Neuroscience 234 (2013) 53–68

BUTYRYLCHOLINESTERASE AND THE CHOLINERGIC SYSTEM

G. A. REID, a N. CHILUKURI b AND S. DARVESH a,c*

aDepartment of Medical Neuroscience Dalhousie University, Halifax,

Nova Scotia, CanadabResearch Division, Physiology & Immunology Branch, US

Army Medical Research, Institute of Chemical Defense,

Aberdeen Proving Ground, MD 21010, USA

cDepartment of Medicine (Neurology and Geriatric Medicine),

Dalhousie University, Halifax, Nova Scotia, Canada

Abstract—The cholinergic system plays important roles in

neurotransmission inboth theperipheral andcentral nervous

systems. The cholinergic neurotransmitter acetylcholine is

synthesized by choline acetyltransferase (ChAT) and its

action terminated by acetylcholinesterase (AChE) and butyr-

ylcholinesterase (BuChE). The predominance of AChE has

focused much attention on understanding the relationship

of this enzyme to ChAT-positive cholinergic neurons. How-

ever, there is ample evidence thatBuChEalsoplays an impor-

tant role in cholinergic regulation. To elucidate the

relationship of BuChE to neural elements that are producing

acetylcholine, the distribution of this enzyme was compared

to that of ChAT in the mouse CNS. Brain tissues from

0306-4522 � 2013 IBRO. Published by Elsevier Ltd.http://dx.doi.org/10.1016/j.neuroscience.2012.12.054

*Correspondence to: S. Darvesh, Department of Medical Neuroscience, DaVeterans’ Memorial Lane, Halifax, Nova Scotia, Canada B3H 2E1. Tel: +1-

E-mail address: sultan.darvesh@dal.ca (S. Darvesh).Abbreviations: AChE, acetylcholinesterase; Ad-moBuChE, adenovirus containAA, anterior amygdaloid area; ac, anterior commissure; ACo, anterior corticalAHP, anterior posterior hypothalamic nucleus; APT, anterior pretectal nucleusauditory cortex; BL, basolateral amygdala; BM, basomedial amygdala; BST,butyrylcholinesterase; CPu, caudate putamen nucleus; Ce, central amygdala;cortex; Cl, claustrum; cc, corpus collosum; DpMe, deep mesencephalic nuclegeniculate nucleus; 10N, dorsal motor nucleus of the vagus; DR, dorsal raimmunosorbent assay; En, endopiriform cortex; ec, external capsule; ECu, exfasciculus retroflexus; FBS, fetal bovine serum; fi, fimbria; f, fornix; g7, genupallidus; H, hippocampal formation; HC, histochemical; HDB, horizontabutyrylcholinesterase; 12N, hypoglossal nucleus; IgG, immunoglobulin G; icpnucleus; IS, inferior salivatory nucleus; I, insular cortex; InG, intermediate graintermediate white layer of the superior colliculus; ic, internal capsule; IP, intCalleja; La, lateral amygdala; LDTg, lateral dorsal tegmental nucleus; LD,hypothalamus; LPGi, lateral paragigantocellular nucleus; LP, lateral posterior tlateral vestibular nucleus; LC, locus coeruleus; MCPO, magnocellular preopticMD, medial dorsal thalamus; MEnt, medial entorhinal cortex; MG, medial geniclongitudinal fasciculus; MS, medial septal nucleus; MT, medial terminal nucleuraphe nucleus; MiTg, microcellular tegmental nucleus; M, motor cortex; Mo5,nigrostriatal bundle; A5, noradrenaline cell group A5; LL, nucleus of lateralhypothalamic nucleus; PPTg, pedunculopontine tegmental nucleus; PAG, periaPir, piriform cortex; PoDG, polymorphic cell layer of the dentate gyrus; Pn, pcommissure; PH, posterior hypothalamic nucleus; PMCo, posteromedial corticanucleus, ventral; PO, preoptic nucleus; Pr, prepositus nucleus; Pr5, principal stract; rmoBuChE, recombinant mouse BuChE; R, red nucleus; Rt, reticular ncortex; Re, reuniens; RI, rostral interstitial nucleus of mlf; RPO, rostral paraosodium dodecyl sulfate–polyacrylamide gel electrophoresis; sol, solitary tract;Sp5OVL, spinal trigeminal nucleus, ventrolateral oralis; sp5, spinal trigeminamedullaris; st, stria terminalis; SubC, subcoeruleus nucleus; SMT, submammillosuperior cerebellar peduncle; str, superior thalamic radiation; SuVe, superitectospinal tract; TeA, temporal association cortex; TMB, 3,30,5,50-tetramethylbeanterior thalamic nucleus; VLG, ventral lateral geniculate; VP, ventral pallidumband of Broca; V, visual cortex; ZI, zona incerta.

