A Genetically Encoded Fluorescent Sensor for Rapid and Specific In Vivo Detection of Norepinephrine.

Norepinephrine (NE) is a key biogenic monoamine neurotransmitter involved in a wide range of physiological processes. However, its precise dynamics and regulation remain poorly characterized, in part due to limitations of available techniques for measuring NE in vivo. Here, we developed a family of GPCR activation-based NE (GRAB) sensors with a 230% peak ΔF/F response to NE, good photostability, nanomolar-to-micromolar sensitivities, sub-second kinetics, and high specificity.

Layer 4 fast-spiking interneurons filter thalamocortical signals during active somatosensation.

We rely on movement to explore the environment, for example, by palpating an object. In somatosensory cortex, activity related to movement of digits or whiskers is suppressed, which could facilitate detection of touch. Movement-related suppression is generally assumed to involve corollary discharges.

Procedures for behavioral experiments in head-fixed mice.

The mouse is an increasingly prominent model for the analysis of mammalian neuronal circuits. Neural circuits ultimately have to be probed during behaviors that engage the circuits. Linking circuit dynamics to behavior requires precise control of sensory stimuli and measurement of body movements.

Whisker dynamics underlying tactile exploration.

Rodents explore the world by palpating objects with their whiskers. Whiskers interact with objects, causing stresses in whisker follicles and spikes in sensory neurons, which are interpreted by the brain to produce tactile perception. The mechanics of the whisker thus couple self-movement and the structure of the world to sensation.

Neural coding during active somatosensation revealed using illusory touch.

Active sensation requires the convergence of external stimuli with representations of body movements. We used mouse behavior, electrophysiology and optogenetics to dissect the temporal interactions among whisker movement, neural activity and sensation of touch. We photostimulated layer 4 activity in single barrels in a closed loop with whisking.

The mechanical variables underlying object localization along the axis of the whisker.

Rodents move their whiskers to locate objects in space. Here we used psychophysical methods to show that head-fixed mice can localize objects along the axis of a single whisker, the radial dimension, with one-millimeter precision. High-speed videography allowed us to estimate the forces and bending moments at the base of the whisker, which underlie radial distance measurement.

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.

Automated tracking of whiskers in videos of head fixed rodents.

We have developed software for fully automated tracking of vibrissae (whiskers) in high-speed videos (>500 Hz) of head-fixed, behaving rodents trimmed to a single row of whiskers. Performance was assessed against a manually curated dataset consisting of 1.32 million video frames comprising 4.

Imaging neuronal activity with genetically encoded calcium indicators.

Genetically encoded calcium indicators (GECIs), which are based on chimeric fluorescent proteins, can be used to monitor calcium transients in living cells and organisms. Because they are encoded by DNA, GECIs can be delivered to the intact brain noninvasively and targeted to defined populations of neurons and specific subcellular compartments for long-term, repeated measurements in vivo. GECIs have improved iteratively and are becoming useful for imaging neural activity in vivo.

Laminar analysis of excitatory local circuits in vibrissal motor and sensory cortical areas.

Rodents move their whiskers to locate and identify objects. Cortical areas involved in vibrissal somatosensation and sensorimotor integration include the vibrissal area of the primary motor cortex (vM1), primary somatosensory cortex (vS1; barrel cortex), and secondary somatosensory cortex (S2). We mapped local excitatory pathways in each area across all cortical layers using glutamate uncaging and laser scanning photostimulation.

Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.

Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.

Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design.

The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as solution biophysical and spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in two calcium-bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain-swapped dimer with decreased fluorescence. This series of structures provides insight into the mechanism of Ca2+-induced fluorescence change.

Reporting neural activity with genetically encoded calcium indicators.

Genetically encoded calcium indicators (GECIs), based on recombinant fluorescent proteins, have been engineered to observe calcium transients in living cells and organisms. Through observation of calcium, these indicators also report neural activity. We review progress in GECI construction and application, particularly toward in vivo monitoring of sparse action potentials (APs).

Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs.

The binding interface of calmodulin and a calmodulin binding peptide were reengineered by computationally designing complementary bumps and holes. This redesign led to the development of sensitive and specific pairs of mutant proteins used to sense Ca(2+) in a second generation of genetically encoded Ca(2+) indicators (cameleons). These cameleons are no longer perturbed by large excesses of native calmodulin, and they display Ca(2+) sensitivities tuned over a 100-fold range (0.