Article Figures & Data
Figures
- Figure 1.
Status epilepticus induces long-lasting potentiation of mossy fiber evoked field potentials in hippocampal region CA3 in vivo. A, Schematic presents design of experiment in which optical stimulation of dentate granule cell mossy fibers evokes field potentials recorded in the ipsilateral CA3 region in awake, freely moving adult mice. Following baseline recordings collected for 2 d, animals underwent infusion of KA (16 mg/kg; n = 7) or PBS (n = 3). Field potentials evoked by pairs of optical stimuli (60-ms interval between stimuli) were recorded several hours later (“post-SE”) and at daily intervals for the following 7 d. B, Representative field potential recordings are presented for PBS and KA-treated animals at baseline, 1 and 7 d following infusion. Blue bars denote the light stimulations. Individual traces are gray and average of traces are black. C, Each of seven animals infused with KA exhibited striking increases in amplitude of evoked field potential detected several hours after status epilepticus which persisted during the following 7 d. By contrast, small reductions in the amplitude of evoked field potentials were detected in each of three animals undergoing infusion of PBS. Bars reflect the mean ± SEM; small squares reflect values of individual animals. Two-way repeated measures ANOVA with post hoc paired t tests revealed significant differences compared with baseline designated by black asterisks for both PBS and KA groups; red asterisks denote significant increases of amplitude of evoked field potentials in the KA group at days 4–6 compared with measures several hours after SE (post-SE). D, Similar statistical analyses were performed for the PPR data and significant reductions were detected at the post-SE time point in the KA group (paired t test compared with baseline p = 0.0007) as denoted by black asterisk. Although significant reductions compared with baseline in the KA group were also detected at days 1, 4, and 5 following SE, the magnitude of reduced PPRs was maximal immediately post-SE and further reductions were not observed. Apart from a reduction on day 4 (paired t test p < 0.003), no changes of PPR were detected in the PBS group.
- Figure 2.
HFS of mossy fibers in vitro induces LTP of both monosynaptic EPSC and disynaptic IPSC. A, Schematic of local circuit (left) in which activation of granule cell evokes monosynaptic EPSC (black) and disynaptic IPSC (red) recorded in CA3 pyramidal cell (right). B, Responses of a CA3 pyramidal cell evoked by mossy fiber stimuli (0.033 Hz) in which EPSC (left) and IPSC (right) were collected during baseline recordings at holding potentials of −65 and 0 mv, respectively. Mossy fibers underwent HFS (denoted by arrow) at holding potential of 0 mv and EPSCs collected at holding potential of – 65 mv between 0 and 15 min and again at 30–45 min later; IPSCs were collected at holding potential of 0 mv between 15 and 30 and again between 60 and 90 min after HFS. Between 90 and 115 min, EPSCs were recorded at −65 mv and IPSCs at 0 mv in the presence of DCG-IV (1 μm). Top, Representative traces show individual EPSC and IPSC collected during baseline (1), between 0 and 30 min after HFS (2), between 30 and 90 min after HFS (3), and between 90 and 115 min after HFS in the presence of DCG-IV (1 μm). C, Results of individual cells collected before and after HFS reveal LTP of mossy fiber-CA3 EPSC (58% increase, paired t test, p = 0.006). D, Results of individual cells collected before and after HFS reveal LTP of mossy fiber-CA3 IPSC (31% increase, paired t test, p = 0.005). E, E/I ratio of the six cells before versus after HFS reveals values of 0.06 ± 0.03 before HFS, 0.06 ± 0.02 after HFS, paired t test, p = 0.5.
- Figure 3.
