, 1996), and subsequent studies support the hypothesis that opioid ligand effects are not adequately described by a single “dimension” of agonist activity (Whistler et al., 1999; Borgland et al., 2003; Pradhan et al., 2010; Arttamangkul et al., 2006). This concept remains controversial, however, particularly with regard to understanding the effects of morphine (McPherson et al., 2010; Molinari et al., 2010). Nevertheless, the general idea that some drugs promote regulated endocytosis of opioid receptors out of proportion

to conventional estimates of relative agonist activity is increasingly recognized (Rivero et al., 2012). Recent mechanistic data provide independent support for this concept because opioid receptor engagement with arrestins and subsequent clustering in CCPs, key initiating events affecting the rate of agonist-induced endocytosis, require multisite phosphorylation of the receptor’s cytoplasmic tail. Detailed analysis of discrete selleck products phosphorylated receptor forms generated in intact cells, by quantitative mass spectrometry

applied to isotope-labeled cells, indicates that this multisite requirement renders endocytosis inherently nonlinear with respect to receptor activation (Lau et al., 2011). This principle for generating nonlinearity Galunisertib ic50 by multiphosphorylation is reminiscent of how multiphosphorylation can produce “ultrasensitive” responses in other biological contexts (Nash et al., Carnitine dehydrogenase 2001; Ferrell, 1996) and is a particularly attractive strategy for integral membrane proteins such as 7TMRs because significant nonlinearity can occur even in the presence of an excess local concentration of kinase (Dushek et al., 2011). Accordingly, nonlinear control by multisite phosphorylation may underlie how apparently complex differences in the regulatory effects of drugs—variously described in terms of “functional selectivity,” “multidimensional” efficacy, or “agonist bias”—are manifest at the cellular level. One function of 7TMR endocytosis is to initiate a multistep trafficking pathway mediating receptor delivery to lysosomes, a proteolytic organelle in which many

7TMRs are destroyed (Figure 1A). When a sufficient fraction of the overall cellular receptor pool is depleted through this pathway, as can occur under conditions of prolonged or repeated ligand-induced activation, cellular signaling responsiveness to neuromodulator is attenuated or “downregulated” (Tsao et al., 2001). Endocytic downregulation of delta opioid neuropeptide receptors by delivery to lysosomes, first recognized in cultured neuroblastoma cells (Law et al., 1984), has been directly shown in vivo and correlated with development of physiological tolerance to opioid drugs (Pradhan et al., 2009; Scherrer et al., 2006). Individual 7TMRs differ greatly in the efficiency with which they traffic to lysosomes after endocytosis, and this contributes to receptor-specific differences in endocytic regulation (Tsao and von Zastrow, 2000).

I first met George at Atlanta in 1984 while, together with Richard Mahoney, on an extensive study tour of rabies research centers in the US, Europe and Asia with a grant from US-AID and the PATH Foundation of Seattle. We were then interested in replacing the neural tissue buy Rapamycin derived rabies vaccines, used for the public sector in Thailand and neighboring countries, with an affordable tissue culture product. George, together with his friends at the Wistar Institute (Hilary Koprowsky, Charles Rupprecht,

Daniel Fischbein, Jean Smith, Hildegund Ertl and Bernard Dietzschold) put us on the right track by introducing us to Olaf Treanhart of Essen, Piere Sureau at the Institute Pasteur, David and Mary Warrell at Oxford University. Their support led to the introduction of the reduced cost, safe and effective intradermal post-exposure rabies vaccination methods and the introduction of Praphan Phanuphak’s economical Thai Red Cross post-exposure regimen and its 1992 approval by WHO. Nerve tissue derived Semple-type and Suckling Mouse Brain vaccines were soon banished from Thailand. Moreover, Bear and other

