Neurobiology of Aging
Volume 33, Issue 1 , Pages 149-161, January 2012

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

  • Shawn N. Watson

      Affiliations

    • Department of Biological Sciences, Faculty of Science, University of Calgary, Calgary, Alberta, Canada T2N 1N4
    • ,
  • Mark A. Nelson

      Affiliations

    • Department of Biological Sciences, Faculty of Science, University of Calgary, Calgary, Alberta, Canada T2N 1N4
    • ,
  • Willem C. Wildering

      Affiliations

    • Department of Biological Sciences, Faculty of Science, University of Calgary, Calgary, Alberta, Canada T2N 1N4
    • Department of Physiology and Pharmacology, Faculty of Medicine, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada T2N 4N1
    • Corresponding Author InformationCorresponding author at: University of Calgary, 2500 University Drive N.W. Calgary, Alberta, Canada T2N 1N4. Tel.: +1 403 220 5283; fax: +1 403 289 9311.

Received 12 August 2009; received in revised form 15 January 2010; accepted 22 January 2010. published online 15 February 2010.

Article Outline

Abstract 

The high oxygen consumption and post-mitotic nature of the central nervous system (CNS) makes it particularly susceptible to oxidative stress, the impact of which is widely regarded as a root cause of functional impairment of the aging brain in vertebrates and invertebrates alike. Using an invertebrate model system we demonstrate that the lipid soluble antioxidant α-tocopherol can both reverse 2,2-azobis(2-methylpropion-amidine) dihydrochloride (AAPH) induced decline in excitability in young neurons as well as restore the electrical activity and excitability of aged neurons not unlike the level of their younger equivalents. Furthermore, using two analogs of α-tocopherol where either the acyl chain has been removed (Trolox™) or the hydroxyl group of the chromanol ring has been methylated we were able to assert that the restorative effect of α-tocopherol requires both insertion into the plasma membrane as well as an active OH group. Thus, our results indicate peroxidation is an important modulator of neuronal excitability as well as support the growing body of evidence suggesting α-tocopherol's actions may extend well beyond its established role as a lipid domain preventative antioxidant.

Keywords: Oxidation, α-Tocopherol, AAPH, Mollusk, Lymnaea stagnalis, α-Tocopherol analogs

 

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1. Introduction 

Pro- and antioxidants have received much attention as important biological referees of senescence since Harman first proposed that aging arises from accumulation of oxidative damage over an organism's lifetime (Harman, 1956). The nervous system is particularly susceptible to oxidative damage due to its post-mitotic nature, high concentration of polyunsaturated fatty acids, elevated levels of oxygen consumption and its reliance on numerous processes and structures sensitive to redox modulation (Cini and Moretti, 1995, Coyle and Puttfarcken, 1993, Gamper et al., 2006, Hool and Corry, 2007, Stadtman, 1992). Despite the overall plausibility of Harman's theory, piecing together all the fragments of evidence in a coherent framework that explains the molecular events underlying the progressive age-related loss of neurological performance observed across the animal kingdom remains as yet beyond our reach (Dickstein et al., 2007, Droge and Schipper, 2007, Hermann et al., 2007).

We explore the link between the pro- and antioxidant status of the plasmamembrane and neuronal excitability. A common finding in invertebrates and vertebrates is that the excitability and electrophysiological activity of the nervous system and neurons declines with age (Barnes et al., 2000, Disterhoft and Oh, 2006, Fergestad et al., 2006, Hermann et al., 2007, Klaassen et al., 1998, Vanfleteren, 1993). This reduction is often correlated to a general loss of neurological performance, including that of learning and memory functionality (Disterhoft and Oh, 2007, Hermann et al., 2007, Murphy et al., 2004, Wang et al., 1997). Though it is often theorized that this age-related decrease in neuronal excitability is due to accumulating oxidative stress, there is currently limited experimental support for this idea. We therefore aim to test this general hypothesis.

Many components of the neuronal plasmamembrane can, if oxidized, precipitate substantial neurophysiological changes. For example, the (per)oxidation of PUFAs may trigger or advance a variety of processes, including the release of free fatty acids (FFAs), that may potentially alter a variety of intrinsic electrophysiological parameters (Angelova and Muller, 2006, Bittner and Muller, 1999, Erin et al., 1986, Sevanian et al., 1983, Sevanian and Kim, 1985). Also, several types of ion channels are themselves a target of redox modulation (Gamper et al., 2006, Joksovic et al., 2006, Ruppersberg et al., 1991, Sesti et al., 2009).

To evaluate the significance of plasmamembrane redox conditions in the age-related decline in neuronal excitability in Lymnaea, we tested the effects of AAPH, a water-soluble oxidizer known to cause lipid peroxidation (Niki, 1990) and the lipid soluble antioxidant α-tocopherol on spontaneous and evoked action potential activity of young and old Lymnaea neurons. Our data confirms the general significance of the plasmamembrane redox status in the regulation of neuronal excitability. Moreover, we show that α-tocopherol's beneficial role in containing the consequences of oxidative insults in the plasmamembrane may go beyond its well established role as a preventative chain breaking antioxidant (Liebert et al., 1986, Wang and Quinn, 1999).

