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The Beta-1 Adrenergic Antagonist, Betaxolol, Improves Working Memory Performance in Rats and Monkeys
The Beta-1 Adrenergic Antagonist, Betaxolol, Improves Working Memory Performance in Rats and Monkeys Brian P. Ramos, Lesley Colgan, Eric Nou, Shira Ovadia, Steven R. Wilson, and Amy F.T. Arnsten Background: Previous studies have indicated that beta adrenergic receptor stimulation has no effect on the cognitive functioning of the prefrontal cortex (PFC). Blockade of beta-1 and beta-2 receptors in the PFC with the mixed beta-1/beta-2 antagonist, propanolol, had no effect on spatial working memory performance. However, more selective blockade of beta-1 or beta-2 receptors might show efficacy if the two receptors have opposite effects on PFC function. The current study examined the effects of the selective beta-1 antagonist, betaxolol, on working memory in rats and monkeys. Methods: In rats, betaxolol (.0011–1.11 g/.5 L) was infused into the PFC 5 min before delayed alternation testing. Monkeys were systemically injected with betaxolol (.0000011–.11 mg/kg) 2 hours before delayed response testing. Results: Betaxolol produced a dose-related improvement in working memory performance following either direct PFC infusion in rats, or systemic administration in monkeys. However, some aged monkeys developed serious pancreatic problems over the course of this study. Conclusions: These findings suggest that endogenous activation of the beta-1 adrenergic receptor impairs PFC cognitive function. These results may have therapeutic relevance to post-traumatic stress disorder or other disorders with excessive noradrenergic activity and PFC dysfunction. Pancreatic side effects in aged subjects taking betaxolol warrants further investigation.
Key Words: Norepinephrine, prefrontal cortex, aging, pancreatic cancer, diabetes, cognition
T
he prefrontal cortex (PFC) guides behavior and thought using working memory (Goldman-Rakic 1987) and plays an important role in the cognitive behavior of primates. The PFC is engaged during the retrieval and encoding of memories (especially if it is an effortful process) and is critical for inhibition of proactive interference, for protecting memories and thoughts from distraction, and for allowing us to plan and organize behavior (Bunge et al 2001; Kapur et al 1995; Lepage et al 2000; Stuss and Knight 2002). Research on the biological basis of mental illness has revealed the relevance of the PFC dysfunction in many neuropsychiatric disorders. Many of the symptoms that are thought to reflect PFC dysfunction, such as impulsivity, poor concentration, and impaired sensory gating are common in many neuropsychiatric disorders. Some of these disorders involve dysregulation of the noradrenergic system. For example, clinical studies have provided evidence for exaggerated noradrenergic activity in humans with post-traumatic stress disorder (PTSD) (Bremner et al 1997; Southwick et al 1999). In contrast, NE turnover is reduced in the PFC of aged rats (Hadfield and Milio 1990) which may contribute to the PFC deficits seen with age (Arnsten 1999; Friedman et al 1999). Thus, the effects of NE on PFC function are highly relevant to neuropsychiatry. The PFC is very sensitive to levels of norepinephrine (NE). Moderate levels of release engage alpha-2A adrenergic receptors and improve PFC function (Franowicz et al 2002). In contrast, higher levels of NE release, as with stress, engage alpha-1 receptors and impair PFC function (Birnbaum et al 1999). Although we have gained extensive knowledge about NE’s actions at alpha-1 and alpha-2 receptors in the PFC, our under-
From the Department of Neurobiology (BPR, LC, EN, AFTA) and Department of Comparative Medicine (SO, SRW), Yale University School of Medicine, New Haven, Connecticut. Address reprint requests to Amy F.T. Arnsten, Ph.D., Department of Neurobiology, P.O. Box 208001, Yale Medical School, New Haven, CT 065208001; E-mail: [email protected]. Received December 6, 2004; revised May 5, 2005; accepted May 12, 2005.
0006-3223/05/$30.00 doi:10.1016/j.biopsych.2005.05.022
standing of NE’s actions at beta receptors in the PFC is scarce. This contrasts with the rest of the peripheral and central nervous systems, where NE actions at beta adrenergic receptors have powerful effects (e.g. Berridge et al 1996; Cahill et al 1994; Hopkins and Johnston 1988; Sessler et al 1989; Tevaearai and Koch 2004; Waterhouse et al 1980). Beta receptors are differentiated into three subtypes, beta-1, beta-2, and beta-3, and all are found in the nervous system. These subtypes are differentially expressed in various regions of the brain, with beta-1 receptors (Rainbow et al 1984) and beta-3 mRNA (Summers et al 1995) in higher concentrations in the rat cortex. Beta receptor mRNA has also been found in the PFC of rats (Nicholas et al 1993). Previous studies have observed no effect on working memory when beta 1 and 2 receptors are blocked with the mixed beta-1, 2, and 3 antagonist, propanolol. For example, neither microinjection of propanolol into the PFC (Li and Mei 1994), nor systemic administration of propanolol (Arnsten and Goldman-Rakic 1985) altered PFC function in monkeys. This lack of effect was not due to a paucity of beta receptors, as beta receptors have been identified in high concentration in the monkey (Goldman-Rakic et al 1990). It is possible that propranolol has no effect on working memory because beta-1 and beta-2 receptors, like alpha-1 and alpha-2 receptors, have opposing functional effects. Pharmacological agents selective for either beta-1 or beta-2 receptors have become available and may help to bring new light to the issue of whether or not beta receptors modulate PFC function. The current study examined whether the beta-1 adrenergic receptor antagonist, betaxolol, alters PFC function in rats and monkeys. Rats received betaxolol directly into the PFC through guide cannula. Rats were tested on the delayed alternation task in a T-maze, a test of spatial working memory that is impaired by lesions to the prelimbic/infralimbic PFC in rats (Larsen and Divac 1978). A parallel study examined the effects of systemic injections of betaxolol in young and aged monkeys performing a spatial working memory task, delayed response, a task that depends upon dorsolateral PFC function in monkeys (Goldman and Rosvold 1970). The findings from our behavioral studies show that betaxolol improves working memory performance under both conditions, suggesting that endogenous activation of the beta-1 receptor impairs PFC cognitive function. However, BIOL PSYCHIATRY 2005;58:894 –900 © 2005 Society of Biological Psychiatry
B.P. Ramos et al serious pancreatic side effects arose in aged monkeys taking betaxolol, warranting further investigation.