53

Open access under CC BY-NC-N

129S1/SvImJ mice were stained for BuChE and ChAT using

histochemical, immunohistochemical and immunofluorescent

techniques. Both BuChE and ChAT were found in neural

elements throughout theCNS.BuChEstainingwith histochem-

istry and immunohistochemistry produced the same distribu-

tion of labeling throughout the brain and spinal cord.

Immunofluorescent double labeling demonstrated that many

nuclei in themedulla oblongata, aswell as regions of the spinal

cord, had neurons that contained both BuChE and ChAT.

BuChE-positive neurons without ChAT were found in close

proximitywithChAT-positive neuropil in areas suchas the thal-

amus and amygdala. BuChE-positive neuropil was also found

closely associated with ChAT-positive neurons, particularly

in tegmental nuclei of the pons. These observations

provide further neuroanatomical evidenceof a role forBuChE

in the regulation of acetylcholine levels in the CNS.

� 2013 IBRO. Published by Elsevier Ltd.

Key words: cholinesterases, acetylcholinesterase, pseudo-

cholinesterase, choline acetyltransferase, 129S1/SvImJ mice,

acetylcholine.

Open access under CC BY-NC-ND license.

lhousie University, Room 1308, Camp Hill Veterans’ Memorial, 5955902-473-2490; fax: +1-902-473-7133.

ing the gene for mouse butyrylcholinesterase; Amb, ambiguus nucleus;amygdala; AD, anterior dorsal thalamus; AO, anterior olfactory cortex;; ATg, anterior tegmental nucleus; AV, anterior ventral thalamus; Au,bed nucleus of stria terminalis; BSA, bovine serum albumin; BuChE,cp, cerebral peduncle; ChAT, choline acetyltransferase; Cg, cingulateus; DAB, 3,30-diaminobenzidine tetrahydrochloride; DLG, dorsal lateralphe nucleus; DTg, dorsal tegmental nucleus; ELISA, enzyme-linkedternal cuneate; eml, external medullary lamina; 7N, facial nucleus; fr,of the facial nucleus; Gi, gigantocellular reticular nucleus; GP, globusl limb nuclei of the diagonal band of Broca; hBuChE, human, inferior cerebellar peduncle; IC, inferior colliculus; IO, inferior olivaryy layer of superior colliculus; IRt, intermediate reticular nucleus; InWh,erpeduncular nucleus; InC, interstitial nucleus of Cajal; ICj, islands oflateral dorsal thalamus; LEnt, lateral entorhinal cortex; LH, lateral

halamus; LRt, lateral reticular nucleus; LS, lateral septal nucleus; LVe,nucleus; mt, mammillothalamic tract; MeA, medial anterior amygdala;

ulate nucleus; MHb, medial habenula; ml, medial lemniscus; mlf, medials of the accessory tract; MVe, medial vestibular nucleus; MnR, medianmotor trigeminal nucleus; moBuChE, mouse butyrylcholinesterase; ns,lemniscus; Tu, olfactory tubercle; opt, optic tract; Pa, paraventricularqueductal gray; PB, phosphate buffer; PBS, phosphate-buffered saline;ontine nuclei; PnO, pontine reticular nucleus, oral part; pc, posteriorl amygdala; PMD, premammillary nucleus, dorsal; PMV, premammillaryensory trigeminal nucleus; Po, pulvinar thalamic nucleus; py, pyramidalucleus; RtTg, reticulotegmental nucleus of the pons; RS, retrospleniallivary nucleus; rs, rubral spinal tract; S, sensory cortex; SDS–PAGE,Sol, solitary tract nucleus; Sp5I, spinal trigeminal nucleus, interpolaris;l tract; SVe, spinal vestibular nucleus; Or, stratum oriens; sm, striathalamic nucleus; SI, substantia innominata; SN, substantia nigra; scp,or vestibular nucleus; SuMM, supramammillary, medial nucleus; ts,nzidine; m5, trigeminal nerve root; TBS, tris-buffered saline; VA, ventral; VTA, ventral tegmental area; VDB, vertical limb nuclei of the diagonal

D license.

