6, p < 0.001)—i.e., with temporal proximity from the motor response. This is to be expected from a response preparation signal driven by large temporal fluctuations in sensory

input ( Yang and Shadlen, 2007). We carried out additional analyses locked to the onset of the response period, which all confirmed that motor beta-band activity behaved as a response preparation signal (Figure S7): (1) the neural encoding of the sum of response updates distinguished correct choices from errors from more than 500 ms before the onset of the response period selleck (paired t test, t14 = 4.8, p < 0.001); (2) the neural decoding of choice (i.e., left- versus right-handed response) showed similar predictive profiles preceding correct choices and errors (see Supplemental Information); and (3) the between-element variability in neural encoding of response updates

correlated positively with NSC 683864 chemical structure the between-element weighting profile estimated behaviorally (r = +0.44 ± 0.10, t test against zero, t14 = 4.4, p < 0.001). Finally, we assessed whether the neural encoding of DUk in motor beta-band activity also fluctuated rhythmically according to the phase of parietal delta oscillations (Figure 7C), and found that it followed the same phase relationship as its earlier encoding in broadband parietal signals (Rayleigh test, r14 = 0.50, p < 0.01). This phase dependency suggests that motor beta-band activity reflects a computation that occurs downstream from the weighting of momentary evidence according

to the phase of parietal delta oscillations. Together, these findings chart the electrophysiological substrates of the sensorimotor cascade whereby successive samples FGD2 of sensory evidence are processed from lower to higher levels, integrated, and converted into an appropriate response. By linking trial-to-trial fluctuations in neural signals to variability in choice, these findings draw a clear distinction between the computations performed by two neural mechanisms during categorical decision making. First, momentary evidence undergoes a multiplicative weighting according to the phase of ongoing delta oscillations (1–3 Hz) overlying human parietal cortex. Subsequently, lateralized beta-band activity (10–30 Hz) over the motor cortex integrates the weighted evidence in an additive fashion, consistent with the formation of a decision variable. Categorical choices are thus preceded by discrete central and motor stages, both of which follow an early perceptual stage confined to early visual cortex. These findings thus call into question the widely held view that evidence accumulation is indistinguishable from the gradual engagement of a response effector—in other words, that the neural encoding of decision-relevant evidence reduces to a preparatory signal that precedes motor output ( Shadlen and Newsome, 2001; Roitman and Shadlen, 2002; Gold and Shadlen, 2003, 2007).

The process by which the RPE cells acquire a retinal progenitor phenotype appears to be a critical one, since the process after this point resembles that of normal development. This phenomenon has been variously termed metaplasia, transdifferentiation (Okada, 1980), or dedifferentiation (since the RPE cells are reverting to a developmentally earlier state). This phenomenon involves shifting the pattern of gene expression in a highly regulated way and might be called “regulated reprogramming” to distinguish it from the KRX-0401 molecular weight “direct

transdifferentiation” process that occurs in support cells in mechanosensory receptor regeneration in the inner ear or lateral line. Pigmented epithelial cells reprogram to a progenitor state in birds as well; however, this phenomenon is restricted to the earliest stages of

eye development, a few days after the lineages of the retinal progenitors and the RPE progenitors have diverged (Coulombre and Coulombre, 1970). Fish also have considerable ability to regenerate sensory receptor EGFR tumor cells and other retinal neurons from sources within the retina. When the fish retina is damaged (via surgical, neurotoxic, or genetic lesions or excessive light), there is a burst of proliferation. As noted above, the fish retina contains a precursor cell that continues to generate new rod photoreceptors throughout the lifetime of the animal. For many years it was believed that

the primary source of new retinal neurons was the rod precursor (Raymond et al., 1988). More recently, it became clear that the Müller glia were another source, if not the major source Enzalutamide of proliferating cells after retinal damage in the fish (Fausett and Goldman, 2006 and Wu et al., 2001), although rod precursors contribute as well, particularly when only rods are damaged. After retinal damage, the Müller glia in the fish retina undergo a dedifferentiation process (Bernardos et al., 2007, Fausett and Goldman, 2006, Qin et al., 2009, Ramachandran et al., 2010, Raymond et al., 2006 and Thummel et al., 2008), somewhat like that described for the RPE in the amphibian; they re-express many, if not all, of the genes normally expressed in retinal progenitors. Thus, in both fish and amphibians, damage in the central retina causes nonneuronal cells to change their phenotype into retinal progenitors: in amphibians, the progenitors are derived from the RPE cells, while in fish these progenitors are derived from Müller glia. However, damage to cells in the peripheral retina can be repaired by a very different mechanism in these species. In both fish and amphibians, the retina contains a specialized zone of progenitor cells at the periphery, called the ciliary marginal zone (or CMZ), which adds new neurons of all types throughout the lifetime of the animal (for review, see Lamba et al., 2008).

