Differential expression of the GABA transporters GAT-1 and GAT-3 in brains of rats, cats, monkeys and humans

The homeostasis of GABA is critical to normal brain function. Extracellular levels of GABA are regulated mainly by plasmalemmal gamma-aminobutyric acid (GABA) transporters. Whereas the expression of GABA transporters has been extensively studied in rodents, validation of this data in other species, including humans, has been limited. As this information is crucial for our understanding of therapeutic options in human diseases such as epilepsy, we have compared, by immunocytochemistry, the distributions of the GABA transporters GAT-1 and GAT-3 in rats, cats, monkeys and humans. We demonstrate subtle differences between the results reported in the literature and our results, such as the predominance of GAT-1 labelling in neurons rather than astrocytes in the rat cortex. We note that the optimal localisation of GAT-1 in cats, monkeys and humans requires the use of an antibody against the human sequence carboxyl terminal region of GAT-1 rather than against the slightly different rat sequence. We demonstrate that GAT-3 is localised mainly to astrocytes in hindbrain and midbrain regions of rat brains. However, in species such as cats, monkeys and humans, additional strong immunolabelling of oligodendrocytes has also been observed. We suggest that differences in GAT distribution, especially the expression of GAT-3 by oligodendrocytes in humans, must be accommodated in extrapolating rodent models of GABA homeostasis to humans.

Quantitative analysis of immunolabeling for serotonin and for glutamate transporters after administration of imipramine and citalopram

Serotonin (5-hydroxytryptamine, 5-HT) is an amine neurotransmitter derived from tryptophan and is important in brain systems regulating mood, emotional behavior, and sleep. Selective serotonin reuptake inhibitor (SSRI) drugs are used to treat disorders such as depression, stress, eating disorders, autism, and schizophrenia. It is thought that these drugs act to prolong the action of 5-HT by blocking reuptake. This may lead to decreased 5-HT content in the nerve fibers themselves; however, this has not previously been directly demonstrated. We have studied the effects of administration of two drugs, imipramine and citalopram, on levels of 5-HT in nerve fibers in the murine brain. Quantitative analysis of the areal density of 5-HT fibers throughout the brain was performed using ImageJ software. While a high density of fibers was observed in mid- and hind-brain regions and areas such as thalamus and hypothalamus, densities were far lower in areas such as cortex, where SSRIs might be thought to exert their actions. As anticipated, imipramine and citalopram produced a decline in 5-HT levels in nerve fibers, but the result was not uniform. Areas such as inferior colliculus showed significant reduction whereas little, if any, change was observed in the adjacent superior colliculus. The reason for, and significance of, this regionality is unclear. It has been proposed that serotonin effects in the brain might be linked to changes in glutamatergic transmission. Extracellular glutamate levels are regulated primarily by glial glutamate transporters. Qualitative evaluation of glutamate transporter immunolabeling in cortex of control and drug-treated mice revealed no discernable difference in intensity of glutamate transporter immunoreactivity. These data suggest that changes in intracellular and extracellular levels of serotonin do not cause concomitant changes in astroglial glutamate transporter expression, and thus cannot represent a mechanism for the delayed efficacy of antidepressants when administered clinically. © 2005 Elsevier B.V. All rights reserved.

Evaluation of the importance of astrocytes when screening for acute toxicity in neuronal cell systems

Reliable, high throughput, in vitro preliminary screening batteries have the potential to greatly accelerate the rate at which regulatory neurotoxicity data is generated. This study evaluated the importance of astrocytes when predicting acute toxic potential using a neuronal screening battery of pure neuronal (NT2.N) and astrocytic (NT2.A) and integrated neuronal/astrocytic (NT2.N/A) cell systems derived from the human NT2.D1 cell line, using biochemical endpoints (mitochondrial membrane potential (MMP) depolarisation and ATP and GSH depletion). Following exposure for 72 h, the known acute human neurotoxicants trimethyltin-chloride, chloroquine and 6-hydroxydopamine were frequently capable of disrupting biochemical processes in all of the cell systems at non-cytotoxic concentrations. Astrocytes provide key metabolic and protective support to neurons during toxic challenge in vivo and generally the astrocyte containing cell systems showed increased tolerance to toxicant insult compared with the NT2.N mono-culture in vitro. Whilst there was no consistent relationship between MMP, ATP and GSH log IC(50) values for the NT2.N/A and NT2.A cell systems, these data did provide preliminary evidence of modulation of the acute neuronal toxic response by astrocytes. In conclusion, the suitability of NT2 neurons and astrocytes as cell systems for acute toxicity screening deserves further investigation.

