Increased matrix metalloproteinases expression in tuberous sclerosis complex: modulation by microRNA 146a and 147b in vitro.

AIM: Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) control proteolysis within the extracellular matrix (ECM) of the brain. Dysfunction of this enzymatic system due to brain inflammation can disrupt the blood-brain barrier (BBB) and has been implicated in the pathogenesis of epilepsy. However, this has not been extensively studied in the epileptogenic human brain. METHODS: We investigated the expression and cellular localization of major MMPs (MMP2, MMP3, MMP9 and MMP14) and TIMPs (TIMP1, TIMP2, TIMP3 and TIMP4) using quantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemistry in resected epileptogenic brain tissue from patients with tuberous sclerosis complex (TSC), a severe neurodevelopmental disorder characterized by intractable epilepsy and prominent neuroinflammation. Furthermore, we determined whether anti-inflammatory microRNAs, miR146a and miR147b, which can regulate gene expression at the transcriptional level, could attenuate dysregulated MMP and TIMP expression in TSC tuber-derived astroglial cultures. RESULTS: We demonstrated higher mRNA and protein expression of MMPs and TIMPs in TSC tubers compared to control and perituberal brain tissue, particularly in dysmorphic neurons and giant cells, as well as in reactive astrocytes, which was associated with BBB dysfunction. More importantly, IL-1ß-induced dysregulation of MMP3, TIMP2, TIMP3 and TIMP4 could be rescued by miR146a and miR147b in tuber-derived TSC cultures. CONCLUSIONS: This study provides evidence of dysregulation of the MMP/TIMP proteolytic system in TSC, which is associated with BBB dysfunction. As dysregulated MMP and TIMP expression can be ameliorated in vitro by miR146a and miR147b, these miRNAs deserve further investigation as a novel therapeutic approach.

Deep brain stimulation and the role of astrocytes.

Deep brain stimulation (DBS) has emerged as a powerful surgical therapy for the management of treatment-resistant movement disorders, epilepsy and neuropsychiatric disorders. Although DBS may be clinically effective in many cases, its mode of action is still elusive. It is unclear which neural cell types are involved in the mechanism of DBS, and how high-frequency stimulation of these cells may lead to alleviation of the clinical symptoms. Neurons have commonly been a main focus in the many theories explaining the working mechanism of DBS. Recent data, however, demonstrates that astrocytes may be active players in the DBS mechanism of action. In this review article, we will discuss the potential role of reactive and neurogenic astrocytes (neural progenitors) in DBS.

Gene expression analysis in the human hypothalamus in depression by laser microdissection and real-time PCR: the presence of multiple receptor imbalances.

Hyperactivity of corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus (PVN) of the hypothalamus is a prominent feature in depression and may be important in the etiology of this disease. The activity of the CRF neurons in the stress response is modulated by a number of factors that stimulate or inhibit CRF expression, including (1) corticosteroid receptors and their chaperones, heat shock proteins 70 and 90, (2) sex hormone receptors, (3) CRF receptors 1 (CRFR1) and 2, (4) cytokines interleukin 1-beta and tumor necrosis factor-alpha, (5) neuropeptides and receptors, vasopressin (AVP), AVP receptor 1a (AVPR1A) and oxytocin and (6) transcription factor cAMP-response element-binding protein. We hypothesized that, in depression, the transcript levels of those genes that are involved in the activation of the hypothalamo-pituitary-adrenal (HPA) axis are upregulated, whereas the transcript levels of the genes involved in the inhibition of the HPA axis are downregulated. We performed laser microdissection and real-time PCR in the PVN and as a control in the supraoptic nucleus. Snap-frozen post-mortem hypothalami of seven depressed and seven matched controls were used. We found significantly increased CRF mRNA levels in the PVN of the depressed patients. This was accompanied by a significantly increased expression of four genes that are involved in the activation of CRF neurons, that is, CRFR1, estrogen receptor-alpha, AVPR1A and mineralocorticoid receptor, while the expression of the androgen receptor mRNA involved in the inhibition of CRF neurons was decreased significantly. These findings raise the possibility that a disturbed balance in the production of receptors may contribute to the activation of the HPA axis in depression.

Global gene expression profiling of ischemic preconditioning in the rat retina.

