Acetylcholinesterase Inhibitors Are Neither Necessary nor Desirable for Microdialysis Studies of Brain Acetylcholine Junji Ichikawa*, Jin Dai and Herbert Y.Meltzer Acetylcholine (ACh), one of the major neurotransmitters in brain, has an important role in various types of cognition, including attention and Address of affiliations: Departments of Psychiatry memory. ACh is critical to the neurocognitive dysfunction in Alzheimer’s and Pharmacology disease and possibly other psychiatric disorders such as schizophrenia. Psychopharmacology Division, Vanderbilt Furthermore, the development of drugs which increase the availability of University School of brain ACh is a major research goal. Thus, direct measurement of brain Medicine, Nashville, TN 37212 USA ACh release under physiological conditions, using in vivo microdialysis, in laboratory animals is of great importance. Because of the low *Send correspondence to: J. Ichikawa, M.D., Ph.D., concentration of ACh, many of the microdialysis studies published to date 1601 23rd Avenue South, required Acetylcholinesterase (AChE) inhibitors in the perfusion medium The First Floor Laboratory R-1117, The Psychiatric to increase basal ACh. However, the artificially increased concentration Hospital at Vanderbilt, exerts a significant influence on the cholinergic system, thereby making Nashville, TN 37212 USA interpretation of drug effects problematic. We have recently developed a Phone: 615-327-7242 highly sensitive method of ACh measurement, which requires no AChE Fax: 615-322-2522 Email: ichikaj@ inhibitors. Advantages of the new method are discussed. ctrvax.vanderbilt.edu Introduction use of AChE inhibitors may interfere (NAC) and striatum (STR) in the with the study of drug effects on ACh presence of 0.3 µM neostigmine in Acetylcholine (ACh) is important in release. Thus, AChE inhibitors may the perfusion medium (31), whereas normal brain function as well as alter muscarinic/nicotinic ACh re- we have found that clozapine (20 mental illness. Specifically, ACh is ceptor-mediated transmission be- mg/kg), in the absence of neostig- of interest in cognitive dysfunction cause of the huge increases in mine, increases ACh release only in in Alzheimer’s disease and schizo- extracellular ACh levels they pro- the mPFC, and not in the other two phrenia (1), and possibly also nega- duce. It is obviously not possible to regions (4). Similarly, in the absence tive and positive symptoms in study any additional effect of AChE of AChE inhibitors (4), we could not schizophrenia (2). In vivo measure- inhibitors themselves on extracellu- replicate the report that haloperidol ment of brain ACh is also important lar ACh, which may be of interest in (1 mg/kg), a typical APD, increases to animal studies of drugs, e.g. antip- some experimental disease models striatal ACh release in the presence sychotic drugs (APDs) to treat or drug-interaction studies. Thus, we of 0.01 µM neostigmine (6). These Alzheimer’s disease and schizophre- have found that the effect of some results provide a strong rationale for nia (3,4,5). Because of the low sen- drugs on ACh release differs in the ACh microdialysis studies without sitivity of conventional ACh presence and absence of neostig- the use of AChE inhibitors. This re- measurements, most investigators mine, a widely used AChE inhibitor. view further discusses the impor- have used acetylcholinesterase For example, clozapine (20 mg/kg), tance of AChE inhibitor-free (AChE) inhibitors to increase basal the prototypical atypical APD, has microdialysis for ACh. The review of extracellular ACh concentrations to been reported to acutely increase Tsai (7) should also be consulted. readily detectable levels. However, ACh release in rat medial prefrontal there is growing evidence that the cortex (mPFC), nucleus accumbens 37 Current Separations 19:2 (2000) T1 Microdialysis determination of the effect of AChE inhibitors on extracellular neurotransmitters in rat brain. * intraventricular injection ** given to substantia nigra, dl: dorsolateral Effects of AChE inhibitors on However, the AChE inhibitor scopolamine and QNB (quinuclid- ACh and other heptyl-physostigmine increased inyl benzilate), non-selective mus- neurotransmitters (T1) ACh and DA in the frontal cortex carinic ACh receptor antagonists, after both systemic administration dose-dependently decreased electri- Extracellular concentrations of ACh (2-5 mg/kg) and local perfusion (50 cally-evoked [3H]DA in the presence in brain are influenced mostly by the µM) (8,9). Furthermore, local perfu- of physostigmine (1 µM). Physostig- activity of AChE and, to a lesser ex- sion of neostigmine has been re- mine itself increased [3H]DA, tent, by autoreceptor-mediated inhi- ported to increase DA in the STR whereas both scopolamine and