Investigation of aspects of reactive gliosis in human astrocytoma cell lines:application for toxicity assessment

The process of astrogliosis, or reactive gliosis, is a typical response of astrocytes to a wide range of physical and chemical injuries. The up-regulation of the astrocyte specific glial fibrillary acidic protein (GFAP) is a hallmark of reactive gliosis and is widely used as a marker to identify the response. In order to develop a reliable, sensitive and high throughput astrocyte toxicity assay that is more relevant to the human response than existing animal cell based models, the U251-MG, U373-MG and CCF-STTG 1 human astrocytoma cell lines were investigated for their ability to exhibit reactive-like changes following exposure to ethanol, chloroquine diphosphate, trimethyltin chloride and acrylamide. Cytotoxicity analysis showed that the astrocytic cells were generally more resistant to the cytotoxic effects of the agents than the SH-SY5Y neuroblastoma cells. Retinoic acid induced differentiation of the SH-SY5Y line was also seen to confer some degree of resistance to toxicant exposure, particularly in the case of ethanol. Using a cell based ELISA for GFAP together with concurrent assays for metabolic activity and cell number, each of the three cell lines responded to toxicant exposure by an increase in GFAP immunoreactivity (GFAP-IR), or by increased metabolic activity. Ethanol, chloroquine diphosphate, trimethyltin chloride and bacterial lipopolysaccharide all induced either GFAP or MTT increases depending upon the cell line, dose and exposure time. Preliminary investigations of additional aspects of astrocytic injury indicated that IL-6, but not TNF-α. or nitric oxide, is released following exposure to each of the compounds, with the exception of acrylamide. It is clear that these human astrocytoma cell lines are capable of responding to toxicant exposure in a manner typical of reactive gliosis and are therefore a valuable cellular model in the assessment of in vitro neurotoxicity.

Non-neuronal, slow GABA signalling in the ventrobasal thalamus targets δ-subunit-containing GABA(A) receptors

The rodent ventrobasal (VB) thalamus contains a relatively uniform population of thalamocortical (TC) neurons that receive glutamatergic input from the vibrissae and the somatosensory cortex, and inhibitory input from the nucleus reticularis thalami (nRT). In this study we describe ?-aminobutyric acid (GABA)(A) receptor-dependent slow outward currents (SOCs) in TC neurons that are distinct from fast inhibitory postsynaptic currents (IPSCs) and tonic currents. SOCs occurred spontaneously or could be evoked by hypo-osmotic stimulus, and were not blocked by tetrodotoxin, removal of extracellular Ca(2+) or bafilomycin A1, indicating a non-synaptic, non-vesicular GABA origin. SOCs were more common in TC neurons of the VB compared with the dorsal lateral geniculate nucleus, and were rarely observed in nRT neurons, whilst SOC frequency in the VB increased with age. Application of THIP, a selective agonist at d-subunit-containing GABA(A) receptors, occluded SOCs, whereas the benzodiazepine site inverse agonist ß-CCB had no effect, but did inhibit spontaneous and evoked IPSCs. In addition, the occurrence of SOCs was reduced in mice lacking the d-subunit, and their kinetics were also altered. The anti-epileptic drug vigabatrin increased SOC frequency in a time-dependent manner, but this effect was not due to reversal of GABA transporters. Together, these data indicate that SOCs in TC neurons arise from astrocytic GABA release, and are mediated by d-subunit-containing GABA(A) receptors. Furthermore, these findings suggest that the therapeutic action of vigabatrin may occur through the augmentation of this astrocyte-neuron interaction, and highlight the importance of glial cells in CNS (patho) physiology.

