Dual transneuronal tracing in the rat entorhinal-hippocampal circuit by intracerebral injection of recombinant rabies virus vectors

Abstract

Dual transneuronal tracing is a novel viral tracing methodology which employs two recombinant viruses, each expressing a different reporter protein. Peripheral injection of recombinant pseudorabies viruses has been used as a powerful method to defi ne neurons that coordinate outputs to various peripheral targets of motor and autonomic systems. Here, we assessed the feasibility of recombinants of rabies virus (RV) vector for dual transneuronal tracing in the central nervous system. First, we examined whether two different RV-vectors can double label cells in vitro, and showed that effi cient double labeling can be realized by infecting targeted cells with the two RV-vectors within a short time interval. The potential of dual transneuronal tracing was then examined in vivo in the entorhinal-hippocampal circuit, using the chain of projections from CA3 pyramidal cells to CA1 pyramidal cells and subsequently to entorhinal cortex. Six days after the injection of two RV-vectors into the left and right entorhinal cortex respectively, double-labeled neurons were observed in CA3 bilaterally. Some double-labeled neurons showed a Golgi-like labeling. Dual transneuronal tracing potentially provides a powerful and sensitive method to study issues such as the amount of convergence and divergence within and between circuits in the central nervous system. Using this sensitive technique, we established that single neurons in CA3 are connected to the entorhinal cortex bilaterally with only one synaptic relay.

Advances in viral transneuronal tracing

“…By testing dual infection in vitro and in vivo, these authors have shown that the phenomenon of viral interference for replication, noted for isogenic PrV strains (see Section 2.10), is also inherent to isogenic rabies virus recombinants. In other words, when the two viruses infect the same neurons at different times, the efficiency of the second infection decreases exponentially in a time-dependent manner; thus, to achieve efficient double labeling, the two viruses must infect the same neurons within a few hours (Ohara et al, 2009). Moreover, as for PrV strains, the length of the inserted foreign genes can modify the replication and/or propagation speed of the rabies virus recombinants (Ohara et al, 2009).…”

“…Moreover, as for PrV strains, the length of the inserted foreign genes can modify the replication and/or propagation speed of the rabies virus recombinants (Ohara et al, 2009). While these findings stress the importance of using isogenic strains with similar propagation properties, and/or possibly the need to adjust the relative dose of each virus to achieve optimal dual transneuronal labeling, the successful colocalization of the two isogenic rabies virus mutants in vitro and in vivo, demonstrated by Ohara et al (2009) clearly illustrates the potential of genetically engineered rabies viruses for dual transneuronal tracing.…”

“…Because of the great specificity of transfer of rabies virus, it would be highly desirable to develop dual transneuronal tracing methods based on the use of isogenic rabies virus strains. In the first attempt, Ohara et al (2009) have generated three isogenic rabies virus recombinants, by inserting different reporter genes (␤-gal, Venus or EGFP) in the HEP-Flury strain, where the G gene of the attenuated HEP-Flury strain has been replaced with the G gene from the virulent CVS strain in order to mimic the properties of CVS. By testing dual infection in vitro and in vivo, these authors have shown that the phenomenon of viral interference for replication, noted for isogenic PrV strains (see Section 2.10), is also inherent to isogenic rabies virus recombinants.…”

The rat perirhinal cortex: A review of anatomy, physiology, plasticity, and function

“…As well as the hippocampus forming reciprocal connections with the perirhinal cortex (Swanson and Cowan, 1977;Deacon et al, 1983;Kosel et al, 1983;Van Groen and Wyss, 1990;McIntyre et al, 1996;Kloosterman et al, 2003b), it also shows strong interconnectivity with the entorhinal (Steward, 1976;Steward and Scoville, 1976;Swanson and Cowan, 1977;Swanson et al, 1981;Wyss, 1981;Nojyo, 1993, 1995;Ohara et al, 2009) and postrhinal cortices (Kloosterman et al, 2003b). Many of these projections have been shown to be capable of eliciting an electrophysiological response in their respective tissues.…”

Seasonal morphometry of the vomeronasal organ in the marsupial mouse, Antechinus subtropicus

“…Differences in anatomy, social communication and reproduction, as well as the reported morphological diversity of the VNO suggest that direct comparison between even closely related species is risky and may not be meaningful (Salazar and Sánchez Quinteiro, 2009). The limited data in marsupials generally supports the presence of a well‐developed VNO similar to that reported in Rodentia (see e.g., Tarsipes rostratus —Kratzing, 1982; Monodelphis domestica —Poran 1998; Macropus eugenii —Schneider et al, 2008; Antechinus —Toftegaard, 1998).…”