Waves were similarly eliminated in OFF

CBCs and diffuse ACs. Next, we applied meclofenamic acid (MFA, 200 μM), a blocker of gap junctions (Pan et al., 2007 and Veruki and Hartveit, 2009), during dual recordings of CBCs and RGCs. Similar to NBQX and AP5, MFA uniformly (6/6) abolished EPSCs in RGCs as well as depolarizations of ON CBCs and diffuse ACs, and the hyperpolarizations of OFF CBCs (Figures 7G and 7H). In agreement with recent data (Veruki and Hartveit, 2009), even with fast solution exchange, the effects of MFA BMS354825 showed slow onset and recovery kinetics (>20 min). To test whether this accounts for our previous failure to silence stage III waves with MFA in multielectrode array (MEA) recordings (Kerschensteiner and Wong, 2008), we repeated these experiments. Dolutegravir mouse Indeed, when allowing

for prolonged exposure and washout, we confirmed that MFA reversibly suppresses stage III waves irrespective of the recording method (Figures S7A and S7B). Moreover, 18-β-Glycyrrhetinic acid (18-β-GA, 50 μM), another blocker of gap junctions, similarly inhibited stage III waves in MEA recordings (Figures S7C and S7D). Together these data suggest that gap junctions and glutamatergic transmission form interacting circuit mechanisms for lateral excitation of ON CBCs, which are both required for the propagation and/or initiation of stage III waves. In waves of all stages (I–III) bursts of RGC activity spread across the retina separated by periods of silence (Demas et al., 2003 and Wong, 1999). Uniquely during stage III (P10–P14), neighboring ON and OFF RGCs are recruited sequentially (ON before OFF) into passing waves (Kerschensteiner and Wong, 2008). This asynchronous activity

is thought to help segregate ON and OFF circuits in the dLGN and shape emerging ON and OFF columns in geniculocortical projections (Cramer and Sur, 1997, Dubin et al., 1986, Gjorgjieva et al., 2009, Hahm et al., 1991, Jin et al., 2008 and Kerschensteiner and Wong, 2008). At the same time, the lateral propagation of stage III waves mafosfamide and the asynchronous firing of RGCs in both eyes appear to maintain retinotopic organization and eye-specific segregation of retinofugal projections (Chapman, 2000, Demas et al., 2006 and Zhang et al., 2012). RGC spiking during stage III waves is known to depend on glutamate release from BCs and a transient rise in extrasynaptic glutamate in the IPL has been shown to accompany each wave (Blankenship et al., 2009, Firl et al., 2013 and Wong et al., 2000). But how stage III waves are initiated and propagated and what mechanisms offset the activity of ON and OFF RGCs was unclear. Using systematic combinations of dual patch-clamp recordings, we identify intersecting lateral excitatory and vertical inhibitory circuits in the developing retina (Figure 8) and elucidate mechanisms by which neurons in these circuits generate precisely patterned stage III waves.