Anomalous diffusion of synaptic vesicles and its influences on spontaneous and evoked neurotransmission.

Neurons in the central nervous system communicate with each other by activating billions of tiny synaptic boutons distributed along their fine axons. These presynaptic varicosities are very crowded environments, comprising hundreds of synaptic vesicles. Only a fraction of these vesicles can be recruited in a single release episode, either spontaneous or evoked by action potentials. Since the seminal work by Fatt and Katz, spontaneous release has been modelled as a memoryless process. Nevertheless, at central synapses, experimental evidence indicates more complex features, including non-exponential distributions of release intervals and power-law behaviour in their rate. To describe these features, we developed a probabilistic model of spontaneous release based on Brownian motion of synaptic vesicles in the presynaptic environment. To account for different diffusion regimes, we based our simulations on fractional Brownian motion. We show that this model can predict both deviation from the Poisson hypothesis and power-law features in experimental quantal release series, thus suggesting that the vesicular motion by diffusion could per se explain the emergence of these properties. We demonstrate the efficacy of our modelling approach using electrophysiological recordings at single synaptic boutons and ultrastructural data. When this approach was used to simulate evoked responses, we found that the replenishment of the readily releasable pool driven by Brownian motion of vesicles can reproduce the characteristic binomial release distributions seen experimentally. We believe that our modelling approach supports the idea that vesicle diffusion and readily releasable pool dynamics are crucial factors for the physiological functioning of neuronal communication. KEY POINTS: We developed a new probabilistic model of spontaneous and evoked vesicle fusion based on simple biophysical assumptions, including the motion of vesicles before they dock to the release site. We provide closed-form equations for the interval distribution of spontaneous releases in the special case of Brownian diffusion of vesicles, showing that a power-law heavy tail is generated. Fractional Brownian motion (fBm) was exploited to simulate anomalous vesicle diffusion, including directed and non-directed motion, by varying the Hurst exponent. We show that our model predicts non-linear features observed in experimental spontaneous quantal release series as well as ultrastructural data of synaptic vesicles spatial distribution. Evoked exocytosis based on a diffusion-replenished readily releasable pool might explain the emergence of power-law behaviour in neuronal activity.

Neural correlates of metacognition: Disentangling the brain circuits underlying prospective and retrospective second-order judgments through noninvasive brain stimulation.

Metacognition encompasses the capability to monitor and control one's cognitive processes, with metamemory and metadecision configuring among the most studied higher order functions. Although imaging experiments evaluated the role of disparate brain regions, neural substrates of metacognitive judgments remain undetermined. The aim of this systematic review is to summarize and discuss the available evidence concerning the neural bases of metacognition which has been collected by assessing the effects of noninvasive brain stimulation (NIBS) on human subjects' metacognitive capacities. Based on such literature analysis, our goal is, at first, to verify whether prospective and retrospective second-order judgments are localized within separate brain circuits and, subsequently, to provide compelling clues useful for identifying new targets for future NIBS studies. The search was conducted following the preferred reporting items for systematic reviews and meta-analyses guidelines among PubMed, PsycINFO, PsycARTICLES, PSYNDEX, MEDLINE, and ERIC databases. Overall, 25 studies met the eligibility criteria, yielding a total of 36 experiments employing transcranial magnetic stimulation and 16 ones making use of transcranial electrical stimulation techniques, including transcranial direct current stimulation and transcranial alternating current stimulation. Importantly, we found that both perspective and retrospective judgments about both memory and perceptual decision-making performances depend on the activation of the anterior and lateral portions of the prefrontal cortex, as well as on the activity of more caudal regions such as the premotor cortex and the precuneus. Combining this evidence with results from previous imaging and lesion studies, we advance ventromedial prefrontal cortex as a promising target for future NIBS studies.

Autism Spectrum Disorder: Brain Areas Involved, Neurobiological Mechanisms, Diagnoses and Therapies.

