Spatial Organization and Dynamics of the Extracellular Space in the Mouse Retina

Abstract

The extracellular space (ECS) plays an important role in the physiology of neural circuits. Despite our detailed understanding of the cellular architecture of the mammalian retina, little is known about the organization and dynamics of the retinal ECS. We developed an optical technique based on two-photon imaging of fluorescently labeled extracellular fluid to measure the ECS volume fraction (a) in the ex vivo retina of male and female mice. This method has high spatial resolution and can detect rapid changes in a evoked by osmotic challenge and neuronal activity. The measured ECS a varied dramatically in different layers of the adult mouse retina, with a equaling ;0.050 in the ganglion cell layer, ;0.122 in the inner plexiform layer (IPL), ;0.025 in the inner nuclear layer (INL), ;0.087 in the outer plexiform layer, and ;0.026 in the outer nuclear layer (ONL). ECS a was significantly larger early in retinal development; a was 67% larger in the IPL and 100% larger in the INL in neonatal mice compared with adults. In adult retinas, light stimulation evoked rapid decreases in ECS a. Light-driven reductions in ECS a were largest in the IPL, where visual stimuli decreased a values ;10%. These light-evoked decreases demonstrate that a physiological stimulus can lead to rapid changes in ECS a and indicate that activity-dependent regulation of extracellular space may contribute to visual processing in the retina.

Cellular mechanisms mediating activity‐dependent extracellular space shrinkage in the retina

“…We previously demonstrated that a flickering light stimulus evokes a rapid decrease in ECS α in the mouse retina (Kuo et al, 2020). We have now explored the cellular mechanisms leading to this ECS α decrease.…”

“…Assuming that the dye fills all of the ECS and that the dye concentration within the ECS equals the concentration in the aCSF, then where F is the mean calcein fluorescence signal in an imaged plane. Calcein fluorescence measurements were corrected for depth‐dependent signal attenuation, as described previously (Kuo et al, 2020). Light‐evoked changes in ECS α were measured from the peak of the change to the pre‐stimulus baseline.…”

“…Calcein fluorescence measurements were corrected for depthdependent signal attenuation, as described previously (Kuo et al, 2020). Light-evoked changes in ECS α were measured from the peak of the change to the pre-stimulus baseline.…”

“…We have recently demonstrated that neuronal activity evoked by physiological photic stimuli results in a rapid modulation of ECS α in the retina (Kuo et al, 2020). Light stimulation leads to a shrinkage of ECS α of up to 10% within tens of seconds and with latencies of $1 s.…”

“…photon excitation of calcein was achieved using a Ti:sapphire laser (Chameleon Vision, Coherent) tuned to 800 nm (2-6 mW postobjective laser power) with calcein fluorescence emission detected using a 500-550 nm bandpass filter. The z-axis resolution of the microscope, measured by imaging 0.175 μm diameter fluorescent beads, was 1.4 μm (full-width at half-maximum of the bead fluorescence profile;Kuo et al, 2020). Alexa Fluor 594-filled Müller cells were imaged with 810 nm excitation (1.2 mW post-objective laser power) and a 570-640 nm bandpass emission filter.…”