Tive fEPSP traces from NonTg and 3xTg-AD mice for each condition. * = significantly different after 10 mM dantrolene bath application, p,0.05, n denotes number of slices. doi:10.1371/journal.pone.Oltipraz chemical information 0052056.gacute inhibition of the RyR as previously described. As in the earlier studies, we compared the degree of LTP against both a pre-tetanus baseline in aCSF and pre-tetanus baseline in dantrolene. Similar to previously reported observations, acute RyR inhibitionNormalizing ER Ca2+ for AD Treatmentdid not affect baseline responses in either saline-treated (p.0.05) or dantrolene-treated NonTg mice (p.0.05, Figure 5C). LTP was vastly reduced in both NonTg treatment groups when compared with aCSF and dantrolene baseline responses. In saline-treated 3xTg-AD mice exposed to acute dantrolene, baseline responses were reduced (16.661.0 below aCSF baseline, t(1, 7) = 12.3, p,0.05, Figure 5D) and modest LTD expressed when compared with the control aCSF baseline (225.161.6 ). When measured against the acute dantrolene baseline, LTP expression was impaired, with a trend towards depression. However, in the subchronic dantrolene treated 3xTg-AD mice, entirely different baseline and plasticity patterns emerge. In these mice, the above-described LTP deficits were rescued, with normalized levels of LTP expression when compared with aCSF baseline (115.862 over baseline, Figure 5D) with maintenance of potentiation (as opposed to depression) when compared to the dantrolene baseline (56.462 over baseline).Amyloid Deposits are Reduced in Dantrolene-treated TASTPM MiceCircular, feed-forward interactions exist between neuronal Ca2+ dysregulation and amyloid deposition [30,33?8], such that amyloid species can increase and destabilize Ca2+ signaling, while increased Ca2+, particularly through the ER, can facilitate amyloid aggregation. Here, we wished to test the hypothesis that RyRmediated Ca2+ dysregulation can increase amyloid deposition, so therefore normalizing ER Ca2+ signaling can slow this cycle and result in reduced amyloid staining in hippocampal and cortical regions. We focused on TASTPM mice in these experiments as they develop amyloid aggregates and depositions in a consistent and well-documented pattern over brain regions and time [26,28]. We measured the density of beta amyloid peptides using the 4G8 antibody which recognizes residues 18?2 of beta amyloid, and separately, we measured and compared the density of insoluble dense core amyloid plaques using thioflavin-S staining in the cortex and hippocampus of 6-month old TASTPM mice treated with dantrolene or saline. In both brain regions, we found a consistent 41?5 reduction of amyloid in the dantrolene-treated mice, for both the 4G8 and thioflavin-S stained tissue (4G8 hippocampus: t(1, 23) = 3.2, p,0.05; 4G8 cortex, t(1, 24) = 3.9; p,0.05; thioflavin-S hippocampus: t(1, 33) = 2.5; p,0.05; thioflavin-S cortex: t(1,22) = 2.3; p,0.05, Figure 6).DiscussionSustained Ca2+ dysregulation is incorporated into many aspects of AD pathology, as both an early component contributing to synaptic pathology, and a later accelerant of amyloid and tau deposition [30,35,36,39?1]. And recently, there has been increasing consideration given to targeting these Ca2+ sources as a therapeutic PHCCC site strategy for AD treatment. Oules et al., 2012 (25) and Peng et al., 2012 (24) demonstrate a marked improvement in cognitive function and amyloid load in AD mice with chronic systemic treatment with dantrolene, and experiments in neu.Tive fEPSP traces from NonTg and 3xTg-AD mice for each condition. * = significantly different after 10 mM dantrolene bath application, p,0.05, n denotes number of slices. doi:10.1371/journal.pone.0052056.gacute inhibition of the RyR as previously described. As in the earlier studies, we compared the degree of LTP against both a pre-tetanus baseline in aCSF and pre-tetanus baseline in dantrolene. Similar to previously reported observations, acute RyR inhibitionNormalizing ER Ca2+ for AD Treatmentdid not affect baseline responses in either saline-treated (p.0.05) or dantrolene-treated NonTg mice (p.0.05, Figure 5C). LTP was vastly reduced in both NonTg treatment groups when compared with aCSF and dantrolene baseline responses. In saline-treated 3xTg-AD mice exposed to acute dantrolene, baseline responses were reduced (16.661.0 below aCSF baseline, t(1, 7) = 12.3, p,0.05, Figure 5D) and modest LTD expressed when compared with the control aCSF baseline (225.161.6 ). When measured against the acute dantrolene baseline, LTP expression was impaired, with a trend towards depression. However, in the subchronic dantrolene treated 3xTg-AD mice, entirely different baseline and plasticity patterns emerge. In these mice, the above-described LTP deficits were rescued, with normalized levels of LTP expression when compared with aCSF baseline (115.862 over baseline, Figure 5D) with maintenance of potentiation (as opposed to depression) when compared to the dantrolene baseline (56.462 over baseline).Amyloid Deposits are Reduced in Dantrolene-treated TASTPM MiceCircular, feed-forward interactions exist between neuronal Ca2+ dysregulation and amyloid deposition [30,33?8], such that amyloid species can increase and destabilize Ca2+ signaling, while increased Ca2+, particularly through the ER, can facilitate amyloid aggregation. Here, we wished to test the hypothesis that RyRmediated Ca2+ dysregulation can increase amyloid deposition, so therefore normalizing ER Ca2+ signaling can slow this cycle and result in reduced amyloid staining in hippocampal and cortical regions. We focused on TASTPM mice in these experiments as they develop amyloid aggregates and depositions in a consistent and well-documented pattern over brain regions and time [26,28]. We measured the density of beta amyloid peptides using the 4G8 antibody which recognizes residues 18?2 of beta amyloid, and separately, we measured and compared the density of insoluble dense core amyloid plaques using thioflavin-S staining in the cortex and hippocampus of 6-month old TASTPM mice treated with dantrolene or saline. In both brain regions, we found a consistent 41?5 reduction of amyloid in the dantrolene-treated mice, for both the 4G8 and thioflavin-S stained tissue (4G8 hippocampus: t(1, 23) = 3.2, p,0.05; 4G8 cortex, t(1, 24) = 3.9; p,0.05; thioflavin-S hippocampus: t(1, 33) = 2.5; p,0.05; thioflavin-S cortex: t(1,22) = 2.3; p,0.05, Figure 6).DiscussionSustained Ca2+ dysregulation is incorporated into many aspects of AD pathology, as both an early component contributing to synaptic pathology, and a later accelerant of amyloid and tau deposition [30,35,36,39?1]. And recently, there has been increasing consideration given to targeting these Ca2+ sources as a therapeutic strategy for AD treatment. Oules et al., 2012 (25) and Peng et al., 2012 (24) demonstrate a marked improvement in cognitive function and amyloid load in AD mice with chronic systemic treatment with dantrolene, and experiments in neu.
