Estimating the glutamate transporter surface density in distinct sub-cellular compartments of mouse hippocampal astrocytes.

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, point to the existence of stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft.

Late adolescence mortality in mice with brain-specific deletion of the volume-regulated anion channel subunit LRRC8A.

The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is critical for cell volume control, but its broader physiological functions remain under investigation. Recent studies in the field indicate that Lrrc8a disruption in the brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated brain-wide conditional LRRC8A knockout mice (LRRC8A bKO) using NestinCre -driven Lrrc8aflox/flox excision in neurons, astrocytes, and oligodendroglia. LRRC8A bKO animals were born close to the expected Mendelian ratio and developed without overt histological abnormalities, but, surprisingly, all died between 5 and 9 weeks of age with a seizure phenotype, which was confirmed by video and EEG recordings. Brain slice electrophysiology detected changes in the excitability of pyramidal cells and modified GABAergic inputs in the hippocampal CA1 region of LRRC8A bKO. LRRC8A-null hippocampi showed increased immunoreactivity of the astrocytic marker GFAP, indicating reactive astrogliosis. We also found decreased whole-brain protein levels of the GABA transporter GAT-1, the glutamate transporter GLT-1, and the astrocytic enzyme glutamine synthetase. Complementary HPLC assays identified reduction in the tissue levels of the glutamate and GABA precursor glutamine. Together, these findings suggest that VRAC provides vital control of brain excitability in mouse adolescence. VRAC deletion leads to a lethal phenotype involving progressive astrogliosis and dysregulation of astrocytic uptake and supply of amino acid neurotransmitters and their precursors.

Neuronal Glutamate Transporters Control Dopaminergic Signaling and Compulsive Behaviors.

There is an ongoing debate on the contribution of the neuronal glutamate transporter EAAC1 to the onset of compulsive behaviors. Here, we used behavioral, electrophysiological, molecular, and viral approaches in male and female mice to identify the molecular and cellular mechanisms by which EAAC1 controls the execution of repeated motor behaviors. Our findings show that, in the striatum, a brain region implicated with movement execution, EAAC1 limits group I metabotropic glutamate receptor (mGluRI) activation, facilitates D1 dopamine receptor (D1R) expression, and ensures long-term synaptic plasticity. Blocking mGluRI in slices from mice lacking EAAC1 restores D1R expression and synaptic plasticity. Conversely, activation of intracellular signaling pathways coupled to mGluRI in D1R-containing striatal neurons of mice expressing EAAC1 leads to reduced D1R protein level and increased stereotyped movement execution. These findings identify new molecular mechanisms by which EAAC1 can shape glutamatergic and dopaminergic signals and control repeated movement execution.SIGNIFICANCE STATEMENT Genetic studies implicate Slc1a1, a gene encoding the neuronal glutamate transporter EAAC1, with obsessive-compulsive disorder (OCD). EAAC1 is abundantly expressed in the striatum, a brain region that is hyperactive in OCD. What remains unknown is how EAAC1 shapes synaptic function in the striatum. Our findings show that EAAC1 limits activation of metabotropic glutamate receptors (mGluRIs) in the striatum and, by doing so, promotes D1 dopamine receptor (D1R) expression. Targeted activation of signaling cascades coupled to mGluRIs in mice expressing EAAC1 reduces D1R expression and triggers repeated motor behaviors. These findings provide new information on the molecular basis of OCD and suggest new avenues for its treatment.

NMDA receptor blockade ameliorates abnormalities of spike firing of subthalamic nucleus neurons in a parkinsonian nonhuman primate.

N-methyl-D-aspartate receptors (NMDARs) are ion channels comprising tetrameric assemblies of GluN1 and GluN2 receptor subunits that mediate excitatory neurotransmission in the central nervous system. Of the four different GluN2 subunits, the GluN2D subunit-containing NMDARs have been suggested as a target for antiparkinsonian therapy because of their expression pattern in some of the basal ganglia nuclei that show abnormal firing patterns in the parkinsonian state, specifically the subthalamic nucleus (STN). In this study, we demonstrate that blockade of NMDARs altered spike firing in the STN in a male nonhuman primate that had been rendered parkinsonian by treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. In accompanying experiments in male rodents, we found that GluN2D-NMDAR expression in the STN was reduced in acutely or chronically dopamine-depleted animals. Taken together, our data suggest that blockade of NMDARs in the STN may be a viable antiparkinsonian strategy, but that the ultimate success of this approach may be complicated by parkinsonism-associated changes in NMDAR expression in the STN.