PURPOSE: To obtain and analyze the gene expression changes after ischemic preconditioning (IPC) in the rat retina. METHODS: Ischemic damage to the inner retina can be prevented by a short, non-deleterious, ischemic insult of 5 min applied 24 h preceding a full ischemic insult of 60 min; a phenomenon termed tolerance or IPC. The time course of changes in gene expression after induction of IPC was assessed by 22K oligonucleotide microarrays, followed by real-time quantitative polymerase chain reaction (qPCR) validation. Functional pathways of interest were identified by Gene Ontology-term analysis. RESULTS: Histology confirmed that IPC induction by 5 min of retinal ischemia results in a complete protection against the neurodegenerative effects of a 60 min ischemic period applied 24 or 48 h later. The microarray analysis revealed differential expression of 104 known genes at one or more time points between 1 h and 7 days after IPC. The group of altered genes contained a significant overrepresentation of genes involved in aminoacyl-tRNA synthetase activity (Iars, Lars, Cars, Yars, Gars, Tars), amino acid transport (Slc3a2, Slc6a6, Slc7a1, Slc38a2), regulation of transcription (including Egr1, Egr4, Nr4a1, Nr4a3, c-fos), and cell death (including Anxa1, Trib3). qPCR assays on cDNA of individual animals confirmed the microarray results. CONCLUSIONS: Endogenous neuroprotection, provoked by ischemic preconditioning is associated with changes in transcript levels of several functionally-related groups of genes. During the time window of effective protection, transcript levels of genes encoding for aminoacyl-tRNA synthetases and for amino acid transport are reduced. These changes suggest that a reduction of translational activity may play a significant role in preconditioning-mediated neuroprotection.

Different stress-related gene expression in depression and suicide.

OBJECTIVE: Suicide occurs in some, but not all depressed patients. So far, it remains unknown whether the studied stress-related candidate genes change in depression, suicide or both. The prefrontal cortex (PFC) is involved in, among other things, impulse control and inhibitory behavior and plays an important role in both suicide and depression. METHODS: We have employed qPCR to study 124 anterior cingulate cortex (ACC) and dorsolateral PFC (DLPFC) brain samples, obtained from two brain banks, from: i) young depressed patients (average age 43 years) who committed suicide (MDD-S) and depressed patients who died from causes other than suicide (MDD-NS) and from ii) elderly depressed patients (average age 75 years) who did not commit suicide (DEP). Both cohorts were individually matched with non-psychiatric non-suicide control subjects. We determined the transcript levels of hypothalamic-pituitary-adrenal axis-regulating molecules (corticotropin-releasing hormone (CRH), CRH receptors, CRH binding protein, mineralocorticoid receptor/glucocorticoid receptor), transcription factors that regulate CRH expression, CRH-stimulating cytokines, chaperone proteins, retinoid signaling, brain-derived neurotrophic factor and tropomyosin-related kinase B, cytochrome proteins, nitric oxide synthase (NOS) and monoamines. RESULTS: In the MDD-S group, expression levels of CRH and neuronal NOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) were increased. Other changes were only present in the DEP group, i.e. decreased NIDD, and increased and 5-hydroxytryptamine receptor 1A (5-HT1A) expression levels. Changes were found to be more pronounced in the anterior cingulate cortex than in the dorsolateral PFC. CONCLUSION: Depressed patients who committed suicide have different gene expression patterns than depressed patients who died of causes other than suicide.

Immunocytochemical localization of the glutamate transporter GLT-1 in goldfish (Carassius auratus) retina.

Glutamate is the major excitatory neurotransmitter in the retina of vertebrates. Electrophysiological experiments in goldfish and salamander have shown that neuronal glutamate transporters play an important role in the clearance of glutamate from cone synaptic clefts. In this study, the localization of the glutamate transporter GLT-1 has been investigated immunocytochemically at the light and electron microscopical levels in the goldfish retina using a GLT-1-specific antibody. GLT immunoreactivity (IR) was observed at the light microscopical level in Müller cells, bipolar cells, the outer plexiform layer (OPL), and the inner plexiform layer (IPL). At the electron microscopical level, membrane-bound and cytoplasmic GLT-IR in the OPL was located in finger-like protrusions of the cone terminal located near the invaginating postsynaptic processes of bipolar and horizontal cells. GLT-IR was not observed in the vicinity of synaptic ribbons. This location of GLT-1 allows modulation of the glutamate concentration in the synaptic cleft, thereby shaping the dynamics of synaptic transmission between cones and second-order neurons. In the inner IPL, GLT-IR was observed in the cytoplasm and was membrane bound in mixed rod/cone bipolar cell terminals and cone bipolar cell terminals. The membrane-bound GLT-1 was generally observed at some distance from the synaptic ribbon. The morphology of the bipolar cell terminal together with the localization of GLT-1 suggests that at least these glutamate transporters are not primarily involved in rapid uptake of glutamate release by the bipolar cells. The GLT-IR in the cytoplasm of Müller cells was located throughout the entire goldfish retina from the outer limiting membrane to the inner limiting membrane. The location of GLT-1 in Müller cells is consistent with the role of Müller cells in converting glutamate to glutamine.

