Pretty good read! Their results call the ‘leaky gate’ model proposed by Sun et al. 2017 into question, will be interesting to see how things evolve from here
Thanks for your thoughts @alex_naka.
What do you think about conclusions in light of the relatively low overlap between Cre and Grp+ neurons? More specifically, the fact that about 75% of Grp+ neurons are tdTom (and hence Cre) negative?
Also, the ablation proportion was pretty weak, consistent with the low proportion of Grp+ neurons that express Cre. I need to dig in more but this caught my eye immediately.
On some other notes, things I was happy to see:
- Use of Cre-dependent HSV129 for anterograde tracing from spinal cord. I think this is the first time this tool has been used in spinal cord. Glad to see it works. I tried from DRG retrogradely and it didn’t work for me.
- CLARITY for imaging of complete morphology of DH neurons. This is nice.
Good catch. Looking back at Sun et al. 2017 again, it looks like the correspondence is only okay between the GRP-eGFP and the GRP-Cre line as well.
GENSAT has another Grp line with EGFP expression under the same promoter. Of neurons from the Grp EGFP line, 93% were reported to express Grp mRNA (Solorzano et al., 2015). We crossed the EGFP line with Grp Cre ; ROSA26 LSL-tdTomato . 90.3% of Grp Cre -positive neurons also expressed Grp EGFP (Figures 1C and S1), while 64.1% of Grp EGFP neurons colocalized with Grp Cre , showing that the Grp EGFP line labeled most Grp Cre -positive neurons.
So I guess the takeaway is GRP-Cre more or less labels a subset of GRP-eGFP, which more or less labels a subset of the population of cells that have observable GRP transcripts in the adult. Not sure what to say about it, this is just one of the weaknesses of BAC transgenics I think - the patterns of labeling you get don’t necessarily have to recapitulate endogenous patterns of gene expression.
I think the relatively low levels of efficiency in ablation might be a choice they’re making here
One potentially relevant difference in the experimental design, however, is the route of administration of DTX. In our experiments, we injected DTX locally into the intrathecal space of the lumbar spinal cord and in a hyperbaric solution to prevent diffusion to the brain. S. Sun et al. (2017), by contrast, applied DTX systemically via (intraperitoneal) injections. This difference may be relevant, as the presence of GRP neurons is not restricted to the spinal cord.
But it does seem possible that maybe the discrepancies in their results could also just be from differences in how many GRP neurons are being ablated in the spinal cord.
I did like the anterograde tracing. But I think people think the GRP neurons are all interneurons right? So the things they show must be at least two synapses away. Might have been interesting to do an earlier timepoint and see what the labeling looked like locally