, 2013). These examples illustrate how cargo receptors and dedicated auxiliary subunits may regulate channel traffic, thereby controlling channel density and composition. As channels assemble in the ER and traffic through the secretory pathway and endosomal pathway, they are exposed to different chaperones

and modifying enzymes as well as different pHs ranging from pH 7.2 in the ER lumen, to pH 6.0–6.7 in the Golgi, and pH 5.5 in secretory vesicles (Mindell, 2012 and Stauber and Jentsch, 2013). Retrieval of channels from the cell surface for recycling or degradation also takes channels from a neutral to a low pH environment on the extracellular/luminal side. The sensitivity of various channels to pH on the extracellular and luminal side of the membrane may be one of the mechanisms to modulate channel

activity in different intracellular compartments and seems to be a fundamental GW786034 property of the channel life see more cycle that deserves increased scrutiny. In the final paragraphs of this Perspective, we offer some thoughts for key challenges that remain for the field. Since the first characterization of the squid axonal sodium and potassium conductances and their voltage dependence 60 years ago by Hodgkin and Huxley (Hodgkin and Huxley, 1952), a desire to understand the nature and mechanics of ion channels has driven the field to devise novel approaches, such as the patch clamp (Hamill et al., 1981), and to harness challenging technologies including crystallography and real-time monitoring of channel conformational changes, in order to study how ion channels work and how they mediate neuronal signaling. These studies have uncovered because the molecular motions of sensing and gating most completely in voltage-gated (Chowdhury and Chanda, 2012, Tombola et al.,

2006 and Vargas et al., 2012), acetylcholine-gated (Changeux, 2012, Corringer et al., 2012 and Unwin, 2013), and glutamate-gated (Mayer, 2011 and Paoletti et al., 2013) channels and revealed the modular construction of many channel types, both within the membrane portions (Minor, 2006 and Yu and Catterall, 2004) and in the extramembranous parts (Mayer, 2011 and Minor, 2007). Understanding how such multicomponent devices act to integrate input signals that regulate the basic function of opening a hole for ions to pass remains a major challenge. There are many channel families in which the gating mechanisms are still very obscure, including thermosensation by TRP channels (Nilius and Owsianik, 2011 and Ramsey et al., 2006a), mechanosensation by the TRP channel NOMPC (Yan et al., 2013) and Piezo channels (Coste et al., 2012 and Kim et al., 2012), and the gating of CRAC channels via formation of multiprotein complexes that involve both plasma and intracellular membrane components (McNally and Prakriya, 2012).

We hypothesized that greater reduction of eversion ROM and peak eversion velocity would be observed in the sport ankle brace compared to the soft ankle brace and in CAI participants compared to healthy participants. It was also hypothesized that the ankle braces would yield greater reduction of eversion ROM and velocity in CAI participants compared to healthy participants. Ten control subjects with no history of previous ankle sprains (age: 24.1 ± 5.4 years, mass: 72.4 ± 12.0 kg, height: 1.74 ± 0.08 m) and 10 CAI subjects who had multiple ankle

sprains (age: 24.8 ± 5.7 years, mass: 73.03 ± 9.31 kg, height: 1.75 ± 0.09 m) were recruited to participate in the study. In each subject group, five females and five males were recruited. The CAI subjects were age and body mass PD0325901 purchase index matched by the

subjects in the control group. Potential subjects were asked to participate in a screening session for ankle functions and instability using Ankle Joint Functional Assessment Tool (AJFAT)20 and arch index measurements. If a subject met the inclusion criteria (multiple ankle sprains in past 12 months and beyond, and no ankle sprains in past 3 months) for CAI group, he/she was then asked to participate in a biomechanical testing session. All participants signed selleck kinase inhibitor an informed consent form approved by the Institution Review Board. The session began with the subject filling out the AJFAT survey21 to document the condition of the reported CAI. Arch index was measured with the subjects in sitting (unloaded) and standing (loaded) positions in barefoot and in both ankle braces using an AHIMS (Arch

Height Index Measurement System; JAK Tool and Model, LLC, Matawan, NJ, USA). The measurements were used to compute arch index (AID)22 and arch deformity (AD)23 using the following equations: AID=DorsumheightTruncatedfootlength AD=AIunloaded−AIloadedAD=AIunloaded−AIloadedwhere dorsum height is the height of dorsum found of the foot at 50% of foot length and the truncated foot length is measured from heel to the head of 1st metatarsal head.22 The biomechanical testing session began with a 5-min warm-up of jogging on a treadmill followed by a stretching routine of major muscle groups. Participants performed five trials in each of the three testing conditions: drop landing from an over-head bar from a height of 0.6 m, wearing NB (NB, lab running shoe: Grid Triumph, Saucony), Element™ (DeRoyal Industries, Inc., Rowell, TN, USA; Fig. 1A) and ASO (ASO, Medical Specialties, Charlotte, NC, USA; Fig. 1B). The Element™ ankle brace is a semi-rigid brace with a hinge joint at the ankle allowing sagittal plane rotation and a heel strapping system designed to strap and stabilize the calcaneus with two cross-pattern straps to restrict ankle frontal-plane motion.