QNB bition of ACh release from the fundus (11), and NE in the hippo- had no effect in the absence of neuron terminals. Inhibition of campus (12), respectively. These re- physostigmine (16). Taken together, AChE activity results in a marked sults suggest that AChE inhibitors in these considerations provided evi- increase in extracellular ACh con- the perfusion medium have appre- dence that the effect of drugs on ex- centrations, and permits reliable ciable effects on other neurotrans- tracellular ACh in the presence of measurement of ACh in dialysate mitter systems as well as ACh, AChE inhibitors may not reflect in samples which would otherwise not possibly mediated via increased vivo effects in the brain under physi- be possible due to insufficient assay ACh. In fact, ACh by itself has been ological conditions. detection limits. However, recent reported to increase DA release in microdialysis studies suggested that the STR in the absence of physostig- Alterations of drug effects on AChE inhibitors may alter extracel- mine (13); however, these increases extracellular ACh by AChE lular levels of neurotransmitters in were attenuated in the presence of 10 inhibitors (T2) the brain other than ACh. For exam- µM physostigmine, and completely ple, systemic administration of abolished by further increases in its The ability of AChE inhibitors to physostigmine (0.03-0.3 mg/kg), an- concentration (50 µM) (13). alter drug effects on extracellular other widely used AChE inhibitor for Radio-receptor binding studies ACh may depend largely upon their microdialysis studies, has been re- demonstrated that AChE inhibitors concentration in the perfusion me- ported to increase extracellular such as physostigmine displaced the dium. It has been suggested that low dopamine (DA), norepinephrine agonist [3H]oxotremorine-methio- concentrations of AChE inhibitors in (NE) and ACh in rat frontal cortex, dide binding in rat brain (14). Neo- the perfusion medium only mini- whereas physostigmine (50 µM) in stigmine has also been reported to mally modulate drug effects on ex- the perfusion medium increased depress neuronal activity mediated tracellular ACh. For example, a low only ACh in that region (8,9). Simi- by nicotinic ACh receptors by inter- concentration of neostigmine (0.01 larly, systemic administration of acting with the ACh binding sites of µM) in the perfusion medium could physostigmine (0.3 mg/kg), but not the receptors (15). These results sug- produce minimal perturbation of its local perfusion (10 µM), in- gest that the ability of ACh receptor physiological conditions, permitting creased extracellular glutamate in agonists and antagonists to affect the reliable measurement of dialysate the STR (10). Thus, it appears that release of ACh, and perhaps other ACh. Thus, systemic administration inclusion of physostigmine in the transmitters as well, may be altered of physostigmine (0.3 mg/kg) has perfusion medium has minimal ef- in the presence of AChE inhibitors. been reported to increase extracellu- fects on extracellular DA, NE and The slice-superfusion studies of cat lar ACh in the STR in the presence glutamate. caudate putamen demonstrated that of 0-10 µM physostigmine, whereas www.currentseparations.com 38 it decreased ACh in the presence of phetamine (2 mg/kg) had no effect quinpirole-induced decrease in ex- 25-50 µM physostigmine (18). Un- on striatal extracellular ACh, respec- tracellular ACh was not increased der physiological conditions, or in tively (6,19). However, the results, in relative to basal ACh (6,19). Local the presence of low concentration of the presence of high concentrations perfusion of atropine (10 µM), a non- neostigmine (0.01 µM), high dose of neostigmine (0.1 µM), were quite selective muscarinic ACh receptor amphetamine (10 mg/kg) as well as different. Thus, amphetamine, 2 antagonist, has been reported to in- the D2 receptor agonist quinpirole (3 mg/kg, increased striatal extracellu- crease basal and handling-evoked mg/kg) decreased, but low dose am- lar ACh, and the amplitude of the extracellular ACh in rat ventral hip- T2 The effect of AChE inhibitors on the ability of drugs to affect extracellular neurotransmitters in rat brain. 39 Current Separations 19:2 (2000) pocampus only in the presence of a for their interaction with ACh. Thus, the post-IMER (immobilized en- higher concentration of neostig- the development of methods for ACh zyme reactor), and then choline is mine; 0.1 and 1 µM, but not 0.01 µM measurement which requires no oxidized by ChO (choline oxidase) (20). Thus, it should be noted that AChE inhibitors provides a neces- to produce betaine and hydrogen haloperidol (1 mg/kg) had no signifi- sary tool for some types of experi- peroxide (H2O2); 4.) H2O2 is de- cant effect on extracellular ACh