Several transgenes containing GFP-tagged synaptic proteins were previously described: nuIs321 (punc-17::mCherry), nuEx379 (pacr-2::GFP), nuIs152 (punc-129::GFP::SNB-1), nuIs159 (punc-129::SYD-2::YFP), nuIs169 (punc-129::GSNL-1::YFP) ( Sieburth et al., 2005 and Sieburth check details et al., 2007), zdIs5 (pmec-4::GFP) ( Pan et al., 2008), akIs38 (UNC-29::GFP), ( Francis et al., 2005) and nuIs283 (pmyo-3::UNC-49::GFP) (J. Bai and J.M.K., unpublished

data). For real-time PCR experiments, late L4 and early adult worms were transferred to mock treatment plates or plates containing 1 mM aldicarb for 1 hr after which the RNA from these animals was harvested and quantitative PCR performed as detailed in Simon et al. (2008). This work was supported by grants from the NIH (NS32196 to J.M.K. and NS32057 to G.G.). We thank the following for strains, reagents, and advice: C. elegans Genetic Stock Center, Villu Maricq, Jihong Bai, Ed Pym, and Eyleen O’Rourke. We also thank members of the Kaplan laboratory for suggestions and comments on this manuscript. “
“Many brain functions, including memory formation and acquired neuroprotection, are controlled by transient increases in the intracellular calcium concentration induced by synaptic activity (Hardingham

and Bading, 2010 and Silva et al., 1998). Calcium can act locally near the site of entry to switch on signaling mechanisms that modulate several biochemical processes that in turn lead to changes in neuronal excitability and/or the efficacy of synaptic transmission (Bliss selleck et al.,

2007). The long-term maintenance of such activity-induced, functional adaptations requires that calcium transients invade the cell nucleus and activate or repress gene Metalloexopeptidase expression (Hardingham and Bading, 2010 and Greer and Greenberg, 2008). Nuclear calcium is one of the most potent signals in neuronal gene expression and represents a key player in the dialog between synapse and nucleus (Zhang et al., 2009). It controls cAMP response element binding (CREB)- and CREB-binding protein (CBP)-mediated transcription (Hardingham et al., 1997, Hardingham et al., 1999, Hardingham et al., 2001, Chawla et al., 1998 and Hu et al., 1999) and is critical for the acquisition of memories and the build-up of neuroprotective activity in synaptically activated neurons (Limbäck-Stokin et al., 2004, Papadia et al., 2005, Zhang et al., 2007 and Zhang et al., 2009). A picture of how genomic events induced by nuclear calcium signaling regulate persistent neuroprotection is emerging (Zhang et al., 2009, Zhang et al., 2011 and Lau and Bading, 2009). In contrast, nuclear calcium-regulated processes required for memory formation are unknown. Here we considered the possibility that nuclear calcium signaling modulates structural features of neurons, in particular the complexity of the dendritic arbor, that determine their ability to receive and process inputs (Cline and Haas, 2008).

Extracellular polarization of the hair cells produced a transverse motion of the

tectorial membrane toward the neural limb (Figures 7B and 7C). Venetoclax molecular weight A mean negative displacement of beads of 24 ± 16 nm (range 10 to 56 nm; d = 0.36–0.51) was achieved in eight preparations using 100 μA current flowing from the abneural to neural electrodes. These measurements were made on beads located above SHCs and, by focusing through the tectorial membrane, it was possible also to image the bundles ( Figures 7A and 7B). Comparison of the relative displacement of the hair bundle to that of beads lying directly above the bundle indicated that the bundle moved slightly more than the selleck screening library bead ( Figure 7B). The ratio of the bundle to bead displacement for the same polarization was 1.45 ± 1.1 (n = 5), though this is not significantly different from 1 (two-tailed Students t test, p = 0.2), suggesting a tight coupling of the bundles to the tectorial membrane. Tectorial membrane

motion in response to extracellular stimulation was also monitored over the THCs, the mean negative displacement being 25 ± 22 nm (n = 4). The displacements obtained by extracellular polarization were similar to those elicited from individual SHCs in that they were reversibly blocked by 10 mM Na+ salicylate (Figure 7C). This reversible block, shown in four preparations, makes it unlikely that lateral movements produced by extracellular currents stemmed from a direct electrophoretic motion of the tectorial membrane. The dose-response relationship for the action of salicylate on the voltage-evoked movements (Figures 7D and 7E) was fit with a Hill equation with a half-blocking concentration of 3.6 mM, similar to that in OHCs (Tunstall et al., 1995).