Autism spectrum disorder (ASD), affecting over 2% of the pre-school children population, includes an important fraction of the conditions accounting for the heterogeneity of autism. The disease was discovered 75 years ago, and the present review, based on critical evaluations of the recognized ASD studies from the beginning of 1990, has been further developed by the comparative analyses of the research and clinical reports, which have grown progressively in recent years up to late 2023. The tools necessary for the identification of the ASD disease and its related clinical pathologies are genetic and epigenetic mutations affected by the specific interaction with transcription factors and chromatin remodeling processes occurring within specific complexes of brain neurons. Most often, the ensuing effects induce the inhibition/excitation of synaptic structures sustained primarily, at dendritic fibers, by alterations of flat and spine response sites. These effects are relevant because synapses, established by specific interactions of neurons with glial cells, operate as early and key targets of ASD. The pathology of children is often suspected by parents and communities and then confirmed by ensuing experiences. The final diagnoses of children and mature patients are then completed by the combination of neuropsychological (cognitive) tests and electro-/magneto-encephalography studies developed in specialized centers. ASD comorbidities, induced by processes such as anxieties, depressions, hyperactivities, and sleep defects, interact with and reinforce other brain diseases, especially schizophrenia. Advanced therapies, prescribed to children and adult patients for the control of ASD symptoms and disease, are based on the combination of well-known brain drugs with classical tools of neurologic and psychiatric practice. Overall, this review reports and discusses the advanced knowledge about the biological and medical properties of ASD.

Exploiting the molecular diversity of the synapse to investigate neuronal communication: A guide through the current toolkit.

Chemical synapses are tiny and overcrowded environments, deeply embedded inside brain tissue and enriched with thousands of protein species. Many efforts have been devoted to developing custom approaches for evaluating and modifying synaptic activity. Most of these methods are based on the engineering of one or more synaptic protein scaffolds used to target active moieties to the synaptic compartment or to manipulate synaptic functioning. In this review, we summarize the most recent methodological advances and provide a description of the involved proteins as well as the operation principle. Furthermore, we highlight their advantages and limitations in relation to studies of synaptic transmission in vitro and in vivo. Concerning the labelling methods, the most important challenge is how to extend the available approaches to the in vivo setting. On the other hand, for those methods that allow manipulation of synaptic function, this limit has been overcome using optogenetic approaches that can be more easily applied to the living brain. Finally, future applications of these methods to neuroscience, as well as new potential routes for development, are discussed.

Synaptic plasticity mechanisms behind TMS efficacy: insights from its application to animal models.

Neural plasticity is defined as a reshape of communication paths among neurons, expressed through changes in the number and weights of synaptic contacts. During this process, which occurs massively during early brain development but continues also in adulthood, specific brain functions are modified by activity-dependent processes, triggered by external as well as internal stimuli. Since transcranial magnetic stimulation (TMS) produces a non-invasive form of brain cells activation, many different TMS protocols have been developed to treat neurological and psychiatric conditions and proved to be beneficial. Although neural plasticity induction by TMS has been widely assessed on human subjects, we still lack compelling evidence about the actual biological and molecular mechanisms. To support a better comprehension of the involved phenomena, the main focus of this review is to summarize what has been found through the application of TMS to animal models. The hope is that such integrated view will shed light on why and how TMS so effectively works on human subjects, thus supporting a more efficient development of new protocols in the future.

Facilitation of dopamine-dependent long-term potentiation in the medial prefrontal cortex of male rats follows the behavioral effects of stress.

