By contrast, tagging NLF-1 with HDEL, a known ER-retention signal

By contrast, tagging NLF-1 with HDEL, a known ER-retention signal ( Basham and Rose, 2001; Denecke et al., 1992; Semenza et al., 1990) did not alter its ER localization (

Figure 2A, HDEL) or affect its rescuing ability ( Figure S2F). Therefore, NLF-1’s ER localization is critical for its function. Collectively, these data indicate that NLF-1 is a novel ER resident protein that functions in the same biological process as the NCA channel. To determine the physiological deficit that underlies the fainting phenotype shared by nlf-1 and nca mutants, we first identified the minimal neural network that contributes most critically to this movement deficit. While NLF-1 is expressed in sensory neurons, interneurons ( Figure 2E) and excitatory motor neurons ( Figure S2G), the restored expression of NLF-1 in the premotor interneurons, see more a subset of interneurons that input directly onto the motor neurons, was necessary to restore the initiation and continuity of rhythmic locomotion in nlf-1 mutants ( Figures 3A, 3B, and S3B; Pnmr-1+Psra-11), and to prevent frequent halting ( Figure S3A; Pnmr-1+Psra-11). By contrast, restoring the NLF-1 expression in motor neurons did not rescue fainting ( Figures 3A, 3B, S3A, and S3B; Pacr-5+Punc-4). Similarly, the locomotion defect of unc-79 fainters was only rescued by restoring their expression in premotor interneurons, not in motor neurons

(data not shown). These results point to dysfunctional premotor interneurons, rather than a lack of motor activity, being the primary cause of the failure in the initiation and maintenance of rhythmic Adriamycin datasheet locomotion exhibited by fainters. Among all premotor interneurons, restoring NLF-1 expression in a subgroup, Oxalosuccinic acid including AVA and AVE, led to the most significant, partial rescue of fainting (Figures 3A, 3B, and S3B; Pnmr-1). Through real-time calcium imaging, we recently demonstrated that the AVA and AVE premotor interneurons exhibit similar activity profiles during spontaneous movements ( Kawano et al., 2011). In wild-type animals, coimaged AVA and AVE exhibited large and periodic rise in intracellular

Ca2+ that temporally correlated with the initiation and duration of backing ( Kawano et al., 2011; Figures 3C and 3D). In both nca(lf) and nlf-1 mutants, pulses of Ca2+ transients in AVA and AVE exhibited a significantly reduced amplitude ( Figures 3C and 3D), which corresponded with shorter backing, and indicates a reduced premotor interneuron activity. Restoring NLF-1 expression only in these neurons fully restored the Ca2+ transient profile ( Figures 3C and 3D). These transgenic animals exhibited Ca2+ transients that were slightly higher than wild-type animals ( Figure 3D), which may be caused by NLF-1 overexpression. These results imply that NLF-1 and the NCA channel potentiate premotor interneurons, whose activity is most critical to maintain the continuity of locomotion.

The average training duration of participants here was 73 hr, wit

The average training duration of participants here was 73 hr, with up to 10 hr devoted to learning to read using the SSD. As part of the training program, the participants were taught (using verbal explanations and palpable images; see Figure 1D

and Supplemental Experimental Procedures) how to process 2D still (static) images, including hundreds of images of seven structured categories: geometric shapes, Hebrew letters and digital numbers, body postures, everyday objects, textures (sometimes with geometric shapes placed over visual texture, used to teach object-background segregation), faces, and houses (see Figure 1E; see Movie S1 for a demo of the visual stimuli and their soundscape representations). For full details on the training technique and protocol, see the Supplemental Experimental Procedures.

After the structured training, participants could tell upon hearing a soundscape which category it represented. This required Gestalt object perception and generalization of the category principles and shape perception to novel stimuli. They could also determine multiple features of the stimulus, enabling them to differentiate between objects within categories. For an example, see Movie S2, depicting one congenitally blind check details participant reading a three-letter word and another participant recognizing emotional facial expressions. In order to assess the efficiency of training in terms of visual recognition, six of the participants in the training protocol underwent a psychophysical evaluation of their Ketanserin ability to identify different object categories. They were required to categorize 35 visual images (in pseudorandomized order) as belonging to the seven object categories.

