Antioxidant protection of cerebellar β-adrenergic receptor function in aged F344 rats

Neuroscience Letters 250 (1998) 165–168

Antioxidant protection of cerebellar b-adrenergic receptor function in aged F344 rats Thomas J. Gould a ,*, Kathy Chadman b, Paula C. Bickford b , c a

Institute for Behavioral Genetics, Box 447, University of Colorado, Boulder, CO 80309, USA Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262, USA c Veterans Administration Medical Center, University of Colorado Health Sciences Center, Denver, CO 80262, USA b

Received 7 April 1998; received in revised form 15 May 1998; accepted 27 May 1998

Abstract We examined whether a 2-week treatment with the spin-trapping agent MDL 101.002 (MDL) or a diet supplemented with vitamin E (Vit E) would alleviate age-related deficits in cerebellar noradrenergic function of male 18–20 month old F344 rats compared to age-matched controls. Cerebellar b-adrenergic receptor function was assessed using extracellular recordings of Purkinje cells during iontophoresis of GABA and isoproterenol (ISO). Noradrenergic receptor function of MDL and Vit E-treated rats was similar to young rats whereas for non-treated rats it was typical of that previously recorded in aged rats. Thus, treatment with MDL or Vit E reverses age-related deficits in cerebellar noradrenergic receptor function.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Aging; Cerebellum; Free radicals; Norepinephrine; Vitamin E; Purkinje cells

Free radical damage may be an important factor in agerelated declines in central nervous system neurotransmission. The tenets of the free radical theory of aging state that free radicals are a product of oxidative metabolism and it is the accumulation of free radicals during an organism’s life span that may lead to deterioration of cell function [7]. In support, K + -evoked release of acetylcholine (ACh) and Ca2 + -ionophore-evoked release of ACh are decreased both in aged rats and in synaptosomes prepared from young rats exposed to the free-radical generating Fenton reaction [12]. The similarity between age-related deficits and free radical-related deficits does suggest that free radical damage may contribute to age-related deficits. If this is true, then alterations of free radical levels during aging should alter the development of age-related deficits. For example, rats fed a vitamin E (Vit E)-deficient diet had an increase in the concentration of lipofuscin in the central nervous system and decreased memory function [9]. Vita-

* Corresponding author. Tel.: +1 303 4925664; fax +1 303 4928063.

min E is an antioxidant, and thus decreased Vit E levels may increase free radical levels. This suggests that increased Vit E levels could protect against free radical damage. In mice, a diet high in Vit E prevented or delayed age-related damage to anion transporter and structural proteins [15]. Another compound that appears promising for treatment of age-related deficits is the nitrone spin-trapping agent Ntert-butyl-a-phenylnitrone (PBN). Ischemia injury to the brain and aging both produce an inactivation of glutamine synthetase (GS), a highly oxidative-sensitive enzyme [18]. The decrease of active GS is associated with an increase of protein oxidation [13]. In aged gerbils, increased levels of oxidized protein and decreased levels of GS and neutral protease activity were found. Chronic treatment with the spin-trapping agent PBN reduced oxidized protein levels and increased GS and neutral protein activity in aged gerbils [2]. In addition, Carney et al. found that chronic treatment with PBN also improved performance in a radial arm maze [2]. Furthermore, treatment with PBN also reduced agerelated loss of muscarinic ACh receptor sensitivity [8] and improved cognitive performance and reduced neuronal oxidative damage in aged rats [5,16]. Thus, treatment with

