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Changes in cerebral neurotransmitters and metabolites induced by acute donepezil and memantine administrations: A microdialysis study
Brain Research Bulletin 69 (2006) 204–213
Changes in cerebral neurotransmitters and metabolites induced by acute donepezil and memantine administrations: A microdialysis study E. Shearman a , S. Rossi a , B. Szasz b , Z. Juranyi c , S. Fallon a , N. Pomara a , H. Sershen a,∗ , A. Lajtha a b
a Nathan Kline Institute, Orangeburg, NY 10962, USA Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest, Hungary c Egis Pharmaceuticals, Budapest, Hungary
Received 7 September 2005; received in revised form 1 November 2005; accepted 1 December 2005 Available online 21 December 2005
Abstract Cholinesterase inhibitors including donepezil, rivastigmine, and galantamine and the N-methyl-d-aspartate (NMDA) antagonist, memantine are the medications currently approved for the treatment of Alzheimer’s disease (AD). In addition to their beneficial effects on cognitive and functional domains typically disrupted in AD, these agents have also been shown to slow down the emergence of behavioral and psychotic symptoms associated with this disease. However, the underlying mechanisms for these therapeutic effects remain poorly understood and could involve effects of these medications on non-cholinergic or non-glutamatergic neurotransmitter systems respectively. These considerations prompted us to initiate a series of investigations to examine the acute and chronic effects of donepezil (Aricept (±)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4piperidinyl]methyl]-1H-inden-1-1 hydrochloride and memantine (1-amino-3,5-dimethyladamantane hydrochloride C12 H21 N·HCl)). The present study focuses on the acute effects of donepezil and memantine on brain extracellular levels of acetylcholine, dopamine, serotonin, norepinephrine and their metabolites. We assayed changes in the ventral and dorsal hippocampus and the prefrontal and medial temporal cortex by microdialysis. Memantine resulted in significant increases in extracellular dopamine (DA), norepinephrine (NE), and their metabolites, in the cortical regions, and in a reduction of DA in the hippocampus. Donepezil produced an increase in extracellular DA in the cortex and in the dorsal hippocampus. Norepinephrine increased in the cortex; with donepezil it increased in the dorsal hippocampus and the medial temporal cortex, and decreased in the ventral hippocampus. Interestingly both compounds decreased extracellular serotonin (5HT) levels. The metabolites of the neurotransmitters were increased in most areas. We also found an increase in extracellular acetylcholine (ACh) by memantine in the nucleus accumbens and the ventral tegmental area. Our results suggest both region and drug specific neurotransmitter effects of these agents as well as some similarities. We conclude that drugs influencing cognitive mechanisms induce changes in a number of neurotransmitters with the changes being both region and drug specific. Release and metabolism are altered and extracellular neurotransmitter levels can be increased or decreased by the drugs. Other studies are in progress to determine the pharmacological effects associated with chronic treatment with these compounds, which may be more pertinent to the clinical situation in which patients take these medications for months or years. © 2006 Elsevier Inc. All rights reserved. Keywords: Neurotransmitter release; Donepezil; Memantine effects; Regional heterogeneity of drug effects
1. Introduction Many studies indicate the major role of neurotransmitter systems in cognitive processes. There are related findings that compounds, such as nicotine, that affect learning, induce changes in
∗
Corresponding author. E-mail address: [email protected] (H. Sershen).
0361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2005.12.001
the extracellular levels, or release in a number of neurotransmitters. Clearly, a complex set of neurotransmitters participates in the mechanism of learning and changes in any one of these may influence it. Similarly, it can be expected that various drugs that influence cognitive processes act at least in part by altering neurotransmitters and such alterations may be drug specific. However, not much has been established on the specificity of the actions of these drugs on mechanisms involved in learning and memory. To better evaluate the role and interactions
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of the different neurotransmitters in cognitive processes, it is important to examine the effect of compounds in addition to nicotine that effect learning or memory. In the present paper we report the changes of extracellular neurotransmitters in selected brain areas induced by acute administration of donepezil and memantine, two compounds that are presently widely used in the treatment of Alzheimer’s disease (AD) patients and which are believed to exert their therapeutic effects by two distinct neurotransmitter mechanisms; donepezil affecting the cholinergic and memantine, the glutamatergic system. Our hypothesis is that these drug’s well-established pharmacological actions may be associated with changes in a number of other neurotransmitters and that the pattern of changes is drug specific and regionally heterogeneous. As part of on-going studies to examine the role of neurotransmitters in cognitive mechanisms, we recently assayed nicotineinduced changes in several brain areas in rats after high- and low-dose nicotine. We assayed the levels of DA, NE, 5HT and their metabolites: dihydroxyphenylacetic acid (DOPAC) homovanillic acid (HVA), hydroxymethoxyphenylglycol (MHPG), and 5-hydroxyindolacetic acid (5-HIAA). These were measured via microdialysis in conscious freely moving adult male rats. The areas studied were the dorsal and ventral hippocampus, the prefrontal and medial temporal cortex, the ventral tegmental area and the nucleus accumbens. Some of these areas may play a role in reward processes, but it is most likely that reward and learning or memory mechanisms are interrelated and connected. At higher nicotine concentrations (0.5 mg/kg s.c.) in most areas tested, the three neurotransmitters and their metabolites were increased and we concluded that nicotine administration causes an alteration in the level, and in the metabolism of DA, NE and 5HT in brain areas involved in cognitive processes and that such changes are partly responsible for the effect of nicotine on learning [21]. At lower nicotine concentrations the effects were somewhat different such as 5HT was decreased rather than increased in some areas. The changes indicate significant regional variation and in sensitivity to nicotine-induced changes between areas of cognitive versus reward functions [15]. Nicotine-induced effects were inhibited by nicotinic, muscarinic and dopaminergic antagonists [16] indicating that the induced changes involve direct and indirect actions, interactions of receptors with the mechanisms involved being regionally heterogeneous. The nicotineinduced changes in transmitter levels observed in areas of significant reward functions differed in some respects from the patterns of changes induced and the receptors involved in the effects seen with cocaine [25]. We hope that by extending this line of investigation to comparing the effects of several compounds (in the present paper that of donepezil and memantine), it will better help us in elucidating the underlying mechanisms for the therapeutic effects of these agents in AD and identifying the role of neurotransmitters in cognitive processes. Here, we report on their effect on DA, NE and 5HT levels and on acetylcholine (ACh)—in specific brain areas. Work is in progress to compare their effect and that of nicotine on other transmitters.
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2. Methods Experiments were conducted using adult male Sprague Dawley rats (250–350 g) bred and raised in our animal facility. All studies were conducted in accordance with the principles and procedures outlined in the NIH guide for the care and use of laboratory animals, and were approved by the animal care committee of the institute.