colleagues from France, Switzerland, Wistar, WHO-Geneva and the US-CDC formed a close working relationship with the growing Thai rabies research community that led to the appointment of two WHO collaborating centers at Bangkok. I was a house guest at the Atlanta Baer residence, lastly some time in the late 1980s, and can vividly remember the KRX-0401 order visit with great pleasure. George was much more than just an outstanding scientist. He spoke fluent French, German and Spanish and often acted as chairman, translator and interpreter at international conferences; always with tact and humor. He also had a profound knowledge of art, literature, international politics and even music. His family dinner table resounded with discussions of all

kinds of topics that often changed from English to German and Spanish in which his family was equally fluent and which they used casually and alternatingly at home. George truly was one of the “Greats” of rabies and a good friend to many colleagues. Adenylyl cyclase They and his many students from around the world will miss him greatly. “
“Flaviviruses comprise more than 70 different viruses, many of which are arthropod-borne and transmitted by either mosquitoes or ticks [1]. Taxonomically, they form a genus in the family Flaviviridae which in addition includes the genera hepacivirus and pestivirus [2]. With respect to disease impact, the most important human pathogenic flaviviruses are yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV) and tick-borne encephalitis virus (TBEV). Several others can also cause severe and even lethal disease in humans but potential exposure to these viruses is apparently limited and the reported case numbers are relatively small. Examples are St.

This method proved itself in confirming the central postulate of direction selectivity, where special attention was paid to a particular set of amacrine-to-ganglion cell synapses. But it can also be used in less focused ways. For example, a patch of mouse retina 200 μm2, which is well within the capability of reconstruction technology, contains ∼1,500 bipolar cells (Jeon et al., 1998). On average, this would amount to 125 bipolar cells of each of 12 types, more than enough for an independent verification of the types defined using light microscopy and

an analysis of their synaptic connectivity. The same could be done for narrow find more field amacrine and ganglion cells. I thank Dr. Steven

DeVries for the electrophysiological traces shown in Figure 3 and for reading the section on bipolar cells. Members of the Jakobs/Masland lab made helpful comments. The figures were made by Michael Becker. Susan Cardoza copyedited and helped with the references. The author is supported by NIH grant www.selleckchem.com/products/bmn-673.html EY13399 and the Harvard Neurodiscovery Center. “
“Long ago defined by William James as “the focusing of the mind,” selective attention is simultaneously one of our most pervasive and most baffling cognitive functions. On one hand attention is recruited for nearly every behavior and has been investigated in humans, monkeys, mice, and rats. On the other hand despite this wealth of research, significant questions remain about the nature of attention, its purpose and neural mechanisms. In humans and nonhuman primates, much of our knowledge of the mechanisms of attention comes from the system of vision and eye movement control. Intensive research into this system has shown that attention affects sensory representations

at all levels of the visual hierarchy, starting from low-level areas such as the lateral geniculate nucleus, through high-level cortical areas in the inferior temporal lobe (Reynolds Thymidine kinase and Heeger, 2009; Saalmann and Kastner, 2011). These studies also suggest that the source of attentional modulations lies, at least in part, in sensorimotor areas associated with rapid eye movements (saccades). Two areas that have been particularly well investigated are the lateral intraparietal area and the frontal eye field (shown in Figure 1A for the macaque monkey brain). Neurons in these areas have spatial receptive fields and saccade-related responses and respond selectively to stimuli that are likely to attract attention in a variety of tasks. Not specifically sensory or motor, these cells seem to encode the specific act of target selection, and can provide feedback regarding this selection both to earlier visual areas and to downstream movement structures that generate shifts of gaze.

