Spontaneous opening of T-type Ca2+ channels contributes to the irregular firing of dopamine neurons in neonatal rats

Abstract

During early postnatal development, midbrain dopamine (DA) neurons display anomalous firing patterns and amphetamine response. Spontaneous miniature hyperpolarizations (SMHs) are observed in DA neurons during the same period but not in adults. These hyperpolarizations have been shown to be dependent on the release of Ca2+ from internal stores and the subsequent activation of Ca2+-sensitive K+ channels. However, the triggering mechanism and the functional significance of SMHs remain poorly understood. To address these issues, using brain slices, we recorded spontaneous miniature outward currents (SMOCs) in DA neurons of neonatal rats. Two types of SMOCs were identified based on the peak amplitude. Both types were suppressed by intracellular dialysis of ruthenium red, a ryanodine receptor (RyR) antagonist, yet none of the known Ca2+-releasing messengers were involved. T-type Ca2+ channel blockers (Ni2+ and mibefradil) inhibited large-amplitude SMOCs without affecting the small-amplitude ones. The voltage dependence of SMOCs displayed a peak of approximately -50 mV, consistent with the involvement of low-threshold T-type Ca2+ channels. Blockade of SMOCs with cyclopiazonic acid or ryanodine converted the irregular firing of DA neurons in neonatal rats into an adult-like pacemaker pattern. This effect was reversed by the injection of artificial currents mimicking SMOCs. Finally, amphetamine inhibited SMOCs and transformed the irregular firing pattern into a more regular one. These data demonstrate that Ca2+ influx through T-type Ca2+ channels, followed by Ca2+-induced Ca2+ release via RyRs, contributes to the generation of SMOCs. We propose that SMOCs-SMHs may underlie the anomalous firing and amphetamine response of DA neurons during the postnatal developmental period.

Figure 2.

SMOCs are dependent on Ca²⁺ release from internal stores via RyRs. A, Summary time graph showing the effect of CPA (10 μm) on SMOCs (n = 4). B, Summary time graph illustrating the effect of ryanodine (Ryn) (20 μm) on SMOCs (n = 6). Both CPA and ryanodine eliminated SMOCs in ∼5 min. C1, Representative traces of SMOCs with a control internal solution or with a solution containing ruthenium red (100 μm). Traces are shown at 1 and 5 min after entering the whole-cell configuration. C2, The SMOC frequency is plotted against time after going into the whole-cell configuration. Recordings were made with a pipette containing a control internal solution (n = 22), ruthenium red (RR) (100 μm; n = 11), both heparin (Hep) (1 mg/ml) and 8-NH₂-cADPR (8AcADPR) (50 μm) (n = 8), and NAADP (1 mm; n = 9). Only ruthenium red induced significant inhibition of SMOCs. ^*p < 0.05; ^**p < 0.01 versus control.

Figure 3.

Large-amplitude SMOCs are triggered by Ca²⁺ influx through T-type Ca²⁺ channels. A1, Representative traces of SMOCs, mGluR-induced outward currents, and AHP currents before and during a brief perfusion (5 min) of Cd²⁺ (200 μm). Aspartate iontophoresis was made at the time indicated by the arrow to evoke mGluR-mediated currents. Slices were pretreated with MK-801 (50 μm) to block NMDA-mediated currents. A2, Superimposed amplitude histograms of SMOCs before and during Cd²⁺ application from the same experiment as in A1. A3, Summary bar graph depicting the effects of Cd²⁺ on the SMOC frequency (n = 7), the mGluR-induced current amplitude (n = 5), and the AHP current amplitude (n = 6). Cd²⁺ significantly inhibited SMOCs and AHP currents but had a marginal effect on mGluR-induced currents. B1, C1, Representative traces of SMOCs showing the effects of Ni²⁺ (100 μm) and mibefradil (100 μm) on SMOCs. B2, C2, Superimposed amplitude histograms of SMOCs from the same experiments as in B1 and C1, respectively. D, Summary bar graph showing the effects of Ni²⁺ (100 μm; n = 9), mibefradil (Mib) (100 μm; n = 8), nifedipine (Nif) (10 μm; n = 5), ω-conotoxin GVIA (Cono) (1 μm; n = 3), ω-agatoxin IVA (Aga) (200 nm; n = 3), and SNX-482 (SNX) (200 nm; n = 3) on SMOCs. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 4.

