Person: Saulnier, Jessica
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Saulnier
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Jessica
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Saulnier, Jessica
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Publication Temporal dynamics of a homeostatic pathway controlling neural network activity(Frontiers Media S.A., 2013) Bateup, Helen S.; Denefrio, Cassandra L.; Johnson, Caroline; Saulnier, Jessica; Sabatini, BernardoNeurons use a variety of mechanisms to homeostatically regulate neural network activity in order to maintain firing in a bounded range. One such process involves the bi-directional modulation of excitatory synaptic drive in response to chronic changes in network activity. Down-scaling of excitatory synapses in response to high activity requires Arc-dependent endocytosis of glutamate receptors. However, the temporal dynamics and signaling pathways regulating Arc during homeostatic plasticity are not well understood. Here we determine the relative contribution of transcriptional and translational control in the regulation of Arc, the signaling pathways responsible for the activity-dependent production of Arc, and the time course of these signaling events as they relate to the homeostatic adjustment of network activity in hippocampal neurons. We find that an ERK1/2-dependent transcriptional pathway active within 1–2 h of up-regulated network activity induces Arc leading to a restoration of network spiking rates within 12 h. Under basal and low activity conditions, specialized mechanisms are in place to rapidly degrade Arc mRNA and protein such that they have half-lives of less than 1 h. In addition, we find that while mTOR signaling is regulated by network activity on a similar time scale, mTOR-dependent translational control is not a major regulator of Arc production or degradation suggesting that the signaling pathways underlying homeostatic plasticity are distinct from those mediating synapse-specific forms of synaptic depression.Publication High Content Image Analysis Identifies Novel Regulators of Synaptogenesis in a High-Throughput RNAi Screen of Primary Neurons(Public Library of Science, 2014) Nieland, Thomas; Logan, David J.; Saulnier, Jessica; Lam, Daniel; Johnson, Caroline; Root, David E.; Carpenter, Anne E.; Sabatini, BernardoThe formation of synapses, the specialized points of chemical communication between neurons, is a highly regulated developmental process fundamental to establishing normal brain circuitry. Perturbations of synapse formation and function causally contribute to human developmental and degenerative neuropsychiatric disorders, such as Alzheimer's disease, intellectual disability, and autism spectrum disorders. Many genes controlling synaptogenesis have been identified, but lack of facile experimental systems has made systematic discovery of regulators of synaptogenesis challenging. Thus, we created a high-throughput platform to study excitatory and inhibitory synapse development in primary neuronal cultures and used a lentiviral RNA interference library to identify novel regulators of synapse formation. This methodology is broadly applicable for high-throughput screening of genes and drugs that may rescue or improve synaptic dysfunction associated with cognitive function and neurological disorders.Publication A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging(Frontiers Media S.A., 2014) Chen, Yao; Saulnier, Jessica; Yellen, Gary; Sabatini, BernardoNeuromodulators have profound effects on behavior, but the dynamics of their intracellular effectors has remained unclear. Most neuromodulators exert their function via G-protein-coupled receptors (GPCRs). One major challenge for understanding neuromodulator action is the lack of dynamic readouts of the biochemical signals produced by GPCR activation. The adenylate cyclase/cyclic AMP/protein kinase A (PKA) module is a central component of such biochemical signaling. This module is regulated by several behaviorally important neuromodulator receptors. Furthermore, PKA activity is necessary for the induction of many forms of synaptic plasticity as well as for the formation of long-term memory. In order to monitor PKA activity in brain tissue, we have developed a 2-photon fluorescence lifetime imaging microscopy (2pFLIM) compatible PKA sensor termed FLIM-AKAR, which is based on the ratiometric FRET sensor AKAR3. FLIM-AKAR shows a large dynamic range and little pH sensitivity. In addition, it is a rapidly diffusible cytoplasmic protein that specifically reports net PKA activity in situ. FLIM-AKAR expresses robustly in various brain regions with multiple transfection methods, can be targeted to genetically identified cell types, and responds to activation of both endogenous GPCRs and spatial-temporally specific