Advancing the path to in-vivo imaging in freely moving mice via multimode-multicore fiber based holographic endoscopy.

Significance: Hair-thin multimode optical fiber-based holographic endoscopes have gained considerable interest in modern neuroscience for their ability to achieve cellular and even subcellular resolution during in-vivo deep brain imaging. However, the application of multimode fibers in freely moving animals presents a persistent challenge as it is difficult to maintain optimal imaging performance while the fiber undergoes deformations. Aim: We propose a fiber solution for challenging in-vivo applications with the capability of deep brain high spatial resolution imaging and neuronal activity monitoring in anesthetized as well as awake behaving mice. Approach: We used our previously developed M3CF multimode-multicore fiber to record fluorescently labeled neurons in anesthetized mice. Our M3CF exhibits a cascaded refractive index structure, enabling two distinct regimes of light transport that imitate either a multimode or a multicore fiber. The M3CF has been specifically designed for use in the initial phase of an in-vivo experiment, allowing for the navigation of the endoscope's distal end toward the targeted brain structure. The multicore regime enables the transfer of light to and from each individual neuron within the field of view. For chronic experiments in awake behaving mice, it is crucial to allow for disconnecting the fiber and the animal between experiments. Therefore, we provide here an effective solution and establish a protocol for reconnection of two segments of M3CF with hexagonally arranged corelets. Results: We successfully utilized the M3CF to image neurons in anaesthetized transgenic mice expressing enhanced green fluorescent protein. Additionally, we compared imaging results obtained with the M3CF with larger numerical aperture (NA) fibers in fixed whole-brain tissue. Conclusions: This study focuses on addressing challenges and providing insights into the use of multimode-multicore fibers as imaging solutions for in-vivo applications. We suggest that the upcoming version of the M3CF increases the overall NA between the two cladding layers to allow for access to high resolution spatial imaging. As the NA increases in the multimode regime, the fiber diameter and ring structure must be reduced to minimize the computational burden and invasiveness.

A septal-ventral tegmental area circuit drives exploratory behavior.

To survive, animals need to balance their exploratory drive with their need for safety. Subcortical circuits play an important role in initiating and modulating movement based on external demands and the internal state of the animal; however, how motivation and onset of locomotion are regulated remain largely unresolved. Here, we show that a glutamatergic pathway from the medial septum and diagonal band of Broca (MSDB) to the ventral tegmental area (VTA) controls exploratory locomotor behavior in mice. Using a self-supervised machine learning approach, we found an overrepresentation of exploratory actions, such as sniffing, whisking, and rearing, when this projection is optogenetically activated. Mechanistically, this role relies on glutamatergic MSDB projections that monosynaptically target a subset of both glutamatergic and dopaminergic VTA neurons. Taken together, we identified a glutamatergic basal forebrain to midbrain circuit that initiates locomotor activity and contributes to the expression of exploration-associated behavior.

Holistic bursting cells store long-term memory in auditory cortex.

The sensory neocortex has been suggested to be a substrate for long-term memory storage, yet which exact single cells could be specific candidates underlying such long-term memory storage remained neither known nor visible for over a century. Here, using a combination of day-by-day two-photon Ca2+ imaging and targeted single-cell loose-patch recording in an auditory associative learning paradigm with composite sounds in male mice, we reveal sparsely distributed neurons in layer 2/3 of auditory cortex emerged step-wise from quiescence into bursting mode, which then invariably expressed holistic information of the learned composite sounds, referred to as holistic bursting (HB) cells. Notably, it was not shuffled populations but the same sparse HB cells that embodied the behavioral relevance of the learned composite sounds, pinpointing HB cells as physiologically-defined single-cell candidates of an engram underlying long-term memory storage in auditory cortex.

Engaging distributed cortical and cerebellar networks through motor execution, observation, and imagery.

