Regulation of presynaptic strength by controlling Ca2+ channel mobility: effects of cholesterol depletion on release at the cone ribbon synapse

Abstract

Synaptic communication requires proper coupling between voltage-gated Ca(2+) (Ca(V)) channels and synaptic vesicles. In photoreceptors, L-type Ca(V) channels are clustered close to synaptic ribbon release sites. Although clustered, Ca(V) channels move continuously within a confined domain slightly larger than the base of the ribbon. We hypothesized that expanding Ca(V) channel confinement domains should increase the number of channel openings needed to trigger vesicle release. Using single-particle tracking techniques, we measured the expansion of Ca(V) channel confinement domains caused by depletion of membrane cholesterol with cholesterol oxidase or methyl-β-cyclodextrin. With paired whole cell recordings from cones and horizontal cells, we then determined the number of Ca(V) channel openings contributing to cone Ca(V) currents (I(Ca)) and the number of vesicle fusion events contributing to horizontal cell excitatory postsynaptic currents (EPSCs) following cholesterol depletion. Expansion of Ca(V) channel confinement domains reduced the peak efficiency of release, decreasing the number of vesicle fusion events accompanying opening of each Ca(V) channel. Cholesterol depletion also inhibited exocytotic capacitance increases evoked by brief depolarizing steps. Changes in efficiency were not due to changes in I(Ca) amplitude or glutamate receptor properties. Replenishing cholesterol restored Ca(V) channel domain size and release efficiency to control levels. These results indicate that cholesterol is important for organizing the cone active zone. Furthermore, the finding that cholesterol depletion impairs coupling between channel opening and vesicle release by allowing Ca(V) channels to move further from release sites shows that changes in presynaptic Ca(V) channel mobility can be a mechanism for adjusting synaptic strength.

Fig. 1.

Cholesterol localization at the cone synapse and effects of pharmacological treatments on retinal cholesterol levels. A: to visualize membrane cholesterol, an enzymatically dissociated double cone was fixed and stained with filipin III (300 μg/ml for 90 min). When visualized under epifluorescence with 380-nm excitation/525-nm emission, cones exhibited generalized cholesterol staining throughout the inner segment (IS), cell body (CB), and terminal, but cholesterol appeared more concentrated at the synapse (arrows). Control cones visualized without filipin III binding showed autofluorescence in the IS but no synaptic fluorescence (B; arrows). C: to specifically target cholesterol-rich lipid rafts, we stained cones with 2 μg/ml FITC-CTX_B for 45 min and visualized them using 488-nm excitation/525-nm emission on a spinning disk confocal microscope. Cones labeled with FITC-CTX_B showed concentrated staining at the cone pedicle (arrow). D: cones without FITC-CTX_B showed no staining at the synaptic terminal (arrow). E: we quantified cholesterol levels in the retina using the Amplex Red cholesterol assay kit. Control retinas exhibited a cholesterol level of 12.6 ± 0.69 ng/μg protein (control), which dropped to 4.94 ± 0.52 ng/μg protein (39% of control; ***P < 0.0001 vs. control) and 7.18 ± 0.66 ng/μg protein (58% of control, ***P < 0.0001) after methyl-β-cyclodextrin (MβCD) or cholesterol oxidase (COase) treatment, respectively. Membrane cholesterol was restored to a level of 11.3 ± 0.41 ng/μg protein (repletion) by repleting tissue with a complex of cholesterol and MβCD (91% of control; P = 0.19 vs. control). Scale bar for immunohistochemistry, 10 μm.

Fig. 2.

Depletion of synaptic cholesterol increased the confinement domains of individual voltage-gated Ca²⁺ (Ca_V) channels. A: mean squared displacement (MSD) calculated using Eq. 1 plotted against time interval. Data were fit with Eq. 2 to determine the size of the confinement area (L²) from the asymptotes of the curves. B: bar graph showing the average confinement areas. Ca_V channels at the synapses of enzymatically dissociated cones (n = 15; black squares in A) were confined to a membrane surface area of 0.18 μm² (control). Depletion of membrane cholesterol with MβCD (n = 10; white triangles in A) or COase (n = 11; black diamonds in A) significantly increased the confinement area to 0.88 μm² (**P = 0.003 vs. control) and 0.37 μm² (*P = 0.014 vs. control), respectively. Repleting membrane cholesterol with the MβCD-cholesterol complex (n = 9; white circles in A) after an initial MβCD treatment reduced the confinement area to 0.26 μm² (P = 0.17 vs. control). C–F: examples of trajectory plots of quantum dot (QD)-labeled Ca_V channels in isolated cones from different conditions: control (C), MβCD treated (D), COase treated (E), and cholesterol repleted (F).

Fig. 3.

Depletion of membrane cholesterol reduced the efficiency of vesicle release. To determine effects of cholesterol manipulation on the ability of Ca_V channel openings to trigger vesicle release, we analyzed synaptic release using simultaneous recordings of I_Ca in cones and postsynaptic currents (PSCs) in horizontal cells. From these paired recordings, we deconvolved the number of presynaptic Ca_V channel openings and vesicle release events. A: a control example of I_Ca from a cone (thick gray trace) overlaid on the simultaneously recorded PSC from a horizontal cell (thin black trace). The rate of Ca_V channel openings per ms per ribbon was obtained by deconvolution of I_Ca. Deconvolution of the PSC using the average mEPSC waveform yielded the release rate. In the example in A, the release rate was normalized for 5 ribbon contacts. Dividing the release rate per ribbon by the rate of Ca_V channel openings per ribbon yielded the number of release events per opening (B). C: PSC from a horizontal cell (thin black trace) and I_Ca from a cone (thick gray trace) in a retinal slice treated with MβCD. D: the number of release events per Ca_V channel opening. In this example, the release rate was normalized for 4 ribbon contacts. E: bar graph showing peak release efficiency (i.e., the number of vesicle fusion events triggered by a Ca_V channel opening at the peak of release) in control (0.32 ± 0.029, n = 20), MβCD (0.22 ± 0.026, n = 13; *P = 0.028 vs. control), COase (0.21 ± 0.033, n = 9; *P = 0.028 vs. control), and cholesterol repletion (0.26 ± 0.030, n = 12; P = 0.21) conditions. F: schematic diagram of the anatomic arrangements used to model release at the cone ribbon synapse as described in the text.

Fig. 4.

Cholesterol depletion diminished vesicle release evoked by 5-ms steps but not by 8-ms steps. Vesicle exocytosis from cones was measured using capacitance techniques. Voltage-clamped cones were stepped from −70 to −10 mV for short pulses to elicit a burst of vesicle fusion. Capacitance measurements were suspended during the test step and for 20 ms afterwards to avoid gating charges and allow time for the phase angle feedback circuitry to settle. A: examples of exocytotic capacitance responses evoked by 5-ms test steps in a control and MβCD-treated cone. B: examples of exocytotic capacitance responses evoked by 8-ms test steps in the same control and MβCD-treated cones. C: using a brief pulse of 5 ms (just faster than the time constant for release of ∼3 ms; Rabl et al. 2005), MβCD treatment caused a significant reduction in capacitance responses (48.4 ± 18.5 fF, n = 5) compared with control cells (143 ± 19.6 fF, n = 6; **P = 0.0072). Release evoked from cones stepped to −10 mV for 8 ms exhibited no significant differences in the 2 conditions (control amplitude: 130 ± 14.8 fF, n = 10; MβCD amplitude: 100 ± 23.5 fF, n = 10; P = 0.27).