Expression of Id proteins increases in astrocytes in the hippocampus of epileptic rats

Reactive gliosis is a prominent morphological feature of temporal lobe epilepsy. The molecular mechanisms underlying glial cell activation remain unclear. We examined expression of Id1-3 protein, a family of helix--loop--helix proteins involved in the regulation of cell proliferation and differentiation, in glial cells after electrically induced status epilepticus (SE) in the rat. In control hippocampus, Id3 was weakly expressed in astrocytes, while Id1-2 were below detection level. After SE, Id1-3 protein expression increased markedly in reactive astrocytes within 1 day and this persisted up to 3 weeks after SE. Three months after SE when rats experience spontaneous seizures, Id expression had returned to control levels. These results support a role of the Id gene family in regulating astrocyte reactivity in epileptic tissue.     

Expression and functional role of mGluR3 and mGluR5 in human astrocytes and glioma cells: opposite regulation of glutamate transporter proteins

We examined the regulation of glutamate transporter protein expression after stimulation with selective metabotropic glutamate receptor (mGluR) agonists in cultured human glial cells. mGluR3 and mGluR5 are expressed in human astrocytes and in human glioma cells in vivo as well as in vitro, as shown by either RT-PCR or western blot analysis. The selective group I agonist (S)-3,5-dihydroxyphenylglycine produced a significant down-regulation of both GLAST and GLT-1 protein expression in astrocytes cultured in the presence of growth factors. This condition mimics the morphology of reactive glial cells in vivo including an increased expression of mGluR5 protein (observed in pathological conditions). In contrast, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine, a selective agonist of group II metabotropic glutamate receptors, positively modulates the expression of GLAST and GLT-1 proteins. A similar opposite effect of (S)-3,5-dihydroxyphenylglycine and (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine was observed for the expression of EAAT3 protein in U373 glioblastoma cell line. Selective group I and II antagonists prevented these effects. Pharmacological inhibition of mitogen-activated protein kinase and phosphatidylinositol-3-K pathways reduces the induction of GLT-1 observed in response to the group II metabotropic glutamate receptor agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine. Thus, mGluR3 and mGluR5 can critically and differentially modulate the expression of glutamate transporters and may represent interesting pharmacological targets to regulate the extracellular levels of glutamate in pathological conditions.     

Distinct regulation of metabotropic glutamate receptor (mGluR1 alpha) in the developing limbic system following multiple early-life seizures

The effects of repeated neonatal seizures on metabotropic glutamate receptors (mGluRs) during critical periods of brain development are unknown. Therefore, we characterized the expression of Group I (mGluR1 and mGluR5) and Group II (mGluR2/3) metabotropic glutamate receptor proteins in the developing limbic system in response to a varied neonatal seizure history. Status epilepticus was induced with kainic acid (KA) either once (1x KA) on postnatal (P) day (P13), twice (2x KA) on P6 and P9 or P13, or three times (3x KA) on P6, P9, and P13. In control hippocampus, mGluR1alpha protein expression differed at all stages of development examined, whereas mGluR2/3 and mGluR5 protein expression patterns were mature by P15. After KA-induced status epilepticus, there was a significant elevation in mGluR1alpha protein expression within a select group of inhibitory interneurons of the CA1 stratum oriens-alveus that was enhanced with increasing number of neonatal seizures. mGluR2/3 and mGluR5 subtypes were unchanged. Increases were also observed within neurons of the amygdala and piriform cortex. Selective increases of mGluR1alpha subtypes within limbic structures may contribute to the resistance and tolerance of the immature hippocampus from damage. This may occur by excessive stimulation of excitatory synapses to collectively enhance the inhibitory drive of the immature brain by increasing GABA release. Data suggest that the mGluR1alpha subtype plays an important role in regulating hippocampal network activity after early-life seizures.     