Autoradiographic analysis of [35S]t-butylbicyclophosphorothionate binding in kindled rat hippocampus shows different changes in CA1 area and fascia dentata.

We studied the binding of [35S]t-butylbicyclophosphorothionate to the GABAA receptor-mediated chloride channel in the CA1 area and fascia dentata of control and Schaffer collateral kindled rats, by means of semi-quantitative autoradiography. The [35S]t-butylbicyclophosphorothionate binding was determined at three stages during kindling acquisition: (i) after six afterdischarges, (ii) after 14 afterdischarges and (iii) after the induction of fully kindled seizures. Furthermore, the binding was studied at the long-term stage, 28 days after the last generalized tonic-clonic seizure [Racine R. J. (1972) Electroenceph. clin. Neurophysiol. 32, 281-294]. The binding was investigated at three [35S]t-butylbicyclophosphorothionate concentrations, 4, 47.5 (KD value) and 180 nM (Bmax value). A significant decrease in [35S]t-butylbicyclophosphorothionate binding in the CA1 area (-6 to -20%) and hilar formation (-17 to -37%), in one or more of the three [35S]t-butylbicyclophosphorothionate concentrations tested at the six and 14 afterdischarges and fully kindled stages was observed, but no significant changes at the long-term kindling stage were found. In contrast, the granular and molecular layers of the fascia dentata presented a significant increase in [35S]t-butylbicyclophosphorothionate binding (+15 to +38%) at the 14 afterdischarges, fully kindled and long-term kindled stages.(ABSTRACT TRUNCATED AT 250 WORDS)

A long-lasting decrease in the inhibitory effect of GABA on glutamate responses of hippocampal pyramidal neurons induced by kindling epileptogenesis.

Experiments were carried out to test whether changes in the sensitivity of hippocampal pyramidal neurons to the neurotransmitters glutamate, GABA and noradrenaline may be associated with the establishment of an epileptogenic focus induced by kindling. The effects of iontophoretically applied neurotransmitters on the firing rate of single units were quantified in the rat hippocampal CA1 area in kindled and control animals. Kindling was induced by electrical tetanic stimulation of the Schaffer collateral/commissural fibers. Firing was evoked by local glutamate iontophoresis while simultaneous GABA or noradrenaline application suppressed this response. A significant reduction of the GABAergic inhibitory action on the firing rate in kindled animals studied around four or around 42 days after the last convulsion was found. In the same neurons, the suppressive effect of noradrenaline was not different from controls. The neurons of kindled animals, investigated around four days after the last seizure, had a reduced sensitivity for glutamate; more glutamate ejection current was needed to evoke firing or to evoke the maximum firing rate. In contrast, the responsiveness for glutamate was significantly increased long-term after the last convulsion. These findings demonstrate that hippocampal Schaffer collateral kindling is associated with a long-lasting reduced effectiveness of the GABA-mediated response on glutamate-evoked firing in CA1.

Long-term and regional specific changes in [3H]flunitrazepam binding in kindled rat hippocampus.