54 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

INTRODUCTION

Cholinergic neurotransmission in the mammalian CNS is

regulated predominantly by the enzyme

acetylcholinesterase (AChE, EC 3.1.1.7) by catalyzing

the hydrolysis of the cholinergic neurotransmitter

acetylcholine (Silver, 1974). Improved histochemical

techniques (Koelle and Friedenwald, 1949) for detecting

this enzyme led to the first map of the distribution of

AChE in the rodent brain (Shute and Lewis, 1963).

Subsequently, antibodies were developed to detect

choline acetyltransferase (ChAT, EC 2.3.1.6), the

enzyme that catalyzes the synthesis of acetylcholine

(Eng et al., 1974). This permitted elucidating the

organization of cholinergic neurons (Kimura et al., 1980;

Armstrong et al., 1983). Combined AChE histochemical

and ChAT immunohistochemical staining studies

demonstrated that most ChAT-positive neurons were

also AChE-positive. (Eckenstein and Sofroniew, 1983;

Levey et al., 1983, 1984).

In addition to AChE, the enzyme butyrylcholinesterase

(BuChE, EC 3.1.1.8) is important in the regulation of the

cholinergic system (Darvesh et al., 1998, 2003;

Mesulam et al., 2002b; Giacobini, 2003; Duysen et al.,

2007). Like AChE, BuChE is able to efficiently catalyze

the hydrolysis of acetylcholine (Silver, 1974). BuChE is

expressed in distinct populations of neurons, some of

which also contain AChE (Friede, 1967; Tago et al.,

1992; Darvesh et al., 1998; Darvesh and Hopkins, 2003;

Geula and Nagykery, 2007). The importance of BuChE

in cholinergic neurotransmission is further supported by

the observation that AChE-knockout mice survive to

adulthood. This indicates BuChE is able to compensate

for the lack of AChE, allowing the continued regulation

of cholinergic neurotransmission (Li et al., 2000; Xie

et al., 2000; Mesulam et al., 2002a).

To date, study of the colocalization of BuChE and

ChAT in the mammalian CNS has been limited to the

spinal cord in the rat (Mis, 2005). Because of the earlier

indications of BuChE co-regulation of cholinergic

neurotransmission (Xie et al., 2000; Mesulam et al.,

2002a,b), the present work was undertaken to examine

the organization of BuChE-expressing neural elements

as they relate to the ChAT-defined cholinergic system in

the mouse CNS.

This work has not been presented elsewhere except

in abstract form (Darvesh et al., 2012b).

EXPERIMENTAL PROCEDURES

Animals

Twenty male, wild-type (129S1/SvImJ) mice were purchased

from The Jackson Laboratory (USA). This mouse strain was

chosen because it has been utilized to examine components of

the cholinergic system in other studies (Li et al., 2000;

Mesulam et al., 2002a; Duysen et al., 2007). Animals were

cared for according to the guidelines set by the Canadian

Council on Animal Care. Formal approval to conduct the

experiments was obtained from the Dalhousie University

Committee on Laboratory Animals.

Materials

Unless otherwise stated, all reagents were purchased from

Sigma–Aldrich (St. Louis, MO).

Preparation of brain tissue

Mice (between 10 and 18 weeks old) were deeply anesthetized

with an intra-peritoneal injection of sodium pentobarbital

(200 mg/kg) and perfused with approximately 25 ml of 0.9%

saline solution containing 0.1% sodium nitrite followed by 50 ml

of 0.1 M phosphate buffer (PB, pH 7.4) containing 4%

paraformaldehyde. Brains were removed and post-fixed in PB

with 4% paraformaldehyde for 1–2 h, cryoprotected and stored

in PB with 30% sucrose and 0.05% sodium azide. Brains were

cut in 40-lm serial sections in a coronal plane on a Leica

SM2000R microtome with Physitemp freezing stage and BFS-

30TC controller. Sections were stained for BuChE or AChE by

histochemical (HC) technique, and for BuChE and ChAT using

immunohistochemical (IHC) methods. Double labeling for

BuChE and ChAT was performed using immunofluorescence

(IF).

Cholinesterase histochemistry

Cholinesterase histochemical staining was performed using a

modified (Darvesh et al., 2012a) Karnovsky–Roots method

(Karnovsky and Roots, 1964). Briefly, tissue sections were

rinsed in 0.1 M maleate buffer (pH 7.4) for 30 min and

incubated for 1 h 45 min in 0.1 M maleate buffer (pH 8.0)

containing 0.5 mM sodium citrate, 0.47 mM cupric sulfate,

0.05 mM potassium ferricyanide, 0.8 mM butyrylthiocholine

iodide and 0.01 mM BW 284 C 51 (to inhibit AChE). All

sections were then rinsed with gentle agitation for 30 min in

dH2O and placed in 0.1% cobalt chloride in water for 10 min.