When we interact with the environment around us, we are sometimes active participants, making directed physical motor movements and other times only mentally engaging with our environment, taking in sensory information and internally planning our next move without directed physical movement. Traditionally, cortical motor regions and key subcortical structures such as the cerebellum have been tightly linked to motor initiation, coordination, and directed motor behavior. However, recent neuroimaging studies have noted the activation of the cerebellum and wider cortical networks specifically during various forms of motor processing, including the observations of actions and mental rehearsal of movements through motor imagery. This phenomenon of cognitive engagement of traditional motor networks raises the question of how these brain regions are involved in the initiation of movement without physical motor output. Here, we will review evidence for distributed brain network activation during motor execution, observation, and imagery in human neuroimaging studies as well as the potential for cerebellar involvement specifically in motor-related cognition. Converging evidence suggests that a common global brain network is involved in both movement execution and motor observation or imagery, with specific task-dependent shifts in these global activation patterns. We will further discuss underlying cross-species anatomical support for these cognitive motor-related functions as well as the role of cerebrocerebellar communication during action observation and motor imagery.

Real-time mechanisms of exacerbated synaptic remodeling by microglia in acute models of systemic inflammation and tauopathy.

Remodeling of synapses by microglia is essential for synaptic plasticity in the brain. However, during neuroinflammation and neurodegenerative diseases, microglia can induce excessive synaptic loss, although the precise underlying mechanisms are unknown. To directly observe microglia-synapse interactions under inflammatory conditions, we performed in vivo two-photon time-lapse imaging of microglia-synapse interactions after bacterial lipopolysaccharide administration to model systemic inflammation, or after inoculation of Alzheimer's disease (AD) brain extracts to model disease-associated neuroinflammatory microglial response. Both treatments prolonged microglia-neuron contacts, decreased basal surveillance of synapses and promoted synaptic remodeling in response to synaptic stress induced by focal single-synapse photodamage. Spine elimination correlated with the expression of microglial complement system/phagocytic proteins and the occurrence of synaptic filopodia. Microglia were observed contacting spines, then stretching and phagocytosing spine head filopodia. Thus, in response to inflammatory stimuli microglia exacerbated spine remodeling through prolonged microglial contact and elimination of spines 'tagged' by synaptic filopodia.

A corticostriatal projection for sound-evoked and anticipatory motor behavior following temporal expectation.

The ability to form predictions based on recent sensory experience is essential for behavioral adaptation to our ever-changing environment. Predictive encoding represented by neuronal activity has been observed in sensory cortex, but how this neuronal activity is transformed into anticipatory motor behavior remains unclear. Fiber photometry to investigate a corticostriatal projection from the auditory cortex to the posterior striatum during an auditory paradigm in mice, and pharmacological experiments in a task that induces a temporal expectation of upcoming sensory stimuli. We find that the auditory corticostriatal projection relays both sound-evoked stimulus information as well as predictive signals in relation to stimulus timing following rhythmic auditory stimulation. Pharmacological experiments suggest that this projection is required for the initiation of both sound-evoked and anticipatory licking behavior in an auditory associative-learning behavioral task, but not for the general recognition of presented auditory stimuli. This auditory corticostriatal projection carries predictive signals, and the posterior striatum is critical to the anticipatory stimulus-driven motor behavior.

Optical Fiber-Based Recording of Climbing Fiber Ca2+ Signals in Freely Behaving Mice.

The olivocerebellar circuitry is important to convey both motor and non-motor information from the inferior olive (IO) to the cerebellar cortex. Several methods are currently established to observe the dynamics of the olivocerebellar circuitry, largely by recording the complex spike activity of cerebellar Purkinje cells; however, these techniques can be technically challenging to apply in vivo and are not always possible in freely behaving animals. Here, we developed a method for the direct, accessible, and robust recording of climbing fiber (CF) Ca2+ signals based on optical fiber photometry. We first verified the IO stereotactic coordinates and the organization of contralateral CF projections using tracing techniques and then injected Ca2+ indicators optimized for axonal labeling, followed by optical fiber-based recordings. We demonstrated this method by recording CF Ca2+ signals in lobule IV/V of the cerebellar vermis, comparing the resulting signals in freely moving mice. We found various movement-evoked CF Ca2+ signals, but the onset of exploratory-like behaviors, including rearing and tiptoe standing, was highly synchronous with recorded CF activity. Thus, we have successfully established a robust and accessible method to record the CF Ca2+ signals in freely behaving mice, which will extend the toolbox for studying cerebellar function and related disorders.

Context value updating and multidimensional neuronal encoding in the retrosplenial cortex.

