It is also possible that neurons

derived from VZ versus OSVZ radial glia are indistinguishable, with a neuron’s timing of origin and migration to the cortical plate being the main determinant of what subtype it will become. Whether oRG cells give rise to distinct neuron subtypes or simply represent a cellular means to amplify neurogenesis is a pivotal question with obvious ramifications for producing specific subtypes of cortical neurons in vitro from human pluripotent stem cells. If we assume that having the correct type of progenitor cell is important for producing neurons of a desired subtype, then this introduces a new challenge for producing excitatory neurons of upper cortical layers from hESCs. As mentioned earlier, upper-layer neurons

may be particularly involved in human diseases of cognitive function including dementia, retardation, schizophrenia, Osimertinib chemical structure and autism. The ability to generate cells with the correct upper-layer identity may be required in order to study the pathophysiology of these disorders. The oRG cells from which most human upper-layer neurons originate represent a new target cell type for hESC derivation. Researchers commonly selleck chemicals use the presence of neural rosettes in differentiating cell cultures as an indication that neural stem cells with properties of neuroepithelial/radial glial cells are abundant and actively producing neurons. In ventricular RG cells, the polarity protein Par3 and the engagement of N-cadherin at apical junctions are required to promote

Notch and β-catenin signaling, respectively, and thus maintain progenitor status (Bultje et al., 2009 and Zhang et al., 2010). Thus, apical-basal polarity is an essential property by which neuroepithelial cells comprise a self-supportive niche that does not require distinct supporting cells. The self-organization of dissociated, differentiating ESCs into a polarized neuroepithelium attests Ergoloid to the intrinsic value of cell-cell interactions for neural stem cell maintenance (Eiraku et al., 2008). Lacking cell-cell interactions of this type, what are the mechanisms by which oRG cells persist in the OSVZ? We presume that there are compensatory mechanisms in the OSVZ that activate some of the same intracellular signaling pathways through alternate means to prevent oRG cell differentiation. The basal fibers of oRG cells are probably critical for receiving signals from the environment that restrain differentiation, induce proliferation, and promote survival. We demonstrated that Notch signaling is required for oRG cell maintenance (Hansen et al., 2010), although why apical polarity is not required for this pathway in oRG cells as it is in ventricular RG is unknown. Integrin binding is also required to maintain Pax6+ OSVZ progenitor cells (Fietz et al., 2010).

(2011). For synaptic blockade, a 0.2 mM CaCl2 and 400 μM CdCl2 ACSF was used. In other cases, a 0 mM CaCl2/3 mM EGTA solution was used, as indicated. Hypothalamic slices were transferred MDV3100 concentration to a recording chamber and superfused with ACSF (30°C–32°C) at a flow rate of ∼3.0 ml min−1. Conventional whole-cell patch-clamp recordings, using a K+-gluconate-based internal solution, were obtained as previously described by Sonner et al.

(2011). When noted, neurons were intracellularly labeled with Alexa Fluor 555 (100 μM) or biocytin (1%). Recordings were obtained from fluorescently labeled PVN-RVLM neurons and from EGFP-VP neurons. For bath-applied drugs, mean firing activity and membrane potential values were calculated from a 2 min period before drug application and in a 2 min period around the peak effect. For briefer applications

(picospritzer) and uncaging, values were calculated from a 1 min period before and 10–20 s period around the peak effect, using Clampfit (Axon Instruments) or miniAnalysis (Synaptosoft) software. Neurons were loaded through the patch pipette with Fluo-5F pentapotassium salt (100 μM; Molecular Probes), as previously described (Filosa et al., 2006 and Sonner et al., 2011). For astrocyte Ca2+ measurements, slices were incubated in ACSF containing Rhod2-AM and pluronic acid (2.5 μg/ml). Imaging was conducted using the Andor Technology Revolution system (iXON EMCCD camera with the Yokogawa CSU 10, confocal scanning unit). Fluorescence images were acquired at 488 nm and emitted light at >495 nm (Fluo5) or 561 nm and emitted light >607 nm (Rhod2). Images were acquired at a rate of 4 Hz. The Volasertib fractional fluorescence (F/F0) was determined by dividing the fluorescence intensity (F) within a region of interest (≈4.8 × 4.8 μm) by a baseline fluorescence value (F0) determined from Adenosine 30 images before

