Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus.

Department of Molecular Biology, Neuroscience Institute, Princeton University, Princeton, New Jersey, United States of America.
PLoS ONE (Impact Factor: 3.53). 02/2009; 4(9):e6923. DOI: 10.1371/journal.pone.0006923
Source: PubMed

ABSTRACT The study of coordinated activity in neuronal circuits has been challenging without a method to simultaneously report activity and connectivity. Here we present the first use of pseudorabies virus (PRV), which spreads through synaptically connected neurons, to express a fluorescent calcium indicator protein and monitor neuronal activity in a living animal. Fluorescence signals were proportional to action potential number and could reliably detect single action potentials in vitro. With two-photon imaging in vivo, we observed both spontaneous and stimulated activity in neurons of infected murine peripheral autonomic submandibular ganglia (SMG). We optically recorded the SMG response in the salivary circuit to direct electrical stimulation of the presynaptic axons and to physiologically relevant sensory stimulation of the oral cavity. During a time window of 48 hours after inoculation, few spontaneous transients occurred. By 72 hours, we identified more frequent and prolonged spontaneous calcium transients, suggestive of neuronal or tissue responses to infection that influence calcium signaling. Our work establishes in vivo investigation of physiological neuronal circuit activity and subsequent effects of infection with single cell resolution.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: An understanding of how the brain processes information requires knowledge of the architecture of its underlying neuronal circuits, as well as insights into the relationship between architecture and physiological function. A range of sophisticated tools is needed to acquire this knowledge, and recombinant rabies virus (RABV) is becoming an increasingly important part of this essential toolbox. RABV has been recognized for years for its properties as a synapse-specific trans-neuronal tracer. A novel genetically modified variant now enables the investigation of specific monosynaptic connections. This technology, in combination with other genetic, physiological, optical, and computational tools, has enormous potential for the visualization of neuronal circuits, and for monitoring and manipulating their activity. Here we will summarize the latest developments in this fast moving field and provide a perspective for the use of this technology for the dissection of neuronal circuit structure and function in the normal and diseased brain.
    Frontiers in Neural Circuits 01/2013; 7:2. · 2.95 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Stimulation of cardiac afferents (CA) increased sympathetic outflow and blood pressure. The goal of the current study is to determine the central autonomic nuclei involved in the regulation of cardiac sympathetic afferent reflex (CSAR) which has been proved in previously functional studies. Neuroanatomical method and pseudorabies virus (PRV) transynaptic retrograde trace technique will be performed to investigate the relationship between kidney and heart and the temporal order of the most PRV-labeled neurons in the central nervous system. Recombinant PRV expressing enhanced green fluorescence protein (EGFP) was injected into the left kidney of rats as a specific trans-synaptic retrograde tracer in neurons. After 2, 3, 4, 5, 6, 7, 8 or 9days, brain, spinal cord and heart were collected for immunofluorescence staining. The temporal order of PRV labeled neurons was found in the ipsilateral intermediolateral nucleus (IML) of T8-T12 spinal segments on day 3; bilateral rostroventrolateral medulla (RVLM), paraventricular nucleus (PVN) and nucleus of the solitary tract (NTS) on day 4; and left and right ventricular walls and ventricular septum of the heart on day 9. In rats with renal denervation, no PRV-infected neurons or cardiomyocytes were found after PRV injection. In conclusion, PRV trans-synaptic retrograde trace confirms that CA, NTS, PVN, RVLM, IML and renal nerves do exist to be involved in the regulation of CSAR and there is a close relationship between heart and kidney. CA is mainly located in the left ventricular wall, right ventricular wall and ventricular septum.
    Journal of the neurological sciences 04/2014; · 2.32 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A clinical hallmark of human alphaherpesvirus infections is peripheral pain or itching. Pseudorabies virus (PRV), a broad host range alphaherpesvirus, causes violent pruritus in many different animals, but the mechanism is unknown. Previous in vitro studies have shown that infected, cultured peripheral nervous system (PNS) neurons exhibited aberrant electrical activity after PRV infection due to the action of viral membrane fusion proteins, yet it is unclear if such activity occurs in infected PNS ganglia in living animals and if it correlates with disease symptoms. Using two-photon microscopy, we imaged autonomic ganglia in living mice infected with PRV strains expressing GCaMP3, a genetically encoded calcium indicator, and used the changes in calcium flux to monitor the activity of many neurons simultaneously with single-cell resolution. Infection with virulent PRV caused these PNS neurons to fire synchronously and cyclically in highly correlated patterns among infected neurons. This activity persisted even when we severed the presynaptic axons, showing that infection-induced firing is independent of input from presynaptic brainstem neurons. This activity was not observed after infections with an attenuated PRV recombinant used for circuit tracing or with PRV mutants lacking either viral glycoprotein B, required for membrane fusion, or viral membrane protein Us9, required for sorting virions and viral glycoproteins into axons. We propose that the viral fusion proteins produced by virulent PRV infection induce electrical coupling in unmyelinated axons in vivo. This action would then give rise to the synchronous and cyclical activity in the ganglia and contribute to the characteristic peripheral neuropathy.
    Proceedings of the National Academy of Sciences 08/2013; · 9.81 Impact Factor

Full-text (3 Sources)

Available from
May 27, 2014