Effects of M2 antagonists on in vivo hippocampal acetylcholine levels

BrainResearchBulletin,Vol.41. No. 4, pp. 221-226, 1996 Copyright© 1996ElsevierScienceInc. Printedin the USA.All rightsreserved 0361-9230/96$15.00 + .00 ELSEVIER

PII S0361-9230(96)00180-3

Effects of M2 Antagonists on In Vivo Hippocampal Acetylcholine Levels MICHAEL J. STILLMAN,.1 BARBARA SHUKITT-HALE,* RACHEL L. GALLI,$ AHARON LEVY-IAND HARRIS R. LIEBERMAN:~2

*GEO-CENTERS, INC., Newton Center, MA 02159 1"Departmentof Pharmacology, Israel Institute for Biological Research, Ness Ziona, 70450 Israel 7tMilitary Performance and Neuroscience Division, United States Army Research Institute of Environmental Medicine, Natick, MA USA 01760-5007 [Received 6 November 1995; Revised 23 April 1996; Accepted 8 May 1996] ABSTRACT: There is evidence that muscarinic receptors of the M2 subtype are presynaptic autoreceptors that modify the release of acetylcholine (ACh) through a negative feedback mechanism. Blocking these receptors by selective antagonists may therefore lead to increased ACh release. This in vivo microdialysis study examined the effects of three M2 antagonists, AFDX 116, AF-DX 384, and AQ-RA 741, on hippocampal cholinergic neurotransmission. Drug (2, 4, 8, or 16/~M) or vehicle (Ringer's solution) was perfused via a microdialysis probe into the CA1 hippocampal region of conscious male Fischer 344 rats. Levels of ACh and choline were assessed by HPLC-EC. When the dose was expressed in I~ multiples, all drugs (except AQ-RA 741 at the two highest concentrations) were found to be on the same linear dose-response curve. Choline levels were not affected by drug administration. All three compounds elevated ACh levels in a similar Krnormalized dose-response fashion, strongly supporting the concept that the proposed presynaptic mechanism of action is indeed based on the same Mz receptor. Such elevations of ACh may not only improve performance on memory tasks, but may also have therapeutic advantages in conditions of cholinergic hypofunction, such as Alzheimer's disease. Copyright © 1996 Elsevier Science Inc.

postsynaptic receptors using cholinergic agonists [5,13]. These strategies have met with limited clinical success. An alternate method to elevate cholinergic neurotransmission involves manipulation of the presynaptic receptor. Evidence from pharmacological [7] and physiological [22] investigations suggests that the M2 receptor serves as a presynaptic autoreceptor, through which ACh regulates its own release into the synaptic gap. Subsequent investigations have demonstrated elevated ACh levels following M2 antagonist administration in vitro [7,14,17,31,32] and in vivo [25,29,33], supporting this hypothesis. We have previously demonstrated a linearly increasing effect of the M2 antagonist, methoctramine, on hippocampal extracellular ACh levels [29]. In the present study, these findings were extended to three additional M2 antagonists, AF-DX 116, AFDX 384, and AQ-RA 741. These drugs also have some activity for other subtypes of muscarinic receptors and therefore have varying degrees of selectivity. The main purpose of this research was to study the mechanism of hippocampal extracellular ACh regulation, through a dose-response examination of the effect of the above comparable compounds in awake and unrestrained rats. By analyzing the effect of these drugs, the postulated role of M2 receptors in ACh release can be further clarified. As in our previous study, solutions were delivered via the microdialysis probe, thereby eliminating the possible effects of blood-brain barrier permeability on drug potency.

KEY WORDS: Microdialysis, Muscarinic, Cholinergic System, Choline, Presynaptic Receptor, HPLC-EC.

INTRODUCTION

METHODS

The role of the central cholinergic system in learning and memory is well established, with many investigations reporting that central cholinergic hypofunction is correlated with impaired performance on various cognitive tasks, whereas improved performance is often observed with elevated cholinergic function [2,19,23]. Attempts to enhance cholinergic activity have generally taken one of the following approaches: (1) precursor manipulation, typically the use of choline-enriched diet [3]; (2) elevation of endogenous acetylcholine (ACh) levels via inhibition of acetylcholinesterase (ACHE) [15,18,30]; and (3) activation of

Animals Sixty male Fischer 344 rats (Charles River Labs, Kingston, NY), weighing 250-360 g at time of testing, were used for the present study. They were housed individually in wire mesh cages, and were maintained on a 12-h light/dark cycle (lights on at 0600 h).

