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Figure Lengend Snippet: Optogenetic labeling and stimulation of dentate gyrus granule cells (GCs) show functional connectivity of GCs and mossy cells (MCs) in organotypic tissue cultures. (A) Schematic representation of the viral construct used to express the fusion protein of channelrhodopsin‐2 (ChR2) variant H134R and enhanced yellow fluorescent protein (eYFP). The construct is delivered via the visually guided local injection of adeno‐associated viruses into the GC layer (GCL) of the dentate gyrus in organotypic entorhino‐hippocampal slice cultures 2–5 days after preparation. The size of the injection was controlled using a pressure application system. Multiple injections along the GCL ensure a high density of labeled granule cells. (B) eYFP expression enables the identification of GCs expressing channelrhodopsin‐2 (ChR2). The cell layer of the dentate gyrus is visualized using TO‐PRO counterstain (blue). At 30 days in vitro (DIV), ChR2 expression is observed in the soma, dendrites, and axons of transduced GCs. The dendrites of ChR2‐expressing GCs densely label the molecular layer (ML), whereas the axons strongly label the hilar region (h). Particularly prominent are the large axon terminals, known as mossy fiber boutons, in the hilus and stratum lucidum of CA3, which display distinct ChR2‐eYFP expression. (C) Whole‐cell path‐clamp recordings demonstrate the firing of action potentials in ChR2‐expressing GCs upon optical stimulation (505‐nm light‐emitting diode [LED], 100 pulses with 10‐ms pulse length at 12.5 Hz). For direct identification of the recorded neurons, the internal solution in the patch pipette contained Alexa 647 dextran. (D) MCs, which are innervated by ChR2‐expressing GCs, exhibit strong suprathreshold synaptic activation during optical stimulation of the GCs ( n = 17 of 17 MCs). During the 100‐pulse train, the MC shows some degree of adaptation or increased inhibition. Nevertheless, every light pulse induced a postsynaptic response in the MCs.

Article Snippet: In cases of pharmacological treatment, the substances were applied to the cell culture medium before the start of the optical stimulation: 10 μmol·L −1 D‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5; Alomone Labs) or 10 μmol·L −1 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; Alomone Labs) dissolved in H 2 O.

Techniques: Labeling, Functional Assay, Construct, Variant Assay, Injection, Expressing, In Vitro, Transferring, Activation Assay, Inhibition

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Figure Lengend Snippet: Chronic optical stimulation of granule cells induces hilar neuronal cell death in the dentate gyrus. (A) A custom‐designed stimulation device was developed for long‐term optical stimulation of organotypic tissue cultures (OTCs) within the incubator. The stimulation box is equipped with six high‐power light‐emitting diodes (LEDs) emitting 505‐nm light. OTC‐containing six‐well plates can be placed inside the box, which fits easily into the incubator. Stimulation parameters are controlled through a Raspberry Pi. (B) Optical stimulation of OTCs expressing channelrhodopsin‐2 (ChR2) over a period of 6 h resulted in cell death indicated by propidium iodide (PI) staining, specifically observed in hilar neurons of the dentate gyrus. Control OTCs, which were either ChR2‐expressing and unstimulated or ChR2‐negative and stimulated for 6 h, did not show signs of cell death. (C) Quantitative analysis of PI‐labeled cells in the hilus following repetitive stimulation protocols conducted for varying durations demonstrates a dose‐dependent effect; an increase in the number of PI‐positive cells is observed with longer stimulation periods. The dotted line represents the least‐squares fit of a sigmoidal dose–response function. n = 6–13 cultures per time point. (D) Immunolabeling against the calcium‐binding protein calretinin, a marker for mossy cells (MCs), reveals that MCs constitute a substantial proportion of the PI‐labeled cells in the hilus. eYFP, enhanced yellow fluorescent protein; GCL, granule cell layer; h, hilus.

Article Snippet: In cases of pharmacological treatment, the substances were applied to the cell culture medium before the start of the optical stimulation: 10 μmol·L −1 D‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5; Alomone Labs) or 10 μmol·L −1 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; Alomone Labs) dissolved in H 2 O.