, 2002, Maffei et al., 2006 and Marik et al., 2010) of GABAergic FS output synapses. These findings add to increasing Dasatinib research buy evidence that FS cells are a site of robust experience-dependent development and

plasticity in vivo (Chittajallu and Isaac, 2010, Jiao et al., 2006, Maffei et al., 2004, Maffei et al., 2006 and Yazaki-Sugiyama et al., 2009). Prior work showed that D-row deprivation reduces feedforward and recurrent excitation into L2/3 of deprived columns (Allen et al., 2003, Bender et al., 2006, Cheetham et al., 2007 and Shepherd et al., 2003), but whether plasticity was coordinated between excitatory and inhibitory circuits was unknown. Because sensory responses in cortical neurons depend strongly on the balance and timing of convergent excitation and inhibition (Pouille et al., 2009, Wehr and Zador, 2003 and Wilent and Contreras, 2005), we simultaneously measured L4-evoked feedforward inhibition and excitation onto single L2/3 pyramidal cells and found that 6–12 days of D-row deprivation caused a coreduction in excitation and inhibition in which the ratio of excitation to inhibition

in single cells was preserved, on average, in the population, relative to spared columns (Figure 8). Deprivation delayed both excitation and inhibition by ∼1 ms but did not alter their relative timing. Thus, Hebbian weakening of deprived inputs in S1 is associated with a coordinated www.selleckchem.com/products/Bafilomycin-A1.html decrease and delay in feedforward excitation and inhibition. Most neurons in L2/3 of S1 respond to whisker deflection with subthreshold depolarization, reflecting sparse spike coding in this region (Crochet et al., 2011). To understand how coreduction of excitation and inhibition affects L4-evoked subthreshold responses, we used a single-compartment parallel Histone demethylase conductance

model (Wehr and Zador, 2003) to predict the PSP produced by the measured L4-evoked Ge and Gi waveforms measured in each pyramidal cell. This model showed that the measured coreduction in feedforward excitation and inhibition will produce a net decrease in L4-evoked PSP amplitude (Figure S5). Thus, this effect is appropriate to explain the Hebbian weakening of L2/3 responses to deprived whiskers. Additional factors mediating reduced L2/3 spiking probability in vivo may include nonlinear amplification of PSP weakening by the spike threshold (Foeller et al., 2005 and Priebe and Ferster, 2008), reduced L2/3 recurrent excitation (Cheetham et al., 2007), or potential changes in feedback inhibition. Whereas the reduction in feedforward excitation is predicted to decrease PSP amplitude, the reduction in feedforward inhibition is expected to increase PSP amplitude and therefore represents a partial, covert compensatory mechanism. This compensation is termed “covert” because it does not result in increased whisker-evoked or spontaneous spikes in vivo (Drew and Feldman, 2009). How coordinated weakening of inhibition and excitation is achieved is an important topic for future work.

Because lentiviral shRNA-HCN1-infected rats displayed increased exploration during 5 min open field test, it is possible that increased exploration attempts might affect Venetoclax solubility dmso passive activity in the forced swim test. However, linear regression analysis from behavior test results indicated that there were no correlation between exploration activity in OFT and duration of passive activity in FST, suggesting specificity for antidepressant-like effect of HCN1 knockdown in the dorsal hippocampal CA1 region. Why does knockdown of HCN1 in a small CA1 region of the dorsal hippocampus produce anxiolytic- and antidepressant-like effects?

It is becoming increasingly clear that small populations of Proteasome inhibitor neurons in specific regions can mediate certain behaviors (Han et al., 2009; Silva et al., 2009). Moreover, it has been shown that chronic electrical stimulation in limbic-cortical region from patients with treatment-resistant depression reduced pathologically elevated metabolic activity, implying that alternation in limbic-cortical activity may be involved in this treatment of severe depression (Mayberg, 2003; Mayberg et al., 2005). In a functional neuroimaging study, patients with severe depression showed

reduced posterior hippocampal volume, suggesting the posterior hippocampus, the equivalent of dorsal hippocampus in rodents, might be involved in both affective status and spatial learning in humans