Sensory-evoked responses in visual cortex vary with spontaneous variations in the levels of network activity, with responses enhanced during the cortical up state of slow oscillations (Haider et al., 2007). This “gain modulation” may be related to the activation of LC neurons just before the fully depolarized cortical state, described above. Released in time with the maximum firing of the cortical neurons, NA would modulate, gate, and tune sensory responses (Berridge and Waterhouse, 2003; Sara, 2009). Active reconfiguration

U0126 of the functional state of networks may underlie attention, sensory-motor coupling, and other cognitive processes. This is in line with data suggesting that LC firing during the transition from down to up states facilitates the achievement of the maximum depolarized state in the cortex (Eschenko et al., 2012). This mechanism of facilitation of transition to the maximum depolarized state by LC may not be limited PF-2341066 to spontaneous oscillations. It may occur each time LC phasic activity is elicited as part of the orienting response or as part of a CR to behaviorally significant stimuli, the equivalent of the cortical TCR. In the following sections, we will see the extent to which this relation between LC activation, cortical arousal, and the conditioned

orienting response to simple environmental challenges extends to cognitive flexibility. There is some evidence that pupil dilation varies with spontaneous activity in

LC neurons (Aston-Jones and Cohen, 2005), in line with several reports relating the firing of LC neurons with autonomic arousal (Jacobs, 1986; Abercrombie and Jacobs, 1987). Several recent studies have used this noninvasive MRIP technique of measuring changes in pupil size in human subjects in an attempt to investigate the role of the LC in cognitive flexibility. A recent example is an experiment aimed at understanding the intrinsic brain mechanisms of bistable perception, a phenomenon in which perception fluctuates between two distinct states when the subject fixates on an ambiguous figure. A typical example is the Necker cube. The state transitions are abrupt and occur spontaneously. The experimental protocol required the subjects to report a state change by pressing a lever. Results showed that pupil dilation occurred just before the change and the amount of dilation predicted the duration of the subsequent perceptual stability (Einhäuser et al., 2008). This experiment does not tell us that LC activation actually caused the abrupt switch in perception, but the loose correlation of the size of the dilation with the duration of the subsequent state suggests a role in maintaining perceptual stability.

The statistical analyses were carried out using the database and the program Statistica 6.0, to obtain the mean and standard deviation of the quantitative variables and the relative and absolute frequencies of the qualitative variables. The positivity for the different variables was analyzed

using Fischer’s exact test, at the 5% significance level. Of the total of children, 51/90 (56.7%) frequented the public squares on one to three days per week, 23/90 (25.5%) on four to five days, and 16/90 (17%) on six to seven days. The seroprevalence rate was substantially higher among children who frequented the squares on six to seven days per week (p < 0.01) ( Table 1). Of 90 children, 16 (17.8%) were seropositive for IgG anti-Toxocara spp. Selleck Nutlin 3 antibodies. Notably, each of them resided in different domiciles, and the

majority (12/16) frequented the squares located on the city outskirts. Most (15/16) of the seropositive children had the habit of geophagy, and half of them (8/16) were between 1 and 4 years of age ( Table 1). Respiratory problems such as asthma and bronchitis were reported by 13/16 (81.2%) (p = 0.02), and problems with skin allergies by 3/16 (18.7%) (p = 0.40). Eosinophilia was observed in all the seropositive children (p < 0.01) ( Table 1); 6/16 children showed Grade I eosinophilia, 8/16 showed Grade II, and 2/16 showed Grade III. The parasitological analysis of the public squares, including both sand and grass turfs, revealed 100% positivity for eggs of Toxocara spp. ( Table 2). Of the 15 public squares examined, 13 (86.7%) consisted of sand and two (13.3%) were composed of grass turfs. Seven (46.7%) of the squares were Selleck Ion Channel Ligand Library fenced, although at all of them the gates were open on the days when visits were made during the study. In 11/15 (73.3%), the presence of wandering dogs was observed at the time of

