Molecular Design of SERTlight: A Fluorescent Serotonin Probe for Neuronal Labeling in the Brain.

The serotonergic transmitter system plays fundamental roles in the nervous system in neurotransmission, synaptic plasticity, pathological processes, and therapeutic effects of antidepressants and psychedelics, as well as in the gastrointestinal and circulatory systems. We introduce a novel small molecule fluorescent agent, termed SERTlight, that specifically labels serotonergic neuronal cell bodies, dendrites, and axonal projections as a serotonin transporter (SERT) fluorescent substrate. SERTlight was developed by an iterative molecular design process, based on an aminoethyl-quinolone system, to integrate structural elements that impart SERT substrate activity, sufficient fluorescent brightness, and a broad absence of pharmacological activity, including at serotonin (5-hydroxytryptamine, 5HT) receptors, other G protein-coupled receptors (GPCRs), ion channels, and monoamine transporters. The high labeling selectivity is not achieved by high affinity binding to SERT itself but rather by a sufficient rate of SERT-mediated transport of SERTlight, resulting in accumulation of these molecules in 5HT neurons and yielding a robust and selective optical signal in the mammalian brain. SERTlight provides a stable signal, as it is not released via exocytosis nor by reverse SERT transport induced by 5HT releasers such as MDMA. SERTlight is optically, pharmacologically, and operationally orthogonal to a wide range of genetically encoded sensors, enabling multiplexed imaging. SERTlight enables labeling of distal 5HT axonal projections and simultaneous imaging of the release of endogenous 5HT using the GRAB5HT sensor, providing a new versatile molecular tool for the study of the serotonergic system.

A non-canonical striatopallidal Go pathway that supports motor control.

In the classical model of the basal ganglia, direct pathway striatal projection neurons (dSPNs) send projections to the substantia nigra (SNr) and entopeduncular nucleus to regulate motor function. Recent studies have re-established that dSPNs also possess axon collaterals within the globus pallidus (GPe) (bridging collaterals), yet the significance of these collaterals for behavior is unknown. Here we use in vivo optical and chemogenetic tools combined with deep learning approaches in mice to dissect the roles of dSPN GPe collaterals in motor function. We find that dSPNs projecting to the SNr send synchronous motor-related information to the GPe via axon collaterals. Inhibition of native activity in dSPN GPe terminals impairs motor activity and function via regulation of Npas1 neurons. We propose a model by which dSPN GPe axon collaterals (striatopallidal Go pathway) act in concert with the canonical terminals in the SNr to support motor control by inhibiting Npas1 neurons.

Adult-born granule cells facilitate remapping of spatial and non-spatial representations in the dentate gyrus.

Adult-born granule cells (abGCs) have been implicated in memory discrimination through a neural computation known as pattern separation. Here, using in vivo Ca2+ imaging, we examined how chronic ablation or acute chemogenetic silencing of abGCs affects the activity of mature granule cells (mGCs). In both cases, we observed altered remapping of mGCs. Rather than broadly modulating the activity of all mGCs, abGCs promote the remapping of place cells' firing fields while increasing rate remapping of mGCs that represent sensory cues. In turn, these remapping deficits are associated with behavioral impairments in animals' ability to correctly identify new goal locations. Thus, abGCs facilitate pattern separation through the formation of non-overlapping representations for identical sensory cues encountered in different locations. In the absence of abGCs, the dentate gyrus shifts to a state that is dominated by cue information, a situation that is consistent with the overgeneralization often observed in anxiety or age-related disorders.

Norepinephrine release in the cerebellum contributes to aversive learning.

The modulation of dopamine release from midbrain projections to the striatum has long been demonstrated in reward-based learning, but the synaptic basis of aversive learning is far less characterized. The cerebellum receives axonal projections from the locus coeruleus, and norepinephrine release is implicated in states of arousal and stress, but whether aversive learning relies on plastic changes in norepinephrine release in the cerebellum is unknown. Here we report that in mice, norepinephrine is released in the cerebellum following an unpredicted noxious event (a foot-shock) and that this norepinephrine release is potentiated powerfully with fear acquisition as animals learn that a previously neutral stimulus (tone) predicts the aversive event. Importantly, both chemogenetic and optogenetic inhibition of the locus coeruleus-cerebellum pathway block fear memory without impairing motor function. Thus, norepinephrine release in the cerebellum is modulated by experience and underlies aversive learning.

An Open-Source Virtual Reality System for the Measurement of Spatial Learning in Head-Restrained Mice.