(Campbell and Macqueen, 2004). Indeed, depressed patients showed deficits in spatial learning and memory assessed by a virtual navigation task as compared to healthy subjects (Gould et al., 2007). In a nonclinical study, Airan et al. (2007) showed that animal models of depression induced by chronic mild stress displayed increased ventral CA1 activity using voltage-sensitive dye imaging, which can be reversed by clinical antidepressant drugs delivered by i.p. injection, indicating that modulation of hippocampal activity might be required for the treatment of depression. In our experiments, knockdown of HCN1 in the dorsal hippocampal CA1 region resulted in a widespread increase of VSD optical signals in response to afferent stimulation, indicating Chlormezanone an enhancement of dorsal hippocampal activity. This discrepancy might be related to brain region, dorsal hippocampus versus ventral hippocampus, or specificity, knockdown of HCN1 versus antidepressant drug delivered by i.p. injection. Another possibility is that we consistently placed the stimulating electrode in the middle of stratum radiatum, close to the border between CA1 and CA2 regions, to activate Shaffer collaterals. In contrast to our experimental configuration, the stratum pyramidale was targeted for stimulation in Airan et al.’s study, which might have activated significantly more inhibitory axons.

The locations of these artifacts can be estimated in each subject and they are summarized MLN8237 order in Figure S4. These artifacts limit our ability to measure a portion of the VWFA in some subjects. The main experiment consisted of separate sessions (on separate days) for each feature type (line contours, motion-dot, luminance-dot, and mixture). Each subject completed six runs (312 s per run) for each feature type. The order of feature types was counterbalanced across subjects. Subjects were asked to keep fixation on a central fixation dot while reading the stimuli and to indicate by button press whether each stimulus was a word or

pseudoword (i.e., lexical decision task). Eye movements were monitored (see above). We measured the BOLD response to words and pseudowords at four different visibility levels for each feature type. In analyzing the data, we grouped words and pseudowords together because they showed similar responses in all regions of interest that we examined. All stimuli used for the main experimental runs were four-letter words or pseudowords (Medler and Binder, 2005). Words were nouns with a frequency of at least four per million (median: 28 per million). All words (n = 480) and pseudowords (n = 480) were unique within each subject, with five words and five pseudowords (× 4 visibility SKI-606 purchase levels × 6 runs/feature × 4 feature types) being assigned randomly to each of four visibility levels within each run (40 stimuli per run). All stimuli

were shown for 2 s. Stimulus presentation and response collection, both for fMRI and TMS (see below), were created using custom Matlab (The MathWorks, Inc.) scripts and controlled using the Psychtoolbox (Brainard, 1997). The stimuli were created as follows: The procedure used for rendering standard words at different visibility not levels was similar to that used by Ben-Shachar and colleagues (2007b). We rendered words in black using the Monospaced (Sans Serif) font within a gray rectangular frame (24 degrees horizontal, 7 degrees vertical). The horizontal and

vertical spans of the word within the frame were approximately 7.5 and 2.5 degrees, respectively (height of an x character was approximately 2°). To obtain different degrees of visibility, we computed the 2D Fourier transform of the word image, randomized the phase, and then applied the inverse Fourier transform. Visibility could be controlled by the degree of offset between the old and new phase. Resulting images ranged from noise (fully phase-scrambled) that contained the same amplitude spectrum as the original images, to highly visible words. To create words defined by dots of spatially varying luminance, we replaced the word image with a field of dots (dot density = 0.3; dot size = 1 pixel, total image size = 600 × 180 pixels), keeping the background color a uniform gray. The luminance of the dots was set separately for dots that fall inside (black) or outside (white) the nominal borders of the word form.

