A paradigm for ethanol consumption in head-fixed mice during prefrontal cortical two-photon calcium imaging.

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".

Intrinsic forebrain arousal dynamics governs sensory stimulus encoding.

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.

A paradigm for ethanol consumption in head-fixed mice during prefrontal cortical two-photon calcium imaging.

The prefrontal cortex (PFC) is a hub for higher-level cognitive behaviors and is a key target for neuroadaptations in alcohol use disorders. Preclinical models of ethanol consumption are instrumental for understanding how acute and repeated drinking affects PFC structure and function. Recent advances in genetically encoded sensors of neuronal activity and neuromodulator release combined with functional microscopy (multiphoton and one-photon widefield imaging) allow multimodal in-vivo PFC recordings at subcellular and cellular scales. While these methods could enable a deeper understanding of the relationship between alcohol and PFC function/dysfunction, they 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 modulated activity rates in a subset of neurons on slow (minutes) and fast (seconds) time scales but the majority of neurons were unaffected. Moreover, ethanol intake did not significantly affect network level interactions in the PFC as assessed through inter-neuronal pairwise correlations. 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.

Astrocyte glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex.

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.

Distinct prefrontal top-down circuits differentially modulate sensorimotor behavior.

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.

Author Correction: Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex.

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

Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex.

Microglia are the brain's resident innate immune cells and also have a role in synaptic plasticity. Microglial processes continuously survey the brain parenchyma, interact with synaptic elements and maintain tissue homeostasis. However, the mechanisms that control surveillance and its role in synaptic plasticity are poorly understood. Microglial dynamics in vivo have been primarily studied in anesthetized animals. Here we report that microglial surveillance and injury response are reduced in awake mice as compared to anesthetized mice, suggesting that arousal state modulates microglial function. Pharmacologic stimulation of ß2-adrenergic receptors recapitulated these observations and disrupted experience-dependent plasticity, and these effects required the presence of ß2-adrenergic receptors in microglia. These results indicate that microglial roles in surveillance and synaptic plasticity in the mouse brain are modulated by noradrenergic tone fluctuations between arousal states and emphasize the need to understand the effect of disruptions of adrenergic signaling in neurodevelopment and neuropathology.

Effects of Developmental Alcohol Exposure on Potentiation and Depression of Visual Cortex Responses.

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.

Fluoxetine modulates breast cancer metastasis to the brain in a murine model.

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.

Spectral sensitivity of the photointrinsic iris in the red-eared slider turtle (Trachemys scripta elegans).

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.

Consensual pupillary light response in the red-eared slider turtle (Trachemys scripta elegans).

Purpose of this study was to determine if the turtle has a consensual pupillary light response (cPLR), and if so, to compare it to its direct pupillary light response (dPLR). One eye was illuminated with different intensities of light over a four log range while keeping the other eye in darkness. In the eye directly illuminated, pupil diameter was reduced by as much as approximately 31%. In the eye not stimulated by light, pupil diameter was also reduced but less to approximately 11%. When compared to the directly illuminated eye, this generated a ratio, cPLR-dPLR, equal to 0.35. Ratio of slopes for log/linear fits to plots of pupil changes versus retinal irradiance for non-illuminated (-1.27) to illuminated (-3.94) eyes closely matched at 0.32. cPLR had time constants ranging from 0.60 to 1.20min; however, they were comparable and not statistically different from those of the dPLR, which ranged from 1.41 to 2.00min. Application of mydriatic drugs to the directly illuminated eye also supported presence of a cPLR. Drugs reduced pupil constriction by approximately 9% for the dPLR and slowed its time constant to 9.58min while simultaneous enhancing constriction by approximately 6% for the cPLR. Time constant for the cPLR at 1.75min, however, was not changed. Results support that turtle possesses a cPLR although less strong than its dPLR.