Three-photon microscopy: an emerging technique for deep intravital brain imaging.

Understanding brain function and pathology requires observation of cellular dynamics within intact neural circuits. Although two-photon microscopy revolutionized mammalian in vivo brain imaging, its limitation to upper cortical layers has restricted access to many important brain regions. Three-photon microscopy overcomes this constraint, enabling minimally invasive yet high-resolution visualization of the deep cortical and subcortical structures that are crucial for higher-order brain functions.

Task-specific regional circuit adaptations in distinct mouse retinal ganglion cells.

In the mouse retina, sustained ON alpha (sONα) retinal ganglion cells (RGCs) have different dendritic and receptive field sizes along the nasotemporal axis, with temporal sONα RGCs likely playing a role in visually guided hunting. Thus, we hypothesized that this cell type also exhibits regional adaptations in dendritic signal processing and that these adaptations are advantageous for prey capture. Here, we measured dendritic signals from individual sONα RGCs at different retinal locations.

Eye saccades align optic flow with retinal specializations during object pursuit in freely moving ferrets.

During prey pursuit, how eye rotations, such as saccades, enable continuous tracking of erratically moving targets while enabling an animal to navigate through the environment is unknown. To better understand this, we measured head and eye rotations in freely running ferrets during pursuit behavior. By also tracking the target and all environmental features, we reconstructed the animal's visual fields and their relationship to retinal structures.

Chasing cortical behavior: designing multiphoton microscopes for imaging neuronal populations in freely moving rodents.

Imaging in the freely moving animal gives unparalleled access to circuit activity as the animal interacts with its environment in a self-guided way. Over the past few years, new imaging technologies have enabled the interrogation of neuronal populations located at any depth of the cortex in freely moving mice while preserving the animal's behavioral repertoire. This commentary gives an updated overview of the recent advances that have enabled the link between behavior and the underlying neuronal activity to be explored.

A three-photon head-mounted microscope for imaging all layers of visual cortex in freely moving mice.

Advances in head-mounted microscopes have enabled imaging of neuronal activity using genetic tools in freely moving mice but these microscopes are restricted to recording in minimally lit arenas and imaging upper cortical layers. Here we built a 2-g, three-photon excitation-based microscope, containing a z-drive that enabled access to all cortical layers while mice freely behaved in a fully lit environment. The microscope had on-board photon detectors, robust to environmental light, and the arena lighting was timed to the end of each line-scan, enabling functional imaging of activity from cortical layer 4 and layer 6 neurons expressing jGCaMP7f in mice roaming a fully lit or dark arena.

Estimation of skeletal kinematics in freely moving rodents.

Forming a complete picture of the relationship between neural activity and skeletal kinematics requires quantification of skeletal joint biomechanics during free behavior; however, without detailed knowledge of the underlying skeletal motion, inferring limb kinematics using surface-tracking approaches is difficult, especially for animals where the relationship between the surface and underlying skeleton changes during motion. Here we developed a videography-based method enabling detailed three-dimensional kinematic quantification of an anatomically defined skeleton in untethered freely behaving rats and mice. This skeleton-based model was constrained using anatomical principles and joint motion limits and provided skeletal pose estimates for a range of body sizes, even when limbs were occluded.

Connectomic analysis of thalamus-driven disinhibition in cortical layer 4.

Sensory signals are transmitted via the thalamus primarily to layer 4 (L4) of the primary sensory cortices. While information about average neuronal connectivity in L4 is available, its detailed higher-order circuit structure is not known. Here, we used three-dimensional electron microscopy for a connectomic analysis of the thalamus-driven inhibitory network in L4.

Measurement of arbitrary scan patterns for correction of imaging distortions in laser scanning microscopy.

Laser scanning microscopy requires beam steering through relay and focusing optics at sub-micron precision. In light-weight mobile systems, such as head mounted multiphoton microscopes, distortion and imaging plane curvature management is unpractical due to the complexity of required optic compensation. Thus, the resulting scan pattern limits anatomical fidelity and decreases analysis algorithm efficiency.

Three-photon head-mounted microscope for imaging deep cortical layers in freely moving rats.

We designed a head-mounted three-photon microscope for imaging deep cortical layer neuronal activity in a freely moving rat. Delivery of high-energy excitation pulses at 1,320 nm required both a hollow-core fiber whose transmission properties did not change with fiber movement and dispersion compensation. These developments enabled imaging at >1.

Impact of visual callosal pathway is dependent upon ipsilateral thalamus.

The visual callosal pathway, which reciprocally connects the primary visual cortices, is thought to play a pivotal role in cortical binocular processing. In rodents, the functional role of this pathway is largely unknown. Here, we measure visual cortex spiking responses to visual stimulation using population calcium imaging and functionally isolate visual pathways originating from either eye.

Behavioral Neuroscience: Who's Afraid of the C57BL/6 Mouse?

Behavioral paradigms in which laboratory rodents express behaviors that their wild counterparts presumably need every day are rare: a novel prey-capture model for laboratory mice has been developed for examining the neurophysiological underpinnings of prey capture in mice.

Primate Thalamus: More Than Meets an Eye.

A recent study shows conclusively that the koniocellular layers of the marmoset dorsal lateral geniculate nucleus have binocularly responsive neurons. This adds a new twist to the traditional view about binocular processing in the primate visual system and raises questions about the role of dorsal lateral geniculate nucleus in early binocular processing.

Robustness of sensory-evoked excitation is increased by inhibitory inputs to distal apical tuft dendrites.

