A bicistronic lentiviral vector-based method for differential transsynaptic tracing of neural circuits

Ohashi, Yohei; Tsubota, Tadashi; Sato, Ayana; Koyano, Konomi; Tamura, Keita; Miyashita, Yasushi

doi:10.1016/j.mcn.2010.08.013

Cited by 19 publications

(14 citation statements)

References 53 publications

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“…In our hands, P2A-mediated cleavage between FGF14 and GFP was quite efficient in CHL cells; however, there was no apparent cleavage in Purkinje neurons in vivo, as suggested by the exact co-localization of the FGF14 and GFP immunostaining at the AIS. A possible explanation for lack of cleavage could be that the P2A nucleotide sequence we used [27], differs slightly from the P2A sequence used by Ohashi and colleagues [57], although the resultant peptide sequences are identical. However, since 2A-mediated cleavage is thought to be a co-translational process in which a peptide bond is “skipped” between the Gly and Pro in the 2A motif (D(V/I)EXNPGP), most of the peptide sequence would have already been generated by the time the skipped peptide bond was reached, and it is unlikely that any small changes in nucleotide sequence would have made a difference.…”

Section: Discussionmentioning

confidence: 98%

“…In the present study, we used a 2A sequence from porcine teschovirus (P2A), which was reported to produce the highest cleavage efficiency compared to other 2A sequences in multiple mammalian cell types, including mouse liver in vivo [27]. P2A sequences have also been used in lentiviral vectors to express multiple proteins in rat Purkinje neurons [57]. In our hands, P2A-mediated cleavage between FGF14 and GFP was quite efficient in CHL cells; however, there was no apparent cleavage in Purkinje neurons in vivo, as suggested by the exact co-localization of the FGF14 and GFP immunostaining at the AIS.…”

Section: Discussionmentioning

confidence: 99%

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Dual Transgene Expression in Murine Cerebellar Purkinje Neurons by Viral Transduction In Vivo

2014

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Viral-vector mediated gene transfer to cerebellar Purkinje neurons in vivo is a promising avenue for gene therapy of cerebellar ataxias and for genetic manipulation in functional studies of animal models of cerebellar disease. Here, we report the results of experiments designed to identify efficient methods for viral transduction of adult murine Purkinje neurons in vivo. For these analyses, several lentiviral and an adeno-associated virus (AAV), serotype 1, vector with various promoter combinations were generated and compared for in situ transduction efficiency, assayed by fluorescent reporter protein expression in Purkinje neurons. Additional experiments were also conducted to identify the optimal experimental strategy for co-expression of two proteins in individual Purkinje neurons. Of the viruses tested, AAV1 with a CAG promoter exhibited the highest specificity for Purkinje neurons. To deliver two proteins to the same Purkinje neuron, several methods were tested, including: an internal ribosome entry site (IRES), a 2A sequence, a dual promoter vector, and co-injection of two viruses. Efficient expression of both proteins in the same Purkinje neuron was only achieved by co-injecting two AAV1-CAG viruses. We found that use of an AAV1-CAG virus outperformed similar lentivirus vectors and that co-injection of two AAV1-CAG viruses could be used to efficiently deliver two proteins to the same Purkinje neuron in adult mice. AAV1 with a CAG promoter is highly efficient and selective at transducing adult cerebellar Purkinje neurons and two AAV-CAG viruses can be used to efficiently express two proteins in the same neuron in vivo.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Dual Transgene Expression in Murine Cerebellar Purkinje Neurons by Viral Transduction In Vivo

2014

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“…RCB2202) were cultured in Dulbecco's modified Eagle's medium (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum at 37°C in a 5% CO 2 atmosphere, and lentiviral vectors were prepared as described previously [46]. We produced two lentiviral vectors, Lenti-sL7-hChR2-EYFP-WPRE (sL7-ChR2) and Lenti-sL7-eNpHR-EYFP-WPRE (sL7-eNpHR), in this study (Fig.…”