In order to ascertain whether the bundle movements were elicited over the same range of membrane potentials as those seen in individual SHCs, we estimated the membrane depolarization evoked by extracellular polarization. To do this, SHCs were patch clamped in a preparation in which the all tectorial membrane had been removed but which was stimulated by extracellular current polarization (Figure 7F). The change in membrane potential increased with the current polarization, as did the size of the hair bundle movement. In multiple SHC recordings, the depolarization (measured in current clamp) was proportional to the magnitude of the external current from 40 to 100 μA, with a proportionality constant of 0.74 mV/μA. If extracellular current stimuli are as effective in the presence of the tectorial membrane, the 100 μA polarization routinely used would depolarize the SHCs to ∼20 mV assuming they have a resting potential of about −55 mV in perilymph (Tan et al., 2013).

Netrins are locally released by the axon terminals of lamina neurons L3 and, instead of forming a gradient, are captured by Fra-expressing target neuron branches in layer M3. Localized Netrins act at short range and are instructive for layer-specific targeting. Our findings provide evidence that localized chemoattractant guidance molecules released not by the synaptic partners but by intermediate target neurons can coordinate layer-specific targeting of axons by providing distinct positional information. To gain insights into the role of the Fra guidance receptor in Rucaparib order adult visual circuit assembly, we examined its expression in the retina and optic lobe. In the retina

(Figures 1C–1F′), colabeling with capricious-Gal4 (caps-Gal4) ( Shinza-Kameda et al., 2006) driving membrane-bound green fluorescent

protein (GFP) expression revealed that at 24 hr after puparium formation (APF), Fra protein is expressed in R8 cells along their cell bodies, and at 42 and 55 hr in their rhabdomeres, the membrane-rich organelles required for phototransduction in adults. Fra was also transiently detected in rhabdomeres Apoptosis Compound Library of R1–R6 cells at 42 hr. In the optic lobe ( Figures 1G–1J′), Fra protein initially accumulates at the distal medulla neuropil border, where R8 axons temporarily pause before proceeding to their final layer M3 during the second half of pupal development. Specific knockdown of fra in the target area by expressing a UAS RNA interference (RNAi) transgene (UAS-fraIR) using the FLPout approach ( Ito et al.,

1997) in conjunction with the transgenes ey-FLP ( Newsome et al., 2000), ey3.5-Gal80 ( Chotard et al., 2005), and longGMR-Gal80 PDK4 (lGMR, kindly provided by C. Desplan) ( Wernet et al., 2003) indicated that this expression can be attributed to R8 growth cones ( Figures 1K–1L′). At 42 and 55 hr, Fra protein is enriched in the emerging and final M3 layer ( Figures 1H–1I′). Expression persists at lower levels in adults ( Figures 1J and 1J′). Moreover, Fra is strongly expressed in R1–R6 axons in the lamina at 42 hr, when their growth cones leave their original bundle and extend stereotypic projections to adjacent columns ( Figures 1H and 1H′). Additional expression was detected in glial cell subtypes in the lamina and medulla. However, within the medulla neuropil, Fra expression is associated with neurons because glial-specific knockdown using reversed polarity (repo)-Gal4 ( Sepp and Auld, 2003) did not alter the expression pattern ( Figures 1M and 1M′). Knockdown of fra specifically in the eye using the FLPout approach in conjunction with the ey3.5-FLP transgene ( Bazigou et al., 2007) further confirmed that Fra protein is associated with target neuron processes (see Figure S1 available online). Thus, Fra is expressed by R8 axons and in neurites of target neuron subtypes extending into the M3 layer. To assess the function of fra in controlling R cell axon targeting, we used the ey3.

utexas.edu/djlab). The apparatus was consisted of opaque- and matte-finished black acrylic sheet (36” × 36” × 24”).

