Microglia complement signaling promotes neuronal elimination and normal brain functional connectivity.

Complement signaling is thought to serve as an opsonization signal to promote the phagocytosis of synapses by microglia. However, while its role in synaptic remodeling has been demonstrated in the retino-thalamic system, it remains unclear whether complement signaling mediates synaptic pruning in the brain more generally. Here we found that mice lacking the Complement receptor 3, the major microglia complement receptor, failed to show a deficit in either synaptic pruning or axon elimination in the developing mouse cortex. Instead, mice lacking Complement receptor 3 exhibited a deficit in the perinatal elimination of neurons in the cortex, a deficit that is associated with increased cortical thickness and enhanced functional connectivity in these regions in adulthood. These data demonstrate a role for complement in promoting neuronal elimination in the developing cortex.

Phospholipid scramblase Xkr8 is required for developmental axon pruning via phosphatidylserine exposure.

The mature mammalian brain connectome emerges during development via the extension and pruning of neuronal connections. Glial cells have been identified as key players in the phagocytic elimination of neuronal synapses and projections. Recently, phosphatidylserine has been identified as neuronal "eat-me" signal that guides elimination of unnecessary input sources, but the associated transduction systems involved in such pruning are yet to be described. Here, we identified Xk-related protein 8 (Xkr8), a phospholipid scramblase, as a key factor for the pruning of axons in the developing mammalian brain. We found that mouse Xkr8 is highly expressed immediately after birth and required for phosphatidylserine exposure in the hippocampus. Mice lacking Xkr8 showed excess excitatory nerve terminals, increased density of cortico-cortical and cortico-spinal projections, aberrant electrophysiological profiles of hippocampal neurons, and global brain hyperconnectivity. These data identify phospholipid scrambling by Xkr8 as a central process in the labeling and discrimination of developing neuronal projections for pruning in the mammalian brain.

Induction of flight via midbrain projections to the cuneiform nucleus.

The dorsal periaqueductal gray is a midbrain structure implicated in the control of defensive behaviors and the processing of painful stimuli. Electrical stimulation or optogenetic activation of excitatory neurons in dorsal periaqueductal gray results in freezing or flight behavior at low and high intensity, respectively. However, the output structures that mediate these defensive behaviors remain unconfirmed. Here we carried out a targeted classification of neuron types in dorsal periaqueductal gray using multiplex in situ sequencing and then applied cell-type and projection-specific optogenetic stimulation to identify projections from dorsal periaqueductal grey to the cuneiform nucleus that promoted goal-directed flight behavior. These data confirmed that descending outputs from dorsal periaqueductal gray serve as a trigger for directed escape behavior.

Sensory-thresholded switch of neural firing states in a computational model of the ventromedial hypothalamus.

The mouse ventromedial hypothalamus (VMH) is both necessary and sufficient for defensive responses to predator and social threats. Defensive behaviors typically involve cautious approach toward potentially threatening stimuli aimed at obtaining information about the risk involved, followed by sudden avoidance and flight behavior to escape harm. In vivo neural recording studies in mice have identified two major populations of VMH neurons that either increase their firing activity as the animal approaches the threat (called Assessment+ cells) or increase their activity as the animal flees the threat (called Flight+ cells). Interestingly, Assessment+ and Flight+ cells abruptly decrease and increase their firing activity, respectively, at the decision point for flight, creating an escape-related "switch" in functional state. This suggests that the activity of the two cell types in VMH is coordinated and could result from local circuit interactions. Here, we used computational modeling to test if a local inhibitory feedback circuit could give rise to key features of the neural activity seen in VMH during the approach-to-flight transition. Starting from a simple dual-population inhibitory feedback circuit receiving repeated trains of monotonically increasing sensory input to mimic approach to threat, we tested the requirement for balanced sensory input, balanced feedback, short-term synaptic plasticity, rebound excitation, and inhibitory feedback exclusivity to reproduce an abrupt, sensory-thresholded reciprocal firing change that resembles Assessment+ and Flight+ cell activity seen in vivo. Our work demonstrates that a relatively simple local circuit architecture is sufficient for the emergence of firing patterns similar to those seen in vivo and suggests that a reiterative process of experimental and computational work may be a fruitful avenue for better understanding the functional organization of mammalian instinctive behaviors at the circuit level.

