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. By comparing the neuronal activity measured from populations in these layers we show that activity in cortical layer 4 and layer 6 is differentially modulated by lit and dark conditions during free exploration.

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. Model-inferred limb positions and joint kinematics during gait and gap-crossing behaviors were verified by direct measurement of either limb placement or limb kinematics using inertial measurement units. Together we show that complex decision-making behaviors can be accurately reconstructed at the level of skeletal kinematics using our anatomically constrained model.

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 >1.1 mm below the cortical surface and stable imaging of layer 5 neuronal activity for >1 h in freely moving rats performing a range of behaviors.

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. Here we show that movements of the two eyes in freely moving rats differ fundamentally from the precisely controlled eye movements used by other mammals to maintain continuous binocular fusion. The observed eye movements serve to keep the visual fields of the two eyes continuously overlapping above the animal during free movement, but not continuously aligned. Overhead visual stimuli presented to rats freely exploring an open arena evoke an immediate shelter-seeking behaviour, but are ineffective when presented beside the arena. We suggest that continuously overlapping visual fields overhead would be of evolutionary benefit for predator detection by minimizing blind spots.

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. To do so we combined in vivo electrophysiology and morphological reconstructions with computational modeling. We show that whisker-evoked membrane depolarization in L2 PNs arises from highly specialized spatiotemporal synaptic input patterns. Temporally L1 INs and L2-5 PNs provide near synchronous synaptic input. Spatially synaptic contacts from L1 INs target distal apical tuft dendrites, whereas PNs primarily innervate basal and proximal apical dendrites. Simulations of such constrained synaptic input patterns predicted that inactivation of L1 INs increases trial-to-trial variability of whisker-evoked responses in L2 PNs. The in silico predictions were confirmed in vivo by L1-specific pharmacological manipulations. We present a mechanism-consistent with the theory of distal dendritic shunting-that can regulate the robustness of sensory-evoked responses in PNs without affecting response amplitude or latency.

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. Neuronal Ca(2+) transients were readily detected, and responses to various static visual stimuli were observed during free movement on a running track. Neuronal activity was sparse and increased when the animal swept its gaze across a visual stimulus. Neurons showing preferential activation by specific stimuli were observed in freely moving animals. These results demonstrate that the multiphoton fiberscope is suitable for functional imaging in awake and freely moving animals.

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. Here, we present a technique that reconstructs the three-dimensional scan path only requiring translation of a simple fluorescent test probe. Our method is applicable to any type of scanning instrument with sectioning capabilities without prior assumptions regarding origin of imaging deviations. Further, we demonstrate that the obtained scan pattern allows analysis of these errors, and allows to restore anatomical accuracy relevant for complementary methods such as motion correction, further enhancing spatial registration and feature extraction.

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. We find that thalamic input drives a subset of interneurons with high specificity, which in turn target excitatory neurons with subtype specificity. These interneurons create a directed disinhibitory network directly driven by the thalamic input. Neuronal activity recordings show that strong synchronous sensory activation yields about 1.5-fold stronger activation of star pyramidal cells than spiny stellates, in line with differential windows of opportunity for activation of excitatory neurons in the thalamus-driven disinhibitory circuit model. With this, we have identified a high degree of specialization of the microcircuitry in L4 of the primary sensory cortex.

Visual pursuit behavior in mice maintains the pursued prey on the retinal region with least optic flow.

Mice have a large visual field that is constantly stabilized by vestibular ocular reflex (VOR) driven eye rotations that counter head-rotations. While maintaining their extensive visual coverage is advantageous for predator detection, mice also track and capture prey using vision. However, in the freely moving animal quantifying object location in the field of view is challenging. Here, we developed a method to digitally reconstruct and quantify the visual scene of freely moving mice performing a visually based prey capture task. By isolating the visual sense and combining a mouse eye optic model with the head and eye rotations, the detailed reconstruction of the digital environment and retinal features were projected onto the corneal surface for comparison, and updated throughout the behavior. By quantifying the spatial location of objects in the visual scene and their motion throughout the behavior, we show that the prey image consistently falls within a small area of the VOR-stabilized visual field. This functional focus coincides with the region of minimal optic flow within the visual field and consequently area of minimal motion-induced image-blur, as during pursuit mice ran directly toward the prey. The functional focus lies in the upper-temporal part of the retina and coincides with the reported high density-region of Alpha-ON sustained retinal ganglion cells.

