Astrocyte structural heterogeneity in the mouse hippocampus.

Astrocytes are integral components of brain circuits, where they sense, process, and respond to surrounding activity, maintaining homeostasis and regulating synaptic transmission, the sum of which results in behavior modulation. These interactions are possible due to their complex morphology, composed of a tree-like structure of processes to cover defined territories ramifying in a mesh-like system of fine leaflets unresolved by conventional optic microscopy. While recent reports devoted more attention to leaflets and their dynamic interactions with synapses, our knowledge about the tree-like "backbone" structure in physiological conditions is incomplete. Recent transcriptomic studies described astrocyte molecular diversity, suggesting structural heterogeneity in regions such as the hippocampus, which is crucial for cognitive and emotional behaviors. In this study, we carried out the structural analysis of astrocytes across the hippocampal subfields of Cornu Ammonis area 1 (CA1) and dentate gyrus in the dorsoventral axis. We found that astrocytes display heterogeneity across the hippocampal subfields, which is conserved along the dorsoventral axis. We further found that astrocytes appear to contribute in an exocytosis-dependent manner to a signaling loop that maintains the backbone structure. These findings reveal astrocyte heterogeneity in the hippocampus, which appears to follow layer-specific cues and depend on the neuro-glial environment.

Genetic Ablation of Inositol 1,4,5-Trisphosphate Receptor Type 2 (IP3R2) Fails to Modify Disease Progression in a Mouse Model of Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by an abnormal polyglutamine expansion within the ataxin-3 protein (ATXN3). This leads to neurodegeneration of specific brain and spinal cord regions, resulting in a progressive loss of motor function. Despite neuronal death, non-neuronal cells, including astrocytes, are also involved in SCA3 pathogenesis. Astrogliosis is a common pathological feature in SCA3 patients and animal models of the disease. However, the contribution of astrocytes to SCA3 is not clearly defined. Inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) is the predominant IP3R in mediating astrocyte somatic calcium signals, and genetically ablation of IP3R2 has been widely used to study astrocyte function. Here, we aimed to investigate the relevance of IP3R2 in the onset and progression of SCA3. For this, we tested whether IP3R2 depletion and the consecutive suppression of global astrocytic calcium signalling would lead to marked changes in the behavioral phenotype of a SCA3 mouse model, the CMVMJD135 transgenic line. This was achieved by crossing IP3R2 null mice with the CMVMJD135 mouse model and performing a longitudinal behavioral characterization of these mice using well-established motor-related function tests. Our results demonstrate that IP3R2 deletion in astrocytes does not modify SCA3 progression.

Astrocyte regulation of neural circuit activity and network states.

Astrocytes are known to influence neuronal activity through different mechanisms, including the homeostatic control of extracellular levels of ions and neurotransmitters and the exchange of signaling molecules that regulate synaptic formation, structure, and function. While a great effort done in the past has defined many molecular mechanisms and cellular processes involved in astrocyte-neuron interactions at the cellular level, the consequences of these interactions at the network level in vivo have only relatively recently been identified. This review describes and discusses recent findings on the regulatory effects of astrocytes on the activity of neuronal networks in vivo. Accumulating but still limited, evidence indicates that astrocytes regulate neuronal network rhythmic activity and synchronization as well as brain states. These studies demonstrate a critical contribution of astrocytes to brain activity and are paving the way for a more thorough understanding of the cellular bases of brain function.

Hippocampal cytogenesis abrogation impairs inter-regional communication between the hippocampus and prefrontal cortex and promotes the time-dependent manifestation of emotional and cognitive deficits.