In addition to neuronal recombination, recombination in nonneuronal cells was evident among astrocytes in midbrain and neocortex ( Figure S3), oligodendrocytes in corpus callosum ( Figure S3) and Bergmann glial cells in

cerebellum (data not shown). No animals exhibited gross congenital defects or tumors in the brain. PTEN KO granule cells exhibited numerous morphological abnormalities characteristic of granule cells from rodents with temporal Onalespib datasheet lobe epilepsy ( Parent et al., 2006; Jessberger et al., 2007; Walter et al., 2007; Kron et al., 2010; Murphy et al., 2011, 2012; Pierce et al., 2011), including neuronal hypertrophy, de novo appearance of basal dendrites, increased dendritic spine density, and ectopically located somata. For illustrative purposes, a small number of PTEN KO animals were crossed into the Thy1-GFP expressing mouse line ( Feng et al., 2000; Vuksic et al., 2008; Danzer et al., 2010), which labels a subset of granule cells with GFP regardless of PTEN expression. GFP expression within adjacent wild-type and PTEN KO cells in these animals revealed

the dramatic morphological impact of PTEN deletion ( Figures 2A and 2B). Quantification of these changes in PTEN KO animals crossed to GFP reporter mice revealed increases in mean soma area from 59.3 ± 3.5 μm2 in control animals to 176.2 ± EPZ-6438 12.1 μm2 in PTEN KO animals (p < 0.001, t test; control n = 4 mice [40 cells]; PTEN KO n = 5 mice [36 cells]).

The percentage of GFP-expressing granule cells ectopically located in the hilus ( Figure 2F) increased from 0.3% ± 0.3% in controls to 3.3% ± below 1.0% in PTEN KO mice (p = 0.049, t test; control n = 5 mice [519 cells examined]; PTEN KO n = 8 mice [1,544 cells]). The number of apical dendrites increased from 1 [range 1.0–1.1] in control mice to 1.8 [1.4–2.3] in PTEN KO mice (p = 0.016, Mann-Whitney rank sum test [RST]; control n = 4 [40 cells], PTEN KO n = 5 mice [36 cells]). Spine density along these dendrites more than doubled ( Figures 2C and 2D), increasing from 2.9 ± 0.4 spines/μm to 7.5 ± 0.5 spines/μm (control, n = 4 mice [12 cells]; PTEN KO, n = 4 mice [12 cells], p < 0.001, t test). The number of basal dendrites/cell increased from an animal median of 0 [range 0–0] in controls to 0.8 [0.4–1.0] in PTEN KO mice ( Figure 3; p = 0.016, RST; control n = 4 mice [40 cells]; PTEN KO n = 5 mice [36 cells]). Basal dendrites, normally lacking in control rodents, are common in several models of temporal lobe epilepsy. 58.1% ± 6.9% (12 dendrites from three mice) of dendritic spines coating these hilar basal dendrites were apposed to puncta immunoreactive for zinc transporter-3 ( Figure 3). Zinc transporter-3 (ZnT-3) labels granule cell mossy fiber terminals ( McAuliffe et al., 2011), and the apposition of presynaptic and postsynaptic components implies that PTEN KO cells receive recurrent excitatory input from neighboring granule cells.

A potential link to preNMDARs thus beckons. Furthermore, dissociative drugs that block NMDARs, such as ketamine, may also act presynaptically. By virtue of its focus on the relatively overlooked preNMDARs, our study offers fresh perspective on neocortical functioning in health and disease. Procedures conformed to the UK Animals (Scientific Procedures) Act 1986 and to the standards and guidelines set in place by the Canadian Council on Animal Care, with appropriate licenses. P12–P20 mice were anesthetized with isoflurane, decapitated, and the brain was swiftly dissected

in ice-cold artificial cerebrospinal fluid (ACSF: 125 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1.25 mM NaH2PO4, selleck inhibitor 2 mM CaCl2, 26 mM NaHCO3, 25 mM dextrose; bubbled with 95% O2/5% CO2). Whole-cell

