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.