Modulation of Ca2+-currents by sequential and simultaneous activation of adenosine A1 and A 2A receptors in striatal projection neurons

Abstract

D(1)- and D(2)-types of dopamine receptors are located separately in direct and indirect pathway striatal projection neurons (dSPNs and iSPNs). In comparison, adenosine A(1)-type receptors are located in both neuron classes, and adenosine A(2A)-type receptors show a preferential expression in iSPNs. Due to their importance for neuronal excitability, Ca(2+)-currents have been used as final effectors to see the function of signaling cascades associated with different G protein-coupled receptors. For example, among many other actions, D(1)-type receptors increase, while D(2)-type receptors decrease neuronal excitability by either enhancing or reducing, respectively, CaV1 Ca(2+)-currents. These actions occur separately in dSPNs and iSPNs. In the case of purinergic signaling, the actions of A(1)- and A(2A)-receptors have not been compared observing their actions on Ca(2+)-channels of SPNs as final effectors. Our hypotheses are that modulation of Ca(2+)-currents by A(1)-receptors occurs in both dSPNs and iSPNs. In contrast, iSPNs would exhibit modulation by both A(1)- and A2A-receptors. We demonstrate that A(1)-type receptors reduced Ca(2+)-currents in all SPNs tested. However, A(2A)-type receptors enhanced Ca(2+)-currents only in half tested neurons. Intriguingly, to observe the actions of A(2A)-type receptors, occupation of A(1)-type receptors had to occur first. However, A(1)-receptors decreased Ca(V)2 Ca(2+)-currents, while A(2A)-type receptors enhanced current through Ca(V)1 channels. Because these channels have opposing actions on cell discharge, these differences explain in part why iSPNs may be more excitable than dSPNs. It is demonstrated that intrinsic voltage-gated currents expressed in SPNs are effectors of purinergic signaling that therefore play a role in excitability.

Fig. 1

Whole-cell Ca²⁺-currents in acutely dissociated neostriatal neurons. a Inward currents elicited by steps of depolarizing voltage commands. b Inward current elicited in the same neuron by a ramp command. c Current-voltage relationships (I-V plots) taken from data in a and b. Note close superimposition. d Time course of absolute current amplitude during application of 200 μM Cd²⁺

Fig. 2

A selective A_2A-type receptor agonist had no actions on Ca²⁺-current by itself. a The time course shows the application of an A_2A-type receptor agonist: 1 µM CGS into the bath saline (horizontal bar). There is no change in current amplitude. b representative I-V plots taken at numbered times from the time course in a. c Box Tukey plots in Fig. 2c show that there is no significant differences between Ca²⁺-currents amplitude with or without the CGS in all neurons tested (n = 6)

Fig. 3

Adenosine actions are biphasic and concentration-dependent. a Addition of low adenosine concentrations (100 nM) to the bath saline decreases absolute current amplitude, whereas application of higher adenosine concentrations (10 μM) partially reversed the current decrease produced by lower adenosine concentrations. b Representative I-V plots taken from the time course in a as indicated by the numbers. c Box plots summarizing a sample of experiments from neurons during both adenosine concentrations (n = 13 for left and middle boxes, n = 7 for right box). d The time course shows that reduction in current produced by low adenosine concentrations was not blocked by SCH 58261 while current enhancement mediated by micromolar adenosine was not present in any recorded cell when SCH 58261 was present. e Representative I-V plots taken from the time course in d as indicated by numbers. f Box plots summarize a sample of similar experiments (n = 13)

Fig. 4

Sequential activation of A₁- and A_2A-type receptors with selective agonists reduces and enhances Ca²⁺ -current, respectively. a Time course of current amplitude showing that CCPA (100 nM) decreases current amplitude (1, 2). Subsequent application of 1 µM CGS 21680 (2, 3) almost completely reverses the reduction induced by CCPA in about half the neurons. b Representative I-V plots taken from the time course in a as indicated by the numbers. c Box plots summarize results from a sample of neurons with responses described in a and b (n = 13 in left and middle boxes and n = 7 in the right box). d Time course similar to that depicted in a but in the presence of 50 nM of the A_2A-receptor antagonist SCH 58261. In these conditions, the action of CGS 21680 could not be observed in any neuron. e Representative I-V plots taken from the time course in d as indicated by the numbers. f Box plots summarize the experimental sample (n = 8)

Fig. 5

A_2A but not A₁ receptor actions are blocked by dihydropyridines. a Time course illustrates the sequential reduction with CCPA and the reversal of current amplitude with CGS 21680. The action of the A_2A-receptor agonist was blocked by 5 μM nicardipine. b Representative I-V plots taken from the time course in a as indicated by the numbers. c Box plots summarizing a sample of similar experiments (n = 10 for control; n = 6 for nicardipine, CCPA and CGS). d Time course shows that 5 μM nicardipine administered first occludes the action of high but not of low concentrations of adenosine. e Representative I-V plots taken from the time course in d as indicated by the numbers. f Box plots summarizing a sample of similar experiments (n = 12)

Fig. 6

Activation of adenosine A₁-receptor mostly reduces current through Ca_V2.2 (N) channels. a Time course illustrates that 1 μM ω-conotoxin GVIA almost completely occluded the action of 100 nM adenosine. b Representative I-V plots taken from the time course in a as indicated by the numbers. c Box plots summarizing a sample of similar experiments (n = 9). d Time course illustrates that 1 μM ω-agatoxin IVA did not occlude the action of 100 nM adenosine. e Representative I-V plots taken from the time course in a as indicated by the numbers. f Box plots summarizing a sample of similar experiments (n = 6)

Fig. 7

Most adenosine A₁-receptors modulation is not voltage-dependent. a Top: standard double pulse protocol. Middle: Evoked currents before (black trace) and after (gray trace) CCPA was added to the bath saline. Bottom: enhanced traces evoked with zero millivolts commands before (P1) and after the 80 mV pre-pulse (P2). Current increases in amplitude and changes its kinetics during P2 suggesting constitutive G-protein action. b However, percent amplitude modulation amplitude before and after the 80 mV pre-pulse was non-significantly different (n = 10). c Ratio of second (P2) to first response (P1) was slightly different, suggesting that most A₁-receptor modulation is mainly non-voltage-dependent

Fig. 8

A₁ and A_2A receptors have synergistic effects on excitability. a Left and right columns illustrate the firing of a putative dSPN and an iSPN, respectively, under a stimulus strength that allowed the firing of the same number of action potentials in the control (step current at the bottom). From top to bottom, these responses were subjected to different conditions without changing the stimulus strength: First, addition of CCPA (100 nM) an agonist of A₁-receptors increased the number of spikes fired in both neurons. Second, addition of CGS 21680 (1 μM) an agonists of A_2A-receptors, further increased the number of spikes fired in iSPNs but not in dSPNs. Graph at the bottom shows the firing behavior of n = 7 neurons. All neurons responded to CCPA, but only four out of seven neurons responded also to CGS