Improving the integrity and reproducibility of research that uses antibodies: a technical, data sharing, behavioral and policy challenge.

Antibodies are one of the most important reagents used in biomedical and fundamental research, used to identify, and quantify proteins, contribute to knowledge of disease mechanisms, and validate drug targets. Yet many antibodies used in research do not recognize their intended target, or recognize additional molecules, compromising the integrity of research findings and leading to waste of resources, lack of reproducibility, failure of research projects, and delays in drug development. Researchers frequently use antibodies without confirming that they perform as intended in their application of interest. Here we argue that the determinants of end-user antibody choice and use are critical, and under-addressed, behavioral drivers of this problem. This interacts with the batch-to-batch variability of these biological reagents, and the paucity of available characterization data for most antibodies, making it more difficult for researchers to choose high quality reagents and perform necessary validation experiments. The open-science company YCharOS works with major antibody manufacturers and knockout cell line producers to characterize antibodies, identifying high-performing renewable antibodies for many targets in neuroscience. This shows the progress that can be made by stakeholders working together. However, their work so far applies to only a tiny fraction of available antibodies. Where characterization data exists, end-users need help to find and use it appropriately. While progress has been made in the context of technical solutions and antibody characterization, we argue that initiatives to make best practice behaviors by researchers more feasible, easy, and rewarding are needed. Global cooperation and coordination between multiple partners and stakeholders will be crucial to address the technical, policy, behavioral, and open data sharing challenges. We offer potential solutions by describing our Only Good Antibodies initiative, a community of researchers and partner organizations working toward the necessary change. We conclude with an open invitation for stakeholders, including researchers, to join our cause.

A hybrid human and machine resource curation pipeline for the Neuroscience Information Framework.

The breadth of information resources available to researchers on the Internet continues to expand, particularly in light of recently implemented data-sharing policies required by funding agencies. However, the nature of dense, multifaceted neuroscience data and the design of contemporary search engine systems makes efficient, reliable and relevant discovery of such information a significant challenge. This challenge is specifically pertinent for online databases, whose dynamic content is 'hidden' from search engines. The Neuroscience Information Framework (NIF; http://www.neuinfo.org) was funded by the NIH Blueprint for Neuroscience Research to address the problem of finding and utilizing neuroscience-relevant resources such as software tools, data sets, experimental animals and antibodies across the Internet. From the outset, NIF sought to provide an accounting of available resources, whereas developing technical solutions to finding, accessing and utilizing them. The curators therefore, are tasked with identifying and registering resources, examining data, writing configuration files to index and display data and keeping the contents current. In the initial phases of the project, all aspects of the registration and curation processes were manual. However, as the number of resources grew, manual curation became impractical. This report describes our experiences and successes with developing automated resource discovery and semiautomated type characterization with text-mining scripts that facilitate curation team efforts to discover, integrate and display new content. We also describe the DISCO framework, a suite of automated web services that significantly reduce manual curation efforts to periodically check for resource updates. Lastly, we discuss DOMEO, a semi-automated annotation tool that improves the discovery and curation of resources that are not necessarily website-based (i.e. reagents, software tools). Although the ultimate goal of automation was to reduce the workload of the curators, it has resulted in valuable analytic by-products that address accessibility, use and citation of resources that can now be shared with resource owners and the larger scientific community. DATABASE URL: http://neuinfo.org.

Prototype of Kepler Processing Workflows For Microscopy And Neuroinformatics.

We report on progress of employing the Kepler workflow engine to prototype "end-to-end" application integration workflows that concern data coming from microscopes deployed at the National Center for Microscopy Imaging Research (NCMIR). This system is built upon the mature code base of the Cell Centered Database (CCDB) and integrated rule-oriented data system (IRODS) for distributed storage. It provides integration with external projects such as the Whole Brain Catalog (WBC) and Neuroscience Information Framework (NIF), which benefit from NCMIR data. We also report on specific workflows which spawn from main workflows and perform data fusion and orchestration of Web services specific for the NIF project. This "Brain data flow" presents a user with categorized information about sources that have information on various brain regions.

Baseline glutamate levels affect group I and II mGluRs in layer V pyramidal neurons of rat sensorimotor cortex.