photolysis of caged NMDA. Data were analyzed using Andor IQ software (Andor Technology). Slices were perfused with MNI-caged NMDA (50 μM). A UV laser excitation (405 nm) was directed to a region of interest drawn on the image of identified PVN-RVLM or EGFP-VP somata. Based on the pixel dwell time (200–800 μs) and the ROI area scanned (∼40 μm2), the uncaging protocol lasted 180–720 ms. For these experiments, an ACSF containing 20 μM Mg2+ was used to facilitate detection of evoked NMDA responses. Rats were anesthetized with urethane (0.75 g/kg i.p.) and α-chloralose (70 mg/kg i.p.) and placed in a stereotaxic apparatus. The coordinates for PVN microinjections were 1.5 mm posterior to the bregma, 0.4 mm lateral to the midline, and 7.8 mm ventral to the dura. To record RSNA, the left kidney was exposed through a retroperitoneal flank incision. A branch of the renal nerve was isolated and placed on a pair of thin bipolar platinum electrodes. The electrical signal was amplified (10,000 times) with a Grass amplifier (P55) with a high- and low-frequency cutoff of 1,000 and 100 Hz, respectively.

With rivalry, the size of this modulatory field can be directly controlled by changing the size of a stimulus in one eye relative to the other. With standard models of binocular normalization, introducing a stimulus in a competing eye should contribute to the this website pooled inhibitory component of normalization (Ding and Sperling, 2006; Moradi and Heeger, 2009), which predicts shifts in contrast gain (strongest effects at

mid-contrasts), but not in response gain (strongest effects at high contrasts), regardless of size (Supplemental Information). However, if rivalry also includes a process that behaves like attention, the shape of contrast response functions for attenuated signals should differ depending on the size of the dominant stimulus in the other eye—a manipulation that would alter the size of the modulatory field. Specifically, when the dominant stimulus is substantially larger than the

stimulus in the other eye, thereby evoking a large modulatory field, the normalization framework of attention predicts a reduction in contrast gain for the probe stimulus (Figure 1A). However, when Navitoclax cell line the dominant stimulus evokes a small modulatory field, the contrast response functions should transition toward a reduction in the response gain (Figure 1B). To explore whether normalization modulates visual competition, we examined how psychometric functions

change for an attenuated stimulus under rivalry, and whether those changes depend on the size of the putative modulatory field. We measured observers’ ability to discriminate fine changes in the orientation of a probe stimulus (4° clockwise or counterclockwise) that was either presented monocularly, or was suppressed under binocular rivalry (Figure 2). To control the size of the modulatory field in the rivalry conditions, we manipulated the size of the dominant competing stimulus such that in some trials, it was either the same size as the probe (small: 1.5°), somewhat larger than the probe (medium: 2.5°), or substantially larger (large: 8°). The rms contrast of the probe stimuli ranged from 0.8%–23%, Rutecarpine allowing us to measure the entire psychometric function, a behavioral measure that scales proportionally to the signal-to-noise ratio of the underlying contrast response function (Herrmann et al., 2010; Pestilli et al., 2009). Specifically, changes in the neural contrast response function under this framework directly impacts an observer’s ability to discriminate orientation changes in the probe, which would, in turn, be reflected in corresponding changes to the behavioral psychometric functions. Rivalry had a substantial impact on psychometric functions (Figure 3A).

To avoid bias, control and dogs in the 1× groups were handled similarly to the other two groups but were not dosed during the administration of the second fraction.

