Galanin interacts with Neuropeptide Y Y1 receptor in the dentate gyrus of the hippocampus through GALR2/NPYY1R heterodimers

We have shown Galanin(GAL) and Neuropeptide Y Y1(NPYY1) interactions at behavioural, cellular and receptor levels through GALR2/NPYY1R heterodimers in the amygdala. The aim of this work was to analyze GAL/NPYY1R interactions in the Dentate Gyrus(DG) of the Hippocampus, using autoradiographic, in situ hybridization and in situ proximity ligation assay(PLA). Rats(n=6) were sacrificed 15 minutes or 5 hours after icv injections of GAL(3nmol) and DG sections were incubated with NPYY1R agonist [I125]-[Leu31,Pro34]PYY(25 pM) or NPYY1R-33PdATP specific probe, for autoradiography and in situ hybridization respectively. Autoradiograms were analyzed using NIH image analysis system and Student’s unpaired t-test was used. For PLA, DG sections were incubated with anti-GALR2 Rabbit(1:100) and anti-NPYY1R Goat(1:200). PLA signals were detected with PLA PLUS or MINUS probes for rabbit or goat/mouse antibodies. PLA signals were visualized by using a confocal microscope Leica TCS-SL confocal microscope(Leica). We observed that GAL significant increased the NPYY1R agonist [I125]-[Leu31,Pro34]PYY binding in the DG by 20% (p<0,05) and the NPYY1R mRNA expression in the granular layer of DG by 31% (p<0,001). Moreover, PLA-positive red clusters were found specifically in the polymorphic layer and subgranular zone of the DG. No PLA clusters were observed neither in the molecular layer of the DG nor in the corpus callosum, an area that seems to lack of GALR2 receptor. These results demonstrate a novel mechanism of interaction between GAL and NPY1R in the DG at receptor level, probably involving the formation of GALR2/NPYY1R heteroreceptor complexes. Study supported by Junta de Andalucia CVI6476.

Positive biocompatibility of nanocrystalline glass-like carbon thin films

The interest in carbon nanomaterials with high transparency and electrical conductivity has grown within the last decade in view of a wide variety of applications, including biocompatible sensors, diagnostic devices and bioelectronic implants. The aim of this work is to test the biocompatibility of particular nanometer-thin nanocrystalline glass-like carbon films (NGLC), a disordered structure of graphene flakes joined by carbon matrix (Romero et al., 2016). We used a cell line (SN4741) from substantia nigra dopaminergic cells derived from transgenic mouse embryo cells (Son et al., 1999). Some cells were cultured on top of NGLC films (5, 20 and 80 nm) and other with NGLC nanoflakes (approx. 5-10 mm2) in increasing concentrations: 1, 5, 10, 20 and 50 μg/ml, during 24 h, 3 days and 7 days. Cells growing in normal conditions were defined under culture with DMEM supplemented with 10% FCS, Glucose (0,6%), penicillin-streptomycin (50U/ml) and L-glutamine (2mM) at 5%CO2 humidified atmosphere. Nanoflakes were resuspended in DMEM at the stock concentration (2 g/l). The experiments were conducted in 96 well plates (Corning) using 2500 cells per well. For MTT analysis, the manufacturer recommendations were followed (Roche, MTT kit assay): a positive control with a 10% Triton X-100 treatments (15 minutes) and a negative control without neither Triton X-100 nor NGLC. As apoptosis/necrosis assay we used LIVE/DEAD® Viability/Cytotoxicity Assay Kit (Invitrogen). In a separate experiment, cells were cultured on top of the NGLC films for 7 days. Primary antibodies: anti-synaptophysin (SYP, clone SY38, Chemicon) and goat anti-GIRK2 (G-protein-regulated inward-rectifier potassium channel 2 protein) (Abcom) following protocol for immunofluorescence. WB for proteins detection performed with a polyclonal anti-rabbit proliferating cell nuclear antigen (PCNA). Results demonstrated the biocompatibility with different concentration of NGLC varying the degree of survival from a low concentration (1 mg/ml) in the first 24 h to high concentrations (20-50 g/ml) after 7 days as it is corroborated by the PCNA analysis. Cells cultured on top of the film showed after 7 days axonal-like alignment and edge orientation as well as net-like images. Neuronal functionality was demonstrated to a certain extent through the analysis of coexistence between SYP and GIRK2. In conclusion, this nanomaterial could offer a powerful platform for biomedical applications such as neural tissue engineering