Analysis of neural activity with fluorescent protein biosensors

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Analysis of neural activity with fluorescent protein biosensors

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Robert D. Burkea,*, Shunsuke Yaguchib a

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada b Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, Japan *Corresponding author: e-mail address: [email protected]

Chapter outline 1 Background......................................................................................................519 2 Methods...........................................................................................................520 2.1 Preparation of RNA............................................................................520 2.2 Imaging............................................................................................521 2.3 Analysis of fluorescent intensity over time.............................................521 2.4 Limitations........................................................................................524 Acknowledgments..................................................................................................526 References............................................................................................................526

Abstract Fluorescent calcium sensors provide a means of detecting and analyzing cytoplasmic calcium levels in embryos and larvae. Conventional RNA injection of eggs results in expression of protein sensors throughout larval tissues. Larvae are immobilized for wide field or confocal recordings and video records reveal recurrent fluctuations in cytoplasmic calcium levels in several cell types. Neurons can be identified by location and form, and continuous records made of their activity. Confocal image stacks are registered and Z-axis, fluorescence intensity profiles of individual neurons generated to provide time/activity plots. These optogenetic methods enable analysis in intact larvae of the activity of identified neurons or effectors, such as muscles or ciliary band cells.

1 Background Echinoid larvae are easily accessible models that have revealed many aspects of planktonic larvae. Their small size, clarity and simple anatomy facilitate studies of cellular level details of swimming behavior, feeding, and larval development. There have been several approaches to the problem of recording cellular activity in Methods in Cell Biology, Volume 151, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2018.10.010 © 2019 Elsevier Inc. All rights reserved.

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larvae—kymographs, extracellular recordings, and video recordings have all provided objective measures of cellular responses (Gustafson, Lundgren, & Treufeldt, 1972; Mackie, Spencer, & Strathmann, 1969; Satterlie & Cameron, 1985; Strathmann, 2007). However, a fuller understanding of cellular functions in planktonic environments requires additional approaches for analysis of cellular activity. Neural activity causes rapid changes in intracellular free calcium, which has been used to track neural activity (Kerr et al., 2000; Tank, Sugimori, Connor, & Llinas, 1988). Although neurons have unusually fast calcium dynamics and low peak calcium accumulations, optical biosensors have been developed to probe neuronal activity. Modern fluorescent biosensors have the specificity, sensitivity, and versatility necessary to provide detailed readouts of the responses of neurons and effector cells and have become a key component of optogenetic monitoring. A range of fluorescent biosensors have been developed (Chen, Truong, & Ai, 2017; Ni, Mehta, & Zhang, 2018) many of which are based on modification of green fluorescent protein (GFP). Several calcium biosensors employ Calmodulin (CaM) and the CaM interacting peptide M13 as an engineered conformational switch that modulates the fluorescent properties of the biosensor when is placed adjacent to the chromophore of a fluorescent protein. GCamPs is such a single protein calcium biosensor, which has undergone several rounds of structure guided mutagenesis and testing to improve sensitivity, dynamic range and kinetics (Chen et al., 2017; Nakai, Ohkura, & Imoto, 2001). GCaMP6s produces large fluorescence transients in response to single action potentials and excellent detection rates over several recording sessions and has achieved widespread application in calcium imaging of neurons. Here we describe methods to monitor calcium-based activity in neurons and effector cells in sea urchin larvae. The approach uses GCaMP6s as a calcium biosensor and enables the extraction of fluorescence profiles from single neurons. The method demonstrates how activity in neurons and effector cells, such as the ciliated cells of the ciliary band, can be correlated and has the potential to permit more detailed experimental analysis of the range of functions of the larval nervous system.

2 Methods 2.1 Preparation of RNA The GCamP6s calcium sensor (Addgene Plasmid 40753; https://www.addgene.org/ 40753/) was amplified (GCaMP6:F1 50 -GACTGGATCCGCCACCATGGGTTCTC ATC-30 , GCaMP6:R1 50 -CTGACTGAATTCTCACTTCGCTGTCATCATTTG-30 ) and cloned between BamHI and EcoRI in pCS2 + (https://www.addgene.org/vec tor-database/2295/). Some preparations of mRNA for injection were made by linearizing purified plasmid pCS2 +:GCamP6s with Not1 and a transcription reaction was prepared using the SP6 mMessage mMachine kit (Ambion). An alternative method used a DNA template amplified from pGP-CMV-GCaMP6s (primers F1 (50 -AAGCAATGCTCGTTTAGG-30 ) and M13 reverse primer). Amplified template

2 Methods

DNA was used directly in an SP6 mMessage mMachine reaction and the abundance of RNA product was determined spectrophotometrically and confirmed by gel electrophoresis. Eggs were prepared for microinjection as described previously (Krupke, Yaguchi, Yaguchi, & Burke, 2014). For detailed methods on microinjection, see also chapter “Microinjection of oocytes and embryos with synthetic mRNA encoding molecular probes” by von Dassow et al. of the companion volume 150, Part A. Injection solutions contained water, 120 mM KCl, and 200–500 ng/μL RNA. Robust expression of GCamp6s was confirmed with epifluorescence after 24 h and embryos were further cultured until early larval stages.