2.1. Microdialysis As was done in our previous studies [16], rats were anesthetized with chloral hydrate (400 mg/kg; i.p., Sigma) and placed in a Kopf stereotaxic apparatus. A small hole was drilled in the skull directly above the sites selected for microdialysis. After puncturing the dura, two guide cannulas, 0.64 mm o.d. (Carnegie Medicine, MA) were lowered above the two selected sites in the brain and fixed to the skull with dental cement and two mounting screws. The stereotaxic coordinates with respect to the bregma and the surface of the skull were used according to the Atlas for Rat Brain (VH; AP: −5.0 ML: 4.8 DV: −6.0, DH; AP: −3.8 ML: 2.4 DV: −2.0, MTC; AP: −3.8 ML: 6.0 DV: −4.0, PFC; AP: +3.2 ML: 3.2 DV: −1.6, NAcc Shell; AP: +1.6 ML: 0.7 DV: −5.0, VTA; AP: −4.8 ML: 0.9 DV: −7.1). After surgery the animals were allowed to recover for a 2-day period. On the day of the experiment animals were placed in a BAS Rat Turn (Bioanalytical Systems; Lafayette, ID) and were attached via a plastic collar to a lever arm. A 1 or 2 mm microdialysis probe was inserted into the guide cannula 2 h before the start of the experiments (Carnegie Medicine, MA), depending on the brain area: 1 mm for the ventral tegmental area and nucleus accumbens, and 2 mm for the hippocampal and cortical regions. Ringer’s solution (125 mM NaCl, 3.3 mM KCl, 2.4 mM Mg2 SO4 , 1.25 mM KH2 PO4 , 1.85 mM CaCl2 ) was perfused at a rate of 1.0 L/min using a BAS baby bee syringe pump (Bioanalytical Systems; Lafayette, ID) to drive the perfusate. Following a 2-h waste period, baseline dialysate was collected at 30-min intervals for 120 min. After baseline stabilization, a subcutaneous injection of either memantine (20 mg/kg; Sigma; St. Louis, MO) or donepezil (2.5 mg/kg; kindly provided by Dr. Nunzio Pomara) was administered, and sample collection continued for the remaining 180 min. In experiments where ACh was assayed, memantine was injected intraperitoneally.
2.2. Testing of the microdialysis probe Microdialysis probes were tested for performance before use. The probe was immersed in a standard solution containing all relevant compounds in concentrations analogous to the expected extracellular fluid and the probe was perfused at a rate of 1 L/min with Ringer’s solution. The concentrations of catecholamines in the dialysates were 20–25% of those in the standard solution, indicating an approximately 23% efficiency of extraction into the dialysate.
2.3. Histological analysis Following each experiment, animals were sedated with chloral hydrate (Sigma; St. Louis, MO), decapitated, and the brains were taken out for verification of probe placement. Postmortem brains of rats were processed histologically in order to verify the location of the dialysis probes and guide cannula. Brains were sectioned, and probe placement was verified by visual analysis.
2.4. Assay of neurotransmitters For the catecholamines and their metabolites, microdialysis samples were analyzed using an HPLC system with electrochemical detection (ESA CoulArray Detector Model 5600A). The mobile phase (ESA 70-1332 Mobile Phase) contained 75 mM sodium dihydrogen phosphate, 1.7 mM 1-octanesulfonic acid sodium salt, 100 L/L triethylamine, 25 M EDTA, and 10% acetonitrile (pH 3). The pump was an ESA Pump 582. Dialysates were injected using a temperaturecontrolled autosampler (ESA Model 542) kept at 4 ◦ C. Compounds were separated on an ESA Microdialysis MD-150 Analytical column (3.2 mm, 15 cm). The cell potentials were set at −175 mV and +250 mV. Average elution time for the compounds, in minutes, are as follows: MHPG (2.10), NE (2.65), DOPAC (3.70), DA (4.6), 5HIAA (5.5), HVA (7.70), and 5-HT (10.0).
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For the determination of ACh, samples were separated in an HPLC system (BAS 200A) equipped with a BAS microbore ACh/Ch analytical drum and ACh/Ch IMER post-column, converting it to H2 O2 . Detection was by BAS peroxidase cross-flow electrode set at −100 mV. Mobile phase is 50 mM sodium phosphate, pH 8.5 ± 0.5, run at 0.13 mL/min. The limit of detections using a new IMER was 1 fmol. The sensitivity was tested with ACh and H2 O2 standards.
2.5. Statistical analysis To determine whether the memantine (20 mg/kg) or donepezil (2.5 mg/kg) subcutaneous injection altered neurotransmitter levels, the first four samples were expressed as baseline and the changes in the remaining samples, 6–10, were expressed as percentage change in baseline (average of the first four time points; mean ± S.E.M., n = 10). Significance was determined using a paired samples ttest (p < 0.05).
3. Results Memantine and donepezil were examined to determine whether or not they have an effect on neurotransmitters found in areas assumed to play major roles in reward mechanisms. Memantine was found to significantly increase the level of extracellular acetylcholine in the ventral tegmental area and as well as in the nucleus accumbens (Fig. 1). Both memantine and donepezil seem to affect DA and 5HT in the nucleus accumbens and ventral tegmentum; as our preliminary assays found significant increases in the DA metabolites DOPAC and HVA and the 5HT metabolite 5HIAA (Table 1) (DA and 5HT level changes were not significant). In the hippocampus, donepezil administration caused changes in each of the three neurotransmitters assayed. In the dorsal hippocampus, DA and NE increased, while in the ventral hippocampus NE and 5HT were decreased, hence changes in the dorsal area were very different from those in the ventral area (Fig. 2). The metabolites of the three neurotransmitters were increased with donepezil. In the dorsal hippocampus, the DA
Table 1 Effect of memantine and donepezil on metabolite levels Memantine
DOPAC HVA 5HIAA
Donepezil
NAc
VTA
NAc
VTA
237 ± 13 209 ± 19 213 ± 28
123 ± 10 146 ± 13 205 ± 66
150 ± 17 120 ± 13 130 ± 11
130 ± 12 142 ± 17
Memantine, 20 mg/kg; Donepezil, 2.5 mg/kg. Values are the averages of assays at 1–3 h after the intraperitoneal injection of the drugs (five animals, five time points in each). Values given are a percent of control (extracellular levels in absence of drugs taken as 100).
metabolites DOPAC and HVA, the NE metabolite MHPG, and the 5HT metabolite 5HIAA were each increased, whereas in the ventral hippocampus HVA, MHPG and 5HIAA were increased. The time course of these changes differed; with HVA increase becoming less, and 5HIAA increases intensifying over longer time periods (Fig. 3). It is of interest that while in the ventral hippocampus, NE and 5HT decreased (Fig. 2); their metabolites MHPG and 5HIAA respectively increased (Fig. 3). Donepezil also altered the extracellular level of the three neurotransmitters in the cortex, DA and 5HT were decreased and NE was increased in the medial temporal cortex, while in the prefrontal cortex only 5HT was decreased. Of the metabolites: DOPAC, HVA and MHPG, only increases were found in both cortical areas (Fig. 4). The changes following memantine administration differed somewhat from the donepezil-induced changes. In the dorsal hippocampus DA and 5HT decreased, and in the ventral hippocampus, DA decreased, while MHPG increased (Fig. 5). In the cortical areas tested, memantine increased DA and NE and decreased 5HT in both areas. Of the metabolites MHPG was increased in both areas; whereas DOPAC and HVA were increased in only the prefrontal cortex (Fig. 6). 4. Discussion
Fig. 1. Effect of memantine (20 mg/kg i.p.) on acetylcholine release as a function of time in the nucleus accumbens (䊉) and ventral tegmental area (). Data is expressed as percent change from baseline (average of the first four time points; mean ± S.E.M.). Each sample was collected at a flow rate of 1 L/min for 30 min. The arrow indicates the time of injection (20 mg/kg memantine). Significance was determined using paired samples t-test (p < 0.05 for the effect of Memantine, n = 5).
