Tag Archives: Expression

Long-term channelrhodopsin-2 expression can induce abnormal axonal morphology and targeting in cerebral cortex

When using optogenetics to study circuit function or animal behavior, a critical prerequisite is that optogenetic protein expression does not, in itself, perturb the circuit being studied. While short-term expression is very commonly used without observable circuit disruption, whether this is also true for long-term expression is less clear. A recent paper by Miyashita et al. in Frontiers in Neural Circuits shows that long-term, high-level expression of ChR2 can induce abnormal axonal morphology and targeting in cerebral cortex. This underscores the importance of using the lowest expression possible, particularly for long-term studies.

Miyashita et al. expressed a common construct, CAG::hChR2 (H134R)-EYFP-WPRE, in L2/3 pyramidal neurons in rat somatosensory cortex via in utero electroporation (IUE). This same strategy was used in several prior studies of S1 circuit function, with one important difference: Miyashita et al. expressed hChR2 that was codon-optimized for mammalian expression, while prior studies expressed native ChR2 (discussed below). This strategy successfully conferred light-evoked spiking in vivo and in in vitro brain slices. However, long-term expression (> 40 d) also caused major abnormalities in axonal morphology, which included cylinders of axonal membrane that enveloped pyramidal cell proximal dendrites, and spherical, calyx-like axonal swellings that surrounded neuron cell bodies. These … Continue reading

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Stop lights for temporal lobe seizures

Unlike electrical stimulation, optogenetics allows neuronal manipulation with great cell-type specificity, with light directly affecting only those cells expressing opsins. In a recent report in Nature Communications, Krook-Magnuson et al harnessed this specificity to stop seizures in vivo in a mouse model of temporal lobe epilepsy. Mice were implanted with electrodes to record brain activity and 200µm thick optical fibers to deliver light to the brain. A closed-loop, on-demand responsive system detected seizures in real time, allowing temporal specificity, in addition to the cell-type specificity achieved through selective opsin expression. Specifically, the authors either selectively inhibited excitatory principal cells or, alternatively, excited a subpopulation of GABAergic inhibitory neurons in the hippocampus by delivering light at the time of a seizure. Both approaches proved successful, despite the less than 5% of illuminated neurons expressing opsins in the latter approach. Light arrested ongoing electrical seizure activity and reduced the incidence of events progressing to overt behavioral seizures.

Epilepsy, a condition of recurrent, spontaneous seizures, is a prevalent disorder, with 1 out of 26 people developing epilepsy during their lifetime. Unfortunately, for over 40% of patients, seizures cannot be controlled with current treatment options. Temporal lobe epilepsy, the most common form of epilepsy … Continue reading

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KENGE-tet System for Expanding the Repertoire of Optogenetically Targeted Cells

Optogenetics has proven to be a powerful tool capable of manipulating the activity of a specific population of cells in a complex multicellular organism. This approach is enthusiastically pursued in recent neuroscience field and the causal relationship between neural activity and behavior is finally starting to become unveiled. However, most studies utilize virus mediated gene transfer for the induction of light-sensitive proteins, such as channelrhodopsin-2 (ChR2), and such method inevitably introduces surgical injuries and variability of expression between trials. Therefore, transgenic approach has long been sought, however, satisfying the demands of the specificity as well as the abundance of expression were difficult.

In a recent paper published in the Cell Reports, Tanaka and Matsui and their colleagues at the National Institute for Physiological Sciences (Okazaki, Japan) established Knockin-mediated ENhanced Gene Expression by improved tetracycline-controlled gene induction system (KENGE-tet). The authors found that high levels of tTA-mediated transcription can be achieved by knocking in tetO-ChR2 cassette into a locus at a housekeeping gene, beta-actin. The authors crossed this tetO-ChR2 knockin mouse with 7 different tTA lines and achieved ChR2 expression in specific cell-types including sub-populations of neurons, astrocytes, oligodendrocytes, and microglial cells. In all cases, the level of ChR2 expression was … Continue reading

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Transcriptional amplification with two rAAVs using tetracycline-controlled gene induction

