Finally, the surround antagonist component had a broad spatial extent and a time constant similar to that of the antagonistic input in the circle response model. We hypothesize that this component is mediated by lateral inputs from columns in which surround responses occur. Overall, the fits to the six circles and four annuli responses explained 98% of the variance (Figures 4D and 4E). However, fitting responses to annuli with small internal

radii (2° and 4°) that provide partial center stimulation and significant surround stimulation required a distinct weighting of inputs (Figure S4E and Supplemental Galunisertib molecular weight Experimental Procedures). In contrast, most responses to bright circles of different sizes could be captured simply as scaled versions of the same response shape (Figure S4F). A center-surround RF differentially affects the amplitudes of responses to stimuli with different spatial periods (e.g., Dubs, 1982). Thus, the relative strengths of responses to sinusoidal inputs with different periods provide a measure

of acuity. Acuity differences between different axes may represent an early specialization for the detection of motion in a particular orientation (Srinivasan and Dvorak, 1980). We therefore measured L2 responses to sinusoidal gratings with periods ranging from 5° to 90°, presented on a virtual cylinder. Each grating was rotated at a different speed so that the temporal contrast frequency was 0.5 Hz and was oriented to simulate either pitch or yaw rotations of the fly (Figure 5A). L2 responses to these stimuli were MK-8776 cost sinusoidal, as expected

for a linear system (Figure 5B; Clark et al., 2011). Intriguingly, at short spatial periods (10° and 20°), responses to pitch rotations were stronger than CYTH4 responses to yaw rotations (p < 10−5, Figures 5B and 5C). At a 5° spatial period, responses were weak, as expected from retinal optics and an RF center of approximately 5° (Järvilehto and Zettler, 1973; Stavenga, 2003), while spatial periods around 40° drove the strongest responses (Figure 5C). Only slight attenuation by surround inhibition was observed at larger spatial periods (Figure S5A). This could be for physiological reasons, arising, for example, from effects of the relative timing of center and surround stimulation on antagonism. However, this could also result from technical limitations, as our display spanned slightly less than 60° of visual space in each direction. Nevertheless, as responses at short spatial periods clearly show higher sensitivity with pitch rotations, visual acuity must be higher around this axis, making the L2 RF spatially anisotropic. Analogous results were obtained using a moving bright bar stimulus, which weakly stimulated the surround prior to entering the RF center, and induced a stronger surround response when it moved upward across the screen than when it moved medially (Figures 1B, S1A, S5B, and S5C).

How could bipolar cells continuously drive excitatory input to the ganglion cell but independently instruct inhibition through wide-field amacrine cells in a discontinuous, switch-like way? To investigate whether the excitatory input to the PV1 ganglion cell and the inhibitory switch encompassing amacrine cells is mediated by the same or different mechanisms, we blocked glutamate signaling using CPP and NBQX, which are antagonists of the ionotropic glutamate

receptors. As expected, the excitation to PV1 cells was blocked. However, at light levels when the switch is ON, the inhibitory input remained, suggesting that the excitatory drive to the amacrine and ganglion cells is acting through a different mechanism (Figures 6D, 6E, and S5). In the presence of NBQX and CPP, the inhibitory current was Gefitinib cost blocked by APB, which stops the response of those bipolar cells that respond to contrast increments (Figure 6E). As amacrine cells could be driven by electrical Ku 0059436 synapses rather than chemical synapses (Deans et al., 2002), we created a triple transgenic line in which both alleles of connexin36 were knocked out (Deans and Paul, 2001) and the PV cells were labeled with EYFP. In this knockout animal, we performed the same

functional experiments as those that showed the switching filtering properties. Since connexin36 is needed for the rod signals to reach the amacrine and ganglion cells (Deans et al., 2002), there were no inhibitory or excitatory responses at low light levels, as expected. More importantly, the inhibitory input to PV1 cells decreased significantly (Figures 6F and S5) and the spiking responses of the PV1 cell to large and small

spots remained similar across higher light intensities (Figures 6G and 6H). These results, the taken together with the voltage-clamp recordings (Figures 6D and 6E), suggest that the switching amacrine cells receive excitatory input via electrical synapses incorporating connexin36. These experiments are consistent with cone bipolar cells providing input to switching amacrine and PV1 cells using different mechanisms but do not explain why the excitatory input to PV1 cells does not show a stepwise increase in strength at the critical light level (Figure 4D). In order to understand this, we examined the time course of the excitation to PV1 cells. The quantification of responses thus far incorporated a long timescale, using average responses across a 0.5 s time window. When we quantified excitation in a shorter time window after stimulus onset, the strength of excitation also showed a stepwise increase at the critical light level (Figures 6I and 6J) and a few spikes were detectable transiently after the onset of the light stimulus (Figures 1A and S4).

