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Receptive fields (RFs) of most retinal ganglion cells (RGCs) consist of an excitatory center and suppressive surround. The RF center arises from the summation of excitatory bipolar cell glutamatergic inputs, whereas the surround arises from lateral inhibitory inputs. In the retina, both gamma amino butyric acid (GABA) and glycine are inhibitory neurotransmitters. A clear role for GABAergic inhibition modulating the RGC RF surround has been demonstrated across species. Glycinergic inhibition is more commonly associated with RF center modulation, although there is some evidence that it may contribute to the RF surround. The synaptic glycinergic chloride channels are formed by three homomeric β and two homomeric α subunits that can be glycine receptor (GlyR) α1, α2, α3, or α4. GlyRα composition is responsible for currents with distinct decay kinetics. Their expression within the inner plexiform laminae and neuronal subtypes also differ. We studied the role of GlyR subunit selective modulation of RGC RF surrounds, using mice lacking GlyRα2 (Glra2−/−), GlyRα3 (Glra3−/−), or both (Glra2/3−/−). We chose this molecular genetic approach instead of pharmacological manipulation because there are no subunit selective antagonists and strychnine blocks all GlyRs. Comparisons of annulus-evoked responses among wild type (WT) and GlyRα knockouts (Glra2−/−, Glra3−/− and Glra2/3−/−) show that GlyRα2 inhibition enhances RF surround suppression and post-stimulus excitation in only WT OFF RGCs. Similarities in the responses in Glra2−/− and Glra2/3−/− RGCs verify these conclusions. Based on previous and current data, we propose that GlyRα2-mediated input uses a crossover inhibitory circuit. Further, we suggest that GlyRα2 modulates the OFF RGC RF center and surround independently. In summary, our results define a selective GlyR subunit-specific control of RF surround suppression in OFF RGCs.

In the retina, the receptive fields (RFs) of almost all ganglion cells (GCs) are comprised of an excitatory center and a suppressive surround. The RF center arises from local excitatory bipolar cell (BC) inputs and the surround from lateral inhibitory inputs. Selective antagonists have been used to define the roles of GABAAand GABACreceptor-mediated input in RF organization. In contrast, the role of glycine receptor (GlyR) subunit-specific inhibition is less clear because the only antagonist, strychnine, blocks all GlyR subunit combinations. We used mice lacking the GlyRα2 (Glra2−/−) and GlyRα3 (Glra3−/−) subunits, or both (Glra2/3−/−), to explore their roles in GC RF organization. By comparing spontaneous and visually evoked responses of WT withGlra2−/−,Glra3−/−andGlra2/3−/−ON- and OFF-center GCs, we found that both GlyRα2 and GlyRα3 modulate local RF interactions. In the On pathway, both receptors enhance the excitatory center response; however, the underlying inhibitory mechanisms differ. GlyRα2 participates in crossover inhibition, whereas GlyRα3 mediates serial inhibition. In the Off pathway, GlyRα2 plays a similar role, again using crossover inhibition and enhancing excitatory responses within the RF center. Comparisons of single and double KOs indicate that GlyRα2 and GlyRα3 inhibition are independent and additive, consistent with the finding that they use different inhibitory circuitry. These findings are the first to define GlyR subunit-specific control of visual function and GlyRα2 subunit-specific control of crossover inhibition in the retina.

In the retina, the receptive fields (RFs) of most neurons are comprised of an excitatory center and a suppressive surround. Retinal ganglion cell (RGC) RF center excitatory input arises from bipolar cell (BC) inputs, while their surround arises from lateral inhibitory inputs. Because of the availability of selective antagonists the role of GABAergic inputs has been well defined. In contrast, the role of individual glycine receptor (GlyR) subunit inhibition is less clear because the antagonist, strychnine, blocks all GlyR subunit combinations. To define individual retinal circuits that utilize specific glycinergic subunits, I examined maintained and visually-evoked responses of ON- and OFF-center GCs from mice lacking expression of the GlyRa2 (Glra2-/-) or GlyRa3 (Glra3-/-) subunits to those of C57Bl/6J (WT) RGCs using an in vivo extracellular approach. Previous observations have defined glycine and GABA inputs across BC classes and in a variety of amacrine and RGCs. Using this information and by comparing the responses of WT vs. Glra2-/- and Glra3-/- RGCs; I conclude that both subunits modulate local RF interactions. Within the On pathway, GlyRa2 and GlyRa3 inputs play similar roles. Their responses predict that they participate in serial inhibitory circuits that decrease a direct GABAergic inhibition that modulates maintained, but not peak firing rates. In contrast within the Off pathway, GlyRa2 and GlyRa3 inputs define two populations of RGCs. In one, GlyRa2 participates in a serial inhibitory circuit that modulates maintained firing, whereas in the other, GlyRa,3 mediates direct inhibition that controls the peak firing rate. Only GlyRa2 modulates lateral interactions to the RF surround where it mediates a direct inhibitory input to all OFF-center RGCs. My results suggest that GlyRa2 and GlyRa3 inputs define two populations of OFF-center RGCs. In addition, both subunits participate in retinal circuits that can be distinguished not only by the RGC RF center type, but also by the type of inhibitory circuit. These results are the first demonstration of subunit specific control of RGC visual responses and, are the first evidence of serial glycine to GABA as well as glycine to glycine circuits in the retina.

Fetal Alcohol Spectrum Disorders (FASD) describes a wide range of phenotypic defects affecting facial and neurological development associated with ethanol teratogenicity. It affects approximately 1 in 100 children born in the United States each year. Genetic predisposition along with timing and dosage of ethanol exposure are critical in understanding the prevalence and variability of FASD. The zebrafish attributes of external fertilization, genetic tractability, and high fecundity make it a powerful tool for FASD studies. However, a lack of consensus of ethanol treatment paradigms has limited the interpretation of these various studies. Here we address this concern by examining ethanol tissue concentrations across timing and genetic background. We utilize headspace gas chromatography to determine ethanol concentration in the AB, fli1:EGFP , and Tu backgrounds. In addition, we treated these embryos with ethanol over two different developmental time windows, 6–24 h post fertilization (hpf) and 24–48 hpf. Our analysis demonstrates that embryos rapidly equilibrate to a sub-media level of ethanol. Embryos then maintain this level of ethanol for the duration of exposure. The ethanol tissue concentration level is independent of genetic background, but is timing-dependent. Embryos exposed from 6 to 24 hpf were 2.7–4.2-fold lower than media levels, while embryos were 5.7–6.2-fold lower at 48 hpf. This suggests that embryos strengthen one or more barriers to ethanol as they develop. In addition, both the embryo and, to a lesser extent, the chorion, surrounding the embryo are barriers to ethanol. Overall, this work will help tighten ethanol treatment regimens and strengthen zebrafish as a model of FASD.