Reiterated male-to-female violence disrupts hippocampal estrogen receptor ß expression, prompting anxiety-like behavior.

Intimate partner violence (IPV) is a significant public health concern whose neurological/behavioral sequelae remain to be mechanistically explained. Using a mouse model recapitulating an IPV scenario, we evaluated the female brain neuroendocrine alterations produced by a reiterated male-to-female violent interaction (RMFVI). RMFVI prompted anxiety-like behavior in female mice whose hippocampus displayed a marked neuronal loss and hampered neurogenesis, namely reduced BrdU-DCX-positive nuclei and diminished dendritic arborization in the dentate gyrus (DG): effects paralleled by a substantial downregulation of the estrogen receptor ß (ERß). After RMFVI, the DG harbored reduced brain-derived neurotrophic factor (BDNF) pools and tyrosine kinase receptor B (TrkB) phosphorylation. Accordingly, ERß knockout (KO) mice had heightened anxiety and curtailed BDNF levels at baseline while dying prematurely during the RMFVI procedure. Strikingly, injecting an ERß antagonist or agonist into the wild-type (WT) female hippocampus enhanced or reduced anxiety, respectively. Thus, reiterated male-to-female violence jeopardizes hippocampal homeostasis, perturbing the ERß/BDNF axis and ultimately instigating anxiety and chronic stress.

Sunflower seed-derived bioactive peptides show antioxidant and anti-inflammatory activity: From in silico simulation to the animal model.

The evolving field of food technology is increasingly dedicated to developing functional foods. This study explored bioactive peptides from sunflower protein isolate (SPI), obtained from defatted flour, a by-product of the oil processing industry. SPI underwent simulated gastrointestinal digestion and the obtained peptide-enriched fraction (PEF) showed antioxidant properties in vivo, in zebrafish. Among the peptides present in PEF identified by mass spectrometry analysis, we selected those with antioxidant properties by in silico evaluation, considering their capability to interact with Keap1, key protein in the regulation of antioxidant response. The selected peptides were synthesized and evaluated in a cellular model. As a result, DVAMPVPK, VETGVIKPG, TTHTNPPPEAE, LTHPQHQQQGPSTG and PADVTPEEKPEV activated Keap1/Nrf2 pathway leading to Antioxidant Response Element-regulated enzymes upregulation. Since the crosstalk between Nrf2 and NF-κB is well known, the potential anti-inflammatory activity of the peptides was assessed and principally PADVTPEEKPEV showed good features both as antioxidant and anti-inflammatory molecule.

Male violence disrupts estrogen receptor ß signaling in the female hippocampus.

Women are the main target of intimate partner violence (IPV), which is escalating worldwide. Mechanisms subtending IPV-related disorders, such as anxiety, depression and PTSD, remain unclear. We employed a mouse model molded on an IPV scenario (male vs. female prolonged violent interaction) to unearth the neuroendocrine alterations triggered by an aggressive male mouse on the female murine brain. Experimental IPV (EIPV) prompted marked anxiety-like behavior in young female mice, coincident with high circulating/cerebral corticosterone levels. The hippocampus of EIPV-inflicted female animals displayed neuronal loss, reduced BrdU-DCX-positive nuclei, decreased mature DCX-positive cells, and diminished dendritic arborization level in the dentate gyrus (DG), features denoting impaired neurogenesis and neuronal differentiation. These hallmarks were associated with marked down-regulation of estrogen receptor ß (ERß) density in the hippocampus, especially in the DG and dependent prosurvival ERK signaling. Conversely, ERα expression was unchanged. After EIPV, the DG harbored lowered local BDNF pools, diminished TrkB phosphorylation, and elevated glucocorticoid receptor phosphorylation. In unison, ERß KO mice had heightened anxiety-like behavior and curtailed BDNF levels at baseline, despite enhanced circulating estradiol levels, while dying prematurely during EIPV. Thus, reiterated male-to-female violence jeopardizes hippocampal homeostasis in the female brain, perturbing ERß/BDNF signaling, thus instigating anxiety and chronic stress.