We noted that 30%–40% of motor neurons are preserved in laterally located LMC motor pools in the absence of GDE2 at E13.5. This number is remarkably similar to that reported for the gamma motor neuron component of motor pools, which are predicted to begin diversifying from alpha motor neurons by E13.5, given their differential sensitivities to embryonic programmed cell death (Burke et al., 1977, Friese et al., 2009, Buss et al., 2006 and Hui et al., 2008). To examine whether GDE2 selectively

regulates the differentiation of alpha, but not gamma, motor neurons, we compared Gde2−/− animals with WT siblings at postnatal day 5 (P5) and P28, when molecular and somal size differences allow alpha and gamma motor neurons to be distinguished ( Friese et al., 2009). The percentage selleck inhibitor of ChAT+/NeuN+ alpha motor neurons in the ventral outer quadrant of the spinal cord corresponding to the LMC was decreased by approximately 30%–40% at P5 and P28 in Gde2−/− animals; however, the percentage

of ChAT+/NeuN− gamma motor neurons was not significantly altered ( Figures 5A–5F). The expression of Err3 in the ventral horn of the spinal cord appeared to be similar between Gde2−/− and WT littermates, consistent with preserved gamma motor neuron differentiation in the Carfilzomib in vitro absence of GDE2 ( Figures 5G and 5H). Gamma motor neurons have a small somal area compared with alpha motor neurons ( Burke et al., 1977, Friese et al., 2009 and Shneider et al., 2009). The number of putative gamma motor neurons (somal area < 380 μm2) was unchanged between WT and Gde2−/− littermates, but there was a dramatic reduction of putative alpha motor neurons in Gde2−/− animals (somal

area = 380–1,400 μm2) ( Figures 5I and 5J). Using the same criteria discussed above, no significant changes in alpha and gamma motor neuron numbers were observed in the medially located MMC of Gde2−/− and WT animals ( Figures 5K–5O). Thus, the reduction in LMC motor pools in Gde2 null animals correlates with Tryptophan synthase a specific loss of alpha motor neurons, whereas LMC gamma motor neurons and MMC alpha and gamma motor neuron production are intact. At hindlimb levels, GDE2 is first localized to motor neuron cell body areas at the time of motor neuron generation but is subsequently enriched in motor axons from E12.5 (Figure 6B; Figure S5). To define when GDE2 functions in hindlimb motor pool formation, we generated Gde2lox/−; Rosa26:CreER+ animals, which enabled the timed ablation of GDE2 through Cre-dependent recombination via the administration of 4-hydroxytamoxifen (4-OHT) ( Badea et al., 2003). We injected pregnant dams with 4-OHT at E8.5 to ablate GDE2 expression prior to the initiation of motor neuron progenitor differentiation at lumbar levels and at E10.5 to eliminate GDE2 by the end of motor neuron generation ( Nornes and Carry, 1978).

, 2012 for review). To determine whether the excitatory drive onto CCK INs was altered during ITDP, we used fluorescence-guided whole-cell recordings to monitor the SC-evoked EPSPs in CCK INs expressing GFP. GFP was restricted to CCK-expressing GABAergic INs using an intersectional genetic approach (Taniguchi et al.,

2011; Figure S5A, see Experimental Procedures). Temsirolimus We also recorded SC-evoked EPSPs in tdTomato-labeled PV INs. We found that ITDP induction did not alter the magnitude of the EPSP evoked by SC stimulation in either CCK or PV INs (Figures 8A1–8A3), ruling out either general or specific changes in synaptic excitation. Next, we tested whether the postsynaptic GABA response was altered in CA1 PNs using the photoactivatable caged compound RuBi-GABA (Rial Verde et al., 2008). The peak amplitude and rise time of uncaging IPSCs in CA1 PNs evoked by a single 470 nm light pulse on the perisomatic space (using 5 μM RuBi-GABA) was unchanged during ITDP (Figures 8B1–8B3). Thus, ITDP does not alter the postsynaptic GABA response. These results imply that iLTD during ITDP is most likely mediated by a decrease in GABA release from CCK INs. To test this idea, we measured the paired-pulse

ratio (PPR) of IPSCs evoked in CA1 PNs by two closely spaced stimuli (50 ms interpulse interval) because an increase in PPR is thought to reflect a decrease in the probability of transmitter release (Dobrunz and Stevens, 1997). We found that ITDP was indeed associated with an increase in the PPR, either when IPSCs were evoked by electrical stimulation of the Rutecarpine SC pathway (73.13% ± Selleck IPI145 7.6% increase, p < 0.0001, n = 13) or by photostimulation of ChR2+ CCK INs (63.59% ± 14.6% increase,

p < 0.01, paired t test, n = 5; Figures 8C1–8C3). In contrast, the PPR for IPSCs evoked by photostimulation of ChR2+ PV INs was unaltered by ITDP (p = 0.8741, paired t test, n = 4). This supports the view that iLTD during ITDP results from a selective decrease in GABA release from perisomatic-targeting CCK INs. One well-characterized mechanism that decreases GABA release from CCK INs is through the action of endocannabinoids (eCBs), retrograde messengers that act on G protein-coupled CB1 receptors (CB1Rs) abundantly expressed in CCK presynaptic terminals (Castillo et al., 2012). These molecules have been implicated in a form of iLTD induced by high-frequency SC stimulation (Chevaleyre and Castillo, 2003). A recent study found that the induction of ITDP in CA1 PNs also requires eCB release and activation of CB1Rs (Xu et al., 2012). However, this latter study used a protocol that was suited neither for examining FFI nor the iLTD component of ITDP (see Discussion). Given our findings that iLTD accounts for the major synaptic change during ITDP, we investigated the role of eCBs in this process.