recordings in acute visual cortex slices were carried out at 32°C–34°C (see Supplemental Experimental Procedures for details) with the following gluconate-based internal solution: 5 mM KCl, 115 mM K-gluconate, 10 mM K-HEPES, 4 mM MgATP, 0.3 mM NaGTP, 10 mM Na-phosphocreatine, and 0.1% w/v biocytin, adjusted with KOH to pH 7.2–7.4. D/L-AP5 (Sigma) was either bath applied or puffed at a concentration of 200 μM in ACSF. MK801 (Sigma) was applied at a concentration of 2 mM to standard internal solution. For 2PLSM imaging, 10–40 μM Alexa Fluor 594 and/or 180 μM Fluo-5F pentapotassium salt (Invitrogen) were added to the internal solution. INs were targeted by green GFP fluorescence detected by 2PLSM (see below) in transgenic mice specific

for SOM (Jackson AUY-922 nmr Laboratories, 3718; Oliva et al., 2000) or PV IN subclasses (Jackson Laboratories, 7677; Chattopadhyaya et al., 2004). Data were acquired using PCI-6229 boards (National Instruments) with custom software (Sjöström et al., 2001) running in Igor Pro 6 (WaveMetrics). Miniature EPSCs were recorded in voltage clamp at −80mV in the presence of 0.1 μM tetrodotoxin (TTX) and 20 μM bicuculline and were detected offline. Workstations for 2PLSM were custom built (see Supplemental Experimental Procedures). Two-photon excitation Montelukast Sodium was achieved using a MaiTai BB (Spectraphysics) or a Chameleon XR (Coherent) Ti:Sa laser, tuned to 800–820 nm for Fluo-5F and Alexa 594 or to 880–900 nm for GFP. Imaging data were acquired with PCI-6110 boards (National Instruments) using ScanImage v3.5-3.7 running in MATLAB (MathWorks) and was analyzed offline using in-house software running in Igor Pro (see below). Uncaging was achieved using a 405 nm laser (MonoPower-405-150-MM-TEC, Alphalas GmbH). In uncaging experiments (Figures 3, S3, and S4), either 1 mM MNI-Glu or 1 mM MNI-NMDA dissolved in ACSF (see above) and supplemented with 20 mM HEPES was puffed using a patch pipette. Only MNI-NMDA was used for bouton uncaging, however. Neurons were reconstructed from 2PLSM stacks using Neuromantic (http://www.reading.ac.uk/neuromantic) or from slices histologically processed for biocytin using Neurolucida (MicroBrightField).

Next, we assessed the TR4 cKO mice in a battery of behavioral tests of acute and injury-associated

persistent pain. The TR4 cKO mice did not differ from WT mice in the Hargreaves (Figure 2A) and tail immersion reflex withdrawal tests of heat pain sensitivity (Figure 2B). However, in the hot plate test, which triggers a behavioral response (licking of the paw) that involves both spinal cord and supraspinal processing of pain messages (Langerman et al., 1995; Le Bars et al., 2001), the cKO mice had significantly higher response latencies (Figure 2C). Many of the animals went to cut-off, which is the maximal response permitted to avoid injury. Compared to WT mice, the TR4 cKO mice have significantly increased reflex withdrawal thresholds in the von Frey test of mechanical pain (Figure 2D). Also, capsaicin-induced licking/flinching (Figure 2E) and pain behavior following hindpaw injection of formalin (5.0%) are profoundly selleckchem reduced Venetoclax supplier in the cKO mice (Figure 2F). Formalin-evoked Fos expression in the superficial dorsal horn was also decreased, by 44.3% (Figure 2I), mostly in lamina II, in cKO (Figure 2H) compared to WT mice (Figure 2G). Taken together, these results reveal a profound reduction of pain behaviors in response to a variety of noxious stimulus modalities, namely heat, mechanical and chemical, with preservation of the reflex responses

provoked by noxious heat. Both tissue and nerve injury induce a prolonged state of mechanical and thermal hypersensitivity, largely due to changes (central sensitization) generated at the level of the spinal cord dorsal horn (Basbaum et al., 2009). These changes are usually manifest as a decreased mechanical withdrawal threshold and decreased withdrawal latency in response to a heat stimulus. Here, we induced paw inflammation by injection of complete Freund’s adjuvant (CFA) and found that TR4 cKO and WT mice develop comparable heat hypersensitivity (Figure 2J) in the Hargreaves (reflex) test. Furthermore, although mechanical thresholds