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2. Materials and methods 

2.1. Animals 

Animals were bred and raised under controlled conditions as previously described (Hermann et al., 2007). Briefly, snails were kept at a 12:12 light–dark cycle in an ambient temperature of 18–20°C and a pH 7.6–7.9 and fed ad libitum with lettuce and Aquamax carnivorous Grower 600 trout pellets (Purina Mills LLC, St Louis, MO). Survival characteristics of the populations were monitored and evaluated using previously established methods based on the Weibull failure model (Janse et al., 1988, Slob and Janse, 1988). Experimental animals were taken at random from different age-synchronized, healthy, aging populations with survival percentages at the time of sampling ranging from >99 to <20% survival (Fig. 1 and Table 1). The use and care of animals conformed to the University of Calgary Animal Care and Use Policy which adheres to the guidelines, policies and standards of the Canadian Council on Animal Care (CCAC), the Canadian Association of Laboratory Animal Medicine (CALAM), standards of Veterinary Care, and the Alberta Veterinary Association (AVMA) professional codes and standards.

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  • Fig. 1. 

    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).

Table 1. Survival characteristics of Lymnaea populations used in the present study.
Population (months)acs×10−2% survival at samplingAge range
Y15584.5−0.28<995–8
Y25504.0−0.25<996–8
O5504.5−0.28>2020–24

Note: “a” and “c” are Weibull parameters (“a” at 50% survival); “s” is slope of tangent of survival curve at median age.

2.2. Malondialdehyde assay 

Malondialdehyde (MDA) concentration was determined using the ALDetect lipid peroxidation assay kit AK170 (BIOMOL international, Plymouth Meeting PA; for review see Esterbauer et al., 1991). Briefly, 150 young CNS were dissected and subsequently incubated for 30mins in either saline (n=75) or AAPH (5mM, n=75). In addition, aged CNS (n=40) were dissected and incubated for 30min in saline. Following the incubation, CNS were homogenized with butylated hydroxytoluene (BHT) to prevent further oxidation and centrifuged. The supernatant was exposed to the reaction mixtures, and the absorbance was then measured at 586nm. MDA levels were determined by plotting the sample absorbances with a standard curve generated by using known concentrations of MDA.

2.3. Electrophysiology 

CNSs were dissected from anesthetized animals and prepared without the use of proteolytic enzymes as previously described (Hermann and Bulloch, 1998). Dissected CNSs were pinned down in an elastomer covered recording chamber filled with a hydroxyethylpiperazine ethanesulfonic acid (HEPES)-buffered saline (for composition see below). Electrophysiological experiments were performed using intracellular recordings techniques, with the use of either Axoclamp 2A or Axoclamp 2B amplifiers (Axon Instruments, Burlingame, CA) operated in discontinuous current clamp mode at sampling rates ranging from 2.0 to 4.5kHz depending on the capacitive transient settling characteristics of the microelectrodes. Voltage and current outputs of the amplifier were low-pass filtered at 1kHz and digitized at 3kHz using a Digidata 1322A under the control of Axoscope version 9.0 (both Axon Instruments, Burlingame, CA). Spontaneous action potential frequency was quantified by recording the number of action potentials observed over 4min periods at 30, 60 and 90min after application of treatment. Evoked action potential activity was quantified by counting the number of action potentials triggered by a suprathreshold 1nA depolarizing current stimuli over a period of 15s at 30, 60 and 90min after treatment. Microelectrodes were pulled from borosilicate glass (TW150F, World Precision Instruments, Sarasota, FL) and filled with 0.5M potassium acetate (CH3COOK)/0.01M potassium chloride (KCl). Tip resistance of the electrodes ranged between 15 and 30MΩ.