Food was offered to the dogs prior to and after each treatment. Dogs were observed at least hourly for 3 h after the first dose fraction was administered and hourly for 4 h after the second dose fraction was administered. Feces from all dogs were examined for the presence of whole undigested chews on the day after treatment. Personnel involved with recording of the in-life observations were blinded as to treatment. The pathologist performing the necropsy was unaware of dog’s group but the origin of the dogs was unblinded for the histologic evaluation. Physical examinations were performed weekly during the pre-test period and biweekly to Day 125, and included the evaluation of the general appearance, body weight, respiration rate, heart rate, selleck products and body temperature. Daily feed intake was monitored and recorded for analysis beginning on Day −1. In addition, blood hematology, plasma chemistry, and coagulation profiles were determined

twice during pre-test in conjunction with physical examinations on Days 14, 27, 42, 55, 70, 83, 97, 111 and 125. Standard laboratory techniques were used for collection and analysis of the samples. The Merial laboratory conducting the analysis provided reference ranges for the plasma chemistry, coagulation, and hematology profiles. The hematology profile included: red blood cell count (RBC), white blood cell count, white blood cell differentials (absolute count), platelet count, hemoglobin,

hematocrit, Selleckchem Epigenetic inhibitor Bay 11-7085 mean corpuscular volume, mean corpuscular hemoglobin, mean cell hemoglobin concentration, and RBC morphology. The plasma chemistry profile included: alanine aminotransferase, albumin, alkaline phosphatase, amylase, aspartate aminotransferase, calcium, chloride, cholesterol, creatinine, globulin, glucose, phosphorus, potassium, sodium, total bilirubin, total protein, triglycerides, and urea nitrogen. The coagulation profile included activated partial thromboplastin time, prothrombin time, and thrombin clotting time. Urine samples were obtained once during pretest and on Days 27 and 126 using either a metabolism pan or by cystocentesis (Day 126). Urinalysis included determination of urobilinogen, nitrite, glucose, bilirubin, ketones, blood, leukocytes, specific gravity, pH, and protein by use of MULTISTIX® SG Reagent Strips (Bayer Corporation). In addition to the reagent strips, a refractometer was used to determine urine specific gravity. Urine sediment was evaluated microscopically for at least the following: crystals, casts, red blood cells, white blood cells, and epithelial cells. Standard laboratory techniques were used. Reference ranges for the specific gravity was from Stockham and Scott (2002). Dogs were humanely euthanized on Day 126 and a complete post-mortem examination was conducted.

, 2007). Most recently, these insights have been extended to studies Selleckchem FG 4592 of P2X4 receptors within microglia, and it has been shown that both lysosomal secretion and plasma membrane lateral mobility of P2X4 receptors are increased by activation of the microglia (Toulme et al., 2010; Toulme and Khakh, 2012). A second form of activity-dependent regulation has been demonstrated for P2X2 and P2X7 receptors and is mediated by the Ca2+ sensor proteins VILIP1 and calmodulin, respectively (Chaumont et al., 2008; Richler et al., 2011; Roger et al., 2008). In both cases, Ca2+ fluxes mediated by these P2X receptors result in the recruitment of the cognate Ca2+ sensor to the C-terminal

domain of the channel to regulate functional responses. The consequences are subtle for P2X2 receptors, but result in profound facilitation

of P2X7 receptor responses (Roger et al., 2008). The interaction with VILIP1, which occurs during endogenous ATP release, requires slow conformational changes resulting in exposure of a VILIP1 binding site in the cytosolic C-terminal tail of P2X2 receptors (Chaumont et al., 2008; Chaumont and Khakh, 2008). Single-molecule experiments reveal that the interaction between P2X2 receptors and VILIP1 regulates plasma membrane lateral mobility of P2X receptors in neuronal dendrites (Richler et al., 2011), perhaps serving to affect recovery from desensitization by controlling the supply of receptors. Determining the full repertoire of proteins that interact with P2X2, P2X4 and P2X7 will help illuminate how these receptors Mephenoxalone are tuned selleck chemicals to perform their tasks in vivo. Single-molecule imaging experiments now provide accurate and consistent values for P2X2, P2X4, and P2X7 receptor diffusion coefficients in the plasma membrane (0.027, 0.023, and 0.021 μm2/s,