Cell-type specific expression of genetically encoded indicators or optogenetic probes is often hampered by the use of promoters that are specific but drive expression only weakly. In a recent paper of the Wang Lab at Princeton University, the authors describe a means of introducing a calcium indicator using the TET promoter system in the cerebellar cortex to boost expression by about ten-fold, as determined by quantitative determination of intracellar concentration. Kuhn et al. show specific labeling of Purkinje cells and all interneuron types. Together with a previous paper of the Wang Lab where glial cells were targeted, now nearly all cell types of the cerebellar cortex can be selectively labeled. Additionally, Kuhn et al. overcome cell toxicity associated with rAAV injection and/or local GECI overexpression by systemic pre-injection of hyperosmotic D-mannitol, doubling the time window for functional imaging.

Original article by Kuhn et al.

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Novel AAVs for Cre-dependent control of transgene expression

Recombinant adeno-associated viruses (rAAVs) designed to activate transgene expression in only those cells expressing Cre-recombinase (Cre-On) are widely used to introduce optogenetics constructs into specific cell types and brain regions. In many experiments, what functionally distinguishes Cre-expressing cells from their non-Cre expressing neighbors is not fully understood. A recent paper by Saunders et al. (Sabatini lab, Harvard Medical School) published in Frontiers in Neural Circuits describes two rAAV strategies that allow for simultaneous Cre-On and Cre-Off transgene expression. One strategy (Cre-Switch) achieves differential transgene expression with a single rAAV. The second strategy introduces a Cre-Off vector (FAS), built with lox sites that do not efficiently recombine with loxP or lox2272 sites, which allow FAS rAAVs to be used simultaneously with popular Cre-On DIO (double-floxed inverted ORF) a.k.a FLEX (flip-excision) rAAVs. All Cre-On, Cre-Off, and Cre-Switch rAAV vectors in Saunders et al. are freely available from Addgene.

Original article by Saunders et al.

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The GCaMP3 reporter strain published

A couple months ago the latest Cre-dependent optogenetic reporter mouse lines were published in a Nature Neuroscience paper. But one particular line, the GCaMP3 reporter strain, was missing at roll call. This line was published almost at the same time in the Journal of Neuroscience. In the February 29 issue of J. Neurosci, Zariwala et al. show that when crossed with Cre lines the Ai38 line yields stable GCaMP3 expression without the typical toxicity observed with AAV infections (which correlates with GCaMP3 diffusing into the nucleus and is observed as early as 4-6 weeks after infection). For a side-by-side comparison of GCaMP3 and Oregon Green BAPTA-1 (OGB-1), the authors looked at the visual cortex of anesthetized mice. GCaMP3-expressing pyramidal cells of the visual cortex (obtained using a Wfs1-Tg2-Cre line) showed preserved orientation selectivity to moving oriented gratings. Interestingly, the maximum ΔF/F achieved in GCaMP3-expressing neurons was substantially higher than with OGB-1. On the opposite, OGB-1 gave higher ΔF/F for low responder cells. One downside is that imaging from the Ai38 line required about 3 x more laser power than when using AAVs or OGB-1. But overall it looks like GcaMP reporter strains are on the right track and already experiment-ready. Now the … Continue reading

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New Cre-dependent optogenetic reporter lines

Cre-dependent mouse strains that would express optogenetic control tools have been seen as the holy grail for the past 6 years. Not only such strains would simplify experimental procedures by eliminating the need for gene delivery (in utero electroporation or viral infection) but they would potentially yield more reproducible experiments by providing more homogeneous and predictable expression levels in a given neuronal population. Many labs gave it a try and failed. The challenge resided in achieving high expression using a ubiquitous promoter and a Cre-activated cassette (principle below).

Some time ago it seemed like the folks at the Allen Institute were on a promising track and their first ChR2 reporter strain called Ai27 leaked out to several labs. But many were discouraged by the insufficient expression level of this line and the small light-evoked responses obtained with it. But the Ai27 mouse was just the beginning. Last year the Allen Institute released a new line, codename Ai32, with significantly improved ChR2 expression. Apparently the same ones which were disappointed by the Ai27 line were pretty unanimous about the good results of this Ai32 mouse. The word spread quickly and the mouse was made available through the Jackson Laboratory together with … Continue reading

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