The marked diversity of unique cortical enhancers enables neuron-specific tools by Enhancer-Driven Gene Expression

Abstract

SUMMARY 15Understanding neural circuit function requires individually addressing their component parts: specific 16 neuronal cell types. However, the precise genetic mechanisms specifying neuronal cell types remain 17 obscure. While most genes are expressed in the brain, the vast majority are expressed in many different 18 kinds of neurons, suggesting that promoters are not sufficiently specific to distinguish cell types. We 19 therefore examined distal genetic cis-regulatory elements controlling transcription (i.e. enhancers) in 20 closely related mouse cortical subregions. We identified thousands of novel putative enhancers, many 21 unique to particular cortical subregions. Remarkably, following pronuclear injection of constructs 22 containing such enhancers, we obtained transgenic lines driving expression in distinct sets of cells 23 specifically in the targeted cortical subregions. This not only helps illuminate the genetic mechanisms 24 underlying the specification of diverse neuronal cell types, it provides a general strategy for the 25 development of genetic tools targeting any neuronal circuit of interest via Enhancer-Driven Gene 26

Generation of viral vectors specific to neuronal subtypes of targeted brain regions by Enhancer-Driven Gene Expression (EDGE)

“…Our prior work showed that identification of cis-regulatory elements uniquely active in finely dissected cortical subregions allows one to generate genetic tools specific to cells in that subregion, an approach we call EDGE (Enhancer-Driven Gene Expression) 11 . Here we show that one can use the same approach to make rAAVs with similar specificity in both mouse and rat, provided the vector and minimal promoter's innate transcriptional activity is minimized.…”

“…serotype, precise injections of minute quantities), we took steps to ensure that any observed specificity comes from the enhancer element used. First and foremost, we used an enhancer specific to a known subset of neurons in the entorhinal cortex 11 in transgenic animals and analyzed whether the rAAV construct could match this specificity. Figure 1A shows the expression pattern obtained from crossing one of the MEC13-53 tTA driver lines to a payload line expressing the helper transgenes for the ΔG-rabies monosynaptic tracing system 11 .…”

“…First and foremost, we used an enhancer specific to a known subset of neurons in the entorhinal cortex 11 in transgenic animals and analyzed whether the rAAV construct could match this specificity. Figure 1A shows the expression pattern obtained from crossing one of the MEC13-53 tTA driver lines to a payload line expressing the helper transgenes for the ΔG-rabies monosynaptic tracing system 11 . Expression in this cross was limited to reelin-positive (RE+), calbindin-negative (CB-) excitatory projection neurons in LII of the medial and lateral EC (i.e.…”

“…It is based upon identifying the cis-regulatory elements uniquely active in particular brain regions and combining them with a heterologous minimal promoter. When we used this strategy to make transgenic mice, they were indeed significantly more specific than the presumed parent gene, often driving expression in particular sets of neurons in the brain region they were derived from 11 . However, while transgenic animals are powerful tools for the analysis of neural circuits, they do have some serious drawbacks.…”

Inhibitory cell populations depend on age, sex, and prior experience across a neural network for Critical Period learning

“…There are multiple inhibitory cell subtypes in the brain, and diversity within each of them (Murphey et al, 2014;Blankvoort et al, 2018). Here, we focused on subtypes defined by GAD65 (a GABA-synthesizing enzyme) and parvalbumin (a calcium-binding protein) expression because of their central roles in controlling the CP for plasticity in the mammalian primary visual cortex, the best-studied CP (Hensch et al, 1998;Takesian and Hensch, 2013;Davis et al, 2015).…”