Astrocytes and the Warning Signs of Intracerebral Hemorrhagic Stroke.

Two decades into the two thousands, intracerebral hemorrhagic stroke (ICH) continues to reap lives across the globe. In the US, nearly 12,000 people suffer from ICH every year. Half of them survive, but many are left with permanent physical and cognitive disabilities, the severity of which depends on the location and broadness of the brain region affected by the hemorrhage. The ongoing efforts to identify risk factors for hemorrhagic stroke have been instrumental for the development of new medical practices to prevent, aid the recovery and reduce the risk of recurring ICH. Recent efforts approach the study of ICH from a different angle, providing information on how we can limit brain damage by manipulating astrocyte receptors. These results provide a novel understanding of how astrocytes contribute to brain injury and recovery from small ICH. Here, we discuss current knowledge on the risk factors and molecular pathology of ICH and the functional properties of astrocytes and their role in ICH. Last, we discuss candidate astrocyte receptors that may prove to be valuable therapeutic targets to treat ICH. Together, these findings provide basic and clinical scientists useful information for the future development of strategies to improve the detection of small ICH, limit brain damage, and prevent the onset of more severe episodes of brain hemorrhage.

M-Track: A New Software for Automated Detection of Grooming Trajectories in Mice.

Grooming is a complex and robust innate behavior, commonly performed by most vertebrate species. In mice, grooming consists of a series of stereotyped patterned strokes, performed along the rostro-caudal axis of the body. The frequency and duration of each grooming episode is sensitive to changes in stress levels, social interactions and pharmacological manipulations, and is therefore used in behavioral studies to gain insights into the function of brain regions that control movement execution and anxiety. Traditional approaches to analyze grooming rely on manually scoring the time of onset and duration of each grooming episode, and are often performed on grooming episodes triggered by stress exposure, which may not be entirely representative of spontaneous grooming in freely-behaving mice. This type of analysis is time-consuming and provides limited information about finer aspects of grooming behaviors, which are important to understand movement stereotypy and bilateral coordination in mice. Currently available commercial and freeware video-tracking software allow automated tracking of the whole body of a mouse or of its head and tail, not of individual forepaws. Here we describe a simple experimental set-up and a novel open-source code, named M-Track, for simultaneously tracking the movement of individual forepaws during spontaneous grooming in multiple freely-behaving mice. This toolbox provides a simple platform to perform trajectory analysis of forepaw movement during distinct grooming episodes. By using M-track we show that, in C57BL/6 wild type mice, the speed and bilateral coordination of the left and right forepaws remain unaltered during the execution of distinct grooming episodes. Stress exposure induces a profound increase in the length of the forepaw grooming trajectories. M-Track provides a valuable and user-friendly interface to streamline the analysis of spontaneous grooming in biomedical research studies.

Amyloid-ß1-42 slows clearance of synaptically released glutamate by mislocalizing astrocytic GLT-1.

GLT-1, the major glutamate transporter in the adult brain, is abundantly expressed in astrocytic processes enveloping synapses. By limiting glutamate escape into the surrounding neuropil, GLT-1 preserves the spatial specificity of synaptic signaling. Here we show that the amyloid-ß peptide Aß1-42 markedly prolongs the extracellular lifetime of synaptically released glutamate by reducing GLT-1 surface expression in mouse astrocytes and that this effect is prevented by the vitamin E derivative Trolox. These findings indicate that astrocytic glutamate transporter dysfunction may play an important role in the pathogenesis of Alzheimer's disease and suggest possible mechanisms by which several current treatment strategies could protect against the disease.

The number and organization of Ca2+ channels in the active zone shapes neurotransmitter release from Schaffer collateral synapses.

Fast synaptic transmission requires tight colocalization of Ca(2+) channels and neurotransmitter vesicles. It is generally thought that Ca(2+) channels are expressed abundantly in presynaptic active zones, that vesicles within the same active zone have similar release properties, and that significant vesicle depletion only occurs at synapses with high release probability. Here we show, at excitatory CA3→CA1 synapses in mouse hippocampus, that release from individual vesicles is generally triggered by only one Ca(2+) channel and that only few functional Ca(2+) channels may be spread in the active zone at variable distances to neighboring neurotransmitter vesicles. Using morphologically realistic Monte Carlo simulations, we show that this arrangement leads to a widely heterogeneous distribution of release probability across the vesicles docked at the active zone, and that depletion of the vesicles closest to Ca(2+) channels can account for the Ca(2+) dependence of short-term plasticity at these synapses. These findings challenge the prevailing view that efficient synaptic transmission requires numerous presynaptic Ca(2+) channels in the active zone, and indicate that the relative arrangement of Ca(2+) channels and vesicles contributes to the heterogeneity of release probability within and across synapses and to vesicle depletion at small central synapses with low average release probability.