The binding of the GABAA/benzodiazepine receptor agonist [3H]flunitrazepam was studied in the hippocampus of rats kindled by daily stimulation of the Schaffer collaterals, using semi-quantitative autoradiography. Two kindled stages were investigated: (i) 24 h after the last generalized tonic-clonic seizure (fully kindled) and (ii) 28 days after the last generalized seizure (long-term). The binding of [3H]flunitrazepam was determined at two concentrations, 3 and 16 nM. In the CA1 area, we found a small but significant decrease (ca. 10%), both in the 3 and 16 nM [3H]flunitrazepam binding at the fully kindled stage. In contrast, there was a significant increase in the 3 nM binding (c. 15%) at the long-term stage. The 16 nM binding was not significantly different from control binding at this stage. In the granular and molecular layers of the fascia dentata, we found at both kindled stages a significantly increased 3 nM (ca. 9 and 19%, respectively) and 16 nM (ca. 19 and 14%, respectively) binding. Furthermore, we found that muscimol was still able to enhance the [3H]flunitrazepam binding in kindled animals, indicating that the GABAA receptor agonist binding site and benzodiazepine agonist binding site are still functionally coupled. The changes in [3H]flunitrazepam binding at the fully kindled stage are in agreement with the recently observed kindling-induced changes in [3H]muscimol binding in the hippocampal formation of the same animals [Titulaer M. N. G. et al. (1994) Neuroscience 59, 817-826] and extend these observations to the benzodiazepine modulatory site of the GABAA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of changes in hippocampal GABA immunoreactivity in the rat kindling model of epilepsy: a light microscopic study with GABA antibodies.

Immunocytochemical techniques were used to study changes of GABA immunoreactivity in the rat hippocampal CA1 region during kindling epileptogenesis, gradually developing over a period of two weeks by daily electrical tetanization of Schaffer collaterals/commissural fibres. The number of GABA-immunoreactive somata per mm2 was quantified in CA1 region after 6 and 14 stimulus-induced afterdischarges and in fully kindled animals. The absolute values were compared with those obtained from controls and the relative difference between the side where the stimulations were applied and the contralateral side was determined. In comparison to controls the rats showed, after 6 afterdischarges at the ipsilateral, stimulated side, a slight increase in cell density of about 10%; after 14 afterdischarges, there was a significant increase of 38% together with an enhanced labelling density, while in fully kindled rats (34 afterdischarges) there was no significant difference with controls. At the contralateral side, we observed a different pattern of, respectively, a small decrease (17%), no significant difference, and a significant increase of 22% in fully kindled animals. This result is discussed in relation to observations that fully kindled rats, investigated 24 days after the last seizure of the acquisition phase, showed rather a significant decrease by 35% of cell density at the stimulated side and a regional loss of immunoreactivity of varicosities. At this point in time, no significant changes were found at the contralateral side. The complex time-course of GABA immunoreactivity during epileptogenesis consists of a transient increase in the early phase of kindling acquisition followed by a process of gradual reduction that leads to the long-term decrease of GABA immunoreactivity and high excitability.

Transient increase of cytoplasmic calcium concentration in the rat hippocampus after kindling-induced seizures. An ultrastructural study with the oxalate-pyro-antimonate technique.

Kindling stimulations were applied to the Schaffer collateral/commissural fibers in the CA1 area of the dorsal rat hippocampus. In fully kindled animals the ultrastructural distribution of calcium was studied at different time intervals after an induced generalized seizure, using the oxalate-pyro-antimonate technique. Semi-quantitative analysis of the amount of precipitate revealed no change in the investigated structures analysed after 2 h or 24 h: boutons and spines of the Schaffer-collateral/pyramidal-dendrite synaptic contacts, cytoplasm and mitochondria of terminals on pyramidal cell bodies and smooth dendrites. The major change was found 15 min after a seizure, when calcium precipitate in boutons and spines of stratum radiatum was strongly increased, precipitate in somata terminals only slightly, while smooth dendrites were not affected. These results imply a seizure-related increase of the intracellular calcium concentration. The transient character suggests that the investigated cellular compartments in kindled tissue are still capable of maintaining calcium homeostasis. The observed increase in precipitate density for at least 15 min may initiate the neurochemical mechanisms leading to an enhanced seizure sensitivity in the kindling model of epilepsy.

N-methyl-D-aspartate and kainate receptor gene expression in hippocampal pyramidal and granular neurons in the kindling model of epileptogenesis.

To investigate the changes underlying kindling epileptogenesis in the rat hippocampus, the levels of the messenger RNAs encoding for the subunits of the N-methyl-D-aspartate-receptor (1, 2A-D) and the kainate-receptor (1, -2, GluR-5, -6, -7) were determined in hippocampal principal neurons using in situ hybridization techniques and semi-quantitative analysis of the autoradiograms. Schaffer collateral-commissural pathway kindled rats were investigated at three different stages of kindling acquisition, always 24 h after the last stimulation. Furthermore, fully kindled rats were studied at long-term (28 days) after termination of kindling stimulations. NR1 messenger RNA levels were slightly decreased in CA1 area of fully kindled animals. In the fascia dentata region, a minor increase of NR2A and NR2B transcripts was found at all stages of kindling acquisition. KA-2 messenger RNA was enhanced in all hippocampal subfields during kindling development. However, none of these changes persisted at long-term after the last seizure and only the low-abundant GluR-7 expression was slightly depressed in the fascia dentata. From our observations we conclude that it is unlikely that alterations in N-methyl-Daspartate or kainate receptor gene expression play an important role in kindling acquisition or maintenance.

