Abstract
Here we present a novel tracing technique to stain projection neurons in Golgi-like detail by double viral infection. We used retrograde lentiviral vectors and adeno-associated viral vectors (AAV) to drive “TET-ON/TET-OFF system” in neurons connecting two regions. Using this method, we successfully labeled the corticothalamic (CT) cells of the mouse somatosensory barrel field (S1BF) and motor cortex (M1) in their entirety. We also labeled contra- and ipsilaterally-projecting corticocortical (CC) cells of M1 by targeting contralateral M1 or ipsilateral S1 for retrograde infection. The strength of this method is that we can observe the morphology of specific projection neuron subtypes en masse. We found that the group of CT cells extends their dendrites and intrinsic axons extensively below but not within the thalamorecipient layer in both S1BF and M1, suggesting that the primary target of this cell type is not layer 4. We also found that both ipsi- and contralateral targeting CC cells in M1 commonly exhibit widespread collateral extensions to contralateral M1 (layers 1–6), bilateral S1 and S2 (layers 1, 5 and 6), perirhinal cortex (layers 1, 2/3, 5, and 6), striatum and claustrum. These findings not only strengthened the previous findings of single cell tracings but also extended them by enabling cross-area comparison of CT cells or comparison of CC cells of two different labeling.
Highlights
Classification of neuronal type is prerequisite to the understanding of cortical circuit
By incorporating “TET-Off system” to lentiviral-based retrograde vector, we previously showed that we can visualize the fine morphology of specific projection neuron subtypes (Watakabe et al, 2012)
One is the “neuron-specific retrograde gene transfer (NeuRet)” vector, pseudotyped with fusion glycoprotein C type (FuG-C), a chimeric glycoprotein composed of parts of rabies virus glycoprotein and vesicular stomatitis virus glycoprotein, which exhibits enhanced ability for retrograde gene transfer (Figure 1A; Kato et al, 2011a,b,c)
Summary
Classification of neuronal type is prerequisite to the understanding of cortical circuit. Genetic identification of cell types using transgenic mouse lines is rapidly becoming ever more sophisticated (Fishell and Heintz, 2013; Huang and Zeng, 2013), identification by projection target is still one of the most reliable criteria for cell type classification of the cortical neurons that is applicable across species (Nelson et al, 2006; Molyneaux et al, 2007; Thomson and Lamy, 2007; Sorensen et al, 2013). To fully characterize the “cell type,” there is a need to analyze axon collateral projections. Investigation of axon collaterals is important in understanding the cortical microcircuitry (Thomson and Lamy, 2007; Morishima et al, 2011). Our knowledge on extrinsic and intrinsic collaterals is still limited mainly due to technical difficulty
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