Presynaptic inhibition by dopamine of a discrete component of GABA release in rat substantia nigra pars reticulata

Abstract

1. Whole-cell patch clamp recordings were made from substantia nigra pars reticulata (SNr) neurones in rat midbrain slices. Monosynaptic IPSCs were evoked by electrical stimulation of the cerebral peduncle in the presence of the glutamate receptor antagonists CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and AP5 (2-amino-5-phosphonopentanoic acid). 2. IPSCs were predominantly outward at -70 mV (in 124/135 cells), with a reversal potential of -83 mV, a time to peak of 2.6 ms and a decay time constant of 6.5 ms. Faster inward IPSCs were also observed in thirty-five cells, with a time to peak of 1.0 ms, a decay time constant of 2.3 ms, and a reversal potential of -61 mV. Both IPSCs were sensitive to the GABAA receptor antagonists picrotoxin or bicuculline. 3. In cells recorded with Cs+-filled pipettes, the outward IPSC reversal potential was shifted to -76 mV, closer to the estimated Cl- equilibrium potential of -56 mV, while that of the inward IPSC was unchanged at -64 mV. 4. The outward IPSC was reversibly depressed by up to 100 % by dopamine in a concentration-dependent manner with an IC50 of 10.5 microM, while the inward IPSC was relatively insensitive. 5. Dopamine was without effect on cell holding current, or on outward IPSC reversal potential, but it increased paired-pulse IPSC facilitation, consistent with a presynaptic site of action. 6. The D1-like dopamine receptor agonist SKF 38393 (10 microM) depressed the outward IPSC by 43 %, while the D2-like dopamine receptor agonist quinpirole (10 microM) was without effect. 7. It is concluded that GABA-ergic synaptic input onto distal rather than proximal regions of SNr neurones is susceptible to presynaptic inhibition via a D1-like receptor. These inputs are probably from striato-nigral fibres, and their inhibition by dopamine is likely to influence the patterning of basal ganglia output.

Figure 1. Electrophysiological characteristics of non-dopaminergic substantia nigra pars reticulata neurones

Aa, spontaneous, tonic action potential firing at rest at a rate of 21 Hz (full amplitude of spikes not faithfully represented by chart recording). Ab, a single spontaneous action potential, duration 0.62 ms. Ba, superimposed records of membrane current obtained under voltage clamp during a series of voltage steps (1 s duration) from a holding potential of −70 mV represented in the inset. Bb, steady-state I-V curve plotted from currents in Ba. All records from the same cell.

Figure 2. Outward IPSCs are monosynaptic, and predominantly GABA_A receptor mediated

A, superimposed records of membrane current showing response to a −5 mV step, followed by outward synaptic currents evoked by single-shock electrical stimulation (for 0.1 ms) of the cerebral peduncle at the currents indicated. oIPSC amplitude is graded proportional to stimulus strength. B, oIPSCs evoked by 7 stimuli (0.1 ms, 50 μA) at 100 Hz show no fatigue or failure. C, oIPSC can be completely abolished by the GABA_A receptor antagonist bicuculline (20 μM). D, in some cells, a bicuculline-resistant, strychnine-sensitive portion of the oIPSC was observed. Means of 5 successive events at a holding potential of −70 mV in all records. dl-AP5 (100 μM) and CNQX (20 μM) were present in all cases.

Figure 5. The oIPSC reverses at a less negative potential in cells recorded with caesium gluconate-containing pipettes, suggesting that it may have a distal location

A, upper panel, superimposed records of membrane current from a single cell recorded with a potassium gluconate-containing pipette held at potentials in the range −50 to −110 mV showing a biphasic IPSC. Peak amplitudes of iIPSCs (taken from the vertical continuous line) and oIPSCs (vertical dashed line) are plotted against membrane potential in the lower panel. Reversal potential of the iIPSC is −60 mV, and the oIPSC reverses at −88 mV. B, records from another cell recorded with a caesium gluconate-containing pipette, showing IPSCs recorded in the range −50 to −100 mV. The iIPSC reverses at −69 mV, and the oIPSC at −74 mV.