The aim of our experiment was to study the effects of acute administration of drugs (donepezil and memantine) used in treatment of Alzheimer’s disease on extra-neuronal concentration of different transmitters and their metabolites. Here, we studied six brain regions, assayed four neurotransmitters with two drugs, at one concentration, from which, we can draw a number of tentative conclusions. It is clear that acute doses of donepezil and memantine alter not one, but a number of neurotransmitters irrespective of whether they are primarily cholinergic or glutamatergic compounds. Most likely some of these effects are direct through cholinergic or glutamatergic receptors on noncholinergic or non-glutamatergic cells, but they are also indirect through the released neurotransmitters. These effects can be seen in many brain areas but the changes are regionally heterogeneous and the pattern is different with different drugs. The time course of changes is similarly heterogeneous. Such complex and drug specific patterns have been observed with other drugs such as nicotine or cocaine. The drugs affect neurotransmitter release and also neurotransmitter metabolism. The increase
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Fig. 2. Effect of donepezil (2.5 mg/kg s.c.) on neurotransmitter concentrations as a function of time in the dorsal (䊉) and ventral hippocampus (). DA, dopamine, NE, norepinephrine, and 5-HT, serotonin. Data is expressed as percent change from baseline (average of the first four time points; mean ± S.E.M.). Each sample was collected at a flow rate of 1 L/min for 30 min. The arrow indicates the time of injection (2.5 mg/kg donepezil). Significance was determined using paired samples t-test (p < 0.05 for the effect of Donepezil, n = 5).
in metabolites indicates the increased metabolism. It seems in some instances the effect on metabolism is greater than it is on release and the extracellular level of the neurotransmitters is decreased. We found numerous interactions of receptor systems in nicotine-induced alterations of extracellular neurotransmitters. The dopaminergic and cholinergic systems interacted [17]. Nicotine induced changes in glutamate that in turn affected DA release [27]. Specific nicotine receptors were involved in nicotine-induced NE release [20] and nicotine altered the extracellular levels of a number of amino acids [28]. Clearly both cholinergic and glutamatergic receptors are present on a number of different neurons; among them are DA, NE, and 5HT neurons. The distribution of such neurons is heterogeneous. It is expected, therefore, that cholinergic or glutamatergic compounds affect numerous neurotransmitters in a regionally heterogeneous manner. There are many questions that need to be answered. Are other compounds, particularly other neurotransmitters or neuromodulators altered, also in regionally heterogeneous drug specific
manners? We are examining presently changes in amino acid neurotransmitters. It is also important to assay which changes in neurotransmitters have functional importance and which of the changes influence different cognitive mechanisms or behavior and are their role and effect quantitatively different? Using antagonists to inhibit the extracellular changes in one neurotransmitter, may answer some of these questions. We found previously that the effects of nicotine on neurotransmitter levels are concentration dependent: at higher nicotine concentrations it stimulates the release of 5HT, increasing its level, at lower concentrations, it stimulates the metabolism of 5HT, decreasing its level [21,15]. It is possible that similarly the effects of donepezil and memantine would quantitatively or in specificity be different at different doses. There are several interesting observations reported about compounds of possible use for Alzheimer’s disease. Donepezil belongs to the compounds that inhibit acetylcholinesterase, and therefore, its effects on cerebral ACh levels were primarily studied. Studies using microdialysis in vivo in the hippocampus show
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Fig. 3. Effect of donepezil (2.5 mg/kg) on metabolite concentrations as a function of time in the dorsal (䊉) and ventral hippocampus (). DOPAC, 3,4dihydroxyphenylacetic acid, MHPG, 3-methoxy-4-hydrophenylglycol, HVA, homovanillic acid, and 5-HIAA, 5-hydroxyindole acetic acid. Data is expressed as percent change from baseline (average of the first four time points; mean ± S.E.M.). Each sample was collected at a flow rate of 1 L/min for 30 min. The arrow indicates the time of injection (2.5 mg/kg donepezil). Significance was determined using paired samples t-test (p < 0.05 for the effect of Donepezil, n = 5).
doses of 0.65 and 2 mg/kg i.p. in rats causes a 4 and 12 times increase in ACh levels [7]. A linear relationship between enzyme inhibition and ACh increase has also been illustrated [9]. In the ventral hippocampus, ACh levels were increased 15, 76, 121% respectively by 2, 5 or 10 mg/kg, donepezil p.o. [5]. Effects noted in the cortex after chronic treatment of 21 days (1.5 mg/kg twice a day p.o.) include the increase of 75% in rats [19]. Increases also were shown in vivo in the prefrontal cortex of monkeys. Donepezil effects were more pronounced in young as opposed to aged monkey brains [33]. In the cortex, donepezil, taurine and another ACh esterase inhibitor NXX-066 increased ACh several fold possibly through muscarinic M1 receptor antagonism [22]. A selective muscarinic M2 antagonist SCH5779 increased ACh release in the hippocampus, cortex and striatum indicating the important role of muscarinic cholinergic receptors in donepezil effects. The primary effect of memantine on DA was assayed. In anesthetized rats increase of extracellular DA in the striatum using voltammetry [2] and an increase of DA in the prefrontal cor-
tex and striatum using microdialysis [24] was found. Another study [6] in awake animals did not find elevated DA in the prefrontal cortex although the level of its metabolites: DOPAC and HVA were elevated. In the striatum, DOPAC increase could only be observed in the absence of anesthesia, DOPAC was also increased in the nucleus accumbens [2]. Only a few studies examined the effect of these drugs on other neurotransmitters and it seems various related drugs have different effects. In the cortex of awake rats using microdialysis, 2.0 mg/kg s.c. donepezil caused a 200% increase of acetylcholine, 80% increase of DA and 100% increase of NE. A 0.5 mg/kg dose caused ACh to increase 200%, NE 50%, and DA was not affected and neither dose influenced 5HT levels [4]. As opposed to donepezil, another acetylcholinesterase inhibitor, rivastigmine in freely moving rats significantly decreased NE concentrations in the hippocampus and increased HVA, while DA was not changed [32]. The cholinesterase inhibitor neostigmine was also shown to increase hippocampal ACh level with a resulting increase in release of NE, which is potentiated by the
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Fig. 4. Effect of donepezil (2.5 mg/kg) on neurotransmitter and metabolite concentrations as a function of time in the medial temporal (䊉) and prefrontal cortex (). Data is expressed as percent change from baseline (average of the first four time points; mean ± S.E.M.). Each sample was collected at a flow rate of 1 L/min for 30 min. The arrow indicates the time of injection (2.5 mg/kg donepezil). Significance was determined using paired samples t-test (p < 0.05 for the effect of Donepezil, n = 5).
