Presynaptic mu and delta opioid receptor modulation of GABAA IPSCs in the rat globus pallidus in vitro

Abstract

The role of enkephalin and the opioid receptors in modulating GABA release within the rat globus pallidus (GP) was investigated using whole-cell patch recordings made from visually identified neurons. Two major GP neuronal subtypes were classified on the basis of intrinsic membrane properties, action potential characteristics, the presence of the anomalous inward rectifier (Ih), and anode break depolarizations. The mu opioid receptor agonist [D-Ala2-N-Me-Phe4-Glycol5]-enkephalin (DAMGO) (1 microM) reduced GABAA receptor-mediated IPSCs evoked by stimulation within the striatum. DAMGO also increased paired-pulse facilitation, indicative of presynaptic mu opioid receptor modulation of striatopallidal input. In contrast, the delta opioid agonist D-Pen-[D-Pen2, 5]-enkephalin (DPDPE) (1 microM) was without effect. IPSCs evoked by stimulation within the GP were depressed by application of [methionine 5']-enkephalin (met-enkephalin) (30 microM). Met-enkephalin also reduced the frequency, but not the amplitude, of miniature IPSCs (mIPSCs) and increased paired-pulse facilitation of evoked IPSCs, indicative of a presynaptic action. Both DAMGO and DPDPE reduced evoked IPSCs and the frequency, but not amplitude, of mIPSCs. However, spontaneous action potential-driven IPSCs were reduced in frequency by met-enkephalin and DAMGO, whereas DPDPE was without effect. Overall, these results indicate that presynaptic mu opioid receptors are located on striatopallidal terminals and pallidopallidal terminals of spontaneously firing GP neurons, whereas presynaptic delta opioid receptors are preferentially located on terminals of quiescent GP cells. Enkephalin, acting at both of these receptor subtypes, serves to reduce GABA release in the GP and may therefore act as an adaptive mechanism, maintaining the inhibitory function of the GP in basal ganglia circuitry.

Fig. 1.

Characterization of two GP neuronal subtypes.A, i, Superimposed voltage responses from a type I neuron in response to 300 msec hyperpolarizing currents steps (25 pA increments every 5 sec) from a resting membrane potential of −50.8 mV. This neuron was spontaneously active at 10 Hz.ii, Typical action potential from a type I neuron of 0.78 msec duration and short-lasting AHP of 29 mV amplitude.B, i, Voltage traces from a type II neuron in response to 300 msec current steps (in 25 pA increments every 5 sec) from resting membrane potential of −67 mV. Hyperpolarizing steps elicited time-dependent inward rectification of membrane potential. On removal of the step, there was an anodal break depolarization accompanied by action potential firing.ii, Representative action potential from a type II GP neuron of 0.95 msec duration and a long-lasting AHP of amplitude 27 mV.

Fig. 2.

Synaptic currents evoked by local stimulation.A, At a holding potential of −50 mV, single shock bipolar electrical stimulation evoked a fast inward current that was blocked by CNQX (10 μm) and dl-AP-5 (100 μm) and a slower outward current. Eachtrace is preceded by a 10 mV hyperpolarizing step of 10 msec duration to monitor input conductance and changes in access resistance. B, Time course from a single experiment showing the reversible block of outward synaptic potential by the GABA_A antagonist bicuculline (10 μm) applied to the superfusion medium for the period indicated by the bar. Each point represents the average of four traces evoked every 15 sec.C, The outward current reversed polarity at −69 mV, close to E_Cl.

Fig. 3.