Each stimulus was displayed using headphones for four repetitions (totaling 8 s), followed by a verbal response. The average rate of object classification success in the blind was 78.1% (±8.8% SD), significantly better than chance (14%; see Figure 1F, t test p < 0.00005). Letter category recognition did not differ from that of the other object categories (all p > 0.05, corrected for multiple comparisons). In order to minimize sensory-motor artifacts, no recording of performance was conducted during the fMRI scan. Prior to each scan, we verified that the subjects were able to easily recognize learned stimuli from the tested categories (see more detail in Supplemental Experimental Procedures). The main study included six experimental conditions presented in a block design paradigm. Each condition included ten novel soundscapes representing unfamiliar images from the trained object categories: letters, faces, houses, body shapes, everyday objects, and textures. Each condition was repeated five times, in a pseudorandom order. In each epoch, three different stimuli of the same category were displayed, each for 4 s (two repetitions of a 2 s stimulus). For instance, in each letter epoch, the subject was presented with a novel meaningless three-consonant letter string.

, 2009) ( Figure 2A) The locomotion rate and motile fraction

, 2009) ( Figure 2A). The locomotion rate and motile fraction

of pdf-1; npr-1 and pdfr-1; npr-1 double mutants during the L4/A lethargus were significantly lower than in npr-1 single mutants ( Figures 3D–3F). Inactivating PDF-1 and PDFR-1 had a much less dramatic effect on adult locomotion in pdf-1; npr-1 and pdfr-1; npr-1 double mutants ( Figure S3A). Thus, increased signaling by PDF-1 and PDFR-1 in npr-1 mutants was required for the increased motility during lethargus. The npr-1 foraging Vemurafenib defect was unaltered in pdf-1; npr-1 and pdfr-1; npr-1 double mutants ( Figure S3B), indicating that PDF was not required for other npr-1 phenotypes. Inactivating PDF-2 had little effect on the locomotion of npr-1 mutants during lethargus ( Figures S3C and S3D), indicating that PDF-1 is the major form of PDF involved in lethargus behavior. Collectively, these results suggest that PDF-1 functions as an arousal peptide in npr-1 mutants, preventing locomotion quiescence during lethargus. PDF-1’s effects on arousal were specific, because knockdown of RNA Synthesis inhibitor 14 other neuropeptides expressed in the RMG circuit had no effect on the npr-1 lethargus defect ( Figure S3E). If PDF-1 functions as an arousal peptide, PDF-1 expression or secretion should be inhibited during lethargus, when animals are quiescent. We did several experiments to test this idea.

The abundance of pdf-1 and pdfr-1 mRNAs (assayed by quantitative PCR) was unaltered during the L4/A lethargus, whereas expression of mlt-10 (a gene required for molting) was significantly increased, as expected ( Figure S4A) ( Frand et al., 2005). To assay PDF-1 secretion, we expressed yellow-fluorescent-protein (YFP)-tagged proPDF-1 with the pdf-1 promoter ( Figures 4A and 4B). During DCV maturation, the YFP linked to proPDF-1

is cleaved by proprotein convertases and is subsequently secreted by DCV exocytosis. To assess the level of PDF-1 secretion, we analyzed PDF-1::YFP fluorescence in the endolysosomal compartment of coelomocytes, which are specialized scavenger cells that internalize proteins secreted into the body cavity ( Fares and Greenwald, 2001; Sieburth et al., 2007). Digestive enzyme The PDF-1::YFP secretion reporter produced high levels of coelomocyte fluorescence in both L4 larvae and adults, whereas dramatically lower coelomocyte fluorescence was observed during the L4/A lethargus ( Figures 4A and 4B). Coelomocyte fluorescence produced by a second secretion probe (mCherry-tagged RIG-3 expressed in cholinergic neurons) ( Babu et al., 2011) was unaltered during lethargus ( Figure S4B), indicating that secretion and coelomocyte function were not globally inhibited during lethargus. If decreased PDF-1 secretion during lethargus is a cellular mechanism for inducing quiescence, we would expect that mutants retaining or lacking locomotion quiescence would exhibit reciprocal patterns of PDF-1 secretion during lethargus. We did several experiments to test this idea.