Possible functional roles for glutamate that is detectable at low concentrations in the extracellular space of intact brain and brain slices have not been explored. To determine whether this endogenous glutamate acts on metabotropic glutamate receptors (mGluRs), we obtained whole cell recordings from layer V pyramidal neurons of rat sensorimotor cortical slices. Blockade of mGluRs with (+)-alpha-amino-4-carboxy-alpha-methyl-benzeacetic acid (MCPG, a general mGluR antagonist) increased the mean amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), an effect attributable to a selective increase in the occurrence of large amplitude sEPSCs. 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY341495, a group II antagonist) increased, but R(-)-1-amino-2,3-dihydro-1H-indene-1,5-dicarboxylic acid (AIDA) and (RS)-hexyl-HIBO (group I antagonists) decreased sEPSC amplitude, and (R,S)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG, a group III antagonist) did not change it. The change in sEPSCs elicited by MCPG, AIDA, and LY341495 was absent in tetrodotoxin, suggesting that it was action potential-dependent. The increase in sEPSCs persisted in GABA receptor antagonists, indicating that it was not due to effects on inhibitory interneurons. AIDA and (S)-3,5-dihydroxyphenylglycine (DHPG, a group I agonist) elicited positive and negative shifts in holding current, respectively. LY341495 and (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV, a group II agonist) elicited negative and positive shifts in holding current, respectively. The AIDA and LY341495 elicited currents persisted in TTX. Finally, in current clamp, LY341495 depolarized cells by approximately 2 mV and increased the number of action potentials to a given depolarizing current pulse. Thus ambient levels of glutamate tonically activate mGluRs and regulate cortical excitability.

Activation of metabotropic glutamate receptors by repetitive stimulation in auditory cortex.

To determine whether metabotropic glutamate receptors (mGluRs) contribute to the responses of neurons to repetitive stimulation in the rat auditory cortex in vitro, five stimulus pulses were delivered at 2-100 Hz which elicited five depolarizing synaptic responses, f-EPSPs: f-EPSPs(1-5). Stimulus pulses 2-5 delivered at low frequencies (2-10 Hz) elicited f-EPSPs(2-5) that were about 15% smaller than the response elicited by the first pulse (f-EPSP(1)). In the presence of the nonspecific mGluR agonist, ACPD, the amplitude of all f-EPSPs was 40% smaller than predrug responses. APV, CNQX, or bicuculline (antagonists of NMDA-, AMPA/kainate-, and GABA(A)-receptors, respectively) did not change this effect of ACPD. The mGluR antagonist, MCPG, had no effect on f-EPSPs but did reduce the effect of ACPD. High-frequency stimulation (50-100 Hz) elicited f-EPSPs that were smaller with each successive stimulus. In ACPD, f-EPSP(1) was 40% smaller than predrug, but f-EPSPs(3-5) were not changed compared to pre-ACPD f-EPSPs(3-5), indicating that ACPD occludes the effect of repetitive stimulation. MCPG increased f-EPSP(5) by 15%, indicating that a portion of the reduction of f-EPSPs during high-frequency stimulation is mediated by mGluRs. MCPG also partially blocked the effect of ACPD. In CNQX, ACPD only decreased EPSPs, but APV or bicuculline did not change the effect of ACPD. These results suggest that the successive reduction of f-EPSPs during a high-frequency train is partially a result of mGluR activation.

Cholinergic synaptic potentials in the supragranular layers of auditory cortex.

Receptive-field plasticity within the auditory neocortex is associated with learning, memory, and acetylcholine (ACh). However, the interplay of elements involved in changing receptive-fields remains unclear. Herein, we describe a depolarizing and a hyperpolarizing potential elicited by repetitive stimulation (20-100 Hz, 0.5-2 sec) and dependent on ACh, which may be involved in modifying receptive-fields. These potentials were recorded, using whole cell techniques, in layer II/III pyramidal cells in the rat auditory cortex in vitro. Stimulation at low stimulus intensities can give rise to a hyperpolarizing response and stimulation at higher stimulus intensities can elicit a depolarizing response. The depolarizing response had a reversal potential of -35 mV, and was reduced by the combination of AMPA/kainate and NMDA glutamate receptor antagonists (AMPA/kainate: CNQX, DNQX, and GYKI 52466; NMDA: APV, MK-801) and by the muscarinic ACh receptor antagonist atropine. The hyperpolarizing response had a reversal potential of -73 mV and could be reduced by atropine, GABA(A) receptor antagonists (bicuculline and a Cl(-) channel blocker picrotoxin), and to a small extent a GABA(B) receptor antagonist (saclofen). This suggests that the hyperpolarizing response is likely to be mediated by ACh acting on GABAergic interneurons. Extracellular recordings, also made from layer II/III of cortical slices, yielded a negative-going potential which was reduced by ionotropic glutamate receptor antagonists (same as above) and by the ACh receptor antagonists atropine and scopolamine, suggesting that this potential was the extracellular representation of the depolarizing response.

Tetanic stimulation and metabotropic glutamate receptor agonists modify synaptic responses and protein kinase activity in rat auditory cortex.