Methods and Materials Rat Studies Subjects. Young adult (6 months) male rats from Harlan (Indianapolis, Indiana) were single-housed in filter frame cages. Rats were approximately 9 months old upon initiation of pharmacological testing. Animals were kept on a 12 hour light/dark cycle, and experiments were conducted during the light phase. Rats were slowly habituated to a restricted diet (16 gm/day per rat) of autoclaved Purina (St. Louis, Missouri) rat chow during the first two weeks. Food was given immediately after behavioral testing and water was available ad libitum. Rats were weighed weekly to confirm normal weight gain. Food rewards during cognitive testing were highly palatable miniature chocolate chips. Rats were assigned a single experimenter who handled them extensively before behavioral testing. Delayed Alternation in T-Maze. Rats were habituated to a T-maze (dimensions, 90 ⫻ 65 cm) until they were readily eating chocolate chips placed at the end of each arm and were acclimated to handling. After habituation, rats were trained on the delayed alternation task. On the first trial, animals were rewarded for entering either arm. Thereafter, for a total of 10 trials per session, rats were rewarded only if they entered the maze arm which was not previously chosen. Between trials the choice point was wiped with alcohol to remove any olfactory clues. The delay between trials started at “0” sec (i.e. about 1.5 sec, minimum possible for delayed alternation) and was subsequently raised in 5 sec intervals as needed to maintain performance at about 70% correct. Rats in the study were at delays that ranged from “0” (actually 1.5 sec) to 30 sec with a mean delay of 15.23 ⫾ 2.85 sec. Cannulae Implantation. After training on the delayed alternation task, animals underwent stereotaxic implantation of chronic guide cannulae as described previously (Taylor et al 1999). Guide cannulae (Plastics One, Roanoke, Virginia; 2.8 mm) with stylettes were aimed dorsal to the medial PFC (prelimbic PFC; stereotaxic coordinates- anterioposterior: ⫹3.2 mm; mediolateral: ⫾.75 mm; dorsoventral: stylette reaching to ⫺4.2 mm). Anesthesia was obtained using a mixture of ketamine (80 mg/kg) and xylazine (10mg/kg) injected (intraperitoneally, IP) prior to the start of the procedure. These agents were supplemented with gas anesthesia (isoflurane) administered during surgery via nosecone. Sterile stylettes were inserted in the cannula to maintain patency. Great care was taken to minimize pain and infection postoperatively to decrease stress to the animal. The region surrounding the cemented guide cannula was treated with triple antibiotic and cleaned daily if needed for a period of about a week. Animals were also acutely treated with Buprenex (.01 mg/kg) to decrease pain. Drug Infusions. Rats were initially adapted to a mock infusion protocol to minimize any stress associated with the procedure. Once the animals were performing normally following mock infusions, actual infusions were done. Rats were gently restrained while the stylets were removed and replaced with 30 gauge sterile infusion needles that extended to 4.5 mm dorsoventral below the skull. Bilateral infusions were driven by a Harvard Apparatus (Hollinston, Massachusetts) syringe pump set at a flow rate of .25 L/min using 25 L Hamilton syringes for an infusion time of 2 min. Needles remained inserted in place for 2 min after completion of the infusion. Stylettes were inserted back
BIOL PSYCHIATRY 2005;58:894 –900 895 into the cannulae, and behavioral testing was performed immediately after the infusion procedure. Drug treatments and vehicle were administered in a counterbalanced order with at least 1 week between each infusion. Counterbalancing ensured that rats received drug infusions both early in the study when delays were short, as well as later in the study when delays were longer. Animals were required to exhibit stable baseline performance (2 consecutive test sessions of 60 –70% correct) prior to drug administration. Betaxolol was purchased from Tocris Cookson Inc. Betaxolol was dissolved in saline to the appropriate dose. A pilot study examined a range of betaxolol doses (.0011, .011, .11, 1.1 g/.5 L). The two lower doses did not have any effect on working memory; thus, the higher doses became the focus of the current study. The experimenter testing the animal was unaware of drug treatment conditions. Histology. Rats were administered an overdose of ketamine and xylazine and sacrificed by decapitation. Brains were removed, stored in formalin, sectioned, and analyzed for histological verification of cannulae placement. All rats had correctly placed cannulae within the PFC (see Figure 2A). Data Analysis. The betaxolol data from rats was analyzed using paired t tests (two-tailed, significance level ⫽ .05). A one-way analysis of variance with repeated measures (1ANOVA-R) was used to analyze the data from the group II rats that had received both doses of betaxolol in the present study. The correlation between drug efficacy and placement of cannula within PFC was also performed using a Pearson r test (two-tailed, significance level ⫽ .05). Monkey Studies Subjects. The animals used in this study were rhesus monkeys (Macaca mulatta) ranging in age from 4 years (postpubescent) to over 30 years. The monkeys were individually housed and maintained on a diet of Purina monkey chow supplemented with fruit. Animals were always tested at the same time of day immediately prior to feeding. Highly palatable food rewards (e.g. peanuts, raisins or chocolate chips) were utilized during testing to minimize the need for dietary regulation. Delayed Response Testing. Cognitive testing occurred in a Wisconsin General Testing Apparatus (WGTA) situated in a sound-attenuating room. Background masking noise (60 dB, wideband) was also used to minimize auditory distractions. Each monkey was assigned to a single experimenter who knew the animal well but was unaware of the drug treatment conditions. The animals were tested twice a week with 3-4 days separating each test session (e.g., Monday and Thursday). The monkeys had been previously trained on the spatial delayed response task as described in Arnsten et al (1988). During delayed response, the animal watched as the experimenter baited one of several foodwells with a food reward. The number of foodwells varied from two to four wells depending on the monkey’s performance level and experience with the task. Care was taken by the experimenter to ensure that the animal attended the baiting procedure. The foodwells were then covered with identical cardboard plaques, and an opaque screen was lowered between the animal and the foodwells for a specified delay. At the end of the delay, the screen was raised and the animal was allowed to choose. Reward was quasi-randomly distributed between the left and right wells over the 30 trials that made up a daily test session. Five different delay lengths (referred to as delays A through E) were quasi-randomly distributed over these 30 trials. The shortest of these delays was less than 1 second (the “0” sec A delay). The remaining delays were in the range which for each individual www.sobp.org/journal
896 BIOL PSYCHIATRY 2005;58:894 –900 monkey yielded baseline performance of about 70% across all delays (i.e. 18-22 trials correct of the possible 30 trials). For example the delays for one animal might be A ⫽ 0, B ⫽ 5, C ⫽ 10, D ⫽ 15, and E ⫽ 20 sec. In the present study, B delay ranged from 2 to 12 sec with a mean B delay of 6.80 ⫾ 1.04 sec. Sedation Assessment. During each cognitive testing session, the experimenter rated the animal’s level of sedation/ agitation and aggression on rating scales. Sedation and agitation were rated using a 9 point scale where ⫺4 ⫽ too agitated to test, ⫺3 ⫽ agitation which interferes with testing, ⫺2 ⫽ slight agitation which does not interfere with testing, ⫺1 ⫽ more alert than usual, 0 ⫽ normal level of arousal, 1 ⫽ quieter than usual, 2 ⫽ sedated (drooping eyelids, slowed movements), 3 ⫽ intermittent sleeping, and 4 ⫽ too sedated to test. Aggression was rated using a similar scale where ⫺3 ⫽ dramatically more aggressive, ⫺2 ⫽ moderately more aggressive, ⫺1 ⫽ mildly more aggressive, 0 ⫽ normal, 1 ⫽ mildly less aggressive, 2 ⫽ moderately less aggressive, 3 ⫽ dramatically less aggressive. Drug Administration. Betaxolol (purchased from Sigma RBI, St. Louis, Missouri) was dissolved in sterile saline to the appropriate concentration for the following doses: .0000011–.11 mg/ kg. Drug solutions were made up fresh each day under sterile conditions. Drug or vehicle was injected intramuscularly 2 hours prior to cognitive testing. The order of dose administration was determined quasi-randomly, and the experimenter testing the animal was unaware of the treatment condition. A washout period of at least 10 days occurred between drug treatments. Monkeys were required to return to stable baseline performance for 2 consecutive testing days prior to new drug treatment. Given these prolonged washout conditions, the research took approximately 24 months to complete. Data Analysis. The betaxolol data from monkeys was analyzed using a one-way analysis of variance with repeated measures (1-ANOVA-R) with a within-subjects factor of betaxolol dosage. Planned comparisons (test of effects) examined the effects of individual doses of betaxolol to saline. A more detailed analysis of the effects of the most effective dose of betaxolol (varied depending on the monkey) at each delay interval was performed on the data (2-ANOVA-R; within subjects factors of betaxolol and delay interval). Planned comparisons examined the effects of the most effective betaxolol doses at each delay length.
B.P. Ramos et al
Figure 1. The effect of betaxolol on PFC cognitive function of rats (n ⫽ 11) performing the delayed alternation task. Data represent the mean percent correct ⫾ SEM (out of 10 trials) following an infusion of either saline or .11 g/.5 L betaxolol. * Significant difference from saline (p ⫽ .03). PFC, prefrontal cortex.