A TRP among the astrocytes.

TRP channels were first identified as membrane proteins mediating phototransduction in fruit flies. Astrocytes were initially referred to as the silent elements of the nervous system. At the time these discoveries were made, few would have suspected TRP channels and astrocytes could contribute significantly to our understanding of brain signalling. Recent findings, however, put TRP channels and astrocytes in the spotlight, describe their ability to modulate the activity of specific sets of synapses, and raise some interesting questions. What makes astrocytes capable of exerting cell-specific effects on interneuronal signals? How do different synapses respond to changes in astrocytic function and in the local micro-structure of the neuropil? Can astrocytes be considered good candidate targets for therapeutic intervention to treat neurological diseases? Here I discuss the recent developments on TRP channels and astrocytes that have made us aware of the many structural and functional features of synapses that still need to be discovered and that could lead a new avant-garde in decoding the cellular and molecular basis of brain (dys)function.

NRN-EZ: an application to streamline biophysical modeling of synaptic integration using NEURON.

One of the fundamental goals in neuroscience is to determine how the brain processes information and ultimately controls the execution of complex behaviors. Over the past four decades, there has been a steady growth in our knowledge of the morphological and functional diversity of neurons, the building blocks of the brain. These cells clearly differ not only for their anatomy and ion channel distribution, but also for the type, strength, location, and temporal pattern of activity of the many synaptic inputs they receive. Compartmental modeling programs like NEURON have become widely used in the neuroscience community to address a broad range of research questions, including how neurons integrate synaptic inputs and propagate information through complex neural networks. One of the main strengths of NEURON is its ability to incorporate user-defined information about the realistic morphology and biophysical properties of different cell types. Although the graphical user interface of the program can be used to run initial exploratory simulations, introducing a stochastic representation of synaptic weights, locations and activation times typically requires users to develop their own codes, a task that can be overwhelming for some beginner users. Here we describe NRN-EZ, an interactive application that allows users to specify complex patterns of synaptic input activity that can be integrated as part of NEURON simulations. Through its graphical user interface, NRN-EZ aims to ease the learning curve to run computational models in NEURON, for users that do not necessarily have a computer science background.

Neuronal glutamate transporters control reciprocal inhibition and gain modulation in D1 medium spiny neurons.

Understanding the function of glutamate transporters has broad implications for explaining how neurons integrate information and relay it through complex neuronal circuits. Most of what is currently known about glutamate transporters, specifically their ability to maintain glutamate homeostasis and limit glutamate diffusion away from the synaptic cleft, is based on studies of glial glutamate transporters. By contrast, little is known about the functional implications of neuronal glutamate transporters. The neuronal glutamate transporter EAAC1 is widely expressed throughout the brain, particularly in the striatum, the primary input nucleus of the basal ganglia, a region implicated with movement execution and reward. Here, we show that EAAC1 limits synaptic excitation onto a population of striatal medium spiny neurons identified for their expression of D1 dopamine receptors (D1-MSNs). In these cells, EAAC1 also contributes to strengthen lateral inhibition from other D1-MSNs. Together, these effects contribute to reduce the gain of the input-output relationship and increase the offset at increasing levels of synaptic inhibition in D1-MSNs. By reducing the sensitivity and dynamic range of action potential firing in D1-MSNs, EAAC1 limits the propensity of mice to rapidly switch between behaviors associated with different reward probabilities. Together, these findings shed light on some important molecular and cellular mechanisms implicated with behavior flexibility in mice.

Regulation of Glutamate, GABA and Dopamine Transporter Uptake, Surface Mobility and Expression.

Neurotransmitter transporters limit spillover between synapses and maintain the extracellular neurotransmitter concentration at low yet physiologically meaningful levels. They also exert a key role in providing precursors for neurotransmitter biosynthesis. In many cases, neurons and astrocytes contain a large intracellular pool of transporters that can be redistributed and stabilized in the plasma membrane following activation of different signaling pathways. This means that the uptake capacity of the brain neuropil for different neurotransmitters can be dynamically regulated over the course of minutes, as an indirect consequence of changes in neuronal activity, blood flow, cell-to-cell interactions, etc. Here we discuss recent advances in the mechanisms that control the cell membrane trafficking and biophysical properties of transporters for the excitatory, inhibitory and modulatory neurotransmitters glutamate, GABA, and dopamine.