Identifying long-range synaptic inputs using genetically encoded labels and volume electron microscopy.

Enzymes that facilitate the local deposition of electron dense reaction products have been widely used as labels in electron microscopy (EM) for the identification of synaptic contacts in neural tissue. Peroxidases, in particular, can efficiently metabolize 3,3'-diaminobenzidine tetrahydrochloride hydrate (DAB) to produce precipitates with high contrast under EM following heavy metal staining, and can be genetically encoded to facilitate the labeling of specific cell-types or organelles. Nevertheless, the peroxidase/DAB method has so far not been reported to work in a multiplexed manner in combination with 3D volume EM techniques (e.g. Serial blockface electron microscopy, SBEM; Focused ion beam electron microscopy, FIBSEM) that are favored for the large-scale ultrastructural assessment of synaptic architecture However, a recently described peroxidase with enhanced enzymatic activity (dAPEX2) can efficienty deposit EM-visible DAB products in thick tissue without detergent treatment opening the possibility for the multiplex labeling of genetically defined cell-types in combination with volume EM methods. Here we demonstrate that multiplexed dAPEX2/DAB tagging is compatible with both FIBSEM and SBEM volume EM approaches and use them to map long-range genetically identified synaptic inputs from the anterior cingulate cortex to the periaqueductal gray in the mouse brain.

Microglia control glutamatergic synapses in the adult mouse hippocampus.

Microglia cells are active players in regulating synaptic development and plasticity in the brain. However, how they influence the normal functioning of synapses is largely unknown. In this study, we characterized the effects of pharmacological microglia depletion, achieved by administration of PLX5622, on hippocampal CA3-CA1 synapses of adult wild type mice. Following microglial depletion, we observed a reduction of spontaneous and evoked glutamatergic activity associated with a decrease of dendritic spine density. We also observed the appearance of immature synaptic features and higher levels of plasticity. Microglia depleted mice showed a deficit in the acquisition of the Novel Object Recognition task. These events were accompanied by hippocampal astrogliosis, although in the absence ofneuroinflammatory condition. PLX-induced synaptic changes were absent in Cx3cr1-/- mice, highlighting the role of CX3CL1/CX3CR1 axis in microglia control of synaptic functioning. Remarkably, microglia repopulation after PLX5622 withdrawal was associated with the recovery of hippocampal synapses and learning functions. Altogether, these data demonstrate that microglia contribute to normal synaptic functioning in the adult brain and that their removal induces reversible changes in organization and activity of glutamatergic synapses.

High-resolution structural and functional deep brain imaging using adaptive optics three-photon microscopy.

Multiphoton microscopy has become a powerful tool with which to visualize the morphology and function of neural cells and circuits in the intact mammalian brain. However, tissue scattering, optical aberrations and motion artifacts degrade the imaging performance at depth. Here we describe a minimally invasive intravital imaging methodology based on three-photon excitation, indirect adaptive optics (AO) and active electrocardiogram gating to advance deep-tissue imaging. Our modal-based, sensorless AO approach is robust to low signal-to-noise ratios as commonly encountered in deep scattering tissues such as the mouse brain, and permits AO correction over large axial fields of view. We demonstrate near-diffraction-limited imaging of deep cortical spines and (sub)cortical dendrites up to a depth of 1.4 mm (the edge of the mouse CA1 hippocampus). In addition, we show applications to deep-layer calcium imaging of astrocytes, including fibrous astrocytes that reside in the highly scattering corpus callosum.

A role for cerebral cortex in the suppression of innate defensive behaviour.

The cerebral cortex is widely accepted to be involved in the control of cognition and the processing of learned information. However, data suggest that it may also have a role in the regulation of innate responses because rodents, cats or primates with surgical removal of cortical regions show excessive aggression and rage elicited by threatening stimuli. Nevertheless, the imprecision and chronic nature of these lesions leave open the possibility that compensatory processes may underlie some of these phenotypes. In the present study we applied a precise, rapid and reversible inhibition approach to examine the contribution of the cerebral cortex to defensive behaviours elicited by a variety of innately aversive stimuli in laboratory mice. Pharmacological treatment of mice carrying the pharmacogenetic inhibitory receptor hM4D selectively in neocortex, archicortex and related dorsal telencephalon-derived structures resulted in the rapid inhibition of cerebral cortex neural activity. Cortical inhibition was associated with a selective increase in defensive behaviours elicited by an aggressive conspecific, a novel prey and a physically stressful stimulus. These findings are consistent with a role for cortex in the acute inhibition of innate defensive behaviours.