Results
group that was improved by the .11 g dose and had anteriorly placed cannula, and a second group with more posteriorly placed cannula that were unaffected by the .11 g dose but were improved by a higher dose of 1.11 g (Group I: 3.56 ⫾ .15 mm vs. Group II: 2.95 ⫾ .17 mm, p ⫽ .022). As can be seen in Figure 2B, group I rats’ performance was increased to 82.9 ⫾ 3.9% following an infusion of the .11 g dose compared to 69.3 ⫾ .8% after vehicle (p ⫽ .009). Conversely, the rats in group II were less sensitive to the effects of betaxolol. These rats were significantly improved by the higher dose (1.11 g) with performance at 87.8 ⫾ 3.0% compared to 67.50 ⫾ 5.53% after the vehicle infusion (1-ANOVA-R effect of betaxolol: F (2,6) ⫽ 21.00, p ⫽ .002; user-defined contrasts: 1.11 g betaxolol vs. saline: F (1,3) ⫽ 81.00, p ⫽ .003). Conversely, group II rats were not significantly improved by the .11 g dose of betaxolol (vehicle: 67.50 ⫾ 5.53%; betaxolol: 65.00 ⫾ 3.33%, user-defined contrasts: .11 g betaxolol vs. saline: F (1,3) ⫽ .27, p ⫽ .64). In contrast to the anterior/posterior dimension, there was very little variation in the dorsal/ventral (DV) placement of the cannulae, as every infusion site was located within the prelimbic cortex (-4.18 ⫾ .12 mm DV). There was also no significant correlation between efficacy of betaxolol and dorsal/ventral placement of the cannulae (r ⫽ .24, n ⫽ 11, p ⬎ .05). Thus, variations in drug efficacy were related to anterior/posterior differences within the prelimbic cortex.
Rats Betaxolol infusions were initiated after stable baseline performance of 60 –70% was established. Intra-PFC infusion of .11 g dose of betaxolol significantly improved the performance of young rats in the T-maze task (Figure 1). The average performance for the 11 rats following treatment was 76.4 ⫾ 3.8% correct compared to 67.7 ⫾ 1.3% after vehicle (p ⫽ .03, paired t test). Futhermore, these cognitive effects following betaxolol treatment were not caused by nonspecific effects on motivation or motor performance as there was no difference in the response times of rats following vehicle (7.4 ⫾ 2.5 sec) versus betaxolol (7.6 ⫾ 1.5 sec) infusions (p ⫽ .89). Further analysis indicated that there was a significant correlation between percent correct following the .11 g dose of betaxolol and placement of the cannula within the PFC along the anterior/posterior axis. Those rats with more anterior placed cannula had greater improvement with drug (Figure 2A;r ⫽ .65, n ⫽ 11, p ⬍ .05). Indeed, the rats divided into two groups: a first
Monkeys A wide range of betaxolol doses were examined in most animals (1.1x10-5 – .11 mg/kg). However, some animals developed pancreatic problems during the betaxolol study (see below), and therefore not every animal was able to receive every dose. Thus, we present only the three doses received by every animal in the current study (.00011, .0011, and .011 mg/kg; Figure 3). Systemic administration of betaxolol had a significant effect on delayed response performance (Figure 3; within subjects effects of betaxolol: F (3, 27) ⫽ 4.81, p ⫽ .008). Both young adult and aged monkeys were improved by betaxolol with similar efficacy and potency. Planned comparisons of individual doses showed that the .001 mg/kg dose of betaxolol produced the greatest improvement in performance of monkeys compared to vehicle (F (1,9) ⫽ 38.72, p ⫽ .0002). In three monkeys the .001 mg/kg dose was repeated to ensure reliability of drug effect; improvement was successfully replicated in all animals. There
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B.P. Ramos et al
BIOL PSYCHIATRY 2005;58:894 –900 897
Figure 3. The effect of betaxolol on delayed response performance of monkeys (n ⫽ 10). Data are shown as mean percent correct ⫾ SEM (out of 30 trials) following systemic administration of saline, .00011, .0011 or .011 mg/kg of betaxolol. *Significant difference from saline (p ⫽ .008).
⫽ .000004]; effect of betaxolol [F (1,7) ⫽ 35.85, p ⫽ .0006]). Betaxolol improved performance at every delay except the 0 sec delay control condition (Saline vs. Betaxolol, 0 sec (A) delay [F (1,7) ⫽ 0, p ⫽ 1.0]). However, as can be seen in Figure 4, the improvement only reached statistical significance at the two lower, intermediate delays (see Methods and Materials) when the PFC is most needed to guide behavior (B delay [F (1,7) ⫽ 27.81, p ⫽ .001]; C delay [F (1,7) ⫽ 7.16, p ⫽ .032]). The improvement was not significantly different at the two longest delay lengths (D delay [F (1,7) ⫽ 3.15, p ⫽ .12]; E delay [F (1,7) ⫽ 1.20, p ⫽ .31]). Given the potential clinical relevance of these results, it is important to mention that 5 of the older female rhesus monkeys (20 –37 years old) developed alterations in glucose homeostasis
Figure 2. Differential sensitivity to betaxolol (Betax) is due to cannula location within the PFC. (A) Correlation between cannula placement (x-axis) and the percent correct following an infusion of a .11 g/.5 L dose of betaxolol (y-axis). (B) Data are shown as mean percent correct ⫾ SEM (out of 10 trials) following an infusion of saline, .11 g or 1.11 g of betaxolol. Rats from Figure 1 were divided into two groups. Group I rats (n ⫽ 7) which were significantly improved by the low dose of betaxolol and the Group II rats (n ⫽ 4) which were not. Group II rats were significantly improved following an infusion of the higher dose of betaxolol. *Significant difference from saline (.11 g: p ⫽ .009; 1.11 g: p ⫽ .003). PFC, prefrontal cortex.
was no evidence of side effects at this dose in any animal (median sedation/aggression score of 0, normal behavior). Similar to the rats, there was no significant difference in total testing time following vehicle (12.9 ⫾ 1.24 min) versus betaxolol (12.9 ⫾ 1.14 min) administration (p ⫽ .921), suggesting that improvement altered PFC cognitive ability rather than producing nonspecific effects on motor performance or motivation. An analysis of the best dose of betaxolol on performance at each delay length confirmed this interpretation (effect of delay [F (4,28) ⫽ 13.12, p
Figure 4. The effect of betaxolol on delayed response performance of monkeys at different delays. The most effective dose was selected for each monkey (n ⫽ 8). Data are shown as mean number correct ⫾ SEM (out of 6) for 5 different delays (A, B, C, D, and E) following betaxolol treatment. *Significant difference from saline.
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898 BIOL PSYCHIATRY 2005;58:894 –900 and/or serious pancreatic pathology while in the betaxolol study. These findings were in higher prevalence compared to other age-matched female macaques in the colony who had not received this drug. Some of the findings included: insulinoma, type 2 diabetes mellitus, necrotic pancreas in conjunction with intestinal adenocarcinoma, and abnormally high or low glucose levels post treatment with betaxolol. Each of the monkeys had received 3 to 11 doses of betaxolol (1.1 ⫻ 10-5-.11 mg/kg). However, they had also received 8 to 25 doses of the partial beta-2 agonist/beta-1 antagonist, clenbuterol (1 ⫻ 10-6-.1 mg/kg) as part of a parallel study on beta-2 receptor influences (unpublished). Thus, it is not clear whether the findings resulted from betaxolol and/or clenbuterol treatment. However, blood glucose levels were found to be altered directly following betaxolol treatment in one monkey who developed diabetes during the study, indicating some direct relationship between betaxolol and pancreatic function.