Cortical inhibitory interneurons (INs) are subdivided into a variety of morphologically and functionally specialized cell types. How the respective specific properties translate into mechanisms that regulate sensory-evoked responses of pyramidal neurons (PNs) remains unknown. Here, we investigated how INs located in cortical layer 1 (L1) of rat barrel cortex affect whisker-evoked responses of L2 PNs.

Imaging neuronal population activity in awake and anesthetized rodents.

Recent advances in in vivo two-photon imaging have extended the technique to permit the detection of action potentials (APs) in populations of spatially resolved neurons in awake animals. Although experimentally demanding, this technique's potential applications include experiments to investigate perception, behavior, and other awake states. Here we outline experimental procedures for imaging neuronal populations in awake and anesthetized rodents.

Miniaturization of two-photon microscopy for imaging in freely moving animals.

This article describes the development and application of miniaturized two-photon-excited fluorescence microscopes ("two-photon fiberscopes"). Two-photon fiberscopes have been developed with the aim of enabling high-resolution imaging of neural activity in freely behaving animals. They use fiber optics to deliver laser light for two-photon excitation.

Rats maintain an overhead binocular field at the expense of constant fusion.

Fusing left and right eye images into a single view is dependent on precise ocular alignment, which relies on coordinated eye movements. During movements of the head this alignment is maintained by numerous reflexes. Although rodents share with other mammals the key components of eye movement control, the coordination of eye movements in freely moving rodents is unknown.

Changing the responses of cortical neurons from sub- to suprathreshold using single spikes in vivo.

Action Potential (APs) patterns of sensory cortex neurons encode a variety of stimulus features, but how can a neuron change the feature to which it responds? Here, we show that in vivo a spike-timing-dependent plasticity (STDP) protocol-consisting of pairing a postsynaptic AP with visually driven presynaptic inputs-modifies a neurons' AP-response in a bidirectional way that depends on the relative AP-timing during pairing. Whereas postsynaptic APs repeatedly following presynaptic activation can convert subthreshold into suprathreshold responses, APs repeatedly preceding presynaptic activation reduce AP responses to visual stimulation. These changes were paralleled by restructuring of the neurons response to surround stimulus locations and membrane-potential time-course.

Functional imaging in freely moving animals.

Uncovering the relationships between animal behavior and cellular activity in the brain has been one of the key aims of neuroscience research for decades, and still remains so. Electrophysiological approaches have enabled sparse sampling from electrically excitable cells in freely moving animals that has led to the identification of important phenomena such as place, grid and head-direction cells. Optical imaging in combination with newly developed labeling approaches now allows minimally invasive and comprehensive sampling from dense networks of electrically and chemically excitable cells such as neurons and glia during self-determined behavior.

Inhibitory interneurons in a cortical column form hot zones of inhibition in layers 2 and 5A.

Although physiological data on microcircuits involving a few inhibitory neurons in the mammalian cerebral cortex are available, data on the quantitative relation between inhibition and excitation in cortical circuits involving thousands of neurons are largely missing. Because the distribution of neurons is very inhomogeneous in the cerebral cortex, it is critical to map all neurons in a given volume rather than to rely on sparse sampling methods. Here, we report the comprehensive mapping of interneurons (INs) in cortical columns of rat somatosensory cortex, immunolabeled for neuron-specific nuclear protein and glutamate decarboxylase.

Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo.

Multiphoton imaging (MPI) is widely used for recording activity simultaneously from many neurons in superficial cortical layers in vivo. We combined regenerative amplification multiphoton microscopy (RAMM) with genetically encoded calcium indicators to extend MPI of neuronal population activity into layer 5 (L5) of adult mouse somatosensory cortex. We found that this approach could be used to record and quantify spontaneous and sensory-evoked activity in populations of L5 neuronal somata located as much as 800 μm below the pia.

Chasing the cell assembly.

Although we know enormous amounts of detailed information about the neurons that make up the cortex, placing this information back into the context of the behaving animal is a serious challenge. The functional cell assembly hypothesis first described by Hebb (The Organization of Behavior; a Neuropsychological Theory. New York: Wiley; 1949) aimed to provide a mechanistic explanation of how groups of neurons, acting together, form a percept.

Timing is not Everything: Neuromodulation Opens the STDP Gate.

Spike timing dependent plasticity (STDP) is a temporally specific extension of Hebbian associative plasticity that has tied together the timing of presynaptic inputs relative to the postsynaptic single spike. However, it is difficult to translate this mechanism to in vivo conditions where there is an abundance of presynaptic activity constantly impinging upon the dendritic tree as well as ongoing postsynaptic spiking activity that backpropagates along the dendrite. Theoretical studies have proposed that, in addition to this pre- and postsynaptic activity, a "third factor" would enable the association of specific inputs to specific outputs.

Visually evoked activity in cortical cells imaged in freely moving animals.

We describe a miniaturized head-mounted multiphoton microscope and its use for recording Ca(2+) transients from the somata of layer 2/3 neurons in the visual cortex of awake, freely moving rats. Images contained up to 20 neurons and were stable enough to record continuously for >5 min per trial and 20 trials per imaging session, even as the animal was running at velocities of up to 0.6 m/s.

Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor.

Measurement of population activity with single-action-potential, single-neuron resolution is pivotal for understanding information representation and processing in the brain and how the brain's responses are altered by experience. Genetically encoded indicators of neuronal activity allow long-term, cell type-specific expression. Fluorescent Ca2+ indicator proteins (FCIPs), a main class of reporters of neural activity, initially suffered, in particular, from an inability to report single action potentials in vivo.