Perisynaptic Location of Metabotropic Glutamate Receptors mGluR1 and mGluR5 on Dendrites and Dendritic Spines in the Rat Hippocampus

Ionotropic and metabotropic (mGluR1a) glutamate receptors were reported to be segregated from each other within the postsynaptic membrane at individual synapses. In order to establish whether this pattern of distribution applies to the hippocampal principal cells and to other postsynaptic metabotropic glutamate receptors, the mGluR1a/b/c and mGluR5 subtypes were localized by immunocytochemistry. Principal cells in all hippocampal fields were reactive for mGluR5, the strata oriens and radiatum of the CA1 area being most strongly immunolabelled. Labelling for mGluR1b/c was strongest on some pyramids in the CA3 area, weaker on granule cells and absent on CA1 pyramids. Subpopulations of non-principal cells showed strong mGluR1 or mGluR5 immunoreactivity. Electron microscopic pre-embedding immunoperoxidase and both pre- and postembedding immunogold methods consistently revealed the extrasynaptic location of both mGluRs in the somatic and dendritic membrane of pyramidal and granule cells. The density of immunolabelling was highest on dendritic spines. At synapses, immunoparticles for both mGluR1 and mGluR5 were found always outside the postsynaptic membrane specializations. Receptors were particularly concentrated in a perisynaptic annulus around type I synaptic junctions, including the invaginations at 'perforated'synapses. Measurements of immunolabelling on dendritic spines showed decreasing levels of receptor as a function of distance from the edge of the synaptic specialization. We propose that glutamatergic synapses with an irregular edge develop in order to increase the circumference of synaptic junctions leading to an increase in the metabotropic to ionotropic glutamate receptor ratio at glutamate release sites. The perisynaptic position of postsynaptic metabotropic glutamate receptors appears to be a general feature of glutamatergic synaptic organization and may apply to other G-protein-coupled receptors.     

Abnormal mGluR2/3 expression in the perforant path termination zones and mossy fibers of chronically epileptic rats

Epilepsy is characterized by hyperexcitability of hippocampal networks, excessive release of glutamate, and progressive neurodegeneration. Presynaptic group II metabotropic receptors (mGluR2 and mGluR3) are among different mechanisms that modulate presynaptic release of glutamate, especially at the mossy fibers in the hippocampus. Here, we explore whether mGluR2/3 expression is affected in a rat model of temporal lobe epilepsy obtained via pilocarpine-induced status epilepticus (SE). Immunohistochemical assays were performed in age-matched controls and two groups of epileptic rats sacrificed at 25-35 days (1 month post-SE) and at 55-65 days (2 months post-SE) following SE onset. A dramatic lessening of mGluR2/3 immunofluorescence was observed at CA1 and CA3 stratum lacunosum/molecular (SLM) declining to 60% and 68% of control values in 1-month and 2-month post-SE, respectively. Additionally, thickness of mGluR2/3-stained SLM layer narrowed up to 70% of controls indicating atrophy at this branch of the perforant path. Epileptic rats exhibited a marked and progressive down-regulation of mGluR2/3 expression in mossy fiber at hilus and CA3 stratum lucidum in contrast with an enhanced expression of vesicular glutamate transporter type 1 (VGluT1) at the mossy fibers. Intense VGluT1 punctated staining was detected at the inner third molecular layer indicating glutamatergic sprouting. In the molecular layer, mGluR2/3 labeling slightly declined in the 1-month post-SE group but then increased in the 2-month post-SE group although it was diffusely distributed. Down-regulation of mGluR2/3 at the mossy fibers and the SLM may render epileptic hippocampal networks hyperexcitable and susceptible to glutamate-mediated excitotoxicity and neurodegeneration.     

Cystatin C, a cysteine protease inhibitor, is persistently up-regulated in neurons and glia in a rat model for mesial temporal lobe epilepsy

Cystatin C (CSTC), a cysteine protease inhibitor, has been implicated in the processes of neuronal degeneration and repair of the nervous system. Using serial analysis of gene expression (SAGE), we recently identified CSTC as one of the genes that are overexpressed after electrically induced status epilepticus (SE). In the present study, Western blot analysis extended the SAGE results, showing increased CSTC protein in the hippocampus and entorhinal cortex. Immunocytochemistry revealed an increase in CSTC expression in glial cells, which was first apparent 24 h after onset of SE, and persisted for at least 3 months. Double immunolabelling confirmed that both reactive astrocytes, and activated microglia were CSTC immunopositive. Within the hippocampus, up-regulation was also observed in neuronal cells within one day after SE. Up-regulation was still present in hippocampal pyramidal cells and surviving interneurons of chronic epileptic rats (3-8 months post-SE). This study demonstrates that status epilepticus leads to a widespread and persistent up-regulation of CSTC in the hippocampus and entorhinal cortex, which may represent an intrinsic neuroprotective mechanism in the course of epileptogenesis that may counteract progression of the disease.     

