Douglas S. Kim

Ultrasensitive fluorescent proteins for imaging neuronal activity

Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5–40-µm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.

Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.

Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

Sensitive red protein calcium indicators for imaging neural activity

Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging. DOI: http://dx.doi.org/10.7554/eLife.12727.001

Labeling of active neural circuits in vivo with designed calcium integrators

Taking a snapshot of active brain circuitry Neuroscientists now have a method to mark active populations of neurons in vivo to study circuit activity in the behaving animal. Fosque et al. designed and thoroughly validated a fluorescent protein–based reagent that allows permanent marking of active cells over short time scales. This indicator, termed CaMPARI, switches from its native green to a red fluorescent state by simultaneous illumination with violet light and exposure to increased levels of intracellular calcium. CaMPARI successfully marked active nerve cells in Drosophila, zebrafish, and mouse brains. Science, this issue p. 755 A fluorescent sensor allows cellular-resolution snapshots of activity across the whole brains of freely moving organisms. The identification of active neurons and circuits in vivo is a fundamental challenge in understanding the neural basis of behavior. Genetically encoded calcium (Ca2+) indicators (GECIs) enable quantitative monitoring of cellular-resolution activity during behavior. However, such indicators require online monitoring within a limited field of view. Alternatively, post hoc staining of immediate early genes (IEGs) indicates highly active cells within the entire brain, albeit with poor temporal resolution. We designed a fluorescent sensor, CaMPARI, that combines the genetic targetability and quantitative link to neural activity of GECIs with the permanent, large-scale labeling of IEGs, allowing a temporally precise “activity snapshot” of a large tissue volume. CaMPARI undergoes efficient and irreversible green-to-red conversion only when elevated intracellular Ca2+ and experimenter-controlled illumination coincide. We demonstrate the utility of CaMPARI in freely moving larvae of zebrafish and flies, and in head-fixed mice and adult flies.

Thy1-GCaMP6 Transgenic Mice for Neuronal Population Imaging In Vivo

Genetically-encoded calcium indicators (GECIs) facilitate imaging activity of genetically defined neuronal populations in vivo. The high intracellular GECI concentrations required for in vivo imaging are usually achieved by viral gene transfer using adeno-associated viruses. Transgenic expression of GECIs promises important advantages, including homogeneous, repeatable, and stable expression without the need for invasive virus injections. Here we present the generation and characterization of transgenic mice expressing the GECIs GCaMP6s or GCaMP6f under the Thy1 promoter. We quantified GCaMP6 expression across brain regions and neurons and compared to other transgenic mice and AAV-mediated expression. We tested three mouse lines for imaging in the visual cortex in vivo and compared their performance to mice injected with AAV expressing GCaMP6. Furthermore, we show that GCaMP6 Thy1 transgenic mice are useful for long-term, high-sensitivity imaging in behaving mice.

Identification of molecular markers of bipolar cells in the murine retina

Retinal bipolar neurons serve as relay interneurons that connect rod and cone photoreceptor cells to amacrine and ganglion cells. They exhibit diverse morphologies essential for correct routing of photoreceptor cell signals to specific postsynaptic amacrine and ganglion cells. The development and physiology of these interneurons have not been completely defined molecularly. Despite previous identification of genes expressed in several bipolar cell subtypes, molecules that mark each bipolar cell type still await discovery. In this report, novel genetic markers of murine bipolar cells were found. Candidates were initially generated by using microarray analysis of single bipolar cells and mining of retinal serial analysis of gene expression (SAGE) data. These candidates were subsequently tested for expression in bipolar cells by RNA in situ hybridization. Ten new molecular markers were identified, five of which are highly enriched in their expression in bipolar cells within the adult retina. Double‐labeling experiments using probes for previously characterized subsets of bipolar cells were performed to identify the subtypes of bipolar cells that express the novel markers. Additionally, the expression of bipolar cell genes was analyzed in Bhlhb4 knockout retinas, in which rod bipolar cells degenerate postnatally, to delineate further the identity of bipolar cells in which novel markers are found. From the analysis of Bhlhb4 mutant retinas, cone bipolar cell gene expression appears to be relatively unaffected by the degeneration of rod bipolar cells. Identification of molecular markers for the various subtypes of bipolar cells will lead to greater insights into the development and function of these diverse interneurons. J. Comp. Neurol. 507:1795–1810, 2008.

