Gene Expression Profiling with Cre-Conditional Pseudorabies Virus Reveals a Subset of Midbrain Neurons That Participate in Reward Circuitry.

TitleGene Expression Profiling with Cre-Conditional Pseudorabies Virus Reveals a Subset of Midbrain Neurons That Participate in Reward Circuitry.
Publication TypeJournal Article
Year of Publication2017
AuthorsPomeranz, LE, Ekstrand, MI, Latcha, KN, Smith, GA, Enquist, LW, Friedman, JM
JournalJ Neurosci
Volume37
Issue15
Pagination4128-4144
Date Published2017 04 12
ISSN1529-2401
KeywordsAnimals, Female, Gene Expression Profiling, Integrases, Male, Mesencephalon, Mice, Mice, Inbred C57BL, Mice, Transgenic, Neurons, Pseudorabies, Reward
Abstract

<p>The mesolimbic dopamine pathway receives inputs from numerous regions of the brain as part of a neural system that detects rewarding stimuli and coordinates a behavioral response. The capacity to simultaneously map and molecularly define the components of this complex multisynaptic circuit would thus advance our understanding of the determinants of motivated behavior. To accomplish this, we have constructed pseudorabies virus (PRV) strains in which viral propagation and fluorophore expression are activated only after exposure to Cre recombinase. Once activated in Cre-expressing neurons, the virus serially labels chains of presynaptic neurons. Dual injection of GFP and mCherry tracing viruses simultaneously illuminates nigrostriatal and mesolimbic circuitry and shows no overlap, demonstrating that PRV transmission is confined to synaptically connected neurons. To molecularly profile mesolimbic dopamine neurons and their presynaptic inputs, we injected Cre-conditional GFP virus into the NAc of (anti-GFP) nanobody-L10 transgenic mice and immunoprecipitated translating ribosomes from neurons infected after retrograde tracing. Analysis of purified RNA revealed an enrichment of transcripts expressed in neurons of the dorsal raphe nuclei and lateral hypothalamus that project to the mesolimbic dopamine circuit. These studies identify important inputs to the mesolimbic dopamine pathway and further show that PRV circuit-directed translating ribosome affinity purification can be broadly applied to identify molecularly defined neurons comprising complex, multisynaptic circuits. The mesolimbic dopamine circuit integrates signals from key brain regions to detect and respond to rewarding stimuli. To further define this complex multisynaptic circuit, we constructed a panel of Cre recombinase-activated pseudorabies viruses (PRVs) that enabled retrograde tracing of neural inputs that terminate on Cre-expressing neurons. Using these viruses and Retro-TRAP (translating ribosome affinity purification), a previously reported molecular profiling method, we developed a novel technique that provides anatomic as well as molecular information about the neural components of polysynaptic circuits. We refer to this new method as PRV-Circuit-TRAP (PRV circuit-directed TRAP). Using it, we have identified major projections to the mesolimbic dopamine circuit from the lateral hypothalamus and dorsal raphe nucleus and defined a discrete subset of transcripts expressed in these projecting neurons, which will allow further characterization of this important pathway. Moreover, the method we report is general and can be applied to the study of other neural circuits.</p>

DOI10.1523/JNEUROSCI.3193-16.2017
Alternate JournalJ Neurosci
PubMed ID28283558
PubMed Central IDPMC5391685
Grant ListP40 OD010996 / OD / NIH HHS / United States
R01 DA018799 / DA / NIDA NIH HHS / United States
R01 AI056346 / AI / NIAID NIH HHS / United States
R01 NS060699 / NS / NINDS NIH HHS / United States
P40 RR018604 / RR / NCRR NIH HHS / United States