Figure 7. oIPSC reversal potential is unaffected when depressed by dopamine, in accordance with a presynaptic site of dopamine action

A, IPSCs from a single cell recorded at membrane potentials in the range −60 to −100 mV under control conditions (left), and in dopamine (30 μM; right), which depresses the IPSCs in a voltage-independent manner. B, plot of pooled data from 3 experiments such as that in A, with IPSC amplitude in each experiment normalized to that seen under control conditions at −60 mV. The IPSC amplitude in dopamine (□) was reduced in a voltage-independent manner relative to control (▪), but with its reversal potential (of −78 mV) essentially unchanged from that in control (−80 mV).

Figure 10. While the oIPSC can be completely depressed by dopamine, the iIPSC is relatively unaffected

A, superimposed records of a biphasic IPSC at potentials in the range −40 to −100 mV. The peak of the oIPSC is indicated by the vertical dashed line, and that of the iIPSC by the vertical continuous line. B, in the presence of dopamine (100 μM), the oIPSC is undetectable, while the iIPSC persists. C, digital subtraction of currents in B from those in A, showing that the dopamine-sensitive component of the IPSC is principally the late oIPSC. D, dopamine-sensitive portions of both the oIPSC (•) and the iIPSC (○), derived from C, plotted against membrane potential. The effect of dopamine is predominantly upon the oIPSC, whereas the iIPSC is only minimally affected.

Figure 3. Inward IPSCs are predominantly GABA_A receptor mediated

A and B, iIPSCs in 2 different cells are depressed in amplitude by > 85 % by GABA_A receptor antagonists picrotoxin (50 μM) or bicuculline (20 μM). In B, the outward component of the IPSC is also depressed by bicuculline. Means of 5 successive events at a holding potential of −70 mV.

Figure 4. The iIPSC shows a faster time to peak and decay time constant than the oIPSC, suggesting that it may have a more proximal origin

IPSCs from 2 different cells where electrical stimulation evoked either a pure iIPSC (left) or a pure oIPSC (right) at the holding potential of −70 mV. Time to peak of the iIPSC is 0.8 ms, while that of the oIPSC is 2.7 ms. Decay time constants, estimated by fitting to a single exponential curve (dashed lines), were 2.0 ms (iIPSC) and 6.9 ms (oIPSC), respectively.

Figure 6. Dopamine depresses the oIPSC in a concentration-dependent manner, and by up to 100 %

A, data pooled from 6 cells showing (upper panel) the time course of depression of the oIPSC by dopamine (30 μM; filled bar), and recovery on washout. Lower panel, sample records (mean of 5 successive events) from 1 of these cells, taken at the time points indicated (1, 2 and 3). Ba and b, superimposed records of oIPSCs from a single cell showing concentration-dependent depression by dopamine (10, 30 and 100 μM; Ba) and recovery from complete oIPSC depression by dopamine (100 μM; Bb) on washout. Bc, pooled concentration-effect data showing the extent of oIPSC depression by dopamine (0.1–100 μM). The maximal effect (100 % depression) is obtained with 100 μM dopamine, and the half-maximal effect (IC₅₀) by 10.5 μM dopamine. Each point represents the mean from 5 or 6 different cells. IPSCs recorded at −70 mV in all cases.

Figure 8. Paired-pulse IPSC facilitation is enhanced by dopamine, consistent with a presynaptic site of action

A, a pair of oIPSCs, evoked at an interval of 30 ms with the same stimulus at −70 mV. The amplitude of the second is facilitated relative to the first. B, in the presence of dopamine (30 μM), both oIPSCs are depressed, but the second less so than the first. C, the records in B (dopamine, 30 μM) are scaled up in amplitude so that the first IPSC matches the first IPSC in A (Control; superimposed records). The facilitation of the second IPSC is enhanced by dopamine, relative to control.

Figure 9. oIPSC depression by dopamine is mimicked by the D₁-like receptor agonist SKF 38393

Superimposed records of oIPSC from a single cell showing depression by dopamine (30 μM) relative to control, and recovery on wash (left panel), and then subsequent depression by SKF 38393 (10 μM) and recovery on rewash (right panel).