Comparative Biochemistry and Physiology, Part C 137 (2004) 349 – 353

www.elsevier.com/locate/cbpc

Muscarinic acetylcholine receptors in the brain of the zebrafish

(Danio rerio) measured by radioligand binding techniques
Frederick E. Williams *, William S. Messer Jr.
Department of Medicinal Chemistry, College of Pharmacy, University of Toledo, 2801 W. Bancroft 2237, Toledo, OH 43606, USA
Department of Pharmacology, College of Pharmacy, University of Toledo, 2801 W. Bancroft 2237, Toledo, OH 43606, USA
Received 2 October 2003; received in revised form 18 February 2004; accepted 8 March 2004

Abstract

Muscarinic acetylcholine receptors (mAChRs) play a role in learning, memory and behavior in vertebrate animals. We measured the
muscarinic cholinergic receptor levels in extracts from zebrafish (Danio rerio) brain by radioligand binding techniques. Saturation binding
experiments with the radioligand [3H]-quinuclidinyl benzilate (QNB) were used to determine receptor number and relative affinity for several
agonists and antagonists. Affinity at zebrafish brain receptors was relatively high with a Kd of 40 F 5 pM. The number of receptors,
represented by Bmax, was 63 F 16 fmol/mg protein. Oxotremorine and carbachol, agonists at muscarinic acetylcholine receptors, bound with
displacement curves indicating multiple binding sites. In addition, oxotremorine bound with a higher affinity than did carbachol. The
antagonist potency profile at zebrafish receptors in brain was determined to be atropineHpirenzipine>p-fluoro-hexahydro-sila-
difenidolHotenzepad. The results obtained with zebrafish brain compare favorably to those found in insect, fish and mammalian species.
Taken together, the binding results and favorable comparisons to mammalian systems indicate that zebrafish may provide a useful model
organism for evaluating the role of cholinergic systems in learning, memory and behavior.
D 2004 Elsevier Inc. All rights reserved.

Keywords: Brain; Zebrafish; Muscarinic; Cholinergic; Receptors; Radioligand; Binding; Agonist; Antagonist

1. Introduction The zebrafish (Danio rerio) represents a potentially

Muscarinic acetylcholine receptors (mAChRs) are known
to be at least partially responsible for mediating many
functions in the brain regarding behavior, particularly learn-
ing and memory (Thomas and Gash, 1986; Messer et al.,
1990, 1991). The cholinergic system has been implicated as
important in several medical disorders including Alzheim-
er’s disease (Levy et al., 2000; Mega, 2000) and autism
(Perry et al., 2001), as well as disorders arising from
exposure to environmental toxins. Changes in the choliner-
gic system were also noted in larval trout that were exposed
to carbaryl pesticides (Beauvais et al., 2001). These changes
were positively correlated to behavioral changes in exposed
animals. The cholinergic system would be a useful tool in the
development of an animal model for looking at behavior and
correlating this to receptor binding and activation.
* Corresponding author. Department of Pharmacology, College of
Pharmacy, University of Toledo, 2801 West Bancroft 2237, Toledo, OH
43606, USA. Tel.: +1-419-530-1991; fax: +1-419-530-1909.
E-mail address: fwilia2@utnet.utoledo.edu (F.E. Williams).
useful organism to be used in developing animal models
of many disorders, due to its size, ease of handling and
powerful system of genetics. In addition, zebrafish may be
utilized to ascertain functional differences among animals
using saturation genetics (Jiang et al., 1996; Schier et al.,
1996) and reverse genetics (Linney et al., 1999). An assay
system for muscarinic receptors and their function in zebra-
fish brain would assist in identifying changes induced in the
cholinergic system of the zebrafish, and potentially, tie these
changes in the cholinergic system to potential neurological/
behavioral problems in the zebrafish. Classic in situ staining
using antibodies could only be part of an analysis of such a
system because an antibody will only show whether there is
protein expressed and does not identify if the protein is
functional; therefore, a binding and functional assay of the
cholinergic receptors and their activity would be beneficial
for studies of muscarinic acetylcholine receptors.
While mAChRs have been well characterized in mam-
malian tissues by pharmacological or molecular biological
means, few extensive studies exist regarding non-mamma-
lian vertebrate species (Hulme et al., 1990). Besides the