After further rinsing in dH2O, sections were placed in PB

containing 1.39 mM 3,30-diaminobenzidine tetrahydrochloride

(DAB). After 5 min in the DAB solution, 50 ll of 0.15% H2O2 in

dH2O was added per ml of DAB solution, and the reaction was

carried out for approximately 3 min. Sections were then washed

in 0.01 M acetate buffer (pH 3.3), mounted on slides, cleared in

xylene and cover-slipped. Control experiments to demonstrate

specificity of BuChE staining were performed as described

previously (Darvesh et al., 1998) and indicated the staining

pattern observed was specific for BuChE activity.

The procedure for the visualization of AChE activity was

similar to that for BuChE except that the reaction solution was

24.99 mM sodium citrate, 14.72 mM cupric sulfate, 2.43 mM

potassium ferricyanide, 2.46 mM acetylthiocholine iodide and

0.13 mM ethopropazine (to inhibit BuChE), at room temperature

in 0.1 M maleate buffer (pH 6.0) for 15 min with gentle agitation.

Generation and characterization of polyclonalantibodies to BuChE

Rabbit polyclonal antibodies to recombinant mouse BuChE

(rmoBuChE) were generated for this study. Production of

rmoBuChE (immunogen) was accomplished in human

embryonic kidney epithelial cells (293A cells) using an

adenovirus containing the gene for mouse BuChE (Ad-

moBuChE) (Parikh et al., 2011). The mouse BuChE

(moBuChE) gene contained a 6x histidine tag at its carboxyl

terminus suggesting that the rmoBuChE used was a fusion

protein. Large-scale expression of rmoBuChE was

accomplished by overnight culturing of 293A cells (10 � 106) in

150-cm2 tissue culture dishes and infecting them for 1 h with

10 ll of 4th cycle crude viral lysate (high titer CVL) of Ad-

moBuChE in 10 ml of infection medium (DMEM containing 2%

fetal bovine serum (FBS), antibiotics penicillin and streptomycin

G. A. Reid et al. / Neuroscience 234 (2013) 53–68 55

and sodium pyruvate) at 37 �C. Fifteen ml of growth medium

(DMEM containing 10% FBS, 50 lg/ml penicillin and

streptomycin, and 50 lg/ml sodium pyruvate) was then added

and plates were returned to the incubator for 5–7 days.

Seventy to one hundred culture dishes were infected in a single

experiment. When BuChE activity in the culture medium

reached between 3–4 units/ml, it was collected and cleared of

cells and debris by centrifugation at 2500 rpm for 15 min, 4 �C.Culture media containing �60,000 units (or 70 mg) of the

recombinant enzyme was used for purification of rmoBuChE.

Ellman assay (Ellman et al., 1961), using butyrylthiocholine as

substrate was employed to monitor moBuChE activity during

various steps of the enzyme purification.

The purification scheme for rmoBuChE involved ammonium

sulfate fractionation followed by affinity chromatography using

procainamide and nickel-affinity resins. The culture media was

fractionated with ammonium sulfate (0–50% saturation). The

precipitate was collected by centrifugation at 10,000 rpm for

15 min and discarded as the supernatant contained all the

enzyme activity. Ammonium sulfate was added to the

supernatant to 80% saturation and the precipitate containing

the entire recombinant enzyme was collected as described

above. The enzyme was dissolved in 100 ml of 50 mM PB (pH

8.0), desalted by dialysis against 30 volumes of the same

buffer and the solution was then applied to a 50-ml column of

procainamide Sepharose equilibrated with the same buffer. The

bound enzyme was eluted with 50 mM PB (pH 8.0) containing

1 M NaCl and 0.2 M choline chloride. The active fractions were

combined and dialyzed against 25 mM PB (pH 8.0) containing

500 mM NaCl and 2.5 mM imidazole and loaded onto a 10-ml

column of nickel Sepharose resin equilibrated against the same

buffer. The bound enzyme was eluted with 25 mM PB (pH 8.0)

containing 500 mM NaCl and 500 mM imidazole. The enzyme

fractions were combined and dialyzed against 25 mM PB (pH

8.0). For long-term storage of the enzyme, glycerol was added

(50%) and stored in �20 �C freezer.

