Short Communication

External Monitoring of Cerebral NicotinicAcetylcholine Receptors in Living Mice

XIANG LIU,1 JOHN L. MUSACHIO,1 HENRY N. WAGNER, JR.,1 TAKAO MOCHIZUKI,1ROBERT F. DANNALS,1 AND EDYTHE D. LONDON2*

1Division of Nuclear Medicine and Radiation Health Sciences, Johns Hopkins Medical Institutions,Baltimore, Maryland

2Brain Imaging Center, National Institute on Drug Abuse, Baltimore, Maryland

Central nicotinic acetylcholine receptors (nAChRs)are fundamental to brain function, and are affected byseveral neuropathological processes. During the lastdecade, several advanced, complicated nuclear medi-cine procedures, such as positron emission tomography(PET) and single photon emission computed tomogra-phy (SPECT), have been applied to the in vivo study ofneurotransmitter receptors, such as nAChRs, in humansubjects and nonhuman primates. Although these ex-pensive technologies are not available to many investi-gators, simpler radiation detector devices, often called‘‘probes,’’ can be used to study neurotransmitter sys-tems in vivo. For example, Sasaki et al. (1993) demon-strated that a simple probe system and [125I]-4-iododexetimide can be used to study cerebral muscarinicacetylcholine receptors in living mice.

The present report describes the feasibility of using asimple procedure for labeling and monitoring nAChRsin the mouse brain. Epibatidine (exo-2-(6-chloro-3-pyridyl)-7-azabicyclo[2.2.1]heptane), an alkaloid firstisolated from the skin of the Ecuadoran frog Epipedo-bates tricolor, has extremely high affinity for nAChRs(Badio and Daly, 1994). Several in vitro and ex vivostudies have demonstrated the utility of radiolabeledforms of this compound and its analogues as tracers tostudy nAChRs (Badio and Daly, 1994; Gerzanich et al.,1995; Houghtling et al., 1995; London et al., 1995). Herewe report that the probe system and a radioiodinatedanalogue of epibatidine, (6)-exo-2-(2-[125I]iodo-5-pyri-dyl)-7-azabicyclo[2.2.1]heptane ([125I]IPH) (Musachio etal., 1997) (Fig. 1), can be used to assess brain nAChRsin living mice. In vitro assays have shown that thistracer has high affinity for nAChRs (Kd 5 90 pM inpreparations of rat brain membranes), and autoradiog-raphy with [125I]IPH demonstrated clear localization ofnAChRs in brain and adrenal gland (Davila-Garcıa etal., 1997).

Male CD-1 mice (22–29 g, Charles River, Wilmington,MA) were anesthetized by intraperitoneal injection ofurethane (1.6 g/kg). Anesthesia was maintained withadditional urethane (0.16 g/kg) as necessary. [125I]IPH

(13.7–18.1 µCi) was administered intravenously (i.v.) in0.9% saline (0.15 ml) through the tail vein. For displace-ment studies, unlabeled (6)epibatidine (2 µg/kg in 0.2ml saline), cytisine (1 mg/kg in 0.2 ml saline), or saline(0.2 ml) was administered i.v. (tail vein) 40 min afterthe radioligand. [125I]IPH was prepared as previouslydescribed (Musachio et al., 1997). The radioligand wasof 97% radiochemical purity, and had specific activity ofapproximately 2,000 mCi/µmol. Epibatidine was pur-chased from Research Biochemicals International(Natick, MA) and cytisine from Sigma Chemical Com-pany (St. Louis, MO).

Brain radioactivity was measured with a computer-based probe system (Capintec, Ramsey, NJ), equippedwith a NaI crystal (5.08 3 5.08 3 5.08 cm3), and fittedwith a removable, single-hole (5-mm diameter) leadcollimator of 30-mm thickness. The data acquisitionprocedures were similar to those described previously(Sasaki et al., 1993). Briefly, the anesthetized mousewas placed in an open 50-ml syringe tube that was cutin half longitudinally, positioned on its right side, andsecured with transparent tape. The immobilized mousewas placed on a table, below the hole of the collimator.The probe was positioned at a point between the left eyeand the upper margin of the auditory track. Althoughwe assumed that the probe did not have adequateresolving power to discriminate between regions of highand low receptor density in the mouse brain, we placedthe animal in such a way as to enhance the probabilitythat the field of view extended from left to the rightthrough brain regions (i.e., the thalamus and superiorcolliculi), where high densities of nAChRs occur in therodent brain (Wada et al., 1989; London et al., 1995).Data were acquired continuously, with radioactivityrecorded as average counts/min. The mouse was re-moved from the probe for i.v. injection of an unlabelednicotinic agonist (cytisine, epibatidine) or saline about

*Correspondence to: Dr. Edythe D. London, Brain Imaging Center, NationalInstitute on Drug Abuse, 5500 Nathan Shock Drive, Baltimore, MD 21224.

Received 17 January 1997; accepted 19 April 1997

SYNAPSE 27:378–380 (1997)

r 1997 WILEY-LISS, INC.

