DRUG-seq Provides Unbiased Biological Activity Readouts for Neuroscience Drug Discovery.

Unbiased transcriptomic RNA-seq data has provided deep insights into biological processes. However, its impact in drug discovery has been narrow given high costs and low throughput. Proof-of-concept studies with Digital RNA with pertUrbation of Genes (DRUG)-seq demonstrated the potential to address this gap. We extended the DRUG-seq platform by subjecting it to rigorous testing and by adding an open-source analysis pipeline. The results demonstrate high reproducibility and ability to resolve the mechanism(s) of action for a diverse set of compounds. Furthermore, we demonstrate how this data can be incorporated into a drug discovery project aiming to develop therapeutics for schizophrenia using human stem cell-derived neurons. We identified both an on-target activation signature, induced by a set of chemically distinct positive allosteric modulators of the N-methyl-d-aspartate (NMDA) receptor, and independent off-target effects. Overall, the protocol and open-source analysis pipeline are a step toward industrializing RNA-seq for high-complexity transcriptomics studies performed at a saturating scale.

Scaled, high fidelity electrophysiological, morphological, and transcriptomic cell characterization.

The Patch-seq approach is a powerful variation of the patch-clamp technique that allows for the combined electrophysiological, morphological, and transcriptomic characterization of individual neurons. To generate Patch-seq datasets at scale, we identified and refined key factors that contribute to the efficient collection of high-quality data. We developed patch-clamp electrophysiology software with analysis functions specifically designed to automate acquisition with online quality control. We recognized the importance of extracting the nucleus for transcriptomic success and maximizing membrane integrity during nucleus extraction for morphology success. The protocol is generalizable to different species and brain regions, as demonstrated by capturing multimodal data from human and macaque brain slices. The protocol, analysis and acquisition software are compiled at https://githubcom/AllenInstitute/patchseqtools. This resource can be used by individual labs to generate data across diverse mammalian species and that is compatible with large publicly available Patch-seq datasets.

Visual physiology of the layer 4 cortical circuit in silico.

Despite advances in experimental techniques and accumulation of large datasets concerning the composition and properties of the cortex, quantitative modeling of cortical circuits under in-vivo-like conditions remains challenging. Here we report and publicly release a biophysically detailed circuit model of layer 4 in the mouse primary visual cortex, receiving thalamo-cortical visual inputs. The 45,000-neuron model was subjected to a battery of visual stimuli, and results were compared to published work and new in vivo experiments. Simulations reproduced a variety of observations, including effects of optogenetic perturbations. Critical to the agreement between responses in silico and in vivo were the rules of functional synaptic connectivity between neurons. Interestingly, after extreme simplification the model still performed satisfactorily on many measurements, although quantitative agreement with experiments suffered. These results emphasize the importance of functional rules of cortical wiring and enable a next generation of data-driven models of in vivo neural activity and computations.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

A NMDA-receptor calcium influx assay sensitive to stimulation by glutamate and glycine/D-serine.

N-methyl-D-aspartate-receptors (NMDARs) are ionotropic glutamate receptors that function in synaptic transmission, plasticity and cognition. Malfunction of NMDARs has been implicated in a variety of nervous system disorders, making them attractive therapeutic targets. Overexpression of functional NMDAR in non-neuronal cells results in cell death by excitotoxicity, hindering the development of cell-based assays for NMDAR drug discovery. Here we report a plate-based, high-throughput approach to study NMDAR function. Our assay enables the functional study of NMDARs with different subunit composition after activation by glycine/D-serine or glutamate and hence presents the first plate-based, high throughput assay that allows for the measurement of NMDAR function in glycine/D-serine and/or glutamate sensitive modes. This allows to investigate the effect of small molecule modulators on the activation of NMDARs at different concentrations or combinations of the co-ligands. The reported assay system faithfully replicates the pharmacology of the receptor in response to known agonists, antagonists, positive and negative allosteric modulators, as well as the receptor's sensitivity to magnesium and zinc. We believe that the ability to study the biology of NMDARs rapidly and in large scale screens will enable the identification of novel therapeutics whose discovery has otherwise been hindered by the limitations of existing cell based approaches.