The effect of stress on animal behavior and brain activity has been attracting growing attention in the last decades. Stress dramatically affects several aspects of animal behavior, including motivation and cognitive functioning, and has been used to model human pathologies such as post-traumatic stress disorder. A key question is whether stress alters the plastic potential of synaptic circuits. In this work, we evaluated if stress affects dopamine (DA)-dependent synaptic plasticity in the medial prefrontal cortex (mPFC). On male adolescent rats, we characterized anxiety- and depressive-like behaviors using behavioral testing before and after exposure to a mild stress (elevated platform, EP). After the behavioral protocols, we investigated DA-dependent long-term potentiation (DA-LTP) and depression (DA-LTD) on acute slices of mPFC and evaluated the activation of DA-producing brain regions by western and dot blot analysis. We show that exposure to the EP stress enhances DA-LTP and that desipramine (DMI) treatment abolishes this effect. We also found that DA-LTD is not affected by EP stress unless when this is followed by DMI treatment. In addition, EP stress reduces anxiety, an effect abolished by both DMI and ketamine, while motivation is promoted by previous exposure to EP stress independently of pharmacological treatments. Finally, this form of stress reduces the expression of the early gene cFOS in the ventral tegmental area. These findings support the idea that mild stressors can promote synaptic plasticity in PFC through a dopaminergic mechanism, an effect that might increase the sensitivity of mPFC to subsequent stressful experiences.

Linear transformation of the encoding mechanism for light intensity underlies the paradoxical enhancement of cortical visual responses by sevoflurane.

KEY POINTS: The mechanisms of action of anaesthetics on the living brain are still poorly understood. In this respect, the analysis of the differential effects of anaesthetics on spontaneous and sensory-evoked cortical activity might provide important and novel cues. Here we show that the anaesthetic sevoflurane strongly silences the brain but potentiates in a dose- and frequency-dependent manner the cortical visual response. Such enhancement arises from a linear scaling by sevoflurane of the power-law relation between light intensity and the cortical response. The fingerprint of sevoflurane action suggests that circuit silencing can boost linearly synaptic responsiveness presumably by scaling the number of responding units and/or their correlation following a sensory stimulation. ABSTRACT: General anaesthetics, which are expected to silence brain activity, often spare sensory responses. To evaluate differential effects of anaesthetics on spontaneous and sensory-evoked cortical activity, we characterized their modulation by sevoflurane and propofol. Power spectra and the bust-suppression ratio from EEG data were used to evaluate anaesthesia depth. ON and OFF cortical responses were elicited by light pulses of variable intensity, duration and frequency, during light and deep states of anaesthesia. Both anaesthetics reduced spontaneous cortical activity but sevoflurane greatly enhanced while propofol diminished the ON visual response. Interestingly, the large potentiation of the ON visual response by sevoflurane was found to represent a linear scaling of the encoding mechanism for light intensity. To the contrary, the OFF cortical visual response was depressed by both anaesthetics. The selective depression of the OFF component by sevoflurane could be converted into a robust potentiation by the pharmacological blockade of the ON pathway, suggesting that the temporal order of ON and OFF responses leads to a depression of the latter. This hypothesis agrees with the finding that the enhancement of the ON response was converted into a depression by increasing the frequency of light-pulse stimulation from 0.1 to 1 Hz. Overall, our results support the view that inactivity-dependent modulation of cortical circuits produces an increase in their responsiveness. Among the implications of our findings, the silencing of cortical circuits can boost linearly the cortical responsiveness but with negative impact on their frequency transfer and with a loss of the information content of the sensory signal.

Novel Approaches for the Treatment of Post-Traumatic Stress Disorder: A Systematic Review of Non-Invasive Brain Stimulation Interventions and Insights from Clinical Trials.

First-line treatments for post-traumatic stress disorder (PTSD) encompass a wide range of pharmacotherapies and psychotherapies. However, many patients fail to respond to such interventions, highlighting the need for novel approaches. Due to its ability to modulate cortical activity, non-invasive brain stimulation (NIBS) could represent a valuable therapeutic tool. Therefore, the aim of this systematic review is to summarize and discuss the existing evidence on the ameliorative effects of NIBS on PTSD and comorbid anxiety and depressive symptoms. Our goal is also to debate the effectiveness of an integrated approach characterized by the combination of NIBS and psychotherapy. This search was conducted following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines in the PubMed, PsycINFO, PsycARTICLES, PSYINDEX, MEDLINE, and ERIC databases. Overall, 31 studies met the eligibility criteria, yielding a total of 26 clinical trials employing transcranial magnetic stimulation (TMS) and 5 making use of transcranial direct-current stimulation (tDCS). From these studies, it emerged that NIBS consistently reduced overall PTSD symptoms' severity as well as comorbid anxiety and depressive symptoms. Moreover, we speculate that combining NIBS with prolonged exposure or cognitive processing therapy might represent a promising therapeutic approach for consistently ameliorating subjects' clinical conditions.

