Commissural neurons were cultured for 24 hr in vitro and subsequently stimulated with VEGF (10 or 25 ng/ml, R&D systems, #493-MV) for 30 min. For immunostaining, neurons were fixed in 4% PFA/4% sucrose (complemented with proteinase and phosphatase inhibitors [Roche]) for 15 min at room temperature. Immunostaining for P-SFK was performed using a Rabbit (polyclonal) anti-Src (pY418) phosphorylation site specific antibody (Invitrogen, #44660G) followed by an Alexa-488 conjugated secondary antibody. For immunoblotting,

neurons were lysed in RIPA buffer complemented with proteinase and phosphatase inhibitors (Roche). An anti-Phospho-Src Family antibody (Cell Signaling, #2101) was used to probe the western blots. Subsequently blots were stripped and reprobed with an anti-Src Dabrafenib solubility dmso (36D10) antibody (Cell Signaling, #2109). The average of the phospho-SFK Selleckchem BMS-754807 fluorescence signal was measured for each growth cone using Image J and normalized to the average fluorescence signal in control growth cones. At least 50 growth cones were analyzed in two independent experiments (performed in triplicates) and statistical differences were assessed by unpaired t test versus control conditions. Floor plates (FPs) isolated from E11.5 mouse embryos were cultured in

three dimensional rat tail collagen in B27-supplemented Neurobasal medium. Conditioned medium from FPs (explants from a single FP were cultured in 300 μl) or control medium were collected after 48 hr and processed for further measurements

of VEGF and Shh protein concentration using the commercial Quantikine human VEGF ELISA kit (R&D Systems) and Shh ELISA kit (Abcam, ab100639), respectively. Flk1 protein expression was determined in lysates of E13 rat dorsal spinal cord tissue using the commercial mouse Flk1 ELISA kit (R&D Systems, Quantikine MVR200B). Expression levels were quantified by real-time RT-PCR, relative to the expression level of β-actin, using the following forward (F) and reverse primers (R) and probes (P), labeled with fluorescent dye (FAM) and quencher (TAMRA). β-actin: F,5′-AGAGGGAAATCGTGCGTGAC-3′; R,5′-CAATAGTGATGACCTGGCCGT-3′; P,5′-FAMCACTGCCGCATCCTCTTCCTCCCTAMRA-3′; next Flk1: F,5′-ACTGCAGTGATTGCCATGTTCT-3′ ; R,5′-TCATTGGCCCGCTTAACG-3′; P,5′-FAMTGGCTCCTTCTTGTCATTGTCCTACGGATAMRA-3′; Vegf: F,5′-AGTCCCATGAAGTGATCAAGTTCA-3′; R,5′-ATCCGCATGATCTGCATGG-3′; P,5′-FAMTGCCCACGTCAGAGAGCAACATCACTAMRA-3′. Reference numbers for primer sequences for mShh and mNetrin-1 are Mm00436528_m1 and Mm00500896_m1, respectively (Applied Biosystems). The percentage of the area occupied by precrossing commissural axons to the total spinal cord area was quantified based on a previously described method (Charron et al., 2003). Briefly, precrossing commissural axon area and total spinal cord area were measured on E11.

, 2011), premotor cortex (Pastor-Bernier and Cisek, 2011), and medial prefrontal cortex (Sohn and Lee, 2007; Seo and Lee, 2009; So and Stuphorn, 2010). Many of these brain areas might in fact encode the signals related to utilities of reward expected from specific actions, even when the probabilities and timing of reward vary. For example, temporally discounted values are encoded by neurons in

the prefrontal cortex (Kim Selleckchem Trichostatin A et al., 2008), posterior parietal cortex (Louie and Glimcher, 2010), and the striatum (Cai et al., 2011). Human neuroimaging experiments have also identified signals related to utilities in multiple brain areas, including the ventromedial prefrontal cortex (VMPFC) and ventral striatum (Kuhnen and Knutson, 2005; Knutson et al., 2005;