Each rat was randomly assigned and placed into the center of the field following 30 min saline, vehicle, and diazepam i.p. injection. Behavior in the open field arena was recorded for 6 min using a CCD camera. The surface of the open field arena was cleaned with 70% EtOH in order to remove permeated odors by previous animals after each trial. To analyze behaviors Selleck JAK inhibitor for the last five minutes, the open field arena was divided into 9 equal-size squares (12” by 12”). Basal exploration activity was measured by total traveled distance (inch) and anxiety level was assessed by the number of center square entries, the duration, Vorinostat nmr and the traveled distance in the center square. A line crossing was defined as the body center crosses a line. The apparatus was made of black acrylic sheet. Four arms (50 cm long and 10 cm wide) are connected and elevated to a height of 50 cm from the floor. Two

arms are open (open arms) and the other two arms are enclosed within 40 cm walls. Each rat was placed into the intersection of the four arms (center area) 30 min following saline, vehicle, or diazepam i.p. injection. Behavior in elevated plus maze was recorded for 6 min using a CCD camera. The surface of the elevated plus maze was cleaned with 70% EtOH in order to remove permeated click here odors by previous animals after each trial. To

analyze behavior test for 6 min, the number of arm entries and the percentage of open arm time (duration in open arm/total time) were assessed. Arm entry was defined as 50% of the body being positioned within the arm. The tank is made of transparent Plexiglas cylinder (80 cm tall × 30 cm in diameter) filled with water (23°C–24°C) to a depth of 45 cm. Water in the tank was changed after each trial. For the first exposure, rats without drug treatment were placed in the water for 15 min (pretest session). Twenty-four hours later, rats were placed in the water again for a 6 min session (test session) 30 min following saline, vehicle, diazepam (1 mg/kg), ketamine (15 mg/kg), or fluoxetine (10 mg/kg) i.p. injection. Forced swim test was recorded by a video cameras positioned on the top of the water tank. A passive activity was defined as floating and making only those movements necessary to keep the nose above the water. Behaviors from forced swim test was quantified by using a time sampling technique to rate the predominant behavior over a 5 s interval as described (Lucki, 1997) and a custom written program. Four- to five-week-old rats were bilaterally microinjected with lentivirus expressing shRNA-control or shRNA-HCN1 in the dorsal hippocampal CA1 region. After behavior test, dorsal hippocampal slices (350 μm) were prepared from 10- to 12-week-old lentivirus-infected male Sprague-Dawley rats.

Changing spike duration can alter the firing pattern, as in the case of BK channels (Madison selleck compound and Nicoll, 1984 and Shao et al., 1999). Not only does the number of action potentials generated during a barrage of synaptic activities dictate the strength of the signal, the message conveyed also depends critically on the temporal pattern of spike firing. We have found that reducing CaCC activity could facilitate the EPSP-spike coupling, causing a short train of synaptic activities to transition from a single spike or no spike at all

to a burst of action potentials, indicating that CaCC modulation could adjust neuronal signaling both quantitatively and qualitatively. Action potentials can back-propagate into the dendrite of hippocampal pyramidal neurons (Hoffman et al., 1997 and Migliore et al., 1999). Modulation of the duration of

back-propagating action potential invading the dendritic tree is likely to have a strong impact not only on dendritic excitability, but also on coincidence detection buy Navitoclax of synaptic inputs—the basis of synaptic plasticity. The relative timing between an incoming synaptic potential and a back-propagating spike can determine whether the synapse giving rise to the synaptic potential is potentiated or depressed (Caporale and Dan, 2008 and Dan and Poo, 2004). A broader spike could conceivably widen the  time window during which a synaptic signal can be potentiated. This study provides

