...CNS: neurons and astrocytes Mice No (LY6C1)   Hanlon et al., 2019 AAV-BI-28 CNS: neurons, astrocytes...astrocytes, but its neuron transduction encompasses more molecularly-defined neuronal subtypes and with ...and is highly specific for neurons, with less bias toward inhibitory neurons than AAV-PHP.eB (Fig. 4c, ...transduce 80% of dorsal root ganglia (DRG) neurons, as well as neurons located in the enteric nervous system...primate nervous systems. Neuron, 110(14), 2242-2257.e6. https://doi.org/10.1016/j.neuron.2022.05.003 Chen, X...-specific labeling. Neuron,109(9), 1449–1464.e13. https://doi.org/10.1016/j.neuron.2021.03.011 Hanlon,...cell type specificity, or retrograde transport in neurons. (b) Genetic cargo can be customized to control...

...individual neurons and in terms of their interactions with one another. In addition to the neuronal networks... (hM3Dq) into a subset of neurons within the LC of mice. When those neurons are chemogenetically activated...reward circuit, and the medial prefrontal cortex (mPFC) are involved. Neurons from the mPFC project to the...findings by controlling neurons in the mPFC with light. Activation of these neurons via the channel Channelrhodopsin...protein that activates neurons upon light stimulation. When they activated the Drd1 neurons 24 hours before ... "Optogenetic stimulation of medial prefrontal cortex Drd1 neurons produces rapid and long-lasting antidepressant...They also don’t give any clues about the types of neurons involved or whether their involvement is activating...

...for genetically manipulating neurons throughout the whole brain. Neurons of the peripheral nervous system...transduced a higher number of neurons overall (GFP+ cells that also stained for a neuronal marker). Check out tables...for?  1. Multi-color labeling of neurons Multi-color labeling of neurons is for more than making pretty ...single-cell anatomical study and neuronal tracing, aka determining where a neuron’s axons and dendrites extend...in order to decrease the number of neurons that get labeled, neurons are more likely to be transduced by...interest, which, in the case of Chan et al, was neurons and astrocytes. Why screen for IV deliverable ...and GFP intensity and transduction efficiency of neurons were measured. On average, AAV.PHP.eB and AAV.PHP.S...

...method that lets you fire neurons with the flick of a light switch. Neurons are not typically persuaded...remains inactive and neurons don't fire. With blue light, ions flow into the neuron and causes it to fire...ChRger. In both experiments, neurons expressing ChRgers produced stronger photocurrents at low light intensity...activate a neuron. ChRgers, however, have a higher conductance, so they can still depolarize neurons at lower...dopaminergic neurons in the VTA, this should propel the mice to increase their nose-poking behavior. Strong nose-poking...shines on excitatory channelrhodopsin-expressing neurons, the channelrhodopsins respond by opening and allowing... allowing an influx of ions into the neuron which generates an action potential (Figure 1). This light-induced...

...periaqueductal gray (PAG), with red neurons expressing tdTomato and green neurons expressing cFos. Image from ...the activity of parvalbumin (PV)-expressing neurons, neurons that the lab previously found had a role in...light activates the neurons (via oChIEF) and a chemogenetic agonist inhibits the neuron (via hM4D). She fused...on the PV neurons is in progress, she has validated the dual virus method to study CR neurons which are...fluorescence based on the release of glutamate from neurons and this new version of the biosensor is faster... to the collaborative and highly interactive environment that Janelia offers,” says Aggarwal.In addition... in electroporated in rat hippocampal cultured neurons, imaged 14 days post-transfection, excited at 505...

...Genetically Targeted Neurons. Neuron 53:639–647 . https://doi.org/10.1016/j.neuron.2007.01.033 Additional...the previous neuron (Wichersham et al., 2007). However, once rabies dG enters this neuron, it cannot infect...However, pseudotyping allows these viruses to infect neurons. Besides altering the host tropism of your virus...reasons VSV-G is used to transduce stem cells and neurons. However, VSV-G’s broad tropism can be undesired...Rabies glycoprotein The rabies glycoprotein targets neuronal cells and can travel in a retrograde direction...the cell body, making it a great tool to study neuronal connectivity. However, because of rabies virus...Lentiviral vectors are also great at infecting neurons and cause minimal inflammatory response, but are...

...they make the inside of the neuron more positive or more negative.  Neuronal stimulation: Depolarization...depolarization. They therefore function as a neuron “on switch.” Neuronal inhibition: Hyperpolarization Conversely...cord neurons using the Gal4/UAS system. This created fish lines with different groups of neurons that ...activity, enables researchers to silence or incite neuronal firing and study subsequent effects on behavior...cross the cell membrane when exposed to light. Neuronal firing depends on these charged particles and ...Depolarization When the inside of the neuron becomes positive over a certain threshold, it becomes depolarized and...Conversely, when the inside of the neuron becomes more negative it becomes less likely to fire and is said...

...reversible PA-FP is the naturally occurring Dronpa protein (named after dron, a ninja term for vanishing, and pa...both activates and inactivates Dronpa is not ideal, since this means Dronpa is quickly inactivated and less... to find a version of Dronpa that doesn't become inactivated as quickly. Padron is a monomeric, reversible... mutating Dronpa (T59M / V60A / N94I / P141L / G155S / V157G / M159Y / F190S) (7). Padron’s “positive”...switching behavior is the opposite of Dronpa; when exposed to blue light, Padron is photoconverted from a dark...Emission (nm) Dronpa Monomer UV-Violet Dark/Green N/A (high) High 503/ 518 Padron Monomer Blue Dark...Correlative light and electron microscopy (CLEM) is an imaging technique that combines electron microscopy (EM...

...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...

...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...

... 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...