the collections, but no viable fecal material of these animals could be located for examination. No cats were present in the public squares at the time of the collections. The squares that did not contain dogs were positively related to the seronegativity of the children (p < 0.05). Similarly, public squares where these animals were present, contributed significantly substrate level phosphorylation to the seropositivity (p < 0.05). All of the 16 seropositive children frequently played in those public squares where the parasite load was above 1.1 eggs of Toxocara spp./g of sand (p < 0.01) ( Table 1). Of the 90 peridomiciles investigated, 38/90 (42.2%) consisted only of sand, 11/90 (12.2%) of grass turf, 17/90 (18.9%) of sand and pavement, 23/90 (25.6%) of grass and pavement, and 1/90 (1.1%) of pavement alone. The parasitological analysis revealed 17/90 (18.9%) peridomiciles with eggs of Toxocara spp., including 12/17 (70.5%) consisting of sand, and 5/17 (29.5%) of grass turf ( Table 2). Seropositivity was positively associated with contamination in the peridomicile ( Table 1).

04–0.6 cycles/degree) to 0 mV (Porciatti et al., 1999). In these experiments, we used VEPs recorded from the surface of the binocular visual cortex, to focus

on synaptic potentials generated in superficial laminae (Katzner et al., 2009). We found that juvenile (P30) NARP−/− mice had an estimated spatial acuity of 0.48 ± 0.04 cycles/degree (average ± SEM, n = 5), which was indistinguishable from age-matched wild-type controls (0.49 ± 0.02 cycles/degree, n = 5; p = 0.86, t test; Figure 5A). Manipulation of the visual stimulus from 20% to 100% contrast revealed similar contrast sensitivity in NARP−/− and wild-type vision (two-way repeated-measures ANOVA, F1,6 = 0.003, p = 0.955; Figure 5B). To ask if the absence of NARP disrupts the organization of the Z-VAD-FMK visual cortex, we quantified ocular preference and retinotopy over the mediolateral extension of V1. To

examine ocular preference, we calculated the ratio of VEP amplitudes in response to separate stimulation of the contralateral and ipsilateral eye (Figure S3). In both wild-type and NARP−/− mice, recordings medial to the binocular region of the primary visual cortex revealed responses to contralateral eye stimulation only, as expected of monocular visual cortex. Recordings from a narrow area, ranging from ∼3.0–3.5 mm lateral to the intersection of lambda and bregma, revealed responses to visual stimulation of both eyes, as expected of binocular visual cortex. Recordings lateral to the binocular region of the primary visual cortex revealed a loss of contralateral preference, as expected for the lateral medial Pembrolizumab in vivo region of secondary visual cortex (Rossi et al., 2001). Retinotopy was also similar in wild-type and NARP−/− mice. The area of visual space resulting in the largest VEP amplitude moved along the visual field azimuth, from contralateral visual

field to the meridian as the recording site was moved laterally across the binocular region of the primary visual cortex and reversed back toward the contralateral Domperidone visual field as the recording site moved laterally from the binocular region of the primary visual cortex into lateral medial (LM) (Figure S3D). The orientation selectivity and orientation tuning of NARP−/− mice was also similar to wild-types (Figure S4). Thus many aspects of visual system organization and function are normal in NARP−/− mice. The binocular primary visual cortex of rodent has a contralateral bias that depends on early binocular visual experience (McCurry et al., 2010). To ask if NARP−/− mice retained normal experience-dependent regulation of VEP contralateral bias, we examined VEP contralateral bias at the site in binocular visual cortex that yielded the largest ipsilateral eye VEP (typically 3.3 mm lateral to the intersection of lambda and bregma). Dark-rearing from birth to postnatal day 30 (P30) prevented the expression of the VEP contralateral bias in both genotypes.

The ineffectiveness of BAPTA in inhibiting mf-LTP in WT slices in ACSF supports the conclusion that events underlying induction reside presynaptically within mf terminals. Whether the locus of induction of mf-LTP is pre- or postsynaptic has been controversial (reviewed by Henze et al., 2000 and Nicoll and Schmitz, 2005), but our conclusion is consistent with evidence implicating a presynaptic locus ( Tong et al., 1996). Our interpretation

that vesicular zinc acts presynaptically raises the question as to what molecular consequences are triggered by the ion that culminate in the increased glutamate Pr that underlies Everolimus price mf-LTP in WT animals. We propose that vesicular zinc, released by HFS of the mf, reenters the mf terminals where it triggers a chain of molecular events. One possibility is that increased concentrations of zinc in the cytosol of the presynaptic terminal transactivate the receptor tyrosine kinase, TrkB (Huang et al.,