evidence for the involvement of Ca2+-activated Cl− channels in the negative feedback to rein in the excitatory synaptic responses. Remarkably, NFA block of CaCC increased synaptic potentials in a way similar to the apamin block of SK channels (Ngo-Anh et al., 2005). Reducing CaCC activity facilitates EPSP summation by leaving the earlier, smaller EPSP intact and most amplifying the later, larger EPSPs (Figure 6A; Table 1). This activity-dependent modulation is more nuanced than simple EPSP modulation and has two important implications (1) CaCC only reins in large EPSPs that have the potential of bringing the neuron to firing an action potential; CaCC acts as a brake, but not on all EPSPs. (2) Once CaCC is activated by Ca2+ influx through NMDA-Rs during a barrage of synaptic responses or Ca2+ from other cellular processes, the neuronal signaling outcome will be influenced by CaCC modulation of EPSP summation and the threshold for spike generation by EPSP. CaCC thus dynamically gates the information flow between neurons, and it only does so when there are sufficient neuronal activities to raise internal calcium level.

Quantification of the proportion of GFP-labeled cells (CreGFP-electroporated cells in RhoAfl/+ animals or GFP-electroporated 3-Methyladenine concentration cells in RhoAfl/fl embryos were used as a control; Figure 4D, yellow bars) confirmed not only the efficient neuronal migration but even revealed an apparently faster migration of RhoA-depleted neurons as a significantly higher proportion of GFP+ cells was located in the upper most bin of five equally bins, corresponding to the cortical plate (CP), compared to controls ( Figure 4D, blue bars). These experiments therefore suggest that RhoA-depleted neurons did not fail to migrate but rather migrated faster than control cells. Some RhoA-depleted cells had migrated even beyond the CP forming ectopic

clusters within and beyond layer 1 in brains analyzed at E19, i.e., 5 days after electroporation ( Figures 4E and 4F), reminiscent of the type II cobblestone lissencephaly observed in the cerebral cortex of cKO mice and described previously. To test the possibility that RhoA may indeed this website slow down migration and release this break in its absence, we electroporated a spontaneously activated (“fast-cycling”) mutant of RhoA (RhoA∗) and quantified the position of GFP+ cells in the cerebral cortex. Indeed, consistent with this scenario, we detected a significant increase of GFP-labeled cells in the lower layers 3 days after electroporating

the fast-cycling RhoA mutant construct ( Figures 4C, 4C′, and 4D, pink bars), suggesting a delay in migration in the condition of activated RhoA. Even though these migrating neurons GPX6 also had a normal polarized morphology, consistent with a normal migration, it would still be possible that RhoA-deficient neurons reach their final position but in a very different or disturbed migration compared to normal. We therefore directly monitored the migration

of electroporated cells by live imaging in slices. E13 Cre-electroporated cortices were sliced and sections were imaged 2 days after transfection for approximately 9 hr to examine the movement of migrating cells (Movie S1; Figures 5A–5D). All cells imaged performed a normal radial migration moving basally and directed by a single leading process, as shown by the traces of tracked cells in Figures 5A–5D. However, despite the early reduction of RhoA protein in electroporated regions, the remnant levels may still be sufficient to allow for migration of these neurons. To examine migration of neurons lacking RhoA protein entirely, we employed transplantation experiments with cells from E14 cKO cortices which had completely lost RhoA protein by E12. E14 cells were dissociated and labeled with cell tracker green prior to transplantation into isochronic WT cortices (Figures 6A and 6B). Similar to control cells also RhoA−/− cells had often reached the IZ or CP 3 days after transplantation ( Figures 6C–6E). Thus, neurons still migrate fairly normal and reach the CP also in the complete absence of RhoA.

