Functional ultrasound (fUS) maps cerebral blood volume (CBV) but lacks molecular and neuronal specificity. By simultaneously integrating fUS with optical imaging, we show that fUS-derived CBV correlates with both optically measured hemoglobin and neuronal calcium activity in awake mice. We further derive hemodynamic response functions linking calcium activity to CBV during spontaneous and sensory-evoked activity. Application to a mouse glioblastoma model demonstrates utility for studying neurovascular dysfunction in complex neuropathologies.

Astrocytes, which are increasingly recognized as pivotal constituents of brain circuits governing a wide range of functions, express GABA transporter 3 (Gat3), an astrocyte-specific GABA transporter responsible for maintenance of extra-synaptic GABA levels. Here, we examined the functional role of Gat3 in astrocyte-mediated modulation of neuronal activity and information encoding. First, we developed a multiplexed CRISPR construct applicable for effective genetic ablation of Gat3 in the visual cortex of adult mice. Using in vivo two-photon calcium imaging of visual cortex neurons in Gat3 knockout mice, we observed changes in spontaneous and visually driven single neuronal response properties such as response magnitudes and trial-to-trial variability. Gat3 knockout exerted a pronounced influence on population-level neuronal activity, altering the response dynamics of neuronal populations and impairing their ability to accurately represent stimulus information. These findings demonstrate that Gat3 in astrocytes profoundly shapes the sensory information encoding capacity of neurons and networks within the visual cortex.

The brain's response to external events depends on its internal arousal states, which are dynamically governed by neuromodulatory systems and have recently been linked to coordinated spike timing cascades in widespread brain networks. At rest, both arousal fluctuations and spiking cascades are evident throughout the forebrain and play out over multisecond time scales. Here, by analyzing large-scale neural recording data collected by the Allen Institute, it is demonstrated that these intrinsic processes persist across the mouse brain even during periods of continuous visual stimulation. In the stationary animal, each quasi-periodic cascade cycle systematically influenced 1) the efficacy of encoding in visually responsive brain areas and 2) the incidence of memory-related hippocampal ripples. During this cycle, the phase of high arousal is marked by high efficiency in visual encoding whereas the phase of low arousal is marked by the occurrence of hippocampal ripples. However, during bouts of active locomotion, this cycle is abolished and brain maintained a state of elevated visual coding efficiency, with ripples being nearly absent. It is hypothesized that the infra-slow cascade dynamics reflect an adaptive cycle of alternating exteroceptive sensory sampling and internal mnemonic function that persistently pervades the forebrain, only stopping during active exploration of the environment.

Updating behavior based on feedback from the environment is a crucial means by which organisms learn and develop optimal behavioral strategies1-3. Norepinephrine (NE) release from the locus coeruleus (LC) has been shown to mediate learned behaviors4-6 such that in a task with graded stimulus uncertainty and performance, a high level of NE released after an unexpected outcome causes improvement in subsequent behavior7. Yet, how the transient activity of LC-NE neurons, lasting tens of milliseconds, influences behavior several seconds later, is unclear. Here, we show that NE acts directly on cortical astrocytes via Adra1a adrenergic receptors to elicit sustained increases in intracellular calcium. Chemogenetic blockade of astrocytic calcium elevation prevents the improvement in behavioral performance. NE-activated calcium invokes purinergic pathways in cortical astrocytes that signal to neurons; pathway-specific astrocyte gene expression is altered in mice trained on the task, and blocking neuronal adenosine-sensitive A1 receptors also prevents post-reinforcement behavioral gain. Finally, blocking either astrocyte calcium dynamics or A1 receptors alters encoding of the task in prefrontal cortex neurons, preventing the post-reinforcement change in discriminability of rewarded and unrewarded stimuli underlying behavioral improvement. Together, these data demonstrate that astrocytes, rather than indirectly reflecting neuronal drive, play a direct, instrumental role in representing task-relevant information and signaling to neurons to mediate a fundamental component of learning in the brain.