Two-Photon Holographic Stimulation of ReaChR.

Optogenetics provides a unique approach to remotely manipulate brain activity with light. Reaching the degree of spatiotemporal control necessary to dissect the role of individual cells in neuronal networks, some of which reside deep in the brain, requires joint progress in opsin engineering and light sculpting methods. Here we investigate for the first time two-photon stimulation of the red-shifted opsin ReaChR. We use two-photon (2P) holographic illumination to control the activation of individually chosen neurons expressing ReaChR in acute brain slices. We demonstrated reliable action potential generation in ReaChR-expressing neurons and studied holographic 2P-evoked spiking performances depending on illumination power and pulse width using an amplified laser and a standard femtosecond Ti:Sapphire oscillator laser. These findings provide detailed knowledge of ReaChR's behavior under 2P illumination paving the way for achieving in depth remote control of multiple cells with high spatiotemporal resolution deep within scattering tissue.

B-lymphocyte-mediated delayed cognitive impairment following stroke.

Each year, 10 million people worldwide survive the neurologic injury associated with a stroke. Importantly, stroke survivors have more than twice the risk of subsequently developing dementia compared with people who have never had a stroke. The link between stroke and the later development of dementia is not understood. There are reports of oligoclonal bands in the CSF of stroke patients, suggesting that in some people a B-lymphocyte response to stroke may occur in the CNS. Therefore, we tested the hypothesis that a B-lymphocyte response to stroke could contribute to the onset of dementia. We discovered that, in mouse models, activated B-lymphocytes infiltrate infarcted tissue in the weeks after stroke. B-lymphocytes undergo isotype switching, and IgM, IgG, and IgA antibodies are found in the neuropil adjacent to the lesion. Concurrently, mice develop delayed deficits in LTP and cognition. Genetic deficiency, and the pharmacologic ablation of B-lymphocytes using an anti-CD20 antibody, prevents the appearance of delayed cognitive deficits. Furthermore, immunostaining of human postmortem tissue revealed that a B-lymphocyte response to stroke also occurs in the brain of some people with stroke and dementia. These data suggest that some stroke patients may develop a B-lymphocyte response to stroke that contributes to dementia, and is potentially treatable with FDA-approved drugs that target B cells.

Leucine-rich repeat transmembrane proteins are essential for maintenance of long-term potentiation.

Leucine-rich repeat transmembrane proteins (LRRTMs) are synaptic cell adhesion molecules that trigger excitatory synapse assembly in cultured neurons and influence synaptic function in vivo, but their role in synaptic plasticity is unknown. shRNA-mediated knockdown (KD) of LRRTM1 and LRRTM2 in vivo in CA1 pyramidal neurons of newborn mice blocked long-term potentiation (LTP) in acute hippocampal slices. Molecular replacement experiments revealed that the LRRTM2 extracellular domain is sufficient for LTP, probably because it mediates binding to neurexins (Nrxs). Examination of surface expression of endogenous AMPA receptors (AMPARs) in cultured neurons suggests that LRRTMs maintain newly delivered AMPARs at synapses after LTP induction. LRRTMs are also required for LTP of mature synapses on adult CA1 pyramidal neurons, indicating that the block of LTP in neonatal synapses by LRRTM1 and LRRTM2 KD is not due to impairment of synapse maturation.

The neurexin ligands, neuroligins and leucine-rich repeat transmembrane proteins, perform convergent and divergent synaptic functions in vivo.