Neurosteroids and epileptogenesis in the pilocarpine model: Evidence for a relationship between P450scc induction and length of the latent period

Purpose:   Cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) catalyzes the initial step in the biosynthesis of neurosteroids within the brain. We sought to determine which cells express P450cc and whether neurosteroids play a role in the regulation of epileptogenesis following pilocarpine-induced status epilepticus (SE).
Methods:   Rats experienced uninterrupted SE or SE terminated with diazepam at 60, 120, and 180 min. P450scc induction in CA3 hippocampus was determined by double immunolabeling with P450scc antiserum and monoclonal antibodies against GFAP (astrocytes), RIP (oligodendrocytes), or heme oxygenase-1 (microglia).
Results:   SE was associated with P450scc induction in many astrocytes and a small number of microglia and oligodendrocytes in the hippocampal CA3 strata radiatum and lacunosum-moleculare. The extent of P450scc induction increased with increasing SE duration. Paradoxically, increased P450scc induction in rats experiencing SE for 180 min or more was associated with the delayed onset of spontaneous recurrent seizures. Treatment with the 5α-reductase inhibitor finasteride (100 mg/kg/day for 25 days), which inhibits the synthesis of γ-aminobutyric acid (GABA)_A receptor modulating neurosteroids such as allopregnanolone, was associated with a significant reduction in time to the onset of spontaneous seizures in rats exposed to 180-min but not 90-min SE.
Discussion:   P450scc is induced by SE in a diverse population of hippocampal glia. Induction of P450scc is associated with the delayed onset of spontaneous seizures. Conversely, inhibition of neurosteroid synthesis accelerated the onset of spontaneous seizures, but only in animals exhibiting significant increases in P450scc. These findings suggest that induction of neurosteroid synthesis in reactive glial cells is associated with delayed onset of spontaneously recurrent seizures.     

Kainate treatment alters TGF-beta3 gene expression in the rat hippocampus

In order to evaluate the role of transforming growth factor (TGF)-beta3 in the neurodegenerative process, we examined the levels of mRNA and immunocytochemical distribution for TGF-beta3 in the rat hippocampus after systemic kainic acid (KA) administration. Hippocampal TGF-beta3 mRNA level was reduced 3 h after KA injection. However, the levels of TGF-beta3 mRNA were elevated 1 day post-KA and lasted for at least 30 days. A mild TGF-beta3 immunoreactivity (TGF-beta3-IR) in the Ammon's horn and a moderate TGF-beta3-IR in the dentate granule cells were observed in the normal hippocampus. The CA1 and CA3 neurons lost their TGF-beta3-IR, while TGF-beta3-positive glia-like cells proliferated mainly throughout the CA1 sector and had an intense immunoreactivity at 7, 15 and 30 days after KA. This immunocytochemical distribution of TGF-beta3-positive non-neuronal populations was similar to that of glial fibrillary acidic protein (GFAP)-positive cells. Double labeling immunocytochemical analysis demonstrated colocalization of TGF-beta3- and GFAP-immunoreactivity in the same cells. These findings suggest a compensatory mechanism of astrocytes for the synthesis of TGF-beta3 protein in response to KA-induced neurodegeneration. In addition, exogenous TGF-beta3 (5 or 10 ng/i.c.v.) significantly attenuated KA-induced seizures and neuronal damages in a dose-related manner. Therefore, our results suggest that TGF-beta3 plays an important role in protective mechanisms against KA-induced neurodegeneration.     