Cranial Window for Acute and Chronic Optical Access to Record Neuronal Network Dynamics in the Olfactory Bulb.

Cranial window implant is the preparation of choice for acute and chronic optical access to a given brain area. The cranial window provides a stable preparation, which can last for months. This window allows to follow the activity of distinct population of neurons expressing genetically encoded fluorescent reporters of activity in awake, behaving, head-fixed animals. The optical access can also be exploited for acute imaging, in anesthetized animals. Here we provide a detailed protocol for acute and chronic cranial window implantations in the olfactory bulb. We also provide the procedure to perform injections of adeno-associated viruses expressing genetically encoded fluorescent sensors in the same area.

Control of circadian rhythm on cortical excitability and synaptic plasticity.

Living organisms navigate through a cyclic world: activity, feeding, social interactions are all organized along the periodic succession of night and day. At the cellular level, periodic activity is controlled by the molecular machinery driving the circadian regulation of cellular homeostasis. This mechanism adapts cell function to the external environment and its crucial importance is underlined by its robustness and redundancy. The cell autonomous clock regulates cell function by the circadian modulation of mTOR, a master controller of protein synthesis. Importantly, mTOR integrates the circadian modulation with synaptic activity and extracellular signals through a complex signaling network that includes the RAS-ERK pathway. The relationship between mTOR and the circadian clock is bidirectional, since mTOR can feedback on the cellular clock to shift the cycle to maintain the alignment with the environmental conditions. The mTOR and ERK pathways are crucial determinants of synaptic plasticity and function and thus it is not surprising that alterations of the circadian clock cause defective responses to environmental challenges, as witnessed by the bi-directional relationship between brain disorders and impaired circadian regulation. In physiological conditions, the feedback between the intrinsic clock and the mTOR pathway suggests that also synaptic plasticity should undergo circadian regulation.

Optogenetic Methods to Investigate Brain Alterations in Preclinical Models.

Investigating the neuronal dynamics supporting brain functions and understanding how the alterations in these mechanisms result in pathological conditions represents a fundamental challenge. Preclinical research on model organisms allows for a multiscale and multiparametric analysis in vivo of the neuronal mechanisms and holds the potential for better linking the symptoms of a neurological disorder to the underlying cellular and circuit alterations, eventually leading to the identification of therapeutic/rescue strategies. In recent years, brain research in model organisms has taken advantage, along with other techniques, of the development and continuous refinement of methods that use light and optical approaches to reconstruct the activity of brain circuits at the cellular and system levels, and to probe the impact of the different neuronal components in the observed dynamics. These tools, combining low-invasiveness of optical approaches with the power of genetic engineering, are currently revolutionizing the way, the scale and the perspective of investigating brain diseases. The aim of this review is to describe how brain functions can be investigated with optical approaches currently available and to illustrate how these techniques have been adopted to study pathological alterations of brain physiology.

Genetically encoded sensors for Chloride concentration.

Insights into chloride regulation in neurons have come slowly, but they are likely to be critical for our understanding of how the brain works. The reason is that the intracellular Cl- level ([Cl-]i) is the key determinant of synaptic inhibitory function, and this in turn dictates all manner of neuronal network function. The true impact on the network will only be apparent, however, if Cl- is measured at many locations at once (multiple neurons, and also across the subcellular compartments of single neurons), which realistically, can only be achieved using imaging. The development of genetically-encoded anion biosensors (GABs) brings the additional benefit that Cl- imaging may be done in identified cell-classes and hopefully in subcellular compartments. Here, we describe the historical background and motivation behind the development of these sensors and how they have been used so far. There are, however, still major limitations for their use, the most important being the fact that all GABs are sensitive to both pH and Cl-. Disambiguating the two signals has proved a major challenge, but there are potential solutions; notable among these is ClopHensor, which has now been developed for in vivo measurements of both ion species. We also speculate on how these biosensors may yet be improved, and how this could advance our understanding of Cl- regulation and its impact on brain function.