The increase in synchrony of both pyramidal cells and interneuron

The increase in synchrony of both pyramidal cells and interneurons from non-REMn to non-REMn+1 was selleck products significantly correlated with the theta and gamma (around

40 Hz) power of the interleaving REM episode but not the power of other frequencies (Figures 4C and 4D). To examine how the rate change of individual neurons across sleep was related to their network pattern-related activity during REM sleep, we introduced the method of spike-weighted spectra (SpWS) by relating the instantaneous firing rates of single cells to the power distribution of the simultaneously detected LFP. LFP spectra and firing rates of individual pyramidal cells were computed in 1 s bins with 0.5 s overlap during REM (Figure S4 and Supplemental Experimental Procedures). For normalization purposes, the LFP spectrograms were Z scored independently for each frequency band and the LFP power spectrum was multiplied bin-by-bin by the neuron’s within-bin firing rate and divided by its overall REM rate (see Figure S4). Since power in each frequency of SpWS is first Z scored, stochastic firing results in power nearing zero, while positive values for a given

SpWS frequency band reflect a cell’s selective firing preference in that band. To quantify the relationship between the neuron’s frequency preference during REM sleep and its firing pattern change across sleep, we normalized the correlation between the neuron’s SpWS in REM selleck inhibitor and its rate change between the first and last non-REM episodes of sleep by the neuron’s REM mean firing rate (see Supplemental Experimental Procedures for the “partialization” procedure). These partial correlations were computed separately for changes occurring across sleep in either within-ripple or between-ripple firing rates (n = 22 sleep sessions). Pyramidal cells with firing rates less than 0.4 Hz during REM (n = 281 of 618 cells) were excluded from the SpWS analysis. The SpWS analyses Resveratrol ( Figure 4E; see also Figure S4) demonstrated that within the

same population of simultaneously recorded pyramidal cells, the across-sleep decrease of between-ripple firing rate was correlated with the pyramidal neurons’ preference to discharge selectively during high-power theta (∼5–10 Hz) and gamma epochs during REM. Similarly, a neuron’s theta and gamma power preference reliably predicted its across-sleep firing rate increase within ripples ( Figure 4E). We found that firing rate changes during sleep display a sawtooth pattern, so that the modest increase in discharge activity within non-REM episodes are overcome by the larger rate deceleration within the intervening REM episodes, resulting in an overall rate decrease during the course of sleep. Theta power of REM sleep is coupled with an increase in synchrony and decrease in rate variability of pyramidal cells during the brief ripple events across sleep.

The suppression effect we measured was statistically significant

The suppression effect we measured was statistically significant only for visual neurons (average response −150–0 ms and 250–400 ms relative to cue onset, Wilcoxon sign-rank test; visual, p < 0.001; visuomovement, p = 0.09; movement, p = 0.39). The differential modulation of responses with attention for the three classes of FEF neurons raised the possibility that the effect of attention on firing rates depended not so much on the cell class, but on the relative size of visual and saccade-related responses for a given cell. Indeed, FEF cells display a continuum

of visual and motor responses (Bruce and Goldberg, find more 1985 and Thompson et al., 2005). We therefore quantified this continuum using a visuomovement index (VMI), and we examined the correlation between the VMI and the attentional effect in firing rate. The VMI could take values between −1 and 1 with positive selleck screening library values indicating stronger visual responses and negative values corresponding to stronger saccade-related responses. The attentional effect was calculated as an attentional index (AI) and could also take values

between −1 and 1, with positive values indicating an increase in activity when attention was directed inside the RF/MF and negative values indicating a stronger response when attention was directed outside the RF/MF. We calculated the correlation between the AI for the time period 100–400 ms after the cue onset and the VMI for all recorded neurons. The correlation between the two variables was statistically significant (r =

