Microdissection of neural networks by conditional reporter expression from a Brainbow herpesvirus

Abstract

Transneuronal transport of neurotropic viruses is widely used to define the organization of neural circuitry in the mature and developing nervous system. However, interconnectivity within complex circuits limits the ability of viral tracing to define connections specifically linked to a subpopulation of neurons within a network. Here we demonstrate a unique viral tracing technology that highlights connections to defined populations of neurons within a larger labeled network. This technology was accomplished by constructing a replication-competent strain of pseudorabies virus (PRV-263) that changes the profile of fluorescent reporter expression in the presence of Cre recombinase (Cre). The viral genome carries a Brainbow cassette that expresses a default red reporter in infected cells. However, in the presence of Cre, the red reporter gene is excised from the genome and expression of yellow or cyan reporters is enabled. We used PRV-263 in combination with a unique lentivirus vector that produces Cre expression in catecholamine neurons. Projection-specific infection of central circuits containing these Cre-expressing catecholamine neurons with PRV-263 resulted in Cre-mediated recombination of the PRV-263 genome and conditional expression of cyan/yellow reporters. Replication and transneuronal transport of recombined virus produced conditional reporter expression in neurons synaptically linked to the Cre-expressing catecholamine neurons. This unique technology highlights connections specific to phenotypically defined neurons within larger networks infected by retrograde transneuronal transport of virus from a defined projection target. The availability of other technologies that restrict Cre expression to defined populations of neurons indicates that this approach can be widely applied across functionally defined systems.

Alphaherpesvirus axon-to-cell spread involves limited virion transmission

“…The importance of this diversity for the fitness of herpesvirus infection is not well known. There is some evidence that transneuronal spread of attenuated strains of PRV involves a limited number of genomes, which would limit diversity (32). Our work demonstrates that a small number of virus particles are involved in ADS, limiting whatever diversity exists during transmission.…”

Orexin neurons suppress narcolepsy via 2 distinct efferent pathways

“…25). This promoter has been used for the same purpose in previous studies (26)(27)(28) and contains a minimal promoter and 8 copies of a promoter sequence from the cis-regulatory region of the dopamine β-hydroxylase (DBH) gene that binds the Phox2a/b transcription factors. These factors are expressed in and critical for noradrenergic neurons, but they are also expressed in several populations of non-noradrenergic neurons in the brainstem.…”

Catecholaminergic C3 Neurons Are Sympathoexcitatory and Involved in Glucose Homeostasis

“…There was no significant difference in the proportion of neurons immunoreactive for VMAT2 between individual C3 subgroups. We also tested immunoreactivity for NPY, a marker of catecholaminergic neurons involved in the glucoregulatory response (Bugarith et al, 2005;). Most C3 neurons were immunoreactive for NPY (92.6 Ϯ 2.3%), with no significant variation between subgroups (data not shown).…”

“…Although the central pathways involved in this response are being elucidated and our understanding remains incomplete, an important role for hindbrain catecholaminergic neurons is clear. Studies over many years by Ritter and colleagues demonstrate the importance of catecholaminergic cell groups in the medulla oblongata in many responses to glucoprivation (Ritter et al, 2001Bugarith et al, 2005;. Lesions of rostrally projecting, catecholamine-synthesizing neurons decrease the food intake response to glucoprivation, whereas lesions of the spinally projecting cells decrease adrenal medullary catecholamine secretion (Ritter et al, 2001;Bugarith et al, 2005;.…”

“…Studies over many years by Ritter and colleagues demonstrate the importance of catecholaminergic cell groups in the medulla oblongata in many responses to glucoprivation (Ritter et al, 2001Bugarith et al, 2005;. Lesions of rostrally projecting, catecholamine-synthesizing neurons decrease the food intake response to glucoprivation, whereas lesions of the spinally projecting cells decrease adrenal medullary catecholamine secretion (Ritter et al, 2001;Bugarith et al, 2005;. Much of the focus resulting from these observations has centered on the role of adrenaline-synthesizing C1 neurons of the rostral ventrolateral medulla (RVLM), which are activated by glucoprivation (Ritter et al, 1998;Verberne and Sartor, 2010;Parker et al, 2013) and contribute to the excitation of adrenal sympathetic nerve activity.…”