The Dysfunctional Mechanisms Throwing Tics: Structural and Functional Changes in Tourette Syndrome.

Tourette Syndrome (TS) is a high-incidence multifactorial neuropsychiatric disorder characterized by motor and vocal tics co-occurring with several diverse comorbidities, including obsessive-compulsive disorder and attention-deficit hyperactivity disorder. The origin of TS is multifactorial, with strong genetic, perinatal, and immunological influences. Although almost all neurotransmettitorial systems have been implicated in TS pathophysiology, a comprehensive neurophysiological model explaining the dynamics of expression and inhibition of tics is still lacking. The genesis and maintenance of motor and non-motor aspects of TS are thought to arise from functional and/or structural modifications of the basal ganglia and related circuitry. This complex wiring involves several cortical and subcortical structures whose concerted activity controls the selection of the most appropriate reflexive and habitual motor, cognitive and emotional actions. Importantly, striatal circuits exhibit bidirectional forms of synaptic plasticity that differ in many respects from hippocampal and neocortical plasticity, including sensitivity to metaplastic molecules such as dopamine. Here, we review the available evidence about structural and functional anomalies in neural circuits which have been found in TS patients. Finally, considering what is known in the field of striatal plasticity, we discuss the role of exuberant plasticity in TS, including the prospect of future pharmacological and neuromodulation avenues.

Neural Correlates of Delay Discounting in the Light of Brain Imaging and Non-Invasive Brain Stimulation: What We Know and What Is Missed.

In decision making, the subjective value of a reward declines with the delay to its receipt, describing a hyperbolic function. Although this phenomenon, referred to as delay discounting (DD), has been extensively characterized and reported in many animal species, still, little is known about the neuronal processes that support it. Here, after drawing a comprehensive portrait, we consider the latest neuroimaging and lesion studies, the outcomes of which often appear contradictory among comparable experimental settings. In the second part of the manuscript, we focus on a more recent and effective route of investigation: non-invasive brain stimulation (NIBS). We provide a comprehensive review of the available studies that applied transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS) to affect subjects' performance in DD tasks. The aim of our survey is not only to highlight the superiority of NIBS in investigating DD, but also to suggest targets for future experimental studies, since the regions considered in these studies represent only a fraction of the possible ones. In particular, we argue that, based on the available neurophysiological evidence from lesion and brain imaging studies, a very promising and underrepresented region for future neuromodulation studies investigating DD is the orbitofrontal cortex.

Poke And Delayed Drink Intertemporal Choice Task (POKE-ADDICT): An open-source behavioral apparatus for intertemporal choice testing in rodents.

Advancements in neuroscience research present opportunities and challenges, requiring substantial resources and funding. To address this, we describe here "Poke And Delayed Drink Intertemporal Choice Task (POKE-ADDICT)", an open-source, versatile, and cost-effective apparatus for intertemporal choice testing in rodents. This allows quantification of delay discounting (DD), a cross-species phenomenon observed in decision making which provides valuable insights into higher-order cognitive functioning. In DD, the subjective value of a delayed reward is reduced as a function of the delay for its receipt. Using our apparatus, we implemented an effective intertemporal choice paradigm for the quantification of DD based on an adjusting delayed amount (ADA) algorithm using mango juice as a reward. Our paradigm requires limited training, a few 3D-printed parts and inexpensive electrical components, including a Raspberry Pi control unit. Furthermore, it is compatible with several in vivo procedures and the use of nose pokes instead of levers allows for faster task learning. Besides the main application described here, the apparatus can be further extended to implement other behavioral tests and protocols, including standard operant conditioning. In conclusion, we describe a versatile and cost-effective design based on Raspberry Pi that can support research in animal behavior, decision making and, more specifically, delay discounting.