1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.cca.2004.03.002
350 F.E. Williams, W.S. Messer Jr. / Comparative Biochemistry and Physiology, Part C 137 (2004) 349–353
extensive mammalian work done, mAChRs have been
identified and studied in brain preparations of birds (Kohler
et al., 1995) and invertebrates such as the fruit fly, housefly,
cockroach and bee (Shapiro et al., 1989; Abdallah et al.,
1991; Orr et al., 1991). Brain mAChRs have been studied in
different fish such as rainbow trout, other trout species,
catfish, minnows, skate and the Torpedo using radiolabelled
binding techniques (Green and Dowdall, 1992; Hyashi and
Fuji, 1993; Nicholson et al., 1994; Jones and King, 1995;
Beauvais et al., 2001). The aim of this work is to charac-
terize muscarinic cholinergic receptor binding in the zebra-
fish brain so that the receptors may be used along with other
tests in developing the zebrafish as an animal model
addressing the cholinergic system.
2. Materials and methods
2.1. Materials
Adult zebrafish (D. rerio) were purchased from Ken’s
Pets (Toledo, OH). Buffers, Lowry reagents and other
miscellaneous chemicals were obtained from Fisher Scien-
tific. Tricaine methanesulfonate (MS222) for anesthesia was
obtained from Sigma. Universol ES scintillant and [3H]-
quinuclidinyl benzilate (QNB) were purchased from ICN.
Oxotremorine and p-fluoro-hexahydro-sila-difenidol were
from RBI. Pirenzipine and atropine were obtained from
Sigma, while the agonist carbachol was purchased from
Aldrich. The M2 antagonist, otenzepad, was courtesy of Dr.
Karl Thomae, GMBH. Fired GF/B filters for filtration of
final binding products were purchased from Brandel.
2.2. Tissue preparation
Zebrafish were euthanized by an overdose of the anes-
thetic tricaine methanesulfonate (MS 222). Zebrafish brains
were removed, snap frozen in liquid nitrogen and stored at
80 jC. Membranes were prepared as in Jones and King
(1995) with some modifications. Approximately 200 mg of
tissue (about 5 –10 adult frozen brains) was thawed in a tube
on ice with 5 ml ice-cold 50 mM Tris –HCl buffer at pH 8.
The tissue was homogenized in three 30-s bursts on a
Brinkmann polytron tissue homogenizer on setting 6. Ice-
cold Tris – HCl pH 8 was added to 25 ml and tissue
homogenates were centrifuged at 1500  g for 10 min.
The supernatant was resuspended in 25 ml ice-cold Tris –
HCl buffer (pH 8) and centrifuged at 33,000  g for 30 min.
The resulting pellet was resuspended in 2 ml of ice-cold
sterile dH2O and samples were removed for a protein
determination. Protein levels of brain membrane extracts
were determined by a modified Lowry method. Briefly, 10
and 20 Al of brain membrane extract homogenate was used
to determine approximate protein concentrations as mea-
sured by Lowry et al. (1951) using a bovine serum albumin
standard curve.
2.3. Binding assays
The binding assay for mAChRs was performed as
previously described (Huang et al., 1998) with some mod-
ifications. Briefly, approximately 200 Al of partially purified
brain-derived membranes (equivalent to approximately 70
Ag of protein) were mixed with buffer (50 mM Tris –HCl,
pH 8) and increasing concentrations of [3H]QNB to measure
total binding. Binding reactions were carried out for 2 h at
RT. Termination of binding occurred with rapid filtration
through Brandel GF/B fired glass fiber filters using a
Brandel M24-R cell harvester. Filters were rinsed three
times with 5 ml of ice-cold Tris – HCl buffer. Filters con-
taining membranes and bound [3H]QNB were transferred to
vials containing 6 ml Universol scintillation fluid. Vials
were counted using a TM-Analytic BetaTrac counter. Non-
specific binding was defined as binding of [3H]QNB in the
presence of a 1000-fold excess of unlabeled QNB. Specific
binding was estimated as the difference between total and
non-specific binding.
Inhibition binding experiments were carried out using 0.1
nM [3H]QNB and increasing concentrations from 1 pM to 1
mM of each competing drug. All other incubation, filtration
and scintillation procedures were as above.
Data were analyzed and graphed using Deltagraph ver-
sion 5.0.1 for Windows. 3H-QNB saturation binding data
were fitted to a one site-binding model in order to determine
Bmax and Kd values. Inhibition binding data were fit into one-
or two-site binding models. Statistics were carried out with
an F-test with P set at 0.05. Ki values were calculated from
IC50 values using the methods of Cheng and Prusoff (1973).
3. Results