, 2005). OR35a-dependent responses to γ-hexalactone persisted in both IR8a and IR25a mutants ( Figures 2B and 2C), indicating independent functioning of this selleck chemicals receptor. The ac2 sensilla neurons respond strongly to acetic acid and 1,4-diaminobutane, and these responses are selectively abolished in IR8a and IR25a mutants, respectively ( Figures 2B and 2C). Finally, ac1 sensilla contain three IR-expressing neurons, but only one strong agonist, ammonia, has been identified ( Yao et al., 2005). Responses to this odor were retained in both IR8a and IR25a mutants,

as well as in IR8a/IR25a double mutants ( Figures 2B and 2C). All defects in odor-evoked responses in IR8a and IR25a mutants were rescued by expression of the corresponding cDNA transgenes using IR8a or IR25a promoters via the GAL4/UAS system ( Figures 2B and 2C; see Figure S1 available online) ( Brand and Perrimon, 1993). The sole exception was our failure to restore ac2 1,4-diaminobutane responses in IR25a mutants (data not shown).

We ascribe this lack of rescue activity to the poor recapitulation of endogenous IR25a expression by our IR25a-GAL4 line ( Figure S1B). Expression of IR25a in IR8a mutant neurons did not rescue electrophysiological responses (data not AT13387 research buy shown), indicating selective functional properties of these two receptors beyond their distinct expression patterns ( Figure 1C). Taken together, the loss of multiple distinct ligand-evoked responses in IR8a and IR25a mutants suggests that these proteins function as coreceptors that act with different subsets of odor-specific IRs. To determine the cellular basis for the loss of electrophysiological responses in these IR coreceptor mutant neurons, we initially focused on the role of IR8a in the correct functioning of the phenylacetaldehyde receptor IR84a (Benton et al., 2009). An EGFP-tagged version of IR84a localizes to the sensory cilium in its endogenous neurons (Figure 3A), defined by the distal distribution relative to the cilium base marker 21A6 (Husain et al., 2006 and Zelhof et al., 2006). By contrast, in IR8a mutants, EGFP:IR84a

is restricted to the inner dendritic segment ( Figure 3A). Restoration of IR8a expression under the control of the IR84a promoter rescues this localization defect, defining a cell-autonomous function almost for IR8a in promoting cilia targeting of IR84a ( Figure 3A). We tested the generality of this requirement for IR8a by examining the cilia localization of a second receptor, IR64a, which is coexpressed with IR8a in morphologically distinct grooved peg sensilla in the third chamber of the sacculus (Ai et al., 2010). EGFP:IR64a is abundant in the outer dendrite of these neurons in wild-type sensilla, and this localization is abolished in IR8a mutants ( Figure S2A). We observed more heterogeneous levels of EGFP:IR64a in IR8a mutant neurons, suggesting that this mislocalized protein is destabilized.

, 2007). Although critical experiments are still needed to address whether T668P phosphorylation causes APP processing in vivo, our study provides additional support to the idea that T668P phosphorylation significantly contributes to APP processing in vivo. We provide compelling evidence that a translational block is a prominent feature in FAD mice and to some see more extent in human AD cases. Since oligomeric Aβ42 induced a translational block in hippocampal neurons in culture, it is highly likely that

oligomeric Aβ42 has a similar effect in vivo. Oligomeric Aβ42 is widely believed to be the central pathologic species that is responsible for inhibiting LTP and memory formation in vivo (Cleary et al., 2005; Walsh et al., 2002). Since inhibiting normal translational processes by disabling eif2α phosphorylation or deleting its kinase, GCN2, resulted in inhibition of LTP ( Costa-Mattioli

