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Glucose attenuation of atropine-induced deficits in paradoxical sleep and memory
BRAIN RESEARCH ELSEVIER
Brain Research 694 (1995) 133-138
Research report
Glucose attenuation of atropine-induced deficits in paradoxical sleep and memory W.S. Stone 1, R.J. Rudd, P.E. Gold * Department of Psychology, University of Virginia, 102 Gilmer Hall, Charlottesville, VA 22903, USA Accepted 13 June 1995
Abstract
When administered systemically, glucose attenuates deficits in memory produced by several classes of drugs, including cholinergic antagonists and opiate agonists. Glucose also enhances memory in aged rats, mice, and humans. In addition, glucose ameliorates age-related reductions in paradoxical sleep. Because deficits in paradoxical sleep are most marked in those individual aged rats that also have deficits in memory, treatments which improve one of these functions may similarly improve the other. The present experiments show that glucose attenuates deficits in paradoxical sleep and memory after atropine administration, with similar dose-response curves for both actions. In the first experiment, rats received saline, atropine (1 mg/kg), glucose (100 mg/kg) or combinations of atropine + glucose (10, 100, 250, and 500 mg/kg) 30 min before assessment on a spontaneous alternation task. In the second experiment, 3-h EEGs were assessed for spontaneous daytime sleep in rats administered saline, atropine (1 mg/kg), glucose (100 mg/kg) or combinations of atropine + glucose (10, 100 and 250 mg/kg). In both experiments, glucose significantly attenuated deficits at an optimal dose of 100 mg/kg. A third experiment assessed blood glucose levels after injections of atropine + glucose (100 mg/kg) and determined that blood glucose levels were similar to those produced by other treatments which enhance memory. These results are consistent with the view that paradoxical sleep and at least one test of memory are similarly influenced by atropine and glucose. Keywords: Atropine; Glucose; Sleep; Memory; Spontaneous alternation; Paradoxical sleep
1. Introduction
Systemic administration of glucose near the time of training or testing enhances memory in rodents and humans (cf. [7,8,31]). In addition, blood glucose levels near the time of training are related to subsequent retention. Under several conditions, treatments which facilitate memory, including optimal doses of glucose, produce increases in blood glucose approximately 30%-40% above baseline levels [9,10]. Among its effects on memory, glucose administration attenuates impairments in retention associated with a variety of circumstances, including old age, pharmacological treatments (e.g. scopolamine, morphine and an NMDA antagonist), prenatal alcohol exposure [22] and amygdaloid kindling [23,25]. When injected systemically, glucose interacts with drugs
* Corresponding author. Fax: (1) (804) 982-4785. E-mail: [email protected] 1 Present address: The Brockton/West Roxbury VAMC, Psychology Service (l16B), 940 Belmont St., Brockton, MA 02401, USA. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V, All rights reserved
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directed at several neurotransmitter systems, including attenuating several behavioral changes after cholinergic antagonists and opiate agonists (cf. [8,16]). The behavioral changes extend beyond measures of memory to include locomotor activity and tremors. Another measure similarly influenced by systemic injections of glucose is sleep. For example, injections of 2-deoxyglucose, which competes with glucose for uptake into cells, reduce paradoxical sleep in cats [14], while peripheral administration of glucose reverses sleep deficits caused by food restriction [3] or injections of morphine [1]. Recent studies with old or extensively kindled rats [23,28] suggest that deficits in paradoxical sleep are particularly susceptible to attenuation by glucose. Evidence that deficits in both paradoxical sleep and memory are ameliorated by glucose injection is consistent with studies relating these variables under a variety of conditions (e.g. [11,20]). In particular, conditions (e.g. aging) which are associated with deficits in paradoxical sleep are also often associated with deficits in memory (e.g. [24]). In addition, both paradoxical sleep and memory are sensitive to effects of cholinergic manipulation (e.g.
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[12,19]) and, as noted, glucose appears to augment cholinergic functions under several conditions. The present experiment demonstrates impairments of both paradoxical sleep and memory by the muscarinic antagonist, atropine. Memory was assessed using a spontaneous alternation procedure. Like trained alternation tasks, spontaneous alternation tasks involve performance in a maze in which the level of performance is related to the ability to remember previous choices (e.g. [2,18,30]), and which is susceptible to disruption by cholinergic antagonists (e.g. [18,27,30]). With the evidence that deficits in both paradoxical sleep and memory occur together under several conditions, and that glucose attenuates deficits in both sleep and memory, the present experiment tested the hypothesis that glucose would reverse deficits in paradoxical sleep and memory after atropine injections. The finding3 indicate that glucose attenuates deficits in both measures with similar dose-response characteristics and with resultant blood glucose levels comparable to those often associated with good memory [10].
2. Experiment 1 -Spontaneous alternation
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Fig. 1. Glucose attenuation of deficits in spontaneous alternation induced by atropine (1 mg/kg). Atropine +saline significantly reduced the percentage of spontaneous alternation behavior (20 trials, 1 rain intertrial intervals). Glucose (100 m g / k g ) + saline did not significantly alter alternation behavior, but glucose + atropine resulted in a percentage of alternation significantly greater than that of atropine + saline, and similar to that of saline controls. In contrast, spontaneous alternation after atropine plus other doses of glucose (10 and 250 m g / k g ) did not differ from levels by atropine +saline. Ns are noted in parentheses over each group. 1st and 2nd injections refer to the two treatments administered at the same time 30 min prior to testing. * P < 0.05 compared to the saline, glucose (100 m g / k g , and atropine + glucose (100 m g / k g ) groups.
