Redox agents modulate neuronal activity and reproduce physiological aspects of neuronal aging

Population survival curve of Lymnaea stagnalis raised under laboratory conditions. Young and old animals used in the present study were sampled from three age-synchronized populations with very similar survival characteristics. The latter is reflected in the near complete overlap of Weibull failure model (dotted line) fitted to the survival data (closed symbols; also see Table 1). At the time of sampling, chronological age of the young animals ranged from 5 to 6 months (>99% population survival) whereas that of the aged animals was over 20 months (<20% population survival; see arrows labeled “Young” and “Old” for the approximate sampling range).

Evoked and spontaneous action potential activity changes with age in an identified neuron. (A) Examples of intracellular recordings of spontaneous electrical activity in young and old RPeD1. Note that the older neuron fires action potentials at a much lower rate than its younger counterpart. (B) Average number of spontaneous action potentials counted over a periods of 10min in young and old preparations. Aged RPeD1 maintained a significantly lower spontaneous action potential firing rate than young RPeD1. (C) Examples of intracellular recordings of electrical activity in young and old RPeD1 evoked a 15s 1.0nA depolarizing current injection. Current injected in RPeD1 of a young animal evoked an initial high-frequency burst of action potentials that within the first few seconds of the response slowed down to steady sustained intermediate rate of action potential firing. In contrast, the evoked response of aged RPeD1 was characterized by a transient rapidly accommodating burst of activity and a general lack of sustained action potential firing after the first few seconds into the stimulus. (D) Average number of evoked action potentials counted over a periods of 15s in young and old preparations. Aged RPeD1 fired significantly fewer action potentials in response to a 1nA suprathreshold stimulus than young RPeD1 (*p<0.05, ***p<0.001).

Validation of 2,2-azobis (2-methylpropion-amidine) dihydrochloride (AAPH) as a tool for the study of oxidative stress dependent electrophysiological phenomena in the Lymnaea brain. (A) Average Malondialdehyde (MDA) concentration per CNS measured in young dissected Lymnaea CNSs after 30min exposure to saline or AAPH (5mM) and old dissected CNSs after 30min exposure to saline. Exposure to AAPH induces an increase in MDA levels of young CNSs that is equivalent to that measured in aged brains. (B) Average number of spontaneous action potentials counted in young RPeD1 over a period of 10min after 90min exposure to vehicle, 5mM AAPH or 0.03% H₂O₂. Data normalized to pretreatment counts. Despite their very different chemistry, both AAPH and H₂O₂ caused a significant reduction in average spontaneous action potential activity whereas the mean activity level of CNSs kept in vehicle over the same period of time did not differ significantly from their pretreatment level (*p<0.05, **p<0.01).

2,2-Azobis (2-methylpropion-amidine) dihydrochloride (AAPH) and α-tocopherol affect spontaneous action potential activity of young RPeD1. (A) Examples of intracellular recordings of spontaneous electrical activity of young RPeD1 before and during a 30min application of 5mM AAPH followed by 60min application of α-tocopherol (upper trace) or vehicle only. (B) Average spontaneous action potential firing rate (normalized to their pretreatment control level) recorded in young RPeD1 treated for 30min with 5mM AAPH followed by a 60min treatment with either vehicle only, Trolox™, methylated α-tocopherol or α-tocopherol (all at final concentration of 0.1mM). AAPH treatment significantly reduced the average number of action potentials. Subsequent treatment with α-tocopherol restored spontaneous action potential activity to pretreatment levels, while neither Trolox™ nor methylated α-tocopherol treatments facilitated recovery beyond the level observed in vehicle controls (*p<0.05, ***p<0.001, ns: not significant).

2,2-Azobis (2-methylpropion-amidine) dihydrochloride (AAPH) and α-tocopherol affect response of young RPeD1 to sustained 15 second 1.0nA suprathreshold stimulation. (A) Examples of evoked response in young RPeD1 in saline (labeled “saline”) and after 30min exposure to 5mM AAPH (labeled “AAPH”) followed by 60min exposure to 0.1mM α-tocopherol (labeled “AAPH/α-tocopherol”) or vehicle only (labeled “AAPH/vehicle”). Exposure to AAPH changes a previously sustained response to one characterized by a brief burst of action potentials followed by irregular or no action potential activity. Treatment with α-tocopherol but not exposure to vehicle only reversed this effect of AAPH. (B) Average evoked action potential firing rate (normalized to their pretreatment control level) recorded in young RPeD1 treated for 30min with 5mM AAPH followed by a 60min treatment with either vehicle only, Trolox™, methylated α-tocopherol or α-tocopherol (all at final concentration of 0.1mM dissolved in saline plus 0.3%, v/v DMSO). AAPH treatment significantly reduced the average number of action potentials recorded in response to the 15s, 1nA depolarizing stimuli. Subsequent treatment with α-tocopherol restored evoked action potential activity to pretreatment levels, while neither Trolox™ nor methylated α-tocopherol treatments facilitated recovery beyond the level observed in vehicle controls (***p<0.001; ns: not significant). (C) Mean response profiles (bin size 1s) observed in young RPeD1 before treatment (curve labeled “pretreatment”), in cells treated with AAPH followed by vehicle only treatment (curve labeled “AAPH/vehicle”) and in cells treated with AAPH followed by 60min exposure to α-tocopherol (curve labeled “AAPH/α-tocopherol”). Note that before treatment the cells start firing action potentials at a mean initial rate of six action potentials per second (i.e., 6Hz) that declines within the first 3s of the response to a steady level of 3Hz. In cells pretreated with AAPH that were subsequently exposed to vehicle only, the initial transient started at 3Hz and dropped to an average rate of approximately 1Hz. In contrast, the mean evoked response profile in cells that received α-tocopherol treatment after they were exposed to AAPH was virtually indistinguishable from that observed before treatment (standard error ranges were omitted from the figure for clarity). The solid lines indicate a single exponential decay model fitted to the each of the three data sets (Y_(t)=Y₀exp^(−kt)+C). The model described the dynamic aspects of the evoked response under all three experimental conditions very well (R²=0.97, 0.96 and 0.97 for pretreated, AAPH/vehicle-treated and AAPH/α-tocopherol-treated cells, respectively).