2.2 Imaging Larvae expressing GCamP6s were pipetted onto NewSilane Adhesive Coated Slides (Newcomer Supply Ltd.) and trapped under a glass coverslip attached along two edges with double-sided adhesive tape (3M Inc.). Paraffin oil was applied to the open edges of the coverslip to reduce evaporation and temperatures maintained at 16°C in the dark, or reduced light. Larvae tolerate 6–8 h in these preparations without apparent deterioration and they can be returned to larger culture dishes by careful removal of the coverslip. Larvae were imaged with a Leica DM6000 epifluorescence microscope fitted with a Hamamatsu Orca camera controlled with μManager (1.4; https://microman ager.org/) to control camera, stage, and shutters. For confocal imaging, a Zeiss LSM700 with Zen 2009 software and a confocal channel (488 nm) and a DIC channel were recorded simultaneously. High numerical aperture objectives are ideal for this application. Scan rates were adjusted so that the interval between images was 1 s. Individual larvae that were brightly fluorescent were assessed for how much they were moving and a region of interest selected. Individual neurons could be identified by their location and form. Recordings were normally up to 5 h or until the specimen moved out of the field of view. Image stacks were saved in .lsm format and converted to multilayered .tif files using either Zen Lite or FIJI (ImageJ 1.51k). For video clips stacks were rendered to MP4 format in Adobe Photoshop CS6 or Wondershare Video Converter Ultimate (ver. 10.1.1).

2.3 Analysis of fluorescent intensity over time Reviews of image stacks reveal that the fluorescence of individual neurons in the ciliary band and apical organ fluctuates (Fig. 1). Cells appear to rapidly increase fluorescence that gradually dissipates (Fig. 1A and B). Fluorescence peaks throughout the cell within a second and decays to background levels over the next 3–4 s (Fig. 1C) and is most likely associated with the cellular depolarizations. Patterns of fluorescence in individual nerve cells over the time course of the recording could be determined from Z-axis fluorescence profiles (Fig. 2). The procedure involves opening an image stack with FIJI and using Image > Color > Split Channels to create a stack of fluorescence images to be used for this analysis. To remove any effects of

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FIG. 1 Excerpts of video records of larvae of Strongylocentrotus purpuratus expressing GCaMP6s. (A) Widefield video record of a 5-day larva. The top arrow indicates a neuron in the apical organ that fluoresces brightly beginning at T ¼ 1 s. The arrowhead indicates an esophageal muscle fiber that fluoresces beginning at 2 s. In the video the muscle can be seen to twitch at the same time as fluorescence begins (see Movie 1 in Supplemental Data). (B) Confocal video record of a 5-day larva. In this sequence cells at the lateral periphery of the apical organ (arrows), where serotonergic neurons are situated, begin to fluoresce at T ¼ 1 s and the fluorescence decays over the next 2 s (see Movie 2 in Supplemental Data). (C) Confocal video record of a 5-day larva. In this sequence a cell that appears to have axonal processes projecting into the neuropil of the apical organ (arrows) begins to fluoresce at T ¼ 1 s and the fluorescence decays over the subsequent 3 s. Bars ¼ 25 μm.

specimen drift or movement, stacks were aligned with Plugins > Registration > Rigid Registration (IOS) or Plugins > Registration > Stackreg > Rigid Body (PC). A cell of interest was selected by moving through the stack manually and using the Oval Selection Tool a small circle or ellipse was made over the region of fluorescence creating a region of interest (ROI). Moving through the stack manually ensures that the cell of interest remains within the ROI. The ROI or the stack registration should be altered if the drifts. Dynamic intensity over time can be determined for that ROI using the Image > Stacks > Plot Z-axis Profile feature. The results of that analysis are presented graphic form in a popup window and the numerical data can be accessed using the List button at the bottom of the window (Fig. 2). Multiple ROIs can be analyzed by opening the ROI manager (Analyze > Tools > ROI Manager) and individual ROIs added to the analysis (Add(t) button). Using the Show All feature avoids analyzing the same cell more than once and a reference image can be created using the More > Draw feature. To generate time/series data for all the ROIs, ensure all the regions are highlighted in the left column of the ROI Manager and select More > Multi Measure and tick the Measure all slices and One row per slice options.