2.1, Methods Subjects. Male, Sprague-Dawley rats were used (300400 g, 80-100 days old at the start of the experiment). Rats were housed individually and maintained on a 12:12 h light/dark cycle (lights on 07.00 h) with free access to food and water throughout the experiment. Drugs. Glucose was obtained from the J.T. Baker Chemical Company, and atropine sulfate was obtained from the Sigma Chemical Company. Both drugs were dissolved in saline on each experimental day and administered i.p. All rats received two injections in succession. Seven independent groups of rats received injections of either saline + saline, atropine (1 m g / k g ) + saline, glucose (100 m g / k g ) + saline, or a combination of atropine (1 m g / k g ) + glucose (10, 100, 250 or 500 m g / k g ) , 30 min before behavioral assessment of spontaneous alternation behavior. The doses of glucose span those effective at enhancing memory and/or sleep in other studies (e.g. [28]). The dose of atropine was selected on the basis of pilot studies showing it would impair alternation performance using a 1-min intertrial interval. Spontaneous alternation. Spontaneous alternation was assessed in a Y-maze, which consisted of 3 trough-shaped arms. The length of each arm was 60 cm, the height of each arm was 17.5 cm, the width of the floor of the maze was 3.5 cm, and the width of the ceiling was 14 cm. The arms converged on a roughly triangular area at the center of the maze which was 4 cm along its longest axis. The ceiling was composed of dark acrylic. At the start of a trial, each rat was placed in the end of one of the arms (arm A), and then allowed to traverse the maze and enter
one of the other arms (arms B or C). When the rat reached the end of arm B or C, it was allowed to remain there for 30 s, removed to its home cage for another 30 s (summing to a 1-min intertrial interval), and then placed back in arm A for the next trial. Each rat received 20 trials. Alternation was defined as the consecutive entry into different arms. With this procedure, possible alternation sequences are equal to the number of arms entered minus one (i.e. 20 - 1 = 19), and the percentage of alternation behavior is equal to the ratio of (actual alternations/possible alternations) × 100. Alternation behavior was analyzed by a oneway ANOVA followed by Student-Newman-Keuls post hoc comparisons [21].
2.2. Results The one-way ANOVA demonstrated significant differences between groups (F(6,51) = 32.05, P < 0.0001). Newman-Keuls comparisons (all P ' s < 0.05) showed that spontaneous alternation scores were lower in atropineversus saline-treated rats (Fig. 1). Glucose, administered at a dose of 100 m g / k g , significantly attenuated the effects of atropine, with alternation scores similar to those of the saline control group. In contrast, glucose administered at doses of 10, 250, or 500 m g / k g , did not significantly attenuate the effects of atropine. In the absence of atropine, glucose (100 m g / k g ) did not significantly change alternation behavior from the level of the saline control group.
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3. Experiment 2 -Sleep 3.1. Methods Subjects. Subjects were 8 male, Sprague-Dawley rats ( 3 0 0 - 4 0 0 g, 8 0 - 1 0 0 days old at the start of the experiment), maintained under conditions identical to those described in Expt. 1. Drugs. Drugs, drug administration and experimental conditions were identical to those described in Expt. 1, except that one atropine + glucose (500 m g / k g ) group was not included, leaving 6 conditions. To reduce the number of animals required for the experiment, 8 rats received all treatments in a counterbalanced order, with recording sessions 2 - 3 days apart to minimize the possibility of proactive drug effects from previous treatments. Drugs were injected 30 min before recording sessions began. Sleep. Cortical electrodes were surgically implanted in all rats to obtain polygraph recordings. Rats were first pretreated with atropine (0.03 m g / k g ) and then anesthetized with sodium pentobarbital (50 m g / k g ) . One cortical electrode (a jeweler's screw) was implanted 1 mm lateral from the midline, and 1 m m anterior to bregma; a second electrode was implanted in the contralateral hemisphere, 4 m m lateral from the midline, and 1 m m anterior to lambda. This placement of the electrodes facilitates the appearance of hippocampal theta rhythms during paradoxical sleep and, at times, during waking states, and aids in the evaluation of both these states of arousal. The placement of the electrodes also enables recordings of synchronized and desynchronized cortical rhythms during nonparadoxical sleep and wakefulness. An additional open (noisy) lead provided a sensitive monitor of behavioral activity. In addition, a separate group of pilot animals (n = 4) had E M G electrodes placed in the nuchal musculature. All electrodes were connected to male Amphenol pins in microminiature connector strips and attached to the skull with dental cement. At the conclusion of surgery, each rat received an injection of Bicillin (60,000 units, i.m.). After recovery from surgery ( 1 - 2 weeks), each rat had a recording cable attached to the connector strip and, while remaining in its home cage, w a s habituated to the procedure for 5 days (3 h / d a y ) in a recording room. Recording sessions began at either 09.00 or 13.00 h, which were both during the light period, and continued for 3 h. We previously determined that sleep measures did not differ on the basis of the recording times, and rats were randomly assigned to the earlier or later recording sessions. The 3-h recording interval was selected in accordance with previous studies showing that glucose at doses of 100-500 m g / k g has minimal effects on sleep in healthy young rats [1,28], and also in accordance with pilot studies determining that atropine (1 m g / k g ) - i n d u c e d effects on sleep are of 6 0 - 9 0 min duration. Movement was recorded simultaneously on an adjacent
polygraph channel through an open lead, and EEG records were classified in 30-s epochs on the basis of the cortical EEG and movement channels as awake, nonparadoxical sleep or paradoxical sleep. As demonstrated in previous studies (e.g. [24,28]), and confirmed again in this experiment, this procedure yields findings which are highly correlated ( r ' s > 0.95 for awake, nonparadoxical sleep and paradoxical sleep) with those obtained using a combination of EEG and E M G electrodes. The following sleep measures were calculated: total sleep time (in minutes), nonparadoxical sleep time (min), paradoxical sleep time (min), the number of bouts of nonparadoxical and paradoxical sleep, the uninterrupted duration of the bouts (in seconds), and the percentage of sleep time spent in paradoxical sleep. Bout numbers and durations were derived from determinations of sleep and wake states. Differences between experimental conditions were assessed with planned, dependent t-tests (two-tailed). 3.2. Results
Sleep data were analyzed in two 90-min periods. One rat was excluded from the study because of recording difficulties, leaving 7 for the statistical analyses. Significant effects of atropine on sleep were limited both to the first 90 min, and also to measures of paradoxical sleep. Measures of nonparadoxical sleep and total sleep were not significantly altered in either the first or second 90-rain 1st INJECTION: m ~
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Fig. 2. Glucose attenuation of deficits in paradoxical sleep in the first 90 min period (beginning 30 min after administration). All rats (n = 7) received all treatments in a counterbalanced design. Atropine+saline significantly reduced the duration of paradoxical sleep bouts. Glucose (100 mg/kg)+saline did not significantly alter paradoxical sleep, but after glucose administration in combination with atropine, the duration of paradoxical sleep bouts was significantly greater than that of the atropine condition, and not significantly different from that of the saline condition. Effects of atropine in combination with other doses of glucose (10 and 250 mg/kg) were intermediate between the effects of saline and atropine, and not significantly different from either condition. The standard errors are included as a measure of variability. PS = paradoxical sleep. 1st and 2nd injections refer to the two treatments administered at the same time 30 min prior to the beginining of EEG recording sessions. * P < 0.05 compared to the saline and atropine +glucose (100 mg/kg) conditions.