Synaptic cell adhesion molecules, including the neurexin ligands, neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs), are thought to organize synapse assembly and specify synapse function. To test the synaptic role of these molecules in vivo, we performed lentivirally mediated knockdown of NL3, LRRTM1, and LRRTM2 in CA1 pyramidal cells of WT and NL1 KO mice at postnatal day (P)0 (when synapses are forming) and P21 (when synapses are largely mature). P0 knockdown of NL3 in WT or NL1 KO neurons did not affect excitatory synaptic transmission, whereas P0 knockdown of LRRTM1 and LRRTM2 selectively reduced AMPA receptor-mediated synaptic currents. P0 triple knockdown of NL3 and both LRRTMs in NL1 KO mice yielded greater reductions in AMPA and NMDA receptor-mediated currents, suggesting functional redundancy between NLs and LRRTMs during early synapse development. In contrast, P21 knockdown of LRRTMs did not alter excitatory transmission, whereas NL manipulations supported a role for NL1 in maintaining NMDA receptor-mediated transmission. These results show that neurexin ligands in vivo form a dynamic synaptic cell adhesion network, with compensation between NLs and LRRTMs during early synapse development and functional divergence upon synapse maturation.

Neuroligins/LRRTMs prevent activity- and Ca2+/calmodulin-dependent synapse elimination in cultured neurons.

Neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs) are postsynaptic cell adhesion molecules that bind to presynaptic neurexins. In this paper, we show that short hairpin ribonucleic acid-mediated knockdowns (KDs) of LRRTM1, LRRTM2, and/or NL-3, alone or together as double or triple KDs (TKDs) in cultured hippocampal neurons, did not decrease synapse numbers. In neurons cultured from NL-1 knockout mice, however, TKD of LRRTMs and NL-3 induced an ∼40% loss of excitatory but not inhibitory synapses. Strikingly, synapse loss triggered by the LRRTM/NL deficiency was abrogated by chronic blockade of synaptic activity as well as by chronic inhibition of Ca(2+) influx or Ca(2+)/calmodulin (CaM) kinases. Furthermore, postsynaptic KD of CaM prevented synapse loss in a cell-autonomous manner, an effect that was reversed by CaM rescue. Our results suggest that two neurexin ligands, LRRTMs and NLs, act redundantly to maintain excitatory synapses and that synapse elimination caused by the absence of NLs and LRRTMs is promoted by synaptic activity and mediated by a postsynaptic Ca(2+)/CaM-dependent signaling pathway.

N-methyl-D-aspartate receptor- and metabotropic glutamate receptor-dependent long-term depression are differentially regulated by the ubiquitin-proteasome system.

Long-term depression (LTD) in CA1 pyramidal neurons can be induced by activation of either N-methyl-D-aspartate receptors (NMDARs) or metabotropic glutamate receptors (mGluRs), both of which elicit changes in synaptic efficacy through alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) endocytosis. To address the role of the ubiquitin-proteasome system in regulating AMPAR endocytosis during these forms of LTD, we examined the effects of pharmacological inhibitors of proteasomal degradation and protein ubiquitination on endocytosis of glutamate receptor 1 (GluR1) -containing AMPARs in dissociated rat hippocampal cultures as well as LTD of excitatory synaptic responses in acute rat hippocampal slices. Our findings suggest that the contribution of the ubiquitin-proteasome system to NMDAR-induced vs. mGluR-induced AMPAR endocytosis and the consequent LTD differs significantly. NMDAR-induced AMPAR endocytosis and LTD occur independently of proteasome function but appear to depend, at least in part, on ubiquitination. In contrast, mGluR-induced AMPAR endocytosis and LTD are enhanced by inhibition of proteasomal degradation, as well as by the inhibitor of protein ubiquitination. Furthermore, the decay of mGluR-induced membrane depolarization and Erk activation is delayed following inhibition of either ubiquitination or proteasomal degradation. These results suggest that, although NMDAR-dependent LTD may utilize ubiquitin as a signal for AMPAR endocytosis, mGluR-induced signaling and LTD are limited by a feedback mechanism that involves the ubiquitin-proteasome system.

Cholinergic modulation of multivesicular release regulates striatal synaptic potency and integration.