Mice have a lot to keep an eye on. To survive, they need to dodge predators looming on land and from the skies, while also hunting down the small insects that are part of their diet. To do this, they are helped by their large panoramic field of vision, which stretches from behind and over their heads to below their snouts. To stabilize their gaze when they are on the prowl, mice reflexively move their eyes to counter the movement of their head: in fact, they are unable to move their eyes independently. This raises the question: what part of their large visual field of view do these rodents use when tracking a prey, and to what advantage? This is difficult to investigate, since it requires simultaneously measuring the eye and head movements of mice as they chase and capture insects. In response, Holmgren, Stahr et al. developed a new technique to record the precise eye positions, head rotations and prey location of mice hunting crickets in surroundings that were fully digitized at high resolution. Combining this information allowed the team to mathematically recreate what mice would see as they chased the insects, and to assess what part of their large visual field they were using. This revealed that, once a cricket had entered any part of the mice's large field of view, the rodents shifted their head ­ but not their eyes ­ to bring the prey into both eye views, and then ran directly at it. If the insect escaped, the mice repeated that behavior. During the pursuit, the cricket's position was mainly held in a small area of the mouse's view that corresponds to a specialized region in the eye which is thought to help track objects. This region also allowed the least motion-induced image blur when the animals were running forward. The approach developed by Holmgren, Stahr et al. gives a direct insight into what animals see when they hunt, and how this constantly changing view ties to what happens in the eyes. This method could be applied to other species, ushering in a new wave of tools to explore what freely moving animals see, and the relationship between behaviour and neural circuitry.

Sensory experience alters specific branches of individual corticocortical axons during development.

Sensory experience can, over the course of days to weeks, produce long-lasting changes in brain function. Recent studies suggest that functional plasticity is mediated by alterations of the strengths of existing synapses or dynamics of dendritic spines. Alterations of cortical axons could also contribute to functional changes, but little is known about the effects of experience at the level of individual corticocortical axons. We reconstructed individual layer (L) 2/3 pyramidal neurons filled in vivo in developing barrel cortex of control and partially sensory-deprived rats. L2 axons had larger field spans than L3 axons but were otherwise equivalently affected by deprivation. Whisker trimming over approximately 2 weeks markedly reduced overall length of axonal branches in L2/3, but individual horizontal axons were as likely to innervate deprived areas as spared ones. The largest effect of deprivation was instead to reduce the length of those axonal branches in L2/3 oriented toward deprived regions. Thus, the location of a branch relative to its originating soma, rather than its own location within any specific cortical column, was the strongest determinant of axonal organization. Individual axons from L2/3 into L5/6 were similarly altered by whisker trimming although to a lesser extent. Thus, sensory experience over relatively short timescales may change the patterning of specific axonal branches within as well as between cortical columns during development.

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. We show that callosal pathway inhibition significantly reduced spiking responses in binocular and monocular neurons and abolished spiking in many cases. However, once isolated by blocking ipsilateral visual thalamus, callosal pathway activation alone is not sufficient to drive evoked cortical responses. We show that the visual callosal pathway relays activity from both eyes via both ipsilateral and contralateral visual pathways to monocular and binocular neurons and works in concert with ipsilateral thalamus in generating stimulus evoked activity. This shows a much greater role of the rodent callosal pathway in cortical processing than previously thought.

Plasticity of representational maps in somatosensory cortex observed by in vivo voltage-sensitive dye imaging.

We investigated the effect of selective whisker trimming on the development of the cortical representation of a whisker deflection in layer 2/3 of rat somatosensory cortex using in vivo voltage-sensitive dye (vsd) imaging. Responses to deflection of D-row whiskers were recorded after trimming of A-row, B-row, and C-row whiskers, referred to as DE pairing, during postnatal development. Animals DE paired from postnatal day (p) 7 to p17 had a significant bias in the spread of the vsd signal, favoring spread toward the concomitantly nondeprived E-row columns. This resulted primarily from a strong decrease in signal spreading into the deprived C-row columns. In contrast, signal spread in control littermates was approximately symmetrical. DE pairing failed to elicit significant changes when begun after p14, thus defining a critical period for this phenomenon. The results suggest that sensory deprivation in this model results in lower connectivity being established between nondeprived columns and adjacent deprived ones.