Head-restrained behavioral experiments in mice allow neuroscientists to observe neural circuit activity with high-resolution electrophysiological and optical imaging tools while delivering precise sensory stimuli to a behaving animal. Recently, human and rodent studies using virtual reality (VR) environments have shown VR to be an important tool for uncovering the neural mechanisms underlying spatial learning in the hippocampus and cortex, due to the extremely precise control over parameters such as spatial and contextual cues. Setting up virtual environments for rodent spatial behaviors can, however, be costly and require an extensive background in engineering and computer programming. Here, we present a simple yet powerful system based upon inexpensive, modular, open-source hardware and software that enables researchers to study spatial learning in head-restrained mice using a VR environment. This system uses coupled microcontrollers to measure locomotion and deliver behavioral stimuli while head-restrained mice run on a wheel in concert with a virtual linear track environment rendered by a graphical software package running on a single-board computer. The emphasis on distributed processing allows researchers to design flexible, modular systems to elicit and measure complex spatial behaviors in mice in order to determine the connection between neural circuit activity and spatial learning in the mammalian brain.

Protocol for in vivo imaging and analysis of brainstem neuronal activity in the dorsal raphe nucleus of freely behaving mice.

In vivo brainstem imaging with miniature microscopy has been challenging due to surgical difficulty, high motion, and correlated activity between neurons. Here, we present a protocol for brainstem imaging in freely moving mice using the dorsal raphe nucleus as an example. We describe surgical procedures to inject a virus encoding GCaMP6m and securely implant a GRIN lens in the brainstem. We then detail motion correction and cell segmentation from the data to parse single-cell activity from correlated networks. For complete details on the use and execution of this protocol, please refer to Paquelet et al. (2022).1.

A non-canonical striatopallidal "Go" pathway that supports motor control.

In the classical model of the basal ganglia, direct pathway striatal projection neurons (dSPNs) send projections to the substantia nigra (SNr) and entopeduncular nucleus to regulate motor function. Recent studies have re-established that dSPNs also possess "bridging" collaterals within the globus pallidus (GPe), yet the significance of these collaterals for behavior is unknown. Here we use in vivo optical and chemogenetic tools combined with deep learning approaches to dissect the roles of bridging collaterals in motor function. We find that dSPNs projecting to the SNr send synchronous motor-related information to the GPe via axon collaterals. Inhibition of native activity in dSPN GPe terminals impairs motor activity and function via regulation of pallidostriatal Npas1 neurons. We propose a model by which dSPN GPe collaterals ("striatopallidal Go pathway") act in concert with the canonical terminals in the SNr to support motor control by inhibiting Npas1 signals going back to the striatum.

Decreased Dorsomedial Striatum Direct Pathway Neuronal Activity Is Required for Learned Motor Coordination.

It has been suggested that the dorsomedial striatum (DMS) is engaged in the early stages of motor learning for goal-directed actions, whereas at later stages, control is transferred to the dorsolateral striatum (DLS), a process that enables learned motor actions to become a skill or habit. It is not known whether these striatal regions are simultaneously active while the expertise is acquired. To address this question, we developed a mouse "Treadmill Training Task" that tracks changes in mouse locomotor coordination during running practice and simultaneously provides a means to measure local neuronal activity using photometry. To measure change in motor coordination over treadmill practice sessions, we used DeepLabCut (DLC) and custom-built code to analyze body position and paw movements. By evaluating improvements in motor coordination during training with simultaneous neuronal calcium activity in the striatum, we found that DMS direct pathway neurons exhibited decreased activity as the mouse gained proficiency at running. In contrast, direct pathway activity in the DLS was similar throughout training. Pharmacological blockade of D1 dopamine receptors in these subregions during task performance demonstrated that dopamine neurotransmission in the direct pathway activity is necessary for efficient motor coordination learning. These results provide new tools to measure changes in fine motor skills with simultaneous recordings of brain activity and reveal fundamental features of the neuronal substrates of motor learning.

Single-cell activity and network properties of dorsal raphe nucleus serotonin neurons during emotionally salient behaviors.