Polyclonal antibodies to rmoBuChE were produced in two

New Zealand white rabbits according to standard polyclonal

antibody production protocols (Washington Biotechnology Inc.,

6200 Seaforth Street, Baltimore, MD 21224). Three mg of the

purified rmoBuChE was supplied to Washington Biotechnology

as lyophilized powder for antibody development. The protocol

was approved by the Institutional Animal Care and Use

Committee of the Johns Hopkins University, Baltimore,

Maryland. Sera collected after the booster injections were used

for purification of anti-moBuChE immunoglobulin G (IgG) by

protein A/G chromatography. Sera were diluted with

phosphate-buffered saline (PBS, 1:10 dilution; pH 7.4) and

loaded onto a 5-ml column of protein A/G Sepharose beads.

The bound IgG were eluted with 50 mM glycine–HCl (pH 2.7)

and fractions containing IgG were neutralized using 1 M dibasic

PB (pH 7.5). Later, sodium azide and glycerol were added to

final concentrations of 0.05% and 10%, respectively for long-

term storage of the antibody.

The reactivity of anti-moBuChE IgG toward rmoBuChE or

human BuChE (hBuChE) was determined by Western blotting.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

(SDS–PAGE) was carried out for 100 ng of each enzyme using

precast 10% Tris–HCl gels. One microgram of bovine serum

albumin (BSA) was included in the sample to prevent non-

specific loss of the enzyme. After electrophoresis, proteins

were transferred to nitrocellulose membrane using IBlot gel

transfer apparatus (InVitrogen, CA). The membrane was

blocked in blocking buffer (Licor Inc., NE) for 2 h at 24 �C,washed once with 50 mM phosphate buffer containing 0.05%

Tween 20 (washing buffer) and kept overnight in blocking buffer

containing anti-moBChE antibody (1:20,000 dilution). The

membrane was then washed five times with intermittent

shaking for 5 min and incubated with secondary antibody

conjugated with infra red dye 680 (Licor Inc., NE, 1:10,000

dilution) made in blocking buffer for 1 h, and protein bands

were detected using Infrared Imager (Licor Inc. NE). The

reactivity of anti-moBuChE IgG toward recombinant moBuChE

or hBuChE was also determined by direct enzyme-linked

immunosorbent assay (ELISA). One hundred ll of recombinant

moBuChE or hBuChE in PBS (5 lg/ml) was added to each well

of a 96-well plate and incubated overnight at 4 �C. After

washing the wells with PBS containing 0.05% Tween 20

(washing buffer), they were blocked with 3% BSA in PBS for

2 h at 24 �C. After washing the plate three times with washing

buffer, 100 ll each of five or fourfold serial dilutions (ranging

from 1:1000 to 1:4,000,000) of anti-moBChE IgG in PBS

containing 0.1% BSA was added and incubated for 2 h at

24 �C, followed by five washes with washing buffer. One

hundred ll of goat anti-rabbit IgG (H + L) conjugated with

horseradish peroxidase diluted 1:10,000 in PBS containing

0.1% BSA was then added to each well and incubated for 1 h

at 24 �C. After eight to 10 washes with washing buffer, 100 llof substrate solution containing 3,30,5,50-tetramethylbenzidine

(TMB) was added and incubated for 15 min in the dark at

24 �C. The reaction was stopped with the addition of 100 ll of2 N sulfuric acid and end point absorbance of each well was

measured in a plate reader at 450 nm. BSA blocked wells were

used as reagent controls.

Butyrylcholinesterase immunohistochemistry

For BuChE immunohistochemistry (IHC), sections were rinsed in

PB for 30 min and placed in 0.3% H2O2 in PB for 30 min to

quench endogenous peroxidase activity. Sections were rinsed

again in PB for 30 min and then incubated in PB containing

0.1% Triton X-100, normal goat serum (NGS, 1:100) and rabbit

anti-BuChE primary antibody (1:10,000) for approximately 16 h

at room temperature. After rinsing, sections were incubated in

PB with 0.1% Triton X-100, NGS (1:1000) and biotinylated goat

anti-rabbit secondary antibody (1:500; Vector) for 1 h. Sections

were rinsed in PB and then placed in PB with 0.1% Triton X-

100 and Vectastain� Elite ABC kit (Vector) according to the

manufacturer’s instructions. After rinsing the sections in PB,

they were placed in a solution of PB containing 1.39 mM DAB.

After 5 min, 50 ll of 0.3% aqueous H2O2 per mL of DAB

staining solution was added and incubated for 5 min. The

reaction was stopped by rinsing the sections in 0.01 M acetate

buffer (pH 3.3). Sections were mounted on slides, cleared in

xylene and cover-slipped.