nicotinic antagonist mecamylamine, but blocked by the muscarinic antagonist atropine [8]. Another acetylcholinesterase inhibitor CHF2819 developed for the treatment of Alzheimer’s disease in the hippocampus decreased DA levels, increased 5HT
and did not effect NE levels. The amino acids tested: glutamate, aspartate, GABA, taurine, arginine and citrullin, were also not affected [30]. The combined increase of ACh and 5HT by this compound may be of therapeutic use in Alzheimer’s disease
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Fig. 5. Effect of memantine (20 mg/kg s.c.) on neurotransmitter and metabolite concentrations as a function of time in the dorsal (䊉) and ventral hippocampus (). Data is expressed as percent change from baseline (average of the first four time points; mean ± S.E.M.). Each sample was collected at a flow rate of 1 L/min for 30 min. The arrow indicates the time of injection (20 mg/kg memantine). Significance was determined using paired samples t-test (p < 0.05 for the effect of Memantine, n = 5).
patients who have a depressive syndrome [31]. Rivastigmine but not CHF2819 decreased glutamate, taurine, arginine and citrullin levels—aspartate was not affected [29]. In rat hippocampal and human cortical slices, galantamine increased GABA release and synaptic transmission, which was not increased by donepezil or rivastigmine [18]. In mouse brain striatal slices, low concentrations of donepezil (1 mM) and galantamine (50 mM) increased action potential-dependent DA release [36]. Another type of cholinergic compound that binds to ACh receptors with higher affinity than nicotine is SIB1533A. This compound influenced neurotransmitter release (examined in brain slices): DA release from striatum, olfactory tubercle and prefrontal cortex, and NE release from hippocampus and prefrontal cortex was inhibited by SIB1533A. It also inhibited NMDA-evoked ACh release from striatum. The effect of SIB1533A was transmitter and region specific and involved multiple receptor affinities and release of several neurotransmitters [14]. Donepezil and other cholinesterase inhibitors also have direct effects on ACh receptors, which is not dependent on their effect on
esterase. Donepezil was shown to modulate nicotinic receptors on dopaminergic neurons [3]. Both donepezil and galantamine elevated the number of nicotinic receptors in the hippocampus and the cortex after chronic treatment altering synaptic plasticity at levels analogous to that used in Alzheimer’s patients [1]. Among cholinesterase inhibitors, galantamine, but not tacrine, acted as an allosterically potentiating ligand sensitizing nicotinic receptors [10]. This allosteric sensitization affects other neurotransmitter systems modulated by nicotinic receptors effecting glutamate release and serotonergic neurotransmission [10]. Memantine effects on release evoked by the glutamate receptor agonist NMDA were studied. In thin layers of isolated rat hippocampal or cortical nerve endings NMDA-evoked NE release was inhibited by memantine [13]. In rat striatal slices memantine inhibited NMDA-evoked DA, ACh and GABA release. It only weakly inhibited NMDA-evoked spermidine release [11]. In synaptosomes prepared from human brain samples removed during neurosurgery, the NMDA elicited NE release was also inhibited by memantine [12]. In these experiments memantine
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Fig. 6. Effect of memantine (20 mg/kg) on neurotransmitter and metabolite concentrations as a function of time in the medial temporal (䊉) and prefrontal cortex (). Data is expressed as percent change from baseline (average of the first four time points; mean ± S.E.M.). Each sample was collected at a flow rate of 1 L/min for 30 min. The arrow indicates the time of injection (20 mg/kg memantine). Significance was determined using paired samples t-test (p < 0.05 for the effect of Memantine, n = 5).
acted as an NMDA antagonist. In isolated rat brain synaptosomes, memantine inhibited both the high and low affinity uptake of 5HT, it also inhibited the binding of 5HT to isolated nerve-ending membranes [35]. The levels of DA, NE and 5HT in the striatum and lumbar spinal cord of rats and of spastic rats was dose-dependently decreased by memantine [23]. Comparing our results with donepezil and memantine, we can find significant differences between their induced changes, especially with DA. In the dorsal hippocampus, donepezil increased and memantine decreased DA. In the medial temporal cortex, donepezil decreased while memantine increased DA. Donepezil in the dorsal hippocampus increased, and in the ventral hippocampus
decreased NE, while memantine in the hippocampus had no effect on NE. In the prefrontal cortex, donepezil had no effect on NE while memantine increased it. The fact that CHF2819, a cholinesterase inhibitor, unlike donepezil in the present study, decreased DA levels and increased 5HT levels in the hippocampus [30], indicates differences in effects among cholinesterase inhibitors. It is not easy to compare the various findings on neurotransmitter release of memantine and donepezil with finding on related drugs as the experimental conditions of various studies differ significantly. It is likely that mechanisms operating in the living brain are significantly altered in various preparations
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such as synaptosomes or brain slices and anesthesia also affects some mechanisms of stimulatory function. In addition, besides classical synaptic transmission responses, the role of nonsynaptic receptors are clearly involved in the observed responses, as shown by the diverse regional and time-dependent interactions of neurons equipped with different transmitters, and support their importance as targets for drug development [34]. Our work clearly indicates that each drug affects several neurotransmitters and such effects can be increases or decreases. There are significant differences among the effects of the drugs including differences among cholinesterase inhibitors. The effects show regional heterogeneity and are concentration dependent, as well as showing that regional heterogeneity and concentration dependence are also drug-dependent. Several receptors and the interactions of such receptors participate in the effects, some which are direct on receptors located on various neurons and some which are affected indirectly by the released or reduced level of neurotransmitters. Such interactions were examined in some detail, showing that changes in the ventral tegmental area affected those in the nucleus accumbens, in which a number of receptors in the ventral tegmental area mediated nicotine-induced DA changes in the nucleus accumbens [26]. The relevance of the current study and some of the aforementioned literature, which has been limited to the effects of single acute doses for the clinical situation in which patients take these medications for months or years, is not known and more studies are in progress to examine this question. Similarly, donepezil and memantine together have been demonstrated to have a better efficacy in the treatment of AD than donepezil alone and more studies will need to be conducted to clarify mechanisms for this observation. Acknowledgement Research described in this article was supported in part by Philip Morris, USA, Inc. and Philip Morris International and the Hungarian Research Fund, OTKA TS 49868. References [1] C.A. Barnes, J. Meltzer, F. Houston, G. Orr, K. McGann, G.L. Wenk, Chronic treatment of old rats with donepezil or galantamine: effects on memory, hippocampal plasticity and nicotinic receptors, Neuroscience 99 (2000) 17–23. [2] H.W. Clement, C. Grote, L. Svensson, J. Engel, P. Zofel, W. Wesemann, In vivo studies on the effects of memantine on dopamine metabolism in the striatum and n. accumbens of the rat, J. Neural Transm. Suppl. 46 (1995) 107–115. [3] S. Di Angelantonio, G. Bernardi, N.B. Mercuri, Donepezil modulates nicotinic receptors of substantia nigra dopaminergic neurones, Br. J. Pharmacol. 141 (2004) 644–652. [4] E. Giacobini, X.D. Zhu, E. Williams, K.A. Sherman, The effect of the selective reversible acetylcholinesterase inhibitor E2020 on extracellular acetylcholine and biogenic amine levels in rat cortex, Neuropharmacology 35 (1996) 205–211. [5] I. Hatip-Al-Khatib, A. Takashi, N. Egashira, K. Iwasaki, M. Fujiwara, Comparison of the effect of TAK-147 (zanapezil) and E-2020 (donepezil) on extracellular acetylcholine level and blood flow in the ventral hippocampus of freely moving rats, Brain Res. 1012 (2004) 169–176.