0.30, p < 0.001; Figure S2A). A similar MYO10 significant correlation was found between the VMI and the AI calculated in a window 400 ms before the color change in the RF (Figure S2B; r = 0.21, p < 0.001). These results indicate that the stronger the visual response of the cell relative to the saccade-related response the larger the increase in firing rate is when attention is directed inside the RF. Thus, cells with predominantly visual responses are more involved in the selection of the target and in the maintenance of attention to a spatial location. In addition to attentional effects on firing rates, we and others have shown that neuronal synchronization is enhanced with attention both within areas which have been implicated in visual attention as well as across distant areas of the attentional network in both humans and monkeys (Bichot et al., 2005, Buschman and Miller, 2007, Fries et al., 2001, Gregoriou et al., 2009a, Lakatos et al., 2008, Saalmann et al., 2007 and Siegel et al., 2008). Recently, we showed that oscillatory coupling between FEF and V4 in the gamma frequency range is enhanced with attention and that this coupling is initiated by the FEF (Gregoriou et al., 2009a).

Basal transmission was monitored every 15 s with PP and SC stimul

Basal transmission was monitored every 15 s with PP and SC stimuli spaced 2 s apart. A 470 nm LED (CoolLED) or a solid-state single-photon laser (OEM lasers) was routed through the 60× objective and two pinholes to optically stimulate ChR2 or uncage RuBiGABA. See Supplemental Experimental Procedures for details. Animals were perfused with 1× PBS followed by 4% paraformaldehyde (PFA) in 1× PBS.

We cut 50 μm sections with a vibratome following an overnight postfixation (4% PFA) of the brain. Slices were permeabilized, stained with antibodies, mounted on slides, and imaged on an inverted laser-scanning confocal microscope (Zeiss LSM 700). ChR2 expression and cell fills in live slices were imaged with a multiphoton microscope (Ultima, Prairie Technologies). See Supplemental Experimental Procedures for all details. Axograph X and ImageJ were used for electrophysiology data analysis and image processing, respectively. Kaleidagraph

(Synergy) and Prism (Graphpad) were used for plotting data and statistical analysis. Time course plots were generated using a box-car average of every four responses (1 min). For calculating the fold change in ITDP, PSP amplitudes were normalized to the GDC-0068 mean PSP amplitude during the first 5 min of baseline recording prior to ITDP induction for each individual experiment and then averaged to generate the mean. For comparing the effect of ITDP induction on response amplitudes, the data were derived from time points corresponding to 5 min before (pre) and 30–40 min after (post) induction. All statistical errors are standard errors of the population mean or boxcar mean (SEM); all p values (significance level set at p < 0.05) for t tests are two tailed and all ANOVAs were corrected for multiple comparisons using post hoc tests why as indicated. Figures were generated with Adobe Illustrator. Neurolucida (MicroBrightField)-based reconstructions of biocytin-filled CA1 PNs were used to generate a compartmental model in the NEURON simulation environment (Hines and Carnevale, 1997) matching the neuron’s digitized anatomy and its

measured τslow (recorded in synaptic and HCN blockers). See Supplemental Information for further details. J.B. and S.A.S. designed the study; J.B. performed the experiments and analyzed the data; K.V.S. performed the computational modeling; S.K.C. and J.B. performed the immunohistochemistry and imaging; H.T. and Z.J.H. generated the CCK-Cre driver and intersectional CCK IN specific transgenic mice; and J.B. and S.A.S. wrote the paper with help from the other authors. We thank K. Deisseroth, G. Fishell, and S. Sternson for generously providing reagents; V. Chevaleyre, J. Dudman, M. Larkum, M. Lovett-Barron, R. Piskorowski, H. Takahashi, P. Trifilieff, and T. Younts for technical advice; and K. Franks, F. Hitti, V. Johnstone, J. Kupferman, A. Losonczy, Z. Rosen, M. Russo, and B. Santoro for helpful comments on previous versions of the manuscript.

