Research PaperSkin suturing and cortical surface viral infusion improves imaging of neuronal ensemble activity with head-mounted miniature microscopes
Introduction
Understanding cortical processing related to vital behavioral functions requires the ability to assess the activity of large populations of cells within the temporal context of behavior. Current methods to study in vivo large-scale neuronal activity during active behavior possess distinct strengths and weaknesses. For example, chronic multi-array electrode recording has high temporal resolution and is applicable to freely moving animals, but sorting out the identity of recorded cells is challenging (Murakami et al., 2014, Rossant et al., 2016, Sul et al., 2011). In vivo two-photon imaging directly reveals the activity of specifically labeled neurons, but it requires animal head-fixation and restricts certain behavioral expression (Chen et al., 2013, Kuhlman et al., 2013, Minderer et al., 2016, Peters et al., 2014).
Recently developed miniature one-photon microscopes can achieve both cell-type specificity and recording in freely moving animals with genetically encoded calcium indicators (Ghosh et al., 2011, Ziv et al., 2013). This technique usually requires the insertion of a small graded-index (GRIN) optical lens inside the brain, and has been successfully used to study the function of deep brain structures such as the hippocampus (Okuyama et al., 2016, Ziv et al., 2013), amygdala (Grewe et al., 2017), striatum (Barbera et al., 2016) and hypothalamus (Jennings et al., 2015). However, the cerebral cortex located at the top of the brain has not yet been extensively studied through this technique (Pinto and Dan, 2015).
An important factor to consider for the application of this head-attached microscope is the trade-off between optical access and tissue damage. Direct insertion of a GRIN lens into the cortex from the pial surface will destroy superficial layer cortical neurons and their projections, which play important roles in integrating columnar and inter-areal neural signals (Harris and Shepherd, 2015, Petersen and Crochet, 2013). While it is possible to obtain a side view of cortical neurons through an inserted prism probe, which rotates the imaging plane 90Ā Ā° from the direction of probe insertion (Andermann et al., 2013, Murayama et al., 2007), this method still leads to significant tissue damage adjacent to the imaged neurons. To avoid any direct damage to cortical neurons, one option is to image through a cranial window implanted above the cortex, as has been done in many two-photon imaging applications (Cao et al., 2015; Fu et al., 2012; Holtmaat et al., 2009; Yang et al., 2010). However, cranial window surgeries can be challenging to conduct successfully, due to the brain dura and pia responding to surgery damage with overgrowth and inflammation. The yield of clear windows is considered dependent on individual operators and sometimes unpredictable (Holtmaat et al., 2009).
The optical clarity of cranial windows is especially important for imaging cortical neurons with head-attached one-photon microscopes, because light-scattering can severely limit optical resolution of cell bodies in this imaging mode. Classic cranial window surgery protocols include the removal of a piece of the scalp, leaving the newly implanted window to recover unprotected by skin (Cao et al., 2013, Goldey et al., 2014, Holtmaat et al., 2009). In addition, viruses encoding calcium indicators are typically injected intracortically in the same surgical procedure as the implantation of the cranial window (Chen et al., 2013, Lowery and Majewska, 2010, Resendez et al., 2016). Variations in intracortical injection-induced damage and viral spread increase unpredictability in the experimental yield. Here, we describe an improved cranial surgery technique that uses scalp skin suturing and cortical surface viral infusion to reduce procedural inconsistencies and enhance the success rate of superficial-layer cortical neuron imaging by head-mounted microscopes in freely moving animals.
Section snippets
Mouse strain
Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the National Institute of Mental Health, Maryland; and LifeSource Biomedical Services, NASA Ames Research Center, California. C57BL/6 wild type mice were used for all experiments.
Pre-Surgery preparation
Mice were first anesthetized with Avertin (1.5% solution given at 0.01Ā ml/g, i.p.) and then mounted in a stereotaxic frame. Ear bars and a nose cone/tooth mount were used to hold the animalās head
Closed-scalp post-operative recovery improves the clarity of chronic cranial windows
To image cortical neuronal activity by head-attached one-photon microscopes over time, high-quality cranial windows lasting days to weeks are necessary. With traditional open-skull cranial window surgery methods, surgery-related inflammation and dura thickening could reduce the clarity of implanted windows after a few days (Holtmaat et al., 2009). Anti-inflammatory drugs, such as dexamethasone and carprofen, have been used to decrease such responses (Cao et al., 2013, Golshani and
Discussion
The cerebral cortex receives multisensory inputs and transforms these signals for decision making, behavioral control and other vital cognitive functions. Superficial cortical layers (Layer II/III) are thought to play an important role in integrating various inputs from cortical and subcortical regions and have been subjected to extensive studies via in vivo two-photon imaging approaches in head-fixed animals (Harris and Shepherd, 2015, Peters et al., 2014, Petersen and Crochet, 2013). Recently
Conclusions
Our studies demonstrate a cortical surface-based viral infusion method to preferentially and efficiently label superficial layer cortical neurons, alongside a closed-scalp post-operative recovery method to improve the clarity of chronic cranial windows. Taken together, these methods significantly improve the efficiency and consistency of animal preparations to study cortical neuronal functions by one-photon miniaturized microscopy in freely moving animals.
Declaration of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. SO and VC are paid employees at Inscopix; SO is Director of Science at Inscopix; and VC is Content and Training Manager at Inscopix. This does not alter the authorsā adherence to the journalās policies and all the authors have abided by the statement of ethical standards for manuscripts submitted to the Journal of
Acknowledgements
This research was supported by the National Institute of Mental Health (NIMH) Division of Intramural Research Programs ZIA MH002897 to KHW, NIMH postdoctoral fellowships (XL, WZ, SM and QL), and the Inscopix Inc (VC and SO). We thank other members of Wang lab for scientific discussion, and Dale Voelker and Zhouyang Ma for helping to rank the clarity of cranial windows.
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2022, Cell ReportsCitation Excerpt :The skull was exposed, and craniotomies were made over different target brain regions after leveling. For drug cannula, fiber optic cannula, or tetrode implantation, dexamethasone (2 mg/kg, i.p., Sigma-Aldrich #D4902, Darmstadt, Germany) and carprofen (5 mg/kg, i.p., Sigma-Aldrich #PHR1452, Darmstadt, Germany) were injected at least 1.5 h before anesthetization to prevent brain swelling and inflammation (Li et al., 2017). All surgery tools were autoclaved before experiments.
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