Discussion The current study is the first to show that a beta adrenergic receptor subtype, specifically the beta-1 adrenergic receptor, indeed has an effect on PFC cognitive function. The results show that blocking the beta-1 receptor in both rats and monkeys improves performance of PFC-dependent tasks, suggesting that endogenous activation of beta-1 receptors impairs PFC function. However, a rigorous test of this hypothesis will require use of a selective, full beta-1 adrenergic agonist to determine whether it will impair PFC cognitive function. To our knowledge, a full, selective beta-1 agonist is not currently available, although a partial agonist (xamoterol) is available. Nevertheless, these data indicate that beta receptor mechanisms are more important to PFC function than previously thought. The rat data are consistent with drug actions in the PFC. Infusion of betaxolol directly into the PFC of rats improved performance of a delayed alternation task. The beneficial effects of betaxolol infusion were especially potent in more anterior regions of the PFC. These results are consistent with noradrenergic innervation being more dense in the anterior regions of the PFC in rats (Morrison et al 1979, 1981) and with cannula placement (i.e. 3.2– 4.2 mm from bregma) in previous pharmacological studies in the laboratory that infused drugs acting on noradrenergic receptors (Arnsten et al 1999; Birnbaum et al 1999). In contrast, dopamine D1 agents were more effective in posterior regions of rat PFC (Zahrt et al 1997). Enhancing effects were also found when betaxolol was administered systemically to monkeys performing a delayed response task. The monkeys performed a variable delay version of the delayed response task, thus providing an opportunity to assess drug effects under control conditions (0 sec delay, when PFC function is not needed), short delays when the PFC, but not the hippocampus is needed, and long delays when both hippocampal and PFC functions are needed for good performance (Zola-Morgan and Squire 1985). Betaxolol did not have any effect at the 0 sec delay, but did improve performance following the shorter, intermediate delays which require PFC regulation of response (Goldman and Rosvold 1970). Interestingly, the improvement was not significant at the longer delays which also depend on hippocampal function. Previous research has shown that blocking beta receptors with propanolol directly into the CA1 region of the hippocampus impairs consolidation of spatial or contextual fear memory (Ji et al 2003a, 2003b). Furthermore, both propanolol and betaxolol impair hippocampal memory www.sobp.org/journal
B.P. Ramos et al retrieval in rodents (Murchison et al 2004). Thus, betaxolol’s impairing effects on hippocampal function may have partially occluded the beneficial influence on PFC function at longer delays (Figure 4: D and E delays). Little is known about the beta-1 receptor’s subcellular location and function, so it is difficult to make any speculations about possible mechanisms by which this receptor can impair PFC function. However, it is known that beta receptors can be coupled to Gs which can lead to an increase in cAMP levels. Previous research has shown that increasing cAMP levels and PKA activation impair PFC function in both young and aged animals (Ramos et al 2003; Taylor et al 1999). Thus, beta-1 receptors may impair PFC cognitive function via this intracellular pathway. The lack of pharmacological agents that could specifically bind to beta adrenergic receptor subtypes has limited previous studies of beta receptor mechanisms. Previous studies did not find any effect when using propanolol, a beta receptor antagonist that binds to beta-1, 2, and 3 receptors (Arnsten and GoldmanRakic 1985; Li and Mei 1994). It is possible that the lack of an effect was due to the opposite effects of beta-1 (impairing) versus beta-2 (improving; unpublished data) adrenergic receptors on working memory. Differences in baseline performance may also contribute to drug sensitivity in the rat studies. In the current study, performance of vehicle treated rats was lower (69.3 ⫾ .8%) compared to that of previous studies using propranolol (⬃80%) (Murphy et al 1996; Roozendaal et al 2004a). Therefore, it is possible that the effects seen in the current study were due to subtly higher stress levels leaving more room for improvement. Stress is known to increase the release of norepinephrine in the PFC (Finlay et al 1995; Goldstein et al 1996) thus betaxolol may enhance cognitive function by preventing high levels of NE from binding to the beta-1 adrenergic receptor. Interestingly, propanolol when given systemically can block working memory impairment induced by corticosterone (Roozendaal et al 2004c), a glucocorticoid that is increased during stress. However, this may be due to drug actions in amygdala that could in turn decrease global NE release (Arnsten 2000; Goldstein et al 1996) and/or decrease the inhibitory effects that the amygdala has on the PFC (Roozendaal et al 2004c). The current study contrasts with neurochemical studies of the emotional enhancement of memory consolidation by the basal lateral amygdala. Early studies demonstrated the critical role of NE, with special emphasis on actions at beta adrenergic receptors (Cahill and McGaugh 1996). Indeed, blockade of beta adrenergic receptors with agents such as propranolol attenuated the emotional enhancement of memory in both rodents and human volunteers (Cahill and McGaugh 1996). However, a more recent study suggests that propanolol or betaxolol treatment does not have a significant effect on hippocampal memory consolidation but impairs retrieval in the absence of an increase in corticosterone (Murchison et al 2004). Interestingly, the impairing effects of elevated levels of glucocorticoids on hippocampal spatial (Roozendaal et al 2004b) or contextual (Roozendaal et al 2004a) memory retrieval are blocked by propranolol treatment similar to what is found for PFC working memory (Roozendaal et al 2004c). Taken together the results suggest that hippocampal memory retrieval is regulated differently than PFC working memory in the absence of elevated levels of glucocorticoids. Interestingly, a number of medications in use for the treatment of PTSD weaken amygdala function while strengthening PFC regulation of behavior. For example, the alpha-1 adrenoceptor antagonist, prazosin, has been reported to reduce night-
B.P. Ramos et al mares and other symptoms of PTSD in combat veterans (Raskind et al 2003). Alpha-1 receptor blockade has been shown to protect PFC function and weaken amygdala function in animal studies (Ferry et al 1999). Similarly, beta receptor blockers such as propranolol are being tested immediately post-trauma in hopes of alleviating the development of PTSD (Vaiva et al 2003). Studies in animals suggest that this drug treatment may prevent amygdala-induced enhancement of traumatic memories, but may not strengthen PFC inhibitory abilities. The current study shows that specific blockade of the beta-1 adrenergic receptor subtype does strengthen the PFC and could weaken amygdala function. Thus, unlike propanolol, betaxolol may prove useful in treating established PTSD. The finding of cognitive enhancement with betaxolol treatment is potentially exciting; however, the serious health problems associated with betaxolol use in the aged monkeys in this study indicate caution is warranted. Betaxolol is commonly used in humans for the treatment of coronary problems, e.g. hypertension, and for the treatment of glaucoma. However, data about the effects of long-term use of betaxolol and its side effects are lacking. Longer studies are required to confirm its lack of serious adverse effects (Beresford and Heel 1986). The possible adverse metabolic effects listed for betaxolol include diabetes and hyperglycemia, although hypoglycemia has been reported with an over dose of the drug (Duplay 2004). Patients with type 1 diabetes are cautioned about use of betaxolol, although it is not known to exacerbate insulin-induced hypoglycemia (Benn et al 1992a, 1992b; Beresford and Heel 1986; Saunders et al 1981a, 1981b; Sinclair et al 1990a, 1990b). In the current study, betaxolol doses were at or below the recommended therapeutic dose for hypertension, and were given on an infrequent basis (maximal frequency of once per week). It should be noted that the macaques received simple carbohydrates as a reward for their performance, which may have contributed to blood glucose problems. It is also important to note that the monkeys who had pancreatic problems also had been treated with clenbuterol (2 adrenergic partial agonist with 1 antagonistic properties) at different time points or in conjunction with betaxolol. However, the very serious nature of these problems (i.e. pancreatic cancer, induction of diabetes) cautions that immediate research is needed to determine whether low doses of betaxolol cause pancreatic dysfunction in elderly primates. In conclusion, the current results suggest that betaxolol could be used as a cognitive enhancer in populations with PFC deficits. Betaxolol is already used clinically to treat glaucoma and hypertension (Duplay 2004; Mann and Gerber 2001), two conditions that are frequently found in the elderly population. It is noteworthy that normal aging (Albert 1997; Rapp and Amaral 1989), and age-related increases in blood pressure (Moore et al 2002; Raz et al 2003; Saxby et al 2003) or glucocorticoid levels (Lupien et al 1994, 1999; Lyons et al 2000) are risk factors for PFC cognitive impairment in humans and monkeys. Thus, the use of betaxolol in the elderly seems logical. Betaxolol treatment may also be useful in treating PTSD patients by enhancing PFC function while weakening amygdala’s ability to strengthen emotionally relevant or traumatic memories, if indeed beta-1 receptors regulate this process. However, the pancreatic side effects seen in some monkeys, in particular the older animals, are a concern that may limit the use of this drug. Future studies should examine whether this is an incidental finding, an adverse effect of betaxolol, clenbuterol or the combination of the two. If these safety factors are addressed sufficiently, then a selective beta-1 antagonist may
BIOL PSYCHIATRY 2005;58:894 –900 899 be useful for enhancing and/or protecting PFC cognitive function.
