To investigate D2R actions on glutamatergic responses in striatopallidal MSNs, we made whole-cell current clamp recordings from GFP-positive cells in acute slices prepared from mice expressing GFP under control of the D2R gene promoter () 15,22
. Membrane potential was held near −60 mV, and experiments were done in the presence of the muscarinic antagonist scopolamine.
Modulation of corticostriatal synaptic responses in striatopallidal MSNs by D2Rs
To examine modulation of corticostriatal synapses, we expressed Channelrhodopsin-2 (ChR2) in motor cortex neurons projecting to the striatum (, see methods). Pulses of blue light applied to the striatum surrounding the current-clamped MSN reliably evoked excitatory postsynaptic potentials (EPSPs, ). Bath application of the D2R agonist quinpirole reduced the average EPSP amplitude from 9.6 ± 1.2 mV to 7.0 ± 1.4 mV (n=7, paired Student’s t-test, p<0.01, ). In voltage-clamp recordings, application of quinpirole reduced light-evoked excitatory postsynaptic currents (EPSCs) from 101.4 ± 23.2 pA to 78.6 ± 19.4 pA (n=8, p<0.01, ). Notably, quinpirole failed to alter EPSPs evoked by local electrical stimulation (Supplemental Fig. 1
), suggesting that electrical stimulation may trigger local release of dopamine, occluding the actions of exogenous agonists 11
Previous studies indicated that D2R activation reduces glutamate release from corticostriatal terminals 10,11
, potentially explaining the inhibition of light-evoked EPSPs and EPSCs. Evidence for direct D2R modulation of postsynaptic glutamate receptors has been less conclusive. We combined 2-photon laser scanning microscopy and glutamate uncaging to bypass presynaptic terminals and examine modulation of glutamatergic responses at single postsynaptic sites. Cells were filled with the Ca-insensitive red fluorophore Alexa Fluor 594 and the Ca-sensitive green fluorophore Fluo-5F. Under current clamp, a 500 μs pulse from the uncaging laser directed 0.5 μm from a dendritic spine located within 60 μm of the cell body produced a small uncaging-evoked EPSP (uEPSP) and a Ca-dependent increase in green fluorescence in the spine head and neighboring dendritic shaft (). Fluorescence transients in the spine and dendrite were quantified as the percent increase in green signal relative to the maximal green fluorescence in saturating Ca (ΔGsp
, respectively, see methods). For each synapse, laser power was adjusted such that the uncaging pulse applied directly to the spine head bleached ~50% of the red fluorescence () 23
and the periphery of the spine head was probed to find the uncaging position that evoked the largest uEPSP 24
D2R modulation of uncaging-evoked Ca transients in active spines
Under control conditions, a single uncaging pulse produced an average (n=36) uEPSP and associated Ca transients in the spine and neighboring dendritic shaft with amplitudes of 1.1 ± 0.1 mV, 7.4 ± 0.5%, and 1.9 ± 0.2%, respectively (). Because MSN dendrites exhibit voltage-dependent conductances that can shape temporal integration of synaptic inputs 25,26
, we also measured the average uEPSP (2.0 ± 0.2 mV) and Ca transients (ΔGsp
= 15.8 ± 1.1%, ΔGden
= 4.7 ± 0.2%) evoked by a burst of uncaging pulses (three stimuli, 20 ms inter-stimulus interval, ). In the presence of quinpirole (n=23), there was no significant change in average peak uEPSP magnitude for either single (1.2 ± 0.1 mV, p=0.61) or triple (1.6 ± 0.2 mV, p=0.14) stimuli () compared to control conditions, although the decay of the triple response was slightly faster, possibly indicating changes in voltage-gated ion channel or NMDAR contributions. In contrast, quinpirole strongly reduced uncaging-evoked Ca influx following both single (ΔGsp
= 4.2 ± 0.4%, p<0.0001, ΔGden
= 0.9 ± 0.1%, p<0.0001) and triple (ΔGsp
= 9.4 ± 0.8%, p<0.0001, ΔGden
= 1.9 ± 0.2%, p<0.001) stimuli (). Quinpirole did not alter the kinetics of recovery following photobleaching of the red fluorophore (50.7 ± 5.2 ms versus 58.6 ± 10.4 ms for control and quinpirole, respectively, p=0.45, ), indicating a lack of change in the diffusional properties of the spine. Furthermore, temporal summation of uEPSPs and Ca transients were not significantly altered by quinpirole (Supplemental Fig. 2
). In summary, when bypassing the presynaptic terminal, activation of D2Rs in striatopallidal MSNs reduces synaptic Ca influx by nearly 50% with minimal effects on somatic potentials.