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periods. Paradoxical sleep and the percentage of paradoxical/total sleep were reduced (t's = 3.00 and 2.86, respectively, df's = 6, P ' s < 0.05) in the first 90 min, as was the duration of paradoxical sleep bouts (t = 3.40, df = 6, P < 0.02). The number of paradoxical sleep bouts was not significantly reduced. In accord with the focus of the experiment, subsequent analyses focused on effects of glucose on atropine-induced deficits in sleep. Glucose (100 mg/kg) + saline did not significantly affect any measure of paradoxical sleep in the first (or second) period. Glucose (100 mg/kg) in combination with atropine, however, produced significantly higher levels of paradoxical sleep (t = 2.48, df = 6, P < 0.05), and longer paradoxical sleep bouts (t = 4.72, df = 6, P < 0.01), compared to atropine + saline, in the first 90-min period. In both measures of paradoxical sleep, glucose (100 mg/kg) attenuated atropine-induced deficits to levels similar to those of the saline control condition (demonstrated in Fig. 2 for the duration of paradoxical sleep bouts). In contrast, glucose administration at other doses (10 and 250 mg/kg) did not significantly attenuate the effects of atropine. Intermediate effects were produced by the 10 and 250 m g / k g doses of glucose, as paradoxical sleep levels and the duration of paradoxical sleep bouts were intermediate to the saline control and atropine conditions.
4. Experiment 3 -Blood glucose
4.1. Methods Subjects. Subjects were 8 male, Sprague-Dawley rats (300-400 g, 80-100 days old at the start of the experiment), maintained under conditions identical to those described in Expts. 1 and 2. Drugs. Drugs and drug administration were similar to those described in Expts. 1 and 2, except that only one atropine+glucose (100 mg/kg) combination was included. Also, rats received saline injections prior to injections of atropine or glucose instead of using a separate saline condition. As in Expt. 2, 8 rats received all treatments in a counterbalanced order, 2-3 days apart to minimize the possibility of proactive drug effects from previous treatments. Blood glucose t,alues. Baseline blood glucose values were assessed 30 min after saline injections by drawing drops of blood from tail nicks for glucose measurements using a reflectance meter (Glucoscan Plus). Rats then received injections of atropine (1 mg/kg), glucose (100 mg/kg), or atropine + glucose. Blood glucose values were again obtained 30 min later. Blood glucose changes from baseline (saline condition) were evaluated with planned, dependent t-tests. 4.2. Results Blood glucose values after saline injections (the baseline condition) did not differ from each other across ses-
sions, and were combined (mean = 96.9 + 2.7 mg/dl) . Relative to baseline levels, mean blood glucose values increased 45.5 + 9.7 m g / d l 30 min after glucose administration (t = 4.69, df = 7, P < 0.01), and 27.1 + 6.2 m g / d l 30 min after a combination of glucose + atropine (t = 4.37, df = 7, P < 0.01). Changes in blood glucose did not differ significantly between the glucose + saline and glucose + atropine combinations, and blood glucose levels did not change significantly after atropine + saline administration.
5. Discussion Consistent with findings of previous studies (e.g. [19,29]), a low dose of atropine (1 mg/kg) selectively impaired paradoxical sleep. Levels of paradoxical sleep and the percentage of paradoxical/total sleep were reduced, mainly through reductions in the duration of paradoxical sleep bouts. These deficits appeared in the first 90 min of the 3-h recording period, but sleep values in the second 90-min period did not differ between the atropine and saline conditions. One of the major findings of this study is that glucose significantly attenuated effects of atropine on paradoxical sleep. These actions followed an inverted-U dose-response curve, in which a dose of 100 mg/kg, but not 10 or 250 mg/kg, significantly attenuated atropine-induced deficits in paradoxical sleep levels, and also in the duration of paradoxical sleep bouts. These results are consistent with other findings showing that glucose, also at an optimal dose of 100 mg/kg, selectively enhanced paradoxical sleep levels and/or the duration of paradoxical sleep bouts in aged [28] and amygdala-kindled rats [23]. Such findings provide evidence that glucose attenuates deficits in paradoxical sleep in a variety of impairing conditions. Effects produced by the 10 and 250 m g / k g doses on these sleep measures were intermediate, and they did not differ significantly from the saline or atropine conditions. These increases in paradoxical sleep may actually reflect modest effects of the 10 and 250 m g / k g doses, but they are more likely indicative of slightly lower than usual values in the saline control group to which they were compared (for example, see [28]). Similar to its effects on sleep, glucose attenuated atropine-induced deficits in memory. These results are consistent with previous studies showing that glucose attenuates scopolamine-induced deficits in spontaneous alternation [15,26,27] and other behaviors involving cholinergic antagonism (cf. [8]). They also add further to evidence that glucose attenuates deficits in memory produced by a variety of conditions in rodents and humans [8]. The effects of glucose on memory showed several similarities to the effects of glucose on sleep. Among these, glucose, at a dose of 100 mg/kg, but not 10, 250 or 500 mg/kg, reduced the effects of atropine on spontaneous alternation behavior. These actions followed the
W.S. Stone et al. /Brain Research 694 (1995) 133-138