3.1. Saturation binding experiments
Saturation binding of [3H]QNB to brain membranes (Fig.
1) indicated a Bmax of the muscarinic acetylcholine receptors
Fig. 1. Saturation binding curve of [3H]-QNB binding to brain membranes
from zebrafish (D. rerio). Points represent an average specific binding at
various concentrations (in pM) of [3H]-QNB determined for three separate
experiments. Error bars represent S.E.M.
 F.E. Williams, W.S. Messer Jr. / Comparative Biochemistry and Physiology, Part C 137 (2004) 349–353
Fig. 2. Agonist inhibition binding curve of [3H]-QNB binding using
zebrafish (D. rerio) brain. (.) Oxotremorine, (5) carbachol. Points
represent an average of three experiments. Error bars represent S.E.M.
of 63 F 16 fmol/mg protein. The zebrafish brain-derived
membranes bound [3H]QNB with a relatively high affinity,
having a Kd of 40 F 5 pM.
3.2. Inhibition binding experiments
Inhibition of the binding of [3H]QNB to brain-derived
membranes was assessed with the two agonists, carbachol
and oxotremorine (Fig. 2) and four antagonists (Fig. 3). All
data are summarized in Table 1. The data from experiments
involving each of the agonists were fit to a two-site curve.
The order of potency of the agonists was oxotremorine>
carbachol. Oxotremorine data showed that 43% of the total
binding sites were of high affinity compared to only 30%
with carbachol. Binding affinities with oxotremorine indi-
cated a high binding affinity Ki of 140 F 11 nM as com-
pared with a low binding affinity Ki of 8.5 F 0.8 AM.
Carbachol displaced [3H]QNB with Ki values of 390 F 32
nM and 33 F 1.1 AM, respectively.
Fig. 3. Antagonist inhibition binding curve of [3H]-QNB binding using
zebrafish (D. rerio) brain. (n) Atropine, (.) pirenzipine, (E) pfHS-
difenidol, (y) otenzepad. Points represent an average of three experiments.
Error bars represent S.E.M.
351
Table 1
Inhibition of [3H]QNB binding by agonists and antagonists of muscarinic
receptors in brain-derived membranes from zebrafish (D. rerio)
Drug used Ki (nM)
H L
Agonists
Carbachol 390 F 32 (43) 33,000 F 1120
Oxotremorine 140 F 11 (30) 8500 F 800
Antagonists
Atropine 1.02 F 0.36
Pirenzipine 31.0 F 7.2
PfHS-difenidol 70 F 10.1
Otenzepad 850 F 103.2
Figures enclosed by ( ) indicate percent receptors in high affinity binding
form. Data represent mean F S.E.M. Each experiment was performed in
triplicate.
Four antagonists were examined for inhibition of the
binding of [3H]QNB to zebrafish brain membranes (Fig. 3).
Atropine, pirenzipine, otenzepad and p-fluoro-hexahydro-
sila-difenidol inhibited the binding of [3H]QNB to zebrafish
brain-derived membranes in a dose dependent manner. The
order of potency of the antagonists was atropineHpirenzi-
pine>p-fluoro-hexahydro-sila-difenidolHotenzepad. The
average Kis of the antagonists, shown in Table 1, were
averages of three separate experiments. The Kis found for
zebrafish membranes in this work are comparable to those
found for other animals in the literature.
4. Discussion
In this study, we used the radioligand [3H]QNB, a non-
selective muscarinic antagonist, to characterize the mAChRs
in the brain of the zebrafish (D. rerio). The [3H]QNB bound
with a high affinity to membranes derived from zebrafish
brain. The Kd of 40 pM achieved from saturation binding
experiments with zebrafish brain membranes agrees well
with the Kd of 47 pM found in carp (Szabo et al., 1989), and
is comparable to the Kd of 96 pM found in brook trout
(Jones and King, 1995), as shown in Table 2. The closeness
of the Kd values of zebrafish and carp could be expected
since Danio and Cyprinus are genera in the same family of
bony fish. The comparison of the Kd values of Danio and
Salvelinus still indicate closeness to each other compared to
other non-related species (Fig. 1), although Salvelinus and
Danio are in different orders. The muscarinic acetylcholine
receptor density in Danio was found to be 63 fmol/mg
protein as compared to 166 fmol/mg in brook trout, 470
fmol/mg in goldfish (Francis and Schechter, 1980) and 630
fmol/mg in carp. The observed Bmax value was comparable
to that in brook trout; however, it was considerably less
dense than those reported for goldfish and carp, showing
differences within the Cyprinus order of minnows. Goldfish
and carp are considered bottom feeders, whereas brook trout
and zebrafish could be considered mid-water fish. This may
352 F.E. Williams, W.S. Messer Jr. / Comparative Biochemistry and Physiology, Part C 137 (2004) 349–353