2.4. Solutions and chemicals 

All experiments were performed in Hepes-buffered saline with the following composition (in mM); 51.3 NaCl, 1.7 KCl, 4.1 CaCl2, 1.5 MgCl2 and 10 HEPES, pH 7.9. The 400μL recording chambers were continuously perfused with saline at a refresh rate of approximately four times per minute. Drugs were added to the perfusing solution. 2,2-Azobis (2-methylpropion-amidine) dihydrochloride (AAPH) was dissolved immediately before use in 100mL of saline at a final concentration of 5mM. Hydrogen peroxide (H2O2) was dissolved to a final concentration of 10mM (i.e., 0.03%, v/v) in saline. α-Tocopherol was dispersed in saline in a final concentration of 0.1mM with the aid of 0.3% (v/v) dimethylsulfoxide (DMSO; final concentration). The α-tocopherol analogs, methylated (methyl group added to the C6 position of the chromanol ring) and Trolox™, were dissolved in an identical vehicle solution. Methylated α-tocopherol was a gift from Dr. B Heyne (Dept. of Chemistry, University of Calgary, Alberta, Canada; see Bitew and Zaremberg, 2009 for details). Acute trials of α-tocopherol for young CNS began with a 10min rinse in saline, followed by a 30min 5mM AAPH application followed by either a 60min application of saline/DMSO (vehicle control) or 60min of either α-tocopherol, Trolox™ or methylated α-tocopherol dissolved in saline (final concentration 0.1mM and 0.3%, v/v DMSO for all treatments). Acute trials in aged CNS began with a 10min wash in saline followed by 90min application of either 0.1mM α-tocopherol, Trolox™, methylated α-tocopherol or of the vehicle control. All chemicals were obtained from Sigma–Aldrich (St Louis, MO).

2.5. Data analysis and statistics 

Statistical analysis was done using version 7.1 of the Statistical data analysis software system (Statsoft Inc.) unless mentioned otherwise. Data analysis was done using either univariate factorial or repeated measures analysis of variance (ANOVA) unless stated differently in the text. Specific hypotheses were tested by means of linear contrast unless stated otherwise. Homogeneity of variance assumption was confirmed for all data sets according to the Hartley F-max test. Log-transformation of the data was performed if the latter indicated violation of the normality assumption. Averages are expressed as the arithmetic mean ± the standard error of the means (sem). Dynamical characteristics of evoked action potential responses were quantified by fitting a single phase exponential decay model (Y(t)=Y0exp(−kt)+C, with Y(t)=number of action potential counted at time t, Y0=value of Y at time zero, k=rate constant, C=constant) to stimulus/response histograms binned at 1s intervals using nonlinear fitting routines implemented in Graphpad Prism version 4.03 (Graphpad Sofware Inc., La Jolla, CA). Note that the rate constant k provides a measure of the rate at which the response's transient phase decays, whereas the constant C quantifies the level of sustained action potential firing during evoked responses.

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3. Results 

3.1. Evoked and spontaneous action potential activity of RPeD1 declines with age 

This study focused on Right Pedal Dorsal 1 (RPeD1), a unique neuron involved in respiratory functions that is located in the right pedal ganglion in the CNS of Lymnaea and that can be unequivocally identified on the basis of its location and physiognomy (Haydon and Winlow, 1981, Winlow and Syed, 1992). First we examined spontaneous and evoked action potential activity in RPeD1 of young and old animals. The two intracellular recordings of spontaneous action potential activity of young (upper trace) and aged (lower trace) RPeD1 shown in Fig. 2A, illustrate the typically much lower rate of activity in the latter. On average, the number of action potentials young RPeD1 generated spontaneously was nearly three times higher than that in old RPeD1 (Fig. 2B; Student's t-test; t=5.868, df=14, p<0.001).

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  • Fig. 2. 

    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).

To assess whether the difference in spontaneous action potential activity of young and old RPeD1 reflected limitations intrinsic to the cells, we tested the ability of young and old RPeD1 to generate action potentials in response to 1nA suprathreshold depolarizing stimuli. Fig. 2C illustrates that young RPeD1 typically responded with a transient burst of high activity followed by a sustained train of action potentials at a lower frequency for the duration of the stimulus. Aged RPeD1, on the other hand, typically displayed a rapidly accommodating response and maintained much lower levels of sustained activity compared to young RPeD1. Consequently, young RPeD1 fired on average twice the number of action potentials than old RPeD1 in response to the same stimulus (Fig. 2D; Student's t-test; t=3.102, df=10, p=0.011). We therefore conclude that the excitability of RPeD1, defined as the ability to generate trains of action potentials, declines with age and that this decline involves changes in electrophysiological parameters intrinsic to the neurons. Note that the 1nA current stimulus consistently depolarized young and aged cells to the same level (average membrane potential levels of −37.1±1.01mV, n=62 and −38.7±1.02mV, n=61, respectively).

3.2. Validation of AAPH as a tool for the creation of oxidative stress 

This study involves creating oxidative stress in an attempt to mimic aging processes. For this purpose we used 2,2-azobis(2-methylpropion-amidine) dihydrochloride (AAPH), a known lipid peroxidizer. To validate the use of AAPH in our model system, we measured the compound's ability to induce the production of malondialdehyde (MDA) a byproduct of lipid peroxidation (Reddy et al., 2007). To this end, two groups of 75 young CNSs were either exposed for 30min to saline (control condition) or saline plus 5mM AAPH. Furthermore, one group of 40 aged CNSs was kept for the same period of time in saline. After 30min all brains were homogenated and analyzed for total MDA content. Each experiment was performed in triplicate. MDA levels in the three test groups differed significantly (Fig. 3A; ANOVA interaction; F2,8=14.22, p=0.0053). Specifically, whereas MDA levels measured in young AAPH-treated and aged untreated CNSs did not differ significantly from each other, they were significantly higher than that measured in young CNSs that were kept in saline (Tukey HSD; young saline vs. young AAPH-treated q=6.142, p<0.05; young saline vs. old saline q=6.860, p<0.01; young AAPH-treated vs. old saline q=0.7180, p>0.05). These data support the conclusion that AAPH creates oxidative stress in the Lymnaea CNS.