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Key Words: Norepinephrine, prefrontal cortex, aging, pancreatic cancer, diabetes, cognition
T
he prefrontal cortex (PFC) guides behavior and thought using working memory (Goldman-Rakic 1987) and plays an important role in the cognitive behavior of primates. The PFC is engaged during the retrieval and encoding of memories (especially if it is an effortful process) and is critical for inhibition of proactive interference, for protecting memories and thoughts from distraction, and for allowing us to plan and organize behavior (Bunge et al 2001; Kapur et al 1995; Lepage et al 2000; Stuss and Knight 2002). Research on the biological basis of mental illness has revealed the relevance of the PFC dysfunction in many neuropsychiatric disorders. Many of the symptoms that are thought to reflect PFC dysfunction, such as impulsivity, poor concentration, and impaired sensory gating are common in many neuropsychiatric disorders. Some of these disorders involve dysregulation of the noradrenergic system. For example, clinical studies have provided evidence for exaggerated noradrenergic activity in humans with post-traumatic stress disorder (PTSD) (Bremner et al 1997; Southwick et al 1999). In contrast, NE turnover is reduced in the PFC of aged rats (Hadfield and Milio 1990) which may contribute to the PFC deficits seen with age (Arnsten 1999; Friedman et al 1999). Thus, the effects of NE on PFC function are highly relevant to neuropsychiatry. The PFC is very sensitive to levels of norepinephrine (NE). Moderate levels of release engage alpha-2A adrenergic receptors and improve PFC function (Franowicz et al 2002). In contrast, higher levels of NE release, as with stress, engage alpha-1 receptors and impair PFC function (Birnbaum et al 1999). Although we have gained extensive knowledge about NE’s actions at alpha-1 and alpha-2 receptors in the PFC, our under-
From the Department of Neurobiology (BPR, LC, EN, AFTA) and Department of Comparative Medicine (SO, SRW), Yale University School of Medicine, New Haven, Connecticut. Address reprint requests to Amy F.T. Arnsten, Ph.D., Department of Neurobiology, P.O. Box 208001, Yale Medical School, New Haven, CT 065208001; E-mail: [email protected]. Received December 6, 2004; revised May 5, 2005; accepted May 12, 2005.
0006-3223/05/$30.00 doi:10.1016/j.biopsych.2005.05.022
standing of NE’s actions at beta receptors in the PFC is scarce. This contrasts with the rest of the peripheral and central nervous systems, where NE actions at beta adrenergic receptors have powerful effects (e.g. Berridge et al 1996; Cahill et al 1994; Hopkins and Johnston 1988; Sessler et al 1989; Tevaearai and Koch 2004; Waterhouse et al 1980). Beta receptors are differentiated into three subtypes, beta-1, beta-2, and beta-3, and all are found in the nervous system. These subtypes are differentially expressed in various regions of the brain, with beta-1 receptors (Rainbow et al 1984) and beta-3 mRNA (Summers et al 1995) in higher concentrations in the rat cortex. Beta receptor mRNA has also been found in the PFC of rats (Nicholas et al 1993). Previous studies have observed no effect on working memory when beta 1 and 2 receptors are blocked with the mixed beta-1, 2, and 3 antagonist, propanolol. For example, neither microinjection of propanolol into the PFC (Li and Mei 1994), nor systemic administration of propanolol (Arnsten and Goldman-Rakic 1985) altered PFC function in monkeys. This lack of effect was not due to a paucity of beta receptors, as beta receptors have been identified in high concentration in the monkey (Goldman-Rakic et al 1990). It is possible that propranolol has no effect on working memory because beta-1 and beta-2 receptors, like alpha-1 and alpha-2 receptors, have opposing functional effects. Pharmacological agents selective for either beta-1 or beta-2 receptors have become available and may help to bring new light to the issue of whether or not beta receptors modulate PFC function. The current study examined whether the beta-1 adrenergic receptor antagonist, betaxolol, alters PFC function in rats and monkeys. Rats received betaxolol directly into the PFC through guide cannula. Rats were tested on the delayed alternation task in a T-maze, a test of spatial working memory that is impaired by lesions to the prelimbic/infralimbic PFC in rats (Larsen and Divac 1978). A parallel study examined the effects of systemic injections of betaxolol in young and aged monkeys performing a spatial working memory task, delayed response, a task that depends upon dorsolateral PFC function in monkeys (Goldman and Rosvold 1970). The findings from our behavioral studies show that betaxolol improves working memory performance under both conditions, suggesting that endogenous activation of the beta-1 receptor impairs PFC cognitive function. However, BIOL PSYCHIATRY 2005;58:894 –900 © 2005 Society of Biological Psychiatry
B.P. Ramos et al serious pancreatic side effects arose in aged monkeys taking betaxolol, warranting further investigation.