40 min after the radioligand injection. It was thenrepositioned as closely as possible to the original loca-tion. The total data acquisition time was 3 h. Forvalidation of the probe measurements, mice were killedby cervical dislocation at approximately 3 h 5 min afterradioligand injection. Regions of the brain were dis-

sected and weighed, and radioactivity was measured inan automated gamma-counter. Radioactivity was mea-sured by the probe 24 h after radioligand injection fromtwo mice (given saline) to determine the amount ofradioligand remaining in the brain.

Data were analyzed by determining the percentagedecrease in [125I]IPH radioactivity after injection ofsaline, epibatidine, or cytisine. Since variation (up to20%) in radioactivity was seen immediately after repo-sitioning the mouse in the probe system in several cases(data acquired during the first min after injection wereaffected by repositioning), the average counts of radioac-tivity at 2–5 min after the injection of the unlabeled testcompounds were taken as baseline. The average countsof radioactivity from each of the 5-min measurementswere compared to the baseline. Differences of thepercentage decrease in radioactivity from the probemeasures, and in radioactivity from different brainregions by the dissection experiments among the micethat received saline, epibatidine, or cytisine were deter-

Fig. 1. Structure of (6)-exo-2-(2-[125I]iodo-5-pyridyl)-7-azabicyclo-[2.2.1]heptane ([125I]IPH).

Fig. 2. Time-activity curve obtained with the probe system afteri.v. injection of [125I]IPH and unlabeled test compounds [saline (n 5 7),epibatidine (n 5 5), and cytisine (n 5 4)]. Values are means 6 SDpercentages of cpm at baseline (average counts at 2–5 min afterinjection of the test compound). Except for baseline, which representsan average of cpm in a 4-min epoch, each point represents an averageof cpm in a 5-min epoch. Counts were not obtained between 25 minand 15 min relative to injections of test compounds. During this time,

each animal was removed from below the collimator for injection, andwas repositioned. Differences between cpm obtained before and afterthis gap reflect a combination of changes in brain radioactivity and aneffect of repositioning. Inset, radioactivity measured in the thalamusdissected from the brains of mice killed approximately 145 min afterinjection of saline, epibatidine, or cytisine, given i.v. 40 min after theradioligand.

379MONITORING OF BRAIN RECEPTORS IN LIVING MICE

mined by analysis of variance (ANOVA) and Student’st-test using SAS software (SAS for Windows V6.10, SASInstitute, Cary, NC).

Figure 2 shows the time–activity curve (mean 1 SDof percentage) of [125I]IPH counted after injection ofsaline (control), epibatidine, or cytisine. At 2 h afterradioligand injection (1 h 20 min after injection ofunlabeled test compounds), the radioactivity measuredby the probe from the mice that received saline (control)was about 76% of the baseline, consistent with theresults of ex vivo studies on the time course of [3H]-epibatidine in mouse brain (London et al., 1995). At 2 hafter injection of saline, epibatidine or cytisine (2 h 40min after radioligand injection), radioactivity mea-sured by the probe was 69%, 47%, and 38% of thebaseline after injection of saline, epibatidine, or cyti-sine, respectively. Mice that received epibatidine orcytisine had significantly lower counts than those thatreceived saline. The differences started to reach statisti-cal significance at the second 5-min epoch after injec-tion of unlabeled test compounds ( p , 0.05) and be-came greater as the studies proceeded. Mice thatreceived cytisine tended to have lower counts thanthose that received epibatidine, but the differences didnot reach statistical significance in any of the 5-minmeasurements. After 24 h, radioactivity measured inthe brain did not differ from background.

Data from dissection experiments supported thoseobtained from the probe measurements. Mice thatreceived epibatidine or cytisine manifested signifi-cantly lower radioactivity than those that receivedsaline in all of the brain regions dissected except for thecerebellum. Mice that received cytisine tended to havelower counts than those that received epibatidine, butthe differences were not statistically significant (see theinset in Fig. 2, which shows radioactivity measured inthe thalamus).

This study demonstrates that [125I]IPH and a simpleprobe system can be used to assess binding to centralnAChRs in living small animals. The lack of detectableradioactivity after 24 h indicates that this compounddid not accumulate within the area assayed by the

probe. Therefore, repeated studies can be performedwith this radioligand on successive days without con-founding effects of residual radioactivity.

Although in vitro and ex vivo studies provide impor-tant information about nAChRs, studies using theprobe system and [125I]IPH in living small animalsprovide a new approach to the study of this receptorsystem, facilitating dynamic in vivo acquisition of dataand repeated studies. As emphasized by Sasaki et al.(1993), the probe approach is particularly useful whenthe questions being addressed do not demand a highdegree of anatomical resolution. With other advan-tages, such as low cost, high sensitivity, minimizing thenumber of animals required for a study, and relativelylong shelf-life of radioligand (several months), theprobe system and [125I]IPH represent a valuable andwidely available tool for the study of central nAChRs.

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380 X. LIU ET AL.