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. Head-fixation has been used for behavioral research, particularly in non-human primates, to facilitate precise stimulus control, behavioral monitoring and neural recording. However, choice-based, perceptual decision tasks by head-fixed mice have only recently been introduced. Training mice relies on motivating mice using water restriction. Here we describe procedures for head-fixation, water restriction and behavioral training for head-fixed mice, with a focus on active, whisker-based tactile behaviors. In these experiments mice had restricted access to water (typically 1 ml/day). After ten days of water restriction, body weight stabilized at approximately 80% of initial weight. At that point mice were trained to discriminate sensory stimuli using operant conditioning. Head-fixed mice reported stimuli by licking in go/no-go tasks and also using a forced choice paradigm using a dual lickport. In some cases mice learned to discriminate sensory stimuli in a few trials within the first behavioral session. Delay epochs lasting a second or more were used to separate sensation (e.g. tactile exploration) and action (i.e. licking). Mice performed a variety of perceptual decision tasks with high performance for hundreds of trials per behavioral session. Up to four months of continuous water restriction showed no adverse health effects. Behavioral performance correlated with the degree of water restriction, supporting the importance of controlling access to water. These behavioral paradigms can be combined with cellular resolution imaging, random access photostimulation, and whole cell recordings.

Many mammals forage and burrow in dark constrained spaces. Touch through facial whiskers is important during these activities, but the close quarters makes whisker deployment challenging. The diverse shapes of facial whiskers reflect distinct ecological niches. Rodent whiskers are conical, often with a remarkably linear taper. Here we use theoretical and experimental methods to analyze interactions of mouse whiskers with objects. When pushed into objects, conical whiskers suddenly slip at a critical angle. In contrast, cylindrical whiskers do not slip for biologically plausible movements. Conical whiskers sweep across objects and textures in characteristic sequences of brief sticks and slips, which provide information about the tactile world. In contrast, cylindrical whiskers stick and remain stuck, even when sweeping across fine textures. Thus the conical whisker structure is adaptive for sensor mobility in constrained environments and in feature extraction during active haptic exploration of objects and surfaces. DOI: http://dx.doi.org/10.7554/eLife.01350.001.

The subcellular locations of synapses on pyramidal neurons strongly influences dendritic integration and synaptic plasticity. Despite this, there is little quantitative data on spatial distributions of specific types of synaptic input. Here we use array tomography (AT), a high-resolution optical microscopy method, to examine thalamocortical (TC) input onto layer 5 pyramidal neurons. We first verified the ability of AT to identify synapses using parallel electron microscopic analysis of TC synapses in layer 4. We then use large-scale array tomography (LSAT) to measure TC synapse distribution on L5 pyramidal neurons in a 1.00 × 0.83 × 0.21 mm(3) volume of mouse somatosensory cortex. We found that TC synapses primarily target basal dendrites in layer 5, but also make a considerable input to proximal apical dendrites in L4, consistent with previous work. Our analysis further suggests that TC inputs are biased toward certain branches and, within branches, synapses show significant clustering with an excess of TC synapse nearest neighbors within 5-15 μm compared to a random distribution. Thus, we show that AT is a sensitive and quantitative method to map specific types of synaptic input on the dendrites of entire neurons. We anticipate that this technique will be of wide utility for mapping functionally-relevant anatomical connectivity in neural circuits.

Mouse visual cortex is subdivided into multiple distinct, hierarchically organized areas that are interconnected through feedforward (FF) and feedback (FB) pathways. The principal synaptic targets of FF and FB axons that reciprocally interconnect primary visual cortex (V1) with the higher lateromedial extrastriate area (LM) are pyramidal cells (Pyr) and parvalbumin (PV)-expressing GABAergic interneurons. Recordings in slices of mouse visual cortex have shown that layer 2/3 Pyr cells receive excitatory monosynaptic FF and FB inputs, which are opposed by disynaptic inhibition. Most notably, inhibition is stronger in the FF than FB pathway, suggesting pathway-specific organization of feedforward inhibition (FFI). To explore the hypothesis that this difference is due to diverse pathway-specific strengths of the inputs to PV neurons we have performed subcellular Channelrhodopsin-2-assisted circuit mapping in slices of mouse visual cortex. Whole-cell patch-clamp recordings were obtained from retrobead-labeled FFV1→LM- and FBLM→V1-projecting Pyr cells, as well as from tdTomato-expressing PV neurons. The results show that the FFV1→LM pathway provides on average 3.7-fold stronger depolarizing input to layer 2/3 inhibitory PV neurons than to neighboring excitatory Pyr cells. In the FBLM→V1 pathway, depolarizing inputs to layer 2/3 PV neurons and Pyr cells were balanced. Balanced inputs were also found in the FFV1→LM pathway to layer 5 PV neurons and Pyr cells, whereas FBLM→V1 inputs to layer 5 were biased toward Pyr cells. The findings indicate that FFI in FFV1→LM and FBLM→V1 circuits are organized in a pathway- and lamina-specific fashion.

Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.

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.

Synaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer’s disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here, using FRET-based glutamate sensor imaging, we show that amyloid-β peptide (Aβ) engages α7 nicotinic acetylcholine receptors to induce release of astrocytic glutamate, which in turn activates extrasynaptic NMDA receptors (eNMDARs) on neurons. In hippocampal autapses, this eNMDAR activity is followed by reduction in evoked and miniature excitatory postsynaptic currents (mEPSCs). Decreased mEPSC frequency may reflect early synaptic injury because of concurrent eNMDAR-mediated NO production, tau phosphorylation, and caspase-3 activation, each of which is implicated in spine loss. In hippocampal slices, oligomeric Aβ induces eNMDAR-mediated synaptic depression. In AD-transgenic mice compared with wild type, whole-cell recordings revealed excessive tonic eNMDAR activity accompanied by eNMDAR-sensitive loss of mEPSCs. Importantly, the improved NMDAR antagonist NitroMemantine, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from Aβ-induced damage both in vitro and in vivo.

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. Mimicking touch-related neural activity caused illusory perception of an object at a particular location, but scrambling the timing of the spikes over one whisking cycle (tens of milliseconds) did not abolish the illusion, indicating that knowledge of instantaneous whisker position is unnecessary for discriminating object locations. The illusions were induced only during bouts of directed whisking, when mice expected touch, and in the relevant barrel. Reducing activity biased behavior, consistent with a spike count code for object detection at a particular location. Our results show that mice integrate coding of touch with movement over timescales of a whisking bout to produce perception of active touch.

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. Mice judged radial object location based on multiple touches. Both the number of touches (1-17) and the forces exerted by the pole on the whisker (up to 573 μN; typical peak amplitude, 100 μN) varied greatly across trials. We manipulated the bending moment and lateral force pressing the whisker against the sides of the follicle and the axial force pushing the whisker into the follicle by varying the compliance of the object during behavior. The behavioral responses suggest that mice use multiple variables (bending moment, axial force, lateral force) to extract radial object localization. Characterization of whisker mechanics revealed that whisker bending stiffness decreases gradually with distance from the face over five orders of magnitude. As a result, the relative amplitudes of different stress variables change dramatically with radial object distance. Our data suggest that mice use distance-dependent whisker mechanics to estimate radial object location using an algorithm that does not rely on precise control of whisking, is robust to variability in whisker forces, and is independent of object compliance and object movement. More generally, our data imply that mice can measure the amplitudes of forces in the sensory follicles for tactile sensation.

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.