same inverted-U dose-response curve, with an optimal dose of glucose at 100 mg/kg, as occurred in the attenuation of atropine-induced deficits in paradoxical sleep. Also, the mean increase in blood glucose (27.1 + 6.2 mg/dl) produced by the optimal dose of glucose (100 mg/kg) in combination with atropine, is within the range of increases in blood glucose often associated with treatments that enhance memory (e.g. [10]). Thus, a treatment which enhanced paradoxical sleep increased blood glucose to levels which predict good memory. Finally, as in previous studies of spontaneous alternation (e.g. [15,27]), sleep [28] and other behaviors (cf. [8]), glucose did not enhance performance in the absence of a deficit. The similarities between effects of treatments for paradoxical sleep and memory adds to a large literature relating these functions in healthy, young animals (e.g. [11,20]). In addition, studies of individual differences in populations characterized by deficits in memory and paradoxical sleep, such as old age (cf. [24]), show that deficits in paradoxical sleep are often highly correlated with deficits in memory in individual animals. The significance of these findings may be conceptualized in terms of two, non-mutually exclusive possibilities. First, deficits in paradoxical sleep may predict deficits in memory or other cognitive functions in vulnerable individuals, suggesting a diagnostic function. Second, deficits in paradoxical sleep may contribute to deficits in memory, suggesting an etiological function. At present, there is more direct evidence for the first possibility. In addition to significant correlations obtained between concurrent deficits in paradoxical sleep and memory in several populations (cf. [24]), a recent investigation determined that deficits in paradoxical sleep at 6 months of age could predict deficits in memory in rats one year later [22]. In this context, the present findings suggest the possibility that the outcome of treatments for deficits in paradoxical sleep may predict the outcome of treatments for deficits in memory, in individuals. At present, the neurochemical substrates underlying the relationship between paradoxical sleep and memory are unclear. However, the correlations between paradoxical sleep and memory, and the common effects of glucose on both functions, provide further evidence that they share common neurobiological mechanisms, including a relative susceptibility to cholinergic manipulation. The apparent increase in the brain's sensitivity to circulating glucose levels in the presence of cholinergic antagonists is consistent with the suggestion that circulating levels of glucose may regulate the synthesis of acetylcholine when oxidative metabolism is reduced [5,6] or transmission is impaired by muscarinic cholinergic antagonists [4,13,17]. Under these circumstances, circulating glucose levels could become the rate-limiting step in the synthesis of acetylcholine by providing the substrate for the synthesis of acetyl-CoA. In summary, the major results of this experiment demonstrate that glucose injections attenuate atropine-in-
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duced deficits in paradoxical sleep and memory. The similarities between effects of glucose on these two functions both confirm and extend relationships between paradoxical sleep and memory, as well as suggest common underlying neurobiological mechanisms.
Acknowledgements Supported by research grants from NSF (BNS-9012239), NLA (AG 07648), NINDS (NS 32914) and ONR (NOOO1489-J-1216).
References [1] Arankowsky-Sandoval, G. and Gold, P.E., Morphine-induced deficits in sleep patterns: Attenuation by glucose, Neurobiol. Learning Memory, (1995) in press. [2] Beracochea, D.J. and Jaffard, R., Effects of ibotenic lesions of mammillary bodies on spontaneous and rewarded spatial alternation in mice, J. Cogn. Neurosci., 2 (1990) 133-140. [3] Danguir, J. and Nicolaidis, S., Dependence of sleep on nutrients availability, Physiol. Behav., 22 (1979) 735-740. [4] Dolezal, V. and Tucek, S., Effects of choline and glucose on atropine-induced alterations of acetyicholine synthesis and content in the caudate nuclei of rats, Brain Res., 240 (1982) 285-293. [5] Gibson, G.E. and Blass, J.P., Impaired synthesis of acetylcholine in brain accompanying mild hypoxia and hypoglycemia, J. Neurochem., 27 (1976) 37-42. [6] Gibson, G.E. and Peterson, C., Aging decreases oxidative metabolism and the release and synthesis of acetylcholine, J. Neurochem., 37 (1981) 978-984. [7] Gold, P.E., An integrated memory system: From blood to brain. In R.C.A. Frederickson, J.L. McGaugh, and D.L. Felton (Eds.), Peripheral Signaling of the Brain: Role in Neural-immune Interactions, Learning and Memory, Hogrefe and Huber, Toronto, 1991, pp. 391-419. [8] Gold, P.E., Modulation of memory processing: Enhancement of memory in rodents and humans. In L.R. Squire and N. Butters (Eds.), Neuropsychology of Memory, second edition, The Guilford Press, New York, 1992, pp. 402-414. [9] Hall, J.L. and Gold, P.E., The effect of training, epinephrine and glucose injections on plasma glucose levels in rats, Behav. Neural Biol., 46 (1986) 156-176. [10] Hall, J.L. and Gold, P.E., Plasma glucose levels predict the disrupting effects of adrenoceptor antagonists on enhancement of memory storage, Eur. J. Pharmacol., 221 (1992) 365-370. [11] Hennevin, E. and Hars, B., Post learning paradoxical sleep: A critical period when new memory is activated? In B.E. Will, P. Schmitt and J.C. Dalrymple-Alford (Eds.), Advances in Behavioral Biology, vol. 4, Plenum Press, New York, 1985. [12] Kesner, R., Reevaluation of the contribution of the basal forebrain cholinergic system to memory, Neurobiol. Aging, 9 (1988) 609-616. [13] Messier, C., Durkin, T., Mrabet, O. and Destrade, C., Memory-improving action of glucose: Indirect evidence for a facilitation of hippocampal acetylcholine synthesis, Behav. Brain Res., 39 (1990) 135-143. [14] Panksepp, J., Jalowiec, J.E., Zolovick, A.J., Stem, W.C. and Morgane, P.J., Inhibition of glycolytic metabolism and sleep-waking states in cats, Pharmacol. Biochem. Behav., 1 (1973) 117-119. [15] Parsons, M.W. and Gold, P.E., Scopolamine-induced deficits in spontaneous alternation performance: Attenuation with lateral ventricle injections of glucose, Behav. Neural Biol., 57 (1992) 90-92.