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00477- 7

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PBN may slow or even reverse central nervous system agerelated deficits by reducing oxidized proteins. In the cerebellum, age-related deficits are seen in the ability of norepinephrine (NE) to modulate GABAergic inhibition of Purkinje cells [1]. The deficit in noradrenergic function is specific for the b1-adrenergic receptor [14] and involves deficits in protein kinase A activation [6]. We have already shown that chronic treatment with PBN can reverse or prevent age-related deficits in b1-adrenergic receptor function in age-rats [5]. Thus, the free radical scavenger PBN is ameliorative for age-related changes in cerebellar b1-adrenergic receptor function. In the present experiments we were specifically interested in whether ameliorative effects on age-related deficits in cerebellar b1-adrenergic receptor function similar to those produced by PBN are produced with other antioxidant/free radical scavenger agents such as Vit E and the spin-trapping agent MDL 101.002 (MDL). Thirty-four male Fisher 344 rats were tested. The rats were between 18 and 20 months old when tested. The rats were housed together in an AAALAC-approved animal care facility with free access to food and water and maintained on a 12 h/12 h light/dark cycle. Twenty rats served as salineinjected or regular chow-fed controls. Seven rats received daily injections of MDL (5 mg/kg i.p., twice daily, freshly made daily; a gift from Marion Merrel Dow) for 2 weeks and seven rats were fed a diet rich in Vit E (500 IU/kg) for 2 weeks. Dietary supplementation with Vit E has been shown to produce significant augmentation of Vit E in the brain [11]. At the end of treatment, rats were anesthetized with urethane (0.75–1.25 g/kg, i.p.), intubated and allow to breath spontaneously. Anesthesia was monitored using the toe-pinch reflex and the corneal blink reflex. A heating pad was used to maintain body temperature at 37°C. An anesthetized animal was secured in a stereotaxic frame and the skin and muscle above the posterior cerebellar vermis was removed. After cisternal drainage, the overlying skull and dura were removed and the surface of the brain was covered with a warm 2% agar in saline. Recordings were made in lobules VI and VII of the cerebellar vermis from Purkinje cells as identified by anatomical location and the characteristic complex spiking of Purkinje cells [3]. Neuronal signals were amplified and filtered (−3 dB at 0.3 and 5 kHz) and displayed on a storage oscilloscope. Action potentials were isolated using a window discriminator and the output was displayed using a strip chart recorder. Multibarrel glass micropipettes were used for single-cell recording and local drug application via microiontophoresis. The resistance of the recording electrodes was 1.9–3.7 MQ. In the multibarrel glass micropipettes, two barrels were filled with 3 M NaCl and the other two barrels were filled with GABA (0.25 M, pH, 4.0–4.5) and with the b-adrenergic agonist isoproterenol (ISO) (0.25 M, pH, 4.0–4.5), respectively. A constant-current source provided ejection and retaining currents for the drug barrels and passed an equal

current of opposite polarity through the balance barrel to neutralize the tip potential [17]. Uniform pulses of drug were applied at regular intervals [4]. GABA was applied at doses that produced approximately a 10–40% change in Purkinje cell response. Isoproterenol was then co-administered with increasing dosage until modulation of the GABA response was seen or a decrease in spontaneous firing rate was observed. After ISO was turned off, the Purkinje cell was recorded from until it could be established if the cell would return to the pre-ISO level of responding. Only cells that showed recovery after exposure to ISO were used in analysis. For each cell, the percent change in firing during the initial GABA application, during ISO and during the recovery period were the average of four samples. Strip chart recordings were digitized using a graphics tablet and analyzed with an IBM computer for the percent changes in firing [4]. Differences in cell responses were analyzed with ANOVA and Tukey HSD post-hoc tests with a significance level of 0.05. Purkinje cells were categorized into one of three possible groups: (1) ISO facilitated GABAergic inhibition (Increase), (2) ISO did not change GABAergic inhibition (No difference) and (3) ISO decreased GABAergic inhibition (Decrease). To be considered an ISO-induced change in firing, the change had to be 15% or greater. Cells not meeting this criterion were considered unchanged by ISO application. A non-directional Chi-square test was performed to test for significant differences in cell response profiles between control and treated rats. Control rats were matched with rats in each treatment group. No difference existed between control rats so the control data was pooled. One hundred and twenty-three cells were recorded from the control group, 42 cells were recorded from the Vit E group, and 35 cells were recorded from the MDL group. Concurrent application of the b-adrenergic agonist ISO with GABA, augments the GABAergic inhibition of Purkinje cell firing in young rats [10]. In aged rats, however, ISO augmentation of GABAergic inhibition is severely decreased [10]. The ability of ISO to modulate GABAergic inhibition of Purkinje cells in the treated group was significantly different than in control rats (x2(4) = 20.8, P , 0.001). As shown in Fig. 1, more Purkinje cells recorded from the MDL-treated and Vit E-treated groups demonstrated ISO-induced facilitation of GABAergic inhibition compared to the control group, whereas more Purkinje cells recorded from the control group were unchanged by ISO or decreased GABAergic inhibition during ISO. In addition, percent modulation of GABAergic inhibition by ISO was significantly greater in treated rats than controls (F(2,198) = 16.35, P , 0.0001). Post-hoc analysis revealed that the control group was significantly different from the treated groups (Fig. 2). No groups were different in the average dose of GABA used, which suggests that the age-related deficits were not related to changes in GABA receptor sensitivity.

