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.

Elevated Hoxb5b Expands Vagal Neural Crest Pool and Blocks Enteric Neuronal Development in Zebrafish.

Neural crest cells (NCCs) are a migratory, transient, and multipotent stem cell population essential to vertebrate embryonic development, contributing to numerous cell lineages in the adult organism. While great strides have been made in elucidating molecular and cellular events that drive NCC specification, comprehensive knowledge of the genetic factors that orchestrate NCC developmental programs is still far from complete. We discovered that elevated Hoxb5b levels promoted an expansion of zebrafish NCCs, which persisted throughout multiple stages of development. Correspondingly, elevated Hoxb5b also specifically expanded expression domains of the vagal NCC markers foxd3 and phox2bb. Increases in NCCs were most apparent after pulsed ectopic Hoxb5b expression at early developmental stages, rather than later during differentiation stages, as determined using a novel transgenic zebrafish line. The increase in vagal NCCs early in development led to supernumerary Phox2b+ enteric neural progenitors, while leaving many other NCC-derived tissues without an overt phenotype. Surprisingly, these NCC-derived enteric progenitors failed to expand properly into sufficient quantities of enterically fated neurons and stalled in the gut tissue. These results suggest that while Hoxb5b participates in vagal NCC development as a driver of progenitor expansion, the supernumerary, ectopically localized NCC fail to initiate expansion programs in timely fashion in the gut. All together, these data point to a model in which Hoxb5b regulates NCCs both in a tissue specific and temporally restricted manner.

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.

The Impact of Steroid Activation of TRPM3 on Spontaneous Activity in the Developing Retina.

In the central nervous system, melastatin transient receptor potential (TRPM) channels function as receptors for the neurosteroid pregnenolone sulfate (PregS). The expression and function of TRPM3 has been explored in adult retina, although its role during development is unknown. We found, during the second postnatal week in mice, TRPM3 immunofluorescence labeled distinct subsets of inner retinal neurons, including a subset of retinal ganglion cells (RGCs), similar to what has been reported in the adult. Labeling for a TRPM3 promoter-driven reporter confirmed expression of the TRPM3 gene in RGCs and revealed additional expression in nearly all Müller glial cells. Using two-photon calcium imaging, we show that PregS and the synthetic TRPM3 agonist CIM0216 (CIM) induced prolonged calcium transients in RGCs, which were mostly absent in TRPM3 knock-out (KO) mice. These prolonged calcium transients were not associated with strong membrane depolarizations but induced c-Fos expression. To elucidate the impact of PregS-activation of TRPM3 on retinal circuits we took two sets of physiological measurements. First, PregS induced a robust increase in the frequency but not amplitude of spontaneous postsynaptic currents (PSCs). This increase was absent in the TRPM3 KO mice. Second, PregS induced a small increase in cell participation and duration of retinal waves, but this modulation persisted in TRPM3 KO mice, indicating PregS was acting on wave generating circuits independent of TRPM3 channels. Though baseline frequency of retinal waves was slightly reduced in the TRPM3 KO mice, other properties of waves were indistinguishable from wildtype. Together, these results indicate that the presence of neurosteroids impact spontaneous synaptic activity and retinal waves during development via both TRPM3-dependent and independent mechanisms.

Implication of Socio-Demographics on Cognitive-Related Symptoms in Sports Concussion Among Children.

BACKGROUND: Sports-related concussion remains a public health challenge due to its morbidity and mortality. One of the consequences of concussion is cognitive impairment (CI) and cognitive-related symptoms (CRS) which determine, to some extent, physical and behavioral functioning of children who sustain concussion. Despite the high prevalence of CI and CRS associated with concussion, the risk factors are not fully understood. We aimed to characterize CRS and to examine its relationship with race, ethnicity, age, insurance, and sex in a pediatric population. METHODS: A retrospective cohort (case-only) design was used to assess CRS prevalence and its relationship with race and sex using a pediatric hospital's electronic medical records. A consecutive sample was used with 1429 cases between 2007 and 2014. Study characteristics were examined using chi-square and log binomial regression for hypothesis-specific testing. RESULTS: Of the 1429 cases, 872 (61.0 %) were boys and 557 (39.0 %) were girls. The racial distribution indicated 1146 (80.2 %) Whites, 170 (11.9 %) Blacks/African Americans, and 113 (7.9 %) others. The prevalence of CRS was 78.0 %. Whereas boys had sustained more concussions, girls were more likely to present with CRS; prevalence risk ratio = 1.07, 95 % CI 1.01-1.13, p = 0.02. The crude analysis indicated no racial disparities in CRS prevalence, but the multivariable analysis did, comparing White to Black/African American children; adjusted prevalence risk ratio (aPRR) = 1.77, 99 % CI 1.02-3.08, p = 0.008. CONCLUSIONS: Racial disparities exist in CRS among children with sports-related concussion, and Black/African American children are more likely, relative to Whites, to suffer CRS. Due to uncertainty in causal inference, we caution the interpretation and application of these data in risk-adapted concussion prevention.