Other synaptic vesicle proteins, SNB-1/synaptobrevin and SNG-1/sy

Other synaptic vesicle proteins, SNB-1/synaptobrevin and SNG-1/synaptogyrin, also showed a similar phenotype to RAB-3 in cyy-1 cdk-5 double mutants ( Figure S2). These results suggest that CYY-1 and CDK-5 are not required for patterning of synapses in the L1 stage, but they are essential for DD synaptic remodeling. Because other synaptic vesicle proteins, SNB-1/synaptobrevin and SNG-1/synaptogyrin, displayed a similar phenotype to RAB-3 in the double

mutants (Figure S2) and GFP::RAB-3 reliably visualized DD synaptic remodeling process (Figure S1), we used GFP::RAB-3 (wyIs202) for further experiments to label synaptic vesicles during the DD remodeling process. To ensure that the GFP::RAB-3 phenotype in the mutants indeed represents synaptic remodeling defects, we colabeled synaptic vesicles Doxorubicin and active zones with RAB-3 and SYD-2/Liprin-α, respectively. In the cyy-1 cdk-5 double mutants, SYD-2 exhibited a similar phenotype as synaptic vesicle proteins—the majority of SYD-2 signals were found in the ventral process, which colocalize with

the RAB-3 puncta in the ventral process at the L4 stage ( Figure 1B). Furthermore, we performed serial electron microscopy (EM) reconstruction to definitively examine the synaptic structural defects in the double mutants. We reconstructed dorsal nerve cord and analyzed the Selleckchem VE822 appearance of synaptic vesicles and active zones of DD neurons in wild-type and cyy-1 cdk-5 double-mutant worms. Consistent with the results from the

fluorophore-tagged synaptic markers ( Figures 1A, and1B; Figure S2), the number of synaptic vesicles and active zones in the DD dorsal process is significantly reduced in the adult double-mutant worms ( Figures 1C–1E). Taken together, our findings suggest that CYY-1 and CDK-5 combined are necessary for eliminating presynapses from the ventral process and forming new synapses in the dorsal process of DD neurons. Because both CYY-1 and CDK-5 are necessary for the completion of synaptic remodeling, we next tested if these two proteins are sufficient to initiate DD synaptic remodeling. In adult cyy-1 cdk-5 double mutants, the majority of the GFP::RAB-3 remains in the ventral processes, suggesting that the DD synaptic remodeling is almost completely blocked in Montelukast Sodium the absence of these two molecules ( Figure S3B, B1; quantified in Figure S3C). To ask whether CYY-1 and CDK-5 play instructive roles in the remodeling process, we tested if expression of CYY-1 and CDK-5 at the mid-L3 stage, a time point long after the normal remodeling process has been completed in wild-type worms, can rescue the remodeling defect in cyy-1 cdk-5 double-mutant animals ( Figure S3A). To induce CYY-1 and CDK-5 expression, we generated transgenic worms expressing CYY-1 and CDK-5 under the control of the heat-shocked promoter (Phs).

, 2009) Tissue was viewed using a 60× (1 0 NA, Olympus) water-im

, 2009). Tissue was viewed using a 60× (1.0 NA, Olympus) water-immersion objective through a 1–4× magnifier onto a digital Rolera XR (Qimaging) or analog Galunisertib solubility dmso OLY-150 (Olympus) camera on a BX51 microscope (Olympus). Tissue was dissected and perfused at rates of 0.35–0.5 ml/min with external solution containing (in mM): 140 NaCl, 2 KCl,