The neural encoding of sensory stimuli is subject to the brain's internal circuit dynamics. Recent work has demonstrated that the resting brain exhibits widespread, coordinated activity that plays out over multisecond timescales in the form of quasi-periodic spiking cascades. Here we demonstrate that these intrinsic dynamics persist during the presentation of visual stimuli and markedly influence the efficacy of feature encoding in the visual cortex. During periods of passive viewing, the sensory encoding of visual stimuli was determined by quasi-periodic cascade cycle evolving over several seconds. During this cycle, high efficiency encoding occurred during peak arousal states, alternating in time with hippocampal ripples, which were most frequent in low arousal states. However, during bouts of active locomotion, these arousal dynamics were abolished: the brain remained in a state in which visual coding efficiency remained high and ripples were absent. We hypothesize that the brain's observed dynamics during awake, passive viewing reflect an adaptive cycle of alternating exteroceptive sensory sampling and internal mnemonic function.

The prefrontal cortex (PFC) is a hub for cognitive behaviors and is a key target for neuroadaptations in alcohol use disorders. Recent advances in genetically encoded sensors and functional microscopy allow multimodal in vivo PFC activity recordings at subcellular and cellular scales. While these methods could enable a deeper understanding of the relationship between alcohol and PFC function/dysfunction, they typically require animals to be head-fixed. Here, we present a method in mice for binge-like ethanol consumption during head-fixation. Male and female mice were first acclimated to ethanol by providing home cage access to 20% ethanol (v/v) for 4 or 8 days. After home cage drinking, mice consumed ethanol from a lick spout during head-fixation. We used two-photon calcium imaging during the head-fixed drinking paradigm to record from a large population of PFC neurons (>1000) to explore how acute ethanol affects their activity. Drinking exerted temporally heterogeneous effects on PFC activity at single neuron and population levels. Intoxication modulated the tonic activity of some neurons while others showed phasic responses around ethanol receipt. Population level activity did not show tonic or phasic modulation but tracked ethanol consumption over the minute-timescale. Network level interactions assessed through between-neuron pairwise correlations were largely resilient to intoxication at the population level while neurons with increased tonic activity showed higher synchrony by the end of the drinking period. By establishing a method for binge-like drinking in head-fixed mice, we lay the groundwork for leveraging advanced microscopy technologies to study alcohol-induced neuroadaptations in PFC and other brain circuits. This article is part of the Special Issue on "PFC circuit function in psychiatric disease and relevant models".

The uptake of glutamate by astrocytes actively shapes synaptic transmission, however its role in the development and plasticity of neuronal circuits remains poorly understood. The astrocytic glutamate transporter, GLT1 is the predominant source of glutamate clearance in the adult mouse cortex. Here, we examined the structural and functional development of the visual cortex in GLT1 heterozygous (HET) mice using two-photon microscopy, immunohistochemistry and slice electrophysiology. We find that though eye-specific thalamic axonal segregation is intact, binocular refinement in the primary visual cortex is disrupted. Eye-specific responses to visual stimuli in GLT1 HET mice show altered binocular matching, with abnormally high responses to ipsilateral compared to contralateral eye stimulation and a greater mismatch between preferred orientation selectivity of ipsilateral and contralateral eye responses. Furthermore, we observe an increase in dendritic spine density in the basal dendrites of layer 2/3 excitatory neurons suggesting aberrant spine pruning. Monocular deprivation induces atypical ocular dominance plasticity in GLT1 HET mice, with an unusual depression of ipsilateral open eye responses; however, this change in ipsilateral responses correlates well with an upregulation of GLT1 protein following monocular deprivation. These results demonstrate that a key function of astrocytic GLT1 function during development is the experience-dependent refinement of ipsilateral eye inputs relative to contralateral eye inputs in visual cortex.

Alcohol use disorder (AUD) exacts a major personal, societal, and economic toll. Top-down control from the prefrontal cortex (PFC), a critical hub for decision making, executive, and other cognitive functions, is key for the regulation of alcohol consumption. Arousal exerts profound effects on cortical processing, allowing it to potentially modulate PFC functions relevant for alcohol consumption and AUD. Despite this, it is unclear whether and how arousal-mediated modulation of PFC circuits relates to voluntary alcohol drinking behaviors. Two-photon microscopy is ideally suited for dissecting the neural circuit mechanisms underlying the effect of alcohol on intact circuits in behaving animals. We addressed a major limitation of this technology by developing a novel behavioral paradigm for voluntary drinking in head-fixed mice. We recorded responses of layer 2/3 excitatory neurons in the anterior cingulate cortex (ACC) subdivision of the PFC as mice voluntarily consumed ethanol, along with video recording of the pupil to track momentary fluctuations in arousal. Ethanol consumption bidirectionally modified the activity of subsets of ACC neurons, both at slow (minutes) and fast (sub-second) time scales. Remarkably, we found that the coupling of arousal to ACC activity before drinking was associated with subsequent ethanol engagement behavior. In turn, ethanol consumption modulated neuronal-arousal coupling. Together, our results suggest neuronal-arousal coupling as a key biomarker for alcohol drinking and lays the groundwork for future studies to dissect the therapeutic potential of this process for AUD and other substance use disorders.