Transplantable human motor networks as a neuron-directed strategy for spinal cord injury.

To repair neural circuitry following spinal cord injury (SCI), neural stem cell (NSC) transplantation has held a primary focus; however, stochastic outcomes generate challenges driven in part by NSC differentiation and tumor formation. The recent ability to generate regionally specific neurons and their support cells now allows consideration of directed therapeutic approaches with pre-differentiated and networked spinal neural cells. Here, we form encapsulated, transplantable neuronal networks of regionally matched cervical spinal motor neurons, interneurons, and oligodendrocyte progenitor cells derived through trunk-biased neuromesodermal progenitors. We direct neurite formation in alginate-based neural ribbons to generate electrically active, synaptically connected networks, characterized by electrophysiology and calcium imaging before transplantation into rodent models of contused SCI for evaluation at 10-day and 6-week timepoints. The in vivo analyses demonstrate viability and retention of interconnected synaptic networks that readily integrate with the host parenchyma to advance goals of transplantable neural circuitry for SCI treatment.

Neuronal transporters regulate glutamate clearance, NMDA receptor activation, and synaptic plasticity in the hippocampus.

In the mammalian brain, the specificity of excitatory synaptic transmission depends on rapid diffusion of glutamate away from active synapses and the powerful uptake capacity of glutamate transporters in astrocytes. The extent to which neuronal glutamate transporters influence the lifetime of glutamate in the extracellular space remains unclear. Here we show that EAAC1, the predominant neuronal glutamate transporter at excitatory synapses in hippocampal area CA1, buffers glutamate released during synaptic events and prolongs the time course of its clearance by astrocytes. EAAC1 does not significantly alter activation of receptors in the synaptic cleft. Instead, it reduces recruitment of perisynaptic/extrasynaptic NR2B-containing NMDARs, thereby facilitating induction of long-term potentiation by short bursts of high-frequency stimulation. We describe novel roles of EAAC1 in regulating glutamate diffusion and propose that NMDARs at different subsynaptic locations can make distinct contributions to the regulation of synaptic strength.

Current knowledge of SLC6A1-related neurodevelopmental disorders.

Advances in gene discovery have identified genetic variants in the solute carrier family 6 member 1 gene as a monogenic cause of neurodevelopmental disorders, including epilepsy with myoclonic atonic seizures, autism spectrum disorder and intellectual disability. The solute carrier family 6 member 1 gene encodes for the GABA transporter protein type 1, which is responsible for the reuptake of the neurotransmitter GABA, the primary inhibitory neurotransmitter in the central nervous system, from the extracellular space. GABAergic inhibition is essential to counterbalance neuronal excitation, and when significantly disrupted, it negatively impacts brain development leading to developmental differences and seizures. Aggregation of patient variants and observed clinical manifestations expand understanding of the genotypic and phenotypic spectrum of this disorder. Here, we assess genetic and phenotypic features in 116 individuals with solute carrier family 6 member 1 variants, the vast majority of which are likely to lead to GABA transporter protein type 1 loss-of-function. The knowledge acquired will guide therapeutic decisions and the development of targeted therapies that selectively enhance transporter function and may improve symptoms. We analysed the longitudinal and cell type-specific expression of solute carrier family 6 member 1 in humans and localization of patient and control missense variants in a novel GABA transporter protein type 1 protein structure model. In this update, we discuss the progress made in understanding and treating solute carrier family 6 member 1-related disorders thus far, through the concerted efforts of clinicians, scientists and family support groups.

Circadian Modulation of Neurons and Astrocytes Controls Synaptic Plasticity in Hippocampal Area CA1.

Most animal species operate according to a 24-h period set by the suprachiasmatic nucleus (SCN) of the hypothalamus. The rhythmic activity of the SCN modulates hippocampal-dependent memory, but the molecular and cellular mechanisms that account for this effect remain largely unknown. Here, we identify cell-type-specific structural and functional changes that occur with circadian rhythmicity in neurons and astrocytes in hippocampal area CA1. Pyramidal neurons change the surface expression of NMDA receptors. Astrocytes change their proximity to synapses. Together, these phenomena alter glutamate clearance, receptor activation, and integration of temporally clustered excitatory synaptic inputs, ultimately shaping hippocampal-dependent learning in vivo. We identify corticosterone as a key contributor to changes in synaptic strength. These findings highlight important mechanisms through which neurons and astrocytes modify the molecular composition and structure of the synaptic environment, contribute to the local storage of information in the hippocampus, and alter the temporal dynamics of cognitive processing.