respectively) (Arizono et al., 2012; Richler et al., 2011; Toulme and Khakh, 2012). In the case of P2X2 and P2X4 receptors, activation by ATP causes the receptors to diffuse twice as fast in a cell- and subunit-specific manner (Richler et al., 2011; Toulme and Khakh, 2012). Accurate P2X receptor diffusion coefficients will be invaluable in modeling receptor movement and plausible roles for lateral mobility in recovery from desensitization during physiological activation such as would occur during point source-like ATP release in vivo. P2X receptors are often expressed at low levels, generally in specific compartments such as the edges of spines and within nerve terminals (Lê et al., 1998; Rubio and Soto, 2001; Vulchanova et al., 1996) and are activated by quite high amounts of ATP. It seems that sufficient ATP to activate extrasynaptic P2X2 receptors is only released during bursts of action potentials (Richler et al., 2008), suggesting that P2X receptors underlie neuromodulatory responses. Also, we are aware of no example in the brain or spinal cord where endogenous ATP release stimulates postsynaptic P2X receptors to trigger action potential firing (i.e., is a primary fast synaptic transmitter).

In vertebrates, H2S drastically increases under hypoxic conditions to levels that are inversely correlated

with tissue O2 levels (Olson, 2011, Olson et al., 2006 and Peng et al., 2010). H2S is endogenously produced by multiple types of enzymes in animals and is constantly oxidized, so its increase might be directly Bafilomycin A1 purchase regulated by local O2 levels to mediate effects of hypoxia (Chen et al., 2004, Kimura, 2010, Olson, 2011, Peng et al., 2010 and Singh et al., 2009). In both C. elegans and mammalian cells, H2S has been shown to promote HIF-1 activity and upregulate HIF-1 target genes ( Budde and Roth, 2010 and Liu et al., 2010). However, the mechanism by which H2S elicits its effects on HIF-1 has been unknown. Our findings demonstrate an essential role of CYSL-1 in mediating H2S upregulation of HIF-1 target genes through CYSL-1 interaction with http://www.selleckchem.com/products/INCB18424.html the EGL-9 C terminus. A recent study found that cysl-1 mutants are sensitive to H2S and hypothesized that CYSL-1 might act in a pathway downstream of HIF-1 to enzymatically assimilate H2S ( Budde and Roth, 2011). Unexpectedly, our studies show that CYSL-1 acts upstream of HIF-1 by directly inhibiting EGL-9 in a manner that is modulated by H2S accumulation.

Interestingly, both H2S and RHY-1 appear to regulate HIF-1 activity in a VHL-1-independent manner ( Budde and Roth, 2010 and Shen et al., 2006), consistent with the notion that CYSL-1 inhibits EGL-9 and mediates H2S activation of HIF-1 independently of EGL-9 hydroxylase activity. Bisulfide is known to bind to an allosteric regulatory site of Salmonella OASS proteins, which are highly similar to CYSL-1 in C. elegans, and can stabilize the interaction between OASS and the SAT C terminus ( Salsi et al., 2010). H2S inhibits mitochondrial