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  • Fig. 3. 

    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).

As will be shown, exposure to AAPH reliably caused substantial changes in spontaneous and evoked action potential activity of RPeD1 (e.g., Fig. 4, Fig. 5). To strengthen the notion that AAPH's effects are due to the compounds pro-oxidant activity, we also tested the effects of H2O2, an oxidizer that is chemically unrelated to AAPH and has entirely different dissociation chemistry, on spontaneous electrical activity of young RPeD1. Thirty minutes of treatment of either AAPH (5mM) or H2O2 (0.03% or 10mM) significantly reduced spontaneous action potential activity of young RPeD1 (ANOVA F2,17=25.29, p<0.001; H2O2 vs. vehicle Dunnett's q=3.071, p<0.05 and AAPH vs. vehicle Dunnett's q=7.110, p<0.01).

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  • Fig. 4. 

    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).

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  • Fig. 5. 

    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).

3.3. Treatment with extracellular pro-oxidants causes a reduction in action potential activity that is reversed by the antioxidant α-tocopherol 

Next, we probed the ability of the antioxidant α-tocopherol to restore spontaneous action potential activity in young AAPH-treated RPeD1 neurons. Because of its molecular geometry (i.e., large, slightly polar headgroup and long apolar acyl tail) α-tocopherol may potentially affect membrane protein function through non-antioxidant actions by altering certain aspects of the plasmamembrane's microarchitecture (i.e., microviscosity and membrane curvature; Erin et al., 1984, Kagan, 1989, Mukherjee et al., 1996, Wang and Quinn, 1999). Therefore, to distinguish between α-tocopherol's antioxidant and non-antioxidant functions, we included two α-tocopherol analogs in these tests. In the first analog α-tocopherol's antioxidant function was disabled by methylation of the hydroxyl group at C6 of α-tocopherol's chromanol ring (methylated α-tocopherol). The second analog we tested, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic (referred to under its trade name Trolox™), on the other hand, retains the chromanol head group of α-tocopherol including the critical C6 hydroxyl group but lacks the hydrophobic acyl tail (Tafazoli et al., 2005). Thus, Trolox™ retains its antioxidant capacity but lacks α-tocopherol's ability to coordinate itself with the lipid bilayer's phospholipid core (Atkinson et al., 2008).

Fig. 4A shows representative examples of recordings illustrating the effect of α-tocopherol and the vehicle control on AAPH-induced suppression of spontaneous action potential activity. Exposure to AAPH induced a transient increase in spiking activity that typically lasted for 5–10min following its introduction in the recording chamber. This transient response was always followed by a gradual decline in spontaneous spiking activity that eventually led to a complete cessation in spiking activity. Introduction of α-tocopherol to the bath (Fig. 4A, upper panel) slowly reversed the effect of AAPH. Although a slight recovery in action potential activity was typically also observed in vehicle controls (“vehicle”, Fig. 4A, lower panel), this reversal of the AAPH effect was always far from complete. The effects of methylated α-tocopherol and Trolox™ were very similar to those of the vehicle control (no example traces shown).

Statistical analysis of the data set confirmed a highly significant difference in the effect of the different treatments on spontaneous action potential activity (Fig. 4B; ANOVA interaction, F6,52=6.462, p<0.001). AAPH treatment invariably caused a highly significant and statistically indistinguishable reduction in spiking activity in all four test groups (ANOVA within treatment contrast for pooled data; F1,26=223.48, p<0.001). With the exception of the methylated α-tocopherol treated group, spontaneous action potential activity recovered to some extent under all treatment conditions over the 60min treatment period (Fig. 4B; ANOVA linear trends within treatment groups; vehicle control, F1,26=7.599, p<0.05; Trolox™, F1,26=10.071, p<0.01; methylated α-tocopherol, F1,26=0.717, p=0.4047; α-tocopherol, F1,26=64.509, p<0.001). However, only in the α-tocopherol treated group did this recovery significantly exceeded that of the vehicle control group (ANOVA linear trend of α-tocopherol vs. linear trend of vehicle control, F1,26=10.530, p<0.01;). Recovery of spontaneous action potential activity in the Trolox™ and methylated α-tocopherol did not differ significantly from the trend observed in the vehicle control group (ANOVA linear trend of Trolox™ vs. linear trend of vehicle control, F1,26=0.869, p=0.771; linear trend of methylated α-tocopherol vs. linear trend of vehicle control F1,26=1.823, p=0.189).