2008; Figure 8). This model is consistent with evidence that TrkB activation can promote transmitter release from presynaptic terminals (Jovanovic et al., 2000, Tyler et al., 2002 and Lohof et al., 1993), that TrkB kinase activity is required for mf-LTP (Huang et al., 2008), and that zinc can transactivate TrkB (Huang et al., 2008). Rapid chelation of synaptically released zinc by ZX1 would inhibit such a process. Our findings establish two important functions for zinc that is localized to synaptic vesicles of the hippocampal mfs: zinc promotes the increased Pr that underlies presynaptic mf-LTP and it also masks induction of postsynaptic mf-LTP. selleck Context-dependent fear conditioning is one behavior potentially related to presynaptic mf-LTP in particular because defects in this behavior have been identified in young adult ZnT3 null mutant mice and following injection of a zinc chelator locally in CA3 of WT mice ( Sindreu et al., 2011). Emergence of a postsynaptic

mf-LTP may help explain the absence of detectable deficits in multiple behaviors examined in young adult stiripentol ZnT3 null mutant mice ( Cole et al., 2001 and Adlard et al., 2010). It seems plausible that dual control of the mf-CA3 synapse by vesicular zinc supports the physiological functions subserved by this synapse while limiting pathologic hyperexcitability mediated by excessive activation of CA3 pyramids. Future investigations will seek to determine the molecular mechanisms underlying these dual functions and whether vesicular zinc exerts similar actions in diverse association cortical circuits in addition to the mf-CA3 synapse. Full details of the preparation, characterization, and physical properties of the new chelator are provided in Supplemental Information. The compound can be obtained from Strem Chemical Co. Potentiometric titrations were performed on a Mettler-Toledo T70 autotitrator, operated by the LabX-light software.

, 1999; Di Stefano et al., 2003; Lee et al., 2000; Polager and Ginsberg, 2008; Goto et al., 2006; Malumbres and Barbacid, 2005). We found evidence that this pathway is directly regulated by Pax6. At the onset of corticogenesis, around embryonic day 12.5 (E12.5), Pax6 is expressed in a gradient across the embryonic cortex with high levels rostrolaterally and low levels caudomedially (Figures 1A–1C;

Bishop et al., 2000; Manuel et al., 2007). We used iododeoxyuridine (IdU) and bromodeoxyuridine (BrdU) double labeling as summarized in Figure 1D to calculate the cell-cycle and S phase times (Tc and Ts, respectively; Martynoga et al., 2005; Figures PLX3397 datasheet 1E and 1F) in regions of E12.5 Pax6+/+ and Pax6−/− cortex expressing high, medium, or low levels of Pax6 ( Figures 1C and 1G–1I). In Pax6+/+ embryos, the mean Tc was longest in areas expressing high or medium levels of Pax6 ( Figures 1G–1I). In Pax6−/− embryos, the mean Tc was significantly shorter by ∼25% in these two areas. Neither the mean Tc in the area of lowest Pax6 expression nor the mean Ts in any area was affected in

Pax6−/− embryos. We also tested the effect of an acute (conditional) loss of Pax6, since the rapid onset of a defect would strengthen the possibility that Pax6 influences the cell cycle directly. We analyzed mice carrying a tamoxifen-induced, cortex-specific deletion of Pax6. Their find more genotypes were as follows: (1) Pax6loxP/loxP; Emx1-CreERT2; R26R-YFP (inducible knockout [iKO] embryos); and (2) Pax6loxP/+; Emx1-CreERT2; R26R-YFP embryos (controls; deletion of only one copy of Pax6 has no detectable effect on cortical progenitor proliferation; Figure S1 available online). Tamoxifen administered at E9.5 resulted in loss of Pax6 from the cortex (sparing the ventral pallium, which does not express Emx1) by E12.5 ( Figures 1J and 1K). The results obtained from iKOs were similar to those from

Pax6−/− embryos, with significant effects of both genotype and cortical region on mean Tc (two-way ANOVA). In rostral and central-lateral areas (i.e., [Pax6]high in controls), the mean Tc was longer than in central-medial and caudal areas in controls (p < 0.0001, Sidak’s multiple-comparisons SDHB test) and was significantly reduced in iKOs ( Figures 1L–1O). These results show an association between the spatial distribution of Pax6 across the cortex and both progenitor Tc in wild-types (WTs) and the effect of Pax6 absence on Tc in mutants at E12.5. The expression of Pax6 across the cortex becomes increasingly uniform with embryonic age, with similar levels being attained in all areas by E15.5 (Figures 2A–2C), suggesting that the regional effects of Pax6 loss on progenitor proliferation might be different at these ages. In a first set of experiments, iKOs were generated by tamoxifen administration at either E10.5 or E13.5 (Figures 2D–2Q). Activation of YFP from the R26R-YFP allele occurred within 48 hr ( Figure S2A).