Sexually dimorphic perineuronal nets in the rodent and primate reproductive circuit.

Perineuronal nets are extracellular glycoprotein structures that have been found on some neurons in the central nervous system and that have been shown to regulate their structural plasticity. Until now work on perineuronal nets has been focused on their role in cortical structures where they are selectively expressed on parvalbumin-positive neurons and are reported to restrict the experience-dependent plasticity of inhibitory afferents. Here, we examined the expression of perineuronal nets subcortically, showing that they are expressed in several discrete structures, including nuclei that comprise the brain network controlling reproductive behaviors (e.g., mounting, lordosis, aggression, and social defense). In particular, perineuronal nets were found in the posterior dorsal division of the medial amygdala, the medial preoptic nucleus, the posterior medial bed nucleus of the stria terminalis, the ventrolateral ventromedial hypothalamus and adjacent tuberal nucleus, and the ventral premammillary nucleus in both the mouse and primate brain. Comparison of perineuronal nets in male and female mice revealed a significant sexually dimorphic expression, with expression found prominently on estrogen receptor expressing neurons in the medial amygdala. These findings suggest that perineuronal nets may be involved in regulating neural plasticity in the mammalian reproductive system.

A neural circuit for competing approach and defense underlying prey capture.

Predators must frequently balance competing approach and defensive behaviors elicited by a moving and potentially dangerous prey. Several brain circuits supporting predation have recently been localized. However, the mechanisms by which these circuits balance the conflict between approach and defense responses remain unknown. Laboratory mice initially show alternating approach and defense responses toward cockroaches, a natural prey, but with repeated exposure become avid hunters. Here, we used in vivo neural activity recording and cell-type specific manipulations in hunting male mice to identify neurons in the lateral hypothalamus and periaqueductal gray that encode and control predatory approach and defense behaviors. We found a subset of GABAergic neurons in lateral hypothalamus that specifically encoded hunting behaviors and whose stimulation triggered predation but not feeding. This population projects to the periaqueductal gray, and stimulation of these projections promoted predation. Neurons in periaqueductal gray encoded both approach and defensive behaviors but only initially when the mouse showed high levels of fear of the prey. Our findings allow us to propose that GABAergic neurons in lateral hypothalamus facilitate predation in part by suppressing defensive responses to prey encoded in the periaqueductal gray. Our results reveal a neural circuit mechanism for controlling the balance between conflicting approach and defensive behaviors elicited by the same stimulus.

Characterization of exploratory patterns and hippocampal-prefrontal network oscillations during the emergence of free exploration.

During free exploration, the emergence of patterned and sequential behavioral responses to an unknown environment reflects exploration traits and adaptation. However, the behavioral dynamics and neural substrates underlying the exploratory behavior remain poorly understood. We developed computational tools to quantify the exploratory behavior and performed in vivo electrophysiological recordings in a large arena in which mice made sequential excursions into unknown territory. Occupancy entropy was calculated to characterize the cumulative and moment-to-moment behavioral dynamics in explored and unexplored territories. Local field potential analysis revealed that the theta activity in the dorsal hippocampus (dHPC) was highly correlated with the occupancy entropy. Individual dHPC and prefrontal cortex (PFC) oscillatory activities could classify various aspects of free exploration. Initiation of exploration was accompanied by a coordinated decrease and increase in theta activity in PFC and dHPC, respectively. Our results indicate that dHPC and PFC work synergistically in shaping free exploration by modulating exploratory traits during emergence and visits to an unknown environment.

Mapping the neural circuitry of predator fear in the nonhuman primate.

In rodents, innate and learned fear of predators depends on the medial hypothalamic defensive system, a conserved brain network that lies downstream of the amygdala and promotes avoidance via projections to the periaqueductal gray. Whether this network is involved in primate fear remains unknown. To address this, we provoked flight responses to a predator (moving snake) in the marmoset monkey under laboratory conditions. We combined c-Fos immunolabeling and anterograde/retrograde tracing to map the functional connectivity of the ventromedial hypothalamus, a core node in the medial hypothalamic defensive system. Our findings demonstrate that the ventromedial hypothalamus is recruited by predator exposure in primates and that anatomical connectivity of the rodent and primate medial hypothalamic defensive system are highly conserved.