Choline acetyltransferase immunohistochemistry

For ChAT-IHC, sections were rinsed in 0.05 M tris-buffered saline

(TBS; pH 7.6) for 30 min and placed in 0.3% H2O2 in TBS for

30 min to quench endogenous peroxidase activity. Sections

were rinsed again for 30 min and incubated in TBS containing

0.3% Triton X-100, normal rabbit serum (NRS, 1:100) and goat

anti-ChAT primary antibody (1:1000; Millipore, AB144P) for

about 16 h at room temperature. After rinsing, sections were

incubated in TBS containing 0.3% Triton X-100, normal rabbit

serum (1:1000) and biotinylated rabbit anti-goat secondary

antibody (1:500; Vector) for 1 h. After another rinse, sections

were placed in TBS containing 0.3% Triton X-100 and

Vectastain� Elite ABC kit according to the manufacturer’s

instructions. After rinsing, sections were placed in a solution of

TBS (pH 8.0) containing 1.39 mM DAB and 0.6% nickel

ammonium sulfate. After 5 min, 50 ll of 0.3% H2O2 in dH2O

was added per mL of DAB staining solution and the mixture

was incubated for 2–5 min. The reaction was stopped by

rinsing the sections in TBS. Sections were mounted on slides,

cleared in xylene and cover-slipped. Control experiments were

performed by omitting the primary antibody and no staining was

observed.

56 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

Butyrylcholinesterase and choline acetyltransferaseimmunofluorescence double labeling

Double labeling for BuChE and ChAT was done on the same

sections using an immunofluorescent method. Sections were

rinsed in TBS for 30 min and incubated for approximately 16 h

at room temperature in TBS containing 0.3% Triton X-100,

normal donkey serum (1:100) and both primary antibodies,

rabbit anti-BuChE (1:7000) and goat anti-ChAT (1:1000). After

rinsing in TBS, sections were incubated for 1 h in TBS

containing 0.3% Triton X-100, donkey anti-goat Alexa Fluor�

488 (1:350; Molecular Probes, A11055) and donkey anti-rabbit

Alexa Fluor� 555 (1:350; Molecular probes, A31572). Sections

were rinsed, mounted on slides, cleared in xylene and cover-

slipped.

Data analysis

Sections stained by HC, IHC or IF were analyzed on a Zeiss

Axioplan 2 microscope and photographed with a Zeiss Axiocam

HRc digital camera and AxioVision 4.6 software.

Photomicrographs of IF sections were also acquired on a Zeiss

LSM 510 laser scanning-confocal microscope. Orthogonal

images were rendered and edited with LSM imaging software

(Zeiss). Photographs were assembled using Adobe�

Photoshop� CS5. Images were contrast enhanced and the

brightness adjusted to match the background from different

images. Images of IF labeling for BuChE and ChAT were

merged to illustrate the relationship between these two

markers. BuChE-IF appeared green while ChAT appeared red.

Neural elements with both BuChE and ChAT appeared yellow

in merged images.

Anatomical maps of the distribution of BuChE or ChAT were

produced by plotting the neuronal distribution on a Wacom

drawing tablet with Microbrightfield Neuroleucida software

linked to a Microfire digital camera on an Olympus BMAX

microscope with Heidenhain linear potentiometers and Prior

Optiscan controller. The same sections were then

photographed at low power and assembled into a single image

using Adobe� Photoshop� CS5. The anatomical parcellation,

based on the mouse brain atlas by Paxinos and Franklin

(2001), and neuronal distributions were then redrawn using

Adobe� Illustrator� CS5 by overlaying and aligning the

neuroleucida drawings on the low power photomicrographs.

RESULTS

The present investigation was undertaken to elucidate the

relationships between BuChE-positive neural elements

and ChAT in the mouse CNS. Using HC and IHC

methods, the distributions of BuChE and ChAT neural

elements were analyzed. Colocalization of these two

markers was confirmed with immunofluorescent (IF)

double labeling.

Characterization of mouse BuChE antibodies

Analysis of rmoBuChE by reducing SDS–PAGE revealed

that it was a pure protein and the molecular weight of

85 kDa for its subunit was similar to that for purified

native hBuChE (Fig. 1A). Polyclonal moBuChE antibody

was characterized for its binding and specificity to

recombinant moBuChE and hBuChE by Western

blotting (Fig. 1B) and direct ELISA (Fig. 1C). Both

assays show that moBuChE antibody preferred binding

to moBuChE than hBuChE. By Western blotting,

antibody binding to moBuChE was 10-fold greater than

its binding to hBuChE (Fig. 1B). ELISA results also

showed a similar trend. Certain dilutions of the antibody

(1:25,000, 1:125,000, and 1:500,000) produced a

fourfold higher absorbance values with moBuChE

compared to hBuChE (Fig. 1C). The specificity of

moBuChE antibody was also confirmed by conducting

BuChE-IHC with the antibody following incubation with

BuChE from mouse serum. No staining was observed

indicating specificity of this antibody (Fig. 1D). All

together, these results demonstrate the specificity of this

antibody toward moBuChE.