Frequency and duration of participation ranged from 2 to 5 days per week and 50–60 min per session. To prevent any potential diurnal variations in performance measures participants were asked to

report to the laboratory at approximately the same time for every session (∼1300–1500 h). Participants were verbally informed of the protocol and then read and signed the informed consent form. This investigation and all procedures utilized was approved by Ohio University’s Institutional Review Board. This investigation used a randomized within subject design to evaluate the effectiveness of a traditional bout of SS, a DS routine (as prescribed by the coaches) and a control (no stretching) session of equal duration on kinetic variables describing the shape of the GRF-time curve during countermovement vertical Galunisertib clinical trial jumping (CMJ) on a force plate. Kinetic parameters that were assessed from the raw vertical GRF trace (Fz) of the force platform were TTT, peak force find more (Fpk), and RFDavg. Because some athletes do not begin competing immediately after their warm-up with stretching routine, we examined the effects of DS and SS post-stretch timeline testing beginning

at 1 min and ending at 15 min ( Fig. 1). Each participant volunteered to participate in four sessions which consisted of one familiarization session and three randomized experimental testing days (Fig. 1). In the first session participants became familiarized to the procedures of each experimental session. This included correct CMJ technique as well as familiarization to the SS procedures. It was assumed that all participants understood the DS procedures, as this was their typical pre-match warm-up routine that was extrapolated from the coaching staff. After the familiarization session the following three randomized experimental testing sessions were conducted: 1) an SS session followed

by three CMJs each at 1 and 15 min after SS, 2) a control session using only a general aerobic warm-up followed by three CMJs each at 1 and 15 min after warm-up, and SDHB 3) a DS session followed by three CMJs each at 1 and 15 min after DS. Prior to each stretching session a brief aerobic warm-up was conducted on a cycle ergometer (Monark, Ergomedic 874E, Vansbro, Sweden) using 1 kg of resistance and cycling at a cadence of 60 RPMs for 5 min. Participants then performed one of three randomly assigned experimental stretching protocols, which lasted for a total duration of 7 min. After stretching, a stop-watch was started in order to monitor testing at 1 and 15 min after the stretch intervention. At each specific timing interval, the participant would position herself on the force platform and begin performing a sequence of three CMJs interspersed with a 1 min standing rest.

Maximum

projections of individual stacks were analyzed using Metamorph software (Universal Imaging Corporation). Results shown correspond to the average of all neurons per condition. Statistical significance was determined by unpaired t test, and error bars represent standard error of the means. FM4-64 (10 μM) uptake was performed using 90 mM KCl loading media containing (in mM) 10 HEPES, 33 NaCl, 90 KCl, 10 D-glucose, 2 CaCl2, 2 MgCl2, 20 μM NBQX, and 50 μM APV for 1 min. Cells were washed three times in washing buffer (WB: E4 with 0.5 mM CaCl2 and 10 mM MgCl2) for 30 s, two times for 15 s in WB containing 1 mM ADVASEP-7 (Sigma), and three times in WB. FM4-64 fluorescence was acquired with 567 nm excitation

and a 647 nm emission filter. GFP imaging was done at the end of each time lapse to minimize photobleaching. www.selleckchem.com/products/z-vad-fmk.html Unloading solution containing (in mM) 10 HEPES, 63 NaCl, 60 KCl, 10 D-glucose, 2 CaCl2, 2 MgCl2, 20 μM NBQX, and 50 μM APV was added with a precision of <2 s. Time constants (τ) of FM4-64 fluorescence decay at boutons corresponding to each condition were pooled and analyzed by a Kolmogorov-Smirnov test. Statistical significance was determined using unpaired t tests. For more details, see Supplemental Experimental Procedures. EPSC internal solution contained (in mM) 30 CsSO4, 70 K2SO4, 25 HEPES, 25 N-methyl-D-glucamine, 0.1 CaCl2, 1 EGTA, 2 (Na)ATP, and 0.1 leupeptin BKM120 nmr (pH 7.2), ∼300 mOsm, and EPSC external solution contained (in mM) 150 NaCl, 5 KCl, 10 HEPES, 1 MgCl2, 30 D-glucose, 2 CaCl2, and 30 μM bicuculline. Only cells with series resistance <30 MΩ and <20% change in input resistance, series resistance, and holding current were analyzed. EPSCs were evoked with a concentric bipolar stimulating electrode (200 μs pulse duration). Paired-pulse responses (100 ms interval) were evoked every 30 s by stimulation of nearby cells. For mEPSC recordings,