Transcranial direct current stimulation (tDCS) over the orbitofrontal cortex reduces delay discounting.

Delay discounting (DD) is a quantifiable psychological phenomenon that regulates decision-making. Nevertheless, the neural substrates of DD and its relationship with other cognitive domains are not well understood. The orbitofrontal cortex (OFC) is a potential candidate for supporting the expression of DD, but due to its wide involvement in several psychological functions and neural networks, its central role remains elusive. In this study, healthy subjects underwent transcranial direct current stimulation (tDCS) while performing an intertemporal choice task for the quantification of DD and a working memory task. To selectively engage the OFC, two electrode configurations have been tested, namely, anodal Fp1-cathodal Fp2 and cathodal Fp1-anodal Fp2. Our results show that stimulation of the OFC reduces DD, independently from electrode configuration. In addition, no relationship was found between DD measures and either working memory performance or baseline impulsivity assessed through established tests. Our work will direct future investigations aimed at unveiling the specific neural mechanisms underlying the involvement of the OFC in DD, and at testing the efficacy of OFC tDCS in reducing DD in psychological conditions where this phenomenon has been strongly implicated, such as addiction and eating disorders.

Nanotopography and Microconfinement Impact on Primary Hippocampal Astrocyte Morphology, Cytoskeleton and Spontaneous Calcium Wave Signalling.

Astrocytes' organisation affects the functioning and the fine morphology of the brain, both in physiological and pathological contexts. Although many aspects of their role have been characterised, their complex functions remain, to a certain extent, unclear with respect to their contribution to brain cell communication. Here, we studied the effects of nanotopography and microconfinement on primary hippocampal rat astrocytes. For this purpose, we fabricated nanostructured zirconia surfaces as homogenous substrates and as micrometric patterns, the latter produced by a combination of an additive nanofabrication and micropatterning technique. These engineered substrates reproduce both nanotopographical features and microscale geometries that astrocytes encounter in their natural environment, such as basement membrane topography, as well as blood vessels and axonal fibre topology. The impact of restrictive adhesion manifests in the modulation of several cellular properties of single cells (morphological and actin cytoskeletal changes) and the network organisation and functioning. Calcium wave signalling was observed only in astrocytes grown in confined geometries, with an activity enhancement in cells forming elongated agglomerates with dimensions typical of blood vessels or axon fibres. Our results suggest that calcium oscillation and wave propagation are closely related to astrocytic morphology and actin cytoskeleton organisation.

Occlusion of dopamine-dependent synaptic plasticity in the prefrontal cortex mediates the expression of depressive-like behavior and is modulated by ketamine.

Unpredictable chronic mild stress (CMS) is among the most popular protocols used to induce depressive-like behaviors such as anhedonia in rats. Differences in CMS protocols often result in variable degree of vulnerability, and the mechanisms behind stress resilience are of great interest in neuroscience due to their involvement in the development of psychiatric disorders, including major depressive disorder. Expression of depressive-like behaviors is likely driven by long-term alterations in the corticolimbic system and by downregulation of dopamine (DA) signaling. Although we have a deep knowledge about the dynamics of tonic and phasic DA release in encoding incentive salience and in response to acute/chronic stress, its modulatory action on cortical synaptic plasticity and the following implications on animal behavior remain elusive. Here, we show that the expression of DA-dependent synaptic plasticity in the medial prefrontal cortex (mPFC) is occluded in rats vulnerable to CMS, likely reflecting differential expression of AMPA receptors. Interestingly, such difference is not observed when rats are acutely treated with sub-anesthetic ketamine, possibly through the recruitment of dopaminergic nuclei such as the ventral tegmental area. In addition, by applying the synaptic activity sensor SynaptoZip in vivo, we found that chronic stress unbalances the synaptic drive from the infralimbic and prelimbic subregions of the mPFC toward the basolateral amygdala, and that this effect is counteracted by ketamine. Our results provide novel insights into the neurophysiological mechanisms behind the expression of vulnerability to stress, as well as behind the antidepressant action of ketamine.