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Changes in cerebral neurotransmitters and metabolites induced by acute donepezil and memantine administrations: A microdialysis study E. Shearman a , S. Rossi a , B. Szasz b , Z. Juranyi c , S. Fallon a , N. Pomara a , H. Sershen a,∗ , A. Lajtha a b
a Nathan Kline Institute, Orangeburg, NY 10962, USA Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest, Hungary c Egis Pharmaceuticals, Budapest, Hungary
Received 7 September 2005; received in revised form 1 November 2005; accepted 1 December 2005 Available online 21 December 2005
Abstract Cholinesterase inhibitors including donepezil, rivastigmine, and galantamine and the N-methyl-d-aspartate (NMDA) antagonist, memantine are the medications currently approved for the treatment of Alzheimer’s disease (AD). In addition to their beneficial effects on cognitive and functional domains typically disrupted in AD, these agents have also been shown to slow down the emergence of behavioral and psychotic symptoms associated with this disease. However, the underlying mechanisms for these therapeutic effects remain poorly understood and could involve effects of these medications on non-cholinergic or non-glutamatergic neurotransmitter systems respectively. These considerations prompted us to initiate a series of investigations to examine the acute and chronic effects of donepezil (Aricept (±)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4piperidinyl]methyl]-1H-inden-1-1 hydrochloride and memantine (1-amino-3,5-dimethyladamantane hydrochloride C12 H21 N·HCl)). The present study focuses on the acute effects of donepezil and memantine on brain extracellular levels of acetylcholine, dopamine, serotonin, norepinephrine and their metabolites. We assayed changes in the ventral and dorsal hippocampus and the prefrontal and medial temporal cortex by microdialysis. Memantine resulted in significant increases in extracellular dopamine (DA), norepinephrine (NE), and their metabolites, in the cortical regions, and in a reduction of DA in the hippocampus. Donepezil produced an increase in extracellular DA in the cortex and in the dorsal hippocampus. Norepinephrine increased in the cortex; with donepezil it increased in the dorsal hippocampus and the medial temporal cortex, and decreased in the ventral hippocampus. Interestingly both compounds decreased extracellular serotonin (5HT) levels. The metabolites of the neurotransmitters were increased in most areas. We also found an increase in extracellular acetylcholine (ACh) by memantine in the nucleus accumbens and the ventral tegmental area. Our results suggest both region and drug specific neurotransmitter effects of these agents as well as some similarities. We conclude that drugs influencing cognitive mechanisms induce changes in a number of neurotransmitters with the changes being both region and drug specific. Release and metabolism are altered and extracellular neurotransmitter levels can be increased or decreased by the drugs. Other studies are in progress to determine the pharmacological effects associated with chronic treatment with these compounds, which may be more pertinent to the clinical situation in which patients take these medications for months or years. © 2006 Elsevier Inc. All rights reserved. Keywords: Neurotransmitter release; Donepezil; Memantine effects; Regional heterogeneity of drug effects
1. Introduction Many studies indicate the major role of neurotransmitter systems in cognitive processes. There are related findings that compounds, such as nicotine, that affect learning, induce changes in
∗
Corresponding author. E-mail address: [email protected] (H. Sershen).
0361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2005.12.001
the extracellular levels, or release in a number of neurotransmitters. Clearly, a complex set of neurotransmitters participates in the mechanism of learning and changes in any one of these may influence it. Similarly, it can be expected that various drugs that influence cognitive processes act at least in part by altering neurotransmitters and such alterations may be drug specific. However, not much has been established on the specificity of the actions of these drugs on mechanisms involved in learning and memory. To better evaluate the role and interactions
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of the different neurotransmitters in cognitive processes, it is important to examine the effect of compounds in addition to nicotine that effect learning or memory. In the present paper we report the changes of extracellular neurotransmitters in selected brain areas induced by acute administration of donepezil and memantine, two compounds that are presently widely used in the treatment of Alzheimer’s disease (AD) patients and which are believed to exert their therapeutic effects by two distinct neurotransmitter mechanisms; donepezil affecting the cholinergic and memantine, the glutamatergic system. Our hypothesis is that these drug’s well-established pharmacological actions may be associated with changes in a number of other neurotransmitters and that the pattern of changes is drug specific and regionally heterogeneous. As part of on-going studies to examine the role of neurotransmitters in cognitive mechanisms, we recently assayed nicotineinduced changes in several brain areas in rats after high- and low-dose nicotine. We assayed the levels of DA, NE, 5HT and their metabolites: dihydroxyphenylacetic acid (DOPAC) homovanillic acid (HVA), hydroxymethoxyphenylglycol (MHPG), and 5-hydroxyindolacetic acid (5-HIAA). These were measured via microdialysis in conscious freely moving adult male rats. The areas studied were the dorsal and ventral hippocampus, the prefrontal and medial temporal cortex, the ventral tegmental area and the nucleus accumbens. Some of these areas may play a role in reward processes, but it is most likely that reward and learning or memory mechanisms are interrelated and connected. At higher nicotine concentrations (0.5 mg/kg s.c.) in most areas tested, the three neurotransmitters and their metabolites were increased and we concluded that nicotine administration causes an alteration in the level, and in the metabolism of DA, NE and 5HT in brain areas involved in cognitive processes and that such changes are partly responsible for the effect of nicotine on learning [21]. At lower nicotine concentrations the effects were somewhat different such as 5HT was decreased rather than increased in some areas. The changes indicate significant regional variation and in sensitivity to nicotine-induced changes between areas of cognitive versus reward functions [15]. Nicotine-induced effects were inhibited by nicotinic, muscarinic and dopaminergic antagonists [16] indicating that the induced changes involve direct and indirect actions, interactions of receptors with the mechanisms involved being regionally heterogeneous. The nicotineinduced changes in transmitter levels observed in areas of significant reward functions differed in some respects from the patterns of changes induced and the receptors involved in the effects seen with cocaine [25]. We hope that by extending this line of investigation to comparing the effects of several compounds (in the present paper that of donepezil and memantine), it will better help us in elucidating the underlying mechanisms for the therapeutic effects of these agents in AD and identifying the role of neurotransmitters in cognitive processes. Here, we report on their effect on DA, NE and 5HT levels and on acetylcholine (ACh)—in specific brain areas. Work is in progress to compare their effect and that of nicotine on other transmitters.
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2. Methods Experiments were conducted using adult male Sprague Dawley rats (250–350 g) bred and raised in our animal facility. All studies were conducted in accordance with the principles and procedures outlined in the NIH guide for the care and use of laboratory animals, and were approved by the animal care committee of the institute.