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Fig. 1. Bar graphs showing the percent of cells that increased, decreased or had no difference in GABA induced inhibition of Purkinje cell spontaneous firing during concurrent ISO application for control rats (n = 123 cells from 20 rats) (stippled bars), and MDL (n = 35 cells from seven rats) (horizontal striped bars) and Vit E (n = 42 cells from seven rats) (vertical striped bars)-treated rats. Cells had to show at least a 15% change in spontaneous firing during ISO application to qualify for either the Increase or Decrease groups. For the MDL and Vit E-treated groups, the majority of cells increased inhibition during ISO application. For the control group, the majority of cells had no difference in inhibition during ISO application.

Previous work has demonstrated PBN-treatment decreases age-related changes in cerebellar noradrenergic function [5]. In this study we demonstrated that a 2-week chronic treatment with MDL or Vit E similarly reversed age-related deficits in cerebellar noradrenergic receptor

function. In aged rats, NE modulation of Purkinje cell inhibition is decreased, compared to young rats [1]. We found that significantly more Purkinje cells in treated rats (i.e. MDL or Vit E) showed NE-induced modulation of GABAergic inhibition than in age-matched control rats. In

Fig. 2. The percent ISO-induced increase in GABAergic inhibition is shown for controls (n = 123 cells from 20 rats) (stippled bar), and MDL (n = 35 cells from seven rats) (horizontal striped bar) and Vit E (n = 42 cells from seven rats) (vertical striped bar)-treated rats. The control group had the lowest percent ISO-induced change in GABAergic inhibition while the treatment groups had similar levels of ISO-induced change in GABAergic inhibition.

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fact, the response profiles of Purkinje cells from the treated groups were similar to the response profiles of Purkinje cells reported for young rats [10]. This suggests that treatment with antioxidants may have reversed age-related deficits in cerebellar noradrenergic receptor function. If free radicals contribute to age-related deficits, the ameliorative properties of the antioxidant/free radical scavengers reported here might be due to a reduction in the concentration of free radicals. In support, maintenance on a diet low in Vit E correlated with decreased learning in multiple tasks [9]. In contrast, treatment with the free radical scavenger PBN improved cognitive and neural function and reduced levels of oxidized proteins [2,5,8,16]. The present study further suggests that free radicals may contribute to aging [7]. Chronic treatment with MDL or Vit E reversed or delayed age-related deficits in Purkinje cell badrenergic receptor function. In addition, even though MDL is a spin-trapping agent and Vit E is an antioxidant, they may work through similar mechanisms, with one possibility being a reduction in free-radical associated damage. This study also suggests that ameliorative treatments for agerelated deficits are not limited to PBN or nitrone effects. Future studies will further address mechanisms of action of these compounds using multiple age-points. Supported in part by USPHS Grants AG14801 (TG), AG04418 (PB), AG00728 (PB) and the VAMRS (PB). [1] Bickford, P.C., Hoffer, B.J. and Freedman, R., Interaction of norepinephrine with Purkinje cell responses to cerebellar afferent inputs in aged rats, Neurobiol. Aging, 6 (1985) 89–94. [2] Carney, J.M., Starke-Reed, P.E., Oliver, C.N., Landum, R.W., Cheng, M.S., Wu, J.F. and Floyd, R.A., Reversal of age-related increase in brain protein oxidation, decrease in enzyme activity, and loss in temporal and spatial memory by chronic administration of the spin-trapping compound N-tert-butyl-aphenylnitrone, Proc. Natl. Acad. Sci. (USA), 88 (1991) 3633– 3636. [3] Eccles, J.C., Ito, M. and Szentogothai, J., The Cerebellum as a Neuronal Machine, Springer, New York, 1967. [4] Freedman, R., Hoffer, B.J. and Woodward, D.J., A quantitative microiontophoretic analysis of the responses of central neurons to norepinephrine: interaction with cobalt, manganese, verapamil, and dichloroisoproterenol, Br. J. Pharmacol., 54 (1975) 529–539.

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