Optogenetic approaches to characterize the long-range synaptic pathways from the hypothalamus to brain stem autonomic nuclei.
Recent advances in optogenetic methods demonstrate the feasibility of selective photoactivation at the soma of neurons that express channelrhodopsin-2 (ChR2), but a comprehensive evaluation of different methods to selectively evoke transmitter release from distant synapses using optogenetic approaches is needed. Here we compared different lentiviral vectors, with sub-population-specific and strong promoters, and transgenic methods to express and photostimulate ChR2 in the long-range projections of paraventricular nucleus of the hypothalamus (PVN) neurons to brain stem cardiac vagal neurons (CVNs). Using PVN subpopulation-specific promoters for vasopressin and oxytocin, we were able to depolarize the soma of these neurons upon photostimulation, but these promoters were not strong enough to drive sufficient expression for optogenetic stimulation and synaptic release from the distal axons. However, utilizing the synapsin promoter photostimulation of distal PVN axons successfully evoked glutamatergic excitatory post-synaptic currents in CVNs. Employing the Cre/loxP system, using the Sim-1 Cre-driver mouse line, we found that the Rosa-CAG-LSL-ChR2-EYFP Cre-responder mice expressed higher levels of ChR2 than the Rosa-CAG-LSL-ChR2-tdTomato line in the PVN, judged by photo-evoked currents at the soma. However, neither was able to drive sufficient expression to observe and photostimulate the long-range projections to brainstem autonomic regions. We conclude that a viral vector approach with a strong promoter is required for successful optogenetic stimulation of distal axons to evoke transmitter release in pre-autonomic PVN neurons. This approach can be very useful to study important hypothalamus-brainstem connections, and can be easily modified to selectively activate other long-range projections within the brain.
Department of Pharmacology and Physiology, The George Washington University, 2300 Eye Street NW, Washington, DC 20037, USA. rpinol@gwmail.gwu.edu
Ramón A
RA
Piñol
Ryan
R
Bateman
David
D
Mendelowitz
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D001426:Bacterial Proteins / Q000235:genetics; D001703:Biophysics; D001933:Brain Stem / Q000166:cytology*; D000075402:Channelrhodopsins; D004558:Electric Stimulation; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects; D005725:Ganglia, Autonomic / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D006321:Heart / Q000294:innervation; D006801:Humans; D007031:Hypothalamus / Q000166:cytology*; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D009434:Neural Pathways / Q000502:physiology*; D009474:Neurons / Q000378:metabolism; D062308:Optogenetics; D010121:Oxytocin / Q000235:genetics / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D026941:Sodium Channel Blockers / Q000494:pharmacology; D013569:Synapses / Q000378:metabolism / Q000502:physiology*; D016704:Synapsins / Q000235:genetics / Q000378:metabolism; D013779:Tetrodotoxin / Q000494:pharmacology; D014161:Transduction, Genetic / Q000379:methods; D014633:Valine / Q000031:analogs & derivatives / Q000494:pharmacology; D014667:Vasopressins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D000075402:Channelrhodopsins; D018691:Excitatory Amino Acid Antagonists; D008164:Luminescent Proteins; D026941:Sodium Channel Blockers; D016704:Synapsins; C054562:yellow fluorescent protein, Bacteria; D014667:Vasopressins; D013779:Tetrodotoxin; D010121:Oxytocin; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; C056639:2-amino-5-phosphopentanoic acid; D014633:Valine