Spontaneous and evoked responses from aged RPeD1 neurons treated with 2,2-azobis (2-methylpropion-amidine) dihydrochloride (AAPH) followed by either vehicle control or 0. 1mM α-tocopherol. (A) Mean spontaneous action potential activity (normalized to pretreatment level) decreased significantly after a 30min exposure to 5mM AAPH. This effect was reversed after a 60min exposure to saline plus 0.1mM α-tocopherol but not by exposure to vehicle only. (B) Evoked action potential firing rate (normalized to their pretreatment levels) decreased significantly in aged RPeD1 following 30min exposure to 5mM AAPH. This effect was reversed following 60min exposure to 0.1mM α-tocopherol but not after exposure to vehicle only for the same period of time (*p<0.05, ***p<0.001, ns: not significant).

α-Tocopherol enhances spontaneous action potential activity in aged, previously untreated RPeD1. (A) Intracellular recordings of two old RPeD1 illustrating the effect of 90min exposure to 0.1mM α-tocopherol (upper trace) or vehicle only (lower trace). In the presence of α-tocopherol, spontaneous action potential firing rate of aged RPeD1 slowly increased whereas activity slowly decreased in the presence of vehicle only. (B) Mean spontaneous action potential firing rates (normalized to pretreatment level) recorded in aged RPeD1 before (labeled “saline”) and after 90min of treatment with either Trolox™ (labeled “Trolox”), methylated α-tocopherol (labeled “met α-tocopherol”) or α-tocopherol (labeled “α-tocopherol”), all at 0.1mM. Exposure to α-tocopherol led to a very substantial increase in mean spiking rates above the cells pretreatment level. No such increase was observed in any of the other three treatment conditions (***p<0.001, ns: not significant).

α-Tocopherol enhances evoked action potential activity in aged RPeD1. (A) Examples of intracellular recordings of the responses of aged RPeD1 to injection of 15s 1.0nA depolarizing currents before (labeled “saline”) and after 90min of exposure to either 0.1mM α-tocopherol (labeled “α-tocopherol”) or vehicle only (labeled “vehicle”). Before treatment the old neurons display a more or less rapidly accommodating response characteristic of old RPeD1 conditions. After treatment with α-tocopherol the same cell is now capable of mounting a vigorous sustained response upon stimulation. Exposure to vehicle only had no such effect. (B) Average evoked action potential firing rate (normalized to their pretreatment control level) recorded in aged RPeD1 after 90min treatment with either vehicle only (labeled “vehicle”), Trolox™ (labeled “Trolox”, methylated α-tocopherol (labeled “met α-tocopherol”) or α-tocopherol (labeled “α-tocopherol”), all at final concentration of 0.1mM dissolved in saline plus 0.3% (v/v) DMSO. Treatment with α-tocopherol increased mean evoked action potential activity to more than 150% of the pretreatment level. Neither Trolox™ nor methylated α-tocopherol treatments facilitated recovery beyond the level observed in vehicle controls (**p<0.01; ns: not significant). (C) Mean response profiles (bin size 1s) observed in old RPeD1 before treatment (curve labeled “pretreatment”) and after 90min of exposure to 0.1mM α-tocopherol (thin dashed lines indicate standard error envelopes of each of the data sets). Treatment with α-tocopherol advanced the average evoked response characteristics of aged RPeD1 from a low level not unlike that observed in young AAPH/vehicle-treated RPeD1 to a high level that reminiscent of the response characteristics of young untreated or young AAPH/α-tocopherol-treated RPeD1 (see Fig. 5C). The solid lines indicate a single exponential decay model fitted to the each of the three data sets (Y_(t)=Y₀exp^(−kt)+C). The model described the dynamic aspects of the evoked response in both groups very well (R²=0.95 and 0.97 for pretreated and α-tocopherol-treated cells, respectively).