Forsaking cre

I read an interesting paper that came out recently, by Garau et al. about an inhibitory AgRP to Orexin connection that mediates exploratory behaviours1. They use optogenetics and photometry to investigate the role of Orexin neurones in some exploratory behaviours, with particular relevance to valence and anxiety.

I’m particularly interested in their use of AAV’s to investigate the Orexin neurones in this paper. I have had serious issue trying to investigate Orexin neurones in my own work, because of the lack of suitable cre-driver lines (we tried an Orexin-cre in the past, but were unable to get any detectable cre recombination using AAV’s or crossing with reporter lines). However, Garau et al. forsake the usual cre-dependent AAV’s when it comes to investigating the Orexin neurones, and instead use Orexin promoter-driven expression of GCaMP6 or inhibitory ArchT.

Trying to copy the promoter region for a gene can be troublesome, particularly if you want to then package it inside an AAV (which can only take around 5-6kb, whereas promoter regions can extend for 10’s of kb). However, it seems to work well for the Orexin gene, and has been done previously by Saito et al. to drive tdTomato in a reportor AAV2. Here, Garau et al. report that their Orexin-ArchT AAV tags around 60% of Orexin neurones, with an extremely high fidelity of over 99% (Figure 1). This is important, because it means that, while they might miss some number of the Orexin neurones, they don’t have off-target expression.

They also demonstrate using current-clamp electrophysiology that the ArchT functions as expected in the Orexin neurones (Figure 1C; always an important control to check in any new optogenetics paradigm you use). They then proceed to show a definitive effect of silencing the Orexin neurones in vivo on real-time place preference (Figure 1E), which is a test I have used as well, and I find very useful in its simplicity.

I won’t go into more detail here, but it is an interesting paper, and I would recommend anyone reading this to go and check it out.

1 Garau et al. (2020) J Physiol 598.19 pp 4371-4383 Orexin neurons and inhibitory AgRP -> orexin circuits guide spatial exploration in mice.

2 Saito et al. (2013) Front Neural Circ 7: 192 GABAergic neurons in the preoptic area send direct inhibitory projections to orexin neurons.