et al., 2005, 2007), it is tempting to speculate that such synaptotoxicity observed with oligomeric Aβ42 is likely to be due to its inhibitory effect on translation. Our data indicate that oligomeric Aβ42 inhibits translation in part by blocking the mTOR pathway. Dysregulation of the mTOR pathway or loss of energy balance has MLN8237 concentration been identified as causative in normal aging as well as type 2-diabetes and obesity (Cohen et al., 2009; Demontis and Perrimon, 2010; Koo et al., 2005; Mair et al., 2011; Song et al., 2010). Our findings that widespread disruption of normal energy balance is prominent in FAD mice and to some extent in human AD cases suggest that in progressive diseases whose symptoms develop

over a long period time, chronic metabolic imbalance becomes a pervasive phenotype. Our data clearly illustrate that oligomeric Aβ42 perturbs energy homeostasis, as indicated by activation of AMPK, a kinase that responds to energy imbalance in the cell (Steinberg and Kemp, 2009). AMPK was shown to play a critical role in aging in yeast and C. elegans, although the loss of snf1p, the yeast homolog of AMPK, increased the life span ( Lin et al., 2001), while mutation in aak-2, the worm Suplatast tosilate homolog of AMPK, decreased the life extension induced by stress ( Apfeld et al., 2004). Besides this apparent species-related difference in homolog roles, the role of AMPK itself in aging appears clear. It is of special interest in this regard that oligomeric Aβ42 activates AMPK, thereby inhibiting the mTOR pathway. Aβ peptides are normally produced and cleared rapidly in human brains ( Bateman et al., 2006). It is plausible that normal production of Aβ peptides contributes to the aging process in part by activating AMPK. AMPK activation was rapid but transient by oligomeric Aβ42, detectable at 10 min, but greatly reduced by 3 hr after Aβ42 addition. Although transiently activated, AMPK substrates Raptor and TSC2 remain phosphorylated up to 16 hr, providing an explanation for a prolonged translational inhibition.

With regards to symptoms at a general level, the majority of parents reported SKI-606 mouse that regular PA positively impacted symptoms. However, there were not uniform effects for all types of ADHD symptoms. The results indicate that there may be more positive benefits for symptoms of inattention and hyperactivity than for those of impulsivity. A comment by one participant reinforces this: “”If the activity is continually fast paced like soccer that seems to bring out the impulsivity because it’s harder for him to control.”" While this may represent

a limitation of PA to address impulse problems it may be that parents/guardians need to find the optimal sport and/or activity that will bring about positive changes in that domain. For example, team sports may not positively impact impulsivity; however an individual sport such as running or cycling may impact impulsivity more profoundly. Alternatively, individual sports such as running or cycling may not present the child with as many opportunities to engage in impulsive behavior due to the inherent nature of those activities. This is supported by evidence that children diagnosed with ADHD display higher levels of aggression and emotional reactivity in team sports

compared to individual sports21 and 22 and have difficulty following rules in team sports.23, 24 and 25 A secondary issue is that Selleck PD0332991 organized sport may not be the optimal way to bring about desired changes in behavior, rather engagement in PA and/or exercise may be more important. This is exemplified by participants who stated: “My

son has a difficult time in organized sports – his coordination does not seem to be on par, and he is not as focused and driven as other children to succeed.” or “There are times when he has a hard time following the rules of games at school in gym and staying focused.” These comments reflect the possibility that organized sports present challenges to children with ADHD that inhibit the benefits of PA on certain behavioral symptoms. Therefore it seems critical for future research to consider PA and/or exercise as separate from sport in order to optimally benefit behavior in children and adolescents with ADHD. For the those questions regarding symptoms broadly, academics, and hyperactivity there were considerable percentages of participants reporting that regular PA does not have an effect on symptoms. These can be interpreted positively in that they demonstrate that PA is not exacerbating symptoms. Another possibility for the reporting of “no effect” might be that parents have not thought about the connection between PA and academic performance and therefore are not able to answer the question adequately. This is supported by one participant’s statement, “Not particularly…we’ll have to pay attention to this (good question!)”.