Aberrant Patterns of Sensory-Evoked Activity in the Olfactory Bulb of LRRK2 Knockout Mice.

The LRRK2 gene is the major genetic determinant of familiar Parkinson's disease (PD). Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein involved in several intracellular signaling pathways. A wealth of evidence indicates that LRRK2 is enriched at the presynaptic compartment where it regulates vesicle trafficking and neurotransmitter release. However, whether the role of LRRK2 affects neuronal networks dynamic at systems level remains unknown. Addressing this question is critical to unravel the impact of LRRK2 on brain function. Here, combining behavioral tests, electrophysiological recordings, and functional imaging, we investigated neuronal network dynamics, in vivo, in the olfactory bulb of mice carrying a null mutation in LRRK2 gene (LRRK2 knockout, LRRK2 KO, mice). We found that LRRK2 KO mice exhibit olfactory behavioral deficits. At the circuit level, the lack of LRRK2 expression results in altered gamma rhythms and odorant-evoked activity with significant impairments, while the spontaneous activity exhibited limited alterations. Overall, our data in the olfactory bulb suggest that the multifaced role of LRRK2 has a strong impact at system level when the network is engaged in active sensory processing.

The role of the odorant receptors in the formation of the sensory map.

In the olfactory system, odorant receptors (ORs) expressed at the cell membrane of olfactory sensory neurons detect odorants and direct sensory axons toward precise target locations in the brain, reflected in the presence of olfactory sensory maps. This dual role of ORs is corroborated by their subcellular expression both in cilia, where they bind odorants, and at axon terminals, a location suitable for axon guidance cues. Here, we provide an overview and discuss previous work on the role of ORs in establishing the topographic organization of the olfactory system and recent findings on the mechanisms of activation and function of axonal ORs.

Whole brain functional recordings at cellular resolution in zebrafish larvae with 3D scanning multiphoton microscopy.

Optical recordings of neuronal activity at cellular resolution represent an invaluable tool to investigate brain mechanisms. Zebrafish larvae is one of the few model organisms where, using fluorescence-based reporters of the cell activity, it is possible to optically reconstruct the neuronal dynamics across the whole brain. Typically, leveraging the reduced light scattering, methods like lightsheet, structured illumination, and light-field microscopy use spatially extended excitation profiles to detect in parallel activity signals from multiple cells. Here, we present an alternative design for whole brain imaging based on sequential 3D point-scanning excitation. Our approach relies on a multiphoton microscope integrating an electrically tunable lens. We first apply our approach, adopting the GCaMP6s activity reporter, to detect functional responses from retinal ganglion cells (RGC) arborization fields at different depths within the zebrafish larva midbrain. Then, in larvae expressing a nuclear localized GCaMP6s, we recorded whole brain activity with cellular resolution. Adopting a semi-automatic cell segmentation, this allowed reconstructing the activity from up to 52,000 individual neurons across the brain. In conclusion, this design can easily retrofit existing imaging systems and represents a compact, versatile and reliable tool to investigate neuronal activity across the larva brain at high resolution.

Spontaneous Afferent Activity Carves Olfactory Circuits.

Electrical activity has a key role in shaping neuronal circuits during development. In most sensory modalities, early in development, internally generated spontaneous activity sculpts the initial layout of neuronal wiring. With the maturation of the sense organs, the system relies more on sensory-evoked electrical activity. Stimuli-driven neuronal discharge is required for the transformation of immature circuits in the specific patterns of neuronal connectivity that subserve normal brain function. The olfactory system (OS) differs from this organizational plan. Despite the important role of odorant receptors (ORs) in shaping olfactory topography, odor-evoked activity does not have a prominent role in refining neuronal wiring. On the contrary, afferent spontaneous discharge is required to achieve and maintain the specific diagram of connectivity that defines the topography of the olfactory bulb (OB). Here, we provide an overview of the development of olfactory topography, with a focus on the role of afferent spontaneous discharge in the formation and maintenance of the specific synaptic contacts that result in the topographic organization of the OB.