The retrosplenial cortex (RSC) has diverse functional inputs and is engaged by various sensory, spatial, and associative learning tasks. We examine how multiple functional aspects are integrated on the single-cell level in the RSC and how the encoding of task-related parameters changes across learning. Using a visuospatial context discrimination paradigm and two-photon calcium imaging in behaving mice, a large proportion of dysgranular RSC neurons was found to encode multiple task-related dimensions while forming context-value associations across learning. During reversal learning requiring increased cognitive flexibility, we revealed an increased proportion of multidimensional encoding neurons that showed higher decoding accuracy for behaviorally relevant context-value associations. Chemogenetic inactivation of RSC led to decreased behavioral context discrimination during learning phases in which context-value associations were formed, while recall of previously formed associations remained intact. RSC inactivation resulted in a persistent positive behavioral bias in valuing contexts, indicating a role for the RSC in context-value updating.

Visual plasticity: Illuminating the role of the hippocampus in cortical sensory encoding.

The primary visual cortex has the capacity to store stimulus-specific information locally. A new study reveals a direct role for the hippocampus in experience-dependent cortical plasticity when visual stimuli are presented in a predictable temporal order.

A cerebellar-thalamocortical pathway drives behavioral context-dependent movement initiation.

Executing learned motor behaviors often requires the transformation of sensory cues into patterns of motor commands that generate appropriately timed actions. The cerebellum and thalamus are two key areas involved in shaping cortical output and movement, but the contribution of a cerebellar-thalamocortical pathway to voluntary movement initiation remains poorly understood. Here, we investigated how an auditory "go cue" transforms thalamocortical activity patterns and how these changes relate to movement initiation. Population responses in dentate/interpositus-recipient regions of motor thalamus reflect a time-locked increase in activity immediately prior to movement initiation that is temporally uncoupled from the go cue, indicative of a fixed-latency feedforward motor timing signal. Blocking cerebellar or motor thalamic output suppresses movement initiation, while stimulation triggers movements in a behavioral context-dependent manner. Our findings show how cerebellar output, via the thalamus, shapes cortical activity patterns necessary for learned context-dependent movement initiation.

Enhanced modulation of cell-type specific neuronal responses in mouse dorsal auditory field during locomotion.

As we move through the environment we experience constantly changing sensory input that must be merged with our ongoing motor behaviors - creating dynamic interactions between our sensory and motor systems. Active behaviors such as locomotion generally increase the sensory-evoked neuronal activity in visual and somatosensory cortices, but evidence suggests that locomotion largely suppresses neuronal responses in the auditory cortex. However, whether this effect is ubiquitous across different anatomical regions of the auditory cortex is largely unknown. In mice, auditory association fields such as the dorsal auditory cortex (AuD), have been shown to have different physiological response properties, protein expression patterns, and cortical as well as subcortical connections, in comparison to primary auditory regions (A1) - suggesting there may be important functional differences. Here we examined locomotion-related modulation of neuronal activity in cortical layers ⅔ of AuD and A1 using two-photon Ca2+ imaging in head-fixed behaving mice that are able to freely run on a spherical treadmill. We determined the proportion of neurons in these two auditory regions that show enhanced and suppressed sensory-evoked responses during locomotion and quantified the depth of modulation. We found that A1 shows more suppression and AuD more enhanced responses during locomotion periods. We further revealed differences in the circuitry between these auditory regions and motor cortex, and found that AuD is more highly connected to motor cortical regions. Finally, we compared the cell-type specific locomotion-evoked modulation of responses in AuD and found that, while subpopulations of PV-expressing interneurons showed heterogeneous responses, the population in general was largely suppressed during locomotion, while excitatory population responses were generally enhanced in AuD. Therefore, neurons in primary and dorsal auditory fields have distinct response properties, with dorsal regions exhibiting enhanced activity in response to movement. This functional distinction may be important for auditory processing during navigation and acoustically guided behavior.

Disynaptic cerebrocerebellar pathways originating from multiple functionally distinct cortical areas.

The cerebral cortex and cerebellum both play important roles in sensorimotor processing, however, precise connections between these major brain structures remain elusive. Using anterograde mono-trans-synaptic tracing, we elucidate cerebrocerebellar pathways originating from primary motor, sensory, and association cortex. We confirm a highly organized topography of corticopontine projections in mice; however, we found no corticopontine projections originating from primary auditory cortex and detail several potential extra-pontine cerebrocerebellar pathways. The cerebellar hemispheres were the major target of resulting disynaptic mossy fiber terminals, but we also found at least sparse cerebrocerebellar projections to every lobule of the cerebellum. Notably, projections originating from association cortex resulted in less laterality than primary sensory/motor cortices. Within molecularly defined cerebellar modules we found spatial overlap of mossy fiber terminals, originating from functionally distinct cortical areas, within crus I, paraflocculus, and vermal regions IV/V and VI - highlighting these regions as potential hubs for multimodal cortical influence.

