2-Cl-MGV-1 Ameliorates Apoptosis in the Thalamus and Hippocampus and Cognitive Deficits After Cortical Infarct in Rats.

BACKGROUND AND PURPOSE: Focal cortical infarction causes neuronal apoptosis in the ipsilateral nonischemic thalamus and hippocampus, which is potentially associated with poststroke cognitive deficits. TSPO (translocator protein) is critical in regulating mitochondrial apoptosis pathways. We examined the effects of the novel TSPO ligand 2-(2-chlorophenyl) quinazolin-4-yl dimethylcarbamate (2-Cl-MGV-1) on poststroke cognitive deficits, neuronal mitochondrial apoptosis, and secondary damage in the ipsilateral thalamus and hippocampus after cortical infarction. METHODS: One hundred fourteen hypertensive rats underwent successful distal middle cerebral artery occlusion (n=76) or sham procedures (n=38). 2-Cl-MGV-1 or dimethyl sulfoxide as vehicle was administrated 2 hours after distal middle cerebral artery occlusion and then for 6 or 13 days (n=19 per group). Spatial learning and memory were tested using the Morris water maze. Secondary degeneration and mitochondrial apoptosis in the thalamus and hippocampus were assessed using Nissl staining, immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling, JC-1 staining, and immunoblotting 7 and 14 days after surgery. RESULTS: Infarct volumes did not significantly differ between the vehicle and 2-Cl-MGV-1 groups. There were more neurons and fewer glia in the ipsilateral thalamus and hippocampus in the vehicle groups than in the sham-operated group 7 and 14 days post-distal middle cerebral artery occlusion. 2-Cl-MGV-1 significantly ameliorated spatial cognitive impairment and decreased neuronal death and glial activation when compared with vehicle treatment (P<0.05). The collapse of mitochondrial transmembrane potential and cytoplasmic release of apoptosis-inducing factors and cytochrome c was prevented within the thalamus. Caspase cleavage and the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling+ or Nissl atrophic cells were reduced within the thalamus and hippocampus. This was accompanied by upregulation of B-cell lymphoma 2 and downregulation of Bax (P<0.05). CONCLUSIONS: 2-Cl-MGV-1 reduces neuronal apoptosis via mitochondrial-dependent pathways and attenuates secondary damage in the nonischemic thalamus and hippocampus, potentially contributing to ameliorated cognitive deficits after cortical infarction.

Altered thalamic GABAA-receptor subunit expression in the stargazer mouse model of absence epilepsy.

PURPOSE: Absence seizures, also known as petit mal seizures, arise from disruptions within the cortico-thalamocortical network. Interconnected circuits within the thalamus consisting of inhibitory neurons of the reticular thalamic nucleus (RTN) and excitatory relay neurons of the ventral posterior (VP) complex, generate normal intrathalamic oscillatory activity. The degree of synchrony in this network determines whether normal (spindle) or pathologic (spike wave) oscillations occur; however, the cellular and molecular mechanisms underlying absence seizures are complex and multifactorial and currently are not fully understood. Recent experimental evidence from rodent models suggests that regional alterations in γ-aminobutyric acid (GABA)ergic inhibition may underlie hypersynchronous oscillations featured in absence seizures. The aim of the current study was to investigate whether region-specific differences in GABAA receptor (GABAAR) subunit expression occur in the VP and RTN thalamic regions in the stargazer mouse model of absence epilepsy where the primary deficit is in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression. METHODS: Immunofluorescence confocal microscopy and semiquantitative Western blot analysis were used to investigate region-specific changes in GABAAR subunits in the thalamus of the stargazer mouse model of absence epilepsy to determine whether changes in GABAergic inhibition could contribute to the mechanisms underlying seizures in this model of absence epilepsy. KEY FINDINGS: Immunofluorescence confocal microscopy revealed that GABAAR α1 and ß2 subunits are predominantly expressed in the VP, whereas α3 and ß3 subunits are localized primarily in the RTN. Semiquantitative Western blot analysis of VP and RTN samples from epileptic stargazers and their nonepileptic littermates showed that GABAAR α1 and ß2 subunit expression levels in the VP were significantly increased (α1: 33%, ß2: 96%) in epileptic stargazers, whereas α3 and ß3 subunits in the RTN were unchanged in the epileptic mice compared to nonepileptic control littermates. SIGNIFICANCE: These findings suggest that region-specific differences in GABAAR subunits in the thalamus of epileptic mice, specifically up-regulation of GABAARs in the thalamic relay neurons of the VP, may contribute to generation of hypersynchronous thalamocortical activity in absence seizures. Understanding region-specific differences in GABAAR subunit expression could help elucidate some of the cellular and molecular mechanisms underlying absence seizures and thereby identify targets by which drugs can modulate the frequency and severity of epileptic seizures. Ultimately, this information could be crucial for the development of more specific and effective therapeutic drugs for treatment of this form of epilepsy.

