Viral Transneuronal Tracing Technology: Defining the Synaptic Organisation of Neural Circuits


Viral transneuronal tracing is a powerful approach that has been widely used to define the cellular organisation of functionally defined systems that underlie brain function. The method exploits the ability of neurotropic viruses to infect neurons and produce infectious progeny that pass through the intimate synaptic connections among neurons to infect polysynaptic pathways. A number of important technological advances in recent years have dramatically improved the ability of this experimental approach to define the synaptology of complex neural circuits. In this chapter we provide an overview of the method as well as mechanisms that contribute to its effectiveness for circuit analysis. We focus on alpha herpesviruses and rabies virus as these pathogens have been most widely applied in transneuronal tracing studies.

Key Concepts:

  • Viral transneuronal tracing exploits the invasive properties of neurotropic viruses.

  • Knowledge of the functional organisation of the viral genome is fundamental to an informed application of the viral transneuronal tracing method.

  • Circuit architecture is influential in determining the progression of viral infection through a neural circuit.

  • Isogenic recombinant viruses expressing unique reporters permit analysis of collateralisation of projections within complex neural circuits.

  • Colocalisation of reporters of infection with neurotransmitters and peptides permits neurochemical phenotyping of neurons within labelled circuits.

Keywords: alpha herpesvirus; pseudorabies; rabies; transneuronal; transsynaptic

Figure 1.

The attributes of ‘classical’ versus viral transneuronal tracing technology are illustrated. Both of these tracing approaches exploit axonal transport capabilities of neurons to establish connections between regions and among neurons. Similarly, both classes of tracers can be separated into different classes based on the direction of transport. In either case, tracers are taken up by either by the cell bodies or axon terminals. Transport from the cell body to the terminal is termed ‘anterograde’ transport whereas transport from axon terminals to the soma is termed ‘retrograde’ transport. The major distinction between classical tracers and viral tracers relates to the extent of labelling of a circuit. Classical tracers (with few exceptions) do not cross synapses and therefore only establish regional associations. In contrast, viral transneuronal tracers cross synapses and, with advancing time, sequentially infect synaptically linked neurons. The replication of virus in each neuron amplifies the signal produced within neurons of a circuit, aiding in their localisation in the brain.

Figure 2.

The life cycles of pseudorabies virus and rabies virus, and their utility for circuit analysis, are illustrated schematically. In spite of the similarity in their common names, the two viruses use distinctly different mechanisms for replication and transneuronal infection. The major distinction in this regard is the fact that pseudorabies virus is a DNA virus and rabies virus is an RNA virus. The major steps in the life cycle of each virus are illustrated and labelled. See the text for a more detailed description.

Figure 3.

The unique advantages of viral transneuronal labelling technology is illustrated in this schematic diagram of the polysynaptic organisation of circuitry revealed by transport of rabies virus through primate neural networks following injection of rabies virus into motor cortex (M1) of nonhuman primate brain. Progressive retrograde transneuronal transport of the virus through multiple synapses reveals all cell groups synaptically linked to M1, including cerebellar components of the network. Reproduced with permission from Kelly and Strick .

Figure 4.

Retrograde transneuronal infection of Purkinje neurons in nonhuman primate brain following injection of rabies virus into motor cortex are illustrated. See Figure for the organisation of the circuitry that leads to this labelling. Immunocytochemical localisation of viral antigens reveals neurons infected by transneuronal passage of virus. Note that vial antigen is present throughout the somatodendritic compartments of neurons, revealing the cytoarchitecture of the infected neurons and further demonstrating that virus passes transneuronally from even the most distal branches of the dendritic tree. Reproduced with permission from Kelly and Strick .



Barnett EM, Cassell MD and Perlman S (1993) Two neurotropic viruses, herpes simplex virus type 1 and mouse hepatitis virus, spread along different neural pathways from the main olfactory bulb. Neuroscience 57(4): 1007–1025.

Boldogkoi Z, Sik A, Denes A et al. (2004) Novel tracing paradigms – genetically engineered herpesviruses as tools for mapping functional circuits within the CNS: present status and future prospects. Progress in Neurobiology 72: 417–445.

Braz JM, Enquist LW and Basbaum A (2009) Inputs to serotonergic neurons revealed by conditional viral transneuronal tracing. Journal of Comparative Neurology 514: 145–160

Brennand KJ, Simone A, Jou J et al. (2011) Modelling schizophrenia using human induced pluripotent stem cells. Nature 473: 221–225.

Campbell RE and Herbison AE (2007) Definition of brainstem afferents to gonadotropin‐releasing hormone neurons in the mouse using conditional viral tract tracing. Endocrinology 148(12): 5884–5890.

Cano G, Card JP and Sved AF (2004) Dual viral transneuronal tracing of central autonomic circuits involved in the innervation of the two kidneys in the rat. Journal of Comparative Neurology 471: 462–481.

