Calcium Indicators and Their Interaction with Physiology

Unlike electrophysiology, calcium imaging does not directly measure changes in local electric potentials (or currents) via the movement of ions. Instead, calcium imaging uses the influx of calcium into a cell, detected by a calcium indicator, as a proxy for neuronal activity. Early experiments employed chemical indicators – molecules that directly chelated calcium – such as fura-2 or Oregon-Green (Grienberger & Konnerth, 2012) which allowed for a relatively fast and direct measure of calcium influx. The downside to chemical indicators is that they must be bath applied to the tissue for each recording session, thus limiting the range of experiments to those that can be performed acutely under head fixation; however, for many researchers this is a relatively easy/robust method for introducing the calcium indicator into cells (Grienberger & Konnerth, 2012; Russell, 2011). Additionally, most chemical indicators do not easily allow introduction into specific cells/cell types. Chemical indicators continued to be employed due to their ease of use and the relative speed of their signal; additionally, many chemical indicators shift their excitation/emission wavelength (see 1.5.3) upon binding calcium, allowing accurate assessment of calcium levels despite uneven indicator levels or photobleaching (Russell, 2011). The predominance of chemical indicators has been surpassed by recent

advances with another class of calcium indicators whose speed and signal-to-noise ratio now surpass that of the more common chemical indicators (Chen et al., 2013; Helassa, Podor, Fine, & Török, 2016) and whose fluorescence is more photostable, allowing longer-term imaging (Tian et al., 2009).

The other method for determining neuronal calcium influx is through the use of a Genetically Encoded Calcium Indicators (GECI), first developed with Cameleon in 1997 (Miyawaki et al., 1997) and re-engineered in 2001 to improve its signal-to-noise ratio as GCaMP (Nakai, Ohkura, & Imoto, 2001). GCaMP has been vastly improved since 2001 to further increase its signal-to-noise ratio and dynamics resulting in an explosion of research employing GCaMP to measure calcium activity (Hamel, Grewe, Parker, & Schnitzer, 2015). GCaMP works by fusing a green fluorescent protein (GFP) molecule, connected to the N-terminus of the M13 fragment of myosin light chain kinase with calmodulin, which is connected to the C-terminus of M13 (Nakai et al., 2001). Subsequent calcium binding with GCaMP introduces a conformational change of the entire molecule which causes a corresponding increase in fluorescence intensity (Nakai et al., 2001). Rapid binding requires that GCaMP have a high affinity for calcium; the trade- off is that while GCaMP increases its fluorescence relatively fast in the presence of calcium, its subsequent decrease after calcium influx stops is slow (Hendel et al., 2008). The entire rise/fall in fluorescence is collectively referred to as a “calcium transient”, though only the rising phase is associated with spiking activity since that is when calcium influx occurs (Chen et al., 2013). The slow decay time of calcium transients is related to both the high affinity of GCaMP for calcium and the relative concentrations of

GCaMP/calcium, and can pose issues for disambiguating fluorescence changes occurring in neighboring neurons (see section 1.5.5 below). This is further complicated by the fact that neurons in which GCaMP has invaded the nucleus exhibit much longer decay times than those without GCaMP in the (Tian et al., 2009). Even the fastest of the most

commonly-used variant (GCaMP6f), however, has a rise-time on the order of 0.1s (Chen et al., 2013), making it difficult to resolve individual action potentials in most

preparations. Careful consideration/evaluation of these factors is vital to producing high- quality data that can be post-processed to obtain interpretable calcium traces (see 1.5.5 below).

How GCaMP is introduced to neurons is a key factor influencing its concentration within the neuron and if/when it will begin to invade the nucleus. There are two main methods for inserting GCaMP into a neuron: via the use of stable mutations in the genome (i.e. transgenic animals) and via viral introduction of the gene for GCaMP. The use of transgenic animals requires careful editing of the genome, generally in mice over rats, to ensure stable expression of GCaMP proteins within a given class of neurons specified by the promoter used. One key advantage of this approach is that, since any genome level changes are introduced at conception, they must result in reliable and stable expression of GCaMP within cells (Dana et al., 2014), which generally precludes any of the deleterious effects related to overexpression, e.g. slower calcium transients and traveling waves (see discussion above/below). Another advantage to this approach is that it can be used to selectively target neurons depending on the promoter used, and/or by cross-breeding GCaMP reporter mice with mice expressing Cre in the neurons of interest

(Zariwala et al., 2012). However, there are a couple disadvantages concerning the use of transgenic mice. First, extensive work must be done to breeding and genotyping these mice. Second, depending on the mouse line and promotor used there can be a variety of expression and brightness levels within neurons, with certain lines exhibiting high levels of expression in one brain region at the expense of others or producing highly fluorescent neurons but in only a sparse subset of cells (Dana et al., 2014). Thus, careful attention to the expression attributes of each mouse line is important to obtain high quality imaging, though many mouse lines are capable of producing adequate data for a wide range of regions (e.g. lines 5.11 or 5.17 in Dana et al. (2014)).

The other ubiquitous method for introducing GCaMP into neurons is through viral transduction. In this method, the gene for GGaMP is packaged into a viral vector which is then introduced into the brain area of interest, usually through stereotactic injection. After introduction, the virus proceeds to insert itself into the cell, resulting in the translation and transcription of the protein of interest (Kaspar et al., 2002) which in this case is GCaMP. Construction of the appropriate viral vector involves combining the GCaMP gene with 1) a virus and 2) a promoter to infect the appropriate cells. Both the virus type and promoter interact to influence the eventual expression profile of GCaMP. For example, the commonly used adeno-associated virus (AAV) has 11 serotypes (Mori, Wang, Takeuchi, & Kanda, 2004), each with a drastically different infection

patterns/expression profile: in conjunction with a synapsin promoter (specific to neurons but excluding glial cells), AAV5 injection results in sparse labeling of cells while AAV9 results in abundant but promiscuous infection of neurons in the mouse hippocampus that

could also include non-specific infection of non-neuronal cells (Aschauer, Kreuz, & Rumpel, 2013), Overexpression of GCaMP can in turn lead to abnormal cell responses or even cell death (Resendez et al., 2016; Tian et al., 2009). Thus, careful attention to viral titer is required for any experiment using virally introduced GCaMP, usually via

performing a study to assess expression levels and cell health at a time point matching the intended time of performing the experiment after injecting virus at various dilutions (Resendez et al., 2016). Despite these potential pitfalls, viral introduction of GCaMP is a robust and relatively easy means of inserting the calcium indicator into neurons.