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The Indirect Neuron-astrocyte Coculture Assay: An <em>In Vitro</em> Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Ruhr-University

The concept of the tripartite synapse proposes that in addition to the presynapse and the postsynaptic membrane closely apposed processes of astrocytes constitute an integral part of the synapse. Accordingly, astrocytes may influence synaptic activity by various ways. Thus glia- and neuron-derived neurotrophins, cytokines and metabolites influence neuronal survival, synaptic activity and plasticity. Beyond these facts, the past years have shown that astrocytes are required for synaptogenesis, the structural maintenance and proper functioning of synapses. In particular, astrocytes seem to play a key role in the organization of the brain's extracellular matrix (ECM) - most prominently the so-called perineuronal nets (PNNs), complex macromolecular assemblies of ECM components. Due to progress in cellular and molecular neurosciences, it has been possible to decipher the composition of ECM structures and to obtain insight into their function(s) and underlying mechanisms. It appears that PNN-related structures are involved in regulating the sprouting and pruning of synapses, which represents an important morphological correlate of synaptic plasticity in the adult nervous system. Perturbation assays and gene elimination by recombinant techniques have provided clear indications that astrocyte-derived ECM components, e.g. the tenascins and chondroitinsulfate proteoglycans (CSPGs) of the lectican family participate in these biological functions. The present review will discuss the glia-derived glycoproteins and CSPGs of the perisynaptic ECM, their neuronal and glial receptors, and in vitro assays to test their physiological functions in the framework of the synapse, the pivotal element of communication in the central nervous system.

Contributions of astrocytes to synapse formation and maturation - Potential functions of the perisynaptic extracellular matrix.

It has been shown that astrocyte-derived extracellular matrix (ECM) is important for formation and maintenance of CNS synapses. In order to study the effects of glial-derived ECM on synaptogenesis, E18 rat hippocampal neurons and primary astrocytes were co-cultivated using a cell-insert system. Under these conditions, neurons differentiated under low density conditions (3500 cells/cm(2) ) in defined, serum-free medium and in the absence of direct, membrane-mediated neuron-astrocyte interactions. Astrocytes promoted the formation of structurally intact synapses, as documented by the co-localisation of bassoon- and ProSAP1/Shank2-positive puncta, markers of the pre- and postsynapse, respectively. The development of synapses was paralleled by the emergence of perineuronal net (PNN)-like structures that contained various ECM components such as hyaluronic acid, brevican and neurocan. In order to assess potential functions for synaptogenesis, the ECM was removed by treatment with hyaluronidase or chondroitinase ABC. Both enzymes significantly enhanced the number of synaptic puncta. Whole-cell voltage-clamp recordings of control and enzyme-treated hippocampal neurons revealed that chondroitinase ABC treatment led to a significant decrease in amplitude and a reduced charge of miniature excitatory postsynaptic currents, whereas inhibitory postsynaptic currents were not affected. When the response to the application of glutamate was measured, a reduced sensitivity could be detected and resulted in decreased currents in response to the excitatory neurotransmitter. These findings are consistent with the interpretation that the ECM partakes in the regulation of the density of glutamate receptors in subsynaptic sites.

Chondroitin sulfate proteoglycans regulate astrocyte-dependent synaptogenesis and modulate synaptic activity in primary embryonic hippocampal neurons.

Intraperitoneal transplantation of human umbilical cord blood (hUCB) cells has been shown to reduce sensorimotor deficits after hypoxic ischemic brain injury in neonatal rats. However, the neuronal correlate of the functional recovery and how such a treatment enforces plastic remodelling at the level of neural processing remains elusive. Here we show by in-vivo recordings that hUCB cells have the capability of ameliorating the injury-related impairment of neural processing in primary somatosensory cortex. Intact cortical processing depends on a delicate balance of inhibitory and excitatory transmission, which is disturbed after injury. We found that the dimensions of cortical maps and receptive fields, which are significantly altered after injury, were largely restored. Additionally, the lesion induced hyperexcitability was no longer observed in hUCB treated animals as indicated by a paired-pulse behaviour resembling that observed in control animals. The beneficial effects on cortical processing were reflected in an almost complete recovery of sensorimotor behaviour. Our results demonstrate that hUCB cells reinstall the way central neurons process information by normalizing inhibitory and excitatory processes. We propose that the intermediate level of cortical processing will become relevant as a new stage to investigate efficacy and mechanisms of cell therapy in the treatment of brain injury.

Human umbilical cord blood cells restore brain damage induced changes in rat somatosensory cortex.

