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The central vocal pathway of the African clawed frog, Xenopus laevis, is a powerful vertebrate model to understand mechanisms underlying central pattern generation. However, fast and efficient methods of introducing exogenous genes into the neurons of adult X. laevis are currently not available. Here, we systematically tested methods of transgene delivery into adult X. laevis neurons. Although successfully used for tadpole neurons for over a decade, electroporation was not efficient in transfecting adult neurons. Similarly, adeno-associated virus (AAV) was not reliable, and lentivirus (LV) failed to function as viral vector in adult Xenopus neurons. In contrast, vesicular stomatitis virus (VSV) was a fast and robust vector for adult X. laevis neurons. Although toxic to the host cells, VSV appears to be less virulent to frog neurons than they are to mice neurons. At a single cell level, infected neurons showed normal physiological properties up to 7 days post infection and vocal circuits that included infected neurons generated normal fictive vocalizations up to 9 days post infection. The relatively long time window during which the physiology of VSV-infected neurons can be studied presents an ideal condition for the use of optogenetic tools. We showed that VSV does not gain entry into myelinated axons, but is taken up by both the soma and axon terminal; this is an attractive feature that drives transgene expression in projection neurons. Previous studies showed that VSVs can spread across synapses in anterograde or retrograde directions depending on the types of glycoprotein that are encoded. However, rVSV did not spread across synapses in the Xenopus central nervous system. The successful use of VSV as a transgene vector in amphibian brains not only allows us to exploit the full potential of the genetic tools to answer questions central to understanding central pattern generation, but also opens the door to other research programs that focus on non-genetic model organisms to address unique questions.
FIGURE 1. Electroporation setup used for tadpoles in vivo (A), for adult brain in vitro (B), and adult brain in vivo (C). A glass pipette containing plasmid suspension was lowered into the brain (AâC), and plasmid was pressure-injected. Following the injection, electrical pulses were applied to a pair of platinum electrodes placed on the two sides of the injection area. A small amount of calcium-free Steinberg solution (tadpoles) or frog saline (adults) were applied to cover the tips of the two electrodes along with the injection area. (C) Transverse section of the telencephalon at the level indicated as (C) in (B).
FIGURE 2. Cells labeled with fluorescent proteins after electroporation and Adeno-associated virus (AAV) injection. (A) Yellow fluorescent protein (YFP)-labeled tadpole neurons 1 day after plasmids were electroporated in vivo. (B) Transverse section of lefttelencephalon of adult X. laevis 5 days after plasmids were electroporated in vivo. LV indicates lateralventricle. (C) Green fluorescent protein (GFP)-expressing cells (inset shown in C) that lack processes of neurons, presumed to be glial cells. (D) Labeled processes of glial-like cells (inset shown in C) that seem to form the ependymal lining of the ventricle. (E) Vimentin-positive radial glial cells in the adult telencephalon. (F) YFP-labeled telencephalic neurons of one of the very few brains that expressed reporter genes in response to AAV injection. (G) Membrane potential of a presumed glial cell electroporated with plasmid (pCS2FA.ChR2.YFP) positive for YFP and Channelrhodopsin 2 (ChR2). In response to blue light exposure (indicated in blue lines below the trace), the membrane potential depolarized, presumably because of the ChR2 expression, but the cell never spiked an action potential.
FIGURE 3. Neuroanatomical evidence that recombinant vesicular stomatitis virus encoding its own glycoprotein [rVSV(VSV-G)] transduce adult X. laevis neurons (i.e., transgene was moved from the virus to the neurons). (A) Adult X. laevis neurons in telencephalon expressing reporter gene (Venus 2) 48 h after injection of rVSV(VSV-G) plaque 21. Transduction efficiency was 86% (24/28 animals). (B) A lone infected neuron in optic tectum away from the injection site with a robust expression of reporter gene in dendrites, axons, dendritic spines and synaptic boutons observed 68 h after injection of rVSV(VSV-G) plaque 21. A dotted area is enlarged in the bottom left. (C) Neurons in laryngeal motor nucleus (n.IX-X) expressing Venus 2 48 h after injection of rVSV(VSV-G) plaque 21. The transduction efficiency in the brainstem with this strain of virus was 50% (9/18 animals). Scale bar is 10 μm, and applies for all three panels.