Microdialysis Using a stereotaxic instrument, a guide cannula (Carnegie Medicin/Bioanalytical Systems [BAS], West Lafayette, IN) was

Michael J. Stillman, Ph.D., is currently a Medical Writer at Project House, Inc., One University Plaza, Suite 210, Hackensack, NJ 07601. 2To whom requests for reprints should be addressed.

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implanted onto the cranium, dorsal to the hippocampus of anesthetized rats (50 mg/kg pentobarbital, IP). Following at least a 48-h recovery period, a dialysis probe with a 2-mm semi-permeable membrane tip (CMA/10, BAS) was inserted into the guide cannula to monitor extracellular concentrations of ACh and choline. The probe membrane was placed in the CA I and DG regions of the hippocampus at stereotaxic coordinates AP - 3 . 8 : L = +1.6 with respect to bregma; and V 2.2-4.2 with respect to dura [24]. Using a microinjection pump (CMA/100, BAS), Ringer's solution ( 147 mM NaC1, 3.4 mM CaCI=, 4.0 mM KCI, pH 6.0) or drug in Ringer's solution perfused the probe at a rate of 2/xL/min. To permit detection of ACh, all dialysis solutions contained 10/JM neostigmine bromide (Sigma, St Louis, MO) to inhibit AChE activity. Throughout the experiments, rats could move li-eely in a transparent acrylic bowl (height = 35 cm; diameter = 39 cm). A liquid swivel mounted on a counterbalanced arm (CMA/120, B AS) was attached to the rim of the bowl, permitting the animal to freely rotate without becoming entangled in the microdialysis tubing. Following probe insertion into the guide cannula, each rat was allowed 30 rain to acclimatize to the sampling bowl. Individual 40 #L samples were subsequently collected every 20 rain, tk)r a total of 13 samples. All microdialysis sessions were conducted between 0800 and 1600 h.

STILLMAN ET AL.

with 4/sL Sudan Black. The animals were then sacrificed by CO2 inhalation. Their brains were removed, immediately frozen in dry ice, then stored at -90°C. Coronal sections (20/~,m) were cut at the level of the hippocampus, placed on microscope slides, and stained with cresyl violet for evaluation.

Statistical Analysis Hippocampal extracellular ACh and choline levels were the dependent variables for this study. Between-subjects analyses of wtriance (ANOVA) using dose as the grouping variable were employed for data analysis. Because of individual variations between baseline values of animals, change-from-baseline neurotransmitter levels were used in statistical analyses, thus allowing each rat to serve as his own control. Sample data were grouped into six blocks of two samples each. To allow for stabilization of neurotransmitter levels following probe insertion, data from the first sample were not included in the analysis. Blocks one and two (samples 2 - 5 ) were averaged to yield baseline values. These baseline values were subtracted from each subsequent block to yield change from baseline values; this change from baseline value served as the repeated measure. All statistical tests utilized a p < 0.05 criterion for hypothesis testing. Post hoc comparisons were performed, when appropriate, with Duncan's multiple range test.

Chromatography High performance liquid chromatography with electrochemical detection (HPLC-EC) was used to analyze the dialysates immediately following their collection. The mobile phase contained 40 mM phosphate buffer solution, pH 8.5, The electrode potential was set to +500 mV vs. Ag/AgCI, 5 nAFS. The chromatograph was fitted with an assay kit (MF-8910, BAS) consisting of an analytical and an enzyme column (ACHE plus choline oxidase). Peak integration and quantitation were performed via computerized software (INJECT, BAS) using an external standard for calibration.

Drug AF-DX 116, AF-DX 384, and AQ-RA 741 (Boehringer In gelheim, Ridgefield, CT) solutions were prepared in Ringer's solution in doses of 2, 4, 8, and 16 pM. (AF-DX 116 was dissolved in DMSO, with subsequent dilutions in Ringer's solution.) Ringer's solution served as vehicle.

Experimental Design Five rats were tested at each dose. After four baseline samples were taken, a liquid switch (CMA/111, BAS) was activated to change the perfusion fluid from Ringer's solution alone to drug in Ringer's solution. An additional four samples were then collected prior to switching back to Ringer's solution for live final samples. Thus, 13 samples were collected from each rat, with a total drug delivery time of 80 rain. It took approximately 16 min for the perfusing fluid to travel from the syringe to the hippocampus, with an additional minute to reach the collecting vial. Although the liquid switch was activated at the end of the fourth sample, the drug solution did not reach the brain until the end of the fifth sampling interval, continuing for the next 80 rain. Therel%re, changes in neurotransmitter concentration after switching perfusates would not begin to be reflected until this time.