The pleiotropic actions of neuromodulators on pre- and postsynaptic targets make disentangling the mechanisms underlying regulation of synaptic transmission challenging. In the striatum, acetylcholine modulates glutamate release via activation of muscarinic receptors (mAchRs), although the consequences for postsynaptic signaling are unclear. Using two-photon microscopy and glutamate uncaging to examine individual synapses in the rat striatum, we found that glutamatergic afferents have a high degree of multivesicular release (MVR) in the absence of postsynaptic receptor saturation. We found that mAchR activation decreased both the probability of release and the concentration of glutamate in the synaptic cleft. The corresponding decrease in synaptic potency reduced the duration of synaptic potentials and limited temporal summation of afferent inputs. These findings reveal a mechanism by which a combination of basal MVR and low receptor saturation allow the presynaptic actions of a neuromodulator to control the engagement of postsynaptic nonlinearities and regulate synaptic integration.

Timing and location of synaptic inputs determine modes of subthreshold integration in striatal medium spiny neurons.

Medium spiny neurons (MSNs) are the principal cells of the striatum and perform a central role in sensorimotor processing. MSNs must integrate many excitatory inputs located across their dendrites to fire action potentials and enable striatal function. However, the dependence of synaptic responses on the temporal and spatial distribution of these inputs remains unknown. Here, we use whole-cell recordings, two-photon microscopy, and two-photon glutamate uncaging to examine subthreshold synaptic integration in MSNs from acute rat brain slices. We find that synaptic responses can summate sublinearly, linearly, or supralinearly depending on the spatiotemporal pattern of activity. Repetitive activity at single inputs leads to sublinear summation, reflecting long-lived AMPA receptor desensitization. In contrast, asynchronous activity at multiple inputs generates linear summation, with synapses on neighboring spines functioning independently. Finally, synchronous activity at multiple inputs triggers supralinear summation at depolarized potentials, reflecting activation of NMDA receptors and L-type calcium channels. Thus, the properties of subthreshold integration in MSNs are determined by the distribution of synaptic inputs and the differential activation of multiple postsynaptic conductances.

Functional interactions at the interface between voltage-sensing and pore domains in the Shaker K(v) channel.

Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.

Synapse-specific plasticity and compartmentalized signaling in cerebellar stellate cells.

Here we demonstrate that cerebellar stellate cells diffusionally isolate synaptically evoked signals in dendrites and are capable of input-specific synaptic plasticity. Sustained activity of parallel fibers induces a form of long-term depression that requires opening of calcium (Ca(2+))-permeable AMPA-type glutamate receptors (CP-AMPARs) and signaling through class 1 metabotropic glutamate receptors (mGluR1) and CB1 receptors. This depression is induced by postsynaptic increases in Ca(2+) concentration ([Ca(2+)]) and is limited to activated synapses. To understand how synapse-specific plasticity is induced by diffusible second messengers in aspiny dendrites, we examined diffusion of Ca(2+) and small molecules within stellate cell dendrites. Activation of a single parallel fiber opened CP-AMPARs, generating long-lived Ca(2+) transients that were confined to submicron dendritic stretches. The diffusion of Ca(2+) was severely retarded due to interactions with parvalbumin and a general restriction of small molecule mobility. Thus stellate cell dendrites spatially restrict signaling cascades that lead from CP-AMPAR activation to endocannabinoid production and trigger the selective regulation of active synapses.

Defining the conductance of the closed state in a voltage-gated K+ channel.

The opening and closing of the ion conduction pathway in ion channels underlies the generation and propagation of electrical signals in biological systems. Although electrophysiological approaches to measuring the flow of ions in the open state have contributed profoundly to our understanding of ion permeation and gating, it remains unclear how much the ion-throughput rate decreases upon closure of the ion conduction pore. To address this fundamental question, we expressed the Shaker Kv channel at high levels and then measured macroscopic K+ currents at negative membrane voltages and counted the number of channels by quantifying the translocation of gating charge. Our results show that the conductance of the closed state is between 0 and 0.16 fS, or at least 100,000 times lower than for the open state of the channel, indicating that the flow of ions is very tightly regulated in this class of K+ channels.