Research Articles: Cellular/Molecular Spatial Organization and Dynamics of the Extracellular Space in the Mouse Retina https://doi.org/10.1523/JNEUROSCI.1717-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.1717-20.2020 Received: 3 July 2020 Revised: 24 August 2020 Accepted: 31 August 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 2 3 4 5 Spatial Organization and Dynamics of the Extracellular Space in the Mouse Retina 6 7 Abbreviated title: Extracellular Space in the Retina 8 9 10 11 12 Sidney P. Kuo, Pei-Pei Chiang, Amy R. Nippert, and Eric A. Newman 13 Department of Neuroscience, University of Minnesota, 321 Church St SE, Minneapolis, MN 14 55455, USA 15 16 Corresponding author: Eric A. Newman, [email protected] 17 18 Number of pages: 26 19 Number of figures: 7 20 Number of words: Abstract 227; Introduction, 648; Discussion, 1424 21 22 The authors declare no competing financial interests. 23 24 25 26 27 Acknowledgements 28 Funded by NIH grants R01-EY026514, R01-EY026882 and P30-EY011374 to EAN, T90- 29 DE0227232 to SPK, and T32-EY025187 to ARN. The authors thank Charles Nicholson for his 30 helpful comments on an earlier version of the manuscript. 31 32 1 33 ABSTRACT 34 The extracellular space (ECS) plays an important role in the physiology of neural circuits. 35 Despite our detailed understanding of the cellular architecture of the mammalian retina, little is 36 known about the organization and dynamics of the retinal ECS. We developed an optical 37 technique based on two-photon imaging of fluorescently-labeled extracellular fluid to measure 38 the ECS volume fraction (D) in the ex vivo retina of male and female mice. This method has high 39 spatial resolution and can detect rapid changes in D evoked by osmotic challenge and neuronal 40 activity. Measured ECS D varied dramatically in different layers of the adult mouse retina, with D 41 equaling ~0.050 in the ganglion cell layer (GCL), ~0.122 in the inner plexiform layer (IPL), 42 ~0.025 in the inner nuclear layer (INL), ~0.087 in the outer plexiform layer (OPL), and ~0.026 in 43 the outer nuclear layer (ONL). ECS D was significantly larger early in retinal development; D 44 was 67% larger in the IPL and 100% larger in the INL in neonatal mice compared to adults. In 45 adult retina, light stimulation evoked rapid decreases in ECS D. Light-driven reductions in ECS 46 D were largest in the IPL, where visual stimuli decreased D ~10%. These light-evoked 47 decreases demonstrate that a physiological stimulus can lead to rapid changes in ECS D and 48 indicate that activity-dependent regulation of extracellular space may contribute to visual 49 processing in the retina. 50 51 52 SIGNIFICANCE STATEMENT 53 The volume fraction of the extracellular space (ECS D), that portion of CNS tissue occupied by 54 interstitial space, influences diffusion of neurotransmitters from the synaptic cleft and volume 55 transmission of transmitters. However, ECS D has never been measured in live retina and little 56 is known about how ECS D varies following physiological stimulation. Here we show that ECS D 57 varies dramatically between different retinal layers and decreases by 10% following light 58 stimulation. ECS D differences within the retina will influence volume transmission and light- 59 evoked D variations may modulate synaptic transmission and visual processing in the retina. 60 Activity-dependent ECS D variations may represent a mechanism of synaptic modulation 61 throughout the CNS. 62 2 63 INTRODUCTION 64 The extracellular space (ECS) is comprised of narrow, interconnected pathways that permit the 65 diffusion of ions, signaling molecules and metabolites between cells within the CNS. By 66 regulating how molecules diffuse through neural tissue, the ECS can strongly influence CNS 67 function. For example, the local geometry of the ECS around synapses affects how 68 neurotransmitters diffuse away from release sites and therefore shapes the spillout of 69 transmitters from the synaptic cleft onto extrasynaptic receptors (receptors localized outside of 70 the cleft), spillover of transmitters between neighboring synapses, and volume transmission of 71 transmitters to distant receptors (Sykova and Nicholson, 2008; Nicholson and Hrabetova, 2017). 72 Studies have demonstrated changes in ECS structure during development (Lehmenkuhler et al., 73 1993; Vorisek and Sykova, 1997), across the sleep-wake cycle (Xie et al., 2013), and during 74 disease progression (Mazel et al., 2002; Reum et al., 2002; Syková et al., 2005; Slais et al., 75 2008; Tonnesen et al., 2018). These studies, along with work showing that electrical stimulation 76 of neuronal activity can drive changes in ECS properties (Svoboda and Sykova, 1991; 77 Prokopova-Kubinova and Sykova, 2000) raise the intriguing possibility that dynamic alterations 78 to ECS structure may provide an unappreciated mechanism for the activity-dependent 79 modulation of synaptic and volume transmission. However, the extent to which physiological 80 activity within a neural circuit can result in altered ECS properties is not known. 