GABA(B), opioid and alpha2 receptor inhibition of calcium channels in acutely-dissociated locus coeruleus neurones

Abstract

1. The effects of GABA(B), opioid and alpha2 receptor activation on different subtypes of calcium channels in acutely-dissociated rat locus coeruleus (LC) neurones were investigated using whole-cell patch clamping. 2. Barium currents through calcium channels could be fractionated into four classes: L-type (nimodipine-sensitive), N-type (omega-conotoxin GVIA-sensitive), P/Q-type (omega-agatoxin IVA-sensitive) and R-type (remaining in the presence of all three blockers). The percentage of each was, respectively, 25+/-2, 34+/-1, 28+/-3 and 12+/-1% (mean+/-s.e.mean, n=4). 3. The GABA(B) receptor agonist, baclofen, and the opioid receptor agonist, enkephalin, partially inhibited the total barium current in a concentration-dependent manner with EC50 values of 2 and 0.3 microm , respectively. Maximal inhibition was 17+/-1% (n=38) for baclofen and 30+/-2% (n=20) for enkephalin. The alpha2-adrenoceptor agonist, UK14304 (10 microM), also inhibited barium current in these neurones (28+/-2%, n=11). The agonists did not shift the current-voltage relationship along the voltage axis. 4. Maximal baclofen inhibition of different calcium channel subtypes was 9+/-7% (L-type, n=4), 11+/-8% (N-type, n=4), 26+/-6% (P/Q-type, n=4), and 6+/-5% (R-type, n=5). The corresponding values for enkephalin inhibition were 5+/-9% (L-type), 30+/-11% (N-type), 37+/-9% (P/Q-type), and 17+/-8% (R-type). 5. In the presence of a saturating concentration of enkephalin, baclofen produced additional inhibition of the barium current. In contrast, in the presence of a saturating concentration of enkephalin, UK14304 produced no further inhibition of the barium current. 6. These results indicate that neuromodulation of calcium channels in LC neurones involves a complex pattern of overlapping and distinct second messenger pathways. Regulation of LC neuronal firing activity by the modulation of calcium channels may be important for LC-mediated behaviour such as alertness and vigilance.

Figure 1

Four different subtypes of calcium channels can be distinguished in acutely-dissociated LC neurones. (a) Example of a barium current recorded during a test pulse to 0 mV from a prepulse of −90 mV (Control). This current was completely blocked by 100 μM cadmium (Cd²⁺). The pulse protocol is shown above. Linear leak currents and capacitance transients have been subtracted using a P/4 protocol. (b) Current-voltage plot of barium current measured in one cell (same as in a). (c) Plot of peak amplitude of barium current at 0 mV versus time during the experiment. Horizontal bars indicate periods of perfusion with nimodipine (Nimo, 3 μM), ω-conotoxin GVIA (CTx, 1 μM), ω-agatoxin IVA (Aga, 500 nM) and Cd²⁺ (100 μM). Numbers 1–5 identify the example traces shown in (e). (d) Summary of the results of a series of four experiments as in (c), showing the fraction of current mediated by each of L-, N-, P/Q- and R-types of calcium channels. Error bars are ±s.e.mean. (e) Example currents at the indicated time points in the experiment shown in (c). (f) Same traces as in (e); asterisks indicate that the traces are normalized to show the similarity of time courses.

Figure 2

Baclofen (Bac) acts via GABA_B receptors to inhibit barium current in a concentration-dependent manner. (a) A typical barium current measured in control solution and after adding a saturating concentration of baclofen (100 μM). (b) Current-voltage (I-V) plot for the same cell as in (a), showing that baclofen does not cause a shift of the I-V plot along the voltage axis. (c) The inhibitory effect of baclofen (10 μM) is blocked by addition of a GABA_B receptor antagonist, CGP56999 (CGP, 100 nM). (d) Time course plot for peak barium current at 0 mV, in the indicated concentrations of baclofen. (e) Summary concentration-response for a series of experiments as in (d). The ordinate is expressed as percentage of maximal inhibition. The smooth curve is a fit of a logistic function, giving an EC₅₀ of 2 μM. The numbers indicate n.

Figure 3

Enkephalin (Enk) acts via opioid receptors to inhibit barium current in a concentration-dependent manner. (a) Barium current measured in control solution and after adding a saturating concentration of enkephalin (100 μM). (b) Current-voltage plot for the same cell as in (a), showing the lack of shift along the voltage axis following addition of enkephalin. (c) Time course plot for peak barium current at 0 mV, in the indicated concentrations of enkephalin. (d) Summary concentration-response plot for a series of experiments as in (c). The ordinate is expressed as percentage of maximal inhibition. The smooth curve is a fit of a logistic function, giving an EC₅₀ of 0.3 μM. The numbers indicate n. (e) Barium current measured in control solution and after adding a saturating concentration of UK14304 (UK, 10 μM). (f) Current-voltage plot for the same cell as in (e).

Figure 4

Baclofen (Bac) and enkephalin (Enk) inhibited different calcium channel subtypes to varying extents. (a) Example traces for the inhibitory effect of baclofen (100 μM, top panels) or enkephalin (100 μM, bottom panels) on the pharmacologically isolated subtypes of calcium channels. (b) Summary of the results of experiments as in (a). Inhibition of pure L-type currents was estimated by subtracting the measured inhibition of R-type currents (Nimo, CTx and Aga present) from that measured for L-type plus R-type currents (CTx and Aga present). A similar method was used to estimate the inhibition of pure N- and P/Q-type currents. A disadvantage of this subtraction procedure is that the error bars (±s.e.mean) are increased. Statistically significant differences in inhibition are indicated by the asterisks (P<0.05). n=5 for R-type measurements, n=4 for all others.

Figure 5

Saturating concentrations of baclofen (Bac) and enkephalin (Enk) produced additive inhibition of barium currents, whereas enkephalin and UK14304 (UK) produced mutually occlusive inhibition. (a and b) Upper panels. Time-course plots for peak barium currents at 0 mV showing the additive (left) and occlusive (right) effects of the indicated agonists. Lower panels. Example currents at the indicated time points on the time course plots. (c and d) Summary of the results of four experiments as in (a and b). The effects of baclofen and enkephalin were partially additive, whereas those of enkephalin and UK14304 were fully occlusive. Asterisks represent a significant difference (P<0.05) between baclofen alone versus baclofen plus enkephalin, and enkephalin alone versus enkephalin plus baclofen.