Time-dependent changes in GLT-1 functioning in striatum of hemi-Parkinson rats

Striatal dopamine loss in Parkinson's disease is accompanied by a dysregulation of corticostriatal glutamatergic neurotransmission. Within this study, we investigated striatal expression and activity of the glial high-affinity Na+/K+-dependent glutamate transporters, GLT-1 and GLAST, in the 6-hydroxydopamine hemi-Parkinson rat model at different time points after unilateral 6-hydroxydopamine injection into the medial forebrain bundle. Using semi-quantitative Western blotting and an ex vivo D-[H-3]-aspartate uptake assay, we showed a time-dependent bilateral effect of unilateral 6-hydroxydopamine lesioning on the expression as well as activity of GLT-1. At 3 and 12 weeks post-lesion, striatal GLT-1 function was bilaterally upregulated whereas at 5 weeks there was no change. Even though our data do not allow a straightforward conclusion as for the role of glutamate transporters in the pathogenesis of the disease, they do clearly demonstrate a link between disturbed glutamatergic neurotransmission and glutamate transporter functioning in the striatum of a rat model for Parkinson's disease. (C) 2010 Elsevier Ltd. All rights reserved

Inactivation of the constitutively active ghrelin receptor attenuates limbic seizure activity in rodents

Ghrelin is a pleiotropic neuropeptide that has been recently implicated in epilepsy. Animal studies performed to date indicate that ghrelin has anticonvulsant properties; however, its mechanism of anticonvulsant action is unknown. Here we show that the anticonvulsant effects of ghrelin are mediated via the growth hormone secretagogue receptor (GHSR). To our surprise, however, we found that the GHSR knockout mice had a higher seizure threshold than their wild-type littermates when treated with pilocarpine. Using both in vivo and in vitro models, we further discovered that inverse agonism and desensitization/internalization of the GHSR attenuate limbic seizures in rats and epileptiform activity in hippocampal slices. This constitutes a novel mechanism of anticonvulsant action, whereby an endogenous agonist reduces the activity of a constitutively active receptor. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13311-012-0125-x) contains supplementary material, which is available to authorized users

Enhanced neuronal survival in senescent cultures of cortical neurons mediated by rAAV2/1-NF8d1-driven GDNF transgene expression.

<p>(<b>A</b>) Embryonic day 16 cortical neurons were maintained in 96 wells plates for 60 days. Cultures were fixed and stained with NeuN (a neuronal specific nuclear protein) or PSD95 (a neuronal postsynaptic density protein) a class of anchoring proteins which serve to localize various neuronal ion channels to post-synaptic densities antibodies. The number of NeuN-positive or PSD95-positive neurons is significantly higher in cultures treated with rAAV2/1-NF8d1-GDNF as compared to rAAV2/1-NF8d1-EGFP (CtrL) (p<0.05; student test, n = 10). Bars represent means ± SEM. (<b>B</b>) Similar cultures seeded on coated glass coverslips were fixed at day 10, day 21 and day 47 and stained with antibodies directed against activated NFkB followed by a secondary antibody coupled with cyanine 2. Shown are confocal microscopy photographs. Bars represent 50 µM. The mean optical density of the nuclei was measured using the Image J software. Differences between day 10 (n = 39) and day 21 (n = 59) or day 47 (n = 57) were respectively highly significant (**; p<0.001) and very highly significant (###; p<0.0001). The experiment was repeated twice with similar results. (<b>C</b>) Culture media from rAAV1-NF8-d1-GDNF- and rAAV1-NF8-d1-EGFP-infected cultures were harvested from day 5 to day 42 post-infection and GDNF concentrations (pg/ml) were measured using ELISA assay. For rAAV1-NF8-d1-GDNF-infected cultures, all values, except at day 5, were significantly higher (***; p<0.0001; n = 10 for rAAV1-NF8-d1-hGDNF and n = 8 for rAAV1-NF8-d1-EGFP) than the basal level (medium from rAAV1-NF8-d1-EGFP-infected cultures). The experiment was repeated twice with similar results.</p

Absence of transgene upregulation in rAAV2/1-NF8d1-EGFP-mediated gene transfer in the cerebellum of kainic acid-treated rats.

<p>Kainic acid (5 mg/kg) was injected intraperitoneally (ip) one month after stereotaxic injection of rAAV2/1-NF8d1-EGFP into the cerebellum. Fifty µm coronal sections were labeled with GFP or GFAP antibodies. Confocal pictures showing GFP (A) or GFAP (B). Bars represent 50 µm. GFAP staining was quantified by measuring the optical density of three area in all sections stained per animal (6 rats for each condition). The difference between KA- and saline-treated rats is not significant (student test, p = 0,8612) (B).</p

Disease-inducible rAAV1-NF-mediated transgene expression in the hippocampus of kainic acid-treated rats.

