Optogenetic approaches to characterize the long-range synaptic pathways from the hypothalamus to brain stem autonomic nuclei

Abstract

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.

Fig. 1

Subpopulation-specific lentiviral vector OXY-ChR2-EYFP expresses ChR2-EYFP in PVN neurons and in distal axons in the brain stem. Confocal images of immunohistochemical staining of the PVN of an animal injected with the OXY-ChR2-YFP lentiviral vector for ChR2-EYFP (a), oxytocin (b), vasopressin (c) and the merged image (d), each with a panel underneath with higher magnification. Scale bar is 90 μm for the top images, and 10 μm for the more detailed panels below (a–d). (e) Distal axons of PVN neurons infected with OXY-ChR2-EYFP in the dorsal vagal complex in the brainstem (scale bar represents 150 μm). (f) Schematic depiction of rat brain in sagital orientation, indicating rostro-caudal levels at which slices are taken. Slices containing the PVN (green) are taken at the level of the rostral gray line and brain stem slices containing CVNs in the DMV and Amb (both nuclei represented in red) at the caudal gray line. Schematic picture was adapted from Paxinos and Watson (2007). Hypoglossal Nucleus; 3V, Third Ventricle; cc, Central Canal; DMV, Dorsal Motor Nucleus of the Vagus; NTS, Nucleus of the Solitary Tract.

Fig. 2

Optogenetic stimulation of PVN neurons expressing ChR2 25–30 days after injection of lentiviral vector OXY-ChR2-EYFP or AVP-ChR2-EYFP. (a) 10 consecutive (gray) and average (black) voltage-clamp traces (3 ms stimulation at 1 Hz) of OXY-ChR2-EYFP expressing neurons in the PVN recorded in the presence of AP5, CNQX and TTX. In this and all subsequent panels the blue bar represents the period of blue laser exposure. (b) Representative current-clamp traces of consecutive stimulations (3 ms, at 1 Hz) of OXY-ChR2-EYFP PVN neurons recorded in the presence of AP-5 and CNQX. (c) Reliable action potential firing during 10s stimulation at 10 Hz in OXY-ChR2-EYFP PVN neurons; detail shows 10 consecutive action potentials, (d) 10 consecutive (gray) and average (black) voltage-clamp traces (3 ms stimulation at 1 Hz) of AVP-ChR2-EYFP PVN neurons in the recorded in the presence of AP5, CNQX and TTX. (e) Representative current-clamp traces of consecutive stimulations (3 ms, at 1 Hz) of AVP-ChR2-EYFP PVN neurons recorded in the presence of AP-5 and CNQX. 12,

Fig. 3

Lentiviral vector with strong neuronal promoter, Syn-ChR2-EYFP, expresses ChR2 in PVN neurons and optogenetic stimulation of long-range PVN projections in brain stem evokes EPSCs in CVNs (a) Syn-ChR2-EYFP expresses ChR2-EYFP in PVN neurons. Scale bar represents 50 μm. (b) 10 consecutive (gray) and average (black) voltage-clamp traces (3 ms stimulation at 1 Hz) of Syn-ChR2-EYFP expressing neurons in the PVN recorded in the presence of AP5, CNQX and TTX. (c) Representative current-clamp traces of 10 consecutive stimulations (3 ms, at 1 Hz) of Syn-ChR2-EYFP PVN neurons recorded in the presence of AP-5 and CNQX. (d) Dense network of distal axons of PVN neurons in brainstem dorsal vagal complex of Syn-ChR2-EYFP rat, 28 days after injection with inset of PVN injection site (scale bars represent 150 μm). (e) Cardiac vagal neurons (red) are surrounded by ChR2-EYFP PVN axons in the nucleus ambiguus (scale bar represents 25 μm). (f) Detail of (e) showing close apposition PVN axons to a CVN (scale represents 10 μm). (g) 7 consecutive (gray) and average (black) responses to 3 ms stimulation (at 2 Hz) of a CVN in the DMV. (h) Bath application of gabazine and strychnine reveals that response is an EPSC, which is completely blocked by AP-5 and CNQX (laser stimulation: 1ms at 2Hz). (i) 12 representative (gray) and average (black) responses to paired-pulse stimulation (3 ms at 10 Hz, 20s between sweeps) of a CVN in the DMV show paired-pulse facilitation. 12, Hypoglossal Nucleus; 3V, Third Ventricle; cc, Central Canal; DMV, Dorsal Motor Nucleus of the Vagus; NTS, Nucleus of the Solitary Tract.

Fig. 4

Sim1-Cre mice crossbred with ChR2 Cre-responder mice yields ChR2 expression in PVN (a) Tile scan of the bilateral PVN of ChR2-tdTomato/Sim1 mouse at P35 (scale bar represents 150 μm). (b) 10 consecutive (gray) and average (black) voltage-clamp traces (3 ms stimulation at 1 Hz) of PVN neuron of ChR2-tdTomato/Sim1 mouse. (c) PVN of P12 ChR2-EYFP/Sim1 mouse (scale bar represents 50 μm). (d) 10 consecutive (gray) and average (black) voltage-clamp traces (3 ms stimulation at 1 Hz) of PVN neuron of ChR2-EYFP/Sim1 mouse. (e) 8 consecutive action potentials of PVN neuron in ChR2-EYFP/Sim1 when stimulated briefly (3ms) at 0.05 Hz. 3V, Third Ventricle