Acetylcholinesterase Inhibitors Are Neither Necessary Nor Desirable for Microdialysis Studies of Brain Acetylcholine

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 in ments. tected, oxidized on a platinum elec- the STR in the absence of neostig- trode with the potential set at e.g. mine (4), whereas De Boer and Ab- AChE inhibitor-free +0.5 V (old system); and 5.) the oxi- ercrombie (6) reported that microdialysis and low dation is monitored with the detector haloperidol (1 mg/kg) increased detection limit ACh signal indicating ACh in the chroma- striatal extracellular ACh in the pres- measurement (T3) togram. Basal dialysate concentra- ence of even low concentration neo- tions of ACh have been reported to stigmine (0.01 µM). These results Several ACh microdialysis studies in be 0.17-10 fmol/µL, although the clearly suggest that the effect of which AChE inhibitors were not size and shape of dialysis probes, some drugs on extracellular ACh dif- used have been reported. Determina- flow rate of perfusion medium, sam- fers, even with a low concentration tion of ACh by radioimmunoassay pling interval and the region receiv- of AChE inhibitors, from the effects (RIA) was used in some of these ing probe implantation are different under physiological conditions or in studies. Although RIA provides suf- in each experiment. The detection the absence of AChE inhibitors. ficient sensitivity for detection of limit based upon the signal-to-noise The use of ACh esterase inhibi- ACh at relevant concentrations ratio (S/N) has not been clearly pro- tors has the major disadvantage that (0.72-1.72 fmol/µL at basal levels), vided in most studies; however, it increased extracellular ACh causes it will not be further discussed in this may range from 20 to 50 fmol per continuous stimulation of presynap- review. Few neuroscience laborato- sample. While this system provides tic and postsynaptic ACh receptors, ries are equipped for this methodol- reliable measurement of ACh in dia- which may result in cholinergic tone ogy, whereas liquid chromatography lysate samples in the presence of significantly different from physi- (LC) is readily available. AChE inhibitors, it is unable to ological conditions. These differ- The principle of ACh measure- measureACh intheabsenceof AChE ences could bias the interpretation of ment with liquid chromatogra- inhibitors. the data obtained from microdialysis phy/electrochemistry (LCEC) is The next generation of ACh studies performed in the presence of shown in F1 and F2: 1.) dialysate measurement has replaced the plati- AChE inhibitors. In the presence of samples are injected onto the LC sys- num electrode with a peroxidase-re- AChE inhibitors, it is also not possi- tem; 2.) ACh is separated on the cat- dox polymer coating on a glassy ble to split dialysate samples to ion exchange column (analytical carbon electrode (F2, new system) measure DA or other substances column); 3.) ACh is hydrolysed by (21,22). Peroxidase is mounted on which can provide direct evidence AChE to form acetate and choline in the surface of a glassy carbon elec-

T3 ACh measurement in the dialysate without AChE inhibitors.