2.1. Microdialysis As was done in our previous studies [16], rats were anesthetized with chloral hydrate (400 mg/kg; i.p., Sigma) and placed in a Kopf stereotaxic apparatus. A small hole was drilled in the skull directly above the sites selected for microdialysis. After puncturing the dura, two guide cannulas, 0.64 mm o.d. (Carnegie Medicine, MA) were lowered above the two selected sites in the brain and fixed to the skull with dental cement and two mounting screws. The stereotaxic coordinates with respect to the bregma and the surface of the skull were used according to the Atlas for Rat Brain (VH; AP: −5.0 ML: 4.8 DV: −6.0, DH; AP: −3.8 ML: 2.4 DV: −2.0, MTC; AP: −3.8 ML: 6.0 DV: −4.0, PFC; AP: +3.2 ML: 3.2 DV: −1.6, NAcc Shell; AP: +1.6 ML: 0.7 DV: −5.0, VTA; AP: −4.8 ML: 0.9 DV: −7.1). After surgery the animals were allowed to recover for a 2-day period. On the day of the experiment animals were placed in a BAS Rat Turn (Bioanalytical Systems; Lafayette, ID) and were attached via a plastic collar to a lever arm. A 1 or 2 mm microdialysis probe was inserted into the guide cannula 2 h before the start of the experiments (Carnegie Medicine, MA), depending on the brain area: 1 mm for the ventral tegmental area and nucleus accumbens, and 2 mm for the hippocampal and cortical regions. Ringer’s solution (125 mM NaCl, 3.3 mM KCl, 2.4 mM Mg2 SO4 , 1.25 mM KH2 PO4 , 1.85 mM CaCl2 ) was perfused at a rate of 1.0 L/min using a BAS baby bee syringe pump (Bioanalytical Systems; Lafayette, ID) to drive the perfusate. Following a 2-h waste period, baseline dialysate was collected at 30-min intervals for 120 min. After baseline stabilization, a subcutaneous injection of either memantine (20 mg/kg; Sigma; St. Louis, MO) or donepezil (2.5 mg/kg; kindly provided by Dr. Nunzio Pomara) was administered, and sample collection continued for the remaining 180 min. In experiments where ACh was assayed, memantine was injected intraperitoneally.
2.2. Testing of the microdialysis probe Microdialysis probes were tested for performance before use. The probe was immersed in a standard solution containing all relevant compounds in concentrations analogous to the expected extracellular fluid and the probe was perfused at a rate of 1 L/min with Ringer’s solution. The concentrations of catecholamines in the dialysates were 20–25% of those in the standard solution, indicating an approximately 23% efficiency of extraction into the dialysate.
2.3. Histological analysis Following each experiment, animals were sedated with chloral hydrate (Sigma; St. Louis, MO), decapitated, and the brains were taken out for verification of probe placement. Postmortem brains of rats were processed histologically in order to verify the location of the dialysis probes and guide cannula. Brains were sectioned, and probe placement was verified by visual analysis.
2.4. Assay of neurotransmitters For the catecholamines and their metabolites, microdialysis samples were analyzed using an HPLC system with electrochemical detection (ESA CoulArray Detector Model 5600A). The mobile phase (ESA 70-1332 Mobile Phase) contained 75 mM sodium dihydrogen phosphate, 1.7 mM 1-octanesulfonic acid sodium salt, 100 L/L triethylamine, 25 M EDTA, and 10% acetonitrile (pH 3). The pump was an ESA Pump 582. Dialysates were injected using a temperaturecontrolled autosampler (ESA Model 542) kept at 4 ◦ C. Compounds were separated on an ESA Microdialysis MD-150 Analytical column (3.2 mm, 15 cm). The cell potentials were set at −175 mV and +250 mV. Average elution time for the compounds, in minutes, are as follows: MHPG (2.10), NE (2.65), DOPAC (3.70), DA (4.6), 5HIAA (5.5), HVA (7.70), and 5-HT (10.0).
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For the determination of ACh, samples were separated in an HPLC system (BAS 200A) equipped with a BAS microbore ACh/Ch analytical drum and ACh/Ch IMER post-column, converting it to H2 O2 . Detection was by BAS peroxidase cross-flow electrode set at −100 mV. Mobile phase is 50 mM sodium phosphate, pH 8.5 ± 0.5, run at 0.13 mL/min. The limit of detections using a new IMER was 1 fmol. The sensitivity was tested with ACh and H2 O2 standards.
2.5. Statistical analysis To determine whether the memantine (20 mg/kg) or donepezil (2.5 mg/kg) subcutaneous injection altered neurotransmitter levels, the first four samples were expressed as baseline and the changes in the remaining samples, 6–10, were expressed as percentage change in baseline (average of the first four time points; mean ± S.E.M., n = 10). Significance was determined using a paired samples ttest (p < 0.05).
3. Results Memantine and donepezil were examined to determine whether or not they have an effect on neurotransmitters found in areas assumed to play major roles in reward mechanisms. Memantine was found to significantly increase the level of extracellular acetylcholine in the ventral tegmental area and as well as in the nucleus accumbens (Fig. 1). Both memantine and donepezil seem to affect DA and 5HT in the nucleus accumbens and ventral tegmentum; as our preliminary assays found significant increases in the DA metabolites DOPAC and HVA and the 5HT metabolite 5HIAA (Table 1) (DA and 5HT level changes were not significant). In the hippocampus, donepezil administration caused changes in each of the three neurotransmitters assayed. In the dorsal hippocampus, DA and NE increased, while in the ventral hippocampus NE and 5HT were decreased, hence changes in the dorsal area were very different from those in the ventral area (Fig. 2). The metabolites of the three neurotransmitters were increased with donepezil. In the dorsal hippocampus, the DA
Table 1 Effect of memantine and donepezil on metabolite levels Memantine
DOPAC HVA 5HIAA
Donepezil
NAc
VTA
NAc
VTA
237 ± 13 209 ± 19 213 ± 28
123 ± 10 146 ± 13 205 ± 66
150 ± 17 120 ± 13 130 ± 11
130 ± 12 142 ± 17
Memantine, 20 mg/kg; Donepezil, 2.5 mg/kg. Values are the averages of assays at 1–3 h after the intraperitoneal injection of the drugs (five animals, five time points in each). Values given are a percent of control (extracellular levels in absence of drugs taken as 100).
metabolites DOPAC and HVA, the NE metabolite MHPG, and the 5HT metabolite 5HIAA were each increased, whereas in the ventral hippocampus HVA, MHPG and 5HIAA were increased. The time course of these changes differed; with HVA increase becoming less, and 5HIAA increases intensifying over longer time periods (Fig. 3). It is of interest that while in the ventral hippocampus, NE and 5HT decreased (Fig. 2); their metabolites MHPG and 5HIAA respectively increased (Fig. 3). Donepezil also altered the extracellular level of the three neurotransmitters in the cortex, DA and 5HT were decreased and NE was increased in the medial temporal cortex, while in the prefrontal cortex only 5HT was decreased. Of the metabolites: DOPAC, HVA and MHPG, only increases were found in both cortical areas (Fig. 4). The changes following memantine administration differed somewhat from the donepezil-induced changes. In the dorsal hippocampus DA and 5HT decreased, and in the ventral hippocampus, DA decreased, while MHPG increased (Fig. 5). In the cortical areas tested, memantine increased DA and NE and decreased 5HT in both areas. Of the metabolites MHPG was increased in both areas; whereas DOPAC and HVA were increased in only the prefrontal cortex (Fig. 6). 4. Discussion
Fig. 1. Effect of memantine (20 mg/kg i.p.) on acetylcholine release as a function of time in the nucleus accumbens (䊉) and ventral tegmental area (). Data is expressed as percent change from baseline (average of the first four time points; mean ± S.E.M.). Each sample was collected at a flow rate of 1 L/min for 30 min. The arrow indicates the time of injection (20 mg/kg memantine). Significance was determined using paired samples t-test (p < 0.05 for the effect of Memantine, n = 5).