2 CaCl2, 2 MgCl2, 10 HEPES, pH = 7.4, at 300–310 mOsm. In addition, an apical perfusion protected the hair bundles from internal solution with rates of 0.07–0.1 ml/min using pipettes with tip sizes 40–200 μm. In all preparations, the tectorial membrane was peeled off the tissue. Experiments occurred on multiple recording stations with comparable capabilities but often-different specific equipment, attesting to the robustness of the data. Whole-cell patch-clamp was achieved on IHCs or first or second row OHCs from middle to apical cochlea turns using an Axon 200A or 200B amplifier (Molecular Devices)

with thick-walled borosilicate Palbociclib chemical structure patch pipettes (<4 MΩ) filled with an intracellular solution containing (in mM): 125 CsCl, 3.5 MgCl2, 5 ATP, 5 creatine phosphate, 10 HEPES, 1 Cesium BAPTA, 3 ascorbate, pH = 7.2, and 280–290 mOsm. For the 10 mM BAPTA solution, removal of an equivalent osmolality of CsCl offset the increased BAPTA concentration. For the EGTA internal (in mM), 1 EGTA replaced cesium BAPTA and ascorbate increased to 4 mM. For Ca2+ imaging, 1 mM Fluo-4 or Fluo-4FF (Invitrogen) and 0.05 mM Alexa 594 hydrazide (Invitrogen) were added to the EGTA internal. 1.4 Ca2+ internal contained (in mM): 121 CsCl, 3.5 MgCl2, 3.5 CaCl2, 3.5 ATP, 5 creatine phosphate, 10 HEPES, 2 ascorbate, pH = 7.2, at 280–290 mOsm. For the 1.4 mM Ca2+ internal, free Ca2+ concentration was measured using

a MI-600 Ca2+ electrode (Microelectrodes) calibrated using Ca2+ buffer standards (CALBUF-2, WPI) and found to be 1.4 mM free Ca2+. Experiments were performed at 18–22°C. Whole cell currents were filtered at 50–100 kHz and sampled at 1 MHz using USB-6356 (National Instruments) through or Personal DAQ3000 (Iotech) controlled by jClamp (SciSoft). Traces were filtered offline at 30 kHz using Origin 8.6 (OriginLab). Voltages were corrected offline for liquid junction potentials. For inclusion, initial MET currents greater than 600 pA in 2 mM external Ca2+ and Mg2+ were required. For a sample of 80 cells recorded, the clamp speed was 28 ± 6 μs with series resistance compensation, whole cell capacitance was 11 ± 1 pF, and leak currents at −84 mV holding potential were −65 ± 40 pA. Borosilicate pipettes were fire polished to shapes that matched the hair bundle structures for IHCs and OHCs.

Interestingly, large patch cells showed the strongest theta-phase

Interestingly, large patch cells showed the strongest theta-phase locking (Figures 7F and 7G; Rayleigh average vector length = 0.35; p < 0.003) and in contrast to superficial neurons,

showed maximal firing on the descending phase of the theta cycle, near the trough (difference in average vector angle: layer 2 versus large patch = 170°, p = 0.006; layer 3 versus large patch = 167°, p = 0.004) (Figures 7F and 7G; Figure S7A). Autocorrelation analysis Quisinostat chemical structure indicated that theta modulation of activity was strong in layer 2 and weak in layer 3 cells (Figures S7A and S7B), consistent with differences of oscillatory discharge behavior described in vitro (Alonso and Klink, 1993 and van der Linden and Lopes da Silva, 1998). In line with the strong theta modulation of the field potentials, the largest fraction of theta-modulated cells was found in large patches (Figure S7B). In order to explore the axonal connectivity scheme across medial entorhinal cortex, we visualized the large-scale architecture of axons traveling in layer 1 in “mass” myelin stains of tangential sections (Figure 8). Large patches (dark brown) were identified

by cell somata clustering and by the clear myelination pattern that surrounded these structures (Figure S8). Figure S8, which shows serial sections through the dorsomedial part of medial entorhinal cortex, also illustrates that large patches seemed to form a continuum with the parasubiculum ( Shipley, 1974, Shipley, 1975, Köhler, 1984, Caballero-Bleda Androgen Receptor Antagonist and Witter, 1993 and Witter and Amaral, 2004). Myelin stainings revealed a striking regularity of layer 1 axonal fibers, organized in axonal bundles running along the dorsomedial to ventrolateral axis ( Figure 8A).