The serotonin system modulates a wide variety of emotional behaviors and states, including reward processing, anxiety, and social interaction. To reveal the underlying patterns of neural activity, we visualized serotonergic neurons in the dorsal raphe nucleus (DRN5-HT) of mice using miniaturized microscopy during diverse emotional behaviors. We discovered ensembles of cells with highly correlated activity and found that DRN5-HT neurons are preferentially recruited by emotionally salient stimuli as opposed to neutral stimuli. Individual DRN5-HT neurons responded to diverse combinations of salient stimuli, with some preference for valence and sensory modality. Anatomically defined subpopulations projecting to either a reward-related structure (the ventral tegmental area) or an anxiety-related structure (the bed nucleus of the stria terminalis) contained all response types but were enriched in reward- and anxiety-responsive cells, respectively. Our results suggest that the DRN serotonin system responds to emotional salience using ensembles with mixed selectivity and biases in downstream connectivity.

Parallel processing of sensory cue and spatial information in the dentate gyrus.

During exploration, animals form an internal map of an environment by combining information about landmarks and the animal's movement, a process that depends on the hippocampus. The dentate gyrus (DG) is the first stage of the hippocampal circuit where self-motion ("where") and sensory cue information ("what") are integrated, but it remains unknown how DG neurons encode this information during cognitive map formation. Using two-photon calcium imaging in mice running on a treadmill along with online cue manipulation, we identify robust sensory cue responses in DG granule cells. Cue cell responses are stable, stimulus-specific, and accompanied by inhibition of nearby neurons. This demonstrates the existence of "cue cells" in addition to better characterized "place cells" in the DG. We hypothesize that the DG supports parallel channels of spatial and non-spatial information that contribute distinctly to downstream computations and affect roles of the DG in spatial navigation and episodic memory.

3D neuronal mitochondrial morphology in axons, dendrites, and somata of the aging mouse hippocampus.

The brain's ability to process complex information relies on the constant supply of energy through aerobic respiration by mitochondria. Neurons contain three anatomically distinct compartments-the soma, dendrites, and projecting axons-which have different energetic and biochemical requirements, as well as different mitochondrial morphologies in cultured systems. In this study, we apply quantitative three-dimensional electron microscopy to map mitochondrial network morphology and complexity in the mouse brain. We examine somatic, dendritic, and axonal mitochondria in the dentate gyrus and cornu ammonis 1 (CA1) of the mouse hippocampus, two subregions with distinct principal cell types and functions. We also establish compartment-specific differences in mitochondrial morphology across these cell types between young and old mice, highlighting differences in age-related morphological recalibrations. Overall, these data define the nature of the neuronal mitochondrial network in the mouse hippocampus, providing a foundation to examine the role of mitochondrial morpho-function in the aging brain.

Quantitative mapping of human hair greying and reversal in relation to life stress.

Background: Hair greying is a hallmark of aging generally believed to be irreversible and linked to psychological stress. Methods: Here, we develop an approach to profile hair pigmentation patterns (HPPs) along individual human hair shafts, producing quantifiable physical timescales of rapid greying transitions. Results: Using this method, we show white/grey hairs that naturally regain pigmentation across sex, ethnicities, ages, and body regions, thereby quantitatively defining the reversibility of greying in humans. Molecularly, grey hairs upregulate proteins related to energy metabolism, mitochondria, and antioxidant defenses. Combining HPP profiling and proteomics on single hairs, we also report hair greying and reversal that can occur in parallel with psychological stressors. To generalize these observations, we develop a computational simulation, which suggests a threshold-based mechanism for the temporary reversibility of greying. Conclusions: Overall, this new method to quantitatively map recent life history in HPPs provides an opportunity to longitudinally examine the influence of recent life exposures on human biology. Funding: This work was supported by the Wharton Fund and NIH grants GM119793, MH119336, and AG066828 (MP).