The data presented above indicate that α-tocopherol, but not Trolox™ or methylated α-tocopherol, is capable of reversing AAPH-induced suppression of spontaneous action potential activity in young RPeD1. Fig. 5 illustrates that very similar results were obtained for evoked action potential activity tests. As shown in Fig. 5A, young RPeD1 respond with a characteristic transient phase of high action potential activity followed by lower level of sustained activity to a 1nA depolarizing current step. A 30min treatment with 5mM AAPH induced substantial change in the cell response characteristics. While the cells still generated a brief transient response, the sustained phase of the response was markedly lower. A 60min treatment with 0.1mM α-tocopherol, but not with Trolox™, methylated α-tocopherol or vehicle only, fully reversed the AAPH-induced decline in excitability (Fig. 5A; no examples for Trolox™, methylated α-tocopherol treated groups shown). Different treatments affected the recovery from AAPH treatment in a different manner (Fig. 5B; ANOVA interaction F2,6=79.202, p<0.005). Further analysis showed that, except for the methylated α-tocopherol treated group, a certain level of recovery occurred in all treatment groups (ANOVA linear trend for; vehicle control, F1,28=6.080, p<0.05; Trolox™, F1,28=1.973, p=0.171; methylated α-tocopherol, F1,28=5.272, p<0.05; F1,28=1.973, p=0.171; α-tocopherol, F1,28=32.767, p<0.001). However, only treatment with α-tocopherol raised the level of the evoked response above that observed in the vehicle control group (ANOVA contrasts; linear trend of α-tocopherol vs. linear trend of vehicle control, F1,28=11.970, p<0.002; linear trend of Trolox™ vs. linear trend of vehicle control, F1,28=0.0328, p=0.857; linear trend of methylated α-tocopherol vs. linear trend of vehicle control F1,28=0.1333, p=0.718).

To facilitate comparison of the response characteristics of cells under different experimental conditions we plotted the average number of action potentials counted per second for each of the 15s of an evoked response test and fitted this data to a one phase exponential decay model. As Fig. 5C illustrates, cells exposed to AAPH were still capable of generating a brief transient burst of action potentials albeit at a, in absolute terms, lower level and, as indicated by a significantly smaller decay rate constant, substantially shorter period of time (k=0.81±0.091s−1 vs. k=1.26±0.198s−1; ANOVA F2,44=3.454, p<0.05, Tukey HSD q=2.401, p<0.05) than cells under control conditions. As reflected in a significantly lower steady state level, action potential firing activity during the sustained phase of the response was significantly lower in AAPH-treated cells as compared untreated control cells (C=1.89±0.053s−1 vs. 0.84±0.036s−1; ANOVA F2,44=8.402, p<0.001, Tukey HSD q=19.10, p<0.01). Treatment with α-tocopherol effectively restored the evoked response characteristic of AAPH-treated cells to that of untreated controls.

Taken, together these data support the conclusion that oxidative stress created by extracellular oxidants cause both spontaneous and evoked action potential activity of young RPeD1 to decline to a level not unlike that observed in their aged counterparts (c.f., Fig. 2, Fig. 4, Fig. 5). In addition, the data also support the conclusion that treatment with α-tocopherol significantly accelerated recovery of both evoked and spontaneous action potential activity from the AAPH-induced decline. This conclusion is somewhat surprising because, as a chain breaking antioxidant, α-tocopherol is thought to work in a preventive manner through limiting the escalation of oxidative damage to the plasmamembrane by “disarming” radical molecules such as lipid peroxide radicals, rather than via a restorative mechanism such as repairing already oxidized lipids.

3.4. Pro-oxidant effects on spontaneous and evoked action potential activity of old RPeD1 

The results described above demonstrate that externally applied oxidative stress reproduces a state of reduced electrophysiological excitability in young RPeD1 that is normally only observed in their aged equivalents. Moreover, the data shows that young cells can be rescued from this pro-oxidant state by treatment with α-tocopherol. In the next set of experiments we investigated the responses of old RPeD1 neurons to pro- and antioxidant treatment. AAPH induced a significant reduction in spontaneous and evoked action potential activity in aged RPeD1 just as it did in young RPeD1 (Fig. 6A, spontaneous activity ANOVA interaction F2,28=7.180, p<0.01, contrast pretreatment vs. AAPH-treatment F1,13=119.91, p<0.001; Fig. 6B, Evoked activity, ANOVA interaction F2,26=5.415, p<0.02, contrast pretreatment vs. AAPH-treatment F1,13=50.313, p<0.001). Moreover, α-tocopherol, but not the vehicle control, effectively reversed the AAPH-induced decrease in spontaneous and evoked action potential activity (spontaneous activity, ANOVA interaction F2,28=7.180, p<0.005; contrast α-tocopherol-treated vs. vehicle control F1,14=8.039, p<0.02; evoked activity ANOVA interaction F2,26=5.415, p<0.005, contrast α-tocopherol-treated vs. vehicle control F1,13=5.642, p<0.05).

