Homeostatic plasticity shapes the visual system’s first synapse

RE Johnson, NW Tien, N Shen, JT Pearson, F Soto… – Nature Communications, 2017

Abstract

Vision in dim light depends on synapses between rods and rod bipolar cells (RBCs). Here, we find that these synapses exist in multiple configurations, in which single release sites of rods are apposed by one to three postsynaptic densities (PSDs). Single RBCs often form multiple PSDs with one rod; and neighboring RBCs share ~13% of their inputs. Rod-RBC synapses develop while ~7% of RBCs undergo programmed cell death (PCD). Although PCD is common throughout the nervous system, its influences on circuit development and function are not well understood. We generate mice in which ~53 and ~93% of RBCs, respectively, are removed during development. In these mice, dendrites of the remaining RBCs expand in graded fashion independent of light-evoked input. As RBC dendrites expand, they form fewer multi-PSD contacts with rods. Electrophysiological recordings indicate that this homeostatic co-regulation of neurite and synapse development preserves retinal function in dim light.

Introduction

The ability of mammals to see in low light depends on the synapses between rods and rod bipolar cells (RBCs)¹. Mutations in genes involved in the formation and function of these synapses cause congenital stationary night blindness (CSNB) in people². Key molecular events in rod-RBC synapse assembly have been uncovered using mouse models of CSNB and other strategies^3,4,5,6,7. A recent electron microscopy study showed that the spherical rod axon terminals (i.e., rod spherules) connect to varying numbers of RBC dendrites⁸, suggesting that rod-RBC synapse configurations might be malleable within molecularly defined boundaries. However, because only a few RBCs were reconstructed⁸, the range of configurations of rod-RBC synapses remains uncertain, and whether plasticity controls their distribution has not been tested.

Developmental plasticity is essential for the emergence of precise circuits; and its dysregulation underlies common neurodevelopmental disorders^{9, 10}. Known plasticity mechanisms include axon and dendrite remodeling^11,12,13, synapse formation and elimination^{14,15,16,17,18}, and changes in the geometry and molecular architecture of synapses^19,20,21. In developing circuits, populations of same-type neurons need to coordinate their connectivity to homogeneously cover input and target cell types, while individual neurons need to adjust their connectivity to avoid saturation and quiescence. Because most studies so far have focused on individual plasticity mechanisms and their underlying signals^22,23,24, how different plasticity mechanisms (e.g., neurite remodeling and synapse formation) are co-regulated during development to optimize wiring of neuronal populations and individuals in vivo is unknown.

Throughout the developing nervous system, many neurons undergo programmed cell death (PCD), adjusting the complement and density of neuronal populations in emerging circuits^{25, 26}. PCD triggers plasticity in the remaining neurons, which take over innervation of vacated inputs and targets. The retina is an ideal system for studying cell density-dependent plasticity, because axons and dendrites of each cell type cover synaptic layers uniformly^{27, 28}. Cell density-dependent plasticity has been shown to regulate axon and dendrite growth of some retinal neurons^{29, 30} but not others^{31, 32}. To what extent RBC axons and dendrites undergo cell density-dependent plasticity is incompletely understood³³, and how cell density-dependent plasticity regulates synaptic development of any neuron is unknown.

To analyze the influence of cell density-dependent plasticity on RBC development and retinal circuit function, we generated mice in which ~53 and ~93% of RBCs, respectively, are removed by transgenic expression of diphtheria toxin concurrent with naturally occurring PCD²⁶. We find that dendritic and axonal territories of the remaining RBCs increase in graded fashion to improve population coverage, whereas multi-PSD synapses on dendrites and synapse density of axons are reduced to restrain connectivity of individual RBCs. This coordinated plasticity of neurites and synapses occurs independent of light-evoked input from rods and preserves retinal output in dim light.

Results

Rod-RBC synapses exist in different configurations

To examine the configurations of rod-RBC synapses, we first sparsely and selectively labeled rods by in vivo electroporation of a plasmid in which the fluorescent protein DsRed is expressed from promoter elements of the rod-specific neural retina leucine zipper (Nrl) transcription factor (Fig. 1a, Nrl-DsRed)^{34, 35}. Each rod contains a single presynaptic ribbon^{36, 37}. By contrast, we observed a range of RBC PSDs containing the probable G-protein coupled receptor 179 (Gpr179)^38,39,40 in individual rod spherules (from one to three receptor clusters in >99% of spherules) (Fig. 1a, b). The distribution of RBC postsynaptic specializations per rod was similar when we stained for the metabotropic glutamate receptor mGluR6^{3, 41} instead of Gpr179, and when super-resolution rather than conventional confocal microscopy was used (Supplementary Fig. 1). We next explored how individual RBCs connect with rods in their dendritic fields. We generated adeno-associated viruses (AAVs) that expressed the fluorescent protein tdTomato from promoter elements of the Grm6 gene, which encodes mGluR6 (Grm6_S-tdTomato)⁴². Intravitreal injections of Grm6_S-tdTomato labeled ON bipolar cells, which include RBCs and ON cone bipolar cells. RBCs could easily be identified by their characteristic morphology^{15, 43}. We flat-mounted retinas of mice injected with Grm6_S-tdTomato and stained synaptic contacts for Gpr179 (Fig. 1c). Rod labeling had shown that overlapping Gpr179 clusters were invariably localized within the same spherule (Fig. 1a). We therefore counted overlapping Gpr179 clusters as synapses with a single rod, and determined whether a given cluster co-localized with a dendritic tip of the labeled RBC. We found that on average RBCs fail to be innervated ~10% of rods in their dendritic fields, assemble a single PSD in ~63% of spherules, and form multi-PSD contacts with ~27% of rods (Fig. 1c, d). Rod-RBC synapses thus exist in different configurations, in which a single presynaptic release site is apposed by one to three PSDs belonging to one or more RBCs.

… Visual stimuli were presented on an organic light-emitting display (OLED, eMagin) and projected onto the photoreceptor side of the retina via a substage condenser (patch clamp recordings) or through a 20 × 0.5 NA water immersion objective (MEA recordings). Photon fluxes at the preparation were calibrated with a photometer (UDT Instruments S471, 268R) and converted to photoisomerization rates based on the spectral output of the OLED measured with a Spectrometer (StellarNet, BLACK Comet), the rod spectral sensitivity, and a collecting area of 0.5 μm²⁷⁰. Scotopic stimuli (mean intensity: 1.5 rhodopsin isomerization/rod/s, 1.5 R*) were centered on the soma of the recorded cell. To test contrast sensitivity, short luminance steps (250 ms) were presented every 2.25 s in a circular area (diameter: 300 μm)⁵⁷. Baseline-subtracted responses (spike rate or conductance) were measured during 100 ms time windows. Spatiotemporal receptive fields were analyzed by presenting circular white noise stimuli, in which the intensity of rings of equal area centered on the recorded cell was chosen at random every 33 ms (refresh rate: 30 Hz) from a Gaussian distribution. Receptive field maps were then constructed by reverse correlation of the response with the stimulus via spike-triggered stimulus averaging^{57, 58}.