Kindling induces time-dependent and regional specific changes in the [3H]muscimol binding in the rat hippocampus: a quantitative autoradiographic study.

To investigate possible changes in the GABAA receptor agonist site in the CA1 area and fascia dentata of rats kindled by stimulation of Schaffer collaterals, a quantitative autoradiographic study of the [3H]muscimol binding was carried out. Two kindled groups were studied, at 24 h (fully kindled stage) and at 28 days (long-term stage) after the last class V seizure. Several concentrations of [3H]muscimol were tested in the range of the high/intermediate (5-40 nM) and low-affinity (60-100 nM) binding sites. In the fully kindled group, the binding over the complete range of tested [3H]muscimol concentrations was significantly increased by 30-50% in the fascia dentata, while the binding was significantly decreased by 10-25% in the CA1 area. The high/intermediate-affinity binding was still significantly increased by 20-30% in the fascia dentata 28 days after the last seizure. In this long-term group there was still a significant decrease of 10-18% of the low-affinity binding in the CA1 area. These results show that kindling epileptogenesis induces long-lasting changes in the GABAA receptor agonist binding sites that are region specific. We hypothesize that the changes encountered at the fully kindled stage, i.e. increased binding in the fascia dentata and decreased binding in the CA1 area, may underly the electrophysiologically observed increased paired-pulse depression of field potentials in the former and the decreased paired-pulse depression in the latter area [Kamphuis et al. (1992) Neurosci. Lett. 141, 101-105; Kamphuis et al. (1988) Brain Res. 440, 205-215; Zhao and Leung (1991) Brain Res. 564, 220-229; Zhao and Leung (1992) Brain Res. 582, 163-167]. We conclude that the observed changes may not only contribute to the induction of kindling epileptogenesis but may also play a role in the maintenance of the kindled state.

Prostanoid receptor gene expression profile in human trabecular meshwork: a quantitative real-time PCR approach.

PURPOSE: To assess the expression pattern of prostanoid receptor-encoding genes in trabecular meshwork (TM) of human donor eyes. METHODS: Disposed human donor eyes (n = 10) were obtained from the Cornea Bank, Amsterdam. The TM was dissected from the scleral tissue and homogenized in lysis buffer, and total RNA was isolated. The RNA was converted into cDNA and used as a template for noncompetitive quantitative real-time polymerase chain reaction (PCR) using green fluorescent dye to quantify the accumulation of double-stranded PCR product. Specific primers for four housekeeping genes and DP, EP(1), EP(2), EP(3,) EP(4), FP, IP, and TP receptor-encoding transcripts were developed and tested for their efficiency. RESULTS: The characterized expression profile was highly reproducible in all samples, with the EP(2) receptor-encoding transcript in the highest abundance, followed by FP, TP, IP, and EP(4) at levels that were approximately 10 to 15 times lower than that of the EP(2) subtype. DP and EP(3) were at the lowest levels, which were, on average, 45 times and 228 times lower than EP(2), respectively. CONCLUSIONS: These data show that all prostanoid receptors are expressed at different levels in human TM tissue. Because the gene expression of the EP(2) receptor is, on average, 15 times more abundant than that of the EP(4) receptor, it may be expected that the increase in flow and cAMP levels in response to the activation of the EP receptors by application of prostaglandin E(1) (PGE(1)), is primarily mediated by the EP(2) receptor. These data should be considered when designing prostanoid receptor mimetics intended to enhance the aqueous humor outflow through the TM and Schlemm's canal.

Expression patterns of voltage-dependent calcium channel alpha 1 subunits (alpha 1A-alpha 1E) mRNA in rat retina.

