We have previously shown that injection of the P/Q-type (Cav2.1/α1A) calcium channel blocker, ω-agatoxin IVA, into the periaqueductal grey (PAG) facilitates meningeal stimulation-evoked trigeminal nociceptive processing (Knight et al. 2001). We sought to determine the mechanism of this pro-nociceptive action. Tonic descending nociceptive inhibition from the PAG to the spinal dorsal horn is modulated through local PAG GABAA receptors. GABAA antagonists in the PAG effectively block excitatory nociceptive transmission in the dorsal horn (Reichling, 1991). In this study we examined whether the inhibition of trigeminal responses mediated by PAG injection of the GABAA antagonist, bicuculline, is affected by P/Q-type calcium channel blockade.
All procedures in this study comply with UK Home Office regulations. Rats were anaesthetised (pentobarbitone 65 mg kg-1 I.P., maintenance with α-chloralose 15 mg kg-1 I.V.), injected with pancuronium (0.4 mg kg-1 I.V.) and artificially ventilated. Depth of anaesthesia was judged from the absence of withdrawal reflexes, and during neuromuscular blockade from the absence of gross fluctuations of blood pressure and heart rate. Rats were killed by pentobarbitone overdose.
The dura adjacent to the middle meningeal artery (MMA) was exposed and stimulated electrically in fifteen Sprague-Dawley rats. Extracellular recordings were made in the deep laminae of the caudal trigeminal nucleus from nociceptive neurons with dural input. A multi-barrelled microinjection unit was driven into the ipsilateral ventrolateral PAG (vlPAG) for injection of 50-400 nl of test drug. Neurons were classified as wide dynamic range (WDR, n = 13) and nociceptive specific (NS; n = 2) which showed latencies in the Aδ- and C-fibre range and showed a cutaneous receptive field within the first trigeminal division. Dural evoked responses were analysed before and after microinjection of test drugs into the PAG: (i) bicuculline (0.4 mM) injected, (ii) recovery from bicuculline, new baseline; (iii) agatoxin (0.1 µM) injected, (iv) new baseline; (v) bicuculline and agatoxin injected; (vi) recovery.
Drugs injected outside the PAG and saline injected into the PAG had no effect on dural responses. Bicuculline microinjection in the vlPAG produced an inhibition of dural responses to 59 ± 4 % of baseline response (mean ± S.E.M.; range 87-30% n = 14). Agatoxin produced a facilitation of trigeminal responses of 137 ± 6 % (mean ± S.E.M.; range 115-200 %, n = 15). Time to onset of facilitation after agatoxin was 5-10 min. Bicuculline and agatoxin administered together produced an inhibition of 63 ± 5 % (mean ± S.E.M.; range 82-25 %, n = 11) over the same time course and characteristics as observed with bicuculline alone. After this inhibition most dural responses returned to baseline at the increased level of activity produced by agatoxin alone.
These results show that bicuculline microinjected into vlPAG inhibits trigeminal responses. This response is unaffected by P/Q-type calcium channel blockade. These data suggest P/Q-type calcium channels in the PAG may be presynaptic to GABAA receptors (as in Purkinje neurons (Stephens et al. 2001). Alternatively, the pro-nociceptive effect of P/Q-type calcium channel blockade in the PAG may not be mediated by GABAA.With thanks to Simon McMullan, David Bulmer and Alex Gourine for technical advice.
- Knight, Y.E., Kaube, H., Bartsch, T. & Goadsby, P.J. (2001). Cephalalgia 21 (in the Press).
Reichling, D.B. (1991). The Midbrain Periaqueductal Gray Matter 1, 329-344.
Stephens, G.J., Morris, N.P., Fyffe, R.E.W. & Robertson, B. (2001). Eur. J. Neurosci. 13, 1902-1912.