cytochrome-C oxidase and can also directly modify target proteins via sulfhydration ( Mustafa et al., 2009). Although CYSL-1 has only weak intrinsic sulfhydrylase activity in vitro, it remains possible that H2S might modify EGL-9 via CYSL-1-modulated sulfhydration to facilitate sequestration of EGL-9 by CYSL-1. The detailed mechanism by which H2S and its in vivo derivatives modulate CYSL-1 and EGL-9 to regulate HIF-1 remains to be investigated. Urease CYSL-1-homologous CBS proteins in mammals are known to be major H2S-biosynthetic enzymes (Chen et al., 2004 and Singh et al., 2009), and we suggest that the pathway we identified is fundamentally similar in nematodes and mammals (Figure 7A). In nematodes, H2S and CYSL-1 regulate HIF-1 through EGL-9. In mammals, H2S also regulates HIF proteins (Li et al., 2011 and Liu et al., 2010), and we propose that CYSL-1-like CBS proteins generate endogenous H2S to modulate HIF. In mammals, HIF activation protects tissues from reperfusion injury (Loor and Schumacker, 2008); we propose that the C.

e., Na+ plus protons). We observed little change in Na+ current (Figure S5), suggesting that most of the current of WT channels in the presence of metal ions is carried by protons (Figure 7A, dashed line). In contrast to WT, R3S channels had currents in Gu+ that were almost 9-fold larger than in Li+ (Figure 7B). Moreover, unlike WT, in the presence of metal ions, R3S current was largest in Li+ (Figure 7B) (e.g., the K+/Li+ ratio was 1.32 ± 0.07 for WT versus 0.24 ± 0.02 for R3S, n = 8 and

n = 11, respectively, p < 0.01, t test). The 100 mM TRIS pH 8 (protons alone) versus 100 mM Na+ pH 8 (i.e., Na+ plus protons) ratio was also close to unity in R3S (Figure S5), suggesting that the R3S mutation increases permeability to Li+ (Figure 7B, bottom, dashed line). To examine D112 we first turned to the D112S see more mutant, but its current was too small (Figure 5). Since the charge conserving D112E mutation did not shift the G-V (Figure 5), the substituted glutamate of this mutant seems

to accommodate the normal interactions of the native aspartate. If the model was correct and pairing between R3 and D112 were important for selectivity, one would expect the D112E mutant to retain normal selectivity. This was indeed the case. The D112E mutant had no appreciable conductance in Gu+ and the order of current amplitudes in the different metal cations closely resembled that of WT (Figure 7D). We therefore turned to the D112S-R3S double mutant. In D112S-R3S, the current of Gu+ was more than 14-fold larger than that of Li+ (Figure 7C). This value is significantly larger than what is seen in R3S alone (Gu+ / Li+ ratio: R3S, 8.69 ± 0.45, Selleck PLX4032 n = 11; D112S-R3S, 14.25 ± 1.77, n = 8; p < 0.01, t test). Strikingly, assessment of the protons alone versus Na+ plus protons ratio indicated that, unlike WT and R3S channels, most of the D112S-R3S current in presence of Na+ is

actually carried by Na+ (Figure S5; the proton/[proton + 100 mM Na+] ratio was 0.22 ± 0.03, n = 5 for D112S-R3S, significantly different from both 0.82 ± 0.06, n = 7 for R3S and 0.80 ± 0.05 for WT, p < 0.01, ANOVA followed by Dunn's method Cediranib (AZD2171) for multiple comparison). To test more precisely the effects on ion selectivity of R3 and D112, we examined the reversal potentials of tail currents under mono- and bi-ionic conditions. In WT channels there was no Gu+ conduction and reversal potentials did not differ between Na+ and Li+ (Erev shift = 0.57 ± 1.20 mV, n = 4, p = 0.67, paired t test), consistent with the analysis above, that indicated that in Na+ and Li+ the current is mainly carried by protons. In R3S the reversal potential shift between Na+ and Li+ was larger and statistically significant (Erev shift = −4.24 ± 1.70 mV, n = 8, p = 0.04, paired t test). In D112S-R3S the reversal potential shift between Na+ and Li+ increased even more (Erev shift = −13.91 ± 2.30 mV, n = 5, p < 0.

