Calcium imaging is commonly used to measure the neural activity of large groups of neurons in mice. Genetically encoded calcium indicators (GECIs) can be delivered for this purpose using non-invasive genetic methods. Compared to viral gene transfer, transgenic targeting of GECIs provides stable long-term expression and obviates the need for invasive viral injections. Transgenic mice expressing the green GECI GCaMP6 are already widely used. Here we present the generation and characterization of transgenic mice expressing the sensitive red GECI jRGECO1a, driven by the Thy1 promoter. Four transgenic lines with different expression patterns showed sufficiently high expression for cellular in vivo imaging. We used two-photon microscopy to characterize visual responses of individual neurons in the visual cortex in vivo. The signal-to-noise ratio in transgenic mice was comparable to, or better than, mice transduced with adeno-associated virus. In addition, we show that Thy1-jRGECO1a transgenic mice are useful for transcranial population imaging and functional mapping using widefield fluorescence microscopy. We also demonstrate imaging of visual responses in retinal ganglion cells in vitro. Thy1-jRGECO1a transgenic mice are therefore a useful addition to the toolbox for imaging activity in intact neural networks.

The anterolateral motor cortex (ALM) and ventromedial (VM) thalamus are functionally linked to support persistent activity during motor planning. We analyzed the underlying synaptic interconnections using optogenetics and electrophysiology in mice (♀/♂). In cortex, thalamocortical (TC) axons from VM excited VM-projecting pyramidal-tract (PT) neurons in layer 5B of ALM. These axons also strongly excited layer 2/3 neurons (which strongly excite PT neurons, as previously shown) but not VM-projecting corticothalamic (CT) neurons in layer 6. The strongest connections in the VM→PT circuit were localized to apical-tuft dendrites of PT neurons, in layer 1. These tuft inputs were selectively augmented after blocking hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. In thalamus, axons from ALM PT neurons excited ALM-projecting VM neurons, located medially in VM. These axons provided weak input to neurons in mediodorsal nucleus, and little or no input either to neurons in the GABAergic reticular thalamic nucleus or to neurons in VM projecting to primary motor cortex (M1). Conversely, M1 PT axons excited M1- but not ALM-projecting VM neurons. Our findings indicate, first, a set of cell-type-specific connections forming an excitatory thalamo-cortico-thalamic (T-C-T) loop for ALM↔VM communication and a circuit-level substrate for supporting reverberant activity in this system. Second, a key feature of this loop is the prominent involvement of layer 1 synapses onto apical dendrites, a subcellular compartment with distinct signaling properties, including HCN-mediated gain control. Third, the segregation of the ALM↔VM loop from M1-related circuits of VM adds cellular-level support for the concept of parallel pathway organization in the motor system.Anterolateral motor cortex (ALM), a higher-order motor area in the mouse, and ventromedial thalamus (VM) are anatomically and functionally linked, but their synaptic interconnections at the cellular level are unknown. Our results show that ALM pyramidal tract neurons monosynaptically excite ALM-projecting thalamocortical neurons in a medial subdivision of VM, and vice versa. The thalamo-cortico-thalamic loop formed by these recurrent connections constitutes a circuit-level substrate for supporting reverberant activity in this system.

Many forms of human and animal behavior involve head movements. A new study reveals the neural code for three-dimensional head movements in the midbrain of freely moving mice.

Tremendous progress has been made since Neuron published our Primer on genetic dissection of neural circuits 10 years ago. Since then, cell-type-specific anatomical, neurophysiological, and perturbation studies have been carried out in a multitude of invertebrate and vertebrate organisms, linking neurons and circuits to behavioral functions. New methods allow systematic classification of cell types and provide genetic access to diverse neuronal types for studies of connectivity and neural coding during behavior. Here we evaluate key advances over the past decade and discuss future directions.

