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

Ohara S, Inoue K, Yamada M, Yamawaki T, Koganezawa N, Tsutsui K, Witter MP, Iijima T - Front Neuroanat (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Systems Neuroscience, Tohoku University Graduate School of Life Sciences Tohoku, Japan.

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 define 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 efficient 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.

No MeSH data available.


Related in: MedlinePlus

Transsynaptic double labeling of neurons in the hippocampus after 6 days following bilateral injections of two RV-vectors into MEC. (A) Schematic representation of the relevant circuitry in the hippocampal formation. The colored panels show the injection site (green: rHEP5.0-CVSG-EGFPx2, magenta: rHEP5.0-CVSG-β-gal). (B) Fluorescence micrograph showing the distribution of labeled neurons in the dorsal hippocampus. In field CA1, neurons are labeled only by the virus vector injected into the ipsilateral MEC, whereas β-gal (magenta) and GFP (green) labels intermingle in CA3 region. Note that double-labeled neurons are observed in CA3. (C–E) Labeled CA3 pyramidal cells taken from the box in (B). Neurons labeled by both β-gal (C) and GFP (D) can be seen in white in the merged image (E). Arrows indicate double-labeled neurons. (F–H) Photomicrographs demonstrating Golgi-like labeling of a double-labeled CA3 pyramidal cell. High magnification image shows that the dendrites and their spines can also be visualized in the double-labeled neuron. MEC, medial entorhinal cortex. Scale bar = 500 μm in (B), 50 μm in (E) [applies to (C,D)], 20 μm in (H) [applies to (F,G)], 10 μm in (I).
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Figure 5: Transsynaptic double labeling of neurons in the hippocampus after 6 days following bilateral injections of two RV-vectors into MEC. (A) Schematic representation of the relevant circuitry in the hippocampal formation. The colored panels show the injection site (green: rHEP5.0-CVSG-EGFPx2, magenta: rHEP5.0-CVSG-β-gal). (B) Fluorescence micrograph showing the distribution of labeled neurons in the dorsal hippocampus. In field CA1, neurons are labeled only by the virus vector injected into the ipsilateral MEC, whereas β-gal (magenta) and GFP (green) labels intermingle in CA3 region. Note that double-labeled neurons are observed in CA3. (C–E) Labeled CA3 pyramidal cells taken from the box in (B). Neurons labeled by both β-gal (C) and GFP (D) can be seen in white in the merged image (E). Arrows indicate double-labeled neurons. (F–H) Photomicrographs demonstrating Golgi-like labeling of a double-labeled CA3 pyramidal cell. High magnification image shows that the dendrites and their spines can also be visualized in the double-labeled neuron. MEC, medial entorhinal cortex. Scale bar = 500 μm in (B), 50 μm in (E) [applies to (C,D)], 20 μm in (H) [applies to (F,G)], 10 μm in (I).

Mentions: In 11 animals, one of the two vectors, rHEP5.0-CVSG-β-gal and rHEP5.0-CVSG-EGFPx2, was injected into MEC of the left and the other into MEC of the right hemisphere (Figure 5A), and the animal was perfused and fixed after 6 days. Successful labeling by the two RV-vectors was observed in bilateral CA3 in two cases (Table 2). In the remaining cases (9 out of 11 rats), sparse or no β-gal labeling was observed in CA3 after 6 days of survival, although numerous EGFP labeled neurons were observed in the bilateral CA3 region. Even in CA1, there were fewer β-gal labeled neurons compared to EGFP labeled neurons. The lack of β-gal labeled neurons in CA3, likely due to low levels of first order infection in CA1, is the main reason for the low percentage of double-labeled animals (2 of 11 rats).


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

Ohara S, Inoue K, Yamada M, Yamawaki T, Koganezawa N, Tsutsui K, Witter MP, Iijima T - Front Neuroanat (2009)

Transsynaptic double labeling of neurons in the hippocampus after 6 days following bilateral injections of two RV-vectors into MEC. (A) Schematic representation of the relevant circuitry in the hippocampal formation. The colored panels show the injection site (green: rHEP5.0-CVSG-EGFPx2, magenta: rHEP5.0-CVSG-β-gal). (B) Fluorescence micrograph showing the distribution of labeled neurons in the dorsal hippocampus. In field CA1, neurons are labeled only by the virus vector injected into the ipsilateral MEC, whereas β-gal (magenta) and GFP (green) labels intermingle in CA3 region. Note that double-labeled neurons are observed in CA3. (C–E) Labeled CA3 pyramidal cells taken from the box in (B). Neurons labeled by both β-gal (C) and GFP (D) can be seen in white in the merged image (E). Arrows indicate double-labeled neurons. (F–H) Photomicrographs demonstrating Golgi-like labeling of a double-labeled CA3 pyramidal cell. High magnification image shows that the dendrites and their spines can also be visualized in the double-labeled neuron. MEC, medial entorhinal cortex. Scale bar = 500 μm in (B), 50 μm in (E) [applies to (C,D)], 20 μm in (H) [applies to (F,G)], 10 μm in (I).
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Related In: Results  -  Collection

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Figure 5: Transsynaptic double labeling of neurons in the hippocampus after 6 days following bilateral injections of two RV-vectors into MEC. (A) Schematic representation of the relevant circuitry in the hippocampal formation. The colored panels show the injection site (green: rHEP5.0-CVSG-EGFPx2, magenta: rHEP5.0-CVSG-β-gal). (B) Fluorescence micrograph showing the distribution of labeled neurons in the dorsal hippocampus. In field CA1, neurons are labeled only by the virus vector injected into the ipsilateral MEC, whereas β-gal (magenta) and GFP (green) labels intermingle in CA3 region. Note that double-labeled neurons are observed in CA3. (C–E) Labeled CA3 pyramidal cells taken from the box in (B). Neurons labeled by both β-gal (C) and GFP (D) can be seen in white in the merged image (E). Arrows indicate double-labeled neurons. (F–H) Photomicrographs demonstrating Golgi-like labeling of a double-labeled CA3 pyramidal cell. High magnification image shows that the dendrites and their spines can also be visualized in the double-labeled neuron. MEC, medial entorhinal cortex. Scale bar = 500 μm in (B), 50 μm in (E) [applies to (C,D)], 20 μm in (H) [applies to (F,G)], 10 μm in (I).
Mentions: In 11 animals, one of the two vectors, rHEP5.0-CVSG-β-gal and rHEP5.0-CVSG-EGFPx2, was injected into MEC of the left and the other into MEC of the right hemisphere (Figure 5A), and the animal was perfused and fixed after 6 days. Successful labeling by the two RV-vectors was observed in bilateral CA3 in two cases (Table 2). In the remaining cases (9 out of 11 rats), sparse or no β-gal labeling was observed in CA3 after 6 days of survival, although numerous EGFP labeled neurons were observed in the bilateral CA3 region. Even in CA1, there were fewer β-gal labeled neurons compared to EGFP labeled neurons. The lack of β-gal labeled neurons in CA3, likely due to low levels of first order infection in CA1, is the main reason for the low percentage of double-labeled animals (2 of 11 rats).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Systems Neuroscience, Tohoku University Graduate School of Life Sciences Tohoku, Japan.

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 define 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 efficient 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.

No MeSH data available.


Related in: MedlinePlus