Basic NeuroscienceOptogenetic approaches to characterize the long-range synaptic pathways from the hypothalamus to brain stem autonomic nuclei
Highlights
► Lentiviral vectors were injected into PVN to express ChR2-EYFP under oxytocin, vasopressin or synapsin promoter. ► Only synapsin promoter was strong enough to optogenetically stimulate long-range axon endings in brainstem autonomic nuclei. ► All three promoters could generate action potential firing at PVN cell bodies. ► Crossbreeding floxed ChR2 with Sim1-Cre mice allowed optogenetic PVN neuron stimulation, but not at brain stem distal axons. ► Useful approach to study important hypothalamus-brainstem connections; easily modifiable for other long-range projections.
Introduction
Electrical stimulation of neurons and fibers in-vivo within the central nervous system (CNS) has been used for identifying and assessing the targets and responses of long-range synaptic pathways for many years. Although much seminal work has been done with this approach, it possesses several insurmountable inherent obstacles, including limited control over the spread and spatial resolution of stimulation, confounding stimulation of a heterogeneous population of neurons or axons within the stimulated region, unpredictable effectiveness of stimulation that depends on different thresholds for fibers and different neurons within the region, and also the potential for tissue damage. In-vivo studies are also hindered by the inability to apply pharmacological agents in a highly localized area surrounding the target neuron that receives these long-range projections.
In-vitro electrophysiological approaches developed over the last 20 years overcome some of these limitations, such as permitting tight control over the external and internal milieu surrounding cells and allowing more precise stimulation and electrophysiological recordings from neurons. However studies of pathways within in-vitro preparations are limited by the viable depth of the tissue, typically 400–800 μm, and therefore many of the important long-range projections to neurons cannot be studied in-vitro.
A new approach, using optogenetic techniques, overcomes many of these obstacles and potentially enables highly selective stimulation of homogenous neuronal populations, high temporal precision, and activation of synapses originating from neurons distant from the neuron of interest while retaining the high degree of control and precision afforded in-vitro (Fenno et al., 2011). This approach utilizes heterologous expression of the microbial opsin channelrhodopsin-2 (ChR2), a light-activated cation channel that is ubiquitously inserted into the plasma membrane of cells. Brief (millisecond scale) light pulses can depolarize the membrane of cell bodies, dendrites or axons of neurons that express ChR2, allowing for excitation of specific compartments of the plasma membrane, including presynaptic terminals distant from the soma (Petreanu et al., 2007).
Several recent reports have shown the feasibility of this optogenetic method to study the neurophysiology of distant synapses by driving expression of ChR2 using different viral vectors (Cruikshank et al., 2010, Varga et al., 2009) or transgenic mice (Ren et al., 2011). However, a comprehensive comparative assessment of the different available methods has not been done. For instance, it is not readily clear which of the different methods could best drive expression of ChR2 into the plasma membrane of distal synaptic terminals. In order to extend the current knowledge of how to successfully and efficiently employ optogenetic stimulation of distant axons and identified synaptic endings, we tested and compared the usefulness of lentivirus, with neuron- and population-specific promoters, as well as two ChR2 Cre responder transgenic mouse lines to express ChR2 and selectively stimulate the important neurotransmission from paraventricular hypothalamus nucleus (PVN) to brainstem cardiac vagal neurons (CVNs) that control heart rate in the brainstem.
More specifically we tested lentiviral vectors expressing ChR2 under two PVN promoters (minimal promoter regions upstream of the origin of transcription) for vasopressin and oxytocin neurons, and evaluated their ability to drive ChR2 expression in distal axons in autonomic brain stem sites after microinjection in the PVN. We also used a lentiviral vector with a strong neuron-specific promoter (human synapsin I fragment) (Kugler et al., 2003). In addition, we assessed the expression of ChR2 in paraventriculo-autonomic projections using crossbred transgenic mice that express ChR2 in the PVN. To this purpose, we employed the Cre/LoxP system and crossbred two different ChR2 Cre responder lines with a Sim1-Cre driver transgenic mouse. Sim1 is a gene strongly expressed during embryonic development in PVN neurons, including in vasopressin and oxytocin parvocellular neurons, that project to the nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus (DMV) (Balthasar et al., 2005, Duplan et al., 2009, Fan et al., 1996). In vitro electrophysiology coupled with laser evoked photoactivation of ChR2 was used to assess the functional connectivity of distal autonomic-related PVN projections and to test the effectiveness of each of these approaches in activation of synaptic terminals and transmitter release from PVN neurons onto CVNs.
Section snippets
Lentiviral vector plasmids and promoter constructs
Lentiviral plasmids pLenti-Syn-hChR2(H134R)-EYFP-WPRE, packaging plasmid pCMV-ΔR8.74 and envelope plasmid pMD2.G were all kindly provided by K. Deisseroth (Stanford University, Stanford, CA). We chose to use the H134R mutant for the current studies for its enhanced photocurrents (Nagel et al., 2005) and higher release probability (Schoenenberger et al., 2011), since our objective was to probe long-range projections and maximize the likelihood of release of neurotransmitter from presynaptic
PVN injection site
To examine long-range and selective PVN projections to caudal brain stem sites two PVN-specific lentiviral vectors, one driving ChR2-EYFP expression by an oxytocin promoter and another using a vasopressin promoter, were tested. Both viral vectors successfully infected PVN neurons, with very few to no infected cell bodies outside the boundaries of the PVN. Immunohistochemical analysis confirmed that a very large fraction (93 ± 2.0%) of neurons infected with OXY-ChR2-EYP stained positive for
Discussion
The results of this study demonstrate that a VSVg pseudotyped lentiviral vector with a strong neuronal promoter (human synapsin1 fragment) is able to drive ChR2-EYFP expression in PVN neurons sufficient to optogenetically stimulate the distal pre-autonomic synaptic endings and evoke transmitter release onto CVNs located in the brainstem. Testing lentiviral vectors with PVN specific promoters (minimal upstream promoter regions of AVP and oxytocin genes), we found a very low number of
Conflict of interest
The authors state they have no conflicts of interest.
Acknowledgments
This work was supported by NIH National Heart, Lung, and Blood Institute grants HL-49965, HL-72006, and HL-59895 to DM and American Heart Association predoctoral fellowship to RP. Confocal images were generated with a grant from The NIH National Center for Research Resources 1S10RR025565-01. We thank Norman Lee for the use of tissue culture facilities, Anastas Popratiloff for assistance with confocal microscopy and Tom Maynard for assistance with transgenic mice.
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