Status epilepticus induces reduction of PPF and reduction of in vitro LTP of mossy fiber CA3 fEPSP. A, GFP fluorescence (green) in coronal section of dorsal hippocampus of DGC ChR mouse costained with DAPI reveals signal restricted to apical dendrites, cell bodies, and mossy fiber axons of dentate granule cells. Schematic depicts location of optical fiber and stimulating electrode. Stimuli administered in pairs (IPI denotes interpulse interval of 60 ms) at frequency of 0.03 Hz. B, Representative traces show EPSPs evoked by electrical (left) or optical (right) stimulation at time points collected 10 min before and between 10 and 20 min after application of HFS in slices isolated from mice infused with either PBS or KA. Artifact of electrical stimulus was subtracted from tracings. C, left panel, Repeated measures ANOVA with post hoc Bonferroni’s revealed that electrical stimulation induced LTP of mossy fiber evoked fEPSP in slices from PBS controls (156 ± 10%, n = 12, post hoc p = 0.001) but not in slices from KA-treated animals (113 ± 8%, n = 16, post hoc p = 0.4). Repeated measures ANOVA with post hoc Bonferroni’s revealed that optical stimulation induced LTP of mossy fiber evoked fEPSP in slices from PBS controls (141 ± 8%, n = 8, post hoc p =0.0002) but not in slices from KA-treated animals (100 ± 2.4%, n = 17, post hoc p = 0.5). Panels in middle and right present time course and mean ± SE of electrical and optimal stimulation, respectively. D, left panel, Repeated measures ANOVA with post hoc Bonferroni’s multiple comparisons revealed that electrical stimulation induced reduction of PPF in slices from PBS controls (before HFS 2.3 ± 0.25; after HFS 1.90 ± 0.27, p = 0.004) but not from KA-treated animals (before HFS 1.73 ± 0.10; after HFS 2.02 ± 0.22, p = 0.03). Likewise repeated measures ANOVA with post hoc Bonferroni’s multiple comparisons revealed that optical stimulation induced reduction of PPF in slices from PBS controls (before HFS 1.94 ± 0.23; after HFS 1.42 ± 0.14, p = 0.01) but not from KA-treated animals (before HFS 1.44 ± 0.06; after HFS 1.34 ± 0.07, p = 0.07). Central and right panels present time course and mean ± SE of electrical and optical stimulation, respectively. Post hoc Bonferroni’s multiple comparisons test revealed significant differences of PPF before HFS between PBS and KA undergoing either electrical (p = 0.02) or optical stimulation (p = 0.01).
- Figure 4.
EPSC/IPSC (E/I) ratio reveals nonsignificant reduction in KA compared with PBS infused animals. Top panels show responses of a CA3 pyramidal cell evoked by mossy fiber stimuli (0.033 Hz) in which EPSC (black) and IPSC (red) were collected during baseline recordings at holding potentials of −65 and 0 mv, respectively, from PBS (left) and KA (right) infused animals. Bottom panels present E/I ratio for each cell; the ratio for each of 12 (KA) and 21 (PBS) cells was averaged to obtain the values presented in this figure (KA 0.12 ± 0.03, n = 12) compared with PBS infused animals (0.20 ± 0.04, n = 21), p = 0.14, Student’s t test. Synaptic responses induced by optical stimulation of mossy fibers were obtained in three PBS and three KA-treated animals; electrical stimulation was used in the remainder.
- Figure 5.
HFS of mossy fibers in vitro induces LTP of feedforward IPSC in both controls and following status epilepticus. A, Schematic of local circuit (left) in which activation of granule cell evokes monosynaptic EPSC and disynaptic IPSC recorded in CA3 pyramidal cell (right). Note the delay between onset of EPSC and IPSC approximates 2.5 ms similar to Torborg et al. (2010). B, top, Representative traces show individual IPSCs recorded at holding potential of 0 mv collected 10 min before and between 10 and 20 min after application of HFS in slices isolated from PBS or KA infused mice. C, left, HFS (denoted by arrow) produced LTP of mossy fiber-CA3 disynaptic IPSC in slices from both PBS (184 ± 24%, n = 9, p = 0.0001, post hoc Bonferroni’s) and KA (164 ± 9%, n = 17, p = 0.0001, post hoc Bonferroni’s) infused animals. Right, Results of individual cells are plotted. D, PPR of mossy fiber evoked IPSC of experiment of C above. Repeated measures ANOVA revealed a p value of 0.0001. Post hoc Bonferroni’s revealed no significant differences between PBS and KA either before or after HFS. Post hoc Bonferroni’s revealed a significant reduction of PPR following HFS in both the PBS (1.76 ± 0.3 before, 1.24 ± 0.1 after, n = 9, p = 0.02) and KA (1.59 ± 0.2 before, 1.26 ± 0.1 after, n = 17, p = 0.04) groups.
Tables
PBS KA p values Rm (MΩ) 105.4 ± 6.7 115.8 ± 5.2 0.133 RMP (mV) 63.1 ± 0.6 61.8 ± 1.0 0.131 MC (pF) 23.9 ± 4.5 18.0 ± 2.1 0.160 TC (MS) 2.23 ± 0.31 2.18 ± 0.24 0.454 Latency to first AP (ms) 161 ± 68.8 133 ± 50 0.367 AP threshold (mV) 39.7 ± 2.9 42.3 ± 1.0 0.27 AP amplitude (mV) 58.7 ± 6.2 68.4 ± 6.2 0.144 AP half width (ms) 2.99 ± 0.26 2.58 ± 0.3 0.142 Afterhyperpolarization potential (mv) 3.6 ± 1.4 5.9 ± 1.5 0.146 Intrinsic properties of CA3 pyramidal cells following status epilepticus (KA, n = 11) or controls (PBS, n = 15). Membrane resistance (MΩ), resting membrane potential (RMP), membrane capacitance (MC), time constant (TC), action potential (AP) threshold, amplitude, and half width are presented as mean ± SE. No significant differences were observed as evident from p values of Student’s t test.
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