Sensorimotor behaviors require processing of behaviorally relevant sensory cues and the ability to select appropriate responses from a vast behavioral repertoire. Modulation by the prefrontal cortex (PFC) is thought to be key for both processes, but the precise role of specific circuits remains unclear. We examined the sensorimotor function of anatomically distinct outputs from a subdivision of the mouse PFC, the anterior cingulate cortex (ACC). Using a visually guided two-choice behavioral paradigm with multiple cue-response mappings, we dissociated the sensory and motor response components of sensorimotor control. Projection-specific two-photon calcium imaging and optogenetic manipulations show that ACC outputs to the superior colliculus, a key midbrain structure for response selection, principally coordinate specific motor responses. Importantly, ACC outputs exert control by reducing the innate response bias of the superior colliculus. In contrast, ACC outputs to the visual cortex facilitate sensory processing of visual cues. Our results ascribe motor and sensory roles to ACC projections to the superior colliculus and the visual cortex and demonstrate for the first time a circuit motif for PFC function wherein anatomically non-overlapping output pathways coordinate complementary but distinct aspects of visual sensorimotor behavior.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Synaptic plasticity is critical for neurodevelopment and proper function of the adult nervous system. Studies show that microglia play critical roles in neurodevelopment, but mechanisms driving these roles are poorly understood. I explored purinergic signaling as a potential mediator between microglia and neurons during synaptic plasticity. Purinergic signaling has been implicated in microglial behavior, but studies focused on inflammatory roles. Non-inflamed microglia highly and selectively express the purinergic receptor, P2Y12, which functions in microglial chemotaxis. I posited that purinergic signaling contributes to the microglial motility underlying synapse surveillance and may be critical for microglial roles in synaptic refinement. My evidence suggests that P2Y12 disruption prevents ocular dominance shifts indicative of synaptic plasticity. P2Y12 disruption also decreases microglial process complexity, without affecting basal microglial process dynamics. In addition, microglial process dynamics appear to be regulated by arousal with increased surveillance during slow-wave sleep-like states. I find that noradrenergic signaling, contributing to arousal, is sufficient to suppress microglial process dynamics and inhibit microglial P2Y12-mediated roles in synaptic plasticity via microglial β2 adrenergic receptors. These data suggest microglia are active participants in cortical network remodeling in adolescent synaptic plasticity primarily during sleep states. These results not only describe novel neuro-immune interactions in the non-pathological brain, but provoke broader considerations of the importance of sleep in microglial roles during neurodevelopment and in neuropathology.

BACKGROUND:Neuronal plasticity deficits are thought to underlie abnormal neurodevelopment in fetal alcohol spectrum disorders and in animal models of this condition. Previously, we found that alcohol exposure during a period that is similar to the last months of gestation in humans disrupts ocular dominance plasticity (ODP), as measured in superficial cortical layers. We hypothesize that exposure to alcohol can differentially affect the potentiation and depression of responses that are necessary for activity-dependent sprouting and pruning of neuronal networks. ODP is an established paradigm that allows the assessment of activity-dependent depression and potentiation of responses in vivo.METHODS:Mouse pups were exposed to 3.6 to 5 g/kg of ethanol in saline daily or every other day between postnatal days 4 and 9. Visual cortex plasticity was then assessed during the critical period for ODP using 2 techniques that separately record in layers 4 (visually evoked potentials [VEPs]) and 2/3 (optical imaging of intrinsic signals [OI]).RESULTS:We discovered a layer-specific effect of early alcohol exposure. Recording of VEPs from layer 4 showed that while the potentiation component of ODP was disrupted in animals treated with alcohol when compared with saline controls, the depression component of ODP (Dc-ODP) was unaltered. In contrast, OI from layers 2/3 showed that Dc-ODP was markedly disrupted in alcohol-treated animals when compared with controls.CONCLUSIONS:Combined with our previous work, these findings strongly suggest that developmental alcohol exposure has a distinct and layer-specific effect on the potentiation and depression of cortical responses after monocular deprivation.