Multiple Ca sources contribute to synaptic Ca transients
To identify potential targets of dopaminergic regulation underlying the inhibition of Ca influx, we examined the Ca sources that contribute to synaptic signaling in striatopallidal MSNs. Blockade of NMDARs with the selective antagonist CPP significantly increased the average (n=21) amplitude of the single stimulus uEPSP to 146.3 ± 20.7% of the control value (p<0.05, ). This counterintuitive result may reflect the role of NMDARs in promoting the opening of SK-type Ca-activated potassium channels that inhibit synaptic potentials in a variety of cell types 23,27
. Indeed, application of the SK channel blocker apamin (0.1 μM) also significantly increased uEPSP amplitude (Supplemental Fig. 3
). A 50 Hz burst stimulus revealed that temporal summation was decreased by NMDAR blockade due to reduced uEPSP duration ( and Supplemental Fig. 2
). CPP reduced the uncaging-evoked Ca influx in the spine head to 14.0 ± 1.3% (p<0.0001) and 9.1 ± 1.0% (p<0.0001) of control values for single and triple stimuli, respectively (), consistent with a dominant contribution of NMDARs to synaptically-evoked Ca transients.
Multiple Ca sources contribute to synaptic Ca signaling
We also analyzed the contribution of VGCCs to synaptic signaling. Selective blockade of P/Q- (n=24), N- (n=26), or L- (n=26) type channels (with 0.2 μM ω-agatoxin-IVA, 1 μM ω-conotoxin GVIA, or 3 μM nimodipine, respectively) had no significant effect on synaptic potentials and Ca transients evoked by single or triple stimuli (, p>0.05 for all comparisons). These blockers also failed to alter the temporal summation of uncaging-evoked potentials and Ca transients (Supplemental Fig. 2
). As shown previously 28
, 20 μM nimodipine did reduce Ca influx (Supplemental Fig. 4
), revealing either a loss of channel selectivity at this high concentration 29
or the presence of relatively dihydropyridine-resistant CaV
1.3 L-type channels30
Combined blockade of R-, L-, and T-type channels with mibefradil had no effect on the average (n=18) uEPSP evoked by a single stimulus, although the uEPSP following the triple stimulus was reduced relative to control (). However, mibefradil reduced the average relative amplitude of the Ca transients evoked by both single (77.2 ± 9.5%, p<0.05) and triple (72.9 ± 9.1%, p<0.01) stimuli (). In contrast, selective blockade of R-type channels with SNX-482 produced a significant increase in average (n=20) uEPSP magnitude (). These results suggest that, similar to the hippocampus, blocking R-type channels disengages a negative feedback loop involving SK channels, thereby boosting synaptic potentials and NMDAR-mediated Ca influx 23
. The lack of uEPSP boosting and the reduction in burst-evoked uEPSPs by mibefradil suggests that the broad actions of this non-selective agent may influence the generation of synaptic potentials. In summary, our results indicate that the largest fraction of spine Ca influx evoked by activation of a single postsynaptic terminal in striatopallidal MSNs arises from NMDAR opening, with additional contributions from R- and either T- or dihydropyridine-resistant L-type VGCCs, and suggest that these sources are potential targets for dopaminergic control of synaptic Ca influx.