Methods and Materials Rat Studies Subjects. Young adult (6 months) male rats from Harlan (Indianapolis, Indiana) were single-housed in filter frame cages. Rats were approximately 9 months old upon initiation of pharmacological testing. Animals were kept on a 12 hour light/dark cycle, and experiments were conducted during the light phase. Rats were slowly habituated to a restricted diet (16 gm/day per rat) of autoclaved Purina (St. Louis, Missouri) rat chow during the first two weeks. Food was given immediately after behavioral testing and water was available ad libitum. Rats were weighed weekly to confirm normal weight gain. Food rewards during cognitive testing were highly palatable miniature chocolate chips. Rats were assigned a single experimenter who handled them extensively before behavioral testing. Delayed Alternation in T-Maze. Rats were habituated to a T-maze (dimensions, 90 ⫻ 65 cm) until they were readily eating chocolate chips placed at the end of each arm and were acclimated to handling. After habituation, rats were trained on the delayed alternation task. On the first trial, animals were rewarded for entering either arm. Thereafter, for a total of 10 trials per session, rats were rewarded only if they entered the maze arm which was not previously chosen. Between trials the choice point was wiped with alcohol to remove any olfactory clues. The delay between trials started at “0” sec (i.e. about 1.5 sec, minimum possible for delayed alternation) and was subsequently raised in 5 sec intervals as needed to maintain performance at about 70% correct. Rats in the study were at delays that ranged from “0” (actually 1.5 sec) to 30 sec with a mean delay of 15.23 ⫾ 2.85 sec. Cannulae Implantation. After training on the delayed alternation task, animals underwent stereotaxic implantation of chronic guide cannulae as described previously (Taylor et al 1999). Guide cannulae (Plastics One, Roanoke, Virginia; 2.8 mm) with stylettes were aimed dorsal to the medial PFC (prelimbic PFC; stereotaxic coordinates- anterioposterior: ⫹3.2 mm; mediolateral: ⫾.75 mm; dorsoventral: stylette reaching to ⫺4.2 mm). Anesthesia was obtained using a mixture of ketamine (80 mg/kg) and xylazine (10mg/kg) injected (intraperitoneally, IP) prior to the start of the procedure. These agents were supplemented with gas anesthesia (isoflurane) administered during surgery via nosecone. Sterile stylettes were inserted in the cannula to maintain patency. Great care was taken to minimize pain and infection postoperatively to decrease stress to the animal. The region surrounding the cemented guide cannula was treated with triple antibiotic and cleaned daily if needed for a period of about a week. Animals were also acutely treated with Buprenex (.01 mg/kg) to decrease pain. Drug Infusions. Rats were initially adapted to a mock infusion protocol to minimize any stress associated with the procedure. Once the animals were performing normally following mock infusions, actual infusions were done. Rats were gently restrained while the stylets were removed and replaced with 30 gauge sterile infusion needles that extended to 4.5 mm dorsoventral below the skull. Bilateral infusions were driven by a Harvard Apparatus (Hollinston, Massachusetts) syringe pump set at a flow rate of .25 L/min using 25 L Hamilton syringes for an infusion time of 2 min. Needles remained inserted in place for 2 min after completion of the infusion. Stylettes were inserted back
BIOL PSYCHIATRY 2005;58:894 –900 895 into the cannulae, and behavioral testing was performed immediately after the infusion procedure. Drug treatments and vehicle were administered in a counterbalanced order with at least 1 week between each infusion. Counterbalancing ensured that rats received drug infusions both early in the study when delays were short, as well as later in the study when delays were longer. Animals were required to exhibit stable baseline performance (2 consecutive test sessions of 60 –70% correct) prior to drug administration. Betaxolol was purchased from Tocris Cookson Inc. Betaxolol was dissolved in saline to the appropriate dose. A pilot study examined a range of betaxolol doses (.0011, .011, .11, 1.1 g/.5 L). The two lower doses did not have any effect on working memory; thus, the higher doses became the focus of the current study. The experimenter testing the animal was unaware of drug treatment conditions. Histology. Rats were administered an overdose of ketamine and xylazine and sacrificed by decapitation. Brains were removed, stored in formalin, sectioned, and analyzed for histological verification of cannulae placement. All rats had correctly placed cannulae within the PFC (see Figure 2A). Data Analysis. The betaxolol data from rats was analyzed using paired t tests (two-tailed, significance level ⫽ .05). A one-way analysis of variance with repeated measures (1ANOVA-R) was used to analyze the data from the group II rats that had received both doses of betaxolol in the present study. The correlation between drug efficacy and placement of cannula within PFC was also performed using a Pearson r test (two-tailed, significance level ⫽ .05). Monkey Studies Subjects. The animals used in this study were rhesus monkeys (Macaca mulatta) ranging in age from 4 years (postpubescent) to over 30 years. The monkeys were individually housed and maintained on a diet of Purina monkey chow supplemented with fruit. Animals were always tested at the same time of day immediately prior to feeding. Highly palatable food rewards (e.g. peanuts, raisins or chocolate chips) were utilized during testing to minimize the need for dietary regulation. Delayed Response Testing. Cognitive testing occurred in a Wisconsin General Testing Apparatus (WGTA) situated in a sound-attenuating room. Background masking noise (60 dB, wideband) was also used to minimize auditory distractions. Each monkey was assigned to a single experimenter who knew the animal well but was unaware of the drug treatment conditions. The animals were tested twice a week with 3-4 days separating each test session (e.g., Monday and Thursday). The monkeys had been previously trained on the spatial delayed response task as described in Arnsten et al (1988). During delayed response, the animal watched as the experimenter baited one of several foodwells with a food reward. The number of foodwells varied from two to four wells depending on the monkey’s performance level and experience with the task. Care was taken by the experimenter to ensure that the animal attended the baiting procedure. The foodwells were then covered with identical cardboard plaques, and an opaque screen was lowered between the animal and the foodwells for a specified delay. At the end of the delay, the screen was raised and the animal was allowed to choose. Reward was quasi-randomly distributed between the left and right wells over the 30 trials that made up a daily test session. Five different delay lengths (referred to as delays A through E) were quasi-randomly distributed over these 30 trials. The shortest of these delays was less than 1 second (the “0” sec A delay). The remaining delays were in the range which for each individual www.sobp.org/journal
896 BIOL PSYCHIATRY 2005;58:894 –900 monkey yielded baseline performance of about 70% across all delays (i.e. 18-22 trials correct of the possible 30 trials). For example the delays for one animal might be A ⫽ 0, B ⫽ 5, C ⫽ 10, D ⫽ 15, and E ⫽ 20 sec. In the present study, B delay ranged from 2 to 12 sec with a mean B delay of 6.80 ⫾ 1.04 sec. Sedation Assessment. During each cognitive testing session, the experimenter rated the animal’s level of sedation/ agitation and aggression on rating scales. Sedation and agitation were rated using a 9 point scale where ⫺4 ⫽ too agitated to test, ⫺3 ⫽ agitation which interferes with testing, ⫺2 ⫽ slight agitation which does not interfere with testing, ⫺1 ⫽ more alert than usual, 0 ⫽ normal level of arousal, 1 ⫽ quieter than usual, 2 ⫽ sedated (drooping eyelids, slowed movements), 3 ⫽ intermittent sleeping, and 4 ⫽ too sedated to test. Aggression was rated using a similar scale where ⫺3 ⫽ dramatically more aggressive, ⫺2 ⫽ moderately more aggressive, ⫺1 ⫽ mildly more aggressive, 0 ⫽ normal, 1 ⫽ mildly less aggressive, 2 ⫽ moderately less aggressive, 3 ⫽ dramatically less aggressive. Drug Administration. Betaxolol (purchased from Sigma RBI, St. Louis, Missouri) was dissolved in sterile saline to the appropriate concentration for the following doses: .0000011–.11 mg/ kg. Drug solutions were made up fresh each day under sterile conditions. Drug or vehicle was injected intramuscularly 2 hours prior to cognitive testing. The order of dose administration was determined quasi-randomly, and the experimenter testing the animal was unaware of the treatment condition. A washout period of at least 10 days occurred between drug treatments. Monkeys were required to return to stable baseline performance for 2 consecutive testing days prior to new drug treatment. Given these prolonged washout conditions, the research took approximately 24 months to complete. Data Analysis. The betaxolol data from monkeys was analyzed using a one-way analysis of variance with repeated measures (1-ANOVA-R) with a within-subjects factor of betaxolol dosage. Planned comparisons (test of effects) examined the effects of individual doses of betaxolol to saline. A more detailed analysis of the effects of the most effective dose of betaxolol (varied depending on the monkey) at each delay interval was performed on the data (2-ANOVA-R; within subjects factors of betaxolol and delay interval). Planned comparisons examined the effects of the most effective betaxolol doses at each delay length.
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Figure 1. The effect of betaxolol on PFC cognitive function of rats (n ⫽ 11) performing the delayed alternation task. Data represent the mean percent correct ⫾ SEM (out of 10 trials) following an infusion of either saline or .11 g/.5 L betaxolol. * Significant difference from saline (p ⫽ .03). PFC, prefrontal cortex.
Results
group that was improved by the .11 g dose and had anteriorly placed cannula, and a second group with more posteriorly placed cannula that were unaffected by the .11 g dose but were improved by a higher dose of 1.11 g (Group I: 3.56 ⫾ .15 mm vs. Group II: 2.95 ⫾ .17 mm, p ⫽ .022). As can be seen in Figure 2B, group I rats’ performance was increased to 82.9 ⫾ 3.9% following an infusion of the .11 g dose compared to 69.3 ⫾ .8% after vehicle (p ⫽ .009). Conversely, the rats in group II were less sensitive to the effects of betaxolol. These rats were significantly improved by the higher dose (1.11 g) with performance at 87.8 ⫾ 3.0% compared to 67.50 ⫾ 5.53% after the vehicle infusion (1-ANOVA-R effect of betaxolol: F (2,6) ⫽ 21.00, p ⫽ .002; user-defined contrasts: 1.11 g betaxolol vs. saline: F (1,3) ⫽ 81.00, p ⫽ .003). Conversely, group II rats were not significantly improved by the .11 g dose of betaxolol (vehicle: 67.50 ⫾ 5.53%; betaxolol: 65.00 ⫾ 3.33%, user-defined contrasts: .11 g betaxolol vs. saline: F (1,3) ⫽ .27, p ⫽ .64). In contrast to the anterior/posterior dimension, there was very little variation in the dorsal/ventral (DV) placement of the cannulae, as every infusion site was located within the prelimbic cortex (-4.18 ⫾ .12 mm DV). There was also no significant correlation between efficacy of betaxolol and dorsal/ventral placement of the cannulae (r ⫽ .24, n ⫽ 11, p ⬎ .05). Thus, variations in drug efficacy were related to anterior/posterior differences within the prelimbic cortex.
