Genetically encoded calcium indicator GCaMP6m for two-photon calcium imaging in newborn mouse cerebral cortex

Abstract

Calcium is a universal messenger regulating essential cellular signaling events in many tissues and organisms. In neurons, action potentials trigger large and rapid changes in cytoplasmic-free calcium concentration. Genetically encoded calcium indicators (GECIs) have been iteratively improved and are becoming useful for quantitative imaging of activity in defined neuronal populations in vivo. Among them, GCaMP6m has been recently introduced as an ultrasensitive calcium sensor for the experiments in vivo. Whether GECIs can be used in developmental studies to characterize early activity patterns in the immature cerebral cortex remains unknown, however. We used in vivo two-photon calcium imaging with GCaMP6m calcium indicator to characterize its developmental expression following adeno-associated virus (AAV) mediated viral induction of GCaMP6m expression in newborn mice (at postnatal days P0-2). Three days after virus injection, the vast majority of neurons showed fluorescent labeling in the neuronal cytoplasm, dendrites and axons. We found that the GCaMP6m calcium sensor is expressed in the cerebral cortex within 3-5 days following transfection at levels that enable monitoring spontaneous and sensory-evoked calcium transients in the intact mice barrel cortex in vivo. Thus, following GCaMP6m transfection at birth, it is possible to monitor neuronal populations in the barrel field of somatosensory cortex in P5-7 mice in vivo.

Keywords

GCaMP6m / genetically encoded calcium indicator / GCaMP6m / adeno-associated virus / in vivo two-photon calcium imaging / somatosensory cortex / barrel cortex

References

[1] Garaschuk O., Linn J., Eilers J. et al. Large-scale oscillatory calcium waves in the immature cortex. Nat. Neurosci. 2000; 3: 452-9.

[2] Khazipov R., Sirota A., Leinekugel X. et al. Early motor activity drives spindle bursts in the developing somatosensory cortex. Nature 2004; 432: 758-61.

[3] Minlebaev M., Colonnese M., Tsintsadze T. et al. Early gamma oscillations synchronize developing thalamus and cortex. Science 2011; 334: 226-9.

[4] Khazipov R., Minlebaev M., Valeeva G. Early gamma oscillations. Neuroscience 2013; 250: 240-52.

[5] Gerasimova E.V., Zakharov A.V., Lebedeva J.A. et al. Sensory evoked responses in somatosensory cortex of neonatal rats. Bull. Exp. Biol. Med. 2013; 268-72.

[6] Dreyfus-Brisac C., Larroche J.C. Discontinuous electroencephalograms in the premature newborn and at term. Electro-anatomo-clinical correlations (in French). Rev. Electroencephalogr. Neurophysiol. Clin. 1971; 1: 95-9.

[7] Stosiek C., Garaschuk O., Holthoff K. et al. In vivo two-photon calcium imaging of neuronal networks. Neuroscience 2003; 100(12): 7319-24.

[8] Kerr J.N., Greenberg D., Helmchen F. Imaging input and output of neocortical networks in vivo. Proc. Natl. Acad. Sci. USA 2005; 102: 14063-8.

[9] Hromadka T., Deweese M.R., Zador A.M. Sparse representation of sounds in the unanesthetized auditory cortex. PLoS Biol. 2008; 6(1): e16.

[10] Milh M., Kaminska A., Huon C. et al. Rapid Cortical Oscillations and Early Motor Activity in Premature Human Neonate. Cerebral Cortex 2007; 17: 1582-94.

[11] Colonnese M.T., Kaminska A., Minlebaev M. et al. A conserved switch in sensory processing prepares developing neocortex for vision. Neuron 2010; 67: 480-98.

[12] Colonnese M., Khazipov R. Spontaneous activity in developing sensory circuits: Implications for resting state fMRI. Neuroimage 2012; 62(4): 2212-21.

[13] Gomez T.M., Spitzer N.C. In vivo regulation of axon extension and pathfinding by growth-cone calcium transients. Nature 1999; 397(6717): 350-5.

[14] Allene C., Cattani A., Ackman J.B. et al. Sequential generation of two distinct synapse-driven network patterns in developing neocortex. J. Neurosci. 2008; 28: 12851-63.

[15] Yuste R., Peinado A., Katz L.C. Neuronal domains in developing neocortex. Science 1992; 257: 665-9.

[16] Fetcho J.R., Cox K.J., O'Malley D.M. Monitoring activity in neuronal populations with single-cell resolution in a behaving vertebrate. Histochem. J. 1998; 30: 153-67.

[17] Tian L., Hires S.A., Mao T. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nature 2009; 6(12): 875-81.

[18] Palmer A.E., Tsien R.Y. Measuring calcium signaling using genetically targetable fluorescent indicators. Nat. Protocols 2006; 1: 1057-65.

[19] Mank M. Santos A.F., Direnberger S. et al. A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat. Methods 2008; 5(9): 805-11.

[20] Chen T.-W., Wardill T.J., SunY. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 2013; 499: 295-300.

[21] Golshani P., Goncalves J.T., Khoshkhoo S. et al. Internally mediated developmental desynchronization of neocortical network activity. J. Neurosci. 2009a; 29: 10890-9.

[22] Khazipov R., Sirota A., Leinekugel X. et al. Early motor activity drives spindle bursts in the developing somatosensory cortex. Nature 2004a; 432: 758-61.

[23] Minlebaev M., Ben-Ari Y., Khazipov R. Network mechanisms of spindle-burst oscillations in the neonatal rat barrel cortex in vivo. J. Neurophys. 2007; 97: 692-700.

[24] Yang J.W., Hanganu-Opatz I.L, Sun J.J. et al. Three patterns of oscillatory activity differentially synchronize developing neocortical networks in vivo. J. Neurosci. 2009a; 29: 9011-25.

[25] Mitrukhina O., Suchkov D., Khazipov R. et al. Imprecise Whisker map in the neonatal rat barrel cortex. Cereb. Cortex. 2014 Aug 6; pii: bhu169.

[26] Yang J.W., An S., Sun J.J. et al. Thalamic network oscillations synchronize ontogenetic columns in the newborn rat barrel cortex. Cereb. Cortex 2013; 23: 1299-316.

[27] Khazipov R., Sirota A., Leinekugel X. et al. Early motor activity drives spindle bursts in the developing somatosensory cortex. Nature 2004b; 432: 758-61.

[28] Golshani P., Goncalves J.T., Khoshkhoo S. et al. Internally mediated developmental desynchronization of neocortical network activity. J. Neurosci. 2009b; 29: 10890-9.

[29] Yang J.W., Hanganu-Opatz I.L, Sun J.J. et al. Three patterns of oscillatory activity differentially synchronize developing neocortical networks in vivo. J. Neurosci. 2009b; 29: 9011-25.

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