Reward Association Enhances Stimulus-Specific Representations in Primary Visual Cortex.

The potential for neuronal representations of external stimuli to be modified by previous experience is critical for efficient sensory processing and improved behavioral outcomes. To investigate how repeated exposure to a visual stimulus affects its representation in mouse primary visual cortex (V1), we performed two-photon calcium imaging of layer 2/3 neurons and assessed responses before, during, and after the presentation of a repetitive stimulus over 5 consecutive days. We found a stimulus-specific enhancement of the neuronal representation of the repetitively presented stimulus when it was associated with a reward. This was observed both after mice actively learned a rewarded task and when the reward was randomly received. Stimulus-specific enhanced representation resulted both from neurons gaining selectivity and from increased response reliability in previously selective neurons. In the absence of reward, there was either no change in stimulus representation or a decreased representation when the stimulus was viewed at a fixed temporal frequency. Pairing a second stimulus with a reward led to a similar enhanced representation and increased discriminability between the equally rewarded stimuli. Single-neuron responses showed that separate subpopulations discriminated between the two rewarded stimuli depending on whether the stimuli were displayed in a virtual environment or viewed on a single screen. We suggest that reward-associated responses enable the generalization of enhanced stimulus representation across these V1 subpopulations. We propose that this dynamic regulation of visual processing based on the behavioral relevance of sensory input ultimately enhances and stabilizes the representation of task-relevant features while suppressing responses to non-relevant stimuli.

High-fidelity multimode fibre-based endoscopy for deep brain in vivo imaging.

Progress in neuroscience relies on new techniques for investigating the complex dynamics of neuronal networks. An ongoing challenge is to achieve minimally invasive and high-resolution observations of neuronal activity in vivo inside deep brain areas. Recently introduced methods for holographic control of light propagation in complex media enable the use of a hair-thin multimode optical fibre as an ultranarrow imaging tool. Compared to endoscopes based on graded-index lenses or fibre bundles, this new approach offers a footprint reduction exceeding an order of magnitude, combined with a significant enhancement in resolution. We designed a compact and high-speed system for fluorescent imaging at the tip of a fibre, achieving a resolution of 1.18 ± 0.04 µm across a 50-µm field of view, yielding 7-kilopixel images at a rate of 3.5 frames/s. Furthermore, we demonstrate in vivo observations of cell bodies and processes of inhibitory neurons within deep layers of the visual cortex and hippocampus of anaesthetised mice. This study paves the way for modern microscopy to be applied deep inside tissues of living animal models while exerting a minimal impact on their structural and functional properties.

The Impact of Visual Cues, Reward, and Motor Feedback on the Representation of Behaviorally Relevant Spatial Locations in Primary Visual Cortex.

The integration of visual stimuli and motor feedback is critical for successful visually guided navigation. These signals have been shown to shape neuronal activity in the primary visual cortex (V1), in an experience-dependent manner. Here, we examined whether visual, reward, and self-motion-related inputs are integrated in order to encode behaviorally relevant locations in V1 neurons. Using a behavioral task in a virtual environment, we monitored layer 2/3 neuronal activity as mice learned to locate a reward along a linear corridor. With learning, a subset of neurons became responsive to the expected reward location. Without a visual cue to the reward location, both behavioral and neuronal responses relied on self-motion-derived estimations. However, when visual cues were available, both neuronal and behavioral responses were driven by visual information. Therefore, a population of V1 neurons encode behaviorally relevant spatial locations, based on either visual cues or on self-motion feedback when visual cues are absent.

Optimization of interneuron function by direct coupling of cell migration and axonal targeting.