Astrocytic GABA transporter GAT-1 dysfunction in experimental absence seizures

An enhanced tonic GABA-A inhibition in the thalamus plays a crucial role in experimental absence seizures, and has been attributed, on the basis of indirect evidence, to a dysfunction of the astrocytic GABA transporter-1 (GAT-1). Here, the GABA transporter current was directly investigated in thalamic astrocytes from a well-established genetic model of absence seizures, the Genetic Absence Epilepsy Rats from Strasbourg (GAERS), and its non-epileptic control (NEC) strain. We also characterized the novel form of GABAergic and glutamatergic astrocyte-to-neuron signalling by recording slow outward currents (SOCs) and slow inward currents (SICs), respectively, in thalamocortical (TC) neurons of both strains. In patch-clamped astrocytes, the GABA transporter current was abolished by combined application of the selective GAT-1 and GAT-3 blocker, NO711 (30µM) and SNAP5114 (60µM), respectively, to GAERS and NEC thalamic slices. NO711 alone significantly reduced (41%) the transporter current in NEC, but had no effect in GAERS. SNAP5114 alone reduced by half the GABA transporter current in NEC, whilst it abolished it in GAERS. SIC properties did not differ between GAERS and NEC TC neurons, whilst moderate changes in SOC amplitude and kinetics were observed. These data provide the first direct demonstration of a malfunction of the astrocytic thalamic GAT-1 transporter in absence epilepsy and support an abnormal astrocytic modulation of thalamic ambient GABA levels. Moreover, while the glutamatergic astrocyte-neuron signalling is unaltered in the GAERS thalamus, the changes in some properties of the GABAergic astrocyte-neuron signaling in this epileptic strain may contribute to the generation of absence seizures.

GABAB receptor-mediated activation of astrocytes by gamma-hydroxybutyric acid

The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABAB receptors (GABABRs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABAA and GABABRs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABABR agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED50: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca2+ in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABABRs and mediated by Ca2+ release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB.

The role of Ca2+ in the generation of spontaneous astrocytic Ca2+ oscillations

Astrocytes in the rat thalamus display spontaneous [Ca2+]i oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca2+]i oscillations using slices loaded with Fluo-4 AM (5 μM) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca2+]i oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca2+]i increases are not due to membrane-coupled PLC activation. Spontaneous [Ca2+]i increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca2+]i oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on store-operated Ca2+ entry. Increasing [Ca2+]o increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca2+ ATPase by cyclopiazonic acid also induced [Ca2+]i transients in astrocytes indicating a role for cytoplasmic Ca2+ in the induction of spontaneous oscillations. Incubation with 20 μM Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases. This study indicates that Ca2+ has a role in triggering Ca2+ release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca2+]i oscillations

Stem cell-derived astrocytes:are they physiologically credible?

Astrocytes are now increasingly acknowledged as having fundamental and sophisticated roles in brain function and dysfunction. Unravelling the complex mechanisms that underlie human brain astrocyte-neuron interactions is therefore an essential step on the way to understanding how the brain operates. Insights into astrocyte function to date, have almost exclusively been derived from studies conducted using murine or rodent models. Whilst these have led to significant discoveries, preliminary work with human astrocytes has revealed a hitherto unknown range of astrocyte types with potentially greater functional complexity and increased neuronal interaction with respect to animal astrocytes. It is becoming apparent, therefore, that many important functions of astrocytes will only be discovered by direct physiological interrogation of human astrocytes. Recent advancements in the field of stem cell biology have provided a source of human based models. These will provide a platform to facilitate our understanding of normal astrocyte functions as well as their role in CNS pathology. A number of recent studies have demonstrated that stem cell derived astrocytes exhibit a range of properties, suggesting that they may be functionally equivalent to their in vivo counterparts. Further validation against in vivo models will ultimately confirm the future utility of these stem-cell based approaches in fulfilling the need for human- based cellular models for basic and clinical research. In this review we discuss the roles of astrocytes in the brain and highlight the extent to which human stem cell derived astrocytes have demonstrated functional activities that are equivalent to that observed in vivo.

