Optical vagus nerve modulation of heart and respiration via heart-injected retrograde AAV.

Vagus nerve stimulation has shown many benefits for disease therapies but current approaches involve imprecise electrical stimulation that gives rise to off-target effects, while the functionally relevant pathways remain poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate targeted neural communication with light-sensitive actuators (opsins) and can be targeted to organs of interest based on the location of viral delivery. Here, we tested whether retrograde adeno-associated virus (rAAV2-retro) injected in the heart can be used to selectively express opsins in vagus nerve fibers controlling cardiac function. Furthermore, we investigated whether perturbations in cardiac function could be achieved with photostimulation at the cervical vagus nerve. Viral injection in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Photostimulation using both one-photon stimulation and two-photon holography with a GRIN-lens incorporated nerve cuff, was tested on the pilot-cohort of injected mice. Changes in heart rate, surface electrocardiogram, and respiratory responses were observed in response to both one- and two-photon photostimulation. The results demonstrate feasibility of retrograde labeling for organ targeted optical neuromodulation.

Intravascular injections of adenoassociated viral vector serotypes rh10 and PHP.B transduce murine sciatic nerve axons.

Adenoassociated viral vectors provide a safe and robust method for expression of transgenes in nondividing cells such as neurons. Intravenous injections of these vectors provide a means of transducing motoneurons of peripheral nerves. Previous research has demonstrated that serotypes 1, rh10 and PHP.B can transduce motor neuron cell bodies in the spinal cord, but has not quantified expression in the peripheral nerve axon. Axonal labeling is crucial for optogenetic stimulation and detection of action potentials in peripheral nerve. Therefore, in this study, serotypes 1, PHP.B, and rh10 were tested for their ability to label axons of the murine sciatic and tibial nerve following intravenous injection. Serotype rh10 elicits expression in 10% of acetylcholine transferase positive axons of the sciatic nerve in immunohistochemically-stained sections. Serotype rh10 transduces a variety of axon diameters from <1-12 µm, while PHP.B transduces larger axons of diameter (4-16 µm). Expression was not seen with serotype 1. These results show the potential of serotypes PHP.B and rh10 delivery of transgenic products to axons of the peripheral nerve.

An automated method for the quantification of transgene expression in motor axons of the peripheral nerve.

BACKGROUND: Determination of transgene expression in motor axons of peripheral nerves is important in evaluating the effectiveness of viral transduction. Currently only manual and semi-automatic methods of quantification have been employed for quantification in immunolabeled nerve sections, but automatic methods exist for axon counting only in brightfield sections. Manual and semi-automatic methods can suffer from inter- and intraobserver bias, sampling bias and can be time consuming to implement. NEW METHOD: A fully automated method using ImageJ and the Nucleus Counter plugin was developed to quantify the fraction of green fluorescent protein (GFP) labeled acetylcholine transferase positive axons in triple immunolabeled peripheral nerve sections. This method utilizes the Nucleus Counter to generate axonal regions of interest which are quantified for colocalization with GFP expression and nonoverlap with Laminin. Thresholding using histograms generated from control animals is used to remove noise. RESULTS: The automated method is able to successfully distinguish transgenic GFP expressing mice from wild type. Using computer generated peripheral nerve sections, the automated method has less than 5% error at signal-to-noise ratios greater than 10% of baseline. COMPARISONS WITH EXISTING METHODS: This method has comparable performance in false positive rates (<1%) and a 95% predictive interval that closely matches existing fully automated methods for quantification in brightfield sections. It outperforms the intra- and interobserver differences of manual and semi-automated methods for quantification. CONCLUSIONS: This automated quantification method provides a fast and robust means of determining the fraction of labeled axons in peripheral nerve sections.

Optical read-out and modulation of peripheral nerve activity.

Numerous clinical and research applications necessitate the ability to interface with peripheral nerve fibers to read and control relevant neural pathways. Visceral organ modulation and rehabilitative prosthesis are two areas which could benefit greatly from improved neural interfacing approaches. Therapeutic neural interfacing, or 'bioelectronic medicine', has potential to affect a broad range of disorders given that all the major organs of the viscera are neurally innervated. However, a better understanding of the neural pathways that underlie function and a means to precisely interface with these fibers are required. Existing peripheral nerve interfaces, consisting primarily of electrode-based designs, are unsuited for highly specific (individual axon) communication and/or are invasive to the tissue. Our laboratory has explored an optogenetic approach by which optically sensitive reporters and actuators are targeted to specific cell (axon) types. The nature of such an approach is laid out in this short perspective, along with associated technologies and challenges.

Imaging of electrical activity in small diameter fibers of the murine peripheral nerve with virally-delivered GCaMP6f.

Current neural interfaces are hampered by lack of specificity and selectivity for neural interrogation. A method that might improve these interfaces is an optical peripheral nerve interface which communicates with individual axons via optogenetic reporters. To determine the feasibility of such an interface, we delivered the genetically encoded calcium indicator GCaMP6f to the mouse peripheral nerve by intramuscular injection of adenoassociated viral vector (AAV1) under the control of the CAG (chicken beta actin- cytomegalovirus hybrid promoter). Small diameter axons in the common peroneal nerve were transduced and demonstrated electrically inducible calcium transients ex vivo. Responses to single electrical stimuli were resolvable, and increasing the number of stimuli resulted in a monotonic increase in maximum fluorescence and a prolongation of calcium transient kinetics. This work demonstrates the viability of using a virally-delivered, genetically-encoded calcium indicator to read-out from peripheral nerve axons.

Anion channels transport ATP into the Golgi lumen.

Anion channels provide a pathway for Cl(-) influx into the lumen of the Golgi cisternae. This influx permits luminal acidification by the organelle's H(+)-ATPase. Three different experimental approaches, electrophysiological, biochemical, and proteomic, demonstrated that two Golgi anion channels, GOLAC-1 and GOLAC-2, also mediate ATP anion transport into the Golgi lumen. First, GOLAC-1 and -2 were incorporated into planar lipid bilayers, and single-channel recordings were obtained. Low ionic activities of K(2)ATP added to the cis-chamber directly inhibited the Cl(-) subconductance levels of both channels, with K(m) values ranging from 16 to 115 microM. Substitution of either K(2)ATP or MgATP for Cl(-) on the cis, trans, or both sides indicated that ATP is conducted by the channels with a relative permeability sequence of Cl(-) > ATP(4-) > MgATP(2-). Single-channel currents were observed at physiological concentrations of Cl(-) and ATP, providing evidence for their importance in vivo. Second, transport of [alpha-(32)P]ATP into sealed Golgi vesicles that maintain in situ orientation was consistent with movement through the GOLACs because it exhibited little temperature dependence and was saturated with an apparent K(m) = 25 microM. Finally, after transport of [gamma-(32)P]ATP, a protease-protection assay demonstrated that proteins are phosphorylated within the Golgi lumen, and after SDS-PAGE, the proteins in the phosphorylated bands were identified by mass spectrometry. GOLAC conductances, [alpha-(32)P]ATP transport, and protein phosphorylation have identical pharmacological profiles. We conclude that the GOLACs play dual roles in the Golgi complex, providing pathways for Cl(-) and ATP influx into the Golgi lumen.