A flexible software framework and post hoc cell type discrimination for in vivo two-photon calcium imaging of neuronal population activity

The neocortex, especially that of humans, is considered to be the most complex structure in the known universe. Functionally, it is involved in higher order brain functions such as sensory processing, generation of motor commands, and cognitive processes. Structurally, it encloses the cerebral hemispheres as a one to four millimeter thick sheet and consists of a highly complex network of interconnected cells. Even though a wealth of anatomical and physiological data has been collected over the past decades, the functional organization of the neocortical microcircuitry is still a mystery. What is known for sure, however, is that information is represented and processed in the neocortex as membrane potential fluctuations travelling through the neuronal network. We refer to such fluctuations as neuronal activity. Due to its tissue penetration capabilities of up to one millimeter combined with threedimensional spatial resolution, two-photon laser-scanning microscopy (2PM) has become the method of choice to image populations of neurons inside the neocortex of living animals. In combination with techniques that allow to load populations of several hundred cells with fluorescent indicators of neural activity (in particular: calcium indicators), it is possible to observe neuronal population activity inside the brain in vivo. Researchers performing in vivo 2PM often use custom-built microscopy setups controlled by custom-written software due to the high flexibility they require. The continuous technical advancement of the field created a need for new control software that is flexible enough to supply both the biological researcher with the newest image acquisition techniques and the developer with a solid platform from which various new extensions can be implemented and ideas quickly realised. In the first part of this thesis, I introduce HelioScan, a software package written in LabVIEW. It serves i) as a component collection allowing to flexibly assemble a microscopy control software according to the own needs, and ii) as a framework within which new functionality can be implemented in a quick and structured manner. A specific HelioScan application assembles at run-time from individual software components and subcomponents, based on user-definable configuration files. This concept allows high flexibility with regard to different hardware or functional requirements. Currently implemented components enable i) camera-based imaging such as intrinsic optical signal (IOS) imaging, as well as different laser-scanning modes employing ii) galvanometric mirrors, iii) acousto-optic deflectors (AODs) or iv) resonant scanners. Components are implemented in an object-oriented fashion, with generic interface classes defining the component types as well as their possible interactions. New components can be implemented by inheriting from existing base classes. Various scaffold classes derived from the generic component classes allow to extend quickly on already existing functionality. Due to its component-based architecture, HelioScan fosters collaborative efforts in which several developers can work in parallel when implementing new functionality. The cellular components of the neocortical neural network are diverse with respect to their precise morphology, physiology and molecular composition. Their diversity does not form a continuum, however. As a consequence, they can be grouped into different classes based on common or at least similar features. Different functional roles can be attributed to these classes (cell types). It is thus important to be able to distinguish different cell types in two-photon calcium imaging. In the second part of this thesis, I present three methods for cell type discrimination in two-photon calcium imaging of the neocortex. The first of these methods allows to distinguish GABAergic cells, astrocytes and excitatory neurons and is based on a three fluorescent labels that can be simultaneously imaged and spectrally resolved. In particular, it involves the GAD67-GFP transgenic mouse line (in which GABAergic neurons express green fluorescent protein (GFP)), uses the red dye sulforhodamine 101 (SR101) for astrocyte-specific labelling and unspecific loading with the green calcium indicator OGB-1. The second method uses post hoc immunostaining to discriminate cells according to their expression of molecular markers. Specifically, after in vivo two-photon calcium imaging and acquisition of a three-dimensional reference image stack, the brain is cut into coronal sections. Slices containing parts of the volume-of-interest are selected based on the blood vessel pattern on their thin stripe of pial surface. After immunostaining, the slices are imaged and rotation-fitted to the reference mage stack. Cell-to-cell assignment between reference image stack and slice image stacks allows cell type discrimation according to different combinations of molecular markers. I demonstrated the applicability of this approach by discriminating different GABAergic cells based on their expression of calciumbinding proteins both in GAD67-GFP mice and in wildtype mice expressing a genetically encoded calcium indicator (GECI). The third method is similar to the second one, but uses a cutting procedure that provides intrinsic rotation-fitting and results in tangential sections. Although further exploration of this approach is still pending, first results indicate that cells previously imaged in vivo can be easily re-identified in the sectioned brain. In summary, I introduced two flexible and extendible tools. I expect HelioScan to become the software of choice in two-photon imaging laboratories that have to deal with diverse and frequently changing hardware and functionality requirements. The rise of chronic preparations based on viral-induced GECI expression allows activity read-outs to be recorded over weeks or months. Due to the associated decrease in the relative overhead, I expect post hoc cell-type discrimination to become a technique that is routinely applied in future experiments.