FIGURE 4. Neurons infected with vesicular stomatitis virus (VSV) are physiologically functional. (A) Differential interference contrast (DIC) image of an adult X. laevis telencephalic neuron expressing reporter gene (enhanced green fluorescent protein, eGFP) in a whole-cell patch-clamp configuration. The brain was injected with recombinant VSV encoding rabies glycoprotein [rVSV(RABV-G)] 2 days prior to the recordings was obtained. (A1) Membrane potential in response to current injections into the neuron shown in (A). (B) A DIC image of a non-labeled telencephalic neuron near the neuron shown in (A) on the same slice preparation. (B1) Membrane potential of neuron B in response to current injection. (C) Schematic diagram illustrating the unilateral injection of recombinant vesicular stomatitis virus encoding its own glycoprotein [rVSV(VSV-G)] into the laryngeal motor nucleus, n.IX-X. (D) Horizontal section of the brainstem showing the labeled neurons in the n.IX-X (encircled in dotted white line) 2 days after rVSV(VSV-G) was injected. Reporter gene expression was observed in the somata and axons (inset) of vocal motoneurons. White arrowheads indicate the midline. (E) A fictive advertisement call elicited from the isolated infected brain shown in (D) in response to the application of serotonin (5-HT). The advertisement call is made of fast and slow trills (labeled below the traces). Extracellular recordings obtained from the left (blue) and right (red) laryngeal nerves are shown. Inset shows enlarged sections of the left and right extracellular recording traces superimposed to demonstrate the temporal synchrony.
FIGURE 5. Neurons transduced by vesicular stomatitis virus (VSV) around the injection site. (A) Neurons with reporter gene expression near the injection site of recombinant vesicular stomatitis virus encoding its own glycoprotein [rVSV(VSV-G)] in the telencephalon 2 days post infection. There was a robust expression of reporter genes about 400 μm surrounding the injection site. A white arrow indicates the injection site. (B) In addition to labeled somata, there are dense processes that are labeled around the injection site. (C) The nerve injection site shown in the isolated brain. (D) Cross section of the laryngeal nerve 24 days post injection. (D1) Enlarged section of perforated rectangle shown in (E). Note that the labeling is concentrated on the periphery of the axons (a, in inset on the right). (E) The horizontal section of the brainstem of a male X. laevis with rVSV(VSV-G) injected into the cranial nerve IX-X 24 days prior. The white line encircle the n.IX-X. There was no labeled neurons nor axons found in the brainstem or the nerve. (E1) Enlarge section of a white perforated rectangle shown in (E). Labeled cells seen are autofluorescent red blood cells in the blood vessels (amphibian red blood cells are nucleated).
FIGURE 6. Retrograde infection of recombinant vesicular stomatitis virus (VSV) missing the glycoprotein (G) gene from its genome, but pseudotyped with the wild-type VSV-G [rVSVÎG(VSV-G)]. VSV infect the neurons via axon terminal. (A) A cartoon showing the dorsal view of the X. laevis brain with the unilateral injection of rVSVÎG(VSV-G) into the left anterior laryngeal motor nucleus (n.IX-X). (B) Nine days after injection, labeled somata with lateral axonal projections were found in ipsilateral dorsal tegmental area of medulla (DTAM). (C) In the contralateral DTAM, there were no labeled neuron somata but some labeled processes (white arrow heads). (D) Left n.IX-X injected with rVSVÎG(VSV-G) shows a large number of labeled neurons. (E) The contralateral n.IX-X show no labeled neurons, but labeled axons are seen (white arrow heads).
FIGURE 7. Recombinant vesicular stomatitis virus encoding VSV glycoprotein [rVSV(VSV-G)] does not spread across axons in anterograde direction in the central nervous system of Xenopus laevis. (A) A dorsal view of the X. laevis forebrain illustrating the injection site of the virus into olfactory bulb (OB). Tel, telencephalon; OT, optic tectum. (B) A coronal section of the olfactory bulb at the rostral-caudal level shown in (A), showing a large number of labeled neurons in the injected side 10 days post injection. The inset shows an enlarged section showing labeled neurons. (C) A lateral view of the left olfactory bulb and left telencephalon in which VSV was injected 10 days prior. Lateral olfactory tract (lot) is visible from the surface of the telencephalon. (C1) An enlarged view of the lateral olfactory tract shown in (C). (D) A coronal section of the caudal telencephalon at the rostral-caudal level shown in A. No labeled neurons were found either in lateral amygdala (D1) or in medial amygdala (D2). White arrow heads show labeled processes. Labeled neurons seen in preoptic area (POA) are considered to have resulted from the intracranial leak of the injected virus from the injection site in the olfactory bulb. ac, anterior commissure; BST, bed nucleus of the stria terminalis; LA, lateral amygdala; lfb, lateral forebrain bundle; LP, lateral pallium; MeA, medial amygdala; Pa, pallidum; POA, preoptic area; VP, ventral pallidum.
FIGURE 8. A test for the transsynaptic spread of recombinant vesicular stomatitis virus encoding rabies glycoprotein [rVSV(RABV-G)]. (A) A dorsal view of the X. laevis forebrain illustrating the connection among laryngeal motor nucleus (n.IX-X), dorsal tegmental area of medulla (DTAM) and the bed nucleus of the stria terminalis (BNST); n.IX-X receives projections from ipsilateral DTAM, which in turn, receives input from the ipsilateral bed nucleus of the BNST. (B) A horizontal section of the anterior brainstem showing labeled neurons in ipsilateral DTAM 10 days post infection. White dotted line encircle DTAM. (C) Horizontal section of the caudal telencephalon showing the location of BNST. (D) An enlarged section in C showing the absence of labeling in the ipsilateral BNST 10 days post injection.
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