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  • Fig. 6. 

    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).

3.5. α-Tocopherol, but not its analogs reverse excitability decline of aged RPeD1 

Next we investigated whether α-tocopherol and its analogs affected spontaneous and evoked action potential activity in aged RPeD1 that had not been pretreated with AAPH, i.e., under conditions prevailing in the aged Lymnaea brain. Comparing the upper and lower panel of Fig. 7A shows that the introduction of α-tocopherol to aged RPeD1 caused a substantial acceleration in spontaneous action potential activity compared to the preparation treated with vehicle only. No such effect was observed in vehicle control, methylated α-tocopherol, or Trolox™ treated test groups. In fact, we observed a slow deceleration of spontaneous activity over the course of a 90min recording in these three test groups. ANOVA confirmed that only α-tocopherol treatment caused a significant increase in spontaneous action potential counts above that observed in the vehicle control group (Fig. 7B, ANOVA interaction, F3,28=15.573, p<0.001, contrast of linear trend of Trolox™ vs. linear trend of vehicle control, F1,28=0.027, p=0.869; contrast of linear trend of methylated α-tocopherol vs. linear trend of vehicle control, F1,28=0.414, p=0.525; contrast of linear trend of α-tocopherol vs. linear trend of vehicle control F1,28=15.131, p<0.001). As shown in Fig. 7B, 90min of treatment with α-tocopherol on average more than doubled spontaneous action potential counts of aged RPeD1.

  • View full-size image.
  • Fig. 7. 

    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).

The resting membrane potential of untreated aged RPeD1 was significantly hyperpolarized compared to that of their young counterparts (−61±1.60mV vs. −51±0.99mV, respectively). Whereas treatment with α-tocopherol had no effect on the resting membrane potential of young RPeD1 it caused a decrease in the resting membrane potential of aged RPeD1 thereby eliminating this difference between young and aged neurons (ANOVA age×treatment interaction; F1,54=8.981, p<0.005; contrast young untreated vs. old untreated, F1,54=26.95, p<0.001; contrast young untreated vs. young α-tocopherol-treated, F1,54=0.1618, p=ns; contrast young α-tocopherol-treated vs. old α-tocopherol-treated, F1,54=0.0082, p=ns). None of the other treatment conditions significantly altered the resting membrane potential of either young or aged cells.

The data presented in the preceding paragraph show that treatment with α-tocopherol but not with either methylated α-tocopherol or Trolox™ doubled the level of spontaneous action potentials generated by aged RPeD1. Fig. 8 shows the analogous effects of α-tocopherol on evoked responses of aged RPeD1. In contrast to young RPeD1, aged RPeD1 typically display a rapidly accommodating response to a 1nA depolarizing stimulus under control conditions (c.f., Fig. 2, Fig. 8). Treatment with α-tocopherol dramatically changed the evoked response of aged RPeD1 to one that typically showed a more robust transient and sustained phase (Fig. 8A upper panels). In contrast, neither treatment with vehicle only (Fig. 8A lower panels) nor treatment with Trolox™ or methylated α-tocopherol (no example recordings shown) brought about any significant changes in the evoked response of aged RPeD1 (Fig. 8B, ANOVA interaction F3,30=11.729, p<0.001; contrast of linear trend of α-tocopherol vs. linear trend of vehicle control, F1,30=22.921, p<0.001).

  • View full-size image.
  • Fig. 8. 

    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).

It is interesting to note that the activity levels of untreated aged RPeD1 look very similar to that of AAPH-treated young RPeD1, whereas α-tocopherol-treated aged RPeD1 are very similar to that of untreated young RPeD1 and α-tocopherol-treated young RPeD1 that were previously challenged with AAPH (c.f., Fig. 5, Fig. 8). Comparison of the evoked response profiles of aged α-tocopherol-treated RPeD1 with aged untreated RPeD1 (Fig. 8C) revealed a very significant difference in both the decay rate constant (0.31±0.041s−1 vs. 1.66±0.349s−1; student t=3.844, df=28, p<0.001) and steady state level (1.91±0.099s−1 vs. 0.97±0.038s−1; student t=8.741, df=28, p<0.001). Interestingly, whereas estimates of the decay model parameters k and C for untreated aged cells were very different from those found in their younger counterparts (k; F2,42=10.61, p<0.001, Tukey HSD q=4.047, p<0.05 and C; F2,42=59.90, p<0.001, Tukey HSD q=9.369, p<0.01; c.f., Fig. 5, Fig. 8), those obtained in α-tocopherol-treated aged cells were not significantly different from the estimates obtained in young untreated RPeD1.