Long‐lasting pro‐ictogenic effects induced in vivo by rat brain exposure to serum albumin in the absence of concomitant pathology

Purpose: Dysfunction of the blood–brain barrier (BBB) is a common finding during seizures or following epileptogenic brain injuries, and experimentally induced BBB opening promotes seizures both in naive and epileptic animals. Brain albumin extravasation was reported to promote hyperexcitability by inducing astrocytes dysfunction. To provide in vivo evidence for a direct role of extravasated serum albumin in seizures independently on the pathologic context, we did the following: (1) quantified the amount of serum albumin extravasated in the rat brain parenchyma during status epilepticus (SE); (2) reproduced a similar concentration in the hippocampus by intracerebroventricular (i.c.v.) albumin injection in naive rats; (3) measured electroencephalography (EEG) activity in these rats, their susceptibility to kainic acid (KA)–induced seizures, and their hippocampal afterdischarge threshold (ADT). Methods: Brain albumin concentration was measured in the rat hippocampus and other forebrain regions 2 and 24 h after SE by western blot analysis. Brain distribution of serum albumin or fluorescein isothiocyanate (FITC)‐albumin was studied by immunohistochemistry and immunofluorescence, respectively. Naive rats were injected with rat albumin or FITC‐albumin, i.c.v., to mimic the brain concentration attained after SE, or with dextran used as control. Inflammation was evaluated by immunohistochemistry by measuring glial induction of interleukin (IL)‐1β. Western blot analysis was used to measure inward rectifying potassium channel subunit Kir4.1 protein levels in the hippocampus. Seizures were induced in rats by intrahippocampal injection of 80 ng KA and quantified by EEG analysis, 2 or 24 h after rat albumin or dextran administration. ADT was measured by electrical stimulation of the hippocampus 3 months after albumin injection. In these rats, EEG was continuously monitored for 2 weeks to search for spontaneous seizures. Key Findings: The hippocampal serum albumin concentration 24 h post‐SE was 0.76 ± 0.21 μm . Similar concentrations were measured in other forebrain regions, whereas no changes were found in cerebellum. The hippocampal albumin concentration was similarly reproduced in naive rats by i.c.v. administration of 500 μg/4 μl rat albumin: albumin was predominantly detected extracellularly 2 h after injection, whereas at 24 h it was visible inside pyramidal neurons and in only a few scattered chondroitin sulphate proteoglycan (NG2)‐positive cells, but not in glial fibrillary acidic protein (GFAP)‐positive astrocytes or CR‐3 complement receptor (OX‐42)‐positive microglia. The presence of albumin in naive rat hippocampus was associated with induced IL‐1β in GFAP‐positive astrocytes and a concomitant tissue down‐regulation of Kir4.1. Spiking activity was evoked by albumin in the hippocampus lasting for 2 h. When KA was intrahippocampally applied either 2 or 24 h after albumin injection, the number of total interictal spikes in 3 h EEG recording was significantly increased by twofold on average. Three months after albumin injection, neither albumin nor inflammation was detected in brain tissue; at this time, the ADT was reduced by 50% but no spontaneous seizures were observed. Significance: Transient hippocampal exposure to albumin levels similar to those attained after prominent BBB breakdown resulted in increased seizure susceptibility and long‐term reduction in seizure threshold, but it did not evoke spontaneous seizures. These effects may be mediated by albumin‐induced astrocytes dysfunction and the associated induction of proinflammatory molecules.     

mGluR5-PLCbeta4-PKCbeta2/PKCgamma pathways in hippocampal CA1 pyramidal neurons in pilocarpine model of status epilepticus in mGluR5+/+ mice

While it is generally accepted that phospholipase C (PLC) and protein kinase C (PKC) are down-stream proteins involved in metabotropic glutamate receptor 5 (mGluR5)-related signal transduction, we still do not know which subtype of PLC or PKC is specifically regulated after mGluR5 activation. In the present study in mGluR5 wild-type (mGluR5+/+) mice, we showed induced PKCbeta2 or PKCgamma expression at the border between the stratum oriens and alveus (O/A border) at 2h during pilocarpine induced status epilepticus (SE), and in the stratum pyramidale in CA1 area at 1 day after pilocarpine induced SE; at 1 day, induced expression of PLCbeta4 in the stratum pyramidale of CA1 area was observed. Furthermore, double labeling revealed the co-localization of induced PKCbeta2 or PKCgamma with mGluR5 or with induced PLCbeta4 in the stratum pyramidale of CA1 area. These induced expression, however, were not found in mGluR5 mutant (mGluR5-/-) mice. It suggests that induced PLCbeta4-PKCbeta2/PKCgamma at 1 day after pilocarpine induced SE in pyramidal neurons or PKCbeta2 or PKCgamma in interneurons at O/A border at 2h during pilocarpine induced SE may be specifically linked to the activation of mGluR5. When compared to mGluR5+/+ mice, significant shorter latency (from pilocarpine injection to the occurrence of status epilepticus) and maintenance period (from beginning to the end of status epilepticus) for status epilepticus in mGluR5-/- mice were also demonstrated. It is possible that mGluR5 may play a negative role in initiation of status epilepticus by interacting with muscarinic acetylcholine receptor in mGluR5+/+ mice.     