In parallel

with technological advances, we need theories to tie together measurements across the spatial and temporal scales and make predictions of the emergent properties of neurons connected in networks. The term emergent property is borrowed from the physics of complex systems, where it refers to phenomena that cannot be directly traced to their individual components, only to how those components interact. Consider the example of weather—the state of the atmosphere. The temperature of the air is not defined at the atomic scale; it is an emergent property of many atmospheric particles. A weather forecast requires a valid theoretical framework: a model. The model incorporates a set of rules worked out by studying interactions among particles; the actual forecast, PI3K inhibitor however, is not predicted

by simulating the position of every molecule. Rather, the forecast is made on the relevant practical scale by means of measurements of the current state of the atmosphere and models formulated with “coarse-grained” variables such as pressure and temperature and parameters such as the physical shapes of landforms. For the most part, this approach works: we can rely on the National Weather Service to predict tomorrow’s rain. While the separation of microscopic and macroscopic scales is less clear in neuroscience than in atmospheric physics, it is nevertheless a useful analogy: using the ability to predict as a surrogate for understanding, understanding higher cortical functions—perception, for example—by quantifying a large number of individual neurons firing across the brain may GSK2118436 ic50 be impractical; instead, it is probably necessary to use intermediary measures and appropriate mathematical models. enough Also, statistical sampling from neurons of known cell type and connectivity would be preferable to merely increasing the numbers of simultaneously captured spikes. This is because our brains, in contrast to those of invertebrates, appear to be built from large populations of neurons performing the same function, collectively and in a probabilistic way. We, humans, can lose neurons from the age of 20

or earlier without a noticeable effect on cognitive performance. For the nematode C. elegans, by contrast, the loss of a single neuron can have catastrophic effects with respect to survival. Thus, intermediary measures reflecting the ensemble activity of neurons of similar types—which can be localized on the cortical sheet—would offer extremely valuable information. Further, a number of different types of measures might be required to provide the critical input to the model. For example, sleep spindles, Up and Down states, and cortical spreading depression could be described by a set of parameters including those related to subthreshold polarization, intracellular concentration of calcium in neurons and glia, blood flow, and energy consumption.

In light of the dysfunctional consequences of the mutant PrP

association with α2δ-1, disrupting their binding might represent a means for therapeutic intervention. The production of Tg mice expressing wild-type, PG14, and D177N/V128 mouse PrPs with an epitope for the monoclonal antibody 3F4 has already been reported by Chiesa et al. (1998) and Dossena et al. (2008). In this study we LY2157299 used Tg mice of the Tg(WT-E1+/+) line, which expresses about four times the endogenous PrP level, referred to throughout the text as Tg(WT); we also used Tg(PG14-A3+/−) and Tg(D177N/V128-A21+/−) mice expressing Tg PrP at approximately one time, referred to as Tg(PG14) and Tg(CJD), respectively. These mice were originally generated on a C57BL/6J X CBA/J hybrid and were then bred with the Zurich I line of Prnp0/0 mice ( Büeler et al., 1992) with a pure C57BL/6J background (European Mouse Mutant Archive, Monterotondo, Rome; EM:01723). C57BL/6J mice were purchased from Charles River Laboratories. All procedures involving animals were conducted according to European Union (EEC Council Directive 86/609, OJ L 358,1;

December 12, 1987) and Italian (D.L. n.116, G.U. suppl. 40, February 18, 1992) laws and policies, and were in accordance with the United States Department of Agriculture Animal Welfare Act and the National Institutes of Health Policy on Humane Care and Use of Laboratory Animals. They were reviewed and approved by the Mario Negri Institute Animal Care and Use Committee that includes ad hoc members for ethical ever issues (18/01-D, see more 18/01-C). Animal facilities meet international standards and are regularly checked by a certified veterinarian who is responsible for health monitoring, animal welfare supervision, experimental protocols, and review of procedures. Animals were anesthetized with 1% isoflurane in a 30%:70% O2:N2O gas mixture and imaged in a horizontal bore 7-Tesla USR preclinical MRI system (BioSpec 70/30; Bruker BioSpin, Germany) with a shielded gradient insert (BGA 12, 400 mT/m; rise time, 110 us). A 7 mm birdcage resonator for RF transmission and a 10 mm diameter single-loop receiver coil were used to receive the signal. T2-weighted anatomical