, 2010). In other cases, bAPs prime the dendrite to produce synaptically evoked calcium spikes which mediate STDP-LTP (Zhou et al., 2005; Kampa et al., 2006) For more on dendritic excitability and STDP, see Sjöström et al. (2008). The decremental propagation of bAPs creates a profound spatial gradient of STDP in neurons. In L5 pyramidal cells in neocortex, brief pre- and postsynaptic spike trains evoke Hebbian STDP at proximal synapses (<100 μm from soma) but progressively less LTP at more distal synapses. The most distal synapses (>500 μm) show only anti-Hebbian LTD in

response to pre-leading-post pairing. Distal LTD can be converted to LTP by supplying sufficient dendritic depolarization to either enhance bAP propagation (Sjöström and Häusser, 2006) or convert the single bAP into a dendritic-somatic spike burst (Letzkus et al., 2006). Smaller L2/3 Selleckchem ISRIB pyramidal cells exhibit a similar OSI-744 mw trend in which distal synapses express less STDP and a broader LTD window than proximal synapses (Froemke et al., 2005). Thus, decremental bAP propagation creates distinct dendritic plasticity zones in which different rules for synapse modification exist ( Figure 4B; Kampa et al., 2007; Spruston, 2008). In general, the most proximal synapses experience the strongest bAPs and are expected to exhibit Hebbian STDP with minimal requirements for synaptic cooperativity

and firing rate. More distal synapses will exhibit LTD-biased Hebbian STDP ( Froemke et al., 2005) or anti-Hebbian LTD ( Sjöström and Häusser, 2006) and will require high firing rates or strong synaptic convergence for Hebbian STDP. These synapses can exhibit anti-Hebbian STDP, if post-leading-pre firing drives synaptically evoked calcium spikes ( Kampa et al., 2006; Letzkus et al.,

new 2006). Very distal synapses may be largely outside the influence of bAPs, so that STDP is absent and plasticity is induced by cooperative firing of neighboring inputs that evokes dendritic sodium or calcium spikes or regenerative NMDA spikes ( Golding et al., 2002; Gordon et al., 2006). The existence of different plasticity rules within dendritic regions may contribute to activity-dependent stabilization of different functional classes of synapses in these regions ( Froemke et al., 2005). Modulation of dendritic excitability will regulate both the shape of STDP rules and the spatial extent of dendritic plasticity zones, including increasing or decreasing the prevalence of STDP relative to local, associative forms of plasticity. Neuromodulation has robust effects on the spike timing dependence of plasticity. This includes gating of STDP, as in adult visual cortex slices, where exogenous activation of receptors coupled to adenylate cyclase (e.g., β-adrenergic receptors) and PLC (e.g., muscarinic acetylcholine receptors) are necessary for LTP and LTD, respectively, within Hebbian STDP (Seol et al., 2007).

At surgery, animals were anesthetized with isoflurane vapor and an intraperitoneal injection of Equithesin (pentobarbital and chloral hydrate; 1.0 ml/250 g body weight; supplementary doses: 0.15 ml/250 g). Local anesthetic (Xylocaine) was applied to skin before making the incision. For MEC implants, tetrodes were inserted 4.6 mm lateral to midline and ∼0.35 mm anterior to the transverse sinus and tilted ∼9° anteriorly in the sagittal plane. For PPC implants, tetrodes were inserted between −3.9

and −4.2 mm Ku-0059436 purchase posterior to bregma, and 2.3–2.6 mm lateral to midline. All PPC implants were in the right hemisphere, all MEC implants were in the left hemisphere. Bone-tapping stainless steel screws were inserted securely in the skull and dental cement was applied to affix the drives to the skull. One screw served as a ground electrode. All rats were housed individually in Plexiglas cages (45 × 44 × 30 cm) in a humidity and temperature-controlled environment, and kept on a 12 hr light/12 hr dark schedule. Training and testing occurred in the dark phase. Experiments were performed in accordance with the Norwegian Animal Welfare Act and the European Convention for the Protection