Rats Betaxolol infusions were initiated after stable baseline performance of 60 –70% was established. Intra-PFC infusion of .11 g dose of betaxolol significantly improved the performance of young rats in the T-maze task (Figure 1). The average performance for the 11 rats following treatment was 76.4 ⫾ 3.8% correct compared to 67.7 ⫾ 1.3% after vehicle (p ⫽ .03, paired t test). Futhermore, these cognitive effects following betaxolol treatment were not caused by nonspecific effects on motivation or motor performance as there was no difference in the response times of rats following vehicle (7.4 ⫾ 2.5 sec) versus betaxolol (7.6 ⫾ 1.5 sec) infusions (p ⫽ .89). Further analysis indicated that there was a significant correlation between percent correct following the .11 g dose of betaxolol and placement of the cannula within the PFC along the anterior/posterior axis. Those rats with more anterior placed cannula had greater improvement with drug (Figure 2A;r ⫽ .65, n ⫽ 11, p ⬍ .05). Indeed, the rats divided into two groups: a first
Monkeys A wide range of betaxolol doses were examined in most animals (1.1x10-5 – .11 mg/kg). However, some animals developed pancreatic problems during the betaxolol study (see below), and therefore not every animal was able to receive every dose. Thus, we present only the three doses received by every animal in the current study (.00011, .0011, and .011 mg/kg; Figure 3). Systemic administration of betaxolol had a significant effect on delayed response performance (Figure 3; within subjects effects of betaxolol: F (3, 27) ⫽ 4.81, p ⫽ .008). Both young adult and aged monkeys were improved by betaxolol with similar efficacy and potency. Planned comparisons of individual doses showed that the .001 mg/kg dose of betaxolol produced the greatest improvement in performance of monkeys compared to vehicle (F (1,9) ⫽ 38.72, p ⫽ .0002). In three monkeys the .001 mg/kg dose was repeated to ensure reliability of drug effect; improvement was successfully replicated in all animals. There
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Figure 3. The effect of betaxolol on delayed response performance of monkeys (n ⫽ 10). Data are shown as mean percent correct ⫾ SEM (out of 30 trials) following systemic administration of saline, .00011, .0011 or .011 mg/kg of betaxolol. *Significant difference from saline (p ⫽ .008).
⫽ .000004]; effect of betaxolol [F (1,7) ⫽ 35.85, p ⫽ .0006]). Betaxolol improved performance at every delay except the 0 sec delay control condition (Saline vs. Betaxolol, 0 sec (A) delay [F (1,7) ⫽ 0, p ⫽ 1.0]). However, as can be seen in Figure 4, the improvement only reached statistical significance at the two lower, intermediate delays (see Methods and Materials) when the PFC is most needed to guide behavior (B delay [F (1,7) ⫽ 27.81, p ⫽ .001]; C delay [F (1,7) ⫽ 7.16, p ⫽ .032]). The improvement was not significantly different at the two longest delay lengths (D delay [F (1,7) ⫽ 3.15, p ⫽ .12]; E delay [F (1,7) ⫽ 1.20, p ⫽ .31]). Given the potential clinical relevance of these results, it is important to mention that 5 of the older female rhesus monkeys (20 –37 years old) developed alterations in glucose homeostasis
Figure 2. Differential sensitivity to betaxolol (Betax) is due to cannula location within the PFC. (A) Correlation between cannula placement (x-axis) and the percent correct following an infusion of a .11 g/.5 L dose of betaxolol (y-axis). (B) Data are shown as mean percent correct ⫾ SEM (out of 10 trials) following an infusion of saline, .11 g or 1.11 g of betaxolol. Rats from Figure 1 were divided into two groups. Group I rats (n ⫽ 7) which were significantly improved by the low dose of betaxolol and the Group II rats (n ⫽ 4) which were not. Group II rats were significantly improved following an infusion of the higher dose of betaxolol. *Significant difference from saline (.11 g: p ⫽ .009; 1.11 g: p ⫽ .003). PFC, prefrontal cortex.
was no evidence of side effects at this dose in any animal (median sedation/aggression score of 0, normal behavior). Similar to the rats, there was no significant difference in total testing time following vehicle (12.9 ⫾ 1.24 min) versus betaxolol (12.9 ⫾ 1.14 min) administration (p ⫽ .921), suggesting that improvement altered PFC cognitive ability rather than producing nonspecific effects on motor performance or motivation. An analysis of the best dose of betaxolol on performance at each delay length confirmed this interpretation (effect of delay [F (4,28) ⫽ 13.12, p
Figure 4. The effect of betaxolol on delayed response performance of monkeys at different delays. The most effective dose was selected for each monkey (n ⫽ 8). Data are shown as mean number correct ⫾ SEM (out of 6) for 5 different delays (A, B, C, D, and E) following betaxolol treatment. *Significant difference from saline.
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898 BIOL PSYCHIATRY 2005;58:894 –900 and/or serious pancreatic pathology while in the betaxolol study. These findings were in higher prevalence compared to other age-matched female macaques in the colony who had not received this drug. Some of the findings included: insulinoma, type 2 diabetes mellitus, necrotic pancreas in conjunction with intestinal adenocarcinoma, and abnormally high or low glucose levels post treatment with betaxolol. Each of the monkeys had received 3 to 11 doses of betaxolol (1.1 ⫻ 10-5-.11 mg/kg). However, they had also received 8 to 25 doses of the partial beta-2 agonist/beta-1 antagonist, clenbuterol (1 ⫻ 10-6-.1 mg/kg) as part of a parallel study on beta-2 receptor influences (unpublished). Thus, it is not clear whether the findings resulted from betaxolol and/or clenbuterol treatment. However, blood glucose levels were found to be altered directly following betaxolol treatment in one monkey who developed diabetes during the study, indicating some direct relationship between betaxolol and pancreatic function.