<p>Recombinant AAV2/1-NF8-d1-EGFP (8×10⁶ vg) was stereotaxically injected in the right hippocampus of male Wistar rats. Animals were kept in two groups: one group was intraperitoneally-injected with kainic acid (n = 8) one month post stereotaxy and the other group received only saline (n = 8). The animals were sacrificed one week after injection. Vibratome brain sections (50 µm) were immunolabeled using an anti-GFP antibody, biotin-streptavidin amplification and Cy2 fluorophore. <b>A.</b> Representative examples of GFP-labeling of the CA hippocampal layers dentate gyrus post-kainic acid injection. Note the massive presence of GFP-positive cells in the CA1 and CA3 layers. Stereological counts of GFP-positive cell numbers per animal demonstrates a highly significant induction of the AAV-NF vector by kainic acid (P = 0,000875; student test; n = 8 for the saline group; n = 6 for the kainic acid group). KA, kainic acid; saline, 0.9% NaCl. The experiment was repeated 3 times with similar results. Bars represent 500 µm and 100µm in lower and higher (1,2,3 subpannels) magnification pictures, respectively. <b>B</b>. Comparison of the staining intensity for activated NFκB in kainic acid- and saline-treated rats. Vibratome brain sections (50 µm) were immunolabeled using anti-activated NFκB antibody followed by streptavidin-biotin-peroxidase staining. Note, in particular, the localized increased staining in the CA hippocampal layers. The intensity of the staining of three random areas in the hipocampal layers on every section was quantified using the Image J program. Data are expressed as the mean optical density ± SEM (n = 8 for each group of animals). The difference between KA and saline-treated groups was significant (student test, P = 0.0010). Bars represent 500 µm. <b>C.</b> Correlation between NFκB activation and GFP expression mediated by rAAV2/1-NF8-d1-EGFP in hippocampal layers. A group of 16 rats was injected with a rAAV1/2-NF8-d1-EGFP in the hippocampus. 8 rats were injected with KA and 8 other animals were injected with the saline solution. Two KA-treated rats were removed from the analysis since they either contained no GFP-positive cells (presumably due to a failure to inject the virus) or had a totally different profile of GFP-expression (no GFP-positive cells in the hippocampal layers, presumably due to wrong stereotaxic coordinates). The total number of GFP-immunoreactive cells per animal (as evaluated by stereology was correlated with the mean optical density of the NFκB stainings in the region containing the transduced cells. There was a significant correlation between the two parameters with a coefficient of 0.7001 (p = 0.0002; n = 6 for KA-treated rats and n = 8 for saline-treated rats). The experiment was repeated a second time (n = 8 for KA-treated rats and n = 7 for saline-treated rats) with similar results.</p

Hippocampal sclerosis and upregulation of inflammatory markers in the hippocampus of kainic acid-treated rats.

<p><b>A.</b> Haematoxylin-eosin staining showing typical hippocampal sclerosis in kainic acid-treated rats (KA) (left) as compared to controls (right). Bar represents 200 µm. <b>B.</b> Comparison of the intensity of the GFAP astrocytic marker staining in kainic acid- and saline-treated rats. Vibratome brain sections (50 µm) were immunolabeled using mouse anti-GFAP followed by a secondary antibody coupled to Cy3 fluorophore. The intensity of the staining of three random areas in the hipocampal layers on every section was quantified using the Image J program. Data are expressed as the mean optical density ± SEM (n = 8 for each group of animals). The difference between kainic acid and saline-treated groups was highly significant (student test, P = 0,00018). Bar represents 500 µm. <b>C.</b> Comparison of the intensity of the CD11b staining of activated microglia in kainic acid- and saline-treated rats. Vibratome brain sections (50 µm) were immunolabeled using mouse anti-CD11b followed by streptavidin-biotin-peroxidase staining. The intensity of the staining of three random areas within the hippocampus on every section was quantified using the Image J program. Arrow shows localized staining at the level of the needle tract. Data are expressed as the mean optical density ± SEM (n = 4 for each group of animals). The difference between kainic acid and saline-treated groups was significant (student test, P = 0,0068). Bar represents 200 µm. <b>D.</b> Comparison of the intensity of the IbaI microglial staining in kainic acid- and saline-treated rats. Vibratome brain sections (50 µm) were immunolabeled using goat anti-IbaI followed by anti-goat antibody coupled with cyanine 3. The intensity of the staining of three random areas within the hippocampus on every section was quantified using the Image J program. Data are expressed as the mean optical density ± SEM (n = 6 for each group of animals). The difference between kainic acid and saline-treated groups was significant (student test, p = 0,00121). Bar represents 500 µm.</p

Cellular specificity of rAAV2/1-NF8d1-EGFP-mediated gene transfer in the hippocampus of kainic acid-treated rats.

<p>Kainic acid (5 mg/kg) was injected intraperitoneally (ip) one month after stereotaxic injection of rAAV2/1-NF8d1-EGFP into the hippocampus. Fifty µm coronal sections were co-labeled with GFP (green fluorescence) and NeuN, GFAP IbaI or Olig2. Confocal pictures showing GFP and cell-specific markers co-labeled cells (yellow). White arrows indicate co-labeled cells. Bars represent 50 µm. The mean percentage of GFP-positive cells co-labeling with the GFAP, NeuN and Olig2 markers are shown. No GFP-positive cell stained positive for IbaI. Analysis was performed using confocal microscopy and counting the number co-labeled cells on 5 sections per animal (n = 5 rats for GFP/NeuN and GFP/GFAP, n = 6 for GFP/IbaI and n = 7 for GFP/Olig2 co-labeling).</p