Serotonin transporter (SERT) and translocator protein (TSPO) expression in the obese ob/ob mouse.

BACKGROUND: An ever growing body of evidences is emerging concerning metabolism hormones, neurotransmitters or stress-related biomarkers as effective modulators of eating behavior and body weight in mammals. The present study sought at examining the density and affinity of two proteins related to neurotransmission and cell metabolism, the serotonin transporter SERT and the cholesterol import-benzodiazepine site TSPO (translocator protein), in a rodent leptin-lacking mutant, the obese ob/ob mouse. Binding studies were thus carried out in brain or peripheral tissues, blood platelets (SERT) and kidneys (TSPO), of ob/ob and WT mice supplied with a standard diet, using the selective radiochemical ligands [3H]-paroxetine and [3H]-PK11195. RESULTS: We observed comparable SERT number or affinity in brain and platelets of ob/ob and WT mice, whilst a significantly higher [3H]-PK11195 density was reported in the brain of ob/ob animals. TSPO binding parameters were similar in the kidneys of all tested mice. By [3H]-PK11195 autoradiography of coronal hypothalamic-hippocampal sections, an increased TSPO signal was detected in the dentate gyrus (hippocampus) and choroids plexus of ob/ob mice, without appreciable changes in the cortex or hypothalamic-thalamic regions. CONCLUSIONS: These findings show that TSPO expression is up-regulated in cerebral regions of ob/ob leptin-deficient mice, suggesting a role of the translocator protein in leptin-dependent CNS trophism and metabolism. Unchanged SERT in mutant mice is discussed herein in the context of previous literature as the forerunner to a deeper biochemical investigation.

Cancer-related overexpression of the peripheral-type benzodiazepine receptor and cytostatic anticancer effects of Ginkgo biloba extract (EGb 761).

The peripheral-type benzodiazepine receptor (PBR) is an 18-kDa high affinity drug- and cholesterol-binding protein that is involved in various cell functions, including cell proliferation and apoptosis. PBR was shown to be overexpressed in certain types of malignant human tumors and cancer cell lines, correlating with enhanced tumorigenicity and cell proliferation rates. The present study was conducted in order to further define the role of PBR in cancer and to extend our recent findings regarding the possible anticancer effects of the standardized Ginkgo biloba extract EGb 761. Treatment with EGb 761 decreased PBR mRNA levels and inhibited the proliferation of breast, glioma and hepatocarcinoma cell lines, further corroborating our previous contention that its mechanism of action is through the modification of PBR expression. In vivo treatment with Ginkgo biloba extract led to dose-dependent decreases in xenograft growth of both MDA-MB-231 breast cancer and U-87 glioma cell lines in nude mice, although the effects were not maintained after 50 days of treatment in the latter. The results obtained in MDA-MB-231 xenografts indicated pronounced inhibition of tumor growth, verified by MRI imaging. These results were obtained using a modified experimental protocol where the animals were treated with the extract before cell inoculation. Although an exact role for PBR in relation to the initiation and progression of various types of cancer remains to be defined, our results indicate that PBR overexpression in certain cancer cells is related to an aggressive phenotype. Since EGb 761 treatment opposes this aggressive phenotype by decreasing PBR overexpression, it could be useful in preventing or treating cancer invasiveness and metastasis.