Table 2 Table 4
Comparison of muscarinic acetylcholine receptor binding in brain tissues in Antagonist inhibition of binding at brain-derived muscarinic acetylcholine
non-mammalian species receptors across various species
Species Kd (nM) Bmax Reference Antagonist Species Ki (nM) for brain Radioligand
Protein Radioligand Atropine Carp 0.55a QNB
(fmol/mg) Goldfish 6.4b,c QNB
Carp 0.047 627 QNB Szabo et al. (1989) Brook trout 0.07d NMS
Brook trout 0.096 166 NMS Jones and King (1995) Zebrafish 1.02e QNB
Cockroach 1.7f QNB
Zebrafish 0.04 63 QNB This study
Honeybee 0.47 108 QNB Abdallah et al. (1991) Housefly 1.0f QNB
Housefly 0.17 65 QNB Abdallah et al. (1991) Honeybee 5.1f QNB
Cockroach 0.13 108 QNB Abdallah et al. (1991) Mouse, neonate 0.26g QNB
Mouse, adult 0.2g QNB
Human, fetal 2.2b,h NMS
explain the difference in densities of muscarinic acetylcho- Human, adult 0.5i QNB
line receptors among these animals. More work on other Pirenzipine Carp 61a QNB
Brook trout 9d NMS
animals in these two categories would be required to Zebrafish 31e QNB
validate this hypothesis. Cockroach 900e, 220j QNB
The pharmacological profile of zebrafish receptors de- Housefly 484f QNB
rived from brain tissue fits very well with profiles from Honeybee 675f QNB
mammalian and other non-mammalian sources. The ago- Mouse, neonate 34g QNB
Mouse, adult 5.7g QNB
nists, oxotremorine and carbachol, were used in inhibition Bovine 18k QNB
binding assays and oxotremorine showed higher affinity for Human, fetal 310b,h NMS
the receptor than did carbachol (see Table 1). This is a trait a
Szabo et al. (1989).
that is seen in brain muscarinic receptors from brook trout as b
IC50.
c
well (see Table 3), indicating that oxotremorine is more Francis and Schechter (1980).
d
potent than carbachol at zebrafish muscarinic receptors as it Jones and King (1995).
e
This study.
has been reported in other species. The antagonist data f
Abdallah et al. (1991).
shows an inhibition profile of atropineHpirenzipine>p- g
Evans et al. (1985).
fluoro-hexahydro-sila-difenidolHotenzepad. This profile h
Aguilar and Lunt (1985).
i
is consistent with the predominance of muscarinic acetyl- Larson et al. (1991).
j
choline receptors in the zebrafish brain being of the M1 Orr et al. (1991).
k
Katz and Marquis (1989).
receptor subtype. Inhibition binding assay data using the
antagonists atropine and pirenzipine are consistent with
other studies and are shown in Table 4. The Ki value of
atropine at receptor proteins derived from zebrafish brain is properties of the muscarinic receptors of the brain may be
1.02 nM, quite close to numbers generated at similar quite similar from fish to mammals. Pirenzipine binding
receptors in both vertebrates and invertebrates. This Ki value data indicate that the Ki at zebrafish brain receptors is 31
for atropine agrees very well with those of carp (0.55 nM) nM. This is in good agreement with carp (61 nM), brook
and goldfish (IC50 = 6.4 nM), indicating that the receptor trout (9 nM), mouse (5.7 nM), mouse neonate (34 nM) and
properties appear similar in all of these fish. However, the bovine (18 nM) studies. The comparison of muscarinic
zebrafish Ki of 1.02 nM for atropine also relates quite well receptors across these different species suggest that zebra-
with numbers generated for mouse (0.2 nM), mouse fetus fish could be useful as a model organism for studying
(0.26 nM), human (0.5 nM) and human fetal tissue muscarinic acetylcholine receptors in the brain and perhaps
(IC50 = 2.2 nM). This may indicate that the pharmacological disease states or syndromes associated with changes in
muscarinic receptors and/or cholinergic activity.

