A miniature head-mounted two-photon microscope small enough for a rat to carry allows researchers to visualize neuronal signaling while the animal freely interacts with its environment.
It is often said that if you want to know how an animal lives, you need to study it in its natural environment. Imagine trying to figure out how a beaver lives if you have to study one sitting in a cage. Could you even determine that it spends much of its time in the water? Neuroscientists must often feel as if they are in a similar position. Sure, they can dissect a brain and perform measurements on isolated cells and tissue sections; or they can perform MRI scans on immobilized subjects exposed to controlled stimuli. But so far nobody has come up with an MRI helmet a subject can wear while they perform natural routines.
Even if a mobile MRI existed, the investigator would be limited to seeing relatively large changes in neuronal activity that correlated with certain stimuli or behaviors. The underlying cellular-level patterns of neuronal signaling that give rise to these larger-scale changes would remain a mystery.
But fluorescence microscopy, with the help of intracellular fluorescent calcium sensors, can visualize cellular-scale neuronal activity in isolated neurons and in situ. Although it would be unacceptable to deliver such probes to humans, rodents are another matter and are widely used in neuroscience research. But how do you fit a research-grade fluorescence microscope on the head of a mouse or rat?
One widely used method of in vivo fluorescence imaging in the brain is to remove or thin the region of the skull covering the area to be examined and then affix the rodent's head to the objective of a two-photon microscope. This typically involves an anesthetized animal, but recent work in David Tank's laboratory at Princeton University has used head-restrained, awake mice held on top of a Styrofoam ball floating on a cushion of air where they can walk and run in place or navigate inside a virtual reality environment (Dombeck et al., 2007). “David's system is fantastic,” remarks Jason Kerr of the Max Planck Institute in Germany. But it's still head-restrained.
To eliminate the head restraint that prevents an animal from interacting with its environment in a natural manner, a number of investigators have been pushing the boundaries of microscope miniaturization. The extent to which a microscope can be miniaturized was recently demonstrated by Mark Schnitzer from Stanford University using a single-photon head-mounted fiberscope to visualize neuronal firing in the deep brain of a freely moving mouse (Flusberg, et al., 2008). But single-photon illumination can only image cells immediately under the microscope objective and necessitates inserting the fiber-optic objective directly into the brain tissue.
In an effort to develop a less invasive microscope, Jason Kerr and colleagues developed a head-mounted two-photon microscope that could be carried by a freely moving rat (Sawinski et al., 2009). All the microscope optics reside in the miniature microscope, while the excitation and emission light are delivered and collected by optical fibers. “The microscope is small enough to be carried around by the animal and doesn't inhibit its behavior whatsoever,” says Kerr. The animals displayed normal running, digging, feeding and pouncing behaviors while carrying the fiberscope.
To demonstrate the unique capabilities of the system, Kerr and colleagues visualized calcium transients in populations of neurons in layer 2/3 of the visual cortex—a two-photon-microscope–accessible region beneath the surface of the brain—as the animal explored a raised unlit track containing three display monitors positioned to show the rat static images at different locations along the route. Infrared LEDs attached to the fiberscope tracked the movement of the animal's head to determine when it was viewing the images. Neuronal activity increased when the animals were viewing the images, and 7 of the 35 neurons the investigators were recording showed a significantly higher rate of firing for a particular monitor.
Kerr says, “It's like natural vision. [The rat] interacts with the environment freely and in any way it pleases.” Kerr envisages their device being used to study things like social behaviors and prey capture that involve complex motor and visual tasks and require the animal to move about freely. In preparation for this, “we are working on a miniaturized version with more bells and whistles which will make it more applicable to complex behaviors,” says Kerr. One can imagine that one day one of these future behaviors may very well be two animals examining the strange devices attached to one another's heads.