The aim of our experiment was to study the effects of acute administration of drugs (donepezil and memantine) used in treatment of Alzheimer’s disease on extra-neuronal concentration of different transmitters and their metabolites. Here, we studied six brain regions, assayed four neurotransmitters with two drugs, at one concentration, from which, we can draw a number of tentative conclusions. It is clear that acute doses of donepezil and memantine alter not one, but a number of neurotransmitters irrespective of whether they are primarily cholinergic or glutamatergic compounds. Most likely some of these effects are direct through cholinergic or glutamatergic receptors on noncholinergic or non-glutamatergic cells, but they are also indirect through the released neurotransmitters. These effects can be seen in many brain areas but the changes are regionally heterogeneous and the pattern is different with different drugs. The time course of changes is similarly heterogeneous. Such complex and drug specific patterns have been observed with other drugs such as nicotine or cocaine. The drugs affect neurotransmitter release and also neurotransmitter metabolism. The increase
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Fig. 2. Effect of donepezil (2.5 mg/kg s.c.) on neurotransmitter concentrations as a function of time in the dorsal (䊉) and ventral hippocampus (). DA, dopamine, NE, norepinephrine, and 5-HT, serotonin. Data is expressed as percent change from baseline (average of the first four time points; mean ± S.E.M.). Each sample was collected at a flow rate of 1 L/min for 30 min. The arrow indicates the time of injection (2.5 mg/kg donepezil). Significance was determined using paired samples t-test (p < 0.05 for the effect of Donepezil, n = 5).
in metabolites indicates the increased metabolism. It seems in some instances the effect on metabolism is greater than it is on release and the extracellular level of the neurotransmitters is decreased. We found numerous interactions of receptor systems in nicotine-induced alterations of extracellular neurotransmitters. The dopaminergic and cholinergic systems interacted [17]. Nicotine induced changes in glutamate that in turn affected DA release [27]. Specific nicotine receptors were involved in nicotine-induced NE release [20] and nicotine altered the extracellular levels of a number of amino acids [28]. Clearly both cholinergic and glutamatergic receptors are present on a number of different neurons; among them are DA, NE, and 5HT neurons. The distribution of such neurons is heterogeneous. It is expected, therefore, that cholinergic or glutamatergic compounds affect numerous neurotransmitters in a regionally heterogeneous manner. There are many questions that need to be answered. Are other compounds, particularly other neurotransmitters or neuromodulators altered, also in regionally heterogeneous drug specific
manners? We are examining presently changes in amino acid neurotransmitters. It is also important to assay which changes in neurotransmitters have functional importance and which of the changes influence different cognitive mechanisms or behavior and are their role and effect quantitatively different? Using antagonists to inhibit the extracellular changes in one neurotransmitter, may answer some of these questions. We found previously that the effects of nicotine on neurotransmitter levels are concentration dependent: at higher nicotine concentrations it stimulates the release of 5HT, increasing its level, at lower concentrations, it stimulates the metabolism of 5HT, decreasing its level [21,15]. It is possible that similarly the effects of donepezil and memantine would quantitatively or in specificity be different at different doses. There are several interesting observations reported about compounds of possible use for Alzheimer’s disease. Donepezil belongs to the compounds that inhibit acetylcholinesterase, and therefore, its effects on cerebral ACh levels were primarily studied. Studies using microdialysis in vivo in the hippocampus show
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Fig. 3. Effect of donepezil (2.5 mg/kg) on metabolite concentrations as a function of time in the dorsal (䊉) and ventral hippocampus (). DOPAC, 3,4dihydroxyphenylacetic acid, MHPG, 3-methoxy-4-hydrophenylglycol, HVA, homovanillic acid, and 5-HIAA, 5-hydroxyindole acetic acid. Data is expressed as percent change from baseline (average of the first four time points; mean ± S.E.M.). Each sample was collected at a flow rate of 1 L/min for 30 min. The arrow indicates the time of injection (2.5 mg/kg donepezil). Significance was determined using paired samples t-test (p < 0.05 for the effect of Donepezil, n = 5).
doses of 0.65 and 2 mg/kg i.p. in rats causes a 4 and 12 times increase in ACh levels [7]. A linear relationship between enzyme inhibition and ACh increase has also been illustrated [9]. In the ventral hippocampus, ACh levels were increased 15, 76, 121% respectively by 2, 5 or 10 mg/kg, donepezil p.o. [5]. Effects noted in the cortex after chronic treatment of 21 days (1.5 mg/kg twice a day p.o.) include the increase of 75% in rats [19]. Increases also were shown in vivo in the prefrontal cortex of monkeys. Donepezil effects were more pronounced in young as opposed to aged monkey brains [33]. In the cortex, donepezil, taurine and another ACh esterase inhibitor NXX-066 increased ACh several fold possibly through muscarinic M1 receptor antagonism [22]. A selective muscarinic M2 antagonist SCH5779 increased ACh release in the hippocampus, cortex and striatum indicating the important role of muscarinic cholinergic receptors in donepezil effects. The primary effect of memantine on DA was assayed. In anesthetized rats increase of extracellular DA in the striatum using voltammetry [2] and an increase of DA in the prefrontal cor-
tex and striatum using microdialysis [24] was found. Another study [6] in awake animals did not find elevated DA in the prefrontal cortex although the level of its metabolites: DOPAC and HVA were elevated. In the striatum, DOPAC increase could only be observed in the absence of anesthesia, DOPAC was also increased in the nucleus accumbens [2]. Only a few studies examined the effect of these drugs on other neurotransmitters and it seems various related drugs have different effects. In the cortex of awake rats using microdialysis, 2.0 mg/kg s.c. donepezil caused a 200% increase of acetylcholine, 80% increase of DA and 100% increase of NE. A 0.5 mg/kg dose caused ACh to increase 200%, NE 50%, and DA was not affected and neither dose influenced 5HT levels [4]. As opposed to donepezil, another acetylcholinesterase inhibitor, rivastigmine in freely moving rats significantly decreased NE concentrations in the hippocampus and increased HVA, while DA was not changed [32]. The cholinesterase inhibitor neostigmine was also shown to increase hippocampal ACh level with a resulting increase in release of NE, which is potentiated by the
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Fig. 4. Effect of donepezil (2.5 mg/kg) on neurotransmitter and metabolite concentrations as a function of time in the medial temporal (䊉) and prefrontal cortex (). Data is expressed as percent change from baseline (average of the first four time points; mean ± S.E.M.). Each sample was collected at a flow rate of 1 L/min for 30 min. The arrow indicates the time of injection (2.5 mg/kg donepezil). Significance was determined using paired samples t-test (p < 0.05 for the effect of Donepezil, n = 5).