Histology To verily probe placement, animals were anesthetized with methoxyflurane and the microdialysis probe site was perfused

RESULTS

Ety~cts ~/" M: Antagonists on Hippocampal Extracellular ACh Figure 1 illustrates the overall dose effect as changes from baseline of hippocampal extracellular ACh levels following AFDX 384, AF-DX 116, and AQ-RA 741 administration. All drugs increased significantly hippocampal extracellular ACh levels compared with vehicle. ACh levels in rats that received vehicle did not deviate significantly from baseline. AF-DX 384 elevated hippocampal ACh dialysate levels in a dose-dependent fashion (Fig. IA). A N O V A indicated significant effects of dose (F(4,20) = 4.78, p < 0.01) and time (F(3,60) 6.78, p < 0.001); the dose-by-time interaction was not significant. Post hoc testing revealed that ACh levels following 8 and 16/IM AF-DX 384 were significantly greater than levels following vehicle; moreover, ACh levels tbllowing 16 #M AF-DX 384 were significantly greater than those following 2 and 4 pM. AF-DX 116 clearly increased ACh levels, although the dose response effect was indeterminate (Fig. I B). A N O V A indicated significant effect of dose (F(4,20) = 2.93, p < 0.05) and time I F(3,60) = 9.42, p < 0.0001 ). Post hoc testing revealed that ACh levels li)llowing 2 and 8 #M AF-DX 116 were significantly greater than levels following vehicle. AQ-RA 741 produced an inverse U-shaped dose response curve, with the maximal effect at 4 #M, and intermediate effects at 2 #M and 8 #M (Fig. 1C). A N O V A indicated significant effects of dose (F(4,20) = 3.29, p < 0.05); however, neither the time nor the dose-by-time interaction effects were significant. Post hoc testing indicated that ACh levels following 4 pM AQRA 741 were significantly greater than levels following vehicle. Figure 2 illustrates the dose effect over time of the three M2 antagonists on hippocampal ACh. In order to more fully explore the data, individual between-subjects ANOVAs and post hoc tests were performed to determine dose effects at particular sampiing times. AF-DX 384 elevated hippocampal ACh dialysate levels from the time it was administered until the conclusion of lhe experiment (Fig. 2A). The lowest dose in this study, 2 pM, yielded the smallest increase in ACh, with greater effects occurring as the doses increased. The highest dose, 16/xM, produced

M2 A N T A G O N I S T S A N D HIPPOCAMPAL ACETYLCHOLINE

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FIG. 1. Dose effect of M2 antagonists on hippocampal extracellular ACh levels: (A) AF-DX 384; (B) AF-DX 116; (C) AQ-RA 741. Each bar represents the sum (+ SEM) of the four postbaseline data points (0-40, 40- 80, 80-120, 120-160). * = significantly different from vehicle Co < 0.05).

FIG. 2. Dose by time effect of M2 antagonists on hippocampal extracellular ACh levels: (A) AF-DX 384; (B) AF-DX 116; (C) AQ-RA 741. Each point represents the mean ( - SEM) of 2 × 40 #L dialysates (20 min each). * = significantly different from vehicle Co < 0.05).

a robust increase in ACh levels, with its maximum occurring 0 40 min post drug onset. Overall, increased ACh was observed initially 0 - 4 0 rain after drug onset (F(4,20) = 50.02, p < 0.01) and was maintained throughout the experiment, at 4 0 - 8 0 min post drug onset (F(4,20) = 3.83, p < 0.05), at 8 0 - 1 2 0 min (even though drug delivery was terminated at 80 min) (F(4,20) = 4.59, p < 0.01), and at 1 2 0 - 1 6 0 min post drug onset (F(4,20) = 3.46, p < 0.05). Post hoc testing revealed that 16 #M was the only dose significantly different from vehicle at 0 - 4 0 and 4 0 - 8 0 min post drug onset, whereas both 8 and 1 6 / z M AF-DX 384 were significantly different from vehicle at 8 0 - 1 2 0 and 1 2 0 - 1 6 0 min. For the lowest dose of AF-DX 116 (2 #M), the maximal elevation of ACh was observed during drug administration ( 0 - 8 0 min), with a slight decline after 8 0 - 1 6 0 min (Fig. 2B). In contrast, 4 and 8 #M AF-DX 116 produced steeper increases to and decreases from the 4 0 - 8 0 min value, ultimately approximating basal levels. ACh levels following 16 #M initially increased, then fell to near basal levels. The only significant difference from vehicle was at 1 2 0 - 1 6 0 min post drug onset (F(4,20) = 30.06, p < 0.05), where post hoc testing revealed that ACh was increased following 2 #M. Even though elevations in ACh were observed following AQRA 741 relative to vehicle for all doses and times (Fig. 2C), none of these differences achieved statistical significance.