We investigated whether tetanic-stimulation and activation of metabotropic glutamate receptors (mGluRs) can modify field-synaptic-potentials and protein kinase activity in rat auditory cortex, specifically protein kinase A (PKA) and protein kinase C (PKC). Tetanic stimulation (50 Hz, 1 s) increases PKA and PKC activity only if the CNQX-sensitive field-EPSP (f-EPSP) is also potentiated. If the f-EPSP is unchanged, then PKA and PKC activity remains unchanged. Tetanic stimulation decreases a bicuculline-sensitive field-IPSP (f-IPSP), and this occurs whether the f-EPSP is potentiated or not. Potentiation of the f-EPSP is blocked by antagonists of mGluRs (MCPG) and PKC (calphostin-C, tamoxifen), suggesting that the potentiation of the f-EPSP is dependent on mGluRs and PKC. PKC antagonists block the rise in PKC and PKA activity, which suggests that these may be coupled. In contrast, ACPD (agonist at mGluRs) decreases both the f-EPSP and the f-IPSP, but increases PKC and PKA activity. Quisqualate (group I mGluR agonist), decreases the f-IPSP, and increases PKA activity, suggesting that the increase in PKA activity is a result of activation of group I mGluRs. Additionally, the increase in PKC and PKA activity appears to be independent of the decrease of the f-EPSP and f-IPSP, because PKC antagonists block the increase in PKC and PKA activity levels but do not block ACPD's effect on the f-EPSP or f-IPSP. These data suggest that group I mGluRs are involved in potentiating the f-EPSP by a PKC and possibly PKA dependent mechanism which is separate from the mechanism that decreases the f-EPSP and f-IPSP.

Metabotropic glutamate receptors modify ionotropic glutamate responses in neocortical pyramidal cells and interneurons.

In neocortex glutamate activates ionotropic and metabotropic receptors (mGluRs). Whole-cell current-clamp recordings in the in vitro rat auditory cortex at 32 degrees C were used to explore the role that mGluRs have in regulation of AMPA/kainate, NMDA, and GABA receptor-mediated synaptic transmission. Our findings are: (a) The fast EPSP (AMPA/kainate), slow EPSP (NMDA), and IPSPs (GABAA, GABAB), elicited in pyramidal neurons are reduced in the presence of (1S,3R)-ACPD (mGluR agonist) with greatest effect on the slow IPSP>fast IPSP>>fast EPSP. The effect is likely the result of ACPD acting at presynaptic mGluRs because the probability of release of glutamate and GABA is reduced in the presence of ACPD, intracellular infusion of a G protein antagonist (GDPPS) did not block the effect of ACPD, nor were iontophoretic kainic acid or NMDA-induced depolarizations reduced by ACPD. (b) The slow EPSP is enhanced following washout of ACPD and enhancement is not due to disinhibition because it is present in the absence of IPSPs, but if IPSPs are present, its magnitude can be influenced. Iontophoretic NMDA responses are enhanced in the presence of ACPD, an effect blocked by GDPbetaS and heparin (intracellular inositol 1,4,5-trisphosphate receptor antagonist). Taken together, this evidence suggests that enhancement is a result of group I postsynaptic mGluR activation. (c) In fast-spiking cells ACPD reduces the EPSP (AMPA/kainate and NMDA-mediated). This action is likely presynaptic because it persists when GDPbetaS is in the cells. (d) The rate of spike discharge recorded from fast-spiking cells is accelerated in ACPD but does not change in the presence of GDPbetaS, suggesting a postsynaptic effect. Our data indicate that mGluRs can influence neocortical synaptic transmission in complex ways by acting presynaptically and postsynaptically.

Role of muscarinic receptors, G-proteins, and intracellular messengers in muscarinic modulation of NMDA receptor-mediated synaptic transmission.

Previously, we reported that activation of muscarinic receptors modulates N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission in auditory neocortex [Aramakis et al. (1997a) Exp Brain Res 113:484-496]. Here, we describe the muscarinic subtypes responsible for these modulatory effects, and a role for G-proteins and intracellular messengers. The muscarinic agonist oxotremorine-M (oxo-M), at 25-100 microM, produced a long-lasting enhancement of NMDA-induced membrane depolarizations. We examined the postsynaptic G-protein dependence of the modulatory effects of oxo-M with the use of the G-protein activator GTP gamma S and the nonhydrolyzable GDP analog GDP beta S. Intracellular infusion of GTP gamma S mimicked the facilitating actions of oxo-M. After obtaining the whole-cell recording configuration, there was a gradual, time-dependent increase of the NMDA receptor-mediated slow-EPSP, and of iontophoretic NMDA-induced membrane depolarizations. In contrast, intracellular infusion of either GDP beta S or the IP3 receptor antagonist heparin prevented oxo-M mediated enhancement of NMDA depolarizations. The muscarinic receptor involved in enhancement of NMDA iontophoretic responses is likely the M1 receptor, because the increase was prevented by pirenzepine, but not the M2 antagonists methoctramine or AF-DX 116. Oxo-M also reduced the amplitude of the pharmacologically isolated slow-EPSP, and this effect was blocked by M2 antagonists. Thus, muscarinic-mediated enhancement of NMDA responses involves activation of M1 receptors, leading to the engagement of a postsynaptic G-protein and subsequent IP3 receptor activity.