Techniques: Expressing, Staining, Control, Labeling, Immunolabeling, Binding Assay, Marker

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Figure Lengend Snippet: Inhibition of glutamatergic transmission prevents hilar neuronal cell death induced by chronic optical stimulation. (A) Dependence of hilar neuronal cell death on glutamatergic synaptic transmission was demonstrated through propidium iodide (PI) staining. Organotypic tissue cultures subjected to chronic optical stimulation while being treated with D‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) exhibited minimal or absent PI‐labeling of cells in the hilus, indicating reduced cell death. (B) Quantification of PI‐positive cells in cultures treated with 10 μmol·L −1 D‐AP5 and 10 μmol·L −1 CNQX during a 6‐h optical stimulation revealed comparable numbers of PI‐positive cells in the hilus (mean = 4.9 ± 4.9) to baseline levels observed in channelrhodopsin‐2 (ChR2)‐negative cultures that were stimulated with the same stimulation protocol (mean = 4.0 ± 2.6). Differences across all groups were analyzed using the Kruskal–Wallis test with post hoc Dunn's multiple comparisons performed for pairwise comparisons; ** p < .01, **** p < .0001 relative to stimulation without blockers; if not indicated in the graph, there was no significant difference between groups. n = 8–22 cultures per condition. Bars represent mean ± SD. eYFP, enhanced yellow fluorescent protein; GCL, granule cell layer; h, hilus; LED, light‐emitting diode.

Article Snippet: In cases of pharmacological treatment, the substances were applied to the cell culture medium before the start of the optical stimulation: 10 μmol·L −1 D‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5; Alomone Labs) or 10 μmol·L −1 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; Alomone Labs) dissolved in H 2 O.

Techniques: Inhibition, Transmission Assay, Staining, Labeling

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Figure Lengend Snippet: Retigabine protects hilar neurons from death following chronic optical stimulation. (A) Sample images of organotypic tissue cultures stained with propidium iodide (PI) after 4 h of optical stimulation. Treatment with retigabine during the optical stimulation prevented cell death, indicated by a reduction in PI staining. (B) The number of PI‐positive cells in the hilus was quantified with semiautomated cell counting. The addition of 5 μmol·L −1 retigabine to the culture medium reduced the number of PI‐positive cells by 75% (26.0 PI‐positive cells in the hilus ± 19.0 untreated vs. 6.5 ± 4.3 with 5 μmol·L −1 retigabine), indicating a protective effect of retigabine on hilar neurons against optical stimulation‐induced cell death. n = 10–20 cultures per condition. Bars represent mean ± SD. (C) The mean number of PI‐positive cells in the hilus for different retigabine concentrations was normalized to the untreated control and fitted with a nonlinear regression curve. Retigabine reduced the number of PI‐positive cells in the hilus in a dose‐dependent manner, with a median effective concentration of ~616 nmol·L −1 . eYFP, enhanced yellow fluorescent protein; ChR2, channelrhodopsin‐2.

Article Snippet: In cases of pharmacological treatment, the substances were applied to the cell culture medium before the start of the optical stimulation: 10 μmol·L −1 D‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5; Alomone Labs) or 10 μmol·L −1 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; Alomone Labs) dissolved in H 2 O.

Techniques: Staining, Cell Counting, Control, Concentration Assay

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Figure Lengend Snippet: Repeated optogenetic stimulation of granule cells elicits excitatory responses and induces degeneration in mossy cells. (A) Schematic representation of the experimental procedure. Optogenetic stimulation of channelrhodopsin‐2 (ChR2)‐expressing granule cells (GCs) was performed using a 505‐nm light‐emitting diode (LED). Action potentials (APs) elicited in the GCs are transmitted via the mossy fibers to the mossy cells (MCs). Via whole‐cell patch‐clamp experiments, spontaneous and optically induced excitatory postsynaptic potentials (EPSPs) were recorded in identified hilar MCs. (B) Representative traces from a current‐clamp recording of an MC. During the initial 20‐min baseline period, spontaneous EPSPs were recorded. MCs exhibit a high frequency of EPSPs, with occasional large‐amplitude single events (10–21 mV). The same stimulation protocol used to induce hilar neuronal cell death in previous experiments was then applied (100 pulses with 10‐ms pulse length, at 12.5 Hz every minute). During the optical stimulation train, MCs displayed a pronounced depolarizing shift of the membrane potential, on top of which APs were generated. The stimulation was repeated 30 times, with a 52‐s intertrain interval. Following stimulation, spontaneous EPSPs were recorded for a maximum of 20 min. The first and last insets show a 2‐s time window of representative spontaneous activity. The second and third insets show the first 10 optically induced APs of the first and last stimulation train, respectively. Cyan lines indicate when the 505‐nm LED was switched on. (C) Confocal z‐stacks of the recorded MCs were captured at the beginning and end of the 70‐min recording period. After 30 min of optical stimulation, the MCs exhibited the first signs of degeneration, such as the formation of dendritic blebs (indicated by arrows). (D) Quantification of spontaneous EPSPs before and after stimulation within the same cell, showing a significant reduction in the mean EPSP frequency after optical stimulation (*** p <.001, paired Brunner–Munzel test), whereas on average EPSP amplitudes stayed the same (ns p =.1406, paired Brunner–Munzel test). n = 7 cells. eYFP, enhanced yellow fluorescent protein; ns, not significant.