The tested compounds differ in their affinity and selectivity to the M2 receptor (Table 1). It may be possible to account for the varying in vivo dose-response profiles of these compounds based on their respective affinities for the M2 receptor in vitro. The data for the various drugs were therefore reanalyzed by replacing molar doses with multiples of the K~ molar values, to permit a normalized comparison across the four different drugs. A plot of Ki multiples versus ACh changes from baseline is shown in Fig. 3 (Methoctramine data [29] are included for comparison). The four abscissa values for each drug represent its doses of 2, 4, 8, and 16 #M. Based on relative affinity concentrations, Fig. 3 shows that all drugs were found to be on a similar "normalized" dose-reTABLE 1 Mz ANTAGONIST AFFINITIES AND K~ VALUES. SELECTIVITY VALUES FOR THE Mz RECEPTOR WERE CALCULATED BY DIVIDING K~(M~) BY K~(M2). DATA ARE FROM [10,11].

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STILLMAN ET AL.

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effect on ACh levels as a function not of the actual doses, but Eather of the doses as K~ multiples. It appears that the dose range employed in this study ( 0 - 1 6 / a M ) was below the linear range for AF-DX 116: specifically, the maximum dose of 16 /aM is below the minimum dose (2/aM) for both AF-DX 384 and AQRA 741, and similar in effectiveness only to the lowest dose of methoctramine. The results obtained in the current study seem to reflect fluctuations at the low end of the dose-response function, which are within the experimental error of the technique employed. A more definite dose-response relationship would likely be obtained at higher doses of AF-DX 116, better approximating K, unultiples of the other Me antagonists. AQ-RA 741 presents a specific deviation because, at the two highest concentrations, the dose-response curve changes to a downward slope. It is possible that the compound activates mainly Me receptors at the two lower doses in this study, whereas other receptor subtypes are activated at the higher doses, resulting in shutting down of ACh release. One possibility is that excitaIo U M~ receptors are being blocked to a lesser degree by M~ administration [10,17], perhaps because of the considerable similarity in their binding profiles [6l. Bhattacharya and Sen [4] noted receptor specificity loss at higher doses for the putative Me receptor agonist carbachol. If M~ receptors, which are primarily postsynaptic [20,27], are activated at these higher doses, they might cause a decrease in released ACh.

E.ffects qf M: Antagonists on Hil)pocampal Extracellular Choline Choline levels in rats that received vehicle decreased significantly from baseline (F(3,12) = 4.18, p < 0.05). Time effects for all drugs were statistically significant [AF-DX 384: (F(3,60) = 68.89, p < 0.0001): AF-DX 116: (F(3,60) = 730.(19, p < 0.0001); AQ-RA 741: (F(3,60) - 18.35, p < 0.0001 l]. Dose and dose-by-time interaction effects for all drugs on hippocampal extracellular choline levels failed to achieve statistical significance. The decline in hippocampal extracellular choline continued throughout the experiment and remained unaltered by changes in dose or drug (Fig. 4).

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it has been postulated that synaptic ACh binds to presynaptic Me receptors, activating a negative feedback mechanism to yield a decrease in ACh release [20,21,27l. The current experiments demonstrated that hippocampal extracellular ACh levels of awake, unrestrained rats were elevated following intrahippocampal application of the M2 antagonists AF-DX 384, AF-DX II 6, and AQ-RA 741, within the dose range utilized. AF-DX 384 produced the greatest and most sustained increase in hippocampal ACh. Over all doses tested, AF-DX 384 was also more efficacious than AF-DX 116, with the maximal increase of the former approximately three times greater than that of the latter. This is consistent with the affinity relationship of the two compounds because AF-DX 384 was developed as a successor to its predecessor, AF-DX 116, with a higher affinity for the Me receptor

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FIG. 4. Dose by time effect of Me antagonists on hippocampal extracelhdar choline levels: (A) AF-DX 384; (B) AF-DX 116; (C) AQ-RA 741. Each point represents the mean ( - SEM) of 2 x 40 ,uL dialysates (20 rain each).