Section: Methodsmentioning

confidence: 99%

Optogenetic Manipulation of Cerebellar Purkinje Cell Activity In Vivo

et al. 2011

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Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex. Although their anatomical connections and physiological response properties have been extensively studied, the causal role of their activity in behavioral, cognitive and autonomic functions is still unclear because PC activity cannot be selectively controlled. Here we developed a novel technique using optogenetics for selective and rapidly reversible manipulation of PC activity in vivo. We injected into rat cerebellar cortex lentiviruses expressing either the light-activated cationic channel channelrhodopsin-2 (ChR2) or light-driven chloride pump halorhodopsin (eNpHR) under the control of the PC-specific L7 promoter. Transgene expression was observed in most PCs (ChR2, 92.6%; eNpHR, 95.3%), as determined by immunohistochemical analysis. In vivo electrophysiological recordings showed that all light-responsive PCs in ChR2-transduced rats increased frequency of simple spike in response to blue laser illumination. Similarly, most light-responsive PCs (93.8%) in eNpHR-transduced rats decreased frequency of simple spike in response to orange laser illumination. We then applied these techniques to characterize the roles of rat cerebellar uvula, one of the cardiovascular regulatory regions in the cerebellum, in resting blood pressure (BP) regulation in anesthetized rats. ChR2-mediated photostimulation and eNpHR-mediated photoinhibition of the uvula had opposite effects on resting BP, inducing depressor and pressor responses, respectively. In contrast, manipulation of PC activity within the neighboring lobule VIII had no effect on BP. Blue and orange laser illumination onto PBS-injected lobule IX didn't affect BP, indicating the observed effects on BP were actually due to PC activation and inhibition. These results clearly demonstrate that the optogenetic method we developed here will provide a powerful way to elucidate a causal relationship between local PC activity and functions of the cerebellum.

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“…Transsynaptic viral labeling such as pseudorabies virus (PRV) (Lee et al, 2014) and wheat germ agglutinin (WGA) expressing vectors (Fujimoto et al, 2012) can be used to trace the entire circuit, but alternate routes of viral transmission should be considered when interpreting data from incomplete injuries. Although the available data supports the hypothesis that both PRV and WGA are transferred at active synaptic connections, many of those studies have been conducted in vitro (Hogue et al, 2014) or intact CNS (Ohashi et al, 2011) rather than a relay model of SCI repair. The release of vesicles from growth cones (Sabo and McAllister, 2003; Tojima et al, 2007), or the establishment and retraction of functional synapses within a relay could lead to labeling of neurons without permanent synaptic connections.…”

Section: Introductionmentioning

confidence: 99%

Repair of spinal cord injury with neuronal relays: From fetal grafts to neural stem cells

Bonner

Steward

2015

Brain Research

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Spinal cord injury (SCI) disrupts the long axonal tracts of the spinal cord leading to devastating loss of function. Cell transplantation in the injured spinal cord has the potential to lead to recovery after SCI via a variety of mechanisms. One such strategy is the formation of neuronal relays between injured long tract axons and denervated neurons. The idea of creating a neuronal relay was first proposed over 25 years ago when fetal tissue was first successfully transplanted into the injured rodent spinal cord. Advances in labeling of grafted cells and the development of neural stem cell culturing techniques have improved the ability to create and refine such relays. Several recent studies have examined the ability to create a novel neuronal circuit between injured axons and denervated targets. This approach is an alternative to long-distance regeneration of damaged axons that may provide a meaningful degree of recovery without direct recreation of lost pathways. This brief review will examine the contribution of fetal grafting to current advances in neuronal grafting. Of particular interest will be the ability of transplanted neurons derived from fetal grafts, neural precursor cells and neural stem cells to reconnect long distance motor and sensory pathways of the injured spinal cord.

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A bicistronic lentiviral vector-based method for differential transsynaptic tracing of neural circuits

Cited by 19 publications

References 53 publications

Dual Transgene Expression in Murine Cerebellar Purkinje Neurons by Viral Transduction In Vivo

Dual Transgene Expression in Murine Cerebellar Purkinje Neurons by Viral Transduction In Vivo

Optogenetic Manipulation of Cerebellar Purkinje Cell Activity In Vivo

Repair of spinal cord injury with neuronal relays: From fetal grafts to neural stem cells

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