of Vertebrate Animals used for Experimental and Other Scientific Purposes. Rats were connected via AC-coupled unity-gain operational amplifiers and counterbalanced cables to an Axona recording system. Tetrodes were lowered in 50 μm steps while the rat rested on a towel in a flower pot on a pedestal. Turning stopped when grid cells MK-8776 order appeared on the MEC

drive (≥1,800 μm) or when well-separated units appeared in PPC (500–1,800 μm). Data collection started when signal amplitudes exceeded approximately five times the noise level (root mean square 20–30 μV) and units were stable for >3 hr. Recordings were performed as rats foraged randomly for crumbs of vanilla cookies on a black mat in a black open-field arena (1.5 × 1.5 × 0.5 Casein kinase 1 m) surrounded by a black curtain. A white cue-card (95 × 45 cm) hung above the south end of the arena. The animals’ movements were tracked with dual infrared LEDs, spaced 6 cm apart on the head stage (sampling rate of 50 Hz). When the rat regularly covered the entire open field in a 20 min trial (typically after 1–2 weeks), it was trained in a hairpin maze constructed by removing the mat and inserting nine black 135 × 30 × 1 cm Perspex walls in parallel grooves 14 cm apart in the underlying floor (Derdikman et al., 2009). Rats were trained to run from east to west and west to east. Food crumbs were initially administered by the experimenter at the south end of each arm. Once the rats ran regularly (after 2–3 weeks) the food protocol was winnowed to 1 crumb in the final arms.

First, the lateral PFC is a large expanse of cortex and the drugs were injected virtually in the middle of it. Second, the effects began immediately during the long, slow, injection. If the site of action were elsewhere, it would take time for the drug to diffuse to those sites. Instead, we observed that the effects began a few minutes after the start of the injection. Third, not all sites in the lateral PFC produced an effect, as might be expected if the drug had widespread actions (see Experimental

Procedures); most vlPFC, but not dlPFC, sites resulted in impairment. There are similar levels of D1R expression in these areas (de Almeida et al., 2008), so this may reflect the greater number of CB-839 price vlPFC than dlPFC neurons with selectivity during conditional visuomotor

tasks (Wilson et al., 1993), and thus suggests the vlPFC as the site of action. Finally, while SCH23390 has a preference for binding to D1Rs, it also binds LGK-974 in vivo to serotonin 5-HT2 and 5-HT1C receptors (Bischoff et al., 1988; Alburges et al., 1992). Thus, we cannot disregard the possibility that some of the SCH23390 effects are mediated in part by these receptors. The specificity of D1 receptors could be established by injecting several 5-HT antagonists, but conducting these experiments would be difficult in monkeys. Importantly, our neurophysiological results are in line with previous studies in which D1R specificity could be established. For example, a previous study in monkeys (Sawaguchi and Goldman-Rakic, 1994) showed that local injections of ketanserin, a selective antagonist of 5-HT2 receptors, near PFC sites affected by SCH23390 failed to induce any clear changes in the monkeys’ performance, suggesting that SCH23390 was acting on D1Rs. Studies in humans indicate that prefrontal D1Rs are involved in working memory (Robbins, 2000). Müller et al. (1998) demonstrated performance-enhancing effects of a mixed D1-D2 agonist on spatial working

memory, but no effect of a selective D2 agonist, suggesting a role for D1Rs. Studies in monkeys and rodents have shown that Thymidine kinase modulation by D1Rs in the dlPFC and prelimbic cortex during spatial working memory follows an inverted-U-shaped curve: too little or too much D1R stimulation causes cognitive impairment, while moderate levels of D1R stimulation strengthen and sculpt selectivity to optimize PFC function (Williams and Goldman-Rakic, 1995; Zahrt et al., 1997; Seamans et al., 1998; Granon et al., 2000; Chudasama and Robbins, 2004; Vijayraghavan et al., 2007). Our results suggest that prefrontal D1Rs in the monkey vlPFC play a relevant role in associative learning, perhaps by sculpting neural selectivity of prefrontal neurons. D1Rs in the lateral PFC may play a role in cognitive flexibility as well.

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.