5 DT or VT retinal explants were cocultured with dissociated chiasm cells (Figure S2) in the presence of a function-blocking Sema6D antibody (αSema6D) or a control antibody (αcontrol) (Figure S3). Whereas αSema6D had no effect on VT explant outgrowth, application Selleckchem KU-55933 of αSema6D significantly reduced DT explant neurite outgrowth on chiasm cells by 50% compared to cocultures with αcontrol (DT plus chiasm plus αSema6D was 0.50 ± 0.03 versus DT plus chiasm plus αCtr 1.02 ± 0.05; p < 0.01) (Figures 2A and 2B). These data support the hypothesis that Sema6D is important for growth of contralaterally projecting RGCs at

the chiasm midline. To further test the effect of Sema6D on RGC outgrowth, we measured neurite growth from E14.5 DT and VT explants cultured on HEK cells expressing full-length Sema6D (Figures 2C and 2D). We observed a 55% reduction in DT explant neurite outgrowth on Sema6D+ HEK cells compared to explants growing on control HEK cells with vector

alone (DT plus HEK Sema6D plus αCtr was 0.45 ± 0.03 versus DT plus HEK Ctr plus αCtr 1.0 ± 0.03; p < 0.01) (Figures 2C–2E). This reduction was attenuated by αSema6D, leading to a reduction of growth only to 10% of control values (Figures 2D and 2E) (DT plus HEK Sema6D plus αSema6D was 0.90 ± 0.05 versus DT plus HEK selleck inhibitor Sema6D plus αCtr 0.45 ± 0.03; p < 0.01). As in coculture with chiasm cells, VT explant neurite outgrowth on Sema6D+ HEK cells was similar with or without αSema6D, indicating that uncrossed RGC axons do not respond to Sema6D (Figures 2D and 2E). Thus, whereas Sema6D presented

alone in HEK cells is inhibitory to crossed RGCs, Sema6D is important for RGC midline crossing in the context of the optic chiasm. The finding that Sema6D supports crossed RGC outgrowth on chiasm cells suggests that factors at the chiasm midline convert Sema6D from an inhibitory to a growth-promoting factor. Sema6D is coexpressed with Nr-CAM by radial glial cells at the chiasm midline, and Plexin-A1 is expressed by SSEA-1+ chiasm neurons that extend into the chiasm midline (Figure 1E). We therefore considered whether Nr-CAM Adenosine and/or Plexin-A1, in the context of the optic chiasm environment, modulate the repulsive effect of Sema6D on crossed axons. Because HEK cells that are singly transfected do not fully recapitulate the cellular composition of the optic chiasm, we designed a HEK-retina coculture system to present Sema6D, Plexin-A1, and Nr-CAM in a manner that best mimics their expression in the different cell types at the optic chiasm in vivo: Sema6D and Nr-CAM were coexpressed in one set of HEK cells (to mimic radial glia cells), and Plexin-A1 was expressed in a separate population of HEK cells (to mimic SSEA-1+ chiasm neurons). When DT explants were grown on Sema6D+/Nr-CAM+ HEK cells, or Sema6D+ HEK cells mixed with Plexin-A1+ HEK cells, neurite outgrowth was significantly improved compared to explants grown on Sema6D+ HEK cells (DT plus HEK Sema6D/Nr-CAM was 0.76 ± 0.

, 2001 and Weihl et al., 1999); and (2) Aβ can both directly and indirectly interfere with canonical Wnt signaling and that this interference compromises neuronal survival ( Caricasole et al., 2004 and Scali et al., 2006). Wnt signaling therefore may provide a bridge between

neurodevelopment and neurodegeneration ( Geschwind and Miller, 2001 and Jackson et al., 2002). It is still an open question as to whether loss of GRN causes FTD pathology through a cell autonomous or noncell autonomous mechanism. Loss of GRN may increase neuronal vulnerability, conferring an intrinsic property in which neurodegeneration is more likely. Alternatively, microglia lacking GRN may become hyperactive, creating a poor extrinsic Perifosine environment

that leads to neuronal death. This is a complex issue because GRN is a secreted protein, the form of GRN that is clinically relevant to FTD is currently unknown, and GRN transcripts are decreased in blood, but are paradoxically increased in GRN+ diseased brain (Chen-Plotkin et al., 2010). The data that we present here, in which GRN loss is sufficient to produce cell death in the absence of microglia, shows that neuronal GRN deficiency is sufficient to significantly reduce neuronal survival, a finding important for potential therapeutics development. While these data argue that GRN loss can indeed increase neuronal vulnerability, they do not preclude the compounding GABA receptor signaling involvement of microglia in the pathophysiology of FTD in patient brain, which may form a vicious cycle (Pickford et al., 2011 and Yin et al., 2010). Here we provide data from multiple systems showing that FZD2 expression increases with GRN loss. We then performed not a proof of principle experiment, indicating that this increase may be protective. Classically, signaling through the FZD2 receptor activates the noncanonical Wnt signaling pathway ( Oishi et al., 2003). This suggests

that modulation of this pathway may have therapeutic relevance; previous work has shown that noncanonical Wnt agonists can be protective in other forms of dementia ( Inestrosa and Toledo, 2008). As opposed to canonical Wnt signaling, which has already been established as a major player in neurodegeneration ( Hooper et al., 2008 and Toledo et al., 2008), these data indicate a role for Wnt signaling independent of GSK-3β and β-catenin in neurodegenerative pathology. Additionally, FZD2 is the initial change that precedes alterations in other Wnt pathway members in mouse in vivo at stages well prior to neurodegeneration or neuronal loss. Thus, FZD2 represents a primary target in that it is a consistent and early upregulated gene in the context of GRN loss.