Complementary roles of cholecystokinin- and parvalbumin-expressing GABAergic neurons in hippocampal network oscillations.

In the hippocampal CA1 area, a relatively homogenous population of pyramidal cells is accompanied by a diversity of GABAergic interneurons. Previously, we found that parvalbumin-expressing basket, axo-axonic, bistratified, and oriens-lacunosum moleculare cells, innervating different domains of pyramidal cells, have distinct firing patterns during network oscillations in vivo. A second family of interneurons, expressing cholecystokinin but not parvalbumin, is known to target the same domains of pyramidal cells as do the parvalbumin cells. To test the temporal activity of these independent and parallel GABAergic inputs, we recorded the precise spike timing of identified cholecystokinin interneurons during hippocampal network oscillations in anesthetized rats and determined their molecular expression profiles and synaptic targets. The cells were cannabinoid receptor type 1 immunopositive. Contrary to the stereotyped firing of parvalbumin interneurons, cholecystokinin-expressing basket and dendrite-innervating cells discharge, on average, with 1.7 +/- 2.0 Hz during high-frequency ripple oscillations in an episode-dependent manner. During theta oscillations, cholecystokinin-expressing interneurons fire with 8.8 +/- 3.3 Hz at a characteristic time on the ascending phase of theta waves (155 +/- 81 degrees), when place cells start firing in freely moving animals. The firing patterns of some interneurons recorded in drug-free behaving rats were similar to cholecystokinin cells in anesthetized animals. Our results demonstrate that cholecystokinin- and parvalbumin-expressing interneurons make different contributions to network oscillations and play distinct roles in different brain states. We suggest that the specific spike timing of cholecystokinin interneurons and their sensitivity to endocannabinoids might contribute to differentiate subgroups of pyramidal cells forming neuronal assemblies, whereas parvalbumin interneurons contribute to synchronizing the entire network.

Ions and amino acid analysis of Cyperus articulatus L. (Cyperaceae) extracts and the effects of the latter on oocytes expressing some receptors.

Extracts from rhizomes of Cyperus articulatus L. (Cyperaceae) used in Africa and Amazonia to treat many diseases has been shown to possess sedative and anticonvulsant properties. The aim of this study is to determine the mechanism of action of Cyperus articulatus extracts. In Xenopus oocytes expressing receptors, using electrophysiological measurement, extracts of rhizomes of Cyperus articulatus (300 microg/ml) inhibited 50% of the EC(50) and EC(80) of glutamate (1.3 and 2.9 microM, respectively) induced inward current through hNMDAR1A/2A receptors. Extracts induced very small current through rGluR3 receptors. The largest current induced by the extract (30 mg/ml) represents 128% of the EC(100) of glutamate induced inward current, through rGluR3 receptors. The excess 28% current could be induced by aspartate and/or glutamate in the extracts. The effect on Xenopus oocytes expressing heteromeric GABA(B)R1b/R2 receptors and rectifying potassium channels (Kir3) is clear. A decoction and water extract of Cyperus articulatus induced a large inward current that represented 71 and 57% (respectively) of the EC(100) of gaba (30 microM) induced inward current. The water extract induced also a large current through rectifying potassium channels (Kir3). Part of the current induced through GABA(B) receptors could be related to rectifying potassium channels and GABA(B) site receptors. Cyperus articulatus extracts possessed components that could decrease excitation (NMDA receptor antagonists) and increase inhibition (GABA(B) receptor agonists) in the central nervous system.

Positive regulation of GABA(B) receptors dually coupled to cyclic AMP by the allosteric agent CGP7930.

