Figure 4. CRY2olig-Geph_FingR clustering impairs evoked and spontaneous synaptic transmission through GABA_AR displacement within the postsynaptic membrane.

(A) Plotted are electrically evoked IPSC amplitudes measured from individual sweeps before and after 2-s light exposures at 0 min and 5 min (denoted by blue bars) recorded from a dissociated hippocampal neuron expressing CRY2olig-Geph_FingR-GFP. Series resistance measurements are plotted above.

(B) Averaged evoked IPSC amplitudes are plotted as a function of time before and after blue light illumination (2-s pulse, denoted by blue arrows) in dissociated hippocampal neurons expressing Geph_FingR-GFP (black) or CRY2olig-Geph_FingR-GFP (blue). Error bars are 95% CI. Example IPSC traces before (red) and 10 min following light exposure (blue) are shown above. Scale bar left, 250 pA, 35 ms. Scale bar right, 150 pA, 45 ms. *p < 0.05; two-way ANOVA. n = 7 cells each condition, from 5 independent cultures.

(C) Paired comparisons of IPSC peak amplitudes before (pre) and 10 min following blue light illumination (post) are shown for hippocampal neurons expressing Geph_FingR-GFP (cntrl) or CRY2olig-Geph_FingR-GFP (CRY2olig). **p < 0.01, n.s. non-significant; paired t test, n = 7 cells, from 5 independent cultures.

(D) Left, spontaneous IPSC (sIPSC) example traces within the inter-stimulus intervals from cells in (A)–(C). Right, paired comparison of sIPSC amplitude from the same cells expressing either Geph_FingR-GFP (cntrl) or CRY2olig-Geph_FingR-GFP (CRY2olig), before (pre) and 10 min following blue light exposure (post). p = 0.07, n.s. non-significant; paired t test. Comparison of population means for before light (pre) conditions on top. n.s. non-significant; Student’s t test. n = 7 cells, from 5 independent cultures.

(E) mIPSC amplitude (left) and frequency (right) are shown, paired from the same cells expressing either Geph_FingR-GFP (cntrl) or CRY2olig-Geph_FingR-GFP (CRY2olig) before (pre) and 10 min following blue light exposure (post). **p < 0.01; paired t test. n.s. non-significant. Comparison of population means for before light (pre) conditions on top. n.s. non-significant; Student’s t test. n = 8 cells.

(F) Tonic GABA_AR currents were measured by the change in holding current before and after the addition of picrotoxin in light-treated cells expressing Geph_FingR-GFP (cntrl) or CRY2olig-Geph_FingR-GFP (CRY2olig). Error bars represent SEM. n.s. non-significant; Student’s t test, n = 7–9 cells.

(G) Paired-pulse ratios (PPRs) were measured (100-msec interval) from cells expressing Geph_FingR-GFP (cntrl) or CRY2olig-Geph_FingR-GFP (CRY2olig) before (pre) and 10 min following blue light exposure (post). n.s. non-significant; paired t test, n = 5–7 cells.

(H) Paired comparisons of IPSC peak amplitudes before (pre) and 10 min after blue light activation (post) are shown for CA1 pyramidal neurons expressing Geph_FingR-GFP (cntrl) or CRY2olig-Geph_FingR-GFP (CRY2olig). Data were taken from biolistically transfected organotypic slices (left) or acute slices from AAV-infected mice (right). *p < 0.05; **p < 0.01; paired t test, n = 5 cells organotypic slice, from 5 independent cultures and 8 cells acute slices, from 3 animals.

(I) Model for activation of receptors within subsynaptic domains apposed to GABA release sites (black dashed circles) before and after expansion of the postsynaptic membrane by Geph_FingR clustering. If electrically evoked IPSCs are reduced due to nanoscale displacement within the postsynapse, uncaging-evoked IPSCs should remain unperturbed as the diffraction-limited uncaging volume releases GABA across the entire postsynaptic membrane (green dashed circle).

(J) Top, images of a hippocampal neuron expressing CRY2olig-Geph_FingR-Halo (labeled with JF646) before and after light-induced clustering. White asterisk marks location of focal GABA uncaging. Bottom, representative uIPSCs (average of two sweeps) are shown before (red) and 8 min following CRY2olig-Geph_FingR-Halo clustering (blue).

(K) Paired comparisons of normalized CRY2-Geph_FingR-Halo intensity (left), raw uIPSC amplitudes (middle), and normalized uIPSC amplitudes (right) before and after CRY2olig-Geph_FingR-Halo clustering. n = 21 synapses from 7 neurons from 3 independent cultures. Statistical comparisons were only carried out on the raw, non-normalized uIPSC amplitude data. *p < 0.05, paired t test.