Differential Encoding of Predator Fear in the Ventromedial Hypothalamus and Periaqueductal Grey.

The ventromedial hypothalamus is a central node of the mammalian predator defense network. Stimulation of this structure in rodents and primates elicits abrupt defensive responses, including flight, freezing, sympathetic activation, and panic, while inhibition reduces defensive responses to predators. The major efferent target of the ventromedial hypothalamus is the dorsal periaqueductal gray (dPAG), and stimulation of this structure also elicits flight, freezing, and sympathetic activation. However, reversible inhibition experiments suggest that the ventromedial hypothalamus and periaqueductal gray play distinct roles in the control of defensive behavior, with the former proposed to encode an internal state necessary for the motivation of defensive responses, while the latter serves as a motor pattern initiator. Here, we used electrophysiological recordings of single units in behaving male mice exposed to a rat to investigate the encoding of predator fear in the dorsomedial division of the ventromedial hypothalamus (VMHdm) and the dPAG. Distinct correlates of threat intensity and motor responses were found in both structures, suggesting a distributed encoding of sensory and motor features in the medial hypothalamic-brainstem instinctive network.SIGNIFICANCE STATEMENT Although behavioral responses to predatory threat are essential for survival, the underlying neuronal circuits remain undefined. Using single unit in vivo electrophysiological recordings in mice, we have identified neuronal populations in the medial hypothalamus and brainstem that encode defensive responses to a rat predator. We found that both structures encode both sensory as well as motor aspects of the behavior although with different kinetics. Our findings provide a framework for understanding how innate sensory cues are processed to elicit adaptive behavioral responses to threat and will help to identify targets for the pharmacological modulation of related pathologic behaviors.

Dynamic encoding of social threat and spatial context in the hypothalamus.

Social aggression and avoidance are defensive behaviors expressed by territorial animals in a manner appropriate to spatial context and experience. The ventromedial hypothalamus controls both social aggression and avoidance, suggesting that it may encode a general internal state of threat modulated by space and experience. Here, we show that neurons in the mouse ventromedial hypothalamus are activated both by the presence of a social threat as well as by a chamber where social defeat previously occurred. Moreover, under conditions where the animal could move freely between a home and defeat chamber, firing activity emerged that predicted the animal's position, demonstrating the dynamic encoding of spatial context in the hypothalamus. Finally, we found that social defeat induced a functional reorganization of neural activity as optogenetic activation could elicit avoidance after, but not before social defeat. These findings reveal how the hypothalamus dynamically encodes spatial and sensory cues to drive social behaviors.

CLARITY analysis of the Cl/pH sensor expression in the brain of transgenic mice.

Genetically encoded biosensors are widely used in cell biology for the non-invasive imaging of concentrations of ions or the activity of enzymes, to evaluate the distribution of small molecules, proteins and organelles, and to image protein interactions in living cells. These fluorescent molecules can be used either by transient expression in cultured cells or in entire organisms or through stable expression by producing transgenic animals characterized by genetically encoded and heritable biosensors. Using the mouse Thy1 mini-promoter, we generated a line of transgenic mice expressing a genetically encoded sensor for the simultaneous measurements of intracellular Cl- and pH. This construct, called ClopHensor, consists of a H+- and Cl--sensitive variant of the enhanced green fluorescent protein (E2GFP) fused with a red fluorescent protein (DsRedm). Stimulation of hippocampal Schaffer collaterals proved that the sensor is functionally active. To reveal the expression pattern of ClopHensor across the brain of Thy1::ClopHensor mice, we obtained transparent brain samples using the CLARITY method and imaged them with confocal and light-sheet microscopy. We then developed a semi-quantitative approach to identify brain structures with high intrinsic sensor fluorescence. This approach allowed us to assess cell morphology and track axonal projection, as well as to confirm E2GFP and DsRedm fluorescence colocalization. This analysis also provides a map of the brain areas suitable for non-invasive monitoring of intracellular Cl-/pH in normal and pathological conditions. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction.