D2Rs inhibit NMDAR- and R-type VGCC-mediated Ca influx
To examine the potential modulation of NMDAR-mediated synaptic responses, MSNs were voltage-clamped at −70 mV using a cesium-based internal solution and bathed in ACSF containing nominally zero extracellular Mg, tetrodotoxin, a full cocktail of VGCC antagonists (see methods), and the AMPA-type glutamate receptor (AMPAR) antagonist NBQX. Under these conditions, glutamate uncaging evoked an average (n=30) excitatory postsynaptic current (uEPSC, 13.0 ± 1.4 pA) and associated Ca transient in the spine head (ΔGsp/Gsat = 26.5 ± 3.0%, ). Activation of D2Rs produced no change in the average (n=27) NMDAR-mediated uEPSC (11.1 ± 1.3 pA, p=0.31) but significantly decreased the Ca transient (ΔGsp/Gsat = 17.0 ± 2.1%, p<0.05, ). Similar analysis was performed for non-NMDAR-mediated responses using ACSF with control Mg concentration and in the presence of the NMDAR antagonist CPP. The average (n=19) non-NMDAR-mediated uEPSC (26.0 ± 3.6 pA) and associated Ca transient (ΔGsp/Gsat = 0.7 ± 0.1%) were not altered in the presence of quinpirole (n=16; 30.2 ± 2.9 pA, p=0.39; ΔGsp/Gsat = 0.8 ± 0.1%, p=0.62, ), indicating that D2Rs selectively reduce Ca accumulation resulting from activation of NMDARs.
Activation of D2Rs modulates NMDAR- but not non-NMDAR-mediated synaptic responses
The actions of D2Rs on VGCCs were determined by examining the modulation of Ca transients evoked by back-propagating action potentials (bAPs, ). On average (n=30), a single bAP evoked by brief somatic current injection resulted in Ca influx within the spine head (ΔGsp
= 8.8 ± 0.01%) and neighboring dendrite (ΔGden
= 6.7 ± 0.4%, ). During a brief burst of three bAPs, Ca further increased in both compartments (ΔGsp
= 22.0 ± 1.6%, ΔGden
= 17.4 ± 1.2%, , Supplemental Fig. 2
). Activation of D2Rs reduced the average (n=34) bAP-evoked Ca transient for both single (ΔGsp
= 5.4 ± 0.5%, p<0.0001; ΔGden
= 4.2 ± 0.4%, p<0.0001) and triple (ΔGsp
= 15.0 ± 1.2%, p<0.001; ΔGden
= 11.9 ± 0.9%, p<0.001; ) stimuli, suggesting that VGCCs are targets of D2R modulation. Quinpirole did not alter the temporal summation (Supplemental Fig. 2
) or exponential decay time constants of the bAP-evoked spine Ca transients (75.6 ± 6.3 ms versus 82.6 ± 9.4 ms for control and quinpirole, respectively, p=0.55), suggesting a lack of effect on Ca clearance.
Activation of D2Rs modulates R-type voltage-gated Ca channels
In the presence of mibefradil, the average (n=24) single bAP-evoked Ca signal was reduced to 33.4 ± 2.8% (p<0.0001) and 42.6 ± 3.9% (p<0.0001) of control values for spines and dendrites, respectively, indicating a large contribution of R-, T-, and/or L-type channels (). The selective R-type blocker SNX-482 also reduced single bAP-evoked Ca influx to 70.0 ± 5.6% (n=23, p<0.0001) and 77.8 ± 7.3% (p<0.01) of control values for spines and dendrites, respectively (). In contrast, no change in Ca influx was observed in the presence of ω-agatoxin IVA (n=23), ω-conotoxin-GVIA (n=22), or 3 μM nimodipine (n=20) (). Similar results were seen following bursts of three bAPs (), and none of the VGCC blockers altered the temporal summation of bAP-evoked Ca signals (Supplemental Fig. 2
). These results suggest that R-type as well as T- and/or dihydropyridine-resistant L-type channels contribute to bAP-evoked Ca influx.