M2 ANTAGONISTS AND HIPPOCAMPAL ACETYLCHOLINE

The present study corroborated others that showed elevated cholinergic function following M2 antagonist administration in vitro. Enhanced ACh release from hippocampal; striatal, and frontal cortical brain slices was observed following application of the M2 antagonist AF-DX 116, but not after pirenzepine [17]. Enhanced release of [3H]ACb from cerebral cortex in vitro was observed following treatment with AF-DX 116 [32]. Torocsik and Vizi [31] showed that the M2 agonist oxotremorine decreased [3H]ACh release from rat frontal cortex in vitro, whereas methoctramine antagonized this effect, in agreement with their respective designations as M2 agonist and M2 antagonist. Hoss and colleagues [14] demonstrated that M2 antagonists not only regulate ACh release in forebrain synaptosomes, but also produce corresponding behavioral changes, including piloerection and hyperreactivity. These symptoms and neurochemical changes were not observed following pirenzepine administration. The present results are in agreement with other investigations examining the effects of M2 antagonists on central cholinergic neurotransmission in vivo. Quirion, Richard, and Wilson [25] reported increased dialysate ACh levels following cortical administration of AF-DX 116, AF-DX 384, and AQ-RA 741. Our laboratory demonstrated elevated levels of hippocampal ACh following administration of the M2 antagonist methoctramine [29]. Peripheral administration of BIBN-99, a novel M2 antagonist, increased hippocampal dialysate ACh levels in rats [26,33]. The decline in choline over time observed in this research has been reported previously [9,16,28]. However, because choline is both a precursor and metabolite of ACh, as well as a component of cellular membrane phospholipids, dialysate choline data are difficult to interpret at the present time. The present study employed the AChE inhibitor neostigmine in the perfusate fluid to permit detection of ACh. Although it may be argued that neostigmine enhances the negative feedback modulation of ACh release, the requirement of an AChE inhibitor in the perfusates reflects current detection methodology, as its omission results in undetectable levels of ACh [8]. Future improvements in detector electrochemistry will in all probability eliminate the need for cholinesterase inhibitors. In summary, the results of the present study demonstrated that M2 antagonists increase hippocampal extracellular ACh levels in conscious rats. The data from this study support the hypothesis that Me receptors in the rat hippocampus function as inhibitory autoreceptors because they are apparently blocked by a series of M2 antagonists including AF-DX 384, AF-DX 116, AQ-RA 741 and methoctramine, suppressing this inhibitory activity, and leading to enhanced hippocampal cholinergic transmission. Furthermore, the fact that all four M2 antagonists appear on the same "normalized" dose-response curve strengthens the role of the subset of muscarinic receptors defined as the M2 subtype in ACh release, because these M2 Ki values were obtained independently from this study. The development of M2 antagonists with improved selectivity, potency, and, most importantly, the ability to penetrate the blood-brain barrier is currently underway. Such compounds could alleviate conditions of cholinergic hypofunction that occur in normal aging, as well as provide potential treatments of cholinergic disorders such as Alzheimer's disease [ 10]. The relationship between Alzheimer's disease and M2 receptor density is unclear at present. Mash, Flynn, and Potter [20] noted a decrease of cortical M2 receptor density in Alzheimer's disease. Aubert and colleagues [1 ] recently reported increased M2 binding sites in aged cognitively impaired rats. Differences in binding assay conditions (e.g., nonselective binding agents) may explain such discrepancies. The nature of such studies, however, precludes a causal relationship between changes in binding sites and disease

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state. An increase in M2 receptor density might represent the means through which impairments in cholinergic function are manifest by the disease. Alternatively, decreases in M2 receptor density may reflect compensatory mechanisms to alleviate cholinergic hypofunction. Perhaps this question will be resolved in future investigations. It is encouraging that BIBN-99 was recently shown to be effective both at elevating bippocampal ACh levels and at impairing cognitive deficits in rats [26]. ACKNOWLEDGEMENTS We thank Boehringer Ingelheim for supplying AF-DX 116, AF-DX 384, and AQ-RA 741. Grateful appreciation is extended to Bryan P. Coffey, SGT Jennifer Seymour, and SPC Jason Irwin for their assistance in data collection. In conducting the research described in this report, the investigators adhered to the "Guide for the Care and Use of Laboratory Animals," as prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. The views, opinions, and/or findings contained in this report are those of the authors and should not be construed as an official Department of the Army position, policy, or decision, unless so designated by other official documentation.

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