Background: Despite advances in the treatment of primary breast tumors, the outcome of metastatic breast cancer remains dismal. Brain metastases present a particularly difficult therapeutic target due to the "sanctuary" status of the brain, with resulting inability of most chemotherapeutic agents to effectively eliminate cancer cells in the brain parenchyma. A large number of breast cancer patients receive various neuroactive drugs to combat complications of systemic anti-tumor therapies and to treat concomitant diseases. One of the most prescribed groups of neuroactive medications is anti-depressants, in particular selective serotonin reuptake inhibitors (SSRIs). Since SSRIs have profound effects on the brain, it is possible that their use in breast cancer patients could affect the development of brain metastases. This would provide important insight into the mechanisms underlying brain metastasis. Surprisingly, this possibility has been poorly explored.Methods: We studied the effect of fluoxetine, an SSRI, on the development of brain metastatic breast cancer using MDA-MB-231BR cells in a mouse model.Results: The data demonstrate that fluoxetine treatment increases the number of brain metastases, an effect accompanied by elevated permeability of the blood-brain barrier, pro-inflammatory changes in the brain, and glial activation. This suggests a possible role of brain-resident immune cells and glia in promoting increased development of brain metastases.Conclusion: Our results offer experimental evidence that neuroactive substances may influence the pathogenesis of brain metastatic disease. This provides a starting point for further investigations into possible mechanisms of interaction between various neuroactive drugs, tumor cells, and the brain microenvironment, which may lead to the discovery of compounds that inhibit metastasis to the brain.

Our goal in this study was to examine the red-eared slider turtle for a photomechanical response (PMR) and define its spectral sensitivity. Pupils of enucleated eyes constricted to light by ∼11%, which was one-third the response measured in alert behaving turtles at ∼33%. Rates of constriction in enucleated eyes that were measured by time constants (1.44-3.70 min) were similar to those measured in turtles at 1.97 min. Dilation recovery rates during dark adaptation for enucleated eyes were predicted using line equations and computed times for reaching maximum sizes between 26 and 44 min. Times were comparable to the measures in turtles where maximum pupil size occurred within 40 min and possessed a time constant of 12.78 min. Hill equations were used to derive irradiance threshold values from enucleated hemisected eyes and then plot a spectral sensitivity curve. The analysis of the slopes and maximum responses revealed contribution from at least two different photopigments, one with a peak at 410 nm and another with a peak at 480 nm. Fits by template equations suggest that contractions are triggered by multiple photopigments in the iris including an opsin-based visual pigment and some other novel photopigment, or a cryptochrome with an absorbance spectrum significantly different from that used in our model. In addition to being regulated by retinal feedback via parasympathetic nervous pathways, the results support that the iris musculature is photointrinsically responsive. In the turtle, the control of its direct pupillary light response (dPLR) includes photoreceptive mechanisms occurring both in its iris and in its retina.

When we move our hands, both visual and proprioceptive input provides information about the motion. We show that subjects integrate these two modalities in a Bayesian optimal way in a two-part study. In the first part, we measured the reliabilities of subjects' estimates of movement direction using only proprioceptive or only visual motion information. In the proprioceptive condition, a robot arm moved a manipulandum held by subjects 15 cm back and forth along linear trajectories sampled uniformly from −25 to −35 and 55 to 65 degrees relative to the midsagittal plane. Subjects judged whether the motion direction presented in a second interval was clockwise to that of the first interval. In the visual condition, subjects made similar judgments of spatial-temporally correlated noise patterns moving with the same velocity profiles as the robot in the proprioceptive condition. We modulated the reliability of the visual information by using two different signal-to-noise ratios in the visual stimuli. In the second part, the robot moved subjects' hands behind a mirror while they viewed similar visual patterns (spatially co-aligned with the manipulandum held by subjects). The visual motion either equaled that of the robot or deviated in direction by ±10 degrees. Subjects adjusted a dial to indicate their perceived motion direction. Subjects finished two sessions of counter-balanced proprioceptive and visual discrimination trials and two sessions of the visual-prioprioceptive adjustment task. The reliability of each modality was computed from fitted psychometric functions from proprioceptive and vision-only discrimination sessions. Relative cue weights were estimated by regressing subjects' direction judgments in the last two sessions against the directions suggested by each cue. Subjects gave slightly more weight to vision in the high visual SNR condition but relied less on the visual cue in the low SNR condition as predicted by the threshold data.