The transcript levels of the genes encoding for the different alpha1 (alpha1A-alpha1E) subunits of voltage-dependent calcium channels (VDCCs) were studied in the retina of the rat using RT-PCR, Northern blotting, and in situ hybridization. Abundant expression of alpha1A and alpha1B was found with RT-PCR and on Northern blots of total retina RNA, corresponding with high expression levels in all nuclear layers (outer and inner nuclear layers and the ganglion cell layer) of the retina. VDCC alpha1D mRNA was also present in all nuclear layers of the retina but at less abundant levels than alpha1A or alpha1B. Expression level of alpha1C in the retina was low as deduced from a faint Northern blot signal and a moderate yield after PCR amplification. VDCC alpha1E specific amplification of retinal cDNA yielded a longer product (designated alpha1E-L) than obtained from the hippocampus. Nucleotide sequencing of this PCR product revealed a 129 bp insert which is largely homologous (97%) with a previously described insert in the same position in human alpha1E cDNA. In situ hybridization in rat brain showed a differential expression pattern of the long and short variants of alpha1E mRNA. Northern blotting of retinal RNA confirmed the absence of the short variant (alpha1E-S), while alpha1E-L was present at low levels. In situ hybridization detected a significant level of expression of alpha1E-L in the inner nuclear layer. The prevalent expression of alpha1A and alpha1B, and to a lesser extent, of alpha1D, indicates that P/Q-, N-, and L-type calcium currents play a prominent role in the various cell types involved in the retinal signal-transduction pathway. The absence of alpha1C transcript in the retina suggests that the slowly inactivating L-type calcium currents involved in neurotransmitter release from the terminals of photoreceptors and bipolar cells may be encoded by the alpha1D isoform.

Editing status at the Q/R site of glutamate receptor-A, -B, -5 and -6 subunit mRNA in the hippocampal kindling model of epilepsy.

The editing status of mRNA at the Q/R site of the glutamate receptor subtypes -A, -B, -5 and -6 modulates channel conductivity and ion selectivity of glutamate operated ion channels [4,15,26,30]. In order to investigate whether a modification of this editing process may be involved in kindling epileptogenesis, the percentage of edited variant was determined in the hippocampus of kindled rats and compared to the percentage in control animals. In the latter, GluR-A mRNA was detected only in the unedited form (with detection threshold for edited form < 0.7%), whereas GluR-B was completely edited (> 99%). For percentages were not significantly changed in Schaffer collateral/commissural pathway kindled animals that were sacrificed 24 h after the last generalized seizure. It is concluded that the increased sensitivity for the induction of seizures characteristic for Schaffer collateral kindled animals is not related to a less selective or less efficient mRNA editing process of the different glutamate receptor subunits in the hippocampus.

Expression of GABAA receptor subunit mRNAs in hippocampal pyramidal and granular neurons in the kindling model of epileptogenesis: an in situ hybridization study.

To investigate the molecular changes underlying kindling epileptogenesis in the rat hippocampus, the expression levels of the genes encoding for 13 different gamma-aminobutyric acid type-A receptor (GABAAR) subunits were measured in hippocampal principal neurons using in situ hybridization techniques and semi-quantitative analysis of the autoradiograms. Schaffer collateral-commissural pathway kindled rats were investigated at three different stages of kindling acquisition, at 24 h after the last seizure and at long-term (28 days) after termination of kindling stimulations. Changes were distinct for the different subunits in the three analyzed regions (CA1, CA3, fascia dentata) and also different for the various kindling stages. In all hippocampal areas at the early phases of kindling epileptogenesis, before the appearance of generalized seizures, an increase was found of those transcripts that constituted the majority of the expressed variants in control animals (alpha 1, alpha 2, alpha 4, beta 1, beta 2, beta 3, gamma 2/gamma 2L mRNA). In these stages, the increased levels of different variants in the granular neurons of the fascia dentata were more pronounced when compared to the pattern of changes in pyramidal cells of CA1 and CA3. In fully kindled animals, the expression levels of several subunits returned to control levels, whereas beta 3 and gamma 2/gamma 2L mRNA expression was still significantly enhanced in all areas. At long-term, few changes were encountered. The long-splice variant of gamma 2 was decreased within pyramidal and granular neurons while the total level of gamma 2 mRNA was not different from controls. The increased GABAAR subunit expression in the fascia dentata may underly the reported increased GABAAR ligand binding and the increased GABA mediated inhibition. However, the decreased GABAAR binding and the attenuation of GABAergic inhibition in CA1, could not be explained by a decrement of receptor subunit expression.