Epileptogenic roles of astroglial death and regeneration in the dentate gyrus of experimental temporal lobe epilepsy

Recent studies have demonstrated that blockade of neuronal death in the hippocampus cannot prevent epileptogenesis in various epileptic models. These reports indicate that neurodegeneration alone is insufficient to cause epilepsy, and that the role of astrocytes in epileptogenesis should be reconsidered. Therefore, the present study was designed to elucidate whether altered morphological organization or the functionalities of astrocytes induced by status epilepticus (SE) is responsible for epileptogenesis. Glial responses (reactive microgliosis followed by astroglial death) in the dentate gyrus induced by pilocarpine-induced SE were found to precede neuronal damage and these alterations were closely related to abnormal neurotransmission related to altered vesicular glutamate and GABA transporter expressions, and mossy fiber sprouting in the dentate gyrus. In addition, newly generated astrocytes showed down-regulated expressions of glutamine synthase, glutamate dehydrogenase, and glial GABA transporter. Taken together, our findings suggest that glial responses after SE may contribute to epileptogenesis and the acquisition of the properties of the epileptic hippocampus. Thus, we believe that it is worth considering new therapeutic approaches to epileptogenesis involving targeting the inactivation of microglia and protecting against astroglial loss.     

Down-regulation of mGluR8 in pilocarpine epileptic rats

Activation of presynaptic metabotropic glutamate receptors (mGluRs) leads to a powerful inhibition of glutamate release from many synaptic terminals throughout the CNS. mGluRs as autoreceptors are believed to provide a negative feedback system that prevents potentially toxic accumulation of glutamate in the extracellular space during synchronous synaptic activity such as epileptic seizures. In this study we analyzed the function of presynaptic mGluR8 on terminals of the lateral perforant pathway in the pilocarpine model of limbic epilepsy. Field excitatory postsynaptic potentials (fEPSPs) recorded in hippocampal slices of rats that developed spontaneous recurrent seizures after pilocarpine-induced status epilepticus (SRS group) showed a significantly reduced sensitivity to Group III mGluR agonists and severe mossy fiber sprouting. The Group III mGluR agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4, 10 microM) depressed fEPSPs in the SRS group only by 26 +/- 21% compared to 50 +/- 18% in untreated rats. Similarly, the mGluR8 preferring agonist (R,S)-4-phosphonophenylglycine (PPG, 5 microM) was significantly less effective in slices from SRS rats (43 +/- 4% vs. 83 +/- 5%). Concentration-response curves for L-AP4 revealed that the EC(50) values were not different between the control and SRS group (13 +/- 7 microM vs. 9 +/- 9 microM), while the maximal depressing effect was significantly reduced. The remaining depressing effect of L-AP4 in the SRS group could be blocked by the Group III specific antagonists (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG) and alpha-methyl-L-AP4 (MAP4). Rats that did not develop SRS following pilocarpine-induced status epilepticus were indistinguishable from control rats: fEPSPs were highly sensitive to L-AP4 and there was no mossy fiber sprouting. The results show that pilocarpine-induced status epilepticus can lead to a downregulation of mGluR8 and suggest that the condition of SRS is associated with a deteriorated autoregulation of glutamate release.     

