A Dual Infection Pseudorabies Virus Conditional Reporter Approach to Identify Projections to Collateralized Neurons in Complex Neural Circuits

Replication and transneuronal transport of pseudorabies virus (PRV) are widely used to define the organization of neural circuits in rodent brain. Here we report a dual infection approach that highlights connections to neurons that collateralize within complex networks. The method combines Cre recombinase (Cre) expression from a PRV recombinant (PRV-267) and Cre-dependent reporter gene expression from a second infecting strain of PRV (PRV-263). PRV-267 expresses both Cre and a monomeric red fluorescent protein (mRFP) fused to viral capsid protein VP26 (VP26-mRFP) that accumulates in infected cell nuclei. PRV-263 carries a Brainbow cassette and expresses a red (dTomato) reporter that fills the cytoplasm. However, in the presence of Cre, the dTomato gene is recombined from the cassette, eliminating expression of the red reporter and liberating expression of either yellow (EYFP) or cyan (mCerulean) cytoplasmic reporters. We conducted proof-of-principle experiments using a well-characterized model in which separate injection of recombinant viruses into the left and right kidneys produces infection of neurons in the renal preautonomic network. Neurons dedicated to one kidney expressed the unique reporters characteristic of PRV-263 (cytoplasmic dTomato) or PRV-267 (nuclear VP26-mRFP). Dual infected neurons expressed VP26-mRFP and the cyan or yellow cytoplasmic reporters activated by Cre-mediated recombination of the Brainbow cassette. Differential expression of cyan or yellow reporters in neurons lacking VP26-mRFP provided a unique marker of neurons synaptically connected to dual infected neurons, a synaptic relationship that cannot be distinguished using other dual infection tracing approaches. These data demonstrate Cre-enabled conditional reporter expression in polysynaptic circuits that permits the identification of collateralized neurons and their presynaptic partners

Centrally Projecting Edinger-Westphal Nucleus in the Control of Sympathetic Outflow and Energy Homeostasis

The centrally projecting Edinger-Westphal nucleus (EWcp) is a midbrain neuronal group, adjacent but segregated from the preganglionic Edinger-Westphal nucleus that projects to the ciliary ganglion. The EWcp plays a crucial role in stress responses and in maintaining energy homeostasis under conditions that require an adjustment of energy expenditure, by virtue of modulating heart rate and blood pressure, thermogenesis, food intake, and fat and glucose metabolism. This modulation is ultimately mediated by changes in the sympathetic outflow to several effector organs, including the adrenal gland, heart, kidneys, brown and white adipose tissues and pancreas, in response to environmental conditions and the animal’s energy state, providing for appropriate energy utilization. Classic neuroanatomical studies have shown that the EWcp receives inputs from forebrain regions involved in these functions and projects to presympathetic neuronal populations in the brainstem. Transneuronal tracing with pseudorabies virus has demonstrated that the EWcp is connected polysynaptically with central circuits that provide sympathetic innervation to all these effector organs that are critical for stress responses and energy homeostasis. We propose that EWcp integrates multimodal signals (stress, thermal, metabolic, endocrine, etc.) and modulates the sympathetic output simultaneously to multiple effector organs to maintain energy homeostasis under different conditions that require adjustments of energy demands

Angiotensin modulation of rostral ventrolateral medulla (RVLM) in cardiovascular regulation

The rostral ventrolateral medulla (RVLM) and the presympathetic bulbospinal neurons in this region play a critical role in cardiovascular regulation. However, there is ambiguity regarding the precise anatomical coordinates of the RVLM and much still needs to be learned regarding the regulation and neurochemistry of this region. This brief review discusses some of these issues and focuses on the role of angiotensin-mediated signaling in the RVLM in blood pressure regulation