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[16] Ragozzino, M.E., Wenk, G.L. and Gold, P.E., Glucose attenuates morphine-induced decrease in hippocampal acetylcholine output: an in vivo microdialysis study in rats, Brain Res., 655 (1994) 77-82. [17] Ricny, J., Tucek, S. and Novakova, J., Acetylcarnitine, carnitine and glucose diminish the effect of muscarinic antagonist quinuclidinyl benzilate on striatal acetylcholine content, Brain Res., 576 (1992) 215-219. [18] Sarter, M., Bodewitz, G. and Stephens, D.N., Attenuation of scopolamine-induced impairment of spontaneous alternation behavior by antagonist but not inverse agonist and b-carbolines. Psychopharmacology, 94 (1988) 491-495. [19] Shiromani, P.J., Gillin, J.C. and Henriksen, S.J., Acetylcholine and the regulation of REM sleep: Basic mechanisms and clinical implications for affective illness and narcolepsy, Annu. Re~,. Pharmacol. Toxicol., 27 (1987) 137-156. [20] Smith, C., Sleep states and learning: A review of the literature. Neurosci. Biobehat.. Rez~., 9 (1985) 157-168. [21] SPSS, S P S S / P C + Statistics 4.0, Marija J. Norusis/SPSS, Chicago, 1990. [22] Stone, W.S., Altman, H.J., Hall, J.L., Arankowsky-Sandoval, G., Parekh, P. and Gold, P.E., Prenatal exposure to alcohol in adult rats: Relationships between sleep and memory deficits, and effects of glucose administration on memory, Brain Res., (1995) in press. [23] Stone, W.S., Cotter, E., Beare, D., Rudd, RJ. and Gold, P.E., Relationships between deficits in sleep and spontaneous alternation in kindled rats: Effects of glucose, Submitted (1995). [24] Stone, W.S. and Gold, P.E., Sleep and memory relationships in
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intact old and amnestic young rats, Neurobiol. Aging, 9 (1988) 719-727. Stone, W.S. and Gold, P.E., Amygdala kindling effects on sleep and memory in rats, Brain Res., 449 (1988) 135-140. Stone, W.S., Rudd, R.J. and Gold, P.E., Glucose attenuation of scopolamine- and age-induced deficits in spontaneous alternation behavior and regional brain [3H]2-deoxyglucose uptake in mice, Psychobiology, 20 (1992) 270-279. Stone, W.S., Walser, B., Gold, S.D. and Gold, P.E., Morphine- and scopolamine-induced impairments of spontaneous alternation in mice: Reversal with glucose and with cholinergic and adrenergic agonists, Behac. Neurosci., 105 (1991) 264-271. Stone, W.S., Wenk, G.L., Stone, S.M. and Gold, P.E., Glucose attenuation of paradoxical sleep deficits in old rats, Behat,. Neural Biol., 57 (1992) 79-86. Szymusiak, R., Danowski, J. and McGinty, D., REM sleep-suppressing effects of atropine in cats vary with environmental temperature, Brain Res., 636 (1994) 115-118. Warburton, D.M. and Heise, G.A., Effects of scopolamine on spatial double alternation in rats, J. Comp. Physiol. Psychol., 81 (1972) 523-532. White, N., Peripheral and central memory-enhancing actions of glucose. In R.C.A. Frederickson, J.L. McGaugh and D.L. Felton (Eds.), Peripheral Signaling of the Brain: Role in Neural-immune Interactions, Learning and Memory, Hogrefe and Huber, Toronto, 1991, pp. 421-441.
Brain Research 694 (1995) 133-138
Research report
Glucose attenuation of atropine-induced deficits in paradoxical sleep and memory W.S. Stone 1, R.J. Rudd, P.E. Gold * Department of Psychology, University of Virginia, 102 Gilmer Hall, Charlottesville, VA 22903, USA Accepted 13 June 1995
Abstract
When administered systemically, glucose attenuates deficits in memory produced by several classes of drugs, including cholinergic antagonists and opiate agonists. Glucose also enhances memory in aged rats, mice, and humans. In addition, glucose ameliorates age-related reductions in paradoxical sleep. Because deficits in paradoxical sleep are most marked in those individual aged rats that also have deficits in memory, treatments which improve one of these functions may similarly improve the other. The present experiments show that glucose attenuates deficits in paradoxical sleep and memory after atropine administration, with similar dose-response curves for both actions. In the first experiment, rats received saline, atropine (1 mg/kg), glucose (100 mg/kg) or combinations of atropine + glucose (10, 100, 250, and 500 mg/kg) 30 min before assessment on a spontaneous alternation task. In the second experiment, 3-h EEGs were assessed for spontaneous daytime sleep in rats administered saline, atropine (1 mg/kg), glucose (100 mg/kg) or combinations of atropine + glucose (10, 100 and 250 mg/kg). In both experiments, glucose significantly attenuated deficits at an optimal dose of 100 mg/kg. A third experiment assessed blood glucose levels after injections of atropine + glucose (100 mg/kg) and determined that blood glucose levels were similar to those produced by other treatments which enhance memory. These results are consistent with the view that paradoxical sleep and at least one test of memory are similarly influenced by atropine and glucose. Keywords: Atropine; Glucose; Sleep; Memory; Spontaneous alternation; Paradoxical sleep
1. Introduction
Systemic administration of glucose near the time of training or testing enhances memory in rodents and humans (cf. [7,8,31]). In addition, blood glucose levels near the time of training are related to subsequent retention. Under several conditions, treatments which facilitate memory, including optimal doses of glucose, produce increases in blood glucose approximately 30%-40% above baseline levels [9,10]. Among its effects on memory, glucose administration attenuates impairments in retention associated with a variety of circumstances, including old age, pharmacological treatments (e.g. scopolamine, morphine and an NMDA antagonist), prenatal alcohol exposure [22] and amygdaloid kindling [23,25]. When injected systemically, glucose interacts with drugs
* Corresponding author. Fax: (1) (804) 982-4785. E-mail: [email protected] 1 Present address: The Brockton/West Roxbury VAMC, Psychology Service (l16B), 940 Belmont St., Brockton, MA 02401, USA. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V, All rights reserved
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directed at several neurotransmitter systems, including attenuating several behavioral changes after cholinergic antagonists and opiate agonists (cf. [8,16]). The behavioral changes extend beyond measures of memory to include locomotor activity and tremors. Another measure similarly influenced by systemic injections of glucose is sleep. For example, injections of 2-deoxyglucose, which competes with glucose for uptake into cells, reduce paradoxical sleep in cats [14], while peripheral administration of glucose reverses sleep deficits caused by food restriction [3] or injections of morphine [1]. Recent studies with old or extensively kindled rats [23,28] suggest that deficits in paradoxical sleep are particularly susceptible to attenuation by glucose. Evidence that deficits in both paradoxical sleep and memory are ameliorated by glucose injection is consistent with studies relating these variables under a variety of conditions (e.g. [11,20]). In particular, conditions (e.g. aging) which are associated with deficits in paradoxical sleep are also often associated with deficits in memory (e.g. [24]). In addition, both paradoxical sleep and memory are sensitive to effects of cholinergic manipulation (e.g.
