Sapap3 deletion anomalously activates short-term endocannabinoid-mediated synaptic plasticity

Abstract

Retrograde synaptic signaling by endocannabinoids (eCBs) is a widespread mechanism for activity-dependent inhibition of synaptic strength in the brain. Although prevalent, the conditions for eliciting eCB-mediated synaptic depression vary among brain circuits. As yet, relatively little is known about the molecular mechanisms underlying this variation, although the initial signaling events are likely dictated by postsynaptic proteins. SAP90/PSD-95-associated proteins (SAPAPs) are a family of postsynaptic proteins unique to excitatory synapses. Using Sapap3 knock-out (KO) mice, we find that, in the absence of SAPAP3, striatal medium spiny neuron (MSN) excitatory synapses exhibit eCB-mediated synaptic depression under conditions that do not normally activate this process. The anomalous synaptic plasticity requires type 5 metabotropic glutamate receptors (mGluR5s), which we find are dysregulated in Sapap3 KO MSNs. Both surface expression and activity of mGluR5s are increased in Sapap3 KO MSNs, suggesting that enhanced mGluR5 activity may drive the anomalous synaptic plasticity. In direct support of this possibility, we find that, in wild-type (WT) MSNs, pharmacological enhancement of mGluR5 by a positive allosteric modulator is sufficient to reproduce the increased synaptic depression seen in Sapap3 KO MSNs. The same pharmacologic treatment, however, fails to elicit further depression in KO MSNs. Under conditions that are sufficient to engage eCB-mediated synaptic depression in WT MSNs, Sapap3 deletion does not alter the magnitude of the response. These results identify a role for SAPAP3 in the regulation of postsynaptic mGluRs and eCB-mediated synaptic plasticity. SAPAPs, through their effect on mGluR activity, may serve as regulatory molecules gating the threshold for inducing eCB-mediated synaptic plasticity.

Figure 1.

Activity-dependent synaptic depression of MSN excitatory synapses is enhanced in Sapap3 KO mice. A–E, A decrease in stimulation interval from 90 s to 10 s causes greater depression of field PS responses in Sapap3 KO mice than WT controls. Each stimulation consists of a pair of pulses 50 ms apart. A, Sample traces of evoked paired stimuli illustrate typical responses at 90 s and 10 s stimulation interval periods in WT and KO mice. B, Time course plot displays relative change in PS amplitude from 90 s stimulation interval period to 10 s stimulation interval period (p = 0.005; rmANOVA). C, Summary bar graph compares WT and KO average values over last 10 min of 10 s interval stimulation normalized to baseline period (p = 0.003; t test). D, E, Time course plot (D) and bar graph (E) demonstrate a concomitant activity-dependent increase in PPR of PS amplitude in Sapap3 KO mice (p = 0.036, rmANOVA; p = 0.038, t test). F–J, Whole-cell, voltage-clamp recordings of eEPSCs from D2 MSNs show greater activity-dependent synaptic depression in Sapap3 KO than WT mice. F, Sample traces illustrate typical responses at 90 s and 10 s stimulation interval periods in WT and KO mice. G, H, Time course plot (G) and summary bar graph (H) of the normalized eEPSC amplitude (p = 0.005, rmANOVA; p = 0.002, t test). I, J, Time course plot (I) and summary bar graph (J) demonstrate a concomitant increase in PPR of eEPSC when stimulation interval is decreased in D2 MSNs of Sapap3 KO mice (p = 0.034, rmANOVA; p = 0.003, t test). Calibration: A, 5 ms, 0.5 mV; F, 20 ms, 200 pA. *p < 0.05; **p < 0.01.

Figure 2.

Excessive synaptic depression in Sapap3 KO mice is mediated by endocannabinoid signaling. A, Sample traces illustrate typical extracellular recording responses in the presence of 3 μm AM251. B, C, AM251 eliminates genotypic differences in activity-dependent depression of PS amplitude (B; p = 0.434, t test) and increase in PPR (C; p = 0.213, t test). D, Room temperature prevents activity-dependent depression of PS in Sapap3 WT and KO mice (p = 0.316; rmANOVA). E, CB1R gene deletion (CB1R KO mice) significantly reduces activity-dependent depression of PS amplitude in Sapap3 KO mice (p = 0.001; rmANOVA). F, CB1R gene deletion significantly reduces activity-dependent increase in PS PPR in Sapap3 KO mice (p = 0.023; rmANOVA). G, Sample traces illustrate typical eEPSC responses in the presence of 3 μm AM251. H, In the presence of 3 μm AM251, there is no difference in activity-dependent synaptic depression of D2 MSN eEPSCs between WT and KO (p = 0.150; t test). I, In the presence of AM251, normalized PPR of KO is no longer greater than WT during the 10 s stimulation interval period (p = 0.137; t test). J, The CB1R agonist, WIN 55212-2 (WIN; 1 μm) causes a similar degree of synaptic depression of WT and KO striatal MSN excitatory synaptic responses (p = 0.791; rmANOVA). The effects of WIN 55212-2 were tested at room temperature and 90 s stimulation interval to avoid unequal contributions by postsynaptically-generated endocannabinoids. K, In WT mice, AM251 has no effect on the degree of activity-dependent depression of D2 MSN eEPSCs (p = 0.472; t test). L, In Sapap3 KO mice, AM251 significantly reduces activity-dependent depression of D2 MSNs eEPSCs (p = 0.0003, t test). Calibration: A, 5 ms, 0.5 mV; G, 20 ms, 200 pA. **p < 0.01.