The results described above are consistent with the idea that α-tocopherol is capable of ameliorating a condition that limits action potential firing frequency in aged RPeD1. If this condition is the result of aging processes, the same effect should not be seen in young untreated RPeD1. To examine this idea we tested the effect of 90min of treatment of 0.1mM α-tocopherol on young RPeD1s. This treatment had no effect on either spontaneous or evoked action potential frequency (ANOVA F1,12=0.31, p=ns and F1,11=2.12, p=ns, respectively; data not shown). Taken together the data presented in this section support the conclusion that α-tocopherol is capable of reversing an age dependent condition that limits the action potential firing ability of aged RPeD1. Moreover, the data supports the conclusion that this effect requires both the presence of the chromanol hydroxyl side group and the acyl tail of α-tocopherol.

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4. Discussion 

In summary, we show that: (1) spontaneous and evoked action potential activity of RPeD1 declined with age; (2) the resting membrane potential of aged RPeD1 is hyperpolarized in comparison to younger RPeD1; (3) treatment with the oxidant AAPH causes a significant increase in lipid peroxidation byproducts; (4) AAPH treatment induced a decline in spontaneous and evoked action potential activity of young neurons resembling the reduced electrical activity observed in aged RPeD1; (5) AAPH-induced changes in spontaneous and evoked action potential activity of young RPeD1 are reversed through treatment with α-tocopherol; (6) treatment with α-tocopherol eliminated the difference in resting membrane potential between young and aged RPeD1 and restored spontaneous and evoked action potential activity to levels similar to those of young neurons; (7) neither Trolox™ nor methylated α-tocopherol reproduced the effects of α-tocopherol in young and aged RPeD1. From these results we conclude that redox mechanisms likely are important factors in the decline in excitability that characterizes the aging process in Lymnaea neurons.

Our conclusion that electrical activity declines with age reiterates findings in other reports on neurophysiological changes occurring in the aging brain of Lymnaea stagnalis (Arundell et al., 2006, Hermann et al., 2007, Klaassen et al., 1998) and for that matter the aging mammalian brain (Barnes, 1994, Disterhoft and Oh, 2007, Kelly et al., 2006). This suggests that this phenomenon may be a fundamental expression of the biological aging process in neurons. Moreover, although our data does not exclude the involvement of subcellular processes and/or organelles, they suggest that a change in redox environment in the plasmamembrane is a likely perpetrator in the hypo-excitability condition that is often associated with impairment of neuronal and neuroendocrine functions of the aging Lymnaea CNS (Frolkis et al., 1995, Hermann et al., 2007, Janse et al., 1999, Klaassen et al., 1998, Patel et al., 2006).

The observation that resting membrane potential of aged RPeD1 is significantly hyperpolarized relative to their younger counterparts is suggestive of possible ionic mechanisms underlying the age-related reduction in excitability of these cells. However, the observation that, despite being depolarized to the same membrane potential, young and aged RPeD1 displayed dramatically different evoked response profiles indicate that the reduced excitability of the latter is not simply a matter of changes in passive membrane properties such as resting membrane potential and input resistances. Rather, it suggests that ionic- and cellular mechanisms involved in the regulation of spiking rates, such as intracellular Ca2+ homeostasis and the activation/inactivation behavior of one or more time-dependent membrane conductances, are substantially changed in aged RPeD1.

A growing body of literature emphasizes the biological implications of the plasmamembrane's redox state. For example, it has been suggested that lipid (per)oxidation may affect biophysically relevant changes in membrane microarchitecture and/or lipid metabolism which, in turn, may alter the function of transmembrane proteins such as ion channels and/or transmembrane signaling molecules (e.g., Angelova and Muller, 2006, Chernomordik et al., 1995, Furber et al., 2009, Meers et al., 1988, Schmitt and Meves, 1995, Sevanian and Kim, 1985, Yeagle et al., 1994; For review see: Boland and Drzewiecki, 2008). In addition there is considerable literature in support of redox-dependent mechanisms in the modulation of ion channel function. For example, much research has focused on redox modulation of voltage-gated K+ currents, including delayed rectifier, “A” and “M” and other K+ current types (Gamper et al., 2006, Giese et al., 1998, Giese et al., 2001, Gulbis, 2002, Hool and Corry, 2007, Sesti et al., 2009). In this context particularly interesting are the results from Ruppersberg et al. (1991) who demonstrated that gating of mammalian transient A-type K+-currents is subject to oxidative modulation of a cysteine residue in the channel's α-subunit N-terminal inactivation gate. Other studies link cellular redox conditions to voltage-gated K+ current gating via the channel auxillary β-subunits (Gulbis, 2002). Both mechanisms predict that oxidative stress will disable the A-current's inactivation mechanisms, the loss of which one would expect to result in a decline of a neuron's excitability. Further studies will be needed to elucidate the ionic basis of the phenomena we reported here.