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[12,19]) and, as noted, glucose appears to augment cholinergic functions under several conditions. The present experiment demonstrates impairments of both paradoxical sleep and memory by the muscarinic antagonist, atropine. Memory was assessed using a spontaneous alternation procedure. Like trained alternation tasks, spontaneous alternation tasks involve performance in a maze in which the level of performance is related to the ability to remember previous choices (e.g. [2,18,30]), and which is susceptible to disruption by cholinergic antagonists (e.g. [18,27,30]). With the evidence that deficits in both paradoxical sleep and memory occur together under several conditions, and that glucose attenuates deficits in both sleep and memory, the present experiment tested the hypothesis that glucose would reverse deficits in paradoxical sleep and memory after atropine injections. The finding3 indicate that glucose attenuates deficits in both measures with similar dose-response characteristics and with resultant blood glucose levels comparable to those often associated with good memory [10].
2. Experiment 1 -Spontaneous alternation
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Fig. 1. Glucose attenuation of deficits in spontaneous alternation induced by atropine (1 mg/kg). Atropine +saline significantly reduced the percentage of spontaneous alternation behavior (20 trials, 1 rain intertrial intervals). Glucose (100 m g / k g ) + saline did not significantly alter alternation behavior, but glucose + atropine resulted in a percentage of alternation significantly greater than that of atropine + saline, and similar to that of saline controls. In contrast, spontaneous alternation after atropine plus other doses of glucose (10 and 250 m g / k g ) did not differ from levels by atropine +saline. Ns are noted in parentheses over each group. 1st and 2nd injections refer to the two treatments administered at the same time 30 min prior to testing. * P < 0.05 compared to the saline, glucose (100 m g / k g , and atropine + glucose (100 m g / k g ) groups.
2.1, Methods Subjects. Male, Sprague-Dawley rats were used (300400 g, 80-100 days old at the start of the experiment). Rats were housed individually and maintained on a 12:12 h light/dark cycle (lights on 07.00 h) with free access to food and water throughout the experiment. Drugs. Glucose was obtained from the J.T. Baker Chemical Company, and atropine sulfate was obtained from the Sigma Chemical Company. Both drugs were dissolved in saline on each experimental day and administered i.p. All rats received two injections in succession. Seven independent groups of rats received injections of either saline + saline, atropine (1 m g / k g ) + saline, glucose (100 m g / k g ) + saline, or a combination of atropine (1 m g / k g ) + glucose (10, 100, 250 or 500 m g / k g ) , 30 min before behavioral assessment of spontaneous alternation behavior. The doses of glucose span those effective at enhancing memory and/or sleep in other studies (e.g. [28]). The dose of atropine was selected on the basis of pilot studies showing it would impair alternation performance using a 1-min intertrial interval. Spontaneous alternation. Spontaneous alternation was assessed in a Y-maze, which consisted of 3 trough-shaped arms. The length of each arm was 60 cm, the height of each arm was 17.5 cm, the width of the floor of the maze was 3.5 cm, and the width of the ceiling was 14 cm. The arms converged on a roughly triangular area at the center of the maze which was 4 cm along its longest axis. The ceiling was composed of dark acrylic. At the start of a trial, each rat was placed in the end of one of the arms (arm A), and then allowed to traverse the maze and enter
one of the other arms (arms B or C). When the rat reached the end of arm B or C, it was allowed to remain there for 30 s, removed to its home cage for another 30 s (summing to a 1-min intertrial interval), and then placed back in arm A for the next trial. Each rat received 20 trials. Alternation was defined as the consecutive entry into different arms. With this procedure, possible alternation sequences are equal to the number of arms entered minus one (i.e. 20 - 1 = 19), and the percentage of alternation behavior is equal to the ratio of (actual alternations/possible alternations) × 100. Alternation behavior was analyzed by a oneway ANOVA followed by Student-Newman-Keuls post hoc comparisons [21].
2.2. Results The one-way ANOVA demonstrated significant differences between groups (F(6,51) = 32.05, P < 0.0001). Newman-Keuls comparisons (all P ' s < 0.05) showed that spontaneous alternation scores were lower in atropineversus saline-treated rats (Fig. 1). Glucose, administered at a dose of 100 m g / k g , significantly attenuated the effects of atropine, with alternation scores similar to those of the saline control group. In contrast, glucose administered at doses of 10, 250, or 500 m g / k g , did not significantly attenuate the effects of atropine. In the absence of atropine, glucose (100 m g / k g ) did not significantly change alternation behavior from the level of the saline control group.
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3. Experiment 2 -Sleep 3.1. Methods Subjects. Subjects were 8 male, Sprague-Dawley rats ( 3 0 0 - 4 0 0 g, 8 0 - 1 0 0 days old at the start of the experiment), maintained under conditions identical to those described in Expt. 1. Drugs. Drugs, drug administration and experimental conditions were identical to those described in Expt. 1, except that one atropine + glucose (500 m g / k g ) group was not included, leaving 6 conditions. To reduce the number of animals required for the experiment, 8 rats received all treatments in a counterbalanced order, with recording sessions 2 - 3 days apart to minimize the possibility of proactive drug effects from previous treatments. Drugs were injected 30 min before recording sessions began. Sleep. Cortical electrodes were surgically implanted in all rats to obtain polygraph recordings. Rats were first pretreated with atropine (0.03 m g / k g ) and then anesthetized with sodium pentobarbital (50 m g / k g ) . One cortical electrode (a jeweler's screw) was implanted 1 mm lateral from the midline, and 1 m m anterior to bregma; a second electrode was implanted in the contralateral hemisphere, 4 m m lateral from the midline, and 1 m m anterior to lambda. This placement of the electrodes facilitates the appearance of hippocampal theta rhythms during paradoxical sleep and, at times, during waking states, and aids in the evaluation of both these states of arousal. The placement of the electrodes also enables recordings of synchronized and desynchronized cortical rhythms during nonparadoxical sleep and wakefulness. An additional open (noisy) lead provided a sensitive monitor of behavioral activity. In addition, a separate group of pilot animals (n = 4) had E M G electrodes placed