Optimization of whole-brain rabies virus tracing technology for small cell populations

Theresia J M Roelofs,Keith M Garner,Vera H Wielenga,Roger A H Adan,Annelijn M Speel,Shanice Menting-Henry,Karl-Klaus Conzelmann,Lieke M Gol,Alexandru A Hennrich,Mieneke C M Luijendijk

doi:10.1038/s41598-021-89862-5

Abstract

The lateral hypothalamus (LH) is critically involved in the regulation of homeostatic energy balance. Some neurons in the LH express receptors for leptin (LepRb), a hormone known to increase energy expenditure and decrease energy intake. However, the neuroanatomical inputs to LepRb-expressing LH neurons remain unknown. We used rabies virus tracing technology to map these inputs, but encountered non-specific tracing. To optimize this technology for a minor cell population (LepRb is not ubiquitously expressed in LH), we used LepRb-Cre mice and assessed how different titers of the avian tumor virus receptor A (TVA) helper virus affected rabies tracing efficiency and specificity. We found that rabies expression is dependent on TVA receptor expression, and that leakiness of TVA receptors is dependent on the titer of TVA virus used. We concluded that a titer of 1.0–3.0 × 107 genomic copies per µl of the TVA virus is optimal for rabies tracing. Next, we successfully applied modified rabies virus tracing technology to map inputs to LepRb-expressing LH neurons. We discovered that other neurons in the LH itself, the periventricular hypothalamic nucleus (Pe), the posterior hypothalamic nucleus (PH), the bed nucleus of the stria terminalis (BNST), and the paraventricular hypothalamic nucleus (PVN) are the most prominent input areas to LepRb-expressing LH neurons.

Highlights

The lateral hypothalamus (LH) is critically involved in the regulation of homeostatic energy balance
We found that careful consideration of the titer of Cre-dependent tumor virus receptor A (TVA) is of utmost importance to avoid nonspecific results when using modified rabies virus tracing technology for mapping neuronal circuitries
We aimed to determine the most optimal titer of Cre-dependent TVA for mapping inputs to LepRb-expressing LH neurons, which still resulted in efficient rabies tracing of inputs in LepRb-Cre mice, but had neglectable infection in wildtype control mice

Summary

Introduction

The lateral hypothalamus (LH) is critically involved in the regulation of homeostatic energy balance. In the modified rabies virus tracing approach used in the current study, two helper adeno-associated viral vectors (AAV vectors) are injected simultaneously; one that carries TVA-mCherry which, once expressed in target neurons, allows EnvA-coated RVdG to enter, and one that carries rabies G which, once expressed in target neurons, leads to normal infectious properties of Center for Image Sciences, University Medical Center Utrecht and Utrecht University, Bolognalaan 50, 3584 CJ Utrecht, The Netherlands. To the best of our knowledge, only one study has investigated the effect of different titers of Cre-dependent TVA on rabies efficiency and s pecificity[30] This is crucial, in particular when only few Cre-positive neurons are present in a brain region, such as in the case of LepRb-expressing neurons in the LH. With the aim to optimize specificity and efficiency of RV tracing, we optimized the TVA titer and used this titer to map inputs to LepRb-expressing LH neurons. Mapping of these inputs will provide more insight into the organization of feeding behaviour-related neural-networks

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