Knutson et al., 2007; Luhmann et al., 2008; Chib et al., 2009; Levy et al., 2011). Consistent with the results from single-neuron recording studies (Sohn and Lee, 2007), signals related to values of reward expected from specific motor actions have been identified in the human supplementary motor area (Wunderlich et al., 2009). Activity in the VMPFC and ventral striatum display additional characteristics of value signals used for decision making. For example, the activity in each of these areas is influenced oppositely by expected gains and losses. In addition, activity in these areas is more enhanced for expected losses than for expected gains, and this difference is related to the level of loss aversion across individuals (Tom et al., 2007). Activity in the VMPFC and ventral BMS-354825 striatum also reflects temporally discounted values for delayed reward during inter-temporal choice (Kable and Glimcher, 2007; Pine et al., 2009). Results from neuroimaging and lesion studies also suggest that the amygdala might play a role in estimating value functions according to potential losses. For example, activity

in the amygdala changes according to whether a particular outcome is framed as a gain or a loss (De Martino et al., 2006), and loss aversion is abolished in patients with focal lesions in the amygdala (De Martino et al., of 2010). Whether decisions are based on values computed for specific goods or their locations, and which brain areas encode the value signals actually used for action selection, might vary depending on the nature of choices to be made (Lee et al., 2012). The DLPFC might contribute to flexible switching between different types of value signals used for decision making. This is possible, since the DLPFC is connected with many other brain areas that encode different types of value signals (Petrides and Pandya, 1984; Carmichael and Price, 1996; Miller and Cohen, 2001). In addition, individual neurons in the DLPFC can modulate their activity according to value signals associated with specific objects as well as their locations (Kim et al., 2012b).

19 Signals were then passed through a BNC adapter chassis that was interfaced with an analog-to-digital board within a personal computer. These signals were then converted to ground reaction force vectors and moments. Data were filtered using a second order recursive low-pass Butterworth digital filter with an estimated optimum cutoff frequency

of 12.53 Hz.19 A customized LabVIEW (National Instruments Corp., Austin, TX, USA) software program computed A/P and M/L TTS. A/P and M/L components of the ground reaction force data were analyzed separately for each subject, but the same procedure Selleckchem R428 was used for both components. First, the last 10 s of the ground reaction forces were analyzed to find the smallest absolute ground reaction force range for each component.19 These ranges were accepted as the optimal range of variation values.19 A/P and M/L components of the ground reaction force data were then rectified.19 An unbounded

third order polynomial was fit from the peak force to the last data point for each component.19 TTS for each component was the point where the unbounded third order polynomial was equal to or less than the respective optimal range of variation value.19 Average A/P and M/L TTS values for each treatment condition were computed in PASW version 18.0 (SPSS, Inc., Chicago, IL, USA). Alpha level was set a priori at p ≤ 0.05 to indicate statistical selleck kinase inhibitor significance. One-tailed paired samples t tests compared SRS to control conditions for A/P and M/L TTS. Effect size d values were calculated for each t test. 22 Average percent improvements for each TTS measure were also computed for all subjects and average improvement of eight subjects who

improved with SRS (subjects who did not improve were removed). No improvements were defined as increased TTS with SRS over a control condition. Lastly, to provide insight on why some subjects did not improve with SRS, we computed effect size d values for comparing responders and non-responders on frequency of sprains, frequency of “giving-way”, and score on the AJFAT. SRS significantly improved A/P TTS over the control condition (SRS = 1.32 ± 0.31 s, Control = 1.74 ± 0.80 s; Thiamine-diphosphate kinase t(11) = −2.04, p = 0.03; d = 0.76). The average percent improvement for A/P TTS with SRS was 24% (n = 12) and increased to 34% (n = 8; SRS = 1.32 ± 0.35 s, Control = 2.01 ± 0.86 s) when four subjects who did not improve were removed. SRS did not affect M/L TTS (SRS = 1.95 ± 0.40 s, Control = 1.92 ± 0.48 s; t(11) = −0.20, p = 0.42; d = −0.07). The average percent improvement for M/L TTS with SRS was 2% (n = 12) and increased to 15% (n = 8; SRS = 1.75 ± 0.30 s, Control = 2.06 ± 0.50 s) when four subjects who did not improve were removed. Using effect size d values to detect mean differences, non-responders had greater mean values than responders on frequency of sprains, frequency of “giving-way”, and score on the AJFAT.

