A paper by researchers from Princeton University, just published in the open access journal PLoS One, describes a new virus-based technique for probing the connections between neurons while simultaneously monitoring their activity in live animals. Various methods are available for studying the activity of neurons and how they are connected to one another, but examining the co-ordinated activity of multiple nerve cells in neural circuits has, until now, posed a big challenge, because none of them can monitor both activity and connectivity at the same time.
The earliest method for tracing neuronal connections involved selective destruction of cell bodies or the axons extending from them. The damaged cells quickly degenerate, leaving a trail of cellular debris which persist for some time before being cleared away by specialized cells of the immune system. These debris can be stained using silver salts, so that the stain reveals the path taken by the fibres when the tissue is seen under the microscope. Another method uses fluorescent dyes which are applied to the nervous tissue. because the dyes are soluble in fats, they are absorbed by the cell membranes, and transported along the length of the nerve fibres.
The most widely used technique for monitoring neuronal activity is electrophysiology. This involves impaling specified neurons with microelectrodes, and enables researchers to measure the electrical currents which flow across the membrane when the cells become active. A characteristic response of active neurons is a transient increase in calcium ion concentration, which takes place in highly localized ‘microdomains’. Neuronal activity can therefore also be monitored using calcium-sensors, which fluoresce when they bind to calcium ions, so that changes in calcium ion concentration can be observed.
In the past few years, several research groups have developed more advanced methods for examining neuronal connections and activity. Ed Callaway and his colleagues at the Salk Institute used a virus-based synaptic tracer to examine neuronal connectivity. They generated a modified rabies virus which expresses green fluorescent protein (GFP), and is engineered in such a way as to be transmitted across only one synapse from infected neuron, enabling them to visualize all the connections formed by each cell harbouring the virus. More recently, Roger Tsien‘s group at the University of California, San Diego, developed a genetically-encoded fluorescent indicator protein, which emits fluorescence when it bind calcium ions.
The new method combines these two approaches – it is a rabies virus containing a genetically-encoded calcium sensor, developed by members of Lynn Enquist‘s lab. It uses a form of herpes virus called psuedorabies virus (PRV), which like other herpes viruses, infects neurons, and is transported along the axon after entering at the peripheral nerve terminal. When it reaches the cell body, the virus hijacks the cellular machinery, so that it’s own genes are expressed along with those in the cell. PRV infects a wide variety of animals, and in all but its natural host (the adult pig), the infection is lethal – the virus is carried along the peripheral nerves into the brain and spinal cord, causing encephalitis.
The strain used here, called PRV-Barth, has been used as a neuroanatomical tracer in earlier studies. These studies used a virus encoding GFP, but time, the GFP gene was replaced with one encoding a fluorescent indicator protein called G-CaMP2. Previously, live animals were infected, then dissected so that the neuronal connectivity, as revealed by the pattern of green fluorescence, could be examined in tissue slices. But in this study, the imaging was done in live animals – mice were anaesthetized, and a solution of viral particles was injected directly into the submandibular ganglion, a cluster of neurons located underneath the tongue, which controls the secretion of saliva, and which can be exposed by performing a simple surgical procedure. The infected neurons could therefore be imaged with two-photon microscopy.
PRV-Barth is transmitted from one cell to the next across synapses, the tiny gaps at which neurons communicate with one another. This new strain expresses the fluorescent calcium sensor in every cell it infects, and can therefore visualize both the connections between them. Spontaneous neuronal activity, and activity elicited by stimuli applied to the cells by microelectrodes, could be observed directly, because the calcium-sensitive protein emits fluorescence in response to the increases in calcium concentration elicited by stimulated cells. The method is highly sensitive to these changes – the fluorescence emitted was found to proportional to the number of nervous impulses generated by infected cells, and single impulses could be detected reliably.
The connections between neurons cannot be visualized directly, but only be inferred from the fluorescence signal. As the virus is transmitted from one neuron to another, each newly infected cell begins to express the fluorescent calcium-sensitive protein. The fluorescence signal can therefore only be detected in adjacent cells which form synapses with one other. Whereas the rabies virus used by Callaway and his colleagues was restricted to crossing just one synapse from infected cells, this one is transmitted across several chains of connections. The fluoresence signals could also still be detected more than 24 hours after initial infection.
This study demonstrates that this system works effectively in the peripheral nervous system, which is far less complex and more easily accessible than the brain. Nevertheless, such a technique could be to study neurons in the brain – some areas of the brain can be infected by direct injection, and then visualized through a cranial window. As it is developed further, this is likely to become a useful method for investigating the connections and activity of neurons. It could also help virologists gain a better understanding of the mechanisms of herpes infection, and how the nervous system responds to it.
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Granstedt, A. et al (2009). Fluorescence-Based Monitoring of In Vivo Neural Activity Using a Circuit-Tracing Pseudorabies Virus. PLoS ONE 4 (9): e6923. DOI: 10.1371/journal.pone.0006923.