Impaired ability to generate new cells in the adult brain has been linked to deficits in multiple emotional and cognitive behavioral domains. However, the mechanisms by which abrogation of adult neural stem cells (NSCs) impacts on brain function remains controversial. We used a transgenic rat line, the GFAP-Tk, to selectively eliminate NSCs and assess repercussions on different behavioral domains. To assess the functional importance of newborn cells in specific developmental stages, two parallel experimental timeframes were adopted: a short- and a long-term timeline, 1 and 4 weeks after the abrogation protocol, respectively. We conducted in vivo electrophysiology to assess the effects of cytogenesis abrogation on the functional properties of the hippocampus and prefrontal cortex, and on their intercommunication. Adult brain cytogenesis abrogation promoted a time-specific installation of behavioral deficits. While the lack of newborn immature hippocampal neuronal and glial cells elicited a behavioral phenotype restricted to hyperanxiety and cognitive rigidity, specific abrogation of mature new neuronal and glial cells promoted the long-term manifestation of a more complex behavioral profile encompassing alterations in anxiety and hedonic behaviors, along with deficits in multiple cognitive modalities. More so, abrogation of 4 to 7-week-old cells resulted in impaired electrophysiological synchrony of neural theta oscillations between the dorsal hippocampus and the medial prefrontal cortex, which are likely to contribute to the described long-term cognitive alterations. Hence, this work provides insight on how newborn neurons and astrocytes display different functional roles throughout different maturation stages, and establishes common ground to reconcile contrasting results that have marked this field.

IP3 R2 null mice display a normal acquisition of somatic and neurological development milestones.

Astrocytes are key players in the regulation of brain development and function. They sense and respond to the surrounding activity by elevating their intracellular calcium (Ca2+ ) levels. These astrocytic Ca2+ elevations emerge from different sources and display complex spatio-temporal properties. Ca2+ elevations are spatially distributed in global (soma and main processes) and/or focal regions (microdomains). The inositol 1,4,5-trisphosphate receptor type 2 knockout (IP3 R2 KO) mouse model lacks global Ca2+ elevations in astrocytes, and it has been used by different laboratories. However, the constitutive deletion of IP3 R2 during development may trigger compensating phenotypes, which could bias the results of experiments using developing or adult mice. To address this issue, we performed a detailed neurodevelopmental evaluation of male and female IP3 R2 KO mice, during the first 21 days of life, as well as an evaluation of motor function, strength and neurological reflexes in adult mice. Our results show that male and female IP3 R2 KO mice display a normal acquisition of developmental milestones, as compared with wild-type (WT) mice. We also show that IP3 R2 KO mice display normal motor coordination, strength and neurological reflexes in adulthood. To exclude a potential compensatory overexpression of other IP3 Rs, we quantified the relative mRNA levels of all 3 subtypes, in brain tissue. We found that, along with the complete deletion of Itpr2, there is no compensatory expression of Itpr1 or Itrp3. Overall, our results show that the IP3 R2 KO mouse is a reliable model to study the functional impact of global IP3 R2-dependent astrocytic Ca2+ elevations.

Region-specific control of microglia by adenosine A2A receptors: uncoupling anxiety and associated cognitive deficits in female rats.

Epidemiologic studies have provided compelling evidence that prenatal stress, through excessive maternal glucocorticoids exposure, is associated with psychiatric disorders later in life. We have recently reported that anxiety associated with prenatal exposure to dexamethasone (DEX, a synthetic glucocorticoid) correlates with a gender-specific remodeling of microglia in the medial prefrontal cortex (mPFC), a core brain region in anxiety-related disorders. Gender differences in microglia morphology, the higher prevalence of anxiety in women and the negative impact of anxiety in cognition, led us to specifically evaluate cognitive behavior and associated circuits (namely mPFC-dorsal hippocampus, dHIP), as well as microglia morphology in female rats prenatally exposed to dexamethasone (in utero DEX, iuDEX). We report that iuDEX impaired recognition memory and deteriorated neuronal synchronization between mPFC and dHIP. These functional deficits are paralleled by microglia hyper-ramification in the dHIP and decreased ramification in the mPFC, showing a heterogeneous remodeling of microglia morphology, both postnatally and at adulthood in different brain regions, that differently affect mood and cognition. The chronic blockade of adenosine A2A receptors (A2A R), which are core regulators of microglia morphology and physiology, ameliorated the cognitive deficits, but not the anxiety-like behavior. Notably, A2A R blockade rectified both microglia morphology in the dHIP and the lack of mPFC-dHIP synchronization, further heralding their role in cognitive function.

Astrocytic signaling supports hippocampal-prefrontal theta synchronization and cognitive function.