GRIP1 regulates AMPA receptor targeting to dendrites and the recycling of AMPA receptors to the plasma membrane following NMDA receptor (NMDAR) activation (Setou et al., 2002 and Mao et al., 2010). We, therefore, hypothesized that GRIP1b palmitoylation might in turn affect GRIP1b’s ability to regulate AMPA receptor recycling. To address this possibility, we transfected hippocampal neurons with wild-type, nonpalmitoylatable, or constitutively membrane-targeted forms of GRIP1b, together with a pHluorin-tagged GluA2 AMPA receptor, to which GRIP1 directly binds (Dong et al., 1997 and Mao et al., 2010). The pHluorin tag fluoresces

brightly at neutral pH, as when the receptor Selleck PFT�� is present on the plasma membrane. Brief treatment with NMDA drives internalization STI571 mw of pHGluA2 to recycling endosomes, whose acidity (pH <6.6) dramatically quenches pHGluA2 fluorescence, while NMDA washout induces pHGluA2 recycling to the plasma membrane and fluorescence recovery (Ashby et al., 2004, Lin and Huganir, 2007, Thomas et al., 2008 and Mao et al., 2010; Figure 6A). Fluorescence of pHGluA2,

therefore, acts as a readout of receptor distribution and can be used to determine rates and degrees of internalization and recycling. In particular the T1/2 of fluorescence recovery time, derived from a single exponential fit of the recycling phase, provides a quantitative measure of recycling rate. In neurons transfected with GRIP1bwt or GRIP1bC11S, rates of pHGluA2 internalization and recycling were highly similar to neurons transfected with vector alone (Figure 6B). However, pHGluA2 PAK6 recycling was markedly accelerated in neurons transfected with Myr-GRIP1b (Figure 6C). This accelerated recycling was also seen in neurons transfected with

DHHC5, which is predicted to increase palmitoylation of endogenous GRIP1b (Figures 6D and Figures S5A). Both Myr-GRIP1b and DHHC5 caused accelerated recycling of both somatic and dendritic pHGluA2 (Figures 6 and Figures S5B–S5E). The effect of transfected DHHC5 is likely due to direct palmitoylation of GRIP1b, as although GluA2 is a known palmitoylated protein (Hayashi et al., 2005), it is not detectably palmitoylated by DHHC5 (Figure S5B). AMPA receptor recycling is, therefore, significantly accelerated under conditions where GRIP1b membrane attachment is enhanced (Figures 6E and Figures S5E). Myr-GRIP1b, which is targeted to trafficking vesicles, also colocalized extensively with pH-GluA2 in dendritic puncta in fixed neurons (Figure S5C), suggesting that effects on trafficking were likely due to a direct GRIP1b-pHGluA2 interaction. Here, we report that two PATs use a novel PDZ domain recognition mechanism to palmitoylate and control the distribution and trafficking of GRIP1b.

, 1999) or inhibition of binding between PSD-95 and

the NMDA receptor with exogenous peptides (Aarts et al., 2002) reduces NO-mediated excitotoxicity, emphasizing the role of PSD-95 in transducing signals from the NMDA receptor to nNOS. PSD-95 also regulates AMPA receptors through its interaction with stargazin (Chen et al., 2000). This binding is required for recruitment of AMPA receptors to the synapse (Schnell et al., 2002). Consistent with this observation, mice deficient in PSD-95 have decreased AMPA receptor-mediated neurotransmission (Béïque et al., 2006). Furthermore, appropriate interactions between PSD-95 and A kinase-anchoring protein (AKAP) are required for NMDA-mediated AMPA receptor endocytosis (Bhattacharyya et al., 2009). PSD-95 function SKI-606 cell line is regulated by dynamic cycling of palmitoylation and depalmitoylation (El-Husseini et al.,

2002). Glutamate receptor activation enhances depalmitoylation of PSD-95 (El-Husseini et al., 2002), while blockade of synaptic activity enhances PSD-95 palmitoylation through regulated translocation of the dendritic palmitoyl acyltransferase (PAT) DHHC2 (Noritake et al., 2009). Palmitoylation influences synaptic dynamics by augmenting clustering of PSD-95 at dendritic spines (Craven et al., 1999). Palmitoylation of PSD-95 takes place at cysteines 3 and 5 (Topinka and Bredt, 1998). Nitric oxide signals in large part by S-nitrosylating (hereafter referred to as “nitrosylating”) cysteines PD0332991 in vivo in a variety of proteins (Hess et al., 2005). Hess et al. (1993) showed that NO donors can inhibit the palmitoylation of several proteins in dorsal root ganglia neurons and suggested that a NO-mediated posttranslational modification might compete with palmitoylation. Because of its close physical proximity to both the NMDA receptor and nNOS, we wondered whether PSD-95