The ability of 2,6 Di-tert-butyl-4-(-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930), a positive allosteric modulator of GABA(B) receptors, to regulate GABA(B) receptor-induced stimulation and inhibition of adenylyl cyclase activity in rat brain was investigated. In olfactory bulb granule cell layer and in frontal cortex, CGP7930 potentiated the stimulatory effects of (-)-baclofen and gamma-aminobutyric acid (GABA) on basal and corticotropin-releasing hormone-stimulated adenylyl cyclase activities, respectively. In these stimulatory responses, CGP7930 enhanced both agonist potencies and maximal effects. When GABA(B) receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity of frontal cortex was examined, CGP7930 increased the agonist potencies but failed to affect the maximal effect of (-)-baclofen and modestly increased that of GABA. Similar results were obtained for the inhibition of Ca(2+)/calmodulin-stimulated adenylyl cyclase in striatum and cerebellum. Western blot analysis of each membrane preparation showed the presence of GABA(B2) receptor subunit, a putative site of action of CGP7930. These data indicate that CGP7930 positively modulates brain GABA(B) receptors coupled to either stimulation or inhibition of cyclic AMP signalling.

Separate domains for desensitization of GABA rho 1 and beta 2 subunits expressed in Xenopus oocytes.

Desensitization of ligand-gated receptor channels is an intrinsic feedback mechanism and prevents the receptor/channels from becoming overly activated thereby maintaining biological function of the nervous system. Desensitization also plays an important role in neuronal plasticity. By taking advantage of biophysical and pharmacological diversities of GABA beta2 subunits from the brain and rho1 subunits from the retina, structural determinants that confer agonist-induced desensitization were identified. A synthetic chimeric receptor/channel was created from the beta2 and rho1 subunits for this investigation. The chimera was constructed from the extracellular N-domain of the beta2 subunit, extending from the amino terminus to the beginning region of the M1 transmembrane segment, and from the C-domain of the rho1 subunit extending from the M1 transmembrane segment to the carboxyl terminus. The C-domain region included the M1 to M4 transmembrane regions and the large intracellular loop between the M3 and M4 transmembrane segments. Homo-oligomeric GABA beta2, rho1, and beta2/rho1 chimeric receptor/channels were individually expressed in Xenopus oocytes, and the desensitization characteristics attributable to each type of subunit were compared. Results from the present study reveal that motifs in the amino-terminal and carboxyl-terminal domains of the beta2 subunit conferred the agonist-induced desensitization; chloroform modulation was linked to specific phases of the GABA-activated current decay.

Cloning and functional expression of a Drosophila gamma-aminobutyric acid receptor.

A cDNA encoding a functional gamma-aminobutyric (GABA)-activated Cl- channel has been isolated from an adult Drosophila head cDNA library. When expressed in Xenopus laevis oocytes, the subunit functions efficiently, presumably as a homooligomeric complex and is activated by GABA or muscimol. GABA-evoked currents are highly sensitive to antagonism by picrotoxin but are insensitive to bicuculline, RU 5135, or zinc. Pentobarbitone greatly enhances GABA-evoked currents, whereas the neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one demonstrates a large reduction in both the potency and maximal effect when compared with its actions upon vertebrate GABA type A receptors. Although zinc-insensitive, the subunit is also insensitive to flunitrazepam. Hence, the GABA receptors formed by this subunit exhibit a unique pharmacology when compared with vertebrate GABA type A receptors or those composed of rho subunits. Because the receptor-channel complex functions as a homooligomer, this subunit may be of value in mutagenesis studies aiming to define drug-binding sites.

Synthesis of functional GABAA receptors in stable insect cell lines.

We have synthesised the beta 1-subunit of the bovine GABAA receptor in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovirus immediate early (IE-1) gene promoter. Transformed cells were obtained by co-transfection of the insect cells with pIEK1.GR beta 1, encoding the beta 1 subunit cDNA, and pIEK1.neo, encoding the neomycin resistance gene. G-418-resistant clones were selected and expanded into continuous cell lines synthesising functional, GABA-gated, homo-oligomeric chloride channels. These cell lines had significant advantages over the transient baculovirus expression system for the characterisation of receptors using electrophysiological recording techniques.