Light-Guided Rabies Virus Tracing for Neural Circuit Analysis (original) (raw)
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Proceedings of the National Academy of Sciences of the United States of America, 2010
We describe a powerful system for revealing the direct monosynaptic inputs to specific cell types in Cre-expressing transgenic mice through the use of Cre-dependent helper virus and a modified rabies virus. We generated helper viruses that target gene expression to Cre-expressing cells, allowing us to control initial rabies virus infection and subsequent monosynaptic retrograde spread. Investigators can use this system to elucidate the connections onto a desired cell type in a high-throughput manner, limited only by the availability of Cre mouse lines. This method allows for identification of circuits that would be extremely tedious or impossible to study with other methods and can be used to build subcircuit maps of inputs onto many different types of cells within the same brain region. Furthermore, by expressing various transgenes from the rabies genome, this system also has the potential to allow manipulation of targeted neuronal circuits without perturbing neighboring cells. transsynaptic | pseudotyped virus | adeno-associated virus | EnvA | TVA O ne of the most intractable problems in systems neuroscience has been the systematic description of neural connectivity in the intact mammalian brain. Many different types of neurons, each with distinct connectivity and function, can inhabit the same brain region. Even within a single neocortical column, dozens of types of projection neurons and local interneurons perform the computations that ultimately lead to the spiking output of that column. Through painstaking studies using molecular and cell biology techniques, electron microscopy, and electrophysiology, we are beginning to understand how a neuron's connectivity contributes to its function in the circuit in which it is embedded, but we lack efficient means for performing circuit-level analyses in vivo. Especially in brain regions with considerable neuronal heterogeneity, we are still greatly limited in our ability to study how groups of cells form their fine-scale connections, how these connections change over time, and how this plasticity affects a cell type's computational role in a dynamic circuit.
Genetically timed, activity-sensor and rainbow transsynaptic viral tools
Nature Methods, 2009
We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions.
Retrograde neuronal tracing with a deletion-mutant rabies virus
Nature Methods, 2007
We have constructed a deletion-mutant rabies virus encoding EGFP and find it to be an excellent tool for studying detailed morphology and physiology of neurons projecting to injection sites within the mammalian brain. The virus cannot spread beyond initially infected cells yet, unlike other viral vectors, replicates its core within them. The cells therefore fluoresce intensely, revealing fine dendritic and axonal structure with no background from partially or faintly labeled cells.
Fluorescence-Based Monitoring of In Vivo Neural Activity Using a Circuit-Tracing Pseudorabies Virus
PLoS ONE, 2009
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.
Transneuronal circuit analysis with pseudorabies viruses
Current protocols in neuroscience / editorial board, Jacqueline N. Crawley ... [et al.], 2014
Our ability to understand the function of the nervous system is dependent upon defining the connections of its constituent neurons. Development of methods to define connections within neural networks has always been a growth industry in the neurosciences. Transneuronal spread of neurotropic viruses currently represents the best means of defining synaptic connections within neural networks. The method exploits the ability of viruses to invade neurons, replicate, and spread through the intimate synaptic connections that enable communication among neurons. Since the method was first introduced in the 1970s, it has benefited from an increased understanding of the virus life cycle, the function of viral genome, and the ability to manipulate the viral genome in support of directional spread of virus and the expression of transgenes. In this unit, we review these advances in viral tracing technology and the way in which they may be applied for functional dissection of neural networks.
Calcium imaging of neuronal circuits in vivo using a circuit-tracing pseudorabies virus
Cold Spring Harbor protocols, 2010
Pseudorabies virus (PRV) is a neuroinvasive virus of the herpes family that has a broad host range but does not infect higher-order primates. PRV characteristically travels along chains of synaptically connected neurons and has been used extensively for elucidating neural circuits in the peripheral and central nervous system in vivo. The recombinant virus PRV369 is an attenuated retrograde tracer that encodes G-CaMP2, a fluorescent calcium sensor protein that is stable at physiological pH and mammalian temperature. This protocol describes the use of PRV369 to express G-CaMP2 in a neuronal circuit and to monitor its activity in a living animal, specifically in the submandibular ganglia (SMG), the peripheral parasympathetic ganglia that innervate the salivary glands. The procedure describes the delivery of PRV369 to the glands and shows how SMG neurons can then be imaged post-inoculation to explore connectivity and activity.
The elegant functions of the brain are facilitated by sophisticated connections between neurons, the architecture of which is frequently characterized by one nucleus connecting to multiple targets via projection neurons. Delineating the sub-nucleus fine architecture of projection neurons in a certain nucleus could greatly facilitate its circuit, computational, and functional resolution. Here, we developed multi-fluorescent rabies virus to delineate the fine organization of corticothalamic projection neuron subsets in the primary visual cortex (V1). By simultaneously labeling multiple distinct subsets of corticothalamic projection neurons in V1 from their target nuclei in thalamus (dLGN, LP, LD), we observed that V1-dLGN corticothalamic neurons were densely concentrated in layer VI, except for several sparsely scattered neurons in layer V, while V1-LP and V1-LD corticothalamic neurons were localized to both layers V and VI. Meanwhile, we observed a fraction of V1 corticothalamic neur...
Presynaptic Inputs to Any CNS Projection Neuron Identified by Dual Recombinant Virus Infection
PLOS ONE, 2015
Although neuroanatomical tracing studies have defined the origin and targets of major projection neurons (PN) of the central nervous system (CNS), there is much less information about the circuits that influence these neurons. Recently, genetic approaches that use Cre recombinase-dependent viral vectors have greatly facilitated such circuit analysis, but these tracing approaches are limited by the availability of Cre-expressing mouse lines and the difficulty in restricting Cre expression to discrete regions of the CNS. Here, we illustrate an alternative approach to drive Cre expression specifically in defined subsets of CNS projection neurons, so as to map both direct and indirect presynaptic inputs to these cells. The method involves a combination of Cre-dependent transneuronal viral tracers that can be used in the adult and that does not require genetically modified mice. To trigger Cre-expression we inject a Creexpressing adenovirus that is retrogradely transported to the projection neurons of interest. The region containing the retrogradely labeled projection neurons is next injected with Credependent pseudorabies or rabies vectors, which results in labeling of poly-and monosynaptic neuronal inputs, respectively. In proof-of-concept experiments, we used this novel tracing system to study the circuits that engage projection neurons of the superficial dorsal horn of the spinal cord and trigeminal nucleus caudalis, neurons of the parabrachial nucleus of the dorsolateral pons that project to the amygdala and cortically-projecting neurons of the lateral geniculate nucleus. Importantly, because this dual viral tracing method does not require genetically derived Cre-expressing mouse lines, inputs to almost any projection system can be studied and the analysis can be performed in larger animals, such as the rat.