external solution contained additional 1 μM TTX. mIPSC internal solution contained (in mM) 150 KCl, 3 MgCl2, 15 HEPES, 0.1 CaCl2, 1 EGTA, 2 Na2ATP, and 0.1 leupeptin [pH 7.2], ∼300 mOsm. mIPSC external solution contained 1 μM TTX, 50 μM D-APV, and 10 μM Camptothecin CNQX instead of bicuculline. All currents were recorded at −70 mV voltage clamp. P60 female FVB or FVB.Cg-Mmp9tm1Tvu/J mice (∼18–25 g) were injected intraperitoneally (i.p.) with 1 mg/kg scopolamine methyl nitrate (Sigma) 30 min prior to the experiment. Status epilepticus was induced by i.p. injection of 315 mg/kg pilocarpine hydrochloride (Sigma), while controls were injected with vehicle alone (sterile 0.9% NaCl). The experiment was terminated 2 hr later, and hippocampal tissue was collected and flash frozen. Only animals with class IV or higher seizures were used for analysis. Male and female FVB or FVB.

, 1995, Lundblad et al., 1995 and Bannister and Kouzarides, 1995) had no effect on cFos regulation or VEGFD transcription ( Figure 2G). We also used RNA interference (RNAi) to specifically decrease CBP mRNA levels in hippocampal neurons ( Figure 2H). This caused a significant reduction of VEGFD mRNA levels ( Figure 2H) confirming the role of CBP in modulating VEGFD transcription. A morphometric analysis revealed that hippocampal neurons expressing E1A have shorter and simplified

dendritic trees compared to neurons expressing E1AΔCR1 ( Figures 2I and 2J). These results indicate that CBP acts downstream of nuclear calcium-CaMKIV signaling to regulate VEGFD expression in hippocampal neurons. selleck chemicals llc To investigate whether VEGFD is involved in mediating the effects of nuclear calcium-CaMKIV signaling on neuronal structure, we either transfected or infected hippocampal neurons, respectively, with an rAAV plasmid (pAAV-VEGFD) or an rAAV (rAAV-VEGFD) containing an expression cassette for HA-tagged VEGFD or we treated the neurons with recombinant VEGFD (rVEGFD). Expression of HA-tagged VEGFD was detected immunocytochemically and by

immunoblotting in rAAV-VEGFD-infected hippocampal neurons and in the culture media ( Figures S1G and S1H). Although VEGFD-HA expression or exogenously applied rVEGFD had no detectable effect on neuronal morphology, both treatments rescued the reduction in dendrite length and complexity caused by expression of CaMBP4 or CaMKIVK75E ( Figures 3A–3C, 3E, and 3F). In contrast, VEGFD-HA and rVEGFD check details failed to restore normal spine density in CaMBP4 or CaMKIVK75E-expressing neurons ( Figures 3D and 3G), indicating that the mechanisms through which nuclear calcium-CaMKIV signaling regulate dendrite geometry

and spine density are distinct. Because ALOX15 VEGFD belongs to a family of closely related factors that in part share the receptors ( Achen and Stacker, 2008), we tested whether VEGF or VEGFC also affect dendrite arborization. However, neither recombinant VEGF (rVEGF) nor recombinant VEGFC (rVEGFC) was able to rescue the reduction in dendrite length and complexity caused by CaMBP4 or CaMKIVK75E expression ( Figure S2), indicating a specific role for VEGFD in the control of dendrite arborization by nuclear calcium-CaMKIV signaling. To determine whether the observed reduction in VEGFD expression that followed blockade of nuclear calcium-CaMKIV signaling is sufficient to alter dendritic architecture, we used RNAi to lower VEGFD expression in hippocampal neurons. DNA sequences encoding short hairpin RNAs (shRNAs) designed to target the mouse VEGFD mRNA were inserted downstream of the U6 promoter of an rAAV vector. The resulting rAAV, rAAV-shVEGFD, also harbors a calcium/calmodulin-dependent protein kinase II (CaMKII) promoter-containing expression cassette for the red fluorescent protein, mCherry ( Figure 4A).