Bradykinin Research
Neuronal Classification
In order to ensure that all of the neurones obtained were the relevant neurones
involved in the trigeminal nerve involvement in migraine, the polymodal C-Fibre,
a number of measurements were taken. The neurones were initially chosen due
to their small size, as this is a good indication of their being polymodal
C-Fibres. This was quantified and further tests were carried out to identify
the neurones’ properties and characteristics before further research.
The measurements taken are explained and analysed below.
- Diameter
- The somal diameter was measured (in µm - micrometers) in order to
aid the classification of the neurones studies. C-Fibres (polymodal nociceptive
neurones) are generally of small somal size (approx. 25mm); A
(delta) fibres tend to be of intermediate size (approx. 36mm); A
(beta) fibres are the largest, with a size of 47µm.
The axon diameters of the 18 neurones tested ranged between 19 and 30, the average being 25.17. As such, the diameters measured of these 18 neurones suggest they were all likely to be Polymodal C-Fibres - Input Resistance
- Input Resistance, measured in M
,
(Megaohms), can also been an indication for the type of fibre being studied.
A higher resistance will indicate a small diameter neurone and a lower resistance
will indicate a larger neurone. Of the 18 neurones the input resistance
ranged from 27 to 338 M,
the average being 153.03 M,
suggesting that they were C-Fibres. - Capacitance
- Capacitance, measured in pF (pico Farads), also indicates neuronal type. Small neurones will tend to have a smaller capacitance (all other factors being equal) and a larger neurone will have a larger capacitance. For the capacitance, 2 of the neurones were not measured, the remaining 16 ranged between 15 & 91 (Average = 29.69), suggesting that they were C-Fibres.
- Spontaneous Firing
- Spontaneous Firing was checked for as this can complicate any measurements made (i.e. A stimulus may appear to produce firing but it may also be due to spontaneous firing and not the stimulus). This was recorded as 0 = no spontaneous firing and 1 = spontaneous firing present. None of the neurones exhibited spontaneous firing before the administration of any chemicals.
- Humps
- A hump is a feature seen in the repolarisation or falling phase of the action potential. Humps are not known to be connected with any specific physical differences, nor do they appear to have any effect on neuronal behaviour but this will be examined in these results; it is thought to be caused by activation of T-Type Ca2+ channels. These were recorded as 0 = no hump and 1 = hump present. Out of the 18 neurones examined 14 displayed a characteristic hump. The effects of the hump will be examined later.
- Sag
- Injection of a negative current can produce a hyperpolarisation of the membrane potential that is known as a time-dependent depolarisation or ‘sag’. Sag is indicative of a holding current (Ih). These were recorded as 0 = no sag and 1 = sag present. 12 out of the 18 neurones displayed this characteristic; the significance of this will be analysed later.
- TTX sensitivity
- TTX (Tetradotoxin) is a toxin that affects some subtypes of sodium channels.
Nociceptive specific channels Nav1.8/1.9 are resistant to the blocking affects
of TTX. Hence, testing TTX sensitivity can give an indication of the neurones
sodium channels and hence its type. TTX effects can be complete blocks or
just a shift in the threshold and response of the neurone. No effect was
recorded as 0, a shift effect as 1 and a complete block as 2.
2 neurones were not affected by TTX, 10 experienced a shift in neuronal response and the remaining 6 were completely blocked. Those displaying TTX sensitivity are certain to be nociceptive in nature, the 2 not responsive may be nociceptive but this test did not clarify it either way. - Capsaicin Sensitivity
- Capsaicin, a chemical mediator that is the active ingredient in chilli peppers, acts on receptors that are specific to nociceptive neurones. The receptors (predominately VR1) are directly coupled to non-selective cation channels, which are highly permeable to Calcium (Ca2+). It stimulates and sensitises nociceptive neurones only. 4 neurones did not respond to capsaicin, 2 had a moderate response and the remaining 7 had an extreme response to capsaicin. Those responsive to capsaicin are certain to be nociceptive in nature.
Out of the four not responsive to capsaicin, none were the same as those
not responsive to TTX. When taken in conjunction with all other measurements
taken for neuronal classification it is highly likely that all 18 neurones
tested were Polymodal C-Fibres.
The variables outlined above were tested before the application of bradykinin
or other chemicals
Graph showing reaction to Capsaicin. Click the thumbnail above for a larger
version.
Graph showing example of a Sag. Click the thumbnail above for a larger version.
Graph showing an example of a Hump. Click the thumbnail above for a larger
version.