these We traced putative centrifugal axons originating above the territory of small layer 2 patches (blue, Figures 8A and 8C), and we drew a large number of axons that surrounded a single large patch (green, Figures 8A and 8B). As in most identified cells from large patches ( Figure 5 and Figure 6), putative circumcurrent axons to dorsolateral neighboring patches were longer and more prominent than axons extending toward the ventromedial ones (green, Figures 8A and 8B). A schematic overview of the position of medial entorhinal patches in the rat brain is shown in Figure 8D. Overall, the circumcurrent axons surrounding large dorsal patches were much more numerous than the circumcurrent axons surrounding medioventral patches. Mass myelin stains appear to be consistent with our single-cell reconstruction data and suggest a global organization of three long-range axon systems in medial entorhinal cortex: (1) centrifugal and (2) centripetal axons, which reciprocally connect large and small patches; and (3) circumcurrent axons, which connect large patches along the mediolateral axis.

L-type voltage-gated calcium channels (L-VGCCs) have previously b

L-type voltage-gated calcium channels (L-VGCCs) have previously been implicated in eCB release (Adermark and Lovinger, 2007, Calabresi et al., 1994, Choi and Lovinger, 1997 and Kreitzer and Malenka, 2005), yet we found that the L-VGCC blocker nitrendipine did not block LFS-LTD (64% ± 6%; Figure 2C). Another L-VGCC blocker, nifedipine, also did not block LFS-LTD (64% ± 10%, n = 6, data not shown). In fact, elevations

in intracellular calcium do not appear to be strictly required for LFS-LTD, since loading MSNs with the calcium-chelator BAPTA did not block LFS-LTD (75% ± 10%; Figure 2C). From these experiments, we conclude that the most likely scenario for LFS-LTD induction is that activation of Gq-coupled mGluRs leads to activation of PLCβ, stimulating the production of DAG, which is then converted to 2-AG by DAGL (Figure 2D). Our initial experiments (Figure 1) showed that the

pathways underlying HFS-LTD and LFS-LTD learn more CT99021 cost diverge after just one step in their induction pathways (activation of Gq by group I mGluRs). Because HFS-LTD is PLCβ-independent (Figure 1C), we predicted it would be DAGL-independent as well. Indeed, as observed previously (Ade and Lovinger, 2007 and Lerner et al., 2010), the DAGL inhibitor THL did not block HFS-LTD (61% ± 10%; Figure 3A). We also tested whether HFS-LTD differed from LFS-LTD in its requirements for calcium. By adding thapsigargin to our intracellular solution to deplete internal calcium stores, we found that, unlike LFS-LTD, HFS-LTD requires these stores (117% ± 17%; p < 0.05 compared to control; Figure 3B). Calcium from internal stores can be released into the cytoplasm via either IP3 receptors or ryanodine from receptors

(RyRs). Since HFS-LTD does not require PLCβ, which produces IP3, we reasoned that the requirement for internal calcium stores in HFS-LTD was more likely to be dependent on RyRs than on IP3 receptors. Indeed, when RyRs were inhibited by including ryanodine in the intracellular solution, HFS-LTD was blocked (108% ± 8%; p < 0.05 compared to control; Figure 3B). An IP3 receptor blocker, 2-APB, did not block HFS-LTD when included in the intracellular solution (63% ± 10%; Figure S2A available online). RyRs are activated by calcium and, once activated, cause the release of more calcium into the cytoplasm. This process of calcium-induced calcium release (CICR) serves to amplify calcium signals initiated by other sources of calcium influx. What is the CICR-initiating source of calcium in HFS-LTD? We consider L-VGCCs to be a likely source, because they are functionally coupled to RyRs (Chavis et al., 1996) and because L-VGCCs have previously been shown to be involved in striatal LTD (Calabresi et al., 1994 and Choi and Lovinger, 1997). In agreement with this hypothesis, the L-VGCC antagonist nitrendipine blocked HFS-LTD (92% ± 4%; p < 0.05 compared to control; Figure 3C).