might be a target for nitrosylation and whether there might be some interaction between putative nitrosylation and palmitoylation of PSD-95. In the present study we show that PSD-95 is physiologically nitrosylated at cysteines 3 and 5 in a reciprocal relationship with palmitoylation. This process impacts the physiologic clustering of PSD-95 at synapses. We examined the possibility that PSD-95 can be nitrosylated by exposing HEK293 cells containing overexpressed PSD-95 to the NO donor cysteine-NO (Cys-NO) (Figure 1A). The NO donor elicits nitrosylation those of PSD-95, monitored by the biotin-switch assay, in a concentration-dependent fashion. To determine whether PSD-95 is physiologically nitrosylated in mammalian brain, we monitored endogenous PSD-95 in mouse brain from wild-type and nNOS-deleted animals (Figure 1B). We observe ascorbate-dependent basal nitrosylation of endogenous PSD-95 that is abolished in nNOS knockout mice. Levels of nitrosylated PSD-95 are comparable to those of the NR2A subunit of the NMDAR (Figure 1C). PSD-95 is palmitoylated at cysteines 3 and 5 (Topinka and Bredt, 1998).

Differences between

axon terminals and cell bodies may also be found in systems that reduce calcium, including mitochondria, plasma membrane calcium pumps, and sodium/calcium exchangers ( Dayanithi et al., 2012). Finally, ATP coreleased from magnocellular neurons exerts different feedback effects on axon terminals and cell bodies, potentially differentially regulating peptide release, in part due to different sets of ATP receptors on axons and cell bodies ( Lemos et al., 2012). Calcium can also be released into the cytoplasm from intracellular stores, particularly the endoplasmic reticulum. Oxytocin, or agents Tofacitinib such as thapsigargin that induce calcium release from intracellular stores into the cytoplasm, can directly evoke dendritic release of oxytocin or vasopressin independent Ruxolitinib of action potentials (Lambert et al., 1994; Ludwig et al., 2002). Release of intracellular calcium can also prime the system for enhanced release upon subsequent increases in electrical activity ( Ludwig et al., 2002; Ludwig and Leng, 2006). Oxytocin receptor activation induces phospholipase C resulting in production of IP3 and subsequent release of calcium from the endoplasmic reticulum. This priming enhances the subsequent release of oxytocin,

potentially related to actin-dependent movement of peptide-containing granules toward the plasma membrane ( Tobin and Ludwig, 2007; Leng et al., 2008). Priming of oxytocin-laden DCVs, in part by movement of the DCVs to a position closer to the plasma membrane, allows a substantial amplification Thymidine kinase of oxytocin release with subsequent electrical activity. Interestingly, priming with thapsigargin can increase the K+-mediated depolarization-induced oxytocin release for an extended period of 90 min ( Ludwig et al., 2002). Priming of DCV release has been studied outside

the brain, particularly in pituitary cells that synthesize luteinizing hormone; axonal release of GnRH from preoptic neurons into the portal blood supply of the median eminence primes the luteinizing hormone cells by multiple mechanisms to show an enhanced release in response to subsequent GnRH stimulation ( Leng et al., 2008; Fink, 1995). Differential expression of proteins involved in exocytosis in dendrites and axon terminals may also account for differences in release. In magnocellular axon terminals in the neurohypophysis, VAMP-2, SNAP-25, and syntaxin-1 are found near oxytocin and vasopressin-containing dense core vesicles; in contrast, the dendrites of the same cell type contain syntaxin-1, but SNAP-25, VAMP-2 and synaptotagmin-1 show no colocalization with oxytocin or vasopressin (Tobin et al., 2012). Synthesis of neuropeptides generally occurs in the cell body, but has also been reported in dendrites.

, 2002), in the skin ( Figure 1E). Interestingly, there was also increased expression of the Wnt-responsive transcription factor Lef1 in a nonautonomous manner in the underlying mesenchymal tissue, including the meninges ( Figure 1F). This made us wonder whether there was Wnt signaling-dependent expression of a Wnt ligand in the skin that was then signaling to the underlying tissues. Wnt6 is normally expressed high laterally and low dorsally in the skin of the head ( Figure 1C) and is absent from the midline. However, in the Msx2-Cre;Ctnnb1lox(ex3) mice, Wnt6 expression covered the entire dorsal surface

of the head. We also found that Wnt6 was elevated about 1.5-fold compared to control by using quantitative UMI-77 concentration real time PCR (qRT-PCR) on mRNA isolated from whole head ( Figure S2C). Expression of Wnt10b, another skin-specific Wnt, was not altered ( Figure 1D), indicating that persistent activation of the Wnt signaling pathway in the skin leads to specific upregulation of Wnt6 expression. Beyond the skin and calvarial defects, we found that in the Msx2-Cre;Ctnnb1lox(ex3) mice, the main cortical commissural pathway, the corpus callosum, failed to form ( Figure 1G). However, in the same mutants, other commissural pathways, including the anterior commissure and hippocampal commissures, were still formed (although the hippocampal commissure was slightly