in the nuchal musculature. All electrodes were connected to male Amphenol pins in microminiature connector strips and attached to the skull with dental cement. At the conclusion of surgery, each rat received an injection of Bicillin (60,000 units, i.m.). After recovery from surgery ( 1 - 2 weeks), each rat had a recording cable attached to the connector strip and, while remaining in its home cage, w a s habituated to the procedure for 5 days (3 h / d a y ) in a recording room. Recording sessions began at either 09.00 or 13.00 h, which were both during the light period, and continued for 3 h. We previously determined that sleep measures did not differ on the basis of the recording times, and rats were randomly assigned to the earlier or later recording sessions. The 3-h recording interval was selected in accordance with previous studies showing that glucose at doses of 100-500 m g / k g has minimal effects on sleep in healthy young rats [1,28], and also in accordance with pilot studies determining that atropine (1 m g / k g ) - i n d u c e d effects on sleep are of 6 0 - 9 0 min duration. Movement was recorded simultaneously on an adjacent
polygraph channel through an open lead, and EEG records were classified in 30-s epochs on the basis of the cortical EEG and movement channels as awake, nonparadoxical sleep or paradoxical sleep. As demonstrated in previous studies (e.g. [24,28]), and confirmed again in this experiment, this procedure yields findings which are highly correlated ( r ' s > 0.95 for awake, nonparadoxical sleep and paradoxical sleep) with those obtained using a combination of EEG and E M G electrodes. The following sleep measures were calculated: total sleep time (in minutes), nonparadoxical sleep time (min), paradoxical sleep time (min), the number of bouts of nonparadoxical and paradoxical sleep, the uninterrupted duration of the bouts (in seconds), and the percentage of sleep time spent in paradoxical sleep. Bout numbers and durations were derived from determinations of sleep and wake states. Differences between experimental conditions were assessed with planned, dependent t-tests (two-tailed). 3.2. Results
Sleep data were analyzed in two 90-min periods. One rat was excluded from the study because of recording difficulties, leaving 7 for the statistical analyses. Significant effects of atropine on sleep were limited both to the first 90 min, and also to measures of paradoxical sleep. Measures of nonparadoxical sleep and total sleep were not significantly altered in either the first or second 90-rain 1st INJECTION: m ~
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Fig. 2. Glucose attenuation of deficits in paradoxical sleep in the first 90 min period (beginning 30 min after administration). All rats (n = 7) received all treatments in a counterbalanced design. Atropine+saline significantly reduced the duration of paradoxical sleep bouts. Glucose (100 mg/kg)+saline did not significantly alter paradoxical sleep, but after glucose administration in combination with atropine, the duration of paradoxical sleep bouts was significantly greater than that of the atropine condition, and not significantly different from that of the saline condition. Effects of atropine in combination with other doses of glucose (10 and 250 mg/kg) were intermediate between the effects of saline and atropine, and not significantly different from either condition. The standard errors are included as a measure of variability. PS = paradoxical sleep. 1st and 2nd injections refer to the two treatments administered at the same time 30 min prior to the beginining of EEG recording sessions. * P < 0.05 compared to the saline and atropine +glucose (100 mg/kg) conditions.
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periods. Paradoxical sleep and the percentage of paradoxical/total sleep were reduced (t's = 3.00 and 2.86, respectively, df's = 6, P ' s < 0.05) in the first 90 min, as was the duration of paradoxical sleep bouts (t = 3.40, df = 6, P < 0.02). The number of paradoxical sleep bouts was not significantly reduced. In accord with the focus of the experiment, subsequent analyses focused on effects of glucose on atropine-induced deficits in sleep. Glucose (100 mg/kg) + saline did not significantly affect any measure of paradoxical sleep in the first (or second) period. Glucose (100 mg/kg) in combination with atropine, however, produced significantly higher levels of paradoxical sleep (t = 2.48, df = 6, P < 0.05), and longer paradoxical sleep bouts (t = 4.72, df = 6, P < 0.01), compared to atropine + saline, in the first 90-min period. In both measures of paradoxical sleep, glucose (100 mg/kg) attenuated atropine-induced deficits to levels similar to those of the saline control condition (demonstrated in Fig. 2 for the duration of paradoxical sleep bouts). In contrast, glucose administration at other doses (10 and 250 mg/kg) did not significantly attenuate the effects of atropine. Intermediate effects were produced by the 10 and 250 m g / k g doses of glucose, as paradoxical sleep levels and the duration of paradoxical sleep bouts were intermediate to the saline control and atropine conditions.
4. Experiment 3 -Blood glucose
4.1. Methods Subjects. Subjects were 8 male, Sprague-Dawley rats (300-400 g, 80-100 days old at the start of the experiment), maintained under conditions identical to those described in Expts. 1 and 2. Drugs. Drugs and drug administration were similar to those described in Expts. 1 and 2, except that only one atropine+glucose (100 mg/kg) combination was included. Also, rats received saline injections prior to injections of atropine or glucose instead of using a separate saline condition. As in Expt. 2, 8 rats received all treatments in a counterbalanced order, 2-3 days apart to minimize the possibility of proactive drug effects from previous treatments. Blood glucose t,alues. Baseline blood glucose values were assessed 30 min after saline injections by drawing drops of blood from tail nicks for glucose measurements using a reflectance meter (Glucoscan Plus). Rats then received injections of atropine (1 mg/kg), glucose (100 mg/kg), or atropine + glucose. Blood glucose values were again obtained 30 min later. Blood glucose changes from baseline (saline condition) were evaluated with planned, dependent t-tests. 4.2. Results Blood glucose values after saline injections (the baseline condition) did not differ from each other across ses-
sions, and were combined (mean = 96.9 + 2.7 mg/dl) . Relative to baseline levels, mean blood glucose values increased 45.5 + 9.7 m g / d l 30 min after glucose administration (t = 4.69, df = 7, P < 0.01), and 27.1 + 6.2 m g / d l 30 min after a combination of glucose + atropine (t = 4.37, df = 7, P < 0.01). Changes in blood glucose did not differ significantly between the glucose + saline and glucose + atropine combinations, and blood glucose levels did not change significantly after atropine + saline administration.