Figure 3.

Anomalous eCB-mediated synaptic depression at Sapap3 KO synapses requires mGluR5. A, Type 5 mGluR antagonist, MPEP (40 μm) significantly reduces activity-dependent depression of D2 MSN eEPSCs in Sapap3 KO mice (p = 0.001; t test). B, MPEP reduces the activity-dependent PPR increase in D2 MSN eEPSCs of Sapap3 KO mice (p = 0.018; t test). C, D, In the presence of MPEP, genotypic differences in activity-dependent synaptic depression (C) and PPR (D) are abolished from D2 MSN eEPSCs, (p = 0.193 and 0.942, respectively; t test). E, F, L-type calcium channel antagonist nifedipine (10 μm) has no effect on activity-dependent changes in synaptic depression (E) or PPR (F) of D2 MSN eEPSCs in Sapap3 KO mice (p = 0.801 and 0.291, respectively; t test). *p < 0.05; **P < 0.01.

Figure 4.

DHPG-induced intracellular calcium transients are increased in striatal MSNs of Sapap3 KO mice. Intracellular calcium transients are monitored using the cell-permeable, calcium indicator dye, fura-2 AM, and two-photon microscopy imaging of acute striatal brain slices. A, Images show, from left to right, overlay of fluorescence from transgenic reporters Drd1a-tdTomato (red) and Drd2-EGFP (green), fura-2 fluorescence (basal condition), and pseudocolored representations of the peak ΔF/F for MSNs in response to DHPG during the 20 s after drug application. The pseudocolor scale represents values for ΔF/F in bins of 0.05. Scale bars, 25 μm. B, Sample traces of individual MSN fluorescent fura-2 AM signal in response to DHPG from WT (black) and KO (gray) mice. C, Post-DHPG peak ΔF/F values are larger in both D1 and D2 MSNs of Sapap3 KO mice (n values refer to cells; p = 0.002 for D1 MSNs; p = 0.001 for D2 MSNs; t test). **p < 0.01.

Figure 5.

Intracellular calcium transients in response to group 1 mGluR activation and surface expression of mGluR5 are increased in Sapap3 KO striatal neurons from corticostriatal cocultures. A, Pseudocolored images of Fluo-4 intensity in corticostriatal cocultured neurons from Sapap3 WT (top) and KO mice (bottom). Sample images correspond to baseline period and 5 s after 100 μm DHPG application. B, Bar graph of the peak Fluo-4 response in the 10 s period following DHPG application demonstrates increased calcium transients in MSNs from Sapap3 KO cultures (n = 131 cells, 12 wells, 3 culture preparations) relative to WT cultures (n = 165 cells, 13 wells, 3 culture preparations; p < 0.0001, Mann–Whitney test). C, Example images of mGluR5 surface immunostaining on DARPP-32-positive dendritic regions in corticostriatal cocultures from Sapap3 WT and KO mice (top). DARPP-32 staining (pseudocolored in green) was used to identify medium spiny neurons (bottom). D, The average mGluR5 intensity is significantly higher in Sapap3 KO cultures compared to WT mice (p = 0.003; Mann–Whitney test). E, Cumulative probability plot shows a rightward shift across all intensities of mGluR5 surface staining of dendritic regions from Sapap3 KO mice compared to WT mice. Scale bars: A, 10 μm; C, 5 μm. p < 0.05; **p < 0.01.

Figure 6.

Augmentation of mGluR5 activity by the positive allosteric mGluR5 modulator, CDPPB, during the activity-dependent protocol increases depression of WT responses to KO levels. A, B, In the presence of CDPPB (0.1 μm), the activity-dependent protocol depresses dorsolateral striatal PS field responses of WT and KO mice similarly. Summary bar graphs show the average response during the last 10 min of 10 s stimulation interval period relative to baseline (p = 0.541, rmANOVA; p = 0.836, t test). C, D, In the presence of CDPPB (0.1 μm), the activity-dependent protocol increases PS PPR similarly in WT and KO mice (p = 0.816, rmANOVA; p = 0.664, t test).

Figure 7.

Sapap3 deletion induces short-term eCB-mediated plasticity, but does not alter magnitude of long-term eCB-mediated plasticity. A, B, In field recordings, AM251 applied after steady-state responses achieved at the 10 s stimulation interval reverses the PS depression (A) and paired-pulse ratio changes (B) of striatal excitatory synapses in Sapap3 KO to levels indistinguishable from WT mice. Bars indicate periods of stimulation interval and drug application. C, Pausing stimulation for 10 min is sufficient to return D2 MSN eEPSCs to basal values when stimulation is resumed at a lower rate (90 s interval, 50 ms IPI paired pulses). D, There is no difference in the magnitude of LTD elicited between WT and KO D2 MSN eEPSCs (p = 0.616; rmANOVA). Arrows indicate time of LTD induction by HFS (4 trains of 100 Hz stimulation paired with 0 mV depolarization). E, Working model depicts SAPAP3 negatively regulating surface levels and activity of mGluR5. In the absence of SAPAP3, increased activity of mGluR5 leads to increased endocannabinoid signaling and inhibition of presynaptic neurotransmitter release.