Topographic organization in the olfactory bulb.

The ability of the olfactory system to detect and discriminate a broad spectrum of odor molecules with extraordinary sensitivity relies on a wide range of odorant receptors and on the distinct architecture of neuronal circuits in olfactory brain areas. More than 1000 odorant receptors, distributed almost randomly in the olfactory epithelium, are plotted out in two mirror-symmetric maps of glomeruli in the olfactory bulb, the first relay station of the olfactory system. How does such a precise spatial arrangement of glomeruli emerge from a random distribution of receptor neurons? Remarkably, the identity of odorant receptors defines not only the molecular receptive range of sensory neurons but also their glomerular target. Despite their key role, odorant receptors are not the only determinant, since the specificity of neuronal connections emerges from a complex interplay between several molecular cues and electrical activity. This review provides an overview of the mechanisms underlying olfactory circuit formation. In particular, recent findings on the role of odorant receptors in regulating axon targeting and of spontaneous activity in the development and maintenance of synaptic connections are discussed.

Altered Cl- homeostasis hinders forebrain GABAergic interneuron migration in a mouse model of intellectual disability.

Impairments of inhibitory circuits are at the basis of most, if not all, cognitive deficits. The impact of OPHN1, a gene associate with intellectual disability (ID), on inhibitory neurons remains elusive. We addressed this issue by analyzing the postnatal migration of inhibitory interneurons derived from the subventricular zone in a validated mouse model of ID (OPHN1-/y mice). We found that the speed and directionality of migrating neuroblasts were deeply perturbed in OPHN1-/y mice. The significant reduction in speed was due to altered chloride (Cl-) homeostasis, while the overactivation of the OPHN1 downstream signaling pathway, RhoA kinase (ROCK), caused abnormalities in the directionality of the neuroblast progression in mutants. Blocking the cation-Cl- cotransporter KCC2 almost completely rescued the migration speed while proper directionality was restored upon ROCK inhibition. Our data unveil a strong impact of OPHN1 on GABAergic inhibitory interneurons and identify putative targets for successful therapeutic approaches.

Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter.

Genetic mosaicism, a condition in which an organ includes cells with different genotypes, is frequently present in monogenic diseases of the central nervous system caused by the random inactivation of the X-chromosome, in the case of X-linked pathologies, or by somatic mutations affecting a subset of neurons. The comprehension of the mechanisms of these diseases and of the cell-autonomous effects of specific mutations requires the generation of sparse mosaic models, in which the genotype of each neuron is univocally identified by the expression of a fluorescent protein in vivo. Here, we show a dual-color reporter system that, when expressed in a floxed mouse line for a target gene, leads to the creation of mosaics with tunable degree. We demonstrate the generation of a knockout mosaic of the autism/epilepsy related gene PTEN in which the genotype of each neuron is reliably identified, and the neuronal phenotype is accurately characterized by two-photon microscopy.

Role of Axonal Odorant Receptors in Olfactory Topography.

A unique feature in the organization of the olfactory system is the dual role of the odorant receptors: they detect odors in the nasal epithelium and they play an instructive role in the convergence of olfactory sensory neuron axons in specific loci, ie, glomeruli, in the olfactory bulb. The dual role is corroborated by the expression of the odorant receptors in 2 specific locations of the olfactory sensory neurons: the cilia that protrude in the nostril, where the odorant receptors interact with odors, and the axon terminal, a suitable location for a potential axon guidance molecule. The mechanism of activation and function of the odorant receptors expressed at the axon terminal remained unknown for almost 20 years. A recent study identified the first putative ligand of the axonal odorant receptors, phosphatidylethanolamine-binding protein1, a molecule expressed in the olfactory bulb. The distinctive mechanisms of activation of the odorant receptors expressed at the opposite locations in sensory neurons, by odors, at the cilia, and by molecules expressed in the olfactory bulb, at the axon terminal, explain the dual role of the odorant receptors and link the specificity of odor perception with its internal representation, in the topographic map.