We next examined the ability of mibefradil and SNX-482 to mimic and occlude the actions of quinpirole (). A one-way ANOVA revealed significant differences between the effects on single bAP-evoked Ca transients of mibefradil, SNX-482, quinpirole alone, and each of the VGCC blockers in the presence of quinpirole (F=13.26, p<0.0001 and F=11.30, p<0.0001 for spines and dendrites, respectively). Tukey’s multiple post-hoc comparisons revealed that SNX-482 mimicked and occluded the actions of quinpirole, while mibefradil produced a significantly larger reduction in Ca influx than either SNX-482 or quinpirole alone (comparisons significant for p<0.05). Similar results were obtained for the triple bAP stimuli, arguing that D2Rs modulate R-type VGCCs with minimal effect on T- or L-type channels.
Previous studies have linked dopaminergic regulation of CaV
1.3 L-type channels with MSN spine stability 31
. To examine whether these channels were present but either inactive or insensitive to dihydropyridine block 30
during brief synaptic or bAP stimuli, we measured spine Ca influx during 300 ms voltage steps from −70 to 0 mV (). In the presence of blockers for R-, N-, and P/Q-type VGCCs, we observed a large Ca transient that rapidly reached steady state and decayed following return of the holding potential to baseline. Nimodipine (3 μM) reduced the step-evoked transients from 60.2 ± 2.4% (n=22) to 43.7 ± 2.2% (n=21) (). Activation of D2Rs decreased the amplitude to 36.7 ± 1.4% (n=20) whereas co-application of quinpirole and nimodipine produced an additional reduction to 21.4 ± 2.6% (n=20, ). A one-way ANOVA comparing control, quinpirole, nimodipine, and quinpirole + nimodipine revealed significant differences between these groups (F=54.6, p<0.0001). Post-hoc analysis revealed that quinpirole and nimodipine both significantly reduced Ca influx relative to control, and that the two together produced significantly greater reduction than either alone (comparisons significant for p<0.05). However, calculation of the nimodipine-sensitive component (the arithmetic difference between pre- and post-nimodipine values) indicated that the contribution of L-type VGCCs was unaffected by quinpirole (16.5 ± 2.3% versus 15.3 ± 2.1% ΔG/Gsat
, baseline in control and quinpirole, respectively, p>0.05). In summary, our data demonstrate that R-, L-, and T-type VGCCs are present in MSN spines, although R-types are the primary channel regulated by D2Rs.
Voltage-steps reveal L-type Ca channels in MSN spines
D2R modulation via PKA- dependent and independent pathways
Studies in hippocampal neurons indicated that a PKA-dependent mechanism augments Ca influx through NMDARs without altering total current flow 32
. We therefore examined whether D2R-mediated inhibition of PKA is responsible for the reduction in NMDAR-mediated Ca influx. We isolated NMDAR-mediated responses as above and tested the ability of PKI(14-22), a membrane permeable specific antagonist of PKA, to mimic and occlude the actions of quinpirole. In the presence of PKI(14-22), the average (n=21) NMDAR-mediated uEPSC was 13.0 ± 1.6 pA and the associated spine Ca transient was 15.8 ± 2.0% and 6.9 ± 1.2% (). Similar results were found using the alternate PKA antagonist H89 (n=19, ). Following co-application of PKI(14-22) and quinpirole (n=20), the uEPSC was 14.9 ± 2.3 pA and the Ca signal was 15.1 ± 1.7% (). A one-way ANOVA comparing control, PKI(14-22), quinpirole, and PKI(14-22) + quinpirole revealed significant differences for Ca transients between these groups (F=6.14, p<0.01). Post-hoc comparisons showed significant differences between control and PKI(14-22) as well as between control and PKI(14-22) + quinpirole, whereas there was no difference between quinpirole alone and in combination with PKI(14-22) (comparisons significant for p<0.05). Thus, antagonism of PKA is sufficient to mimic and occlude the actions of D2Rs on NMDAR-mediated Ca transients.
