The actions of anandamide on rat superficial medullary dorsal horn neurons in vitro

Abstract

Whole-cell patch-clamp recordings were made from neurons in the trigeminal nucleus caudalis and trigeminal ganglion, in vitro, to investigate the cellular actions of the endogenous cannabinoid, anandamide. Anandamide has been shown to act through both the cannabinoid receptor 1 (CB1) and the vanilloid receptor 1 (VR1). Anandamide (30 microM) caused a 54 % increase in the rate of miniature excitatory post-synaptic currents (mEPSCs), without affecting their amplitude. The effect of anandamide was blocked by the VR1 antagonist capsazepine (20 microM), but not by the CB1-specific antagonist AM251 (3 microM). Application of the VR1 receptor agonist capsaicin (300 nM) caused a 4200 % increase in the mEPSC rate. In dissociated trigeminal ganglion neurons, both anandamide and capsaicin caused an outward current in neurons that were voltage clamped at +40 mV. The maximal outward current produced by anandamide (EC50, 10 microM) was 45 % of that produced by capsaicin (10 microM). Co-application of the VR1 antagonist capsazepine (30 microM) completely reversed the effects of both capsaicin and anandamide. The anandamide transport inhibitor, AM404 (30 microM) caused a 40 % increase in mEPSC rate in the slice preparation and an outward current in dissociated neurons. The latter current was reversed by the VR1 antagonist iodoresiniferatoxin (1 microM). The fatty acid amide hydrolase (FAAH) inhibitors phenylmethylsulfonyl fluoride (PMSF) (20 microM) and OL53 (1 microM) did not enhance the effect of anandamide in either the slice or dissociated neuron preparations. These results suggest that within the superficial medullary dorsal horn, anandamide (30 microM) acts presynaptically to enhance the release of glutamate via activation of the VR1 receptor.

Figure 1. Anandamide increases the mEPSC rate without altering the amplitude

A, time plot of mEPSC rate showing an increase after application of anandamide (in emulsion; AEA; 30 μm) and then following application of capsaicin (300 nm). Inset shows the same plot on a different scale to demonstrate the magnitude of the capsaicin response. B, traces of raw data showing mEPSCs in control and following superfusion of anandamide, capsaicin or CNQX (5 μm). C, normalised cumulative histograms of mEPSC amplitude in the absence (ctrl; continuous line) and presence of anandamide (AEA; dotted line). Inset shows averaged mEPSCs before (ctrl; continuous line) and during application of anandamide (AEA; dashed line). D, graph showing the mEPSC rate before (control) and during (AEA) application of anandamide. Data in A-C are taken from one neuron.

Figure 2. The VR1 antagonist capsazepine attenuates the effects of anandamide

A, time plot of mEPSC rate in slices pre-treated with capsazepine (20 μm). The addition of anandamide does not significantly change the mEPSC rate. Note that the capsaicin-induced increase in rate is significantly reduced by capsazepine. B, cumulative plot analysis of mEPSCs in the same neuron showing the peak amplitude; inset shows averaged mEPSCs before (control; continuous line) and during application of anandamide (AEA; dotted line). C, pooled results demonstrating that anandamide has no effect on the mEPSC rate in cells pre-treated with capsazepine.

Figure 3. Effects of agents on the action of anandamide on mEPSC frequency

Pooled data showing the increase in mEPSC frequency in the presence of anandamide (30 μm) and the CB1 antagonist AM251 (3 μm) (A), methanandamide (30 μm) (B), anandamide (mAEA; 30 μm) and the FAAH inhibitor PMSF (20 μm) (C) and the anandamide transport inhibitor AM404 (30 μm) (D). There was no mean mEPSC rate increase in the presence of anandamide and the FAAH inhibitor PMSF.

Figure 4. Anandamide does not alter the evoked EPSC amplitude

A, time plot of the evoked EPSC amplitude before and during application of anandamide (30 μm; AEA). Neither anandamide nor capsaicin (300 nm) altered the eEPSC. Baclofen (10 μm) causes a decrease in amplitude. B, averaged eEPSC before (control; thick line) and during application of anandamide (AEA; grey line), and after application of baclofen (10 μm; thin line). C, normalised averaged responses to identical paired stimuli (inter-stimulus interval, 70 ms), with the eEPSC₁ normalised (left) to demonstrate the lack of change in eEPSC₂ relative to eEPSC₁, following application of anandamide (AEA). Baclofen caused a facilitation of the eEPSC₂ relative to eEPSC₁.

Figure 5. Anandamide activates VR1 in dissociated trigeminal ganglion neurons

A, time plot of the VR1-mediated current at +40 mV illustrating the effects of anandamide (AEA; 10 μm and 100 μm) and the reversal of the 100 μm anandamide-induced current following the addition of capsazepine (30 μm). Also illustrated is the response of the cell to capsaicin (10 μm). B, concentration-response relationship for capsaicin (EC₅₀, 500 nm) and anandamide (EC₅₀, 10 μm) in trigeminal ganglion neurons. C, histogram showing that the outward current induced by AM404 (30 μm) can be reversed by the addition of the VR1 receptor antagonist iodoresiniferatoxin (1 μm). The fatty acid amide hydrolase (FAAH) inhibitor, OL53 (300 nm) has no significant effect on the anandamide-induced outward current, and causes only a small outward current when applied alone.