...www.janelia.org/open-science/voltron-and-positron.  Both Ace2N-mNeon-A122D and Voltron2 are available as Cre-dependent...33714753. Voltron2 voltage sensors By Brian O’Neill New viral vector preps expressing the Voltron2 voltage...performance.      Figure 4: Structure of Voltron or Voltron2 conjugated to a JaneliaFluor dye emitting...HaloTag domain.  Find Voltron2 plasmids here! Abdelfattah, A.S., et al. (2023) Neuron, 111(10), 1547–1563... SARS-CoV-2 research New viral vector preps of Voltron2 voltage sensors AAV vectors for targeting microglia... to facilitate SARS-CoV-2 research   By Ashley Waldron Ready-to-use recombinant antibodies were recently..., are now available! Building on their earlier Voltron indicators (a fusion of Ace2N and HaloTag, Figure...

...Visualizing single neurons in the living brain   Figure 1: Plasmid mix to label neuronal morphology (...labeling of cortical neurons and their synapses.   How can you label a single neuron and its synapses...DNA’, is derived from the yeast S. cerevisiae (Gronostajski and Sadowski 1985). The FLP system works in ...neuroscience Manipulation of specific cell types in neuronal networks Double inverted vectors expressing light...manipulate specific cell types (populations) within neuronal networks in living animals. To accomplish such...electroporation. To achieve sparse labeling of individual neurons one could reduce the plasmids amounts to reduce...efficiency. However, if the cellular structure of a neuron and its synapse should be visualized at the same...

... Inhibition of neuronal activation is a little more complicated since basal neuronal activity in your...advent of optogenetics, ICSS can now be done with neuronal cell-type and temporal precision. In ICSS, a test...actually stimulating or inhibiting the targeted neuronal population prior to behavioral testing. One method...a reliable and widely used molecular marker of neuronal activation and is a sufficient readout to proceed...readout may be necessary to prove inhibition of neuronal activation, but it may still be possible to use...example, as shown in this study [3], hippocampal neurons are activated when the mouse is placed in a novel... the authors were able to show that HR blocked neuronal activation in the novel context as determined ...

...linked to disease, including motor neurons (-3mnx1/Hb9), pan neuronal (elavl3/HuC), microglial/macrophage...specific neuronal subtypes was not available until now. In order to target specific neuronal subtypes ...type SOX2 with either a SOX2-VP16 (strong activator) or SOX2-HP1 (strong repressor) fusion protein, the authors...interest. In order to improve the study of specific neuronal cell types involved in neurodegenerative diseases...), and astrocytic (gfap).This toolbox adds new neuronal tools to the expanding gateway compatible vectors...to express the transcription factors from a polycistronic cassette. However, it was unknown whether variations...variations in gene expression from these polycistronic cassettes could lead to comparable experimental...

...expression in E. coli. This operon is under the transcriptional control of a strong constitutive promoter (...powerful tool used by neuroscientists to interrogate neuronal networks. A major drawback of this method is the... the stray excitation of neighboring neurons in opsin dense regions. To overcome this issue, Shemesh et... C-terminus of the protein. Unlike the diffuse neuronal expression observed with wild type CoChR, soCoChR...describe a novel system for the precise mapping of neuronal networks at the single-cell level. Various soCoChR...PMID: 29184208.        Synthetic decaffeination operon Article contributed by Angela Kaczmarczyk  Listen...Listen to the synthetic decaffeination operon podcast segment With the help of engineered microbes, used...

...cyan light, Dronpa dimers dissociate, Dronpa goes dark, and MEK1 is active or “on.” Dronpa’s color thus...Additionally, dimeric Dronpa is less prone to aggregation in cells compared to the tetrameric Dronpa it was derived... by attaching two dimerizing photoswitchable Dronpa (dron= a ninja term for vanishing, and pa= photoactivation... the Dronpa dimers interact and lock MEK1 in an “off” setting by blocking the active site. Dronpa also...similarity. For these three kinases, photoswitchable Dronpa domains were inserted at sites homologous to the...here. In addition, it could be possible to use pdDronpa to control other kinases and proteins. Zhou et...proteins could potentially be created by inserting pdDronpa domains into loops of a protein of interest. If...

...2011) labels Drosophila neurons at the neuroblast stage, meaning that neurons of the same lineage will...cell types, and research applications. Labeling neurons with Brainbow The Lichtman group developed the ...iteration of this technology in 2007. Used to label neurons in the cerebellum of mice, the team appropriately...2007). Brainbow uses a Cre/lox system to label neurons. Genes encoding the fluorescent proteins are organized...the truncated YFP. In Thy1-Brainbow mice, some neurons expressed as many as 16 copies of the injected ...entire length of the cell, even axons of up to 100 microns in length. Some challenges of the Brainbow system...cells, the expression level of these XFP is not strong enough for all applications. To resolve this, similar...