Card JP (2001) Pseudorabies virus neuroinvasiveness: a window into the functional organization of the brain. Advances in Virus Research 56: 39–71.

Card JP, Santone DJ, Gluhovsky MY and Adelson PD (2005) Plastic reorganization of hippocampal and neocortical circuitry in experimental traumatic brain injury in the immature rat. Journal of Neurotrauma 22(9): 989–1002.

Card JP, Dubin JR, Whealy ME and Enquist LW (1995) Influence of infectious dose upon productive replication and transynaptic passage of pseudorabies virus in rat central nervous system. Journal of NeuroVirology 1: 349–358.

Card JP, Rinaman L, Lynn RB et al. (1993) Pseudorabies virus infection of the rat central nervous system: ultrastructural characterization of viral replication, transport, and pathogenesis. Journal of Neuroscience 13(6): 2515–2539.

Card JP, Kobiler O, McCambridge J et al. (2011) Microdissection of neural networks by conditional reporter expression from a Brainbow herpesvirus. Proceedings of the National Academy of Sciences USA 108(8): 3377–3382.

DeFalco J, Tomishima MJ, Liu H et al. (2001) Virus‐assisted mapping of neural inputs to a feeding center in the hypothalamus. Science 291: 2608–2613.

Denes A, Boldogkoi Z, Hornyak A, Palkovits M and Kovacs KJ (2006) Attenuated pseudorabies virus‐evoked rapid innate immune response in the rat brain. Journal of Neuroimmunology 180: 88–103.

Dolivo M (1980) A neurobiological approach to neurotropic viruses. Trends in Neuroscience 3: 149–152.

Dum RP, Levinthal DJ and Strick PL (2009) The spinothalamic system targets motor and sensory areas in the cerebral cortex of monkeys. Journal of Neuroscience 29: 14223–14235.

Ekstrand MI, Enquist LW and Pomerantz RJ (2008) The alpha‐herpesviruses: molecular pathfinders in nervous system circuits. Trends in Molecular Medicine 14(3): 134–140.

Gong S, DeCuypere M, Zhao Y and Ledoux MS (2005) Cerebral cortical control of orbicularis oculi motoneurons. Brain Research 1047(2): 177–193.

Granstedt AE, Szpara ML, Kuhn B , Wang S‐H and Enquist LW (2009) Fluorescence‐based monitoring of in vivo neural activity using a circut‐tracing pseudorabies virus. PLos One 4: e6923. doi: 6910.1371/journal.pone.0006923.

Grantyn A, Brandi A‐M, Dubayle D et al. (2002) Density gradients of trans‐synaptically labeled collicular neurons after injections of rabies virus in the lateral rectus muscle of the rhesus monkey. Journal of Comparative Neurology 451: 346–361.

Hoover JE and Strick PL (1999) The organization of cerebellar and basal ganglia outputs to primary motor cortex as revealed by retrograde transneuronal transport of herpes simplex virus type 1. Journal of Neuroscience 15(4): 1446–1463

Hoshi E, Tremblay L, Feger J, Carras PL and Strick PL (2005) The cerebellum communicates with the basal ganglia. Nature Neuroscience 8: 1491–1493.

Hubel D (1981)‐lecture.html.

Irnaten M, Neff RA, Wang J et al. (2001) Activity of cardiorespiratory networks revealed by transsynaptic virus expressing GFP. Journal of Neurophysiology 85: 435–438.

Jansen ASP, Van Nguyen X, Karpitskiy V, Mettenleiter TC and Loewy AD (1995) Central command neurons of the sympathetic nervous system: basis of the fight‐or‐flight response. Science 270: 253–260.

Kelly RM and Strick PL (2000) Rabies as a transneuronal tracer of circuits in the central nervous system. Journal of Neuroscience Methods 103: 63–71.

Kelly RM and Strick PL (2003) Cerebellar loops with motor cortex and prefrontal cortex of a nonhuman primate. Journal of Neuroscience 23(23): 8432–8444.

Kelly RM and Strick PL (2004) Macro‐architecture of basal ganglia loops with the cerebral cortex: use of rabies virus to reveal multisynaptic circuits. Progress in Brain Research 143: 449–459.

Klingen Y, Conzelmann KK and Finke S (2008) Double‐labeled rabies virus: live tracking of enveloped virus transport. Journal of Virology 82: 237–245.

Kristensson K (1970) Morphological studies of the neural spread of herpes simplex virus in peripheral nerves. Acta Neuropathologica 16: 54–63.

Lafon M (2005) Rabies virus receptors. Journal of NeuroVirology 11: 82–87.

Lewis P and Lentz TL (2000) Rabies virus entry at the neuromuscular junction in nerve‐muscle cocultures. Muscle Nerve 23: 720–730.