Pilocarpine-induced status epilepticus increases Homer1a and changes mGluR5 expression

Homer1a regulates expression of group I metabotropic glutamate receptors type I (mGluR1 and mGluR5) and is involved in neuronal plasticity. It has been reported that Homer1a expression is upregulated in the kindling model and hypothesized to act as an anticonvulsant. In the present work, we investigated whether pilocarpine-induced status epilepticus (SE) would alter Homer1a and mGluR5 expression in hippocampus. Adult rats were subjected to pilocarpine-model and analyzed at 2h, 8h, 24h and 7 d following SE. mRNA analysis showed the highest expression of Homer1a at 8h after SE onset, while immunohistochemistry demonstrated that Homer1a protein expression was significantly increased in hippocampus, amygdala and piriform and entorhinal cortices at 24h after SE onset when compared to control animals. The increased Homer1a expression coincided with a significant decrease of mGluR5 protein expression in amygdala and piriform and entorhinal cortices. The data suggest that during the critical periods of epileptogenesis, overexpression of Homer1a occurs to counteract hyperexcitability and thus Homer1a may be a molecular target in the treatment of epilepsy.     

Distinct increased metabotropic glutamate receptor type 5 (mGluR5) in temporal lobe epilepsy with and without hippocampal sclerosis

Metabotropic glutamate receptor type 5 (mGluR5) upregulation in temporal lobe epilepsy (TLE) and the correlation of its expression with features of hippocampal sclerosis (HS) remains unclear. Here we characterized mGluR5 immunoreactivity in hippocampus, entorhinal cortex (EC), and subiculum of TLE specimens with confirmed HS, with neocortical TLE (non‐HS) and necropsy controls. We correlated mGluR5 immunoreactivity with neuronal density, mossy fiber sprouting, astrogliosis (GFAP), and dendritic alterations (MAP2). TLE specimens showed increased mGluR5 expression, which was most pronounced in the EC, subiculum, CA2, and dentate gyrus outer molecular layer. Increased mGluR5 expression was seen in hippocampal head and body segments and was independent of neuronal density, astrogliosis, or dendritic alterations. Positive correlation between mGluR5 expression with mossy fiber sprouting and with MAP2 in CA3 and CA1 was found only in HS specimens. Negative correlation between mGluR5 expression with seizure frequency and epilepsy duration was found only in non‐HS cases. Specimens from HS patients without previous history of febrile seizure (FS) showed higher mGluR5 and MAP2 expression in CA2. Our study suggests that mGluR5 upregulation is part of a repertoire of post‐synaptic adaptations that might control overexcitation and excessive glutamate release rather than a dysfunction that leads to seizure facilitation. That would explain why non‐HS cases, on which seizures are likely to originate outside the hippocampal formation, also exhibit upregulated mGluR5. On the other hand, lower mGluR5 expression was related to increased seizure frequency. In addition to its role in hyperexcitability, mGluR5 upregulation could play a role in counterbalance mechanisms along the hyperexcitable circuitry uniquely altered in sclerotic hippocampal formation. Inefficient post‐synaptic compensatory morphological (dendritic branching) and glutamatergic (mGluR5 expression) mechanisms in CA2 subfield could potentially underlie the association of FS with HS and TLE. © 2013 The Authors. Hippocampus Published by Wiley Periodicals, Inc.     

The expression of transforming growth factor-beta1 (TGF-beta1) in hippocampal neurons: a temporary upregulated protein level after transient forebrain ischemia in the rat

Exogenous TGF-beta1 has been shown to protect neurons from damage induced in vitro and in vivo. In this study we attempted to examine the expression of endogenous TGF-beta1 mRNA and protein in the hippocampus of non-ischemic and ischemic rats, and to localize TGF-beta1 protein and DNA fragmentation by double-staining. Transient ischemia was induced for 10 min in Wistar rats by clamping both common carotid arteries and lowering blood pressure to 40 mmHg. Bioactive TGF-beta1 was selectively determined in CA1 pyramidal neurons of non-ischemic rats. It was upregulated after 3 h and 6 h of reperfusion corresponding to the increase in TGF-beta1 mRNA level detected by RT-PCR. Lectin and GFAP staining showed no detectable activated microglial cells and astrocytes in the hippocampus 3 h and 6 h after ischemia. When neuronal damage proceeded through day 2 to day 4 after ischemia as demonstrated by TUNEL-staining, TGF-beta1 immunoreactivity (ir) disappeared in damaged neurons but persisted in viable neurons although TGF-beta1 mRNA levels continuously increased. Double-staining revealed that TUNEL-positive neurons did not express TGF-beta1, while TUNEL-negative neurons in the CA1 subfield exhibited a distinct TGF-beta1 ir. These data indicate that hippocampal CA1 neurons can express TGF-beta1 under physiological conditions and upregulate its expression during the first hours after ischemia, that is independent of the activation of glial cells. The endogenous TGF-beta1 expressed in neurons may play a role in the pathological process of DNA degradation and delayed neuronal death after transient forebrain ischemia.     