...’s lab has enabled progress on both fronts – as a genetic electron microscopy (EM) tag providing superior...presynaptic region of one neuron and postsynaptic region of a different neuron, come into proximity in ...Druckmann et al., Neuron. 2014. Feb 5;81(3):629-40.  Next-Gen Brainbow toolkit for neuronal imaging Joshua...Howarth. Shining a light on channelrhodopsins – Chronos & Chrimson Through de novo sequencing of 127 algal...These tools include a pair of channelrhodopsins, Chronos and Chrimson, which have enabled the group to perform...perform independent two-color optical excitation of neurons. The paper describes five previously unknown ...and Scherffelia dubia (SdChR). ShChR, nicknamed Chronos, is blue and green light-excitable and has faster...

...Additional considerations CMV General expression mRNA Strong mammalian expression promoter from the human cytomegalovirus...some cell types. EF1a General expression mRNA Strong mammalian expression from human elongation factor...promoter hybrids. CAG General expression mRNA Strong hybrid mammalian promoter Constitutive  Contains...activate promoter. Ac5 General expression mRNA Strong insect promoter from Drosophila Actin 5c gene Constitutive...Drosophila. Polyhedrin General expression mRNA  Strong insect promoter from baculovirus Constitutive  ...calmodulin-dependent protein kinase II promoter Specific Used for neuronal/CNS expression. Modulated by calcium and calmodulin...mammalian EF1a promoter. GDS General expression mRNA  Strong yeast expression promoter from glyceraldehyde 3...

...effector cells in the tumor microenvironment. Tumor cells in this environment produce growth factor signals...inflammatory cytokines - interleukin (IL)-12 and interferon gamma (IFNγ) - and eliminate cancer cells. 2....responses that eliminate early stage cancer cells, chronic inflammatory responses can augment tumor growth...development are observed in autoimmune disorders. Chronic inflammation due to autoimmune disorders is also...Strategic immune evasion mechanisms - The tumor microenvironment Through interactions with the immune system...and thrive within what is known as the tumor microenvironment - a vital atmosphere created by the cancer...recruit regulatory T cells (T-regs) into their microenvironment by producing C-C motif chemokine 22 (CCL22...

...AAV1 and AAV9 serotypes have strong neuronal tropism, AAV5 targets both neurons and astrocytes, and AAV4 ...activation of neurons in a specific brain region. One option is to overexpress a gene that alters neuronal function...introduced in neurons and depending on the choice of channel protein, they can trigger neuronal excitation...certain tests and is not always recommended.  Environmental influences like bright light or noise should...(vendor or in-house mouse breeding colony) Environment Olfaction, temperature, humidity, noise level...factors. Another option uses optogentics to control neuronal activity, commonly via light-activated ion channels...broad cell tropism and has a high percentage of neuronal targeting after stereotaxic injection. Find more...

...example, lowers the threshold for neuronal signaling and causes neuronal burst firing. Other DREADDs, such...DREADDs for Neuroscientists. Neuron 89:683–694 . https://doi.org/10.1016/j.neuron.2016.01.040 Saloman, J. ...method for manipulating cell activity, especially neuronal firing. What is chemogenetics? Chemogenetics ...decreased heart rate (Redfern et al., 1999) or altered neuron excitability (Zhu et al., 2014). The system allows...Studies looking at immediate effects of specific neuron firing are better equipped using fast-acting optogenetic...tissues, cell types, or even subcellular regions of a neuron need to be targeted. Targeting is also important...common target for chemogenetic manipulation are neurons where chemogenetic receptors can be used to alter...

... of densely labeled neurons because background fluorescence from inactive neurons is reduced. 105322... of densely-labeled neurons because background fluorescence from inactive neurons is reduced.  Additional...calcium sensors and can be used for imaging of small neuronal processes like dendrites and axons. Find the 7b...Archon1 voltage reporter allows a way to monitor neuronal electrical activity through imaging. Since it’...mDlx enhancer restricts expression to GABAergic interneurons. These vectors can also be used as the control...Plasmid Serotype Name 119036  AAV1  pAAV-hsyn-flex-Voltron-ST Chemogenetics Plasmid Serotype Name ...flex PSAM4 GlyR IRES EGFP Chimeric channels for neuronal manipulation 119742  AAV5  AAV SYN PSAM4 GlyR...