Astrocytes interact with neurons at the cellular level through modulation of synaptic formation, maturation, and function, but the impact of such interaction into behavior remains unclear. Here, we studied the dominant negative SNARE (dnSNARE) mouse model to dissect the role of astrocyte-derived signaling in corticolimbic circuits, with implications for cognitive processing. We found that the blockade of gliotransmitter release in astrocytes triggers a critical desynchronization of neural theta oscillations between dorsal hippocampus and prefrontal cortex. Moreover, we found a strong cognitive impairment in tasks depending on this network. Importantly, the supplementation with d-serine completely restores hippocampal-prefrontal theta synchronization and rescues the spatial memory and long-term memory of dnSNARE mice. We provide here novel evidence of long distance network modulation by astrocytes, with direct implications to cognitive function.

Multi-level therapeutic actions of cannabidiol in ketamine-induced schizophrenia psychopathology in male rats.

Repeated administration of ketamine (KET) has been used to model schizophrenia-like symptomatology in rodents, but the psychotomimetic neurobiological and neuroanatomical underpinnings remain elusive. In parallel, the unmet need for a better treatment of schizophrenia requires the development of novel therapeutic strategies. Cannabidiol (CBD), a major non-addictive phytocannabinoid has been linked to antipsychotic effects with unclear mechanistic basis. Therefore, this study aims to clarify the neurobiological substrate of repeated KET administration model and to evaluate CBD's antipsychotic potential and neurobiological basis. CBD-treated male rats with and without prior repeated KET administration underwent behavioral analyses, followed by multilevel analysis of different brain areas including dopaminergic and glutamatergic activity, synaptic signaling, as well as electrophysiological recordings for the assessment of corticohippocampal and corticostriatal network activity. Repeated KET model is characterized by schizophrenia-like symptomatology and alterations in glutamatergic and dopaminergic activity mainly in the PFC and the dorsomedial striatum (DMS), through a bi-directional pattern. These observations are accompanied by glutamatergic/GABAergic deviations paralleled to impaired function of parvalbumin- and cholecystokinin-positive interneurons, indicative of excitation/inhibition (E/I) imbalance. Moreover, CBD counteracted the schizophrenia-like behavioral phenotype as well as reverted prefrontal abnormalities and ventral hippocampal E/I deficits, while partially modulated dorsostriatal dysregulations. This study adds novel insights to our understanding of the KET-induced schizophrenia-related brain pathology, as well as the CBD antipsychotic action through a region-specific set of modulations in the corticohippocampal and costicostrtiatal circuitry of KET-induced profile contributing to the development of novel therapeutic strategies focused on the ECS and E/I imbalance restoration.

Assessing Different Histological Preparations for Reconstruction of Astrocyte Tridimensional Structure.

Astrocytes are ubiquitous in the brain and spinal cord and display a complex morphology important for the local interactions with neighboring cells, resulting in the modulation of circuit function. Thus, studies focusing on astrocyte physiology in the healthy and diseased brain generally present analyses of astrocytic structure. The labeling method used to visualize the astrocytic structure defines the morphological level to observe and may vary depending on the anatomical sub-regions. The method choice may significantly affect our understanding of their structural diversity. The main goal of this work was to identify a straightforward and efficient protocol for labeling and reconstructing a detailed astrocytic structure to apply and validate in different brain tissue preparations across laboratories. For that, we explored different tissue processing protocols before GFAP labeling to determine the most effective method for reconstructing astrocytic backbones in the mouse hippocampus. Our results show that the reconstruction of astrocytic structure in vibratome sections labeled by free-floating immunofluorescence protocol provides a more practical method to achieve a higher level of detail and arbor complexity in astrocyte backbone reconstruction. Free-floating immunofluorescence labeling is the most reliable method for obtaining better antibody penetration and more detailed astrocyte structure. Finally, we also show that introducing an antigen retrieval step appears useful for visualizing more complete structural details.

Rodent cortical astroglia express in situ functional P2X7 receptors sensing pathologically high ATP concentrations.

