Dear fellow scholars,
We are working with a specific group of projection neurons from the brainstem to the spinal cord, and we want to somehow label (genetically, immuno etc) its target neurons in the spinal cord. Have have 2 mouse lines which could be useful, one with cre expression in this group and some other cells, and one with both cre and flp expression in this group and only very few other cells. How can I map the output neurons?
Some ideas I already thought of - mostly for ex vivo preparations:
1) Stimulate the projection neurons (optogenetically, electrically etc) at high intensity to induce cFos expression in target neurons (possibly ex vivo in ACSF). To predominantly target monosynaptic outputs, mephenesin, a drug that diminishes polysynaptic transmission, or maybe change ACSF ion compositions could be done(?) Thoughts on this?
2) Certain viruses have recently been developed which do anterograde monosynaptic jumps. This has potential, but Im not sure how robust this is,and some issues are correctly targeting the neuron group with virus injection (specificity, coverage), and the need for adult animals (I prefer to study developmentally immature animals). Does anyone know any good viruses, that might be cre or flp or both specific?
3) Cre/Flp sensitive transgene mice that could be used?
Any ideas or pointers are welcome. Also criticism on my current ideas, especially cFos and polysynaptic blocking.
Best regards,
Anders
see this paper.
Wolfe A, Divall S, Singh SP, Nikrodhanond AA, Baria AT, Le WW, Hoffman GE, Radovick S. Temporal and spatial regulation of CRE recombinase expression in gonadotrophin-releasing hormone neurones in the mouse. J Neuroendocrinol. 2008; 20(7):909-16. PMID: 18445125
Hi,
my first thought is pseudotyped rabies virus (idea 2). This system is very robust and used widely. However, this is mostly used in the brain. Gaining access to the brainstem to inject and subsequently have the animals survive long enough to get proper expression in the connected cells might be a challenge. I have no experience in that. This paper should get you started on this method Article Monosynaptic Circuit Tracing with Glycoprotein-Deleted Rabies Viruses
Idea 1 is very creative, but will require a lot of work and controls to limit the responses to only the monosynaptically connected neurons. I am not sure of the results will ever fully convince anyone as it will be very hard to limit excitation to monosynpatic connections.
crossing the mouse line with a Cre reporter line would get you reasonably close to what you want. You could label all the Cre positive cells with a fluorescent protein. Than you would have to reconstruct the axons to find out where they project to. Than the problems start. How to truly determine on which cells they terminate? Proximity is not necessarily a synaptic connection. If you do see a synaptic connection, how do you determine what cell type the upstream cell is? You would have to do immunostaining with a pre- and post-synaptic marker to establish that there is indeed a synaptic connection and stain for a marker, (somatostatin, parvalbumin,...) to determine the identity of the upstream neuron.
good luck
Nils
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