Livet J, Weissman TA, Kang H et al. (2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450: 56–63.

Marchand CF and Schwab ME (1987) Binding, uptake and retrograde axonal transport of herpes virus suis in sympathetic neurons. Brain Research 383: 262–270.

Middleton FA and Strick PL (2001) Cerebellar projections to the prefrontal cortex of the primate. Journal of Neuroscience 21(2): 700–712.

Miyachi S, Lu X, Inoue S et al. (2005) Organization of multisynaptic inputs from prefrontal cortex to primary motor cortex as revealed by retrograde transneuronal transport of rabies virus. Journal of Neuroscience 25(10): 2547–2556.

Moschovakis AK, Gregoriou GG, Ugolini G et al. (2004) Oculomotor areas of the primate frontal lobes: a transneuronal transfer of rabies virus and [14C]‐2‐deoxyglucose functional imaging study. Journal of Neuroscience 24(25): 5726–5740.

Pomeranz LE, Reynolds AE and Hengartner CJ (2005) Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiology and Molecular Biology Reviews 69: 462–500.

Rathelot J‐A and Strick PL (2006) Muscle representation in the primary motor cortex. An anatomical perspective. Proceedings of the National Academy of Sciences USA 103: 8257–8262.

Rinaman L and Schwartz GJ (2004) Anterograde transneuronal viral tracing of central viscerosensory pathways in rats. Journal of Neuroscience 24: 2782–2786.

Rinaman L, Card JP and Enquist LW (1993) Spatiotemporal responses of astrocytes, ramified microglia, and brain macrophages to central neuronal infection with pseudorabies virus. Journal of Neuroscience 13(2): 685–702.

Sams JM, Jansen AS, Mettenleiter TC and Loewy AD (1995) Pseudorabies virus mutants as transneuronal tracers. Brain Research 687: 182–190.

Schnell MJ, McGettigan JP, Wirblich C and Papaneri A (2010) The cell biology of rabies virus: using stealth to reach the brain. Nature Reviews Microbiology 8: 51–61.

Smith BN, Banfield BW, Smeraski CA et al. (2000) Pseudorabies virus expressing enhanced green fluorescent protein: a tool for in vitro electrophysiological analysis of transsynaptically labeled neurons in identified central nervous system circuits. Proceedings of the National Academy of Sciences USA 97: 9264–9269.

Ugolini G (1995) Specificity of rabies virus as a transneuronal tracer of motor networks: transfer from hypoglossal motoneurons to connected second‐order and higher order central nervous system cell groups. Journal of Comparative Neurology 356: 457–480.

Ugolini G, Kuypers HGJM and Simmons A (1987) Retrograde transneuronal transfer of herpes simplex virus type 1 (HSV1) from motoneurons. Brain Research 422: 242–256.

Vahlne A, Nystrom B, Sandberg M, Hamberger A and Lycke E (1978) Attachment of herpes simplex virus to neurons and glial cells. Journal of General Virology 40: 359–371.

Watson HD, Tignor GH and Smith A (1981) Entry of rabies virus into the peripheral nerves of mice. Journal of General Virology 56: 372–382.

Weible AP, Schwarcz L, Wickersham IR et al. (2010) Transgenic targeting of recombinant rabies virus reveals monosynaptic connectivity of specific neurons. Journal of Neuroscience 30(49): 16509–16513.

Wickersham IR, Lyon DC , Barnard RJO et al. (2007) Monosynaptic restriction of transsynaptic tracing from single genetically targeted neurons. Neuron 53(5): 639–647.

Zemanick MC, Strick PL and Dix RD (1991) Direction of transneuronal transport of herpes simplex virus 1 in the primate motor system is strain‐dependent. Proceedings of the National Academy of Sciences USA 88: 8048–8051.

Further Reading

Boldogkoi Z, Balint K, Awatramani GB et al. (2009) Genetically timed, activity‐sensor and rainbow transsynaptic viral tools. Nature Methods 6: 127–130.

Callaway EM (2008) Transneuronal circuit tracing with neurotropic viruses. Current Opinion in Neurobiology 18: 617–623.

Enquist LW and Card JP (2003) Recent advances in the use of neurotropic viruses for circuit analysis. Current Opinion in Neurobiology 13: 603–606.

Enquist LW, Tomishima MJ, Gross MJ and Smith GA (2002) Directional spread of alpha‐herpesvirus in the nervous system. Veterinary Microbiology 86: 5–16.

Kuypers HGJM (1990) Viruses as transneuronal tracers. Trends in Neurosciences 13: 71–75.

Song CK, Enquist LW and Bartness TJ (2005) New developments in tracing neural circuits with herpesviruses. Virus Research 111: 235–249.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Strick, Peter L, and Card, J Patrick(Dec 2011) Viral Transneuronal Tracing Technology: Defining the Synaptic Organisation of Neural Circuits. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0022359]