images of the mouse brain were acquired with the following parameters: TR 2500 ms, TE 50 ms, RARE factor 16, FOV 3 × 1.5 × 1.5 cm, Matrix 256 × 102 × 102, voxel 0.147 × 0.117 × 0.147. The scan time was approximately 25 min. The cerebellar volume was quantified using the ImageJ software (http://rsbweb.nih.gov/ij/). We used an accelerating Rotarod 7650 model (Ugo Basile). Juvenile mice were tested starting from 19 days of age (P19) for 7 consecutive days. On the first day a training session was done during which each mouse was placed on the Rotarod at a constant speed (4 rpm) for a maximum of 60 s. Then they were assessed in three consecutive test sessions with a 10 min intertrial resting period. They were positioned on the rotating bar and allowed to become acquainted with the environment for 30 s.

, 2002, Chevaleyre et al., 2007, Fourcaudot Galunisertib supplier et al., 2008 and Kaeser et al., 2008). Another possibility is that assembly of the autonomous functions of different RIM domains into a single protein ensures the right relative activity of these domains, i.e., a constant ratio of their activities. A third possibility is that this arrangement may be economical in terms of organizing the expression and localization of so many activities mediated by different domains. Crystal structures revealed that the Munc13 C2A domain forms a tight homodimer with nanomolar affinity; this dimer is disrupted by binding of the RIM Zn2+ finger, resulting in Zn2+ finger/C2A domain heterodimers (Dulubova et al.,

2005 and Lu Palbociclib supplier et al., 2006). Our results suggest that Munc13 homodimerized by its C2A domain

is inactive in priming but activated by the RIM Zn2+ finger binding that disrupts the homodimer. The strongest evidence for this conclusion comes from the suppression of the priming phenotype in RIM-deficient synapses by mutant, constitutively monomeric Munc13, but not by wild-type Munc13 (Figure 6 and Figure 7). Note that the constitutively monomeric Munc13 mutants rescued only the priming deficit of RIM-deficient neurons not their Ca2+-triggering phenotype, which manifested in a ∼50% rescue of synaptic strength in the RIM-deficient neurons by the mutant Munc13 (Figure 6E). Furthermore, whereas only mutant, constitutively monomeric Munc13 but not wild-type Munc13 rescued

priming in RIM-deficient neurons, both mutant Munc13 and wild-type Munc13 rescued priming in Munc13-deficient neurons (Figure 8). An alternative hypothesis to the model proposed here is that an as yet unidentified protein binds to the Munc13 C2A domain and inhibits Munc13 function and that this protein is displaced by the RIM Zn2+ finger. However, this hypothesis would require that the putative Munc13-binding protein has nanomolar affinity for Munc13 (since it has be stronger than Munc13 homodimerization) that it is nevertheless displaced from Munc13 by RIM. In addition, the putative Munc13-binding protein would be required to bind to the site of Munc13 homodimerization, effectively suppressing it because the C2A domain would always be either bound to through RIM or to the other protein. Viewed together, these improbable requirements render the alternative hypothesis highly unlikely and nonparsimonious. The autoinhibitory function of the Munc13 C2A domain is surprising since no other C2 domain has been associated with a comparable function. Of four principal synaptic Munc13 isoforms (Munc13-1, ubMunc13-2, bMunc13-2, and Munc13-3), only the first two contain a C2A domain (Brose et al., 1995, Augustin et al., 1999b and Koch et al., 2000), raising the question of how the other two Munc13 isoforms (which are less abundant) are regulated and whether they are possibly controlled by a different RIM-dependent mechanism.