Neurons in multiple brain regions fire trains of action potentials anticipating specific movements, but this 'preparatory activity' has not been systematically compared across behavioral tasks. We compared preparatory activity in auditory and tactile delayed-response tasks in male mice. Skilled, directional licking was the motor output. The anterior lateral motor cortex (ALM) is necessary for motor planning in both tasks. Multiple features of ALM preparatory activity during the delay epoch were similar across tasks. First, majority of neurons showed direction-selective activity and spatially intermingled neurons were selective for either movement direction. Second, many cells showed mixed coding of sensory stimulus and licking direction, with a bias toward licking direction. Third, delay activity was monotonic and low-dimensional. Fourth, pairs of neurons with similar direction selectivity showed high spike-count correlations. Our study forms the foundation to analyze the neural circuit mechanisms underlying preparatory activity in a genetically tractable model organism.Short-term memories link events separated in time. Neurons in frontal cortex fire trains of action potentials anticipating specific movements, often seconds before the movement. This 'preparatory activity' has been observed in multiple brain regions, but has rarely been compared systematically across behavioral tasks in the same brain region. To identify common features of preparatory activity, we developed and compared preparatory activity in auditory and tactile delayed-response tasks in mice. The same cortical area is necessary for both tasks. Multiple features of preparatory activity, measured with high-density silicon probes, were similar across tasks. We find that preparatory activity is low-dimensional and monotonic. Our study forms the foundation to analyze the circuit mechanisms underlying preparatory activity in a genetically tractable model organism.

Vibrations are important cues for tactile perception across species. Whisker-based sensation in mice is a powerful model system for investigating mechanisms of tactile perception. However, the role vibration plays in whisker-based sensation remains unsettled, in part due to difficulties in modeling the vibration of whiskers. Here, we develop an analytical approach to calculate the vibrations of whiskers striking objects. We use this approach to quantify vibration forces during active whisker touch at a range of locations along the whisker. The frequency and amplitude of vibrations evoked by contact are strongly dependent on the position of contact along the whisker. The magnitude of vibrational shear force and bending moment is comparable to quasi-static forces. The fundamental vibration frequencies are in a detectable range for mechanoreceptor properties and below the maximum spike rates of primary sensory afferents. These results suggest two dynamic cues exist that rodents can use for object localization: vibration frequency and comparison of vibrational to quasi-static force magnitude. These complement the use of quasi-static force angle as a distance cue, particularly for touches close to the follicle, where whiskers are stiff and force angles hardly change during touch. Our approach also provides a general solution to calculation of whisker vibrations in other sensing tasks.

Neurons in motor cortex and connected brain regions fire in anticipation of specific movements, long before movement occurs. This neural activity reflects internal processes by which the brain plans and executes volitional movements. The study of motor planning offers an opportunity to understand how the structure and dynamics of neural circuits support persistent internal states and how these states influence behavior. Recent advances in large-scale neural recordings are beginning to decipher the relationship of the dynamics of populations of neurons during motor planning and movements. New behavioral tasks in rodents, together with quantified perturbations, link dynamics in specific nodes of neural circuits to behavior. These studies reveal a neural network distributed across multiple brain regions that collectively supports motor planning. We review recent advances and highlight areas where further work is needed to achieve a deeper understanding of the mechanisms underlying motor planning and related cognitive processes.

Sensory, motor and cognitive operations involve the coordinated action of large neuronal populations across multiple brain regions in both superficial and deep structures. Existing extracellular probes record neural activity with excellent spatial and temporal (sub-millisecond) resolution, but from only a few dozen neurons per shank. Optical Ca(2+) imaging offers more coverage but lacks the temporal resolution needed to distinguish individual spikes reliably and does not measure local field potentials. Until now, no technology compatible with use in unrestrained animals has combined high spatiotemporal resolution with large volume coverage. Here we design, fabricate and test a new silicon probe known as Neuropixels to meet this need. Each probe has 384 recording channels that can programmably address 960 complementary metal-oxide-semiconductor (CMOS) processing-compatible low-impedance TiN sites that tile a single 10-mm long, 70 × 20-μm cross-section shank. The 6 × 9-mm probe base is fabricated with the shank on a single chip. Voltage signals are filtered, amplified, multiplexed and digitized on the base, allowing the direct transmission of noise-free digital data from the probe. The combination of dense recording sites and high channel count yielded well-isolated spiking activity from hundreds of neurons per probe implanted in mice and rats. Using two probes, more than 700 well-isolated single neurons were recorded simultaneously from five brain structures in an awake mouse. The fully integrated functionality and small size of Neuropixels probes allowed large populations of neurons from several brain structures to be recorded in freely moving animals. This combination of high-performance electrode technology and scalable chip fabrication methods opens a path towards recording of brain-wide neural activity during behaviour.

Neurons relevant to a particular behavior are often widely dispersed across the brain. To record activity in groups of individual neurons that might be distributed across large distances, neuroscientists and optical engineers have been developing a new type of microscope called a mesoscope. Mesoscopes have high spatial resolution and a large field of view. This Q&A will discuss this exciting new technology, highlighting a particular instrument, the two-photon random access mesoscope (2pRAM).