2 Methods

FIG. 2 Flow diagram illustrating the procedure for extraction of fluorescence profiles from single neurons. The procedure involves collection of a stack, registration of the stack to correct for specimen movement or drift, selection of a region of interest (ROI), and generation of a z-axis fluorescent profile.

The data are presented in a spreadsheet format that can be transferred to other applications (GraphPad Prism, Excel) for plotting. Ciliated cells of the ciliary band undergo reversals of ciliary beat associated with feeding and coordinated reversals associated with obstacle avoidance (Strathmann, 1971, 2007). Neurophysiological recordings suggest coordinated

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depolarizations involving calcium (Hart, 1990; Mackie et al., 1969; Satterlie & Cameron, 1985; Strathmann, 2007). Records of GCamP6s fluorescence in larvae show recurrent fluctuations in cytoplasmic calcium in individual ciliated cells and episodes in which the entire ciliary band fluoresces. This pattern is consistent with ciliated cell depolarizing and cytoplasmic calcium levels peaking during episodes of ciliary reversal. Similarly, the recordings indicate neurons, identified by position and form, have similar fluctuations in cytoplasmic calcium. Analysis of fluorescence Intensity over time enables correlating neural and effector activity. In an example recording (Fig. 3, Movie 3) the entire band fluoresces twice (73–83 s and 158–168 s). There are two neurons, identified by long axonal processes, in the optical section of this recording (Fig. 3B and C) and these neurons have fluorescence peaks associated with two incidents of ciliary band fluorescence. Neuron 1 has a peak in fluorescence immediately before each of the ciliary band events (70–82 s and 155–167 s). Similarly, Neuron 2 has peaks of fluorescence intensity preceding the peaks in fluorescence of the ciliary band (70–85 s and 155–169 s). There is a less intense ciliary band event at 148 s for which there is no associated activity in these two neurons. These data indicate that neural activity associated with overall depolarization of the ciliated cells of the ciliary band and that it appears to correlate with the activity of the ciliary band cells.

2.4 Limitations One of the major limitations of these methods is the restraining of motile specimens. Larvae are pinned between a glass slide and a glass coverslip, and it is anticipated that the range of behaviors and activities will be constrained. The methods of restraining larvae are not completely satisfactory as the most common reason for a preparation to fail to be useful is for the larva to break free and move out of the field of view of the microscope. It should be noted that some specimens lost vigor over the period of recording, suggesting that the conditions of recording are only marginally satisfactory. In addition to the ciliary band, details of other effectors, such as muscles of the esophagus and those controlling the mouth and arms in older larvae are potentially amenable to this type of analysis. It remains unclear if the nervous system has a role in controlling the localized ciliary reversals of feeding (Strathmann, 2007). Fluorescent biosensors may provide insights into the cellular activity required to understand this process. Detailed understanding of neural activity will need methods of perturbation to enable experimentation. Direct tactile stimulations, light stimulation and food particle tracking could all potentially used to supplement video records. In addition, application of specific chemicals, pharmacological agonists and antagonists could potentially add considerably to our understanding of neural activity in larvae.

2 Methods

FIG. 3 An example of extracted fluorescent profiles from neurons in the ciliary band correlating with events in which the entire ciliary band cells fluoresces. (A–D) Excerpts of a confocal video record of a 5-day larva expressing GCaMP6s. The plane of focus is through the ciliary band of the left postoral arm (see Movie 3 in Supplemental Data). Panel (A) is a DIC image to show the orientation and location of the plane of focus of the images. Panels (B and C) show images in which neurons are identified by their long axonal processes and the region of interest (ROI) is the small circle in each image that was used to generate the z-axis fluorescent profiles. (D) An event in which the cells of the ciliary band fluoresce in unison. The outlined regions show the ROI use to generate the z-axis fluorescent profile. (E–G) Extracted z-axis fluorescent profiles for each of the neurons and a region of ciliary band. Note that for the two major fluorescent events in the ciliary band, there are corresponding fluorescent peaks in each of the neurons. The neurons initiate a rise in fluorescence 1–2 s prior to the initiation of fluorescence in the ciliary bands. Note also that neuron 2 has several events of fluorescence that do not have fluorescence of the ciliary band associated. As well, there is one even of fluorescence in the band that does not have associated neural peaks of fluorescence.

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Acknowledgments This research was supported by an NSERC Discovery Grant to R.D.B. Dan Mellott provided excellent technical assistance throughout the development of these methods. Kerry Delaney provided GCaMP6s DNA.

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