... of densely-labeled neurons because background fluorescence from inactive neurons is reduced. This variant...50457-AAV5): The Synapsin promoter directs broad, neuronal expression. AAV pCAG-FLEX-EGFP-WPRE (51502-AAV5...element directs expression in forebrain GABAergic interneurons. pOTTC1032 - pAAV EF1a Nuc-flox(mCherry)-EGFP...AAV5 and 112677-AAVrg): The EF1a promoter directs strong, ubiquitous expression. This is a Cre-dependent...(jGCaMP7) continue to be useful for monitoring neuronal activity in the form of calcium dynamics. (For...fluorescence and can be used for imaging small neuronal processes (dendrites and axons). jGCaMP7c exhibits...method for understanding how this molecule impacts neuronal circuits. We recently packaged a constitutive ...

...VTA GABA neurons disrupts reward consumption. Neuron. 2012;73(6):1184-94. doi: 10.1016/j.neuron.2012.02.016..., for example, the firing of a specific type of neuron in a specific brain region can result in a particular...wherein mouse initiated optogenetic activation of a neuronal circuit either does or does not result in a reward...consider whether you wish to stimulate or inhibit neuronal firing, the cell-type specificity, the kinetics...suggests that Arch may more effectively silence neurons than HR. Delivering your opsin  The standard opsin...manipulations to help restrict expression to a specific neuronal subtype. However, the length of promoter that ...construct is limited to only a few kb so attaining neuronal specificity through promoter-directed expression...

...to monitor? Neurons, for example, have unusually fast calcium dynamics, whereas non-neuronal systems tend...populations of neurons in vivo to study circuit activity. CaMPARI is available for neuron-specific expression...and target a defined population of cells (e.g., a neuron subtype) and specific subcellular compartments ... rapid measurements, like action potentials in neurons. jGCaMP7 and jGCaMP8 are also available with improved...improved version named CaMPARI2 is available for neuron-specific expression in both zebrafish and mammalian...studies. There are different versions optimized for neuron-specific, astrocyte-specific (GCaMP6 only) or mammalian... to measure changes in calcium specific to the neuronal cell body or the axon (GCaMP6 only). The genetic...

...great in a GABA neuron, for instance, may not transduce the adjacent norepinephrine neuron at all. There... for Defense Behaviors. Neuron, 93(1), 33–47. https://doi.org/10.1016/j.neuron.2016.11.045 More resources...or inducible.  Ubiquitous promoters  These often-strong promoters offer the most flexibility for expression...Promoters such as chicken-beta-actin or CAG can drive strong early gene expression, but can become methylated...expression can still occur (Fig. 3) - for example, some neurons can transiently express TH - so you’ll need to ...expression in both oligodendrocytes (Olig2) and neurons (NeuN). Image from Griffin et al., 2019, Figure...Second, high efficiency transduction coupled with a strong promoter, may result in overexpression of your ...

...whether and how well your FP will function in the environment where it will localize [4]. Most investigators...in or were engineered for use in cytoplasmic environments. However, approximately 40% of human (and most... localize to chemically distinct subcellular environments, including the organelles that compose the secretory...to interfere with the function of resident ER chaperones and may impact cellular function or possibly ...consequences of misfolded dark FP fusions including erroneous quantitation of fusion protein levels. It remains...these compartments using CRISPR systems should strongly consider using moxFPs over standard FPs for the...optimizing fluorescent proteins for subcellular environment. Currently, she is a SPIRE postdoctoral fellow...

...soluble protein CAG: Generic strong promoter. Formulated as AAV-DJ for neuronal expression. CAG-FLEX: CAG... the ability to monitor glutamate release from neurons and other brain cells in vivo. Glutamate plays ...synaptic communication and can trigger other forms of neuronal signaling and regulation. To create iGluSnFR, ...responsiveness, they showed that iGluSnFR could detect neuronal activity in C. elegans, zebrafish, and mice. ...higher expression levels in bacteria and produces stronger fluorescent signals upon sensing glutamate in ...membrane-targeting flavors – PDGFR, GPI, and SGZ, to improve neuronal membrane trafficking and localization. Improved...iGluSnFR3 have higher expression levels and produce stronger fluorescent signals upon sensing glutamate using...

...cell types (astrocytes, microglia, neurons, etc.) and even neuronal phenotype in a mixed population (Burger...have experienced that some vectors are much more prone to recombination and can exhibit recombination levels...your cells.  Some promoters, such as CAG are very strong and may require less viral particles or relatively...tyrosine hydroxylase (TH) promoter for catecholamine neurons or glial fibrillary acidic protein (GFAP) for astrocytes...vectors for off-target expression.  Some classes of neurons transiently express TH during development or have...there is promoter activity. Additionally, some neurons express low levels of GFAP, which can lead to off-target...off-target expression of the packaged transgene in neurons and glia.   Check the recombination rate The first...