Calcium Imaging

Calcium is a crucial component of cellular communication, development, and function. In neurons, calcium influx into the cell results in depolarization, generation of action potentials and neurotransmitter release. Calcium activity provides a measure of real-time neuronal activity in awake and behaving animals. More importantly, the use of genetically encoded calcium sensors (GECIs) allows us to target the indicator to specific types of neurons, and projections, allowing us to determine neuronal activity from just one type of neuron. Classical in vivo electrophysiology methods record activity from multiple types of neurons within a region. The added specificity of GECIs allows us to disentangle the complex neurocircuitry underlying behavior. Currently there are two approaches used in the laboratory for in vivo calcium recordings.

Fiber Photometry

Fiber Photometry is an imaging technique that collects and measures fluorescence in deep brain regions. In the example on the right, a virus encoding GCamP6 was infused into the NAc core and shell subregions, and a fiber optic implanted into the NAc shell on the left side of the brain and another into the NAc core on the right side of the brain for simultaneous measurements in both regions. Once the GCamP expression peaks, the the implant is connected to a control system to deliver light of one wavelength to activate the GCamP and collect the light it generates. The light and signal are passed through a fiber optic to a photodetector and the “bulk” fluorescence is measured. In fiber photometry, the fiber collects the total light being emitted, in essences capturing changes in "bulk" fluorescence. While this approach does not tell us much about specific subpopulations of neurons within the NAc, it does provide a very sensitive and reliable readout of activity. This approach is also very scalable, and easily applied across many species, including rats.

​In the example on the right, we observe a rise in activity in the NAc shell but not the core when the male rat is exposed to "stranger". This rise is evident with a new rat five days later, but seems to decrease when re-exposed to a novel rat less than 1 day later, perhaps indicating habituation. The core is largely unresponsive to this social interaction.

​Simultaneous calcium recording in the nucleus accumbens shell and core of a rat during a social interaction test. An unfamiliar rat is placed in the open field and allowed to interact with the subject for approximately 1-minute. A sharp rise in fluorescence is detected in the shell but not the core. This effect is still present when significant time is allowed between exposures, but habituation occurs when an interaction occurs within 24-hours of the last. 

Miniscopes

This technique, as with fiber photometry, relies on genetically encoded calcium indicators to capture calcium influx. In this case, a GRIN lens is implanted into the brain, and images are taken. After quite a bit of image processing, individual neurons can be distinguished. Miniscope technology is a more difficult approach to implement. It is particularly difficult to port to rats, mainly because the miniscope (based on the UCLA design) has been developed for mice that weigh less than 40-50g. Small movements in the rats can break this rather fragile instument, for example, regular grooming behavior puts great deal of stress on some of the miniscope components. We have thus bulked up certain components of the miniscope to handle these stressors. Another important consideration is that the GRIN lenses must be longer to reach deep regions in the rat. Such lenses are uncommon and because they are custom made they can be quite expensive.

Miniscope technology allows us to parse activity of neurons in a way that fiber photometry cannot. For example, when tagging a specific type of neuron (e.g. dopamine neurons in the VTA), it's possible to observe subpopulations that are active, become inactive, or don't change with different stimuli. If fiber photometry was used, the net result may be little to no change in bulk fluorescence. However with miniscopes individual neurons can be studied over time with miniscopes, allowing us to determine if multiple populations exists, and how they may change over time. Thus, while miniscopes are more difficult to implement, and process the copious amounts of data, they provide a wealth of information on population dynamics within a give field of view.

UCLA designed miniscope with baseplate. 

Miniscope recording from an awake rat

Calcium imaging of the nucleus accumbens in an awake and behaving rat using the miniscope. The rat was infused with a generalized GCamp6f AAV and then implanted with a 12 mm long custom GRIN lens. Rat was awake and freely moving at the time of recording.