Enhanced astroglial Ca2+ signaling increases excitatory synaptic strength in the epileptic brain

The fine‐tuning of synaptic transmission by astrocyte signaling is crucial to CNS physiology. However, how exactly astroglial excitability and gliotransmission are affected in several neuropathologies, including epilepsy, remains unclear. Here, using a chronic model of temporal lobe epilepsy (TLE) in rats, we found that astrocytes from astrogliotic hippocampal slices displayed an augmented incidence of TTX‐insensitive spontaneous slow Ca²⁺ transients (STs), suggesting a hyperexcitable pattern of astroglial activity. As a consequence, elevated glutamate‐mediated gliotransmission, observed as increased slow inward current (SICs) frequency, up‐regulates the probability of neurotransmitter release in CA3‐CA1 synapses. Selective blockade of spontaneous astroglial Ca²⁺ elevations as well as the inhibition of purinergic P2Y1 or mGluR5 receptors relieves the abnormal enhancement of synaptic strength. Moreover, mGluR5 blockade eliminates any synaptic effects induced by P2Y1R inhibition alone, suggesting that the Pr modulation via mGluR occurs downstream of P2Y1R‐mediated Ca²⁺‐dependent glutamate release from astrocyte. Our findings show that elevated Ca²⁺‐dependent glutamate gliotransmission from hyperexcitable astrocytes up‐regulates excitatory neurotransmission in epileptic hippocampus, suggesting that gliotransmission should be considered as a novel functional key in a broad spectrum of neuropathological conditions. GLIA 2015;63:1507–1521     

Reversible and Irreversible Neuronal Injury Induced by Intrahippocampal Infusion of the mGluR Agonist 1S,3R-ACPD in the Rat

Metabotropic glutamate receptor (mGluR)-induced neuronal injury in the brain was further investigated in the rat. The highly selective mGluR agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) was infused stereotaxically into the left dorsal hippocampus of adult rats. Control (2 μl saline injected) rats had minimal tissue injury that was confined to the area around the injection site. In contrast, dose of 250 nmol/2μl 1S,3R-ACPD produced a moderate number of swollen and injured cells in polymorphic, pyramidal and molecular layers of the injected hippocampus which was observed at 4 and 8 h post-injection. However, at 24 h few injured or necrotic cells were found. A dose of 1000 nmol/2μl 1S,3R-ACPD produced severe cellular injury in polymorphic, pyramidal and molecular layers of the hippocampus at 4, 8, or 24 h. At 24 h after this higher dose of 1S,3R-ACPD, a number of necrotic cells (i.e. pyramidal neurons of area CA1) were found. Both doses of 1S,3R-ACPD produced seizures in animals that were charcaterized by multiple episodes of wet dog shakes, staring, immobility, facial automatisms, rearing, bilateral forelimb clonus, and loss of postural control. These data support a possible role for excesive mGluR activation in pathological states of convulsions and neurodegeneration.     

Expression patterns of Group III metabotropic glutamate receptors mGluR4 and mGluR8 in multiple sclerosis lesions

Recent evidence supports a role for metabotropic glutamate receptors (mGluRs) in neuroinflammatory diseases. In the present study, we have investigated whether the group III mGluR subtypes mGluR4 and mGluR8 are expressed in MS lesions at various stages of evolution. In control patient tissue and in normal-appearing MS white matter (NAWM), no microglial or astrocyte staining was detected. In contrast, in active lesions, mGluR8 immunoreactivity (IR) was detected in cells of the microglia/macrophage lineage. Fewer macrophage-like cells were positive for mGluR8 in chronic active and inactive lesions. No mGluR4 IR was detected in cells of the microglia/macrophage lineage in the MS lesions studied. In chronic active lesions, however, a population of reactive astrocytes localized in the rim of the lesions expressed both mGluR4 and mGluR8. Our results suggest a role for these receptor subtypes in the inflammatory response in MS that involves both astrocytes and cells of the microglia/macrophage lineage.