Differential Expression Analysis Identifies Candidate Synaptogenic Molecules for Wiring Direction-Selective Circuits in the Retina.

An organizational feature of neural circuits is the specificity of synaptic connections. A striking example is the direction-selective (DS) circuit of the retina. There are multiple subtypes of DS retinal ganglion cells (DSGCs) that prefer motion along one of four preferred directions. This computation is mediated by selective wiring of a single inhibitory interneuron, the starburst amacrine cell (SAC), with each DSGC subtype preferentially receiving input from a subset of SAC processes. We hypothesize that the molecular basis of this wiring is mediated in part by unique expression profiles of DSGC subtypes. To test this, we first performed paired recordings from isolated mouse retinas of both sexes to determine that postnatal day 10 (P10) represents the age at which asymmetric synapses form. Second, we performed RNA sequencing and differential expression analysis on isolated P10 ON-OFF DSGCs tuned for either nasal or ventral motion and identified candidates which may promote direction-specific wiring. We then used a conditional knock-out strategy to test the role of one candidate, the secreted synaptic organizer cerebellin-4 (Cbln4), in the development of DS tuning. Using two-photon calcium imaging, we observed a small deficit in directional tuning among ventral-preferring DSGCs lacking Cbln4, though whole-cell voltage-clamp recordings did not identify a significant change in inhibitory inputs. This suggests that Cbln4 does not function primarily via a cell-autonomous mechanism to instruct wiring of DS circuits. Nevertheless, our transcriptomic analysis identified unique candidate factors for gaining insights into the molecular mechanisms that instruct wiring specificity in the DS circuit.

The influence of spontaneous and visual activity on the development of direction selectivity maps in mouse retina.

In mice, retinal direction selectivity is organized in a map that aligns to the body and gravitational axes of optic flow, and little is known about how this map develops. We find direction selectivity maps are largely present at eye opening and develop normally in the absence of visual experience. Remarkably, in mice lacking the beta2 subunit of neuronal nicotinic acetylcholine receptors (ß2-nAChR-KO), which exhibit drastically reduced cholinergic retinal waves in the first postnatal week, selectivity to horizontal motion is absent while selectivity to vertical motion remains. We tested several possible mechanisms that could explain the loss of horizontal direction selectivity in ß2-nAChR-KO mice (wave propagation bias, FRMD7 expression, starburst amacrine cell morphology), but all were found to be intact when compared with WT mice. This work establishes a role for retinal waves in the development of asymmetric circuitry that mediates retinal direction selectivity via an unknown mechanism.

Excitatory neurotransmission activates compartmentalized calcium transients in Müller glia without affecting lateral process motility.

Neural activity has been implicated in the motility and outgrowth of glial cell processes throughout the central nervous system. Here, we explore this phenomenon in Müller glia, which are specialized radial astroglia that are the predominant glial type of the vertebrate retina. Müller glia extend fine filopodia-like processes into retinal synaptic layers, in similar fashion to brain astrocytes and radial glia that exhibit perisynaptic processes. Using two-photon volumetric imaging, we found that during the second postnatal week, Müller glial processes were highly dynamic, with rapid extensions and retractions that were mediated by cytoskeletal rearrangements. During this same stage of development, retinal waves led to increases in cytosolic calcium within Müller glial lateral processes and stalks. These regions comprised distinct calcium compartments, distinguished by variable participation in waves, timing, and sensitivity to an M1 muscarinic acetylcholine receptor antagonist. However, we found that motility of lateral processes was unaffected by the presence of pharmacological agents that enhanced or blocked wave-associated calcium transients. Finally, we found that mice lacking normal cholinergic waves in the first postnatal week also exhibited normal Müller glial process morphology. Hence, outgrowth of Müller glial lateral processes into synaptic layers is determined by factors that are independent of neuronal activity.

When it comes to studying the nervous system, neurons often steal the limelight; yet, they can only work properly thanks to an ensemble cast of cell types whose roles are only just emerging. For example, 'glial cells' ­ their name derives from the Greek word for glue ­ were once thought to play only a passive, supporting function in nervous tissues. Now, growing evidence shows that they are, in fact, integrated into neural circuits: their activity is influenced by neurons, and, in turn, they help neurons to function properly. The role of glial cells is becoming clear in the retina, the thin, light-sensitive layer that lines the back of the eye and relays visual information to the brain. There, beautifully intricate Müller glial cells display fine protrusions (or 'processes') that intermingle with synapses, the busy space between neurons where chemical messengers are exchanged. These messengers can act on Müller cells, triggering cascades of molecular events that may influence the structure and function of glia. This is of particular interest during development: as Müller cells mature, they are exposed to chemicals released by more fully formed retinal neurons. Tworig et al. explored how neuronal messengers can influence the way Müller cells grow their processes. To do so, they tracked mouse retinal glial cells 'live' during development, showing that they were growing fine, highly dynamic processes in a region rich in synapses just as neurons and glia increased their communication. However, using drugs to disrupt this messaging for a short period did not seem to impact how the processes grew. Extending the blockade over a longer timeframe also did not change the way Müller cells developed, with the cells still acquiring their characteristic elaborate process networks. Taken together, these results suggest that the structural maturation of Müller glial cells is not impacted by neuronal signaling, giving a more refined understanding of how glia form in the retina and potentially in the brain.

Müller Glia in Retinal Development: From Specification to Circuit Integration.

Müller glia of the retina share many features with astroglia located throughout the brain including maintenance of homeostasis, modulation of neurotransmitter spillover, and robust response to injury. Here we present the molecular factors and signaling events that govern Müller glial specification, patterning, and differentiation. Next, we discuss the various roles of Müller glia in retinal development, which include maintaining retinal organization and integrity as well as promoting neuronal survival, synaptogenesis, and phagocytosis of debris. Finally, we review the mechanisms by which Müller glia integrate into retinal circuits and actively participate in neuronal signaling during development.