ATP is an important neuronal and astroglial signaling molecule in the brain. In the present study, brain slices were prepared from the prefrontal cortex (PFC) of Wistar rats and 2 lines of mice. P2X7 receptor immunoreactivity was colocalized with astro- and microglial but not neuronal markers. Whole-cell patch-clamp recordings showed that, in astroglial cells, dibenzoyl-ATP (BzATP) and ATP caused inward currents, near the resting membrane potential. The inactivity of α,ß-methylene ATP, as well as the potency increases of BzATP and ATP in a low divalent cation (X²(+))-containing superfusion medium suggested the involvement of P2X7 receptors. This idea was corroborated by the inhibition of these current responses by PPADS, Brilliant Blue G, A 438079, and calmidazolium. Ivermectin, trinitrophenyl-adenosine-5'-triphosphate, and cyclopentyl-dipropylxanthine did not alter the agonist effects. The reversal potential of BzATP was near 0 mV, indicating the opening of cationic receptor channels. In a low X²(+) superfusion medium, ATP-induced current responses in PFC astroglial cells of wild-type mice but not of the P2X7 knockouts. Hence, cortical astroglia of rats and mice possess functional P2X7 receptors. These receptors may participate in necrotic/apoptotic or proliferative reactions after stimulation by large quantities of ATP released by central nervous system injury.

Beyond New Neurons in the Adult Hippocampus: Imipramine Acts as a Pro-Astrogliogenic Factor and Rescues Cognitive Impairments Induced by Stress Exposure.

Depression is a prevalent, socially burdensome disease. Different studies have demonstrated the important role of astrocytes in the pathophysiology of depression as modulators of neurotransmission and neurovascular coupling. This is evidenced by astrocyte impairments observed in brains of depressed patients and the appearance of depressive-like behaviors upon astrocytic dysfunctions in animal models. However, little is known about the importance of de novo generated astrocytes in the mammalian brain and in particular its possible involvement in the precipitation of depression and in the therapeutic actions of current antidepressants (ADs). Therefore, we studied the modulation of astrocytes and adult astrogliogenesis in the hippocampal dentate gyrus (DG) of rats exposed to an unpredictable chronic mild stress (uCMS) protocol, untreated and treated for two weeks with antidepressants-fluoxetine and imipramine. Our results show that adult astrogliogenesis in the DG is modulated by stress and imipramine. This study reveals that distinct classes of ADs impact differently in the astrogliogenic process, showing different cellular mechanisms relevant to the recovery from behavioral deficits induced by chronic stress exposure. As such, in addition to those resident, the newborn astrocytes in the hippocampal DG might also be promising therapeutic targets for future therapies in the neuropsychiatric field.

Astrocyte-derived TNF and glutamate critically modulate microglia activation by methamphetamine.

Methamphetamine (Meth) is a powerful illicit psychostimulant, widely used for recreational purposes. Besides disrupting the monoaminergic system and promoting oxidative brain damage, Meth also causes neuroinflammation, contributing to synaptic dysfunction and behavioral deficits. Aberrant activation of microglia, the largest myeloid cell population in the brain, is a common feature in neurological disorders triggered by neuroinflammation. In this study, we investigated the mechanisms underlying the aberrant activation of microglia elicited by Meth in the adult mouse brain. We found that binge Meth exposure caused microgliosis and disrupted risk assessment behavior (a feature that usually occurs in individuals who abuse Meth), both of which required astrocyte-to-microglia crosstalk. Mechanistically, Meth triggered a detrimental increase of glutamate exocytosis from astrocytes (in a process dependent on TNF production and calcium mobilization), promoting microglial expansion and reactivity. Ablating TNF production, or suppressing astrocytic calcium mobilization, prevented Meth-elicited microglia reactivity and re-established risk assessment behavior as tested by elevated plus maze (EPM). Overall, our data indicate that glial crosstalk is critical to relay alterations caused by acute Meth exposure.

Astrocyte-mediated switch in spike timing-dependent plasticity during hippocampal development.