Hair greying is a visible sign of aging that affects everyone. The loss of hair color is due to the loss of melanin, a pigment found in the skin, eyes and hair. Research in mice suggests stress may accelerate hair greying, but there is no definitive research on this in humans. This is because there are no research tools to precisely map stress and hair color over time. But, just like tree rings hold information about past decades, and rocks hold information about past centuries, hairs hold information about past months and years. Hair growth is an active process that happens under the skin inside hair follicles. It demands lots of energy, supplied by structures inside cells called mitochondria. While hairs are growing, cells receive chemical and electrical signals from inside the body, including stress hormones. It is possible that these exposures change proteins and other molecules laid down in the growing hair shaft. As the hair grows out of the scalp, it hardens, preserving these molecules into a stable form. This preservation is visible as patterns of pigmentation. Examining single-hairs and matching the patterns to life events could allow researchers to look back in time through a person's biological history. Rosenberg et al. report a new way to digitize and measure small changes in color along single human hairs. This method revealed that some white hairs naturally regain their color, something that had not been reported in a cohort of healthy individuals before. Aligning the hair pigmentation patterns with recent reports of stress in the hair donors' lives showed striking associations. When one donor reported an increase in stress, a hair lost its pigment. When the donor reported a reduction in stress, the same hair regained its pigment. Rosenberg et al. mapped hundreds of proteins inside the hairs to show that white hairs contained more proteins linked to mitochondria and energy use. This suggests that metabolism and mitochondria may play a role in hair greying. To explore these observations in more detail Rosenberg et al. developed a mathematical model that simulates the greying of a whole head of hair over a lifetime, an experiment impossible to do with living people. The model suggested that there might be a threshold for temporary greying; if hairs are about to go grey anyway, a stressful event might trigger that change earlier. And when the stressful event ends, if a hair is just above the threshold, then it could revert back to dark. The new method for measuring small changes in hair coloring opens up the possibility of using hair pigmentation patterns like tree rings. This could track the influence of past life events on human biology. In the future, monitoring hair pigmentation patterns could provide a way to trace the effectiveness of treatments aimed at reducing stress or slowing the aging process. Understanding how 'old' white hairs regain their 'young' pigmented state could also reveal new information about the malleability of human aging more generally.

A device for stereotaxic viral delivery into the brains of neonatal mice.

The increasing interest in manipulating neural circuits in developing brains has created a demand for reliable and accurate methods for delivering viruses to newborn mice. Here we describe a novel 3D-printed mouse neonatal stereotaxic adaptor for intracerebral viral injection that provides enhanced precision and reliability. Using this device, we injected A2a-Cre mice with a Cre-dependent hM4D-mCherry viral construct at postnatal day 1 (P1) and demonstrated selective expression in the striatal indirect pathway neurons on days P7, P11 and P25. Similarly, dopaminergic midbrain neurons were selectively targeted with a Cre-dependent green fluorescent protein virus in Dat-IRES-Cre neonates and expression examined at P25. Our open-source neonatal stereotaxic mouse adaptor facilitates neonatal neuronal targeting, which should improve the ability to label and modify neural circuits in developing mouse brains.

DIY-NAMIC Behavior: A High-Throughput Method to Measure Complex Phenotypes in the Homecage.

Complex behavioral assessment is necessary to comprehensively assess in vivo manipulations in rodent models for neuropsychiatric disorders. Operant behavioral paradigms provide rich datasets and allow for the careful analysis of behavioral phenotypes. However, one major limitation in these studies is the expense and work-load that are required using traditional methods. The equipment for commercial operant boxes can be prohibitively expensive, and the daily experimenter effort and mouse costs required for these studies is extensive. Rodents are generally trained on task-specific paradigms for months, tested every day for 5-7 d/week. Additionally, appetitive paradigms usually require food restriction and are also commonly run in the non-active light phase of the rodent circadian rhythm. These limitations make operant behavioral testing especially difficult during adolescence, a time period of interest with regards to the development of adult-like phenotypes and a high-risk period for the development of neuropsychiatric disorders, including those which involve impulsive behavior. In order to address these issues, we developed an automated, inexpensive, open-source method which allows the implementation of most standard operant paradigms in the homecage of rodents in shorter time frames without food restriction, and with much less experimenter effort. All construction and code for the do-it-yourself Nautiyal Automated Modular Instrumental Conditioning (DIY-NAMIC) system are open source. We demonstrate their utility here by measuring impulsive behavior in a pharmacology experiment, as well as in adolescent mice.

Contributions of adult neurogenesis to dentate gyrus network activity and computations.

Anatomical observations, theoretical work and lesion experiments have led to the idea that an important function of the dentate gyrus of the mammalian hippocampus is pattern separation, a neural computation that ensures new memories are encoded without interference from previously stored memories that share similar features. The dentate gyrus also exhibits a unique form of neural plasticity that results from the continuous integration of newly born excitatory granule cells, termed adult hippocampal neurogenesis. However, the manner in which adult neurogenesis contributes to dentate gyrus network activity and computations is incompletely understood. Here, we first describe the prevailing models for the role of adult neurogenesis in dentate gyrus network function and then re-evaluate these models in the light of recent findings regarding the in vivo activity of the dentate gyrus and synaptic interactions of adult born granule cells with local circuit components, as well as, inputs, and outputs of the dentate gyrus. We propose that adult neurogenesis provides flexibility for the dentate gyrus to rapidly generate a context specific, distributed representation of important sensory stimuli such as spatial cues, which ultimately gives rise to behavioral discrimination.