Let us consider the specific findings individually. Figure 1 shows a schematic summary of the results. The first result of the paper—that regions showing positive BOLD responses correlate with increases in CBV and CBF—is arguably the most straightforward and easiest to understand. It is well known that, with activation, CBV and CBF increase. Second, they show that adjacent regions associated with a negative BOLD response correspond to a decrease in CBF but an increase in CBV. This result is slightly puzzling. This could be explained selleck chemicals llc if the CBV response, being larger than BOLD, might result

in significant amount of hemodynamic “spillover” from the truly active regions. However, this explanation seems likely to be wrong since multiple papers have shown that CBV, if anything, has a smaller point-spread function GSK1210151A nmr than BOLD, and further, the results here show an exquisite layer specificity of CBV. Goense et al. (2012) also show that with regard to layer specificity, for positive BOLD responses, CBF and CBV both increased in the central layers. This is also an interesting but yet not easily explained finding. It is thought that the center layers, which have the greatest concentration of microvessels, would be most active (and it is heartening to see that both CBF and CBV show selective increases in the center layers). The

most surprising of the study’s findings is their last result, that for negative BOLD responses, CBF decreased near the surface but CBV increased in the central layers. Why and how would only surface CBF decrease and why would only middle-layer CBV increase in these areas of negative BOLD? The CBF decrease at the surface layers appears to be what determines the decrease in BOLD, but is this something that reflects a decrease in neuronal activity? Surface (larger) vessels are presumably less directly controlled by neuronal activity. Why would the middle layers

not show any decrease in CBF with less neuronal activity? Lastly, the increase through in CBV in the middle layers might even suggest a local increase in neuronal activity (increased activity of inhibitory neurons?) in these negative regions. The authors suggest that interneuron inhibitory activity often eludes electrophysiological measures, thus explaining the failure to detect this effect in previous experiments. Other hypotheses to explain this apparently perplexing result invoke more “plumbing”-related autoregulatory or redistribution effects, mechanisms which would be extremely difficult fully unravel. For instance, it is suggested that there might be a decrease in perfusion pressure in center layers (without a decrease in perfusion itself), causing a reduction of flow in superficial vessels and an therefore an increase in venous backpressure, leading to an increase in center layer CBV.

A standard laboratory task of this type therefore Neratinib cell line involves multiple stimulus-response associations (Lalazar and Vaadia, 2008). The microcircuitry connecting sensory and motor cortices described here might help to implement these stimulus-response associations. It has been suggested that vibrissa-based object localization requires the brain to interpret contact between whisker and object in

the context of an internal reference signal indicating whisker location or phase (Curtis and Kleinfeld, 2009 and Diamond et al., 2008). This reference signal might consist of an efference copy generated by the inverse model driving goal-directed whisking. The circuits uncovered here may underlie mixing of whisking ISRIB manufacturer and contact signals and thus, might underlie computation of object location. Experiments were conducted according to National Institutes of Health guidelines for animal research and were approved

by the Institutional Animal Care and Use Committee at Janelia Farm Research Campus. For anterograde tracing we used adeno-associated virus (AAV; serotype 2/1) expressing eGFP (www.addgene.com) or tdTomato (a gift from J. Magee) under the CAG promoter. For sCRACM mapping experiments, we used AAV virus (serotype 2/1; in some experiments serotype 2/10) expressing either ChR2-venus (Petreanu et al., 2009) or ChR2-tdTomato (www.addgene.com). For retrograde tracing we used fluorescent LumaFluor microbeads (LumaFluor Inc.). C57BL/6J mice (Charles River) (13–16 days old) were anesthetized using an isoflurane-oxygen mixture and placed in a custom stereotactic apparatus. A small hole was drilled into the skull,