Activity-based anorexia animal model: a review of the main neurobiological findings.

BACKGROUND: The genesis of anorexia nervosa (AN), a severe eating disorder with a pervasive effect on many brain functions such as attention, emotions, reward processing, cognition and motor control, has not yet been understood. Since our current knowledge of the genetic aspects of AN is limited, we are left with a large and diversified number of biological, psychological and environmental risk factors, called into question as potential triggers of this chronic condition with a high relapse rate. One of the most valid and used animal models for AN is the activity-based anorexia (ABA), which recapitulates important features of the human condition. This model is generated from naïve rodents by a self-motivated caloric restriction, where a fixed schedule food delivery induces spontaneous increased physical activity. AIM: In this review, we sought to provide a summary of the experimental research conducted using the ABA model in the pursuit of potential neurobiological mechanism(s) underlying AN. METHOD: The experimental work presented here includes evidence for neuroanatomical and neurophysiological changes in several brain regions as well as for the dysregulation of specific neurochemical synaptic and neurohormonal pathways. RESULTS: The most likely hypothesis for the mechanism behind the development of the ABA phenotype relates to an imbalance of the neural circuitry that mediates reward processing. Evidence collected here suggests that ABA animals show a large set of alterations, involving regions whose functions extend way beyond the control of reward mechanisms and eating habits. Hence, we cannot exclude a primary role of these alterations from a mechanistic theory of ABA induction. CONCLUSIONS: These findings are not sufficient to solve such a major enigma in neuroscience, still they could be used to design ad hoc further experimental investigation. The prospect is that, since treatment of AN is still challenging, the ABA model could be more effectively used to shed light on the complex AN neurobiological framework, thus supporting the future development of therapeutic strategies but also the identification of biomarkers and diagnostic tools. Anorexia Nervosa (AN) is a severe eating disorder with a dramatic effect on many functions of our brain, such as attention, emotions, cognition and motion control. Since our current knowledge of the genetic aspects behind the development of AN is still limited, many biological, psychological and environmental factors must be taken into account as potential triggers of this condition. One of the most valid animal models for studying AN is the activity-based anorexia (ABA). In this model, rodents spontaneously limit food intake and start performing increased physical activity on a running wheel, a result of the imposition of a fixed time schedule for food delivery. In this review, we provide a detailed summary of the experimental research conducted using the ABA model, which includes extended evidence for changes in the anatomy and function of the brain of ABA rodents. The hope is that such integrated view will support the design of future experiments that will shed light on the complex brain mechanisms behind AN. Such advanced knowledge is crucial to find new, effective strategies for both the early diagnosis of AN and for its treatment.

A novel integrated approach to estimate the mitochondrial content of neuronal cells and brain tissues.

BACKGROUND: Mitochondria and their dynamics fuel most cellular processes both in physiological and pathological conditions. In the central nervous system, mitochondria sustain synaptic transmission and plasticity via multiple mechanisms which include their redistribution and/or expansion to higher energy demanding sites, sustaining activity changes and promoting morphological circuit adaptations. NEW METHOD: To be able to evaluate changes in mitochondrial number and protein phenotype, we propose a novel methodological approach where the simultaneous analysis of both mitochondrial DNA and protein content is performed on each individual microsample, avoiding non-homogeneous loss of material. RESULTS: We validated this method on neuronal-like cells and tissue samples and obtained estimates for the mitochondrial/genomic DNA ratio as well as for the abundance of protein counterparts. When the mitochondrial content per cell was evaluated in different brain areas, our results matched the known regional variation in aerobic-anaerobic metabolism. When long-term potentiation (LTP) was induced on hippocampal neurons, we detected increases in the abundance of mitochondria that correlated with the degree of synaptic enhancement. CONCLUSIONS: Our approach can be effectively used to study the mitochondrial content andits changes in different brain cells and tissues.