Multiplexed aberration measurement for deep tissue imaging in vivo.

We describe an adaptive optics method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine the sample-induced aberration. Applicable to fluorescent protein–labeled structures of arbitrary complexity, it allowed us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improved structural and functional imaging of fine neuronal processes over a large imaging volume.

A Core Paired-Type and POU Homeodomain-Containing Transcription Factor Program Drives Retinal Bipolar Cell Gene Expression

The diversity of cell types found within the vertebrate CNS arises in part from action of complex transcriptional programs. In the retina, the programs driving diversification of various cell types have not been completely elucidated. To investigate gene regulatory networks that underlie formation and function of one retinal circuit component, the bipolar cell, transcriptional regulation of three bipolar cell-enriched genes was analyzed. Using in vivo retinal DNA transfection and reporter gene constructs, a 200 bp Grm6 enhancer sequence, a 445 bp Cabp5 promoter sequence, and a 164 bp Chx10 enhancer sequence, were defined, each driving reporter expression specifically in distinct but overlapping bipolar cell subtypes. Bioinformatic analysis of sequences revealed the presence of potential paired-type and POU homeodomain-containing transcription factor binding sites, which were shown to be critical for reporter expression through deletion studies. The paired-type homeodomain transcription factors (TFs) Crx and Otx2 and the POU homeodomain factor Brn2 are expressed in bipolar cells and interacted with the predicted binding sequences as assessed by electrophoretic mobility shift assay. Grm6, Cabp5, and Chx10 reporter activity was reduced in Otx2 loss-of-function retinas. Endogenous gene expression of bipolar cell molecular markers was also dependent on paired-type homeodomain-containing TFs, as assessed by RNA in situ hybridization and reverse transcription-PCR in mutant retinas. Cabp5 and Chx10 reporter expression was reduced in dominant-negative Brn2-transfected retinas. The paired-type and POU homeodomain-containing TFs Otx2 and Brn2 together appear to play a common role in regulating gene expression in retinal bipolar cells.

Adenosine receptor blockade reverses hypophagia and enhances locomotor activity of dopamine-deficient mice

Adenosine receptors modulate dopaminergic function by regulating dopamine release in presynaptic neurons and intracellular signaling in postsynaptic striatal neurons. To investigate how adenosine impinges on the action of dopamine in feeding and locomotion, genetically altered, dopamine-deficient mice were treated with adenosine receptor antagonists. Acute administration of the nonselective adenosine receptor antagonist, caffeine (5–25 mg/kg i.p.), reversed the hypophagia of mutant mice and induced hyperactivity in both control and mutant animals. However, caffeine treatment elicited much less hyperactivity in dopamine-deficient mice than did l-3,4-dihydroxyphenylalanine (l-dopa) administration, which partially restores dopamine content. Caffeine treatment enhanced feeding of l-dopa-treated mutants but, unexpectedly, it reduced their hyperlocomotion. Caffeine administration induced c-Fos expression in the cortex of dopamine-deficient mice but had no effect in the striatum by itself. Caffeine attenuated dopamine agonist-induced striatal c-Fos expression. An antagonist selective for adenosine A2A receptors induced feeding and locomotion in mutants much more effectively than an A1 receptor antagonist. l-dopa-elicited feeding and hyperlocomotion were reduced in mutants treated with an A1 receptor agonist, whereas an A2A receptor agonist decreased l-dopa-induced feeding without affecting locomotion. The observations suggest that the hypophagia and hypoactivity of mutants result not only because of the absence of dopamine but also because of the presence of A2A receptor signaling. This study of a genetic model of dopamine depletion provides evidence that A2A receptor antagonists could ameliorate the hypokinetic symptoms of advanced Parkinsons disease patients without inducing excessive motor activity.