Genetic Reporters of Neuronal Activity: c-Fos and G-CaMP6

Abstract

The majority of 20th century investigations into anesthetic effects on the nervous system have used electrophysiology. Yet some fundamental limitations to electrophysiologic recordings, including the invasiveness of the technique, the need to place (potentially several) electrodes in every site of interest, and the difficulty of selectively recording from individual cell types, have driven the development of alternative methods for detecting neuronal activation. Two such alternative methods with cellular scale resolution have matured in the last few decades and will be reviewed here: the transcription of immediate early genes, foremost c-fos, and the influx of calcium into neurons as reported by genetically encoded calcium indicators, foremost GCaMP6. Reporters of c-fos allow detection of transcriptional activation even in deep or distant nuclei, without requiring the accurate targeting of multiple electrodes at long distances. The temporal resolution of c-fos is limited due to its dependence upon the detection of transcriptional activation through immunohistochemical assays, though the development of RT-PCR probes has shifted the temporal resolution of the assay when tissues of interest can be isolated. GCaMP6 has several isoforms that trade-off temporal resolution for signal to noise, but the fastest are capable of resolving individual action potential events, provided the microscope used scans quickly enough. GCaMP6 expression can be selectively targeted to neuronal populations of interest, and potentially thousands of neurons can be captured within a single frame, allowing the neuron-by-neuron reporting of circuit dynamics on a scale that is difficult to capture with electrophysiology, as long as the populations are optically accessible.

Keywords: GCaMP6; Genetically encoded calcium indicators; Immediate early genes; Neuronal activation; c-Fos.

Figure 1

A. Different measures of neuronal activation include intracellular and extracellular electrical recordings, intracellular calcium concentration measured through fluorescent reporters or through downstream transcriptional activation of IEGs like c-fos through CAM kinase (CAMK) and MAP Kinase (MAPK) pathways, and measures of metabolism such as uptake of oxygen in the BOLD response. B. Selected brain regions of interest in anesthesiology and pain research. Deep targets indicated by gray shading. The wide spatial distribution through the central nervous system, makes IEG reporter approaches appealing for simultaneous sampling. Similarly, deep targets are difficult to access with electrophysiology and microscopy methods. Abbreviations: PFC, prefrontal cortex; Thal, thalamus; NB, nucleus basalis of Meynert; VLPO, ventral lateral preoptic nucleus; VTA, ventral tegmental area; PAG, periaqueductal gray; LDT, lateral dorsal tegmentum; LC, locus ceruleus; PBN, parabrachial nucleus; SC, spinal cord. C. Layer 2/3 of frontal cortex in a mouse expressing the red fluorescent protein Td-tomato in parvalbumin expressing interneurons and the green fluorescent protein GCAMP6 in pyramidal neurons. Electrophysiologic methods cannot easily target specific cell types during recording, whereas genetically encoded calcium indicators can target expression to specific cell lines.

Figure 2

Signal transduction pathways involving c-fos. Calcium fluxes into the neuron during action potentials via NMDARs and voltage dependent calcium channels. The influx of Ca²⁺ binds calmodulin, activating CaMKII and then CREB. NMDAR activity also activates RasGRF1, activating the MAPK pathway via Ras and c-Raf activation, with resultant activation of CREB and Elk1. Elk1 binds SRF and acts as a transcription factor at SRE, while activated CREB acts as a transcription factor at CaRE, with resultant expression of c-fos mRNA. Once the c-Fos is transcribed, it binds to jun to form the transcription factor AP-1, which results in transcription of late response genes via the TRE (Chaudhuri et al., 2000; Wang et al., 2007). Abbreviations: NMDAR, N-methyl-D-aspartate glutamine receptor; CaV1.2, L-type Calcium Channel; CaM, calmodulin; CaMKII, calcium/calmodulin dependent protein kinase II; RasGRF1: Ras-guanine nucleotide releasing factor; MEK1/2, mitogen-activated protein kinase kinase 1 and 2; MAPK1/3, mitogen-activated protein kinase 1 and 3; SRF, serum response factor; CREB, cyclic AMP response element-binding protein; AP-1, activating protein 1; SRE, serum response element; CRE, cAMP response elements; TRE, 12-O-Tetradecanoylphorbol-13-acetate response element.

Figure 3

A. A sample c-fos experimental protocol. Animals were divided into three groups. Group one (+ISO with emergence) was exposed to isoflurane for 60 minutes, then allowed to recover for 60 minutes before being sacrificed for c-fos histology (red line). Group 2 (+ISO without emergence) was exposed to isoflurane for 60 minutes and then sacrificed. Group 3 (-ISO oxygen only) was exposed to oxygen alone for 60 minutes prior to sacrifice. B. Sections through the parabrachial nucleus were stained for DAPI and c-fos, showing that exposure to isoflurane or oxygen alone were insufficient for increasing c-fos expression. Neurons in parabrachial nucleus increased c-fos expression when the animals were anesthetized and then emerged from anesthesia (seen as the cluster of cells in +ISO with emergence ii and iii). Data from (Muindi et al., 2016). Abbreviations: ISO, isoflurane; cb, cerebellum; vsc, ventral spinocerebellar tract; scp, superior cerebellar peduncle.

Figure 4

Image registration for suppression of motion artifacts. Two frames of an acquired video of a fluorophore are shown with motion occurring between the acquisition of frame 17 (A) and frame 18 (B). C. The motion results in blurring of the mean image. D. The difference image, resulting from B–A. E. A schematic drawing of D, showing that the movement occurred while the laser was scanning through the frame. This is indicated by the thin horizontal line, as those neurons above the line moved before the scan path reached them, while those below the line did not. F. Assuming that all motion between frames occurs as a rigid translation does not substantially sharpen the mean image when compared to C. G. Using a spline approach to model movement as a non-rigid deformation of the image results in a notable sharpening of the mean image compared to C, suggesting that superior registration has been obtained.

Figure 5

A. Selective expression of GCaMP6 is a cell line and tissue of interest will require titration studies, where serial dilutions are injected into different mice that are then imaged at the latest time point that will be imaged in the study. B. Adequate expression is indicated by fluorescence signal that shows nuclear sparing (circle), whereas complete filling of the neuron (arrow) indicates overexpression.

Figure 6

A. After registration, a neuron with appropriate expression levels of GCaMP6 (the circled neuron from 5A) is identified, and the correlation amongst pixels during the movie is used to isolate the pixels of interest in order to extract the fluorescence signal attributed to a single neuron, highlighted in the blue area. B. The fluorescence signal is first normalized to is mean level (top), and the generating spike rate can be estimated through a deconvolution operation (bottom) to within a proportionality constant, assuming stable kinetics of the GCaMP6 signal.