81 The ECS plays an important role in retinal physiology. For example, the spatial extent of 82 neuromodulatory signaling by transmitters such as dopamine (Witkovsky, 2004), and the 83 recruitment of extrasynaptic receptors following synaptic release (Chen and Diamond, 2002; 84 Zhang and Diamond, 2009) is shaped by the properties of the ECS in the retina. Despite our 85 detailed understanding of the cellular architecture of the mammalian retina, little is known 86 regarding the organization or dynamics of the retinal ECS. This is in part because the compact, 87 layered structure of the retina poses a particular challenge to the traditional method of 88 measuring ECS properties in live tissue, termed ‘real-time iontophoresis’ (Nicholson and 89 Hrabetova, 2017). In this approach, an ion, typically tetramethylammonium (TMA), is 90 iontophoretically injected into a tissue. The ion diffuses through the ECS and its buildup is 91 measured at a distance of ~150 Pm by an ion selective microelectrode. Values of ECS volume 92 fraction (D and tortuosity (O are derived from the time course and magnitude of the rise in TMA 93 concentration at the ion selective microelectrode. An important assumption of the real-time 94 iontophoresis technique is that the tissue being studied is isotropic; that is, the properties of the 95 ECS are uniform in all directions (but see Rice et al., 1993; Saghyan et al., 2012). This 3 96 assumption does not hold in the retina, which is comprised of discrete layers (e.g. somatic 97 versus synaptic) with very different cellular organizations. The spatial resolution of the real-time 98 iontophoresis technique, which is limited by the distance between iontophoretic and ion- 99 selective electrodes (~150 μm), is also not compatible with the dimensions of the retina (~180 100 Pm thick in mouse). 101 We report here an optical method for measuring the ECS D the fraction of the total volume of 102 tissue occupied by the ECS, in the retina. This technique, based on two-photon excitation of a 103 fluorescent dye that labels the ECS, allows us to determine the precise values of D in different 104 retinal layers with high spatial resolution and to measure changes in D during development and 105 during light stimulation. We find that D varies nearly 5-fold within different layers of the retina, 106 from ~0.025 in the inner nuclear layer to ~0.122 in the inner plexiform layer and that D is 46 - 107 100% larger in different layers of the developing retina than in the adult. We also show that D 108 can decrease by >10% in response to visual stimulation. These light-evoked decreases 109 demonstrate that a physiological stimulus can evoke rapid changes in ECS D and indicate that 110 activity-dependent regulation of ECS may contribute to visual processing in the retina. 4 111 METHODS 112 Ethics Statement 113 All experimental procedures were approved by and adhered to the guidelines of the Institutional 114 Animal Care and Use Committee of the University of Minnesota. 115 116 Retinal preparations 117 All experiments were performed on tissue from 0 to 12 week old male and female C57BL/6 118 mice. Mice were anesthetized with isoflurane and killed by cervical dislocation. The eyes were 119 enucleated and the cornea, lens and vitreous was removed in room temperature, oxygenated 120 (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF). The ventral portion of eyecups were 121 then used to obtain either of two experimental preparations. Eyecup preparation: Experiments 122 that assessed osmotic and light-driven changes in ECS Dutilized an eyecup preparation in 123 which the retina remained attached to the underlying pigment epithelium, choroid and sclera. 124 The eyecup was trimmed down to a ~3 x 3 mm square and mounted flat onto a poly-L-lysine 125 coated coverglass on the bottom of a perfusion chamber, vitreal surface up. Tissue was secured 126 in place with a harp made from platinum wire and nylon threads. Isolated retina preparation: The 127 isolated retina preparation was used when determining the depth-dependent fluorescence 128 signal attenuation and when measuring ECS D in different layers in order to minimize the 129 amount of dye-labeled ACSF between the tissue and microscope objective and to achieve more 130 uniform tissue flatness than was possible in the eyecup preparation.