Discussion The current study is the first to show that a beta adrenergic receptor subtype, specifically the beta-1 adrenergic receptor, indeed has an effect on PFC cognitive function. The results show that blocking the beta-1 receptor in both rats and monkeys improves performance of PFC-dependent tasks, suggesting that endogenous activation of beta-1 receptors impairs PFC function. However, a rigorous test of this hypothesis will require use of a selective, full beta-1 adrenergic agonist to determine whether it will impair PFC cognitive function. To our knowledge, a full, selective beta-1 agonist is not currently available, although a partial agonist (xamoterol) is available. Nevertheless, these data indicate that beta receptor mechanisms are more important to PFC function than previously thought. The rat data are consistent with drug actions in the PFC. Infusion of betaxolol directly into the PFC of rats improved performance of a delayed alternation task. The beneficial effects of betaxolol infusion were especially potent in more anterior regions of the PFC. These results are consistent with noradrenergic innervation being more dense in the anterior regions of the PFC in rats (Morrison et al 1979, 1981) and with cannula placement (i.e. 3.2– 4.2 mm from bregma) in previous pharmacological studies in the laboratory that infused drugs acting on noradrenergic receptors (Arnsten et al 1999; Birnbaum et al 1999). In contrast, dopamine D1 agents were more effective in posterior regions of rat PFC (Zahrt et al 1997). Enhancing effects were also found when betaxolol was administered systemically to monkeys performing a delayed response task. The monkeys performed a variable delay version of the delayed response task, thus providing an opportunity to assess drug effects under control conditions (0 sec delay, when PFC function is not needed), short delays when the PFC, but not the hippocampus is needed, and long delays when both hippocampal and PFC functions are needed for good performance (Zola-Morgan and Squire 1985). Betaxolol did not have any effect at the 0 sec delay, but did improve performance following the shorter, intermediate delays which require PFC regulation of response (Goldman and Rosvold 1970). Interestingly, the improvement was not significant at the longer delays which also depend on hippocampal function. Previous research has shown that blocking beta receptors with propanolol directly into the CA1 region of the hippocampus impairs consolidation of spatial or contextual fear memory (Ji et al 2003a, 2003b). Furthermore, both propanolol and betaxolol impair hippocampal memory www.sobp.org/journal
B.P. Ramos et al retrieval in rodents (Murchison et al 2004). Thus, betaxolol’s impairing effects on hippocampal function may have partially occluded the beneficial influence on PFC function at longer delays (Figure 4: D and E delays). Little is known about the beta-1 receptor’s subcellular location and function, so it is difficult to make any speculations about possible mechanisms by which this receptor can impair PFC function. However, it is known that beta receptors can be coupled to Gs which can lead to an increase in cAMP levels. Previous research has shown that increasing cAMP levels and PKA activation impair PFC function in both young and aged animals (Ramos et al 2003; Taylor et al 1999). Thus, beta-1 receptors may impair PFC cognitive function via this intracellular pathway. The lack of pharmacological agents that could specifically bind to beta adrenergic receptor subtypes has limited previous studies of beta receptor mechanisms. Previous studies did not find any effect when using propanolol, a beta receptor antagonist that binds to beta-1, 2, and 3 receptors (Arnsten and GoldmanRakic 1985; Li and Mei 1994). It is possible that the lack of an effect was due to the opposite effects of beta-1 (impairing) versus beta-2 (improving; unpublished data) adrenergic receptors on working memory. Differences in baseline performance may also contribute to drug sensitivity in the rat studies. In the current study, performance of vehicle treated rats was lower (69.3 ⫾ .8%) compared to that of previous studies using propranolol (⬃80%) (Murphy et al 1996; Roozendaal et al 2004a). Therefore, it is possible that the effects seen in the current study were due to subtly higher stress levels leaving more room for improvement. Stress is known to increase the release of norepinephrine in the PFC (Finlay et al 1995; Goldstein et al 1996) thus betaxolol may enhance cognitive function by preventing high levels of NE from binding to the beta-1 adrenergic receptor. Interestingly, propanolol when given systemically can block working memory impairment induced by corticosterone (Roozendaal et al 2004c), a glucocorticoid that is increased during stress. However, this may be due to drug actions in amygdala that could in turn decrease global NE release (Arnsten 2000; Goldstein et al 1996) and/or decrease the inhibitory effects that the amygdala has on the PFC (Roozendaal et al 2004c). The current study contrasts with neurochemical studies of the emotional enhancement of memory consolidation by the basal lateral amygdala. Early studies demonstrated the critical role of NE, with special emphasis on actions at beta adrenergic receptors (Cahill and McGaugh 1996). Indeed, blockade of beta adrenergic receptors with agents such as propranolol attenuated the emotional enhancement of memory in both rodents and human volunteers (Cahill and McGaugh 1996). However, a more recent study suggests that propanolol or betaxolol treatment does not have a significant effect on hippocampal memory consolidation but impairs retrieval in the absence of an increase in corticosterone (Murchison et al 2004). Interestingly, the impairing effects of elevated levels of glucocorticoids on hippocampal spatial (Roozendaal et al 2004b) or contextual (Roozendaal et al 2004a) memory retrieval are blocked by propranolol treatment similar to what is found for PFC working memory (Roozendaal et al 2004c). Taken together the results suggest that hippocampal memory retrieval is regulated differently than PFC working memory in the absence of elevated levels of glucocorticoids. Interestingly, a number of medications in use for the treatment of PTSD weaken amygdala function while strengthening PFC regulation of behavior. For example, the alpha-1 adrenoceptor antagonist, prazosin, has been reported to reduce night-
B.P. Ramos et al mares and other symptoms of PTSD in combat veterans (Raskind et al 2003). Alpha-1 receptor blockade has been shown to protect PFC function and weaken amygdala function in animal studies (Ferry et al 1999). Similarly, beta receptor blockers such as propranolol are being tested immediately post-trauma in hopes of alleviating the development of PTSD (Vaiva et al 2003). Studies in animals suggest that this drug treatment may prevent amygdala-induced enhancement of traumatic memories, but may not strengthen PFC inhibitory abilities. The current study shows that specific blockade of the beta-1 adrenergic receptor subtype does strengthen the PFC and could weaken amygdala function. Thus, unlike propanolol, betaxolol may prove useful in treating established PTSD. The finding of cognitive enhancement with betaxolol treatment is potentially exciting; however, the serious health problems associated with betaxolol use in the aged monkeys in this study indicate caution is warranted. Betaxolol is commonly used in humans for the treatment of coronary problems, e.g. hypertension, and for the treatment of glaucoma. However, data about the effects of long-term use of betaxolol and its side effects are lacking. Longer studies are required to confirm its lack of serious adverse effects (Beresford and Heel 1986). The possible adverse metabolic effects listed for betaxolol include diabetes and hyperglycemia, although hypoglycemia has been reported with an over dose of the drug (Duplay 2004). Patients with type 1 diabetes are cautioned about use of betaxolol, although it is not known to exacerbate insulin-induced hypoglycemia (Benn et al 1992a, 1992b; Beresford and Heel 1986; Saunders et al 1981a, 1981b; Sinclair et al 1990a, 1990b). In the current study, betaxolol doses were at or below the recommended therapeutic dose for hypertension, and were given on an infrequent basis (maximal frequency of once per week). It should be noted that the macaques received simple carbohydrates as a reward for their performance, which may have contributed to blood glucose problems. It is also important to note that the monkeys who had pancreatic problems also had been treated with clenbuterol (2 adrenergic partial agonist with 1 antagonistic properties) at different time points or in conjunction with betaxolol. However, the very serious nature of these problems (i.e. pancreatic cancer, induction of diabetes) cautions that immediate research is needed to determine whether low doses of betaxolol cause pancreatic dysfunction in elderly primates. In conclusion, the current results suggest that betaxolol could be used as a cognitive enhancer in populations with PFC deficits. Betaxolol is already used clinically to treat glaucoma and hypertension (Duplay 2004; Mann and Gerber 2001), two conditions that are frequently found in the elderly population. It is noteworthy that normal aging (Albert 1997; Rapp and Amaral 1989), and age-related increases in blood pressure (Moore et al 2002; Raz et al 2003; Saxby et al 2003) or glucocorticoid levels (Lupien et al 1994, 1999; Lyons et al 2000) are risk factors for PFC cognitive impairment in humans and monkeys. Thus, the use of betaxolol in the elderly seems logical. Betaxolol treatment may also be useful in treating PTSD patients by enhancing PFC function while weakening amygdala’s ability to strengthen emotionally relevant or traumatic memories, if indeed beta-1 receptors regulate this process. However, the pancreatic side effects seen in some monkeys, in particular the older animals, are a concern that may limit the use of this drug. Future studies should examine whether this is an incidental finding, an adverse effect of betaxolol, clenbuterol or the combination of the two. If these safety factors are addressed sufficiently, then a selective beta-1 antagonist may
BIOL PSYCHIATRY 2005;58:894 –900 899 be useful for enhancing and/or protecting PFC cognitive function.
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