www.currentseparations.com 40 trode and coated with a redox poly- peak from the huge choline peak can decreasing the detection limit for the mer material. This type of electrode be obtained with a reversed phase ACh measurement. The extent of de- increases the signal-to-noise ratio by column, a microbore column, which composition of H2O2 in the pre- providing a stabilized electrode sur- is best fitted to the analysis of dia- IMER may vary based on the activity face on which H2O2 is reduced to lysate ACh, is not available at present of the catalase, leaving some amount form H2O. For example, a platinum because of technical difficulties that of H2O2 in the analytical column. electrode requires higher potential, are being remedied. The H2O2 left over in the injected e.g. +0.5 V versus the reference elec- Since it is difficult in most cases dialysate samples increases the front trode, compared with +0.1 V for the to separate the ACh peak from the peak in the chromatogram which new type of electrode. This low po- huge choline peak with the cation tails and may interfere with the ACh tential contributes, in part, to lower exchange column, Kato et al. (23,24) peak. However, this interference has noise levels. However, a high con- and Tsai et al. (25) used a pre-IMER been shown to be minimal as long as centration of choline remains in the (ChO/peroxidase and ChO/catalase, the catalase remains active (F3). injected samples and interferes with respectively) set prior to the analyti- Thus, the use of the pre-IMER cou- the peak due to ACh, which elutes cal column (F2). Choline in the dia- pled with the cation exchanger mi- prior to the choline peak in the chro- lysate samples is oxidized by the crobore column is essential for the matogram. The ACh peak is well ChO to form betaine and H2O2. The method to achieve sufficient sensi- separated from the choline peak in H2O2 is then converted by peroxi- tivity to permit AChE inhibitor free the authentic standard. In dialysate dase/catalase to H2O before entering determination of ACh in dialysates. samples, the ACh peak appears on the analytical column. The pre- We modified and improved the the slope of a huge choline peak. IMER completely eliminates method of Kato et al. (24) so that This chromatographic interference choline and H2O2 from the injected dialysate ACh can be reliably and markedly increases the detection samples. This procedure eliminates consistently measured in microdia- limit for the ACh measurement. Al- overlap of choline with the ACh peak lysis experiments in the absence of though clear separation of the ACh in the chromatogram (F3), thereby AChE inhibitors (4,5). The proce- dure, in brief, is as follows: Three to F1 MOBILE PHASE MICRODIALYSIS five days following cannulation sur- Scheme of bioanalysis of microdialysis dialysate gery, a concentric-shaped dialysis samples for ACh. The PUMP probe (2 mm length, polyacryloni- arrow indicates the direction of sample trile/sodium methalylsulfonate poly- analysis. IMER: mer membrane, AN69H; Hospal) is immobilized enzyme INJECTOR SAMPLES reactor. implanted into either mPFC, NAC or STR of rats under slight anesthesia PRE-IMER with methoxyflurane (Metofane; Pitman-Moore, Mundelein, IL), CATION ION EXCHANGER ACh DATA which make them temporarily im- mobilized. The rats quickly recover from slight anesthesia and are al- POST-IMER lowed to move freely in the cage. Perfusion flow rate of the implanted ELECTRODE/DETECTOR WORK-STATION probes is set at 1.5 µL/min and then dialysate samples (45 µL) are col- lected every 30 min. The perfusion F2 medium is Dulbecco’s phosphate Post-IMER Separation Samples buffered saline solution (Sigma, St. Scheme comparing the ACh/Choline 2+ new system with the old AChesterase Louis, MO) including Ca (NaCl system. The arrow Choline ACh indicates the direction of 138 mM, Na2HPO4 8.1 mM, KCl 2.7 Pre-IMER sample analysis IMER: Choline Oxidase mM, KH2PO4 1.5 mM, MgCl2 0.5 immobilized enzyme Choline Oxidase/Catalase reactor. M: mediator of mM, CaCl2 1.2 mM, pH = 7.4). electron transfer. Three hours after probe implantation Peroxidase H2O2 H2O and its perfusion, dialysate samples are directly injected onto the LCEC [M] [M+] system assisted by a chromatogra- + 2H +2e+O2 phy manager (Millennium; Waters, Milford, MA), and analyzed for ACh. ACh is separated on a coiled Platinum Electrode Glassy Carbon Electrode Old System New System ion exchanger ACh column (UniJet