Validation of BuChE immunohistochemical staining

The mouse BuChE antibody was evaluated for its ability

to recapitulate the distribution of BuChE activity through

established histochemical analysis. To this end,

sections, at representative levels throughout the brain,

were stained by BuChE-HC, BuChE-IHC or AChE-HC

and compared. Fig. 2 provides two examples, the

piriform cortex (Pir; Fig. 2A–C) and the motor trigeminal

nucleus (Mo5; Fig. 2D–F), that illustrate the

comparability of BuChE-HC (Fig. 2A, D) and BuChE-

IHC (Fig. 2B, E) staining and emphasizes differences in

distribution of this enzyme relative to that of AChE-HC

staining (Fig. 2C, F). These areas were chosen as

examples because of the distinct distribution of BuChE

and AChE neural elements that occur therein.

Throughout the brain, the patterns of staining for BuChE

were comparable whether HC or IHC was used, except

that neuropil staining by BuChE-HC was generally more

diffuse than that revealed by BuChE-IHC. These

experiments confirmed the ability of the BuChE antibody

to accurately visualize the location of BuChE that is

conventionally obtained by HC analysis.

Distribution of butyrylcholinesterase and cholineacetyltransferase

Within the mouse CNS, BuChE staining labeled distinct

populations of neurons, neuropil, glia and white matter

while ChAT labeled neurons and neuropil (see Fig. 3 for

examples). The glial perikarya were distinguished from

the neuronal perikarya based on their size and

morphology (Kettenmann and Ransom, 2005). Staining

patterns for each marker were consistent in all brains

examined. Comparative photomicrographs and

corresponding schematic maps (Fig. 4) of BuChE-HC

(left) and ChAT-IHC (right), show the presence of

stained neural elements in each region, proceeding from

the rostral to the caudal levels of the brain. In these

maps, each neuron stained for BuChE or ChAT is

represented by a black circle. Stained neuropil and

white matter, not dealt with in the maps, can be seen

directly for each marker in the corresponding

photomicrographs (Fig. 4). A summary of structures in

the mouse brain containing neuronal somata or neuropil

stained for BuChE and ChAT is presented in Table 1

with an estimate of soma numbers and density of

neuropil. In this tables, boxes shaded in gray indicate

nuclei that contain neurons stained for both BuChE and

ChAT as demonstrated by IF double labeling. The

Fig. 1. Characterization of recombinant mouse butyrylcholinesterase (rmoBuChE) and its antibodies. (A) Reducing sodium dodecyl sulfate–

polyacrylamide gel electrophoresis (SDS–PAGE) comparison of purified native human butyrylcholinesterase (hBuChE; Lane 1) to purified

rmoBuChE (Lane 2) revealed that rmoBuChE was a pure protein with the same molecular weight of 85 kDa as hBuChE. Lane M shows molecular

weight markers. (B) Western Blot showing 10-fold higher specificity of moBuChE antibodies to rmoBuChE (Lane 1) than with hBuChE (lane 2). (C)

Direct ELISA showing the binding curves of the antibody with rmoBuChE (line with squares), hBuChE (line with triangles), and bovine serum

albumin (BSA) (line with diamonds). Most dilutions of the antibodies gave significantly higher absorbance values with rmoBuChE compared with

hBuChE. (D) Butyrylcholinesterase immunohistochemistry in the mouse brain showing neuronal staining (top) and lack of such staining in the

adjacent section after preincubation of the moBuChE antibodies with serum moBuChE (bottom). Scale bar = 100 lm.

G. A. Reid et al. / Neuroscience 234 (2013) 53–68 57

distribution of BuChE- and ChAT-stained neural elements

in specific regions of the mouse brain is described below

in more detail.