Automated axon length quantification for populations of labelled neurons.

Virus-based methods for labelling populations of cortical neurons, when combined with cell-type specific recombinant promoters and techniques allowing temporal control of gene expression, provide neuroscience with new opportunities to examine the connectivity between brain regions and how this connectivity is modified by experience or disease. However, to take full advantage of these technical advances, it is necessary to develop new methods for quantification of the axonal projections revealed. Here we describe a method for quantitative analysis of axonal projection patterns emanating from populations of labelled cells, using transmitted light bright field microscopy. A single high resolution image of an area to be analysed is first acquired using mosaic extended focus image microscopy. This image is then analysed by specifically developed image processing algorithms that identify and track axon segments present. For quantitative analysis, measurement grids consisting of a user-defined number of individual elements are placed over an area of interest, with the computer-based method then returning the summed length of the axon segments in each element. Axon density plots can thus be generated. We present an example from rat brain showing, over a whole coronal section, axon densities emanating from a population of layer 2/3 somatosensory neurons.

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.

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.

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. Details are provided on habituation to head fixation, surgery, head plate design, and dye injection. Determination of AP detection accuracy through simultaneous optical and electrophysiological recordings is also discussed. Basic problems of data analysis are considered, such as correction of signal background and baseline drift, AP detection, and motion correction. As an application of the method, the comparison of neuronal activity across arousal states is considered in detail, and some future directions are discussed.

Perceptual judgements and chronic imaging of altered odour maps indicate comprehensive stimulus template matching in olfaction.

Lesion experiments suggest that odour input to the olfactory bulb contains significant redundant signal such that rodents can discern odours using minimal stimulus-related information. Here we investigate the dependence of odour-quality perception on the integrity of glomerular activity by comparing odour-evoked activity maps before and after epithelial lesions. Lesions prevent mice from recognizing previously experienced odours and differentially delay discrimination learning of unrecognized and novel odour pairs. Poor recognition results not from mice experiencing an altered concentration of an odour but from perception of apparent novel qualities. Consistent with this, relative intensity of glomerular activity following lesions is altered compared with maps recorded in shams and by varying odour concentration. Together, these data show that odour recognition relies on comprehensively matching input patterns to a previously generated stimulus template. When encountering novel odours, access to all glomerular activity ensures rapid generation of new templates to perform accurate perceptual judgements.

Multimodal vessel mapping for precise large area alignment of functional optical imaging data to neuroanatomical preparations in marmosets.

Imaging technologies, such as intrinsic optical imaging (IOI), functional magnetic resonance imaging (fMRI) or multiphoton microscopy provide excellent opportunities to study the relationship between functional signals recorded from a cortical area and the underlying anatomical structure. This, in turn, requires accurate alignment of the recorded functional imaging data with histological datasets from the imaged tissue obtained after the functional experiment. This alignment is complicated by distortions of the tissue which naturally occur during histological treatment, and is particularly difficult to achieve over large cortical areas, such as primate visual areas. We present here a method that uses IOI vessel maps revealed in the time course of the intrinsic signal, in combination with vascular casts and vascular lumen labeling techniques together with a pseudo three dimensional (p3D) reconstruction of the tissue architecture in order to facilitate alignment of IOI data with posthoc histological datasets. We demonstrate that by such a multimodal vessel mapping approach, we are able to constitute a hook in anatomical-functional data alignment that enables the accurate assignment of functional signals over large cortical regions. As an example, we present precise alignments of IOI responses showing orientation selectivity of primate V1 with anatomical sections stained for cytochrome-oxidase-reactivity.

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. In addition, we found that RAMM could be used to simultaneously image activity from large (80) populations of apical dendrites and follow these dendrites down to their somata of origin.