41 Current Separations 19:2 (2000) 10 µm ID 530 × 1.0 mm; BAS, West origin. With this newly developed 3. Jentsch J.D., Dazzi L., Chhatwal J.P., Verrico C.D., Roth R.H. (1998) Lafayette, IN). The pre-IMER (MF- method, it is now possible to reliably Reduced prefrontal cortical 8907) purchased from BAS consists and accurately measure ACh in dia- dopamine, but not acetylcholine, of ChO/catalase, and the post-IMER lysate samples in the absence of release in vivo after repeated, intermittent phencyclidine (BAS) consists of ChO/AChE. The AChE inhibitors. administration to rats. Neurosci Lett mobile phase (Na2HPO4 50 mM, pH In conclusion, AChE inhibitors 258: 175-178. 8.2) including ProClin (BAS), a mi- have long been used to investigate 4. Ichikawa J., O’Laughlin I.A., Dai J., crobiocide, is pumped at 0.14 ACh by pharmacologically increas- Fowler W.L., Meltzer H.Y.(1999) Clozapine increased extracellular mL/min by a LC-10AD pump (Shi- ing its basal concentrations, in push- acetylcholine (ACh-ext) levels in rat madzu, Kyoto, Japan). ACh is de- pull, slice-superfusion, in vivo medial prefrontal cortex (mPFC) tected using a UniJet amperometric voltammetry and in vivo microdia- but not striatum (STR) or nucleus accumbens (NAC) in the absence detector cell with the peroxidase-re- lysis studies. AChE inhibitors have of AChesterase (AChE) inhibition. dox polymer-coated glassy carbon significant effects on the ACh trans- Soc Neurosci Abstr 25: 178.12. electrode (MF-2098; BAS), set at mission and alter physiological con- 5. Meltzer H.Y., O’Laughlin I.A., Dai J., +0.1 V (LC-4C; BAS) versus AgCl- ditions by themselves. Thus, caution Fowler W.L., Ichikawa J. (1999) Atypical antipsychotic drugs (APD) coated Ag reference electrode. The is indicated in the interpretation of but not typical APD increased ACh peak is clearly resolved in the potentially biased data in the pres- extracellular acetylcholine chromatograph (F3). ence of AChE inhibitors. Highly sen- (ACh-ext) levels in rat medial prefrontal cortex (mPFC) in the The ACh peak is clearly identi- sitive measurement of ACh, which absence of AChesterase (AChE) fied in all the chromatograms be- has been recently developed, permits Inhibition. Soc Neurosci Abstr 25: cause there is no interference (F3). measurement of ACh under physi- 178.11. Areas and the heights of ACh peaks ological conditions. This new 6. DeBoer P., Abercrombie E.D. (1996) Physiological release of striatal showed a linear correlation with the method may contribute to progress acetylcholine in vivo: modulation by concentration. Due to the markedly in ACh pharmacology and neurosci- D1 and D2 dopamine receptor increased S/N ratio, the detection ence, and help to develop new thera- subtypes. J Pharmacol Exp Ther 277: 775-783. limit is about 1-2 fmol per sample peutic drugs for Alzheimer’s disease 7. Tsai T.H. (2000) Separation methods (10 µL sample loop). Thus, we can and cognitive dysfunction in schizo- used in the determination of obtain stable baseline values of ACh phrenia or other mental illness. choline and acetylcholine. J in dialysates in all the experiments Chromatography B 747: 111-122. for these three regions. The basal References 8. Cuadra G., Summers K., Giacobini E. (1994) Cholinesterase inhibitor levels of ACh are 0.6-2.74 fmol/µL effects on neurotransmitters in rat 1. Meltzer H.Y., McGurk S.R. (1999) for the mPFC, NAC and STR (T3). cortex in vivo. J Pharmacol Exp The effects of clozapine, Ther 270: 277-284. The basal ACh and its increase pro- risperidone, and olanzapine on duced by clozapine (20 mg/kg) were cognitive function in schizophrenia. 9. Cuadra G., Giacobini E. (1995) Schizophr Bull 25: 233-255. Coadministration of cholinesterase completely eliminated by co-perfu- inhibitors and idazoxan: effects of 2. Tandon R. (1999) Cholinergic aspects sion of the sodium channel blocker neurotransmitters in rat cortex in of schizophrenia. Br J Psychiat vivo. J Pharmacol Exp Ther 273: tetrodotoxin (1 µM) (4), indicating 174, Suppl 37: 7-11. 230-240. dialysate ACh is entirely of neuronal

F3 nA 0.75 nA 0.75 nA 0.75 Separation of acetylcholine (ACh) peak in the chromatogram obtained from an authentic standard solution (10 fmol, left panel) and dialysate samples (center and right panels) in rat medial prefrontal cortex (mPFC). The ACh peak was clearly separated and reliably detected in the chromatogram. Clozapine (20 mg/kg, s.c.) significantly increased the ACh height and area of ACh ACh peak compared to the ACh basal peak. Detection limit was about 1-2 fmol per sample. Vertical axis indicates nA and horizontal axis indicates time after injection (min), respectively. 0.25 0.25 0.25 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 www.currentseparations.com 42 10. Dijk S.N., Francis P.T., Stratmann 21. Huang T., Yang L., Gitzen J., 30. Izurieta-Sánchez P., Jonkers N., G.C., Bowen D.M. (1995) Kissinger P.T., Vreeke M., Heller A. Sarre S., Ebinger G., Michotte Y. 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43 Current Separations 19:2 (2000)