Microelectrode arrays (MEAs) are widely used to investigate neuronal network activity in vitro at multiple sites. While this system has been successfully used with primary embryonic rat hippocampal or cortical neurons, its applicability for mouse hippocampal neurons has so far not been reported in detail. As mouse genetics offer a large variety of models, it is highly desirable to close this gap. For that purpose, we established and characterized an indirect co-culture assay of mouse hippocampal neurons in the presence of astrocytes on MEAs. Embryonic day 15.5 (E15.5) mouse hippocampal neurons were cultivated on MEAs in completely defined medium. We show, that the co-culture with postnatal primary mouse astrocytes allows the establishment and the maintenance of neuronal networks under these conditions. We were able to cultivate the neurons for at least 28 days in vitro (DIV) and observed the first neuronal network activity around 7 DIV. Hippocampal neurons showed early bursting behavior and synchronous activity that evolved further with increasing time in culture. The application of bicuculline increased network activity, which revealed the presence of active GABAergic interneurons. Taken together, this study provides a novel MEA-based assay for investigating the activity in neuronal networks in an indirect neuron-astrocyte co-culture setting, and leads to first insights into the physiological development of mouse hippocampal neurons under these conditions in vitro.

A new indirect co-culture set up of mouse hippocampal neurons and cortical astrocytes on microelectrode arrays.

The extracellular matrix (ECM) of the brain plays crucial roles during the development, maturation, and regeneration of the CNS. In a subpopulation of neurons, the ECM condenses to superstructures called perineuronal nets (PNNs) that surround synapses. Camillo Golgi described PNNs a century ago, yet their biological functions remain elusive. Here, we studied a mouse mutant that lacks four ECM components highly enriched in the developing brain: the glycoproteins tenascin-C and tenascin-R and the chondroitin sulfate proteoglycans brevican and neurocan. Primary embryonic hippocampal neurons and astrocytes were cultivated using a cell insert system that allows for co-culture of distinct cell populations in the absence of direct membrane contacts. The wild-type and knock-out cells were combined in the four possible permutations. Using this approach, neurons cultivated in the presence of mutant astrocytes displayed a transient increase of synapses after 2 weeks. However, after a period of 3 weeks or longer, synapse formation and stabilization were compromised when either neuron or astrocyte cell populations or both were of mutant origin. The development of PNN structures was observed, but their size was substantially reduced on knock-out neurons. The synaptic activity of both wild-type and knock-out neurons was monitored using whole-cell patch clamping. The salient observation was a reduced frequency of IPSCs and EPSCs, whereas the amplitudes were not modified. Remarkably, the knock-out neuron phenotypes could not be rescued by wild-type astrocytes. We conclude that the elimination of four ECM genes compromises neuronal function.

Primary hippocampal neurons, which lack four crucial extracellular matrix molecules, display abnormalities of synaptic structure and function and severe deficits in perineuronal net formation.

The therapy of patients suffering the psychiatric disorder schizophrenia requires the usage of antipsychotic drugs that are classified into two different groups, the first-generation (FGAs) and the second-generation antipsychotics (SGAs). This study compares the effects of the two FGAs haloperidol and flupentixol with those of the SGA olanzapine on synapse formation and synaptic activity of embryonic rat hippocampal neurons. To this end, the development of perineuronal nets (PNNs), the formation of synapses and the resulting spontaneous network activity under control and treatment conditions were studied using an indirect co-culture system of neurons and astrocytes in completely defined media. The number and extent of PNNs that consist of extracellular matrix superstructures surrounding synapses was not altered in hippocampal neurons by exposure to antipsychotic drugs. In contrast treatment of hippocampal neurons with haloperidol led to a slight decrease whereas olanzapine induced a significant increase of the number of structural synapses after 13days. This differential effect concerning synapse numbers was also reflected in the spontaneous activity of neuronal networks, as monitored on multielectrode arrays (MEAs). In that context, application of haloperidol reduced while olanzapine significantly enhanced network activity. Unexpectedly, flupentixol that is regarded as an FGA caused similar effects than the SGA olanzapine in that it augmented synapse number as well as network activity. Our pilot study provides a proof of concept that the neuron-astrocyte co-culture model can be used to investigate the impact of antipsychotics on pivotal parameters of neuronal cell biology. Thereby, it may support the comparative analysis of antipsychotics applied in the therapy of schizophrenia.

Article	Year
Contributions of astrocytes to synapse formation and maturation - Potential functions of the perisynaptic extracellular matrix. Brain research reviews\| PubMed ID: 20096729	2010
Chondroitin sulfate proteoglycans regulate astrocyte-dependent synaptogenesis and modulate synaptic activity in primary embryonic hippocampal neurons. The European journal of neuroscience\| PubMed ID: 21615557	2011
Human umbilical cord blood cells restore brain damage induced changes in rat somatosensory cortex. PloS one\| PubMed ID: 21673795	2011
A new indirect co-culture set up of mouse hippocampal neurons and cortical astrocytes on microelectrode arrays. Journal of neuroscience methods\| PubMed ID: 22182586	2012
Primary hippocampal neurons, which lack four crucial extracellular matrix molecules, display abnormalities of synaptic structure and function and severe deficits in perineuronal net formatio... The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 23637166	2013
First and second generation antipsychotics differentially affect structural and functional properties of rat hippocampal neuron synapses. Neuroscience\| PubMed ID: 27615033	2016

Maren Geissler

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