Micropatterning of Substrates for the Culture of Cell Networks by Stencil-Assisted Additive Nanofabrication.

The fabrication of in vitro neuronal cell networks where cells are chemically or electrically connected to form functional circuits with useful properties is of great interest. Standard cell culture substrates provide ensembles of cells that scarcely reproduce physiological structures since their spatial organization and connectivity cannot be controlled. Supersonic Cluster Beam Deposition (SCBD) has been used as an effective additive method for the large-scale fabrication of interfaces with extracellular matrix-mimicking surface nanotopography and reproducible morphological properties for cell culture. Due to the high collimation of SCBD, it is possible to exploit stencil masks for the fabrication of patterned films and reproduce features as small as tens of micrometers. Here, we present a protocol to fabricate micropatterned cell culture substrates based on the deposition of nanostructured cluster-assembled zirconia films by stencil-assisted SCBD. The effectiveness of this approach is demonstrated by the fabrication of micrometric patterns able to confine primary astrocytes. Calcium waves propagating in the astrocyte networks are shown.

Behavioral assessment of activity-based-anorexia: how cognition can become the drive wheel.

Anorexia Nervosa (AN) is a disorder with a dramatic impact on both the individual and society. Besides severe weight loss, excessive physical exercise and cognitive disturbances can be present in patients with AN as primary symptoms of the pathology or as secondary effects induced by physical and metabolic alterations. Mechanistic research in this field has taken advantage of a well characterized animal model, the activity-based anorexia model (ABA). ABA rodents and subjects with AN show clear behavioral and physiological similarities, but a throughout neurocognitive assessment of the model is still missing. Here, we review the available literature in the ABA field, highlighting similarities between ABA and AN at the behavioral, neurophysiological and cognitive level. Furthermore, based on availability, feasibility and adaptability of rodent behavioral protocols, we propose a set of neurocognitive assays that can be performed on the ABA. The proposed assessment represents an important step forward in the validation and extension of the ABA model, opening several routes of investigation related to AN and other eating disorders.

Neuronal Cells Confinement by Micropatterned Cluster-Assembled Dots with Mechanotransductive Nanotopography.

Artificially grown neuronal cultures of brain cells have been used for decades in the attempt to reproduce and study in vitro the complexity of brain circuits. It soon became evident that this alone was insufficient, because of the random architecture of these artificial networks. Important groundwork therefore resulted in the development of methods to confine neuronal adhesion at specific locations to match predefined network topologies and connectivity. Despite this notable progress in neural circuitry engineering, there is still need for micropatterned substrates that recapitulate better biophysical cues of the neuronal microenvironment, taking into account recent findings of their significance for neuronal differentiation and functioning. Here, we report the development and characterization of a novel approach that, by using supersonic cluster beam deposition of zirconia nanoparticles, allows the patterning of small nanostructured cell-adhesive areas according to predefined geometries onto elsewhere nonadhesive antifouling glass surfaces. As distinguishing features, compared to other micropatterning approaches in this context, the integrated nanostructured surfaces possess extracellular matrix-like nanotopographies of predetermined roughness; previously shown to be able to promote neuronal differentiation due to their impact on mechanotransductive processes, and can be used in their original state without any coating requirements. These micropatterned substrates were validated using (i) a neuron-like PC12 cell line and (ii) primary cultures of rat hippocampal neurons. After initial uniform plating, both neuronal cells types were found to converge and adhere specifically to the nanostructured regions. The cell-adhesive areas effectively confined cells, even when these were highly mobile and repeatedly attempted to cross boundaries. Inside these small permissive islands, cells grew and differentiated, in the case of the hippocampal neurons, up to the formation of mature, functionally active, and highly connected synaptic networks. In addition, when spontaneous instances of axon bridging between nearby dots occurred, a functional interdot communication between these subgroups of cells was observed.