Receptor actions of synaptically released glutamate: the role of transporters on the scale from nanometers to microns.

Actions of the excitatory neurotransmitter glutamate inside and outside the synaptic cleft determine the activity of neural circuits in the brain. However, to what degree local glutamate transporters affect these actions on a submicron scale remains poorly understood. Here we focus on hippocampal area CA1, a common subject of synaptic physiology studies. First, we use a two-photon excitation technique to obtain an estimate of the apparent (macroscopic) extracellular diffusion coefficient for glutamate, approximately 0.32 mum(2)/ms. Second, we incorporate this measurement into a Monte Carlo model of the typical excitatory synapse and examine the influence of distributed glutamate transporter molecules on signal transmission. Combined with the results of whole-cell recordings, such simulations argue that, although glutamate transporters have little effect on the activation of synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, this does not rule out the occurrence of up to several dozens of transporters inside the cleft. We further evaluate how the expression pattern of transporter molecules (on the 10-100 nm scale) affects the activation of N-methyl-D-aspartic acid or metabotropic glutamate receptors in the synaptic vicinity. Finally, we extend our simulations to the macroscopic scale, estimating that synaptic activity sufficient to excite principal neurons could intermittently raise extracellular glutamate to approximately 1 muM only at sparse (microns apart) hotspots. Greater rises of glutamate occur only when <5% of transporters are available (for instance, when an astrocyte fails). The results provide a quantitative framework for a better understanding of the relationship between glutamate transporters and glutamate receptor signaling.

Astrocytic Coverage of Dendritic Spines, Dendritic Shafts, and Axonal Boutons in Hippocampal Neuropil.

Distal astrocytic processes have a complex morphology, reminiscent of branchlets and leaflets. Astrocytic branchlets are rod-like processes containing mitochondria and endoplasmic reticulum, capable of generating inositol-3-phosphate (IP3)-dependent Ca2+ signals. Leaflets are small and flat processes that protrude from branchlets and fill the space between synapses. Here we use three-dimensional (3D) reconstructions from serial section electron microscopy (EM) of rat CA1 hippocampal neuropil to determine the astrocytic coverage of dendritic spines, shafts and axonal boutons. The distance to the maximum of the astrocyte volume fraction (VF) correlated with the size of the spine when calculated from the center of mass of the postsynaptic density (PSD) or from the edge of the PSD, but not from the spine surface. This suggests that the astrocytic coverage of small and larger spines is similar in hippocampal neuropil. Diffusion simulations showed that such synaptic microenvironment favors glutamate spillover and extrasynaptic receptor activation at smaller spines. We used complexity and entropy measures to characterize astrocytic branchlets and leaflets. The 2D projections of astrocytic branchlets had smaller spatial complexity and entropy than leaflets, consistent with the higher structural complexity and less organized distribution of leaflets. The VF of astrocytic leaflets was highest around dendritic spines, lower around axonal boutons and lowest around dendritic shafts. In contrast, the VF of astrocytic branchlets was similarly low around these three neuronal compartments. Taken together, these results suggest that astrocytic leaflets preferentially contact synapses as opposed to the dendritic shaft, an arrangement that might favor neurotransmitter spillover and extrasynaptic receptor activation along dendritic shafts.

Glutamatergic Tuning of Hyperactive Striatal Projection Neurons Controls the Motor Response to Dopamine Replacement in Parkinsonian Primates.

Dopamine (DA) loss in Parkinson's disease (PD) alters the function of striatal projection neurons (SPNs) and causes motor deficits, but DA replacement can induce further abnormalities. A key pathological change in animal models and patients is SPN hyperactivity; however, the role of glutamate in altered DA responses remains elusive. We tested the effect of locally applied AMPAR or NMDAR antagonists on glutamatergic signaling in SPNs of parkinsonian primates. Following a reduction in basal hyperactivity by antagonists at either receptor, DA inputs induced SPN firing changes that were stable during the entire motor response, in clear contrast with the typically unstable effects. The SPN activity reduction over an extended putamenal area controlled the release of involuntary movements in the "on" state and therefore improved motor responses to DA replacement. These results demonstrate the pathophysiological role of upregulated SPN activity and support strategies to reduce striatal glutamate signaling for PD therapy.