Axonal Odorant Receptors Mediate Axon Targeting.

In mammals, odorant receptors not only detect odors but also define the target in the olfactory bulb, where sensory neurons project to give rise to the sensory map. The odorant receptor is expressed at the cilia, where it binds odorants, and at the axon terminal. The mechanism of activation and function of the odorant receptor at the axon terminal is, however, still unknown. Here, we identify phosphatidylethanolamine-binding protein 1 as a putative ligand that activates the odorant receptor at the axon terminal and affects the turning behavior of sensory axons. Genetic ablation of phosphatidylethanolamine-binding protein 1 in mice results in a strongly disturbed olfactory sensory map. Our data suggest that the odorant receptor at the axon terminal of olfactory neurons acts as an axon guidance cue that responds to molecules originating in the olfactory bulb. The dual function of the odorant receptor links specificity of odor perception and axon targeting.

Oligophrenin-1 regulates number, morphology and synaptic properties of adult-born inhibitory interneurons in the olfactory bulb.

Among the X-linked genes associated with intellectual disability, Oligophrenin-1 (OPHN1) encodes for a Rho GTPase-activating protein, a key regulator of several developmental processes, such as dendrite and spine formation and synaptic activity. Inhibitory interneurons play a key role in the development and function of neuronal circuits. Whether a mutation of OPHN1 can affect morphology and synaptic properties of inhibitory interneurons remains poorly understood. To address these open questions, we studied in a well-established mouse model of X-linked intellectual disability, i.e. a line of mice carrying a null mutation of OPHN1, the development and function of adult generated inhibitory interneurons in the olfactory bulb. Combining quantitative morphological analysis and electrophysiological recordings we found that the adult generated inhibitory interneurons were dramatically reduced in number and exhibited a higher proportion of filopodia-like spines, with the consequences on their synaptic function, in OPHN1 ko mice. Furthermore, we found that olfactory behaviour was perturbed in OPHN1 ko mice. Chronic treatment with a Rho kinase inhibitor rescued most of the defects of the newly generated neurons. Altogether, our data indicated that OPHN1 plays a key role in regulating the number, morphology and function of adult-born inhibitory interneurons and contributed to identify potential therapeutic targets.

Role of the odorant receptor in neuronal connectivity in the olfactory bulb.

Olfaction is a highly sophisticated sensory modality able to detect and discriminate thousands of different odours, even at very low concentration. How such a challenging task is achieved remains to be fully understood. A unique feature of the olfactory system is the dual role of the odorant receptor: it does detect odours in the olfactory epithelium but it also contributes to neuronal circuit formation in the olfactory bulb. The odorant receptors are indeed expressed on the cilia that protrude in the nasal cavity, where they bind odorants, and at the axon termini, where they could act as axon guidance molecules. In this review we discuss findings that show how the odorant receptor contributes in regulating neuronal connectivity.

Circuit formation and function in the olfactory bulb of mice with reduced spontaneous afferent activity.

The type of neuronal activity required for circuit development is a matter of significant debate. We addressed this issue by analyzing the topographic organization of the olfactory bulb in transgenic mice engineered to have very little afferent spontaneous activity due to the overexpression of the inwardly rectifying potassium channel Kir2.1 in the olfactory sensory neurons (Kir2.1 mice). In these conditions, the topography of the olfactory bulb was unrefined. Odor-evoked responses were readily recorded in glomeruli with reduced spontaneous afferent activity, although the functional maps were coarser than in controls and contributed to altered olfactory discrimination behavior. In addition, overexpression of Kir2.1 in adults induced a regression of the already refined connectivity to an immature (i.e., coarser) status. Our data suggest that spontaneous activity plays a critical role not only in the development but also in the maintenance of the topography of the olfactory bulb and in sensory information processing.