allowing insertion of a pulled glass pipette (Drummond) (tip diameter: 10–20 μm for virus; 40–60 μm for LumaFluor microbeads). For sCRACM experiments, coordinates were as follows (in mm, from bregma): vS1, 0.5 to 0.8 posterior, 2.9–3.3 lateral; vM1, 1.0−1.1 anterior, 0.60–0.75 lateral. Injections sites Isotretinoin were confirmed by post hoc histological analysis (Figures 1B, 3, 4, 5, 6, 7, and S1B–S1H). See Supplemental Experimental Procedures for further details. Brain slices were prepared as described (Bureau et al., 2006) 14 to 24 days after viral infections (see Supplemental Experimental Procedures). For vM1, the brain was tilted ∼10° to 15° forward during slicing to optimize the alignment of apical dendrites with the slice surface. When cutting from rostral to caudal, 1∼2 slices, ∼0.8–1.3 mm anterior to bregma, corresponding to the first and/or second slice containing a fused corpus callosum, were used. For vS1 slices, the brain was cut in the coronal plane. Only slices with prominent barrels (Figure S9B) were used (Petreanu et al., 2009). All recordings were performed at room temperature in circulating ACSF. For most experiments (except Figures 6B, S6F, S8B, and S8C) TTX (1 μM), 4-AP (100 μM), and CPP (5 μM) were added (Petreanu et al., 2009).

1 CaCl2, 15 HEPES (pH7.2), osmolarity 300 ± 2 mOsm/l. Dissected hippocampal CA1-CA3 regions were placed into a holding chamber containing protease type XIV (1 mg/ml, Sigma-Aldrich) dissolved in oxygenated HEPES-buffered Hank’s balanced salt solution (HBSS 6136: Sigma-Aldrich) and maintained at 37°C, pH 7.4, osmolarity 300 ± 5 mOsm/l. After 30 min incubation in the enzyme solution, learn more the tissue was rinsed three times with the Low-Ca2+ HBS and triturated using fire-polished Pasteur pipettes. The cell suspension was placed into a 50 mm plastic petri dish for electrophysiological recordings. Hippocampal pyramidal neurons were selected on the basis of their characteristic morphology. Agonist-evoked currents were recorded

from

transfected HEK293T cells, acutely isolated neurons, and primary hippocampal cultures as described (Kato et al., 2008). Recordings were made using thick-walled borosilicate glass electrodes pulled and fire-polished to a resistance of 2–5 MΩ. All cells were voltage-clamped at −80 mV and data were collected and digitized using Axoclamp 200 and Axopatch software and hardware (Molecular Devices, Sunnyvale, CA). For whole cell recordings, the transfected Navitoclax datasheet HEK293T cells were bathed in external solution containing the following (in mM): 117 TEA, 13 NaCl, 5 BaCl2, 1 MgCl2, 20 CsCl, 5 glucose, and 10 Na-HEPES pH 7.4 ± 0.03. For acutely isolated and cultured primary neurons, 10 μM CPP, 10 μM bicuculline, 1 μM TTX, and 300 nM 7-chlorokynurenic acid were added in the external solution and the extracellular concentration of NaCl was increased to 130 mM and TEA was omitted. 7-Chlorokynurenic acid (7-CK) was omitted for acutely isolated neurons. The intracellular electrode solution contained the following (in mM): 160 N-methyl-D-glucamine, 4 MgCl2, 40.0 Na-HEPES pH 7.4, 12 phosphocreatine, 2.0 Na2-ATP pH7.2 ± 0.02 adjusted by H2SO4. For neuronal

recordings, 1 mM QX314 were added to the internal solution. For outside-out patches and whole cell recordings using fast perfusion, the internal solution contained (in mM): 130 CsCl, 10 CsF, 10 Cs-HEPES pH 7.3, 10 ethylene glycol tetraacetic acid (EGTA), 1 MgCl2, and 0.5 PD184352 (CI-1040) CaCl2 and was adjusted to ∼290 mOsm. The transfected HEK293T cell or the acutely isolated neuron was lifted and perfused with ligand-containing solutions from a sixteen-barrel glass capillary pipette array positioned 100–200 μm from the cells (VitroCom). Each gravity-driven perfusion barrel is connected to a syringe ∼30 cm above the recording chamber. The solutions were switched by sliding the pipette array with an exchange rate of less than 20 ms. For fast application experiments with a junction potential rise time of less than 300 μs, rapid solution exchange (1 and 200 ms application for deactivation and desensitization, respectively) from a θ tube containing external solution (in mM: 140 NaCl, 3 KCl, 10 glucose, 10 HEPES pH 7.