smaller in size than normal) ( Figure 1G). At E17.5, a day before the mutant embryos die, it was BMS-354825 cost apparent that the callosal axons stopped at the cortical midline, rather than crossing formed Probst bundles (asterisk in Figure 1G), which are aberrant axonal tracts made up of callosal axons that fail to cross the midline ( Paul et al., 2007). These callosal defects were also observed in horizontal sections

of mutant animals ( Figure S1A) and showed full penetrance from 14 mutant embryos that we analyzed. Because the failure of corpus callosum formation is a dramatic midline structural defect, we wondered how excess Wnt signaling in the dorsal skin might cause this phenotype. There have also been numerous studies showing strain differences in the appearance of all corpus callosum defects in mice with some strains (e.g., 129 and Balb/c); however, our colonies of Msx2-Cre and Ctnnb1lox(ex3) mice have been extensively crossed into the CD-1 background, not noted for defects in the corpus callosum. One possible cause of agenesis of the corpus callosum could be defects in the development of the cortical projection neurons. This phenotype has often been observed in mutant animals for the transcription factors governing maturation of the cortical callosal neurons that comprise layer II/III (Alcamo et al., 2008, Armentano et al., 2006, Britanova et al., 2008, Molyneaux et al., 2007, Paul et al., 2007, Piper et al., 2009 and Shu et al., 2003).

The foregoing results are consistent with the idea that future and atemporal imagined events are represented similarly, but other recent data indicate differences between temporal and atemporal imagined scenarios. For example, de Vito et al. (2012b) http://www.selleckchem.com/products/dinaciclib-sch727965.html report that patients with Parkinson’s disease exhibit deficits when asked to imagine future events, but perform normally when asked to imagine atemporal scenarios. Rendell et al. (2012), using a task based on previous work by Hassabis et al. (2007a, 2007b), found that older adults exhibited deficits when imagining future and atemporal scenarios compared with younger adults, but showed a significantly greater impairment for the

future than the atemporal scenarios. Klein et al. (2010) demonstrated that encoding of new information benefits

from creating imagined scenarios that involve planning for the future, but the same encoding benefit is not observed when people encode information by calling up past scenarios or imagining atemporal scenarios. Andrews-Hanna et al. (2010b) reported fMRI evidence that distinct regions within the default network were associated with imagining future scenarios involving oneself versus reflecting about oneself in the present. However, it is not clear that this contrast specifically see more isolated temporal factors, because as noted by the authors, the future and present conditions differed in other ways (e.g., greater use of mental imagery in the future self condition). Another recent fMRI study examined the neural basis of chronesthesia, or the capacity to be aware of subjective time ( Tulving, 2002b; for related ideas, see Dalla Barba and Boissé, 2010; Szpunar, 2011). Chronesthesia is invoked whenever

people remember the past or imagine the future, but isolating the cognitive processes or brain regions associated with chronesthesia requires an experimental design that controls for nontemporal cognitive activities. That is, an appropriate experimental paradigm should contrast tasks that involve chronesthesia (e.g., remembering the past, imagining the future) with a task that is matched to the past and future tasks on nontemporal features, such as imagining oneself interacting with people and locations, without requiring “movement” Bay 11-7085 in subjective time. Nyberg et al. (2010) scanned participants using fMRI during experimental tasks that, they contended, require chronesthesia—remembering a recent short walk along a familiar route or imagining a future short walk along the same route. Brain activity during these tasks was compared with activity during a matched task that, according to the authors, does not require chronesthesia: participants were instructed to take a mental walk through the same route in the present moment, without any thoughts about specific personal past or future happenings.