delivery of glutamate. Initial experiments reveal differential regulation of PKA activity across subcellular compartments in response to neuromodulator inputs. Therefore, the reporter FLIM-AKAR, coupled with 2pFLIM, enables the study of PKA activity in response to neuromodulator inputs in genetically identified neurons in the brain, and sheds light on the intracellular dynamics of endogenous GPCR activation.Publication Spatial Organization of Local Inputs to Spiny Projection Neurons in the Striatum(2015-10-19) Saulnier, Jessica; Morris, James; Sabatini, Bernardo L.; Straub, ChristophGABAergic interneurons are important for balanced activity of the principal projection neurons of the striatum (spiny projection neurons, SPNs) and dysfunction of striatal GABAergic interneurons can lead to movement-related disorders. Despite this importance, very little is known about the connectivity of striatal interneurons and their functional spatial arrangement. In preliminary experiments our group optogenetically identified a group of interneuron connections that had not previously been seen in paired recordings. Here we tested the hypothesis that this finding is due to long-range connections of genetically defined interneuron classes. Using a pseudotyped Rabies Viral (RV) monosynaptic retrograde tracing strategy in sparsely identified SPNS, followed by three-dimensional reconstruction, we tested the spatial attributes of connections from different striatal interneuron classes, and lateral connections between SPNs. Our results demonstrate the feasibility of an RV-dependent approach for local distance mapping and for the first time identify distinct projection properties of different striatal neuron classes. Importantly, our experiments reveal short, local connections of Fast Spiking (FS), but long-ranging projections of Low-Threshold Spiking (LTS) interneurons, which together form the majority of striatal GABAergic interneurons. These findings can resolve the opposing results from paired and optogenetic recordings and also suggest distinct signaling modalities for these two types of interneurons.Publication CRISPR/Cas9-Mediated Gene Knock-Down in Post-Mitotic Neurons(Public Library of Science, 2014) Straub, Christoph; Granger, Adam; Saulnier, Jessica; Sabatini, BernardoThe prokaryotic adaptive immune system CRISPR/Cas9 has recently been adapted for genome editing in eukaryotic cells. This technique allows for sequence-specific induction of double-strand breaks in genomic DNA of individual cells, effectively resulting in knock-out of targeted genes. It thus promises to be an ideal candidate for application in neuroscience where constitutive genetic modifications are frequently either lethal or ineffective due to adaptive changes of the brain. Here we use CRISPR/Cas9 to knock-out Grin1, the gene encoding the obligatory NMDA receptor subunit protein GluN1, in a sparse population of mouse pyramidal neurons. Within this genetically mosaic tissue, manipulated cells lack synaptic current mediated by NMDA-type glutamate receptors consistent with complete knock-out of the targeted gene. Our results show the first proof-of-principle demonstration of CRISPR/Cas9-mediated knock-down in neurons in vivo, where it can be a useful tool to study the function of specific proteins in neuronal circuits.Publication All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins(2014) Hochbaum, Daniel; Zhao, Yongxin; Farhi, Samouil; Klapoetke, Nathan; Werley, Christopher A.; Kapoor, Vikrant; Zou, Peng; Kralj, Joel M.; Maclaurin, Dougal; Smedemark-Margulies, Niklas; Saulnier, Jessica; Boulting, Gabriella; Straub, Christoph; Cho, Yong Ku; Melkonian, Michael; Wong, Gane Ka-Shu; Harrison, D. Jed; Murthy, Venkatesh; Sabatini, Bernardo; Boyden, Edward S.; Campbell, Robert E.; Cohen, AdamAll-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and 2, which show improved brightness and voltage sensitivity, microsecond response times, and produce no photocurrent. We engineered a novel channelrhodopsin actuator, CheRiff, which shows improved light sensitivity and kinetics, and spectral orthogonality to the QuasArs. A co-expression vector, Optopatch, enabled crosstalk-free genetically targeted all-optical electrophysiology. In cultured neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials in dendritic spines, synaptic transmission, sub-cellular microsecond-timescale details of action potential propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In brain slice, Optopatch induced and reported action potentials and subthreshold events, with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without use of conventional electrodes.Publication Corrigendum: A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging(Frontiers Media S.A., 2016) Chen, Yao; Saulnier, Jessica; Yellen, Gary; Sabatini, Bernardo