Microglia are highly motile glial cells that are proposed to mediate synaptic pruning during neuronal circuit formation. Disruption of signaling between microglia and neurons leads to an excess of immature synaptic connections, thought to be the result of impaired phagocytosis of synapses by microglia. However, until now the direct phagocytosis of synapses by microglia has not been reported and fundamental questions remain about the precise synaptic structures and phagocytic mechanisms involved. Here we used light sheet fluorescence microscopy to follow microglia-synapse interactions in developing organotypic hippocampal cultures, complemented by a 3D ultrastructural characterization using correlative light and electron microscopy (CLEM). Our findings define a set of dynamic microglia-synapse interactions, including the selective partial phagocytosis, or trogocytosis (trogo-: nibble), of presynaptic structures and the induction of postsynaptic spine head filopodia by microglia. These findings allow us to propose a mechanism for the facilitatory role of microglia in synaptic circuit remodeling and maturation.

Dorsal Raphe Serotonin Neurons Mediate CO2-Induced Arousal from Sleep.

Arousal from sleep in response to CO2 is a critical protective phenomenon. Dysregulation of CO2-induced arousal contributes to morbidity and mortality from prevalent diseases, such as obstructive sleep apnea and sudden infant death syndrome. Despite the critical nature of this protective reflex, the precise mechanism for CO2-induced arousal is unknown. Because CO2 is a major regulator of breathing, prevailing theories suggest that activation of respiratory chemo- and mechano-sensors is required for CO2-induced arousal. However, populations of neurons that are not involved in the regulation of breathing are also chemosensitive. Among these are serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) that comprise a component of the ascending arousal system. We hypothesized that direct stimulation of these neurons with CO2 could cause arousal from sleep independently of enhancing breathing. Dialysis of CO2-rich acidified solution into DRN, but not medullary raphe responsible for modulating breathing, caused arousal from sleep. Arousal was lost in mice with a genetic absence of 5-HT neurons, and with acute pharmacological or optogenetic inactivation of DRN 5-HT neurons. Here we demonstrate that CO2 can cause arousal from sleep directly, without requiring enhancement of breathing, and that chemosensitive 5-HT neurons in the DRN critically mediate this arousal. Better understanding mechanisms underlying this protective reflex may lead to interventions to reduce disease-associated morbidity and mortality.SIGNIFICANCE STATEMENT Although CO2-induced arousal is critical to a number of diseases, the specific mechanism is not well understood. We previously demonstrated that serotonin (5-HT) neurons are important for CO2-induced arousal, as mice without 5-HT neurons do not arouse to CO2 Many have interpreted this to mean that medullary 5-HT neurons that regulate breathing are important in this arousal mechanism. Here we found that direct application of CO2-rich aCSF to the dorsal raphe nucleus, but not the medullary raphe, causes arousal from sleep, and that this arousal was lost with genetic ablation or acute inhibition of 5-HT neurons. We propose that 5-HT neurons in the dorsal raphe nucleus can be activated directly by CO2 to cause arousal independently of respiratory activation.

Errant gardeners: glial-cell-dependent synaptic pruning and neurodevelopmental disorders.

The final stage of brain development is associated with the generation and maturation of neuronal synapses. However, the same period is also associated with a peak in synapse elimination - a process known as synaptic pruning - that has been proposed to be crucial for the maturation of remaining synaptic connections. Recent studies have pointed to a key role for glial cells in synaptic pruning in various parts of the nervous system and have identified a set of critical signalling pathways between glia and neurons. At the same time, brain imaging and post-mortem anatomical studies suggest that insufficient or excessive synaptic pruning may underlie several neurodevelopmental disorders, including autism, schizophrenia and epilepsy. Here, we review current data on the cellular, physiological and molecular mechanisms of glial-cell-dependent synaptic pruning and outline their potential contribution to neurodevelopmental disorders.

Prefrontal cortical control of a brainstem social behavior circuit.

The prefrontal cortex helps adjust an organism's behavior to its environment. In particular, numerous studies have implicated the prefrontal cortex in the control of social behavior, but the neural circuits that mediate these effects remain unknown. Here we investigated behavioral adaptation to social defeat in mice and uncovered a critical contribution of neural projections from the medial prefrontal cortex to the dorsal periaqueductal gray, a brainstem area vital for defensive responses. Social defeat caused a weakening of functional connectivity between these two areas, and selective inhibition of these projections mimicked the behavioral effects of social defeat. These findings define a specific neural projection by which the prefrontal cortex can control and adapt social behavior.