Blood-brain barrier in vitro model: A tissue engineering approach and validation

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

Characterization of a Novel Mouse Phenotype with Marked Hydrocephalus

In multigenic diseases, disorders where mutations in multiple genes affect the expressivity of the disease, genetic interactions play a major role in prevalence and phenotypic severity. While studying the genetic interactions between Pax3 and EdnrB in the melanocyte lineage, a new phenotype was noted in 80% of Pax3 mutants that we believe to be a novel murine model for hydrocephalus. Hydrocephalus, an accumulation of cerebrospinal fluid in the cranial cavity due to obstruction of flow in and out of the cavity, is one of the most common birth defects surpassing Down syndrome. Characteristic to hydrocephalus is a "domed" head appearance, expansion of the ventricles of the brain, and loss of neurons with hyperproliferation of glial cell types all three of which were seen in the mutant mice. The phenotype also consisted of craniofacial deformities coupled with skeletal defects including, but not limited to kyphosis, lordosis, and an apparent shortening of the some limbs. For the cellular analysis of the hydrocephalus phenotype, brains were removed and stained with two antibodies: Glial Fibrillary Acidic Protein (GFAP) and Neurofilament (NF), which are astrocyte- and neuron- specific respectively. A higher number of cells expressing GF AP and a lower number of cells expressing NF were seen in the mutant brain, when compared to control. For skeletal deformity analysis, affected mice skeletons were stained with Alizarin Red and Alcian Blue showing no apparent difference in ossification. Future genetic analysis of these mutant mice has the potential to identify novel gene modifiers involved in the promotion of this particular phenotype.

Glia-augmented artificial neural networks: foundations and applications

Information processing in the human brain has always been considered as a source of inspiration in Artificial Intelligence; in particular, it has led researchers to develop different tools such as artificial neural networks. Recent findings in Neurophysiology provide evidence that not only neurons but also isolated and networks of astrocytes are responsible for processing information in the human brain. Artificial neural net- works (ANNs) model neuron-neuron communications. Artificial neuron-glia networks (ANGN), in addition to neuron-neuron communications, model neuron-astrocyte con- nections. In continuation of the research on ANGNs, first we propose, and evaluate a model of adaptive neuro fuzzy inference systems augmented with artificial astrocytes. Then, we propose a model of ANGNs that captures the communications of astrocytes in the brain; in this model, a network of artificial astrocytes are implemented on top of a typical neural network. The results of the implementation of both networks show that on certain combinations of parameter values specifying astrocytes and their con- nections, the new networks outperform typical neural networks. This research opens a range of possibilities for future work on designing more powerful architectures of artificial neural networks that are based on more realistic models of the human brain.

Modifications de l'expression génique du système nerveux central lors de l'insuffisance hépatique aiguë : rôle dans les mécanismes pathophysiologiques responsables de l'œdème cérébral

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

RNA Localization and Translational Regulation on the Endoplasmic Reticulum

mRNA localization is emerging as a critical cellular mechanism for the spatiotemporal regulation of protein expression and serves important roles in oogenesis, embryogenesis, cell fate specification, and synapse formation. Signal sequence-encoding mRNAs are localized to the endoplasmic reticulum (ER) membrane by either of two mechanisms, a canonical mechanism of translation on ER-bound ribosomes (signal recognition particle pathway), or a poorly understood direct ER anchoring mechanism. In this study, we identify that the ER integral membrane proteins function as RNA-binding proteins and play important roles in the direct mRNA anchoring to the ER. We report that one of the ER integral membrane RNA-binding protein, AEG-1 (astrocyte elevated gene-1), functions in the direct ER anchoring and translational regulation of mRNAs encoding endomembrane transmembrane proteins. HITS-CLIP and PAR-CLIP analyses of the AEG-1 mRNA interactome of human hepatocellular carcinoma cells revealed a high enrichment for mRNAs encoding endomembrane organelle proteins, most notably encoding transmembrane proteins. AEG-1 binding sites were highly enriched in the coding sequence and displayed a signature cluster enrichment downstream of encoded transmembrane domains. In overexpression and knockdown models, AEG-1 expression markedly regulates translational efficiency and protein functions of two of its bound transcripts, MDR1 and NPC1. This study reveals a molecular mechanism for the selective localization of mRNAs to the ER and identifies a novel post-transcriptional gene regulation function for AEG-1 in membrane protein expression.

Modifications de l'expression génique du système nerveux central lors de l'insuffisance hépatique aiguë : rôle dans les mécanismes pathophysiologiques responsables de l'œdème cérébral

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.