Voltage dependence of SMOCs. A1, Representative traces of SMOCs at three different holding potentials. All of the traces are from the same cell. A2, Superimposed amplitude histograms of SMOCs from the same recording as in A1. B, Summary graph showing the voltage dependence of SMOC frequency (n = 26). The frequency was normalized to the value at -55 mV in each cell. Each data point, except for the one at -55 mV, represents data from 7-18 cells. The frequency-voltage relationship displayed a bell-shaped curve that peaked at -55 to -45 mV.

Figure 1.

Basal properties of SMOCs. A1, A2, A representative trace of SMOCs (A1) and their amplitude histogram (A2) from a DA neuron showing two peaks in the SMOC amplitude distribution. A3, Relationship between the amplitude and the rise time constant of SMOCs from the same recording as in A1 and A2. There was no correlation between the amplitude and the rise time constant (r = 0.25). A4, Bar graph depicting the average rise time constant of S-SMOCs (<22 pA) and L-SMOCs (≥22 pA) from the same recording. The line dividing S-SMOCs and L-SMOCs is shown in A2 and A3. B1, B2, Representative trace of SMOCs (B1) and their amplitude histogram (B2) from a DA neuron showing only one peak in the amplitude distribution. C, Summary bar graph depicting the effects of apamin, TTX, and various neurotransmitter antagonists on SMOCs. The SMOC frequency in the presence of drugs was normalized to the control frequency before drug application. The drugs tested included apamin (100 nm; n = 5), TTX (300 nm; n = 4), a mixture of NBQX (AMPA antagonist; 5 μm), MK-801 (NMDA antagonist; 50 μm), and picrotoxin (GABA_A antagonist; 100 μm) (n = 4), MCPG (mGluR antagonist; 1 mm; n = 4), hexamethonium (nicotinic acetylcholine receptor antagonist; 200 μm; n = 4), scopolamine (muscarinic acetylcholine receptor antagonist; 1 μm; n = 7), and prazosin (α1 adrenergic receptor antagonist; 100 nm; n = 3). Only apamin suppressed SMOCs. ^***p < 0.001.

Figure 5.

SMOCs contribute to the irregular firing pattern of DA neurons in neonatal rats. A, Representative traces of spontaneous firing before and during perfusion of CPA (10 μm). The bottom trace was obtained when simulated SMOCs were injected during CPA treatment. B, ISI histograms from the corresponding recordings in A. Note then arrow peak with a Gaussian distribution in CPA. C, Autocorrelograms from the corresponding recordings in A. Note multiple identifiable peaks in CPA.

Figure 6.

Amphetamine suppresses SMOCs via activation of α1 adrenergic receptors. A1, Representative traces of SMOCs in a control extracellular solution, amphetamine (Amph) (10 μm), and amphetamine plus prazosin (100 nm). A2, Superimposed amplitude histograms of SMOCs from the same experiment as in A1. B, Summary time graph illustrating the effect of amphetamine on SMOCs and its reversal by prazosin (n = 4). C, Representative traces of firing before and during perfusion of amphetamine (10 μm).

Figure 7.

Proposed model illustrating two types of SMOCs. L-SMOCs are initially triggered by Ca²⁺ influx through T-type Ca²⁺ channels (1), which by itself is not sufficient to activate SK channels but which can activate RyRs in close apposition to the plasma membrane and induce CICR. The Ca²⁺ signal amplified by CICR (2) is now sufficient to activate nearby SK channels. S-SMOCs are also produced by Ca²⁺ release via RyRs. However, the opening of RyRs is not triggered by T-type channel-mediated Ca²⁺ influx in S-SMOCs. A larger amount of Ca²⁺ is mobilized in L-SMOCs than in S-SMOCs.