Bradykinin Research
Practical Methodology
The trigeminal neurones studied were obtained from adult Wister rats and kept in culture, after dissociation, for up to 7 days prior to use. The rats, of 175-250g and of either sex, were killed in accordance with regulations via CO2 inhalation. Following dissection the cells were maintained in an F-12 culture media that was supplemented with nerve growth factor (mNGF 2.5S, 50 ng ml-1) and plated onto coverslips (22 mm) that were poly-D-lysine and laminin pre-coated.
The culture was kept at 37°C in a dehumidified atmosphere (5% CO2). The cells were periodically supplemented with Ara-C. The culture was then neurone enriched following visual morphological assessment. Neurones appearing to have a small (19-28 µm) somal diameter and processes suitable for electrophysiological examination were chosen. These are known to predominately be Polymodal C-Fibres and neuronal classification was later undertaken to ensure this.
The cells were then chopped and centrifuged to obtain a pure culture. The final culture was maintained in an F-12 medium, which was supplemented with an inhibitor of mitosis, to avoid growth of non-neuronal tissues. The final number of neurones tested was 18. Experiments followed the Whole-Cell Perforated Patch Clamp technique (which is explained in depth elsewhere on this site).
The patch pipettes were filled with a solution that contained (in mM): 55
KCl, 75 K2SO4, 5 MgSO4, 10 HEPES, 0.5 EGTA, pH 7.2 and had resistances of
~0.5 M
. Amphotericin
B was the antibiotic used to obtain perforation, after which the mean series
resistance = 5.0±0.7 M,
and neuronal capacitance = 25±1.4 pF. During experimentation neurones
were continually perfused with a solution comprising of (in mM): 135 NaCl,
3.5 KCl, 1 MgCl2, 2.5 CaCl2, 22.5 HEPES, 3.3 glucose, 0.01 % (w/v) BSA, pH
7.4.
Experiments were performed at 32°C and drug additions were made by bath
perfusion. Current injections were used in order to determine both active
and passive electrical properties of the neurones and to simplify recordings
of responses the resting membrane potential for all neurones was maintained
at ~-60 mV (by continuous current injection).
All of the results are indicated as representative readings, measurements
are given as the mean ±S.E.M (Standard Error of the Mean). Significance
was statistically determined using either Student’s t-test or 
[49, 50]