Push-pull modulation of NMDARs by D2Rs and A2ARs is dependent on PKA activity
Striatopallidal MSNs also express A2ARs, which are positively coupled to PKA via Gαs
and oppose the actions of D2Rs on long-term synaptic plasticity in the striatum 19
. We examined whether these two modulatory pathways also interact in the control of NMDAR-mediated Ca influx. Application of the specific A2AR agonist CGS-21680 (n=16) resulted in a uEPSC magnitude of 27.8 ± 4.5 pA and a spine Ca transient of 28.5 ± 3.2% (). Co-application of CGS-21680 and quinpirole (n=15) resulted in a uEPSC magnitude of 22.7 ± 3.0 pA and spine Ca transient of 29.5 ± 3.0% (). A one-way ANOVA comparing control, quinpirole, CGS-21680, and CGS-21680 + quinpirole revealed significant differences between these groups (F=3.9, p<0.05). Post-hoc analyses indicated that the uEPSC magnitudes for A2AR and combined A2AR+D2R activation were significantly increased, whereas spine Ca transients did not differ from control values (significant for p<0.05).
We next examined whether basal activation of A2ARs influences Ca influx through NMDARs. Application of the specific A2AR antagonist SCH-58261 (n=19) resulted in a uEPSC magnitude of 8.8 ± 1.8 pA and a spine Ca transient of 8.5 ± 0.7% (). Co-application of SCH-58261 and quinpirole (n=18) resulted in a uEPSC magnitude of 7.6 ± 1.7 pA and a spine Ca transient of 12.7 ± 2.3% (). A one-way ANOVA with post-hoc tests comparing control, quinpirole, SCH-58261 and SCH-58261 + quinpirole revealed no significant change in uEPSC magnitude. However, A2AR antagonism alone or in combination with quinpirole significantly reduced synaptic Ca influx, while there was no difference between these two groups. In summary, basal levels of A2AR activation enhance Ca influx through NMDARs and reduction of this activity is sufficient to mimic and occlude the effects of D2R activation. Furthermore, exogenous activation of A2ARs can counteract the D2R-mediated PKA-dependent reduction in synaptic Ca influx, suggesting that modulatory control of PKA exerts a bidirectional influence on NMDAR Ca signaling.
In the hippocampus, NMDARs containing the NR2B-type subunit contribute to particularly large Ca influx 33
, and removal of these subunits might explain the reduction in synaptic Ca influx. However, application of the selective NR2B-containing NMDAR antagonist ifenprodil (n=18) revealed no significant change in uEPSC or Ca transient magnitude (), suggesting that this subgroup of receptors does not contribute substantially to Ca influx under control conditions.
Finally, we examined whether similar PKA-dependent mechanisms underlie the D2R-mediated reduction in bAP-evoked Ca signals. A one-way ANOVA revealed significant differences between spine Ca transients in control, quinpirole, PKI(14-22) (n=20), PKI(14-22) + quinpirole (n=17), and CGS-21680 + quinpirole (n=11) (F=10.3, p<0.0001, ). Post-hoc tests indicated that, in the presence of PKI(14-22), Ca influx did not significantly differ from control conditions. Furthermore, co-application of quinpirole and either PKI(14-22) or CGS-21680 produced a reduction in signal that did not differ from that seen with quinpirole alone. Similar results were obtained for dendrite Ca signals (). These findings indicate that the actions of D2Rs on bAP-evoked VGCC-mediated Ca transients occur independently of PKA and A2AR activation and demonstrate that D2Rs couple to two independent pathways that converge on a similar endpoint of reduced Ca signaling in dendritic spines.
Modulation of VGCCs by D2Rs is independent of A2AR and PKA activity