Presynaptic depression of evoked striatopallidal IPSCs by μ opioid receptor activation. A, Time course showing the effect of the μ opioid receptor agonist DAMGO (1 μm) on the normalized IPSC amplitude evoked by stimulation of striatum in parasagittal slices in the presence of CNQX (10 μm) and dl-AP-5 (100 μm). DAMGO reduces the IPSC by 53 ± 6% (n = 7).B, Time course showing the effect of the δ opioid receptor agonist DPDPE (1 μm) on the normalized IPSC amplitude evoked by stimulation of the striatum in parasagittal slices in the presence of CNQX (10 μm) and dl-AP-5 (100 μm). DPDPE has no significant effect on the evoked IPSC (n = 5). However, the IPSC evoked in each of these five cells was inhibited by DAMGO (1 μm).C, Paired IPSCs recorded from the same cell in response to the same stimulation at an interval of 50 msec in control (i) and DAMGO (1 μm) (ii). iii, The pair of IPSCs recorded in the presence of DAMGO have been scaled so that the conditioning (first) responses with and without drug are of similar amplitude. The ratio of the test (second) response in DAMGO has increased, indicative of a presynaptic site of action. Paired IPSCs recorded in the presence of the μ opioid receptor antagonist CTOP (1 μm) (iv) and CTOP plus DAMGO (1 μm) (v), and ratio (vi). In the presence of CTOP, there was no depression of the conditioning IPSC by DAMGO and no change in the paired-pulse ratio. Currents resulting from a 10 mV hyperpolarizing step precede all IPSC pairs.

Fig. 4.

Enkephalin reduces GABA_A IPSCs. IPSCs recorded at a holding potential of −50 mV in the presence of CNQX (10 μm) and dl-AP-5 (100 μm).A, i, IPSCs (mean of 4 evoked every 15 sec) before (a) and during (b) the application of met-enkephalin (30 μm) and after wash (c).A, ii, Time course showing the effect of bath application of met-enkephalin (30 μm) on normalized IPSC amplitude. B, Paired IPSCs recorded from a type II GP neuron in control (i) and met-enkephalin (10 μm) (ii). iii, The pair of IPSCs recorded in the presence of met-enkephalin has been scaled so that the conditioning (first) responses with and without drug are of similar amplitude. The increase in ratio of the test (second) response is indicative of a presynaptic site of action of met-enkephalin. C, mIPSCs were recorded from chloride loaded cells (E_Cl, +1 mV) in the presence of TTX (1 μm), CNQX (10 μm), and dl-AP-5 (100 μm) at a holding potential of −80 mV. mIPSCs were collected over a period of 120 sec. The same time frame was taken 3 min after drug application.i, Control mIPSCs and those recorded after application of met-enkephalin (30 μm). ii, Cumulative frequency and amplitude distributions (derived from 120 sec of data) before and after met-enkephalin. Met-enkephalin reduced the frequency of mIPSCs (p < 0.01) but not their amplitude.

Fig. 5.

The δ agonist DPDPE rather than the μ opioid agonist DAMGO preferentially reduced IPSCs evoked by stimulation within the GP. A, Time course showing the effect of the δ opioid receptor agonist DPDPE (1 μm) (i) and the μ opioid receptor agonist DAMGO (1 μm) (ii) on normalized IPSC amplitude evoked by stimulation within the GP. DPDPE reduced the IPSC by 49.9 ± 6.7% (n = 8), but DAMGO only reduced the IPSC by 29 ± 9.8% (n = 6).B, Cumulative frequency (i) and amplitude (ii) distributions of mIPSCs before and after the application of DPDPE and DAMGO from two representative experiments. Both agonists reduced the frequency of the events (p < 0.01) without change in amplitude.

Fig. 6.

Depression of sIPSCs by activation of μ opioid receptors. A, sIPSCs in a single GP neuron recorded at a holding potential of −50 mV in the presence of CNQX (10 μm) and dl-AP-5 (100 μm). Bath application of met-enkephalin (30 μm) suppressed the frequency and amplitude of sIPSCs. This effect was replicated by the μ opioid receptor agonist DAMGO (1 μm), whereas the δ opioid receptor agonist DPDPE (1 μm) was without effect. sIPSCs were blocked by TTX (1 μm). B, Cumulative frequency distributions of sIPSCs before and after the addition of met-enkephalin (p < 0.05) (i), DAMGO (p < 0.05) (ii), and DPDPE (non significant) (iii). Data were collected over a period of 120 sec. Same cell asA.