at baseline are higher in the cKO mice, these animals did develop mechanical hypersensitivity. The magnitude of the mechanical all hyperalgesia was somewhat less than in WT mice and the sensitized threshold in the cKO mice was considerably greater than in their WT littermates (Figure 2K). To model nerve injury-induced neuropathic pain, we used the chronic-constriction injury (CCI), as this results in both heat and mechanical hyperalgesia (Bennett and Xie, 1988; Urban et al., 2011). We found that cKO and WT mice developed comparable thermal hyperalgesia; however, the magnitude of the change in the cKO mice decreased slightly by 7 days, compared to WT mice (Figure 2L). By contrast, although mechanical hyperalgesia was readily observed in the WT mice, this was absent in the TR4 cKO mice (Figure 2M).

, 2013). We selected 26 target genes (Table S16) and tested Trichostatin A in vivo their expression in C9ORF72 autopsied CNS tissue against non-ALS control tissue using nanostring gene expression methodologies. Sixteen of the target genes tested were also aberrantly expressed in C9ORF72 ALS patient tissue (Figures 5C, S7A, and S7B), of which seven showed the same direction of dysregulation

(up or down) when compared to iPSNs (Figure 5D). These genes could be potential candidates for the future development of a pharmacodynamic biomarker to monitor C9ORF72 therapy in human CSF and/or blood. Glutamate toxicity has been shown to play a major role in ALS as sporadic ALS and C9ORF72 patients exhibit a loss of astroglial glutamate transporter 1 (GLT-1/EAAT2), which buffers synaptic glutamate thus preventing excitotoxicity (Lin et al., 1998, Rothstein

et al., 1995 and Renton et al., 2011). Notably, C9ORF72 iPSNs express glutamate receptors (GluR2), NMDA receptors (NR2B), and postsynaptic marker protein postsynaptic density protein-95 (PSD95), comparable to control iPSNs, thus suggesting that these cells form functional synapses and are capable of responding to glutamate-induced excitotoxicity (Figure 6A). To 5-Fluoracil solubility dmso determine whether the C9ORF72 mutation leads to an altered physiology in iPSC neurons, we explored sensitivity to glutamate excitotoxicity. Glutamate treatment resulted in a dose- and time-dependent increase of cell death in control and C9ORF72 iPSN cultures as determined by cellular propidium iodide (PI) uptake (Sattler et al., 1997 and Sattler et al., 1999) (Figures 6B–6D and S8). Notably, C9ORF72 ALS iPSNs are almost 100-fold more sensitive to glutamate treatment, as toxicity at 3 μM glutamate in healthy control iPSNs was comparable to death at 100 μM

glutamate in C9ORF72 iPSNs (Figures 6B and S8). Glutamate-induced cell death was blocked by inhibitors of glutamate receptors and calcium channels (MK-801, 10 μM; CNQX, 10 μM; nimodipine, 2 μM; Sattler until et al., 1999), confirming that C9ORF72 iPSN cell death was glutamate-receptor dependent (Figure 6E). To test whether the sequestration of ADARB2 plays a role in the observed glutamate susceptibility, we knocked down ADARB2 levels in healthy control iPSNs via siRNA treatment (Figure 6F) and investigated their susceptibility to glutamate-mediated cell death. Interestingly, a loss of about 50% of ADARB2 RNA significantly enhances control iPSC neurons’ susceptibility to glutamate (30 μM at 4 hr) to levels similar to those observed in C9ORF72 iPSNs (Figures 6F and 6G). This suggests that a partial loss of ADARB2 via sequestration to GGGGCC RNA might play a role in C9ORF72-mediated RNA toxicity. If an RNA-dominant pathogenic mechanism exists for C9ORF72 ALS, it could be a candidate for antisense oligonucleotide therapeutics.