Hat the rise in GluN1 and GluN2A subunits in the hippocampus of those rats which spent 5 minutes in the OF (59?09), would not be related to exposure to novelty. To evaluate if habituation to a new environment, exploration or locomotion could be responsible for GluN1 and GluN2A changes, NMDAR subunits were analyzed in the hippocampus of rats twice exposed to 5 minutes OF sessions 24 h apart and sacrificed 70 minutes after the second session. These results were compared with those from rats exposed to a unique 5 minutes OF session and sacrificed either immediately, 70 minutes or 24 h later. As it is shown in 1531364 Figure 1D, 70 minutes after the second 5 minutes session (Test), GluN1 and GluN2A levels were similar to those in 59-09 rats and in rats sacrificed 24 h after the OF, without a second OF session. Therefore, NMDAR subunits Fruquintinib change observed 70 minutes after a single 5 minutes OF session was not observed in rats that explored twice the OF for 5 minutes each. These results confirmed that there were selective increases in hippocampal GluN1 and GluN2A subunits after a unique 5 minutes session in the OF and showed that these increases were transient since NMDAR subunits levels were similar to control rats in the following day (Figure 1D). Since rats exploring twice the same OF for 5 minutes have similar subunits levels than control animals, this strongly suggests that habituation, rather than just exploration or locomotion, would be related to the NMDAR subunits increase.To induce “plastic-like” changes, repeated pulses of KCl were applied [26,30?2]. First, it was verified that the already reported LTP-induced increase of NMDAR puncta in dendritic spines of hippocampal neurons [12], also took place in neurites in the KCl stimulated cultures. As it is shown in Figure 2A, GluN1 and GluN2A puncta increased significantly at neurites 30 and 70 minutes after KCl treatment, compared to controls fixed immediately after KCl treatment (Figure 2A, photos). There were about 1.5 and 2 fold increases of GluN1 puncta in neurites, 30 and 70 minutes after KCl pulses respectively (861 puncta/10 mm neurite in control cultures, 1261 puncta/10 mm neurite in 30 minutes cultures and 1562 puncta/10 mm neurite in 70 minutes cultures), indicating that a “plastic-like” change was already established in these neurons (Figure 2A). GluN2 expression is required for GluN1 membrane expression [12]. Accordingly, after repeated depolarization by KCl there was also a MedChemExpress Deslorelin significant increase of GluN2A puncta in neurites (1361 and 1461 puncta/10 mm neurite after 30 and 70 minutes, respectively, compared to 1061 puncta/10 mm neurite immediately after depolarization [control]) (Figure 2A). Then, total immunofluorescence was also assessed immediately (control), 30 and 70 minutes after KCl stimulation (Figure 2B). GluN1, GluN2A and GluN2B immunofluorescence in control cultures was not statistically different from cultures without stimulation (data not shown). Conversely, total immunofluorescence significantly increased for GluN1 (1.4260.06 fold) and GluN2A (1.2660.04 fold) 70 minutes after stimulation, compared to control cultures (Figure 2B). There was no significant difference in total immunofluorescence for any subunit 30 minutes after KCl stimulation. In addition, there were not significant changes in GluN2B total immunofluorescence at the times analyzed (Figure 2B). These results indicate that changes in total immunofluorescence for each subunit in mature cultures are analogous.Hat the rise in GluN1 and GluN2A subunits in the hippocampus of those rats which spent 5 minutes in the OF (59?09), would not be related to exposure to novelty. To evaluate if habituation to a new environment, exploration or locomotion could be responsible for GluN1 and GluN2A changes, NMDAR subunits were analyzed in the hippocampus of rats twice exposed to 5 minutes OF sessions 24 h apart and sacrificed 70 minutes after the second session. These results were compared with those from rats exposed to a unique 5 minutes OF session and sacrificed either immediately, 70 minutes or 24 h later. As it is shown in 1531364 Figure 1D, 70 minutes after the second 5 minutes session (Test), GluN1 and GluN2A levels were similar to those in 59-09 rats and in rats sacrificed 24 h after the OF, without a second OF session. Therefore, NMDAR subunits change observed 70 minutes after a single 5 minutes OF session was not observed in rats that explored twice the OF for 5 minutes each. These results confirmed that there were selective increases in hippocampal GluN1 and GluN2A subunits after a unique 5 minutes session in the OF and showed that these increases were transient since NMDAR subunits levels were similar to control rats in the following day (Figure 1D). Since rats exploring twice the same OF for 5 minutes have similar subunits levels than control animals, this strongly suggests that habituation, rather than just exploration or locomotion, would be related to the NMDAR subunits increase.To induce “plastic-like” changes, repeated pulses of KCl were applied [26,30?2]. First, it was verified that the already reported LTP-induced increase of NMDAR puncta in dendritic spines of hippocampal neurons [12], also took place in neurites in the KCl stimulated cultures. As it is shown in Figure 2A, GluN1 and GluN2A puncta increased significantly at neurites 30 and 70 minutes after KCl treatment, compared to controls fixed immediately after KCl treatment (Figure 2A, photos). There were about 1.5 and 2 fold increases of GluN1 puncta in neurites, 30 and 70 minutes after KCl pulses respectively (861 puncta/10 mm neurite in control cultures, 1261 puncta/10 mm neurite in 30 minutes cultures and 1562 puncta/10 mm neurite in 70 minutes cultures), indicating that a “plastic-like” change was already established in these neurons (Figure 2A). GluN2 expression is required for GluN1 membrane expression [12]. Accordingly, after repeated depolarization by KCl there was also a significant increase of GluN2A puncta in neurites (1361 and 1461 puncta/10 mm neurite after 30 and 70 minutes, respectively, compared to 1061 puncta/10 mm neurite immediately after depolarization [control]) (Figure 2A). Then, total immunofluorescence was also assessed immediately (control), 30 and 70 minutes after KCl stimulation (Figure 2B). GluN1, GluN2A and GluN2B immunofluorescence in control cultures was not statistically different from cultures without stimulation (data not shown). Conversely, total immunofluorescence significantly increased for GluN1 (1.4260.06 fold) and GluN2A (1.2660.04 fold) 70 minutes after stimulation, compared to control cultures (Figure 2B). There was no significant difference in total immunofluorescence for any subunit 30 minutes after KCl stimulation. In addition, there were not significant changes in GluN2B total immunofluorescence at the times analyzed (Figure 2B). These results indicate that changes in total immunofluorescence for each subunit in mature cultures are analogous.
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