Article Snippet: In cases of pharmacological treatment, the substances were applied to the cell culture medium before the start of the optical stimulation: 10 μmol·L −1 D‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5; Alomone Labs) or 10 μmol·L −1 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; Alomone Labs) dissolved in H 2 O.

Techniques: Expressing, Patch Clamp, Membrane, Generated, Activity Assay

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Article Title: Role of spinal NMDA receptors, protein kinase C and nitric oxide synthase in the hyperalgesia induced by magnesium deficiency in rats

doi: 10.1038/sj.bjp.0704354

Figure Lengend Snippet: Effect of intrathecal injections of a non-competitive NMDA receptor antagonist, MK-801 (0.6, 6.0, 60 nmol rat−1, n=8/dose) or saline (10 μl rat−1, n=8) on the time-course of the vocalization threshold of Mg-deficient rats submitted to the paw pressure test (A). AUC was calculated throughout the period of 120 min for each dose in Mg-deficient rats (B). Results are expressed as means±s.e.mean. *P<0.05, **P<0.01 and ***P<0.001; compared to the saline-treated group of Mg-deficient rats.

Article Snippet: Two competitive NMDA receptor antagonists: D -2-amino-5-phosphono-valeric acid (AP-5) binding to the glutamate site (ICN Pharmaceuticals France, Orsay, France), was dissolved in saline and 5,7-dichlorokynurenic acid (DCKA) binding to the glycine site (RBI, Sigma-Aldrich Co., Saint Quentin Fallavier, France), was dissolved in 50% dimethyl sulphoxide (DMSO, Sigma-Aldrich Co., Saint Quentin Fallavier, France) (vehicle).

Techniques: Saline, Randall–Selitto Test

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Article Title: Role of spinal NMDA receptors, protein kinase C and nitric oxide synthase in the hyperalgesia induced by magnesium deficiency in rats

doi: 10.1038/sj.bjp.0704354

Figure Lengend Snippet: Effect of intrathecal injections of two competitive NMDA receptor antagonists, AP5 (10.2, 40.6, 162.3 nmol rat−1, n=8/dose) or saline (10 μl rat−1, n=8) (A) and DCKA, (0.97, 9.7, 97 nmol rat−1, n=7/dose) or vehicle (10 μl rat−1, n=7) (C) on the time-course of the vocalization threshold of Mg-deficient and control rats submitted to the paw pressure test. AUC was calculated throughout the period of 120 min for each dose in Mg-deficient and control rats (B,D). Results are expressed as means±s.e.mean. *P<0.05, **P<0.01 and ***P<0.001; compared to the saline or vehicle-treated groups of Mg-deficient rats.

Article Snippet: Two competitive NMDA receptor antagonists: D -2-amino-5-phosphono-valeric acid (AP-5) binding to the glutamate site (ICN Pharmaceuticals France, Orsay, France), was dissolved in saline and 5,7-dichlorokynurenic acid (DCKA) binding to the glycine site (RBI, Sigma-Aldrich Co., Saint Quentin Fallavier, France), was dissolved in 50% dimethyl sulphoxide (DMSO, Sigma-Aldrich Co., Saint Quentin Fallavier, France) (vehicle).

Techniques: Saline, Control, Randall–Selitto Test