Presynaptic spike timing-dependent long-term depression (t-LTD) at hippocampal CA3-CA1 synapses is evident until the 3rd postnatal week in mice, disappearing during the 4th week. At more mature stages, we found that the protocol that induced t-LTD induced t-LTP. We characterized this form of t-LTP and the mechanisms involved in its induction, as well as that driving this switch from t-LTD to t-LTP. We found that this t-LTP is expressed presynaptically at CA3-CA1 synapses, as witnessed by coefficient of variation, number of failures, paired-pulse ratio and miniature responses analysis. Additionally, this form of presynaptic t-LTP does not require NMDARs but the activation of mGluRs and the entry of Ca2+ into the postsynaptic neuron through L-type voltage-dependent Ca2+ channels and the release of Ca2+ from intracellular stores. Nitric oxide is also required as a messenger from the postsynaptic neuron. Crucially, the release of adenosine and glutamate by astrocytes is required for t-LTP induction and for the switch from t-LTD to t-LTP. Thus, we have discovered a developmental switch of synaptic transmission from t-LTD to t-LTP at hippocampal CA3-CA1 synapses in which astrocytes play a central role and revealed a form of presynaptic LTP and the rules for its induction.

Identification of region-specific astrocyte subtypes at single cell resolution.

Astrocytes, a major cell type found throughout the central nervous system, have general roles in the modulation of synapse formation and synaptic transmission, blood-brain barrier formation, and regulation of blood flow, as well as metabolic support of other brain resident cells. Crucially, emerging evidence shows specific adaptations and astrocyte-encoded functions in regions, such as the spinal cord and cerebellum. To investigate the true extent of astrocyte molecular diversity across forebrain regions, we used single-cell RNA sequencing. Our analysis identifies five transcriptomically distinct astrocyte subtypes in adult mouse cortex and hippocampus. Validation of our data in situ reveals distinct spatial positioning of defined subtypes, reflecting the distribution of morphologically and physiologically distinct astrocyte populations. Our findings are evidence for specialized astrocyte subtypes between and within brain regions. The data are available through an online database (https://holt-sc.glialab.org/), providing a resource on which to base explorations of local astrocyte diversity and function in the brain.

The Role of Astrocytic Calcium Signaling in the Aged Prefrontal Cortex.

Aging is a lifelong process characterized by cognitive decline putatively due to structural and functional changes of neural circuits of the brain. Neuron-glial signaling is a fundamental component of structure and function of circuits of the brain, and yet its possible role in aging remains elusive. Significantly, neuron-glial networks of the prefrontal cortex undergo age-related alterations that can affect cognitive function, and disruption of glial calcium signaling has been linked with cognitive performance. Motivated by these observations, we explored the possible role of glia in cognition during aging, considering a mouse model where astrocytes lacked IP3R2-dependent Ca2+ signaling. Contrarily to aged wild-type animals that showed significant impairment in a two-trial place recognition task, aged IP3R2 KO mice did not. Consideration of neuronal and astrocytic cell densities in the prefrontal cortex, revealed that aged IP3R2 KO mice present decreased densities of NeuN+ neurons and increased densities of S100ß+ astrocytes. Moreover, aged IP3R2 KO mice display refined dendritic trees in this region. These findings suggest a novel role for astrocytes in the aged brain. Further evaluation of the neuron-glial interactions in the aged brain will disclose novel strategies to handle healthy cognitive aging in humans.

Employing an open-source tool to assess astrocyte tridimensional structure.

Astrocytes display important features that allow them to maintain a close dialog with neurons, ultimately impacting brain function. The complex morphological structure of astrocytes is crucial to the role of astrocytes in brain networks. Therefore, assessing morphologic features of astrocytes will help provide insights into their physiological relevance in healthy and pathological conditions. Currently available tools that allow the tridimensional reconstruction of astrocytes present a number of disadvantages, including the need for advanced computational skills and powerful hardware, and are either time-consuming or costly. In this study, we optimized and validated the FIJI-ImageJ, Simple Neurite Tracer (SNT) plugin, an open-source software that aids in the reconstruction of GFAP-stained structure of astrocytes. We describe (1) the loading of confocal microscopy Z-stacks, (2) the selection criteria, (3) the reconstruction process, and (4) the post-reconstruction analysis of morphological features (process length, number, thickness, and arbor complexity). SNT allows the quantification of astrocyte morphometric parameters in a simple, efficient, and semi-automated manner. While SNT is simple to learn, and does not require advanced computational skills, it provides reproducible results, in different brain regions or pathophysiological states.