5. Discussion Consistent with findings of previous studies (e.g. [19,29]), a low dose of atropine (1 mg/kg) selectively impaired paradoxical sleep. Levels of paradoxical sleep and the percentage of paradoxical/total sleep were reduced, mainly through reductions in the duration of paradoxical sleep bouts. These deficits appeared in the first 90 min of the 3-h recording period, but sleep values in the second 90-min period did not differ between the atropine and saline conditions. One of the major findings of this study is that glucose significantly attenuated effects of atropine on paradoxical sleep. These actions followed an inverted-U dose-response curve, in which a dose of 100 mg/kg, but not 10 or 250 mg/kg, significantly attenuated atropine-induced deficits in paradoxical sleep levels, and also in the duration of paradoxical sleep bouts. These results are consistent with other findings showing that glucose, also at an optimal dose of 100 mg/kg, selectively enhanced paradoxical sleep levels and/or the duration of paradoxical sleep bouts in aged [28] and amygdala-kindled rats [23]. Such findings provide evidence that glucose attenuates deficits in paradoxical sleep in a variety of impairing conditions. Effects produced by the 10 and 250 m g / k g doses on these sleep measures were intermediate, and they did not differ significantly from the saline or atropine conditions. These increases in paradoxical sleep may actually reflect modest effects of the 10 and 250 m g / k g doses, but they are more likely indicative of slightly lower than usual values in the saline control group to which they were compared (for example, see [28]). Similar to its effects on sleep, glucose attenuated atropine-induced deficits in memory. These results are consistent with previous studies showing that glucose attenuates scopolamine-induced deficits in spontaneous alternation [15,26,27] and other behaviors involving cholinergic antagonism (cf. [8]). They also add further to evidence that glucose attenuates deficits in memory produced by a variety of conditions in rodents and humans [8]. The effects of glucose on memory showed several similarities to the effects of glucose on sleep. Among these, glucose, at a dose of 100 mg/kg, but not 10, 250 or 500 mg/kg, reduced the effects of atropine on spontaneous alternation behavior. These actions followed the
W.S. Stone et al. /Brain Research 694 (1995) 133-138
same inverted-U dose-response curve, with an optimal dose of glucose at 100 mg/kg, as occurred in the attenuation of atropine-induced deficits in paradoxical sleep. Also, the mean increase in blood glucose (27.1 + 6.2 mg/dl) produced by the optimal dose of glucose (100 mg/kg) in combination with atropine, is within the range of increases in blood glucose often associated with treatments that enhance memory (e.g. [10]). Thus, a treatment which enhanced paradoxical sleep increased blood glucose to levels which predict good memory. Finally, as in previous studies of spontaneous alternation (e.g. [15,27]), sleep [28] and other behaviors (cf. [8]), glucose did not enhance performance in the absence of a deficit. The similarities between effects of treatments for paradoxical sleep and memory adds to a large literature relating these functions in healthy, young animals (e.g. [11,20]). In addition, studies of individual differences in populations characterized by deficits in memory and paradoxical sleep, such as old age (cf. [24]), show that deficits in paradoxical sleep are often highly correlated with deficits in memory in individual animals. The significance of these findings may be conceptualized in terms of two, non-mutually exclusive possibilities. First, deficits in paradoxical sleep may predict deficits in memory or other cognitive functions in vulnerable individuals, suggesting a diagnostic function. Second, deficits in paradoxical sleep may contribute to deficits in memory, suggesting an etiological function. At present, there is more direct evidence for the first possibility. In addition to significant correlations obtained between concurrent deficits in paradoxical sleep and memory in several populations (cf. [24]), a recent investigation determined that deficits in paradoxical sleep at 6 months of age could predict deficits in memory in rats one year later [22]. In this context, the present findings suggest the possibility that the outcome of treatments for deficits in paradoxical sleep may predict the outcome of treatments for deficits in memory, in individuals. At present, the neurochemical substrates underlying the relationship between paradoxical sleep and memory are unclear. However, the correlations between paradoxical sleep and memory, and the common effects of glucose on both functions, provide further evidence that they share common neurobiological mechanisms, including a relative susceptibility to cholinergic manipulation. The apparent increase in the brain's sensitivity to circulating glucose levels in the presence of cholinergic antagonists is consistent with the suggestion that circulating levels of glucose may regulate the synthesis of acetylcholine when oxidative metabolism is reduced [5,6] or transmission is impaired by muscarinic cholinergic antagonists [4,13,17]. Under these circumstances, circulating glucose levels could become the rate-limiting step in the synthesis of acetylcholine by providing the substrate for the synthesis of acetyl-CoA. In summary, the major results of this experiment demonstrate that glucose injections attenuate atropine-in-
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duced deficits in paradoxical sleep and memory. The similarities between effects of glucose on these two functions both confirm and extend relationships between paradoxical sleep and memory, as well as suggest common underlying neurobiological mechanisms.
Acknowledgements Supported by research grants from NSF (BNS-9012239), NLA (AG 07648), NINDS (NS 32914) and ONR (NOOO1489-J-1216).
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[16] Ragozzino, M.E., Wenk, G.L. and Gold, P.E., Glucose attenuates morphine-induced decrease in hippocampal acetylcholine output: an in vivo microdialysis study in rats, Brain Res., 655 (1994) 77-82. [17] Ricny, J., Tucek, S. and Novakova, J., Acetylcarnitine, carnitine and glucose diminish the effect of muscarinic antagonist quinuclidinyl benzilate on striatal acetylcholine content, Brain Res., 576 (1992) 215-219. [18] Sarter, M., Bodewitz, G. and Stephens, D.N., Attenuation of scopolamine-induced impairment of spontaneous alternation behavior by antagonist but not inverse agonist and b-carbolines. Psychopharmacology, 94 (1988) 491-495. [19] Shiromani, P.J., Gillin, J.C. and Henriksen, S.J., Acetylcholine and the regulation of REM sleep: Basic mechanisms and clinical implications for affective illness and narcolepsy, Annu. Re~,. Pharmacol. Toxicol., 27 (1987) 137-156. [20] Smith, C., Sleep states and learning: A review of the literature. Neurosci. Biobehat.. Rez~., 9 (1985) 157-168. [21] SPSS, S P S S / P C + Statistics 4.0, Marija J. Norusis/SPSS, Chicago, 1990. [22] Stone, W.S., Altman, H.J., Hall, J.L., Arankowsky-Sandoval, G., Parekh, P. and Gold, P.E., Prenatal exposure to alcohol in adult rats: Relationships between sleep and memory deficits, and effects of glucose administration on memory, Brain Res., (1995) in press. [23] Stone, W.S., Cotter, E., Beare, D., Rudd, RJ. and Gold, P.E., Relationships between deficits in sleep and spontaneous alternation in kindled rats: Effects of glucose, Submitted (1995). [24] Stone, W.S. and Gold, P.E., Sleep and memory relationships in
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