One of the more remarkable aspects of the present study is the observation that α-tocopherol has the ability to correct deleterious effects of oxidative stress in young and old neurons within a time frame of about 1h. At first glance, this finding appears incongruous with α-tocopherol's classification as a chain breaking antioxidant (Atkinson et al., 2008, Liebert et al., 1986, Traber and Atkinson, 2007). According to their definition, chain breaking antioxidants cannot restore oxidative insults once they have occurred. Yet, we find that treatment with α-tocopherol can reverse AAPH-induced neurophysiological changes and can return the state of excitability of older neurons to that of their younger counterparts. Although we have currently no definite answer to this quandary, several plausible scenarios can be suggested. For instance, it is conceivable that supplementation of α-tocopherol slows progression of the lipid peroxidation cascade down sufficiently to avoid saturation of the cell's resident antioxidant repair systems. Alternatively, the literature provides other interesting leads. Specifically, tocopherols are indispensable components of the lipid bilayer in more than one sense. That is, in addition to acting as preventative antioxidants, they also influence the physicochemical state of the bilayer through interacting with polyenoic fatty acid residues of membrane phospholipids and may act as free fatty acid traps (Erin et al., 1984, Erin et al., 1985, Erin et al., 1986, Kagan, 1989, Lucy, 1972, Mukherjee et al., 1996). These processes critically depend on α-tocopherol's ability to form hydrogen-bridges by way of its hydroxyl residue on the carbon 6 of the chromanol ring and presence of the lipophilic acyl chain (Kagan, 1989, Wang and Quinn, 1999). Some studies suggest that through such mechanisms, α-tocopherol may stabilize the membrane against curvature stresses caused by the formation of excessive free fatty acids and/or lysophospholipids (Atkinson et al., 2008). Other studies provide an alternative scenario for α-tocopherol's beneficial actions that centers on phospholipid metabolism and free fatty acid release. That is, lipid peroxidation triggers an increase in the activity of phospholipase A2, leading to an increased formation of free fatty acids that may in turn cause alterations in the activity of lipid domain signaling pathways, precipitate changes in membrane viscosity, membrane curvature and/or lead to the production of more neurotoxic lipid metabolites such as 4-hydroxynonenal or malondialdehyde (Clapp et al., 1995, Esterbauer et al., 1991, Sevanian and Kim, 1985). α-Tocopherol's ability to complex free fatty acids and lysophospholipids relies on its ability to engage in hydrogen-bond formation between its chromonal hydroxyl group and carbonyl groups on free fatty acids (Kagan, 1989). In addition, α-tocopherol's acyl tail has been shown to play a critical role in its membrane stabilizing effect (Kagan, 1989). Methylation of this critical hydroxyl group disrupts α-tocopherol's ability to engage in hydrogen-bridge formations. Our observations that neither Trolox™ nor methylated α-tocopherol was able to rescue young neurons from the effects of AAPH and to “rejuvenate” membrane excitability of old neurons is consistent with the view that these effects involve intervention with products of phospholipase-mediated lipid hydrolysis. Moreover, since methylated α-tocopherol essentially retains the molecular features that make its parent a potential factor in lipid bilayer microarchitecture, methylated α-tocopherol's inability to restore excitability of AAPH-pretreated young neurons and to “rejuvenate” aged neurons suggest that the impact of α-tocopherol on membrane curvature and/or viscosity are of minor importance in the phenomena we observed.

Taken together, the presented arguments suggest that α-tocopherol has two separate and potentially beneficial effects: first it can function in its well established role as a preventative chain breaking lipophilic antioxidant, and second, as our data demonstrates in live neurons and other authors have observed in in vitro systems, it may prevent the downstream consequences of lipid peroxidation by normalizing the free fatty acid status of oxidatively stressed plasmamembranes. Further studies will need to investigate this hypothesis in more detail by focusing on the interactions between α-tocopherol, PLA2 and free fatty acids on neuronal excitability and the aging process. Notwithstanding, our results establish redox modulation of lipid domains as a determinant of neuronal excitability in L. stagnalis and a plausible parameter in the functional impairment of the aging brain.

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Conflict of interest 

There are no actual or potential conflicts of interest.

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Acknowledgments 

This work was supported by grants to WCW from the National Sciences and Engineering Research Council (NSERC) Canada and the Canadian Institute for Health Research - Institute of Aging (CIHR-IA). The authors thank Drs. P.M. Hermann (Department of Biological Sciences, University of Calgary), V. Zaremberg (Department of Biological Sciences, University of Calgary) and B. Heyne (Department of Chemistry, University of Calgary) for their helpful suggestions on this research and thank Dr. Heyne for generously supplying us with methylated α-tocopherol).

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PII: S0197-4580(10)00068-0

doi:10.1016/j.neurobiolaging.2010.01.017

Neurobiology of Aging
Volume 33, Issue 1 , Pages 149-161, January 2012

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