Table 3
Agonist inhibition of binding at brain-derived muscarinic acetylcholine
receptors across various species References
Agonist used organism H (AM) (%) L (AM) H/L Radioligand
Abdallah, E.A.M., Eldefrawi, M.E., Eldefrawi, A.T., 1991. Pharmacologic
Carbachol characterization of muscarinic receptors of insect brains. Arch. Insect
Zebrafish 0.39 (43) 33 0.012 QNB Biochem. Physiol. 17, 107 – 118.
Brook trout 0.11 (37) 7.2 0.014 NMS Aguilar, J., Lunt, G., 1985. Muscarinic receptor sites in human foetal brain.
Cockroach 7.5 (50) 149 0.05 QNB Neurochem. Int. 7, 509 – 514.
Oxotremorine Beauvais, S., Jones, S., Parris, J., Brewer, S., Little, E., 2001. Cholinergic
Zebrafish 0.14 (30) 8.5 0.017 QNB and behavioral neurotoxicity of carbaryl and cadmium to larval rainbow
Brook trout 0.005 (29) 0.34 0.016 NMS trout (Onchorhyncus mykiss). Ecotoxicol. Environ. Saf. 49, 84 – 90.
Human fetus 0.008 (52) 0.96 0.009 NMS Cheng, Y., Prusoff, W., 1973. Relationship between the inhibition con-
 F.E. Williams, W.S. Messer Jr. / Comparative Biochemistry and Physiology, Part C 137 (2004) 349–353
stant (KI) and the concentration of inhibitor which causes 50 percent
inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22,
3099 – 3108.
Evans, R., Watson, M., Yamamura, H., Roeske, R., 1985. Differential
ontogeny of putative M1 and M2 muscarinic receptor binding sites in
the murine cerebral cortex and heart. J. Pharmacol. Exp. Ther. 235,
612 – 618.
Francis, A., Schechter, N., 1980. Regional and subcellular distribution of
cholinergic enzyme and receptor activity in goldfish brain. Neurosci-
ence 5, 293 – 301.
Green, A., Dowdall, M., 1992. Muscarinic autoreceptors of Torpedo elec-
tric organ are of the M1 subtype: evidence by radioligand binding using
selective antagonists. J. Neurochem. 58, 478 – 484.
Huang, X., Williams, F., Peseckis, S., Messer Jr., W.S., 1998. Pharmaco-
logical characterization of human m1 muscarinic acetylcholine recep-
tors with double mutations at the junction of TM VI and the third
extracellular domain. J. Pharmacol. Exp. Ther. 286, 1129 – 1139.
Hulme, E., Birdsall, N., Buckley, N., 1990. Muscarinic receptor subtypes.
Annu. Rev. Pharmacol. Toxicol. 30, 633 – 673.
Hyashi, H., Fuji, R., 1993. Muscarinic cholinoreceptors that mediate ag-
gregation are present in melanophores of cyprinids (Zacco spp.). Pig-
ment Cell Res. 6, 37 – 44.
Jiang, Y., Brand, M., Heisenberg, C., Beuchle, D., Furatani-Seiki, M.,
Kelsh, R., Warga, R., Granato, M., Haffter, P., Hammerschmidt, M.,
Kane, D., Mullins, M., Odenthal, J., van Eeden, F., Nusslein-Volhard,
C., 1996. Mutations affecting neurogenesis and brain morphology in the
zebrafish (Danio rerio). Development 123, 205 – 216.
Jones, S.B., King, L.B., 1995. Muscarinic cholinergic receptors in brain
and atrial membranes of adult brook trout (Salvelinus fontinalis) mea-
sured by radioligand binding techniques. Comp. Biochem. Physiol.
C112, 43 – 50.
Katz, L., Marquis, J., 1989. Modulation of central muscarinic receptor
