimulate the hairs of these follicles the edge of the pinna was folded back leaving a small saline-filled gap between the apposed skin layers. Advantages of the preparation are: there seem to be no down hairs on the anterior skin; guard hairs are sparse compared to most of the pelage; and whereas most possess a palisade ending few, if any, have other types of presumed low-threshold ending. Thus there is little spontaneous activity allowing the evoked responses from the 1 or 2 stimulated follicles to be readily detected. We confirmed that guard-hair palisade endings are rapidly adapting and found that action potentials show a strong tendency to be locked to a specific phase of the sinusoidal movement.

Citation

Banks, R., Cahusac, P., Watson, S., & Bewick, G. (2012). An ex vivo preparation of mouse pinna for recording afferent responses from guard hair follicles of hairy skin.

• Conference Name: Physiology 2012
• Conference Location: Edinburgh
• Publication Date: 2012
• Deposit Date: Jan 5, 2015
• Volume: 27
• Series Title: Proceedings of the Physiological Society
• Publisher URL: http://www.physoc.org/proceedings/abstract/Proc%20Physiol%20Soc%2027PC264