Neural circuit assembly relies on the precise synchronization of developmental processes, such as cell migration and axon targeting, but the cell-autonomous mechanisms coordinating these events remain largely unknown. Here we found that different classes of interneurons use distinct routes of migration to reach the embryonic cerebral cortex. Somatostatin-expressing interneurons that migrate through the marginal zone develop into Martinotti cells, one of the most distinctive classes of cortical interneurons. For these cells, migration through the marginal zone is linked to the development of their characteristic layer 1 axonal arborization. Altering the normal migratory route of Martinotti cells by conditional deletion of Mafb-a gene that is preferentially expressed by these cells-cell-autonomously disrupts axonal development and impairs the function of these cells in vivo. Our results suggest that migration and axon targeting programs are coupled to optimize the assembly of inhibitory circuits in the cerebral cortex.

Action and learning shape the activity of neuronal circuits in the visual cortex.

Nonsensory variables strongly influence neuronal activity in the adult mouse primary visual cortex. Neuronal responses to visual stimuli are modulated by behavioural state, such as arousal and motor activity, and are shaped by experience. This dynamic process leads to neural representations in the visual cortex that reflect stimulus familiarity, expectations of reward and object location, and mismatch between self-motion and visual-flow. The recent development of genetic tools and recording techniques in awake behaving mice has enabled the investigation of the circuit mechanisms underlying state-dependent and experience-dependent neuronal representations in primary visual cortex. These neuronal circuits involve neuromodulatory, top-down cortico-cortical and thalamocortical pathways. The functions of nonsensory signals at this early stage of visual information processing are now beginning to be unravelled.

Modulation of complex spike activity differs between zebrin-positive and -negative Purkinje cells in the pigeon cerebellum.

The cerebellum is organized into parasagittal zones defined by its climbing and mossy fiber inputs, efferent projections, and Purkinje cell (PC) response properties. Additionally, parasagittal stripes can be visualized with molecular markers, such as heterogeneous expression of the isoenzyme zebrin II (ZII), where sagittal stripes of high ZII expression (ZII+) are interdigitated with stripes of low ZII expression (ZII-). In the pigeon vestibulocerebellum, a ZII+/- stripe pair represents a functional unit, insofar as both ZII+ and ZII- PCs within a stripe pair respond best to the same pattern of optic flow. In the present study, we attempted to determine whether there were any differences in the responses between ZII+ and ZII- PCs within a functional unit in response to optic flow stimuli. In pigeons of either sex, we recorded complex spike activity (CSA) from PCs in response to optic flow, marked recording sites with a fluorescent tracer, and determined the ZII identity of recorded PCs by immunohistochemistry. We found that CSA of ZII+ PCs showed a greater depth of modulation in response to the preferred optic flow pattern compared with ZII- PCs. We suggest that these differences in the depth of modulation to optic flow stimuli are due to differences in the connectivity of ZII+ and ZII- PCs within a functional unit. Specifically, ZII+ PCs project to areas of the vestibular nuclei that provide inhibitory feedback to the inferior olive, whereas ZII- PCs do not. NEW & NOTEWORTHY Although the cerebellum appears to be a uniform structure, Purkinje cells (PCs) are heterogeneous and can be categorized on the basis of the expression of molecular markers. These phenotypes are conserved across species, but the significance is undetermined. PCs in the vestibulocerebellum encode optic flow resulting from self-motion, and those that express the molecular marker zebrin II (ZII+) exhibit more sensitivity to optic flow than those that do not express zebrin II (ZII-).

FISSA: A neuropil decontamination toolbox for calcium imaging signals.

In vivo calcium imaging has become a method of choice to image neuronal population activity throughout the nervous system. These experiments generate large sequences of images. Their analysis is computationally intensive and typically involves motion correction, image segmentation into regions of interest (ROIs), and extraction of fluorescence traces from each ROI. Out of focus fluorescence from surrounding neuropil and other cells can strongly contaminate the signal assigned to a given ROI. In this study, we introduce the FISSA toolbox (Fast Image Signal Separation Analysis) for neuropil decontamination. Given pre-defined ROIs, the FISSA toolbox automatically extracts the surrounding local neuropil and performs blind-source separation with non-negative matrix factorization. Using both simulated and in vivo data, we show that this toolbox performs similarly or better than existing published methods. FISSA requires only little RAM, and allows for fast processing of large datasets even on a standard laptop. The FISSA toolbox is available in Python, with an option for MATLAB format outputs, and can easily be integrated into existing workflows. It is available from Github and the standard Python repositories.