Changes in voltage-dependent calcium channel alpha1-subunit mRNA levels in the kindling model of epileptogenesis.

The establishment of a focus of epileptiform activity in the hippocampus of the rat, using the kindling paradigm, leads to enhanced voltage-dependent calcium conductance of CA1 pyramidal neurones (G.C. Faas, M. Vreugdenhil, W.J. Wadman, Calcium currents in pyramidal CA1 neurones in vitro after kindling epileptogenesis in the hippocampus of the rat, Neuroscience 75 (1996) 57-67; M. Vreugdenhil, W.J. Wadman, Kindling-induced long-lasting enhancement of calcium in hippocampal CA1 area of the rat: relation to calcium-dependent inactivation, Neuroscience 59 (1994) 105-114). Using semi-quantitative in situ hybridization techniques, we investigated whether these changes were associated with an altered expression of the genes that encode for the alpha1A-E-subunits of the voltage-dependent calcium channels (VDCC). Kindling epileptogenesis was induced in rats that received an electrical tetanic stimulation of the Schaffer collateral/commissural fibre pathway in the hippocampus twice daily. Two groups of rats were studied before the appearance of generalized seizures, one group after at least 5 generalized seizures (fully kindled) and one group was investigated at long-term (28 days) after the last seizure. During the initial stages of epileptogenesis, the alpha1A-, alpha1D- and alpha1E-subunit mRNA levels were significantly increased in the different hippocampal subareas in comparison to the levels in control animals. In contrast, alpha1B-subunit gene expression decreased in the CA area and dentate gyrus. No significant change was observed in the alpha1C-I and alpha1C-II expression. At the fully kindled stage, the only significant change was an up-regulation of the alpha1B-subunit mRNA levels in the CA3 area, 24 h after the last seizure. No change in VDCC alpha1-subunit gene expression was found in animals investigated long-term after the establishment of the fully kindled state. Thus, the VDCC alpha1-subunit gene expression is altered in a subclass-specific manner during the early stages of kindling and may play a role in the establishment of a kindled focus, possibly caused by an alteration of the population of VDCCs involved in neurotransmitter release. The absence of long-lasting changes suggests that the maintenance of a kindled focus is not due to persisting alterations in VDCC alpha1 mRNA levels.

Corticosteroid actions on the expression of kainate receptor subunit mRNAs in rat hippocampus.

Previous studies have shown that corticosteroid hormones affect kainate-induced excitotoxic processes in the rat hippocampus. In the present study we employed in situ hybridization to examine the effect of adrenalectomy, and subsequent treatment with a low or a high lose of corticosterone on the mRNA levels for kainate receptor subunits in the hippocampus. We observed that adrenalectomy by itself does not affect the expression pattern for the GluR6, GluR7, KAR1 and KAR2 subunits. However, treatment of the adrenalectomized animals with a low dose of corticosterone (3 micrograms/100 g bodyweight) resulted in an enhanced expression of the KAR1, KAR2 and GluR6 subunit mRNAs, when compared to the expression levels in the untreated rats or the sham operated controls. Treatment with a high dose of corticosterone (1 mg/100 g bodyweight) yielded expression levels which were significantly lower than those observed in animals treated with a low corticosterone dose, for the KAR1, KAR2 and GluR7 subunit mRNAs; the levels did not differ from those in untreated rats or in the sham group. We conclude that changes in corticosteroid receptor occupancy, which may occur daily due to circadian or stress-induced variations in the circulating corticosterone level, potentially regulate high affinity kainate receptor activation and the processes in which these receptors are involved.

Ischaemia does not alter the editing status at the Q/R site of glutamate receptor-A, -B, -5 and -6 subunit mRNA.

The editing status of mRNA at the Q/R site of the AMPA/kainate type glutamate receptor (GluR) subunits A, B, 5 and 6 modulates the ion selectivity of glutamate receptor operated ion channels. We hypothesized that a long-lasting dysfunction of this editing process after an ischeamic insult may be involved in the process of delayed neuronal cell death. In order to test this hypothesis, the editing status of the GluRs was determined in the hippocampus of rats up to 24 h after 10 min of global ischaemia. No statistical significant differences were found in the hippocampus of animals after ischaemia compared with controls. It is concluded that delayed neuronal cell death is not mediated by a less selective or less efficient mRNA editing process of the different glutamate receptor subunits in the hippocampus.