nicotinic antagonist mecamylamine, but blocked by the muscarinic antagonist atropine [8]. Another acetylcholinesterase inhibitor CHF2819 developed for the treatment of Alzheimer’s disease in the hippocampus decreased DA levels, increased 5HT
and did not effect NE levels. The amino acids tested: glutamate, aspartate, GABA, taurine, arginine and citrullin, were also not affected [30]. The combined increase of ACh and 5HT by this compound may be of therapeutic use in Alzheimer’s disease
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Fig. 5. Effect of memantine (20 mg/kg s.c.) on neurotransmitter and metabolite concentrations as a function of time in the dorsal (䊉) and ventral hippocampus (). Data is expressed as percent change from baseline (average of the first four time points; mean ± S.E.M.). Each sample was collected at a flow rate of 1 L/min for 30 min. The arrow indicates the time of injection (20 mg/kg memantine). Significance was determined using paired samples t-test (p < 0.05 for the effect of Memantine, n = 5).
patients who have a depressive syndrome [31]. Rivastigmine but not CHF2819 decreased glutamate, taurine, arginine and citrullin levels—aspartate was not affected [29]. In rat hippocampal and human cortical slices, galantamine increased GABA release and synaptic transmission, which was not increased by donepezil or rivastigmine [18]. In mouse brain striatal slices, low concentrations of donepezil (1 mM) and galantamine (50 mM) increased action potential-dependent DA release [36]. Another type of cholinergic compound that binds to ACh receptors with higher affinity than nicotine is SIB1533A. This compound influenced neurotransmitter release (examined in brain slices): DA release from striatum, olfactory tubercle and prefrontal cortex, and NE release from hippocampus and prefrontal cortex was inhibited by SIB1533A. It also inhibited NMDA-evoked ACh release from striatum. The effect of SIB1533A was transmitter and region specific and involved multiple receptor affinities and release of several neurotransmitters [14]. Donepezil and other cholinesterase inhibitors also have direct effects on ACh receptors, which is not dependent on their effect on
esterase. Donepezil was shown to modulate nicotinic receptors on dopaminergic neurons [3]. Both donepezil and galantamine elevated the number of nicotinic receptors in the hippocampus and the cortex after chronic treatment altering synaptic plasticity at levels analogous to that used in Alzheimer’s patients [1]. Among cholinesterase inhibitors, galantamine, but not tacrine, acted as an allosterically potentiating ligand sensitizing nicotinic receptors [10]. This allosteric sensitization affects other neurotransmitter systems modulated by nicotinic receptors effecting glutamate release and serotonergic neurotransmission [10]. Memantine effects on release evoked by the glutamate receptor agonist NMDA were studied. In thin layers of isolated rat hippocampal or cortical nerve endings NMDA-evoked NE release was inhibited by memantine [13]. In rat striatal slices memantine inhibited NMDA-evoked DA, ACh and GABA release. It only weakly inhibited NMDA-evoked spermidine release [11]. In synaptosomes prepared from human brain samples removed during neurosurgery, the NMDA elicited NE release was also inhibited by memantine [12]. In these experiments memantine
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Fig. 6. Effect of memantine (20 mg/kg) on neurotransmitter and metabolite concentrations as a function of time in the medial temporal (䊉) and prefrontal cortex (). Data is expressed as percent change from baseline (average of the first four time points; mean ± S.E.M.). Each sample was collected at a flow rate of 1 L/min for 30 min. The arrow indicates the time of injection (20 mg/kg memantine). Significance was determined using paired samples t-test (p < 0.05 for the effect of Memantine, n = 5).
acted as an NMDA antagonist. In isolated rat brain synaptosomes, memantine inhibited both the high and low affinity uptake of 5HT, it also inhibited the binding of 5HT to isolated nerve-ending membranes [35]. The levels of DA, NE and 5HT in the striatum and lumbar spinal cord of rats and of spastic rats was dose-dependently decreased by memantine [23]. Comparing our results with donepezil and memantine, we can find significant differences between their induced changes, especially with DA. In the dorsal hippocampus, donepezil increased and memantine decreased DA. In the medial temporal cortex, donepezil decreased while memantine increased DA. Donepezil in the dorsal hippocampus increased, and in the ventral hippocampus
decreased NE, while memantine in the hippocampus had no effect on NE. In the prefrontal cortex, donepezil had no effect on NE while memantine increased it. The fact that CHF2819, a cholinesterase inhibitor, unlike donepezil in the present study, decreased DA levels and increased 5HT levels in the hippocampus [30], indicates differences in effects among cholinesterase inhibitors. It is not easy to compare the various findings on neurotransmitter release of memantine and donepezil with finding on related drugs as the experimental conditions of various studies differ significantly. It is likely that mechanisms operating in the living brain are significantly altered in various preparations
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such as synaptosomes or brain slices and anesthesia also affects some mechanisms of stimulatory function. In addition, besides classical synaptic transmission responses, the role of nonsynaptic receptors are clearly involved in the observed responses, as shown by the diverse regional and time-dependent interactions of neurons equipped with different transmitters, and support their importance as targets for drug development [34]. Our work clearly indicates that each drug affects several neurotransmitters and such effects can be increases or decreases. There are significant differences among the effects of the drugs including differences among cholinesterase inhibitors. The effects show regional heterogeneity and are concentration dependent, as well as showing that regional heterogeneity and concentration dependence are also drug-dependent. Several receptors and the interactions of such receptors participate in the effects, some which are direct on receptors located on various neurons and some which are affected indirectly by the released or reduced level of neurotransmitters. Such interactions were examined in some detail, showing that changes in the ventral tegmental area affected those in the nucleus accumbens, in which a number of receptors in the ventral tegmental area mediated nicotine-induced DA changes in the nucleus accumbens [26]. The relevance of the current study and some of the aforementioned literature, which has been limited to the effects of single acute doses for the clinical situation in which patients take these medications for months or years, is not known and more studies are in progress to examine this question. Similarly, donepezil and memantine together have been demonstrated to have a better efficacy in the treatment of AD than donepezil alone and more studies will need to be conducted to clarify mechanisms for this observation. Acknowledgement Research described in this article was supported in part by Philip Morris, USA, Inc. and Philip Morris International and the Hungarian Research Fund, OTKA TS 49868. References [1] C.A. Barnes, J. Meltzer, F. Houston, G. Orr, K. McGann, G.L. Wenk, Chronic treatment of old rats with donepezil or galantamine: effects on memory, hippocampal plasticity and nicotinic receptors, Neuroscience 99 (2000) 17–23. [2] H.W. Clement, C. Grote, L. Svensson, J. Engel, P. Zofel, W. Wesemann, In vivo studies on the effects of memantine on dopamine metabolism in the striatum and n. accumbens of the rat, J. Neural Transm. Suppl. 46 (1995) 107–115. [3] S. Di Angelantonio, G. Bernardi, N.B. Mercuri, Donepezil modulates nicotinic receptors of substantia nigra dopaminergic neurones, Br. J. Pharmacol. 141 (2004) 644–652. [4] E. Giacobini, X.D. Zhu, E. Williams, K.A. Sherman, The effect of the selective reversible acetylcholinesterase inhibitor E2020 on extracellular acetylcholine and biogenic amine levels in rat cortex, Neuropharmacology 35 (1996) 205–211. [5] I. Hatip-Al-Khatib, A. Takashi, N. Egashira, K. Iwasaki, M. Fujiwara, Comparison of the effect of TAK-147 (zanapezil) and E-2020 (donepezil) on extracellular acetylcholine level and blood flow in the ventral hippocampus of freely moving rats, Brain Res. 1012 (2004) 169–176.
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