Muscarinic reduction of GABAergic synaptic potentials results in disinhibition of the AMPA/kainate-mediated EPSP in auditory cortex.

The present study is concerned with the ability of muscarinic actions of acetylcholine (ACh) to modulate glutamate and gamma-aminobutyric acid (GABA)-mediated synaptic transmission in the in vitro rat auditory cortex. Whole-cell patch clamp recordings were obtained from layer II-III pyramidal neurons, and the fast-EPSP (AMPA/kainate), fast-IPSP (GABA(A)), and slow-IPSP (GABA(B)), were elicited following a stimulus to deep gray/white matter. Acetyl-beta-methylcholine (MCh), a muscarinic receptor agonist, applied by either superfusion or iontophoresis, produced an atropine-sensitive increase or decrease in the amplitude of the fast-EPSP. The effect of MCh could be predicted by the response of the fast-EPSP to paired-pulse stimulation (i.e. a conditioning pulse followed 300 ms later by a test pulse). The fast-EPSP was decreased in amplitude by MCh in cases where the test-EPSP was suppressed in the pre-MCh condition, and increased in amplitude when the test-EPSP was facilitated. The fast- and slow-IPSPs were always reduced by MCh. In several experiments, the strength of synaptic inhibition was systematically modified by varying stimulus intensity. When the fast-EPSP was elicited in the absence of IPSPs, it was decreased in amplitude by MCh. However, when the fast-EPSP was elicited in conjunction with large IPSPs it was increased in amplitude during MCh. Because the magnitude of the fast-EPSP is influenced by the degree of temporal overlap with IPSPs, it was hypothesized that enhancement of the fast-EPSP was the result of disinhibition produced as a consequence of muscarinic reduction of GABAergic IPSPs. This view was supported by the finding that MCh could reduce the amplitude of pharmacologically isolated GABAergic IPSPs (i.e. elicited in the absence of glutamatergic transmission). Our results suggest that ACh at muscarinic receptors can modify fast glutamatergic neurotransmission differently as a function of strength of inhibition, to suppress that produced by 'weak' inputs and enhance that produced by 'strong' inputs.

Activation of muscarinic receptors modulates NMDA receptor-mediated responses in auditory cortex.

The present study examines the ability of muscarinic receptor activation to modulate glutamatergic responses in the in vitro rat auditory cortex. Whole-cell patch-clamp recordings were obtained from layer II-III pyramidal neurons and responses elicited by either stimulation of deep gray matter or iontophoretic application of glutamate receptor agonists. Iontophoresis of the muscarinic agonist acetyl-beta-methylcholine (MCh) produced an atropine-sensitive reduction in the amplitude of glutamate-induced membrane depolarizations that was followed by a long-lasting (at least 20 min) response enhancement. Glutamate depolarizations were enhanced by MCh when elicited in the presence of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 2,3-dihydroxy-6-nitro-7-sulfamoyl, benzo(F)quinoxaline (NBQX) but not the NMDA antagonists D-2-amino-5-phosphonovaleric acid (APV) or MK-801 hydrogen maleate. The magnitude of enhancement was voltage-dependent with the percentage increase greater at more depolarized membrane potentials. An involvement of NMDA receptors in these MCh-mediated effects was tested by using AMPA/kainate receptor antagonists to isolate the NMDA-mediated slow excitatory postsynaptic potential (EPSP) from other synaptic potentials. The slow EPSP and iontophoretic responses to NMDA were similarly modified by MCh, i.e., both being reduced during and enhanced (15-55 min) following MCh application. Cholinergic modulation of NMDA responses involves the engagement of G proteins, as enhancement was prevented by intracellular infusion with the nonhydrolyzable GDP analog guanosine-5'-O-(2-thiodiphosphate) trilithium salt (GDPbetaS). GDPbetaS was without effect on the early MCh-induced response suppression. Our results suggest that acetylcholine, acting at muscarinic receptors, produces a long-lasting enhancement of NMDA-mediated neurotransmission in auditory cortex, and that this modulatory effect is dependent upon a G protein-mediated event.