Michael B. Orger

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.

Brain-wide neuronal dynamics during motor adaptation in zebrafish

A fundamental question in neuroscience is how entire neural circuits generate behaviour and adapt it to changes in sensory feedback. Here we use two-photon calcium imaging to record the activity of large populations of neurons at the cellular level, throughout the brain of larval zebrafish expressing a genetically encoded calcium sensor, while the paralysed animals interact fictively with a virtual environment and rapidly adapt their motor output to changes in visual feedback. We decompose the network dynamics involved in adaptive locomotion into four types of neuronal response properties, and provide anatomical maps of the corresponding sites. A subset of these signals occurred during behavioural adjustments and are candidates for the functional elements that drive motor learning. Lesions to the inferior olive indicate a specific functional role for olivocerebellar circuitry in adaptive locomotion. This study enables the analysis of brain-wide dynamics at single-cell resolution during behaviour.

An optimized fluorescent probe for visualizing glutamate neurotransmission

We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus–evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.

Control of visually guided behavior by distinct populations of spinal projection neurons

A basic question in the field of motor control is how different actions are represented by activity in spinal projection neurons. We used a new behavioral assay to identify visual stimuli that specifically drive basic motor patterns in zebrafish. These stimuli evoked consistent patterns of neural activity in the neurons projecting to the spinal cord, which we could map throughout the entire population using in vivo two-photon calcium imaging. We found that stimuli that drive distinct behaviors activated distinct subsets of projection neurons, consisting, in some cases, of just a few cells. This stands in contrast to the distributed activation seen for more complex behaviors. Furthermore, targeted cell by cell ablations of the neurons associated with evoked turns abolished the corresponding behavioral response. This description of the functional organization of the zebrafish motor system provides a framework for identifying the complete circuit underlying a vertebrate behavior.

Perception of Fourier and non-Fourier motion by larval zebrafish

A moving grating elicits innate optomotor behavior in zebrafish larvae; they swim in the direction of perceived motion. We took advantage of this behavior, using computer-animated displays, to determine what attributes of motion are extracted by the fish visual system. As in humans, first-order (luminance-defined or Fourier) signals dominated motion perception in fish; edges or other features had little or no effect when presented with these signals. Humans can see complex movements that lack first-order cues, an ability that is usually ascribed to higher-level processing in the visual cortex. Here we show that second-order (non-Fourier) motion displays induced optomotor behavior in zebrafish larvae, which do not have a cortex. We suggest that second-order motion is extracted early in the lower vertebrate visual pathway.

Whole-brain activity maps reveal stereotyped, distributed networks for visuomotor behavior.

Most behaviors, even simple innate reflexes, are mediated by circuits of neurons spanning areas throughout the brain. However, in most cases, the distribution and dynamics of firing patterns of these neurons during behavior are not known. We imaged activity, with cellular resolution, throughout the whole brains of zebrafish performing the optokinetic response. We found a sparse, broadly distributed network that has an elaborate but ordered pattern, with a bilaterally symmetrical organization. Activity patterns fell into distinct clusters reflecting sensory and motor processing. By correlating neuronal responses with an array of sensory and motor variables, we find that the network can be clearly divided into distinct functional modules. Comparing aligned data from multiple fish, we find that the spatiotemporal activity dynamics and functional organization are highly stereotyped across individuals. These experiments systematically reveal the functional architecture of neural circuits underlying a sensorimotor behavior in a vertebrate brain.

Vesicular Glutamate Transport at a Central Synapse Limits the Acuity of Visual Perception in Zebrafish

The neural circuitry that constrains visual acuity in the CNS has not been experimentally identified. We show here that zebrafish blumenkohl (blu) mutants are impaired in resolving rapid movements and fine spatial detail. The blu gene encodes a vesicular glutamate transporter expressed by retinal ganglion cells. Mutant retinotectal synapses release less glutamate, per vesicle and per terminal, and fatigue more quickly than wild-type in response to high-frequency stimulation. In addition, mutant axons arborize more extensively, thus increasing the number of synaptic terminals and effectively normalizing the combined input to postsynaptic cells in the tectum. This presumably homeostatic response results in larger receptive fields of tectal cells and a degradation of the retinotopic map. As predicted, mutants have a selective deficit in the capture of small prey objects, a behavior dependent on the tectum. Our studies successfully link the disruption of a synaptic protein to complex changes in neural circuitry and behavior.

Neural Control and Modulation of Swimming Speed in the Larval Zebrafish

Vertebrate locomotion at different speeds is driven by descending excitatory connections to central pattern generators in the spinal cord. To investigate how these inputs determine locomotor kinematics, we used whole-field visual motion to drive zebrafish to swim at different speeds. Larvae match the stimulus speed by utilizing more locomotor events, or modifying kinematic parameters such as the duration and speed of swimming bouts, the tail-beat frequency, and the choice of gait. We used laser ablations, electrical stimulation, and activity recordings in descending neurons of the nucleus of the medial longitudinal fasciculus (nMLF) to dissect their contribution to controlling forward movement. We found that the activity of single identified neurons within the nMLF is correlated with locomotor kinematics, and modulates both the duration and oscillation frequency of tail movements. By identifying the contribution of individual supraspinal circuit elements to locomotion kinematics, we build a better understanding of how the brain controls movement.