Mapping and Analysis of the Connectome of Sympathetic Premotor Neurons in the Rostral Ventrolateral Medulla of the Rat Using a Volumetric Brain Atlas.

Bowen Dempsey,Sheng Le,Radhika Ramadas,Rachael Neve,Anita Turner,Clement Menuet,Ann K Goodchild,Andrew M Allen,Phil Bokiniec,Jan G Bjaalie,Simon Mcmullan

doi:10.3389/fncir.2017.00009

Abstract

Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) play a critical role in the generation of vasomotor sympathetic tone and are thought to receive convergent input from neurons at every level of the neuraxis; the factors that determine their ongoing activity remain unresolved. In this study we use a genetically restricted viral tracing strategy to definitively map their spatially diffuse connectome. We infected bulbospinal RVLM neurons with a recombinant rabies variant that drives reporter expression in monosynaptically connected input neurons and mapped their distribution using an MRI-based volumetric atlas and a novel image alignment and visualization tool that efficiently translates the positions of neurons captured in conventional photomicrographs to Cartesian coordinates. We identified prominent inputs from well-established neurohumoral and viscero-sympathetic sensory actuators, medullary autonomic and respiratory subnuclei, and supramedullary autonomic nuclei. The majority of inputs lay within the brainstem (88–94%), and included putative respiratory neurons in the pre-Bötzinger Complex and post-inspiratory complex that are therefore likely to underlie respiratory-sympathetic coupling. We also discovered a substantial and previously unrecognized input from the region immediately ventral to nucleus prepositus hypoglossi. In contrast, RVLM sympathetic premotor neurons were only sparsely innervated by suprapontine structures including the paraventricular nucleus, lateral hypothalamus, periaqueductal gray, and superior colliculus, and we found almost no evidence of direct inputs from the cortex or amygdala. Our approach can be used to quantify, standardize and share complete neuroanatomical datasets, and therefore provides researchers with a platform for presentation, analysis and independent reanalysis of connectomic data.

Highlights

The expression of TVA, rabies glycoprotein, and a fluorescent reporter (YFP) in neurons that project to the interomediolateral cell column of the spinal cord (IML), a major site of termination of sympathetic premotor neurons
We found that longer intervals between HSV and rabies injections were associated with more off-target infection of bulbospinal (TVA-expressing) neurons outside of the rostral ventrolateral medulla (RVLM)
The distribution of TH-immunoreactive neurons labeled by HSV control vectors was plotted in Waxholm co-ordinates (Figure 1) and was used to define the anatomical boundaries of the RVLM (Supplementary Image 1)

Summary

Introduction

The expression of TVA, rabies glycoprotein, and a fluorescent reporter (YFP) in neurons that project to the interomediolateral cell column of the spinal cord (IML), a major site of termination of sympathetic premotor neurons. SAD1G(EnvA)-mCherry into the RVLM, selectively restricting its access to TVA-expressing bulbospinal neurons This enabled us to map brain-wide sources of synaptic input to bulbospinal. The central tenet of the connectomic approach is that insights regarding both the functional properties of specific neural circuits and general brain organizational principles may be gained by definitively resolving network architecture (Carandini, 2012; Denk et al, 2012; Mitra, 2014) Ultrastructural approaches such as serial block-face electron microscopy can comprehensively identify local synaptic connectivity, but are limited by the enormous time and costs associated with data acquisition and analysis, and are best suited to the examination of small regions of brain in high detail (discussed by Lichtman and Denk, 2011; Wanner et al, 2015). Investigators interested in mapping more diffuse circuits have instead monitored the trans-synaptic spread of replication-competent neurotropic viruses such as rabies (Ugolini, 1995; Kelly and Strick, 2000; Dum et al, 2016) and alpha herpes variants (Strack et al, 1989; Rinaman and Schwartz, 2004; McGovern et al, 2012; reviewed by Nassi et al, 2015; Wojaczynski et al, 2015)

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