Multimodal exercise program contributes to balance and motor functions in men and women with Parkinson's disease differently: an intervention study

Abstract Aim: To verify the effects of a multimodal exercise program on balance and motor functions, and the differences by sex, in people with Parkinson's disease (PD). Methods: The intervention study, was composed of 16 people with PD, that were assessed before and after 16 weeks of interventions with the multimodal exercise program. The effects were analyzed generally and by sex, using the Wilcoxon Test. The significance level was established at 5%. Results: Overall, there was an improvement in the strength of the lower limbs (LL) (p = 0.035) and upper limbs (UL) (p = 0.009), functional mobility (p = 0.003), gait (p = 0.050), balance (p = 0.001) and in motor scores of UPDRS III (p = 0.005), which categorize motor symptoms of the disease. In regards to sex, women affected muscle strength (p = 0.044) and flexibility of LL (p = 0.028), gait (p = 0.018), and motor aspects of the UPDRS III (p = 0.042). The men presented effects in muscle strength of the UL (p = 0.042). Women and men had a significant increase in functional mobility (p = 0.046 and p = 0.027, respectively) and in balance (p = 0.012 and p = 0.042, respectively). There was no significant difference for both sexes, in body mass and the reach behind the backtest. Conclusion: the multimodal exercise program contributed to the improvement in motor function and balance in men and women with PD. Nevertheless, the effects were more significant in women. The comprehension of the differences between men and women grants us a more directional and efficient approach to their treatment.

Superficial siderosis of the central nervous system is a rare and possibly underdiagnosed disorder.

METHODS: Series of cases collected from Brazilian centers. RESULTS: We studied 13 cases of patients presenting with progressive histories of neurological dysfunction caused by SS-CNS. The most frequent clinical findings in these patients were progressive gait ataxia, hearing loss, hyperreflexia and cognitive dysfunction. The diagnoses of SS-CNS were made seven months to 30 years after the disease onset. CONCLUSION: SS-CNS is a rare disease that may remain undiagnosed for long periods. Awareness of this condition is essential for the clinician.

Absence of Tau triggers age-dependent sciatic nerve morphofunctional deficits and motor impairment.

Dementia is the cardinal feature of Alzheimer's disease (AD), yet the clinical symptoms of this disorder also include a marked loss of motor function. Tau abnormal hyperphosphorylation and malfunction are well-established key events in AD neuropathology but the impact of the loss of normal Tau function in neuronal degeneration and subsequent behavioral deficits is still debated. While Tau reduction has been increasingly suggested as therapeutic strategy against neurodegeneration, particularly in AD, there is controversial evidence about whether loss of Tau progressively impacts on motor function arguing about damage of CNS motor components. Using a variety of motor-related tests, we herein provide evidence of an age-dependent motor impairment in Tau-/- animals that is accompanied by ultrastructural and functional impairments of the efferent fibers that convey motor-related information. Specifically, we show that the sciatic nerve of old (17-22-months) Tau-/- mice displays increased degenerating myelinated fibers and diminished conduction properties, as compared to age-matched wild-type (Tau+/+) littermates and younger (4-6 months) Tau-/- and Tau+/+ mice. In addition, the sciatic nerves of Tau-/- mice exhibit a progressive hypomyelination (assessed by g-ratio) specifically affecting large-diameter, motor-related axons in old animals. These findings suggest that loss of Tau protein may progressively impact on peripheral motor system.

Do stars govern our actions? Astrocyte involvement in rodent behavior.

Astrocytes have emerged as important partners of neurons in information processing. Important progress has been made in the past two decades in understanding the role of astrocytes in the generation of neuron-astrocyte network outputs resulting in behavior. We review evidence for astrocyte involvement across four different behavioral domains: cognition, emotion, motor, and sensory processing. Accumulating evidence from animal models has provided a wealth of data that largely supports a direct involvement of astrocytes on diverse aspects of behavior. The development of tools for selectively controlling the temporal and spatial properties of astrocyte activity will help to consolidate our knowledge of the mechanisms underlying this involvement.