It is theoretically possible for the phase alone (Figure 2B, right) or amplitude alone (Figure 2B, left) Bortezomib price to carry all the information, or they can both contribute in part. A central goal of our study is to determine the prevalence of these different response types in the medial temporal and frontal areas of the human brain. We also aim to better understand these electrophysiological signals by asking which component carries the most information about behavioral events. We used the LFP measurements, triggered on the first and second card presentations,

to calculate the discriminability index d  ′ between correct and incorrect trials. This was done using the full LFP signal (dLFP’) and using the amplitude (damp’) and phase (dphase’) of the signal at a given frequency after decomposing the LFP using a wavelet transform ( Figure 2A; see Experimental Procedures). There was a clear dependence www.selleckchem.com/products/E7080.html of d  ′ on frequency ( Figure 3A). Discriminability

was low for phase and amplitude after the first click ( Figure 3A, black lines), but it was substantially higher for phase than amplitude after the second click ( Figure 3A, red lines). The differences between damp’ and dphase’ were greatest for frequencies below 4 Hz (Wilcoxon sign-rank test; p = 1 × 10−36 at 2.14 Hz; see also Figure S2A), and the largest average value for dphase’ occurred at 2.14 Hz. Interestingly, in addition to differences between phase and amplitude classifiers, there were differences between brain regions. The values of dphase’ in the temporal lobe (n = 1,008) Mannose-binding protein-associated serine protease were significantly larger than those in the frontal lobe (n = 644) when measured after the second

click (Figure 3B). Again, the largest average dphase’ value occurred at a frequency of 2.14 Hz, where the difference between temporal and frontal values was greatest (two-sample t test; p = 1 × 10−39; see also Figure S2B). Looking specifically at 2.14 Hz, a scatter plot of all d′ values in the temporal lobe confirms that classification using the phase of the LFP is better than classification using the amplitude, and it demonstrates that the d′ values based on phase rival those obtained using the full LFP signal ( Figure 3C, top left). No such relationships were found in the frontal lobe regions, where the d′ values were lower ( Figure 3C, bottom). To assess the significance of individual d′ values, we employed the technique of permutation resampling. For each electrode, all correct and incorrect trials were pooled together. Then, two new groups (of equal size to the original correct and incorrect groups) were chosen randomly without replacement by random assignment of the correct/incorrect labels to each waveform. These two new groups were used to calculate a classifier and an associated d′ value.

, 1995). Crossing the midline may be an easier option than turning back into the ipsilateral optic tract. The tripartite system described here, as with VEGF/neuropilin signaling, could provide a selleck chemicals llc necessary molecular “boost” that augments this inertial midline tendency. All animal procedures followed the regulatory guidelines of the Columbia University Institutional Animal Care and Use Committee. Noon of the day on which a plug was found was considered E0.5. Generation, breeding, and genotyping of Nr-CAM−/−, Plexin-A1−/−, and Sema6D−/−

mutants were described previously by Sakurai et al., 2001 and Takamatsu et al., 2010, and Yoshida et al. (2006). Mice were maintained on a 129SvEvS6 (Nr-CAM−/−) or a C57BL/6 (Plexin-A1−/− and Sema6D−/−) genetic background. Plexin-A1−/−;Nr-CAM−/− double mutants were generated from these mutants

resulting in a 129SvEvS6/C57BL/6 background. These mice are born at roughly Mendelian ratios, are fertile, and survive to adulthood. Whole-anterograde and retrograde labeling was performed on fixed tissue using DiI (Molecular Probes) as described previously by Pak et al. (2004) and Plump et al. (2002). Selleck Screening Library For quantification of the ipsilateral projection in mutants anterogradely labeled with DiI, pixel intensity of DiI+ ipsilateral and contralateral optic tracts adjacent to chiasm midline in a 500 × 500 μm area Thiamine-diphosphate kinase was measured with MetaMorph image analysis software. The ipsilateral index was obtained by dividing the intensity of the ipsilateral projection as seen in whole mounts by the sum of the contralateral and ipsilateral pixel intensities. Each of the ipsilateral indexes in mutants was normalized to the WT ipsilateral index. Details are shown in Figure 7B. Retinal explants were dissected from E14.5 WT C57BL/6 or mutant embryos as described previously by Wang et al. (1996). To harvest chiasm cells, a 400 × 400 μm area of the ventral diencephalon that included the chiasm midline was dissected, dissociated, and plated at a density

of 140,000 cells/dish shortly after retinal explants were plated, in DMEM/F12 serum-free medium containing 0.4% methylcellulose with 80 μg/ml αSema6D or preimmune serum added. Cultures were grown for 18 hr and then fixed for 30 min with 4% PFA. Neurites were visualized with a monoclonal neurofilament antibody (2H3). The total area covered by neurites of individual explants was quantified by measuring pixel intensity with OpenLab image analysis software. The amount of axon growth was normalized with respect to the outgrowth of DT or VT explants under control conditions, and indicated in the leftmost bar in graphs in Figures 2, 3, and 5. Each experiment was carried out at least three times, and in each experiment at least four explants were treated in each experimental group.