binding in vitro by ultralow levels of the organophosphate paraoxon.
Toxicology and Applied Pharmacology 101, 114 – 123.
Kohler, E., Messer Jr., W.S., Bingman, V., 1995. Evidence for muscarinic
acetylcholine receptor subtypes in the pigeon telencephalon. J. Comp.
Neurol. 362, 271 – 282.
Larson, E., Pfenning, M., Richelson, E., 1991. Selectivity of antimuscarinic
compounds for muscarinic receptors of human brain and heart. Psycho-
pharmacology 103, 162 – 165.
Levy, J., Parasuraman, R., Greenwood, P., Dukoff, R., Sunderland, T.,
353
2000. Acetylcholine affects the spatial scale of attention: evidence from
Alzheimer’s disease. Neuropsychology 14, 288 – 298.
Linney, E., Hardison, N., Lonze, B., Lyons, S., DiNapoli, L., 1999. Trans-
gene expression in zebrafish: a comparison of retroviral-vector and
DNA-injection approaches. Dev. Biol. 213, 207 – 216.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein
measurement with the Folin phenol reagent. J. Biol. Chem. 193,
265 – 275.
Mega, M.S., 2000. The cholinergic deficit in Alzheimer’s disease: impact
on cognition, behaviour and function. Int. J. Neuropsychopharmacol. 3,
3 – 12.
Messer Jr., W.S., Bohnett, M., Stibbe, J., 1990. Evidence for a preferential
involvement of M1 muscarinic receptors in representational memory.
Neurosci. Lett. 116, 184 – 189.
Messer Jr., W.S., Stibbe, J., Bohnett, M., 1991. Involvement of the septo-
hippocampal cholinergic system in representational memory. Brain Res.
564, 66 – 72.
Nicholson, L., Montgomery, J., Faull, R., 1994. GABA, muscarinic cho-
linergic, excitatory amino acid, neurotensin and opiate binding sites in
the octavolateralis column and cerebellum of the skate Raja nasuta
(Pisces: Rajidae). Brain Res. 652, 40 – 48.
Orr, G., Orr, N., Hollingsworth, R., 1991. Distribution and pharmacological
characterization of muscarinic – cholinergic receptors in the cockroach
brain. Arch. Insect Biochem. Physiol. 16, 107 – 118.
Perry, E., Lee, M., Martin-Ruiz, C., Court, J., Volsen, S., Merrit, J., Folly,
E., Iversen, P., Bauman, M., Perry, R., Wenk, G., 2001. Cholinergic
activity in autism: abnormalities in the cerebral cortex and basal fore-
brain. Am. J. Psych. 158, 1058 – 1066.
Schier, A., Neuhauss, S., Harvey, M., Malicki, J., Solnica-Krezel, L., Stain-
ier, D., Zwartkruis, F., Abdelilah, S., Stemple, D., Rangini, Z., Yang, H.,
Driever, W., 1996. Mutations affecting the development of the embry-
onic zebrafish brain. Development 123, 165 – 178.
Shapiro, R., Wakimoto, B., Subers, E., Nathanson, N., 1989. Characteriza-
tion and functional expression in mammalian cells of genomic and
cDNA clones encoding a Drosophila muscarinic acetylcholine receptor.
Proc. Natl. Acad. Sci. U. S. A. 86, 9039 – 9043.
Szabo, A., Nemcsok, J., Kasa, P., Gulya, K., 1989. Muscarinic cholinergic
components in the carp brain. Neurochem. Int. 15, 511 – 516.
Thomas, G., Gash, D., 1986. Differential effects of posterior septal lesions
on dispositional and representational memory. Behav. Neurosci. 100,
712 – 719.