Reinforcement Learning Recruits Somata and Apical Dendrites across Layers of Primary Sensory Cortex.

The mammalian brain can form associations between behaviorally relevant stimuli in an animal's environment. While such learning is thought to primarily involve high-order association cortex, even primary sensory areas receive long-range connections carrying information that could contribute to high-level representations. Here, we imaged layer 1 apical dendrites in the barrel cortex of mice performing a whisker-based operant behavior. In addition to sensory-motor events, calcium signals in apical dendrites of layers 2/3 and 5 neurons and in layer 2/3 somata track the delivery of rewards, both choice related and randomly administered. Reward-related tuft-wide dendritic spikes emerge gradually with training and are task specific. Learning recruits cells whose intrinsic activity coincides with the time of reinforcement. Layer 4 largely lacked reward-related signals, suggesting a source other than the primary thalamus. Our results demonstrate that a sensory cortex can acquire a set of associations outside its immediate sensory modality and linked to salient behavioral events.

Sensation, movement and learning in the absence of barrel cortex.

For many of our senses, the role of the cerebral cortex in detecting stimuli is controversial1-17. Here we examine the effects of both acute and chronic inactivation of the primary somatosensory cortex in mice trained to move their large facial whiskers to detect an object by touch and respond with a lever to obtain a water reward. Using transgenic mice, we expressed inhibitory opsins in excitatory cortical neurons. Transient optogenetic inactivation of the primary somatosensory cortex, as well as permanent lesions, initially produced both movement and sensory deficits that impaired detection behaviour, demonstrating the link between sensory and motor systems during active sensing. Unexpectedly, lesioned mice had recovered full behavioural capabilities by the subsequent session. This rapid recovery was experience-dependent, and early re-exposure to the task after lesioning facilitated recovery. Furthermore, ablation of the primary somatosensory cortex before learning did not affect task acquisition. This combined optogenetic and lesion approach suggests that manipulations of the sensory cortex may be only temporarily disruptive to other brain structures that are themselves capable of coordinating multiple, arbitrary movements with sensation. Thus, the somatosensory cortex may be dispensable for active detection of objects in the environment.

Simultaneous Denoising, Deconvolution, and Demixing of Calcium Imaging Data.

We present a modular approach for analyzing calcium imaging recordings of large neuronal ensembles. Our goal is to simultaneously identify the locations of the neurons, demix spatially overlapping components, and denoise and deconvolve the spiking activity from the slow dynamics of the calcium indicator. Our approach relies on a constrained nonnegative matrix factorization that expresses the spatiotemporal fluorescence activity as the product of a spatial matrix that encodes the spatial footprint of each neuron in the optical field and a temporal matrix that characterizes the calcium concentration of each neuron over time. This framework is combined with a novel constrained deconvolution approach that extracts estimates of neural activity from fluorescence traces, to create a spatiotemporal processing algorithm that requires minimal parameter tuning. We demonstrate the general applicability of our method by applying it to in vitro and in vivo multi-neuronal imaging data, whole-brain light-sheet imaging data, and dendritic imaging data.

Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms.

We report a new 3D microscopy technique that allows volumetric imaging of living samples at ultra-high speeds: Swept, confocally-aligned planar excitation (SCAPE) microscopy. While confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image 3D volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry, and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light-sheet in a single-objective, en-face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless 3D imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae.

Wide-field in vivo neocortical calcium dye imaging using a convection-enhanced loading technique combined with simultaneous multiwavelength imaging of voltage-sensitive dyes and hemodynamic signals.

In vivo calcium imaging is an incredibly powerful technique that provides simultaneous information on fast neuronal events, such as action potentials and subthreshold synaptic activity, as well as slower events that occur in the glia and surrounding neuropil. Bulk-loading methods that involve multiple injections can be used for single-cell as well as wide-field imaging studies. However, multiple injections result in inhomogeneous loading as well as multiple sites of potential cortical injury. We used convection-enhanced delivery to create smooth, continuous loading of a large area of the cortical surface through a solitary injection site and demonstrated the efficacy of the technique using confocal microscopy imaging of single cells and physiological responses to single-trial events of spontaneous activity, somatosensory-evoked potentials, and epileptiform events. Combinations of calcium imaging with voltage-sensitive dye and intrinsic signal imaging demonstrate the utility of this technique in neurovascular coupling investigations. Convection-enhanced loading of calcium dyes may be a useful technique to advance the study of cortical processing when widespread loading of a wide-field imaging is required.