Cerebral cortex. BuChE-positive neuronal somata

were scattered throughout the mouse cerebral cortex

(Table 1; Fig. 4A, C, E, G), as reported previously

(Mesulam et al., 2002a). Labeled neurons were more

numerous rostrally but generally were few in number

and located in layer 6 with an occasional neuron in

layers 1–2. On the other hand, the medial entorhinal

cortex (MEnt) had numerous BuChE-positive neurons

primarily located in layer 2 (Fig. 4I). There was also a

group of BuChE-positive neurons located between the

Pir, the ventral portion of the tenia tecta, the anterior

olfactory cortex (AO) and the olfactory tubercle (Tu).

Caudally, these neurons continued adjacent to the

ventromedial corner of the Pir. There was very little

BuChE-positive neuropil in the cortex with the exception

of the cingulate cortex (Cg), which had distinct

processes localized to layers 2–3. There was also

diffuse neuropil staining in the retrosplenial cortex (RS),

predominantly in layer 4. As reported previously

(Mesulam et al., 2002a), there were numerous ChAT-

positive neurons evident in the cerebral cortex with the

exception of the Cg and MEnt (Table 1; Fig. 4B, D, F,

H, J). ChAT-positive cells were concentrated in layers

2–3, but were also scattered throughout layers 4–6.

Also, as previously reported (Kitt et al., 1994), there was

laminar distribution of ChAT-positive neuropil throughout

the cerebral cortex. Experiments using IF double

labeling for BuChE and ChAT demonstrated that these

two markers labeled different populations of neurons in

the cerebral cortex.

Fig. 2. Staining in the piriform cortex (Pir; A–C) and the trigeminal motor nucleus (Mo5; D–F) comparing butyrylcholinesterase (BuChE)

histochemistry (A, D), BuChE immunohistochemistry (B, E) and acetylcholinesterase (AChE) histochemistry (C, F) demonstrating that BuChE

histochemical and immunohistochemical staining methods are comparable, in contrast with AChE histochemistry staining. Scale bars = 200 lmand 25 lm (inset).

58 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

Basal ganglia. BuChE-positive neurons were very

sparse within the basal ganglia with only a few scattered

neurons in the caudate putamen nucleus (CPu) and the

ventral pallidum (VP) (Table 1; Fig. 4A, C). In contrast,

there were numerous ChAT-positive neurons and

neuropil labeling in these regions of the basal ganglia

(Fig. 4B, D), as reported previously (Kitt et al., 1994;

Mesulam et al., 2002a). There were no BuChE/ChAT

double-labeled neurons in this region.

Basal forebrain. Within the basal forebrain there were

scattered BuChE-positive neuronal somata (Table 1,

Fig. 4A) in the medial septal nucleus (MS), substantia

innominata (SI) and vertical (VDB) and horizontal (HDB)

limb nuclei of the diagonal band of Broca. There was

generally sparse BuChE neuropil staining in these

regions except for islands of Calleja (ICj), which had

intense labeling. In contrast, ChAT staining within the

basal forebrain was much more prominent (Table 1;

Fig. 4B), as shown previously (Kitt et al., 1994).

Immunofluorescent double labeling showed that BuChE

and ChAT were located in different populations of

neuronal somata in the basal forebrain.

Amygdala. The amygdala had several nuclei with

BuChE-positive neurons (see Table 1). There were

numerous BuChE-positive neuronal somata in the

anterior amygdaloid area (AA), medial anterior

amygdala (MeA; Fig. 4C) and in the most caudal tip of

the posteromedial cortical amygdala (PMCo). The

remaining nuclei either had a few scattered BuChE-

positive neurons (Fig. 4C), as in the anterior cortical

(ACo), basolateral (BL), basomedial (BM) and lateral

(La) amygdaloid nuclei or were not positive for BuChE,

as in the central amygdala (Ce). ChAT staining within

the amygdala was limited to neuropil (Fig. 4D), as

reported previously (Kitt, 1994). There were no BuChE-

positive neurons stained for ChAT in this region.

Hypothalamus. A number of nuclei of the

hypothalamus contained BuChE-positive neuronal

somata (Table 1; Fig. 4A, C, E) including the medial

supramammillary (SuMM), ventral premammillary

(PMV), lateroanterior hypothalamic (LA), posterior part

of the anterior hypothalamic (AHP), dorsal

premammillary (PMD), lateral hypothalamic (LH),

magnocellular preoptic (MCPO), terete hypothalamic,

submammillothalamic (SMT), lateral preoptic,

paraventricular hypothalamic (Pa), perifornical and

posterior hypothalamic (PH) nuclei (Fig. 4A, C, E).

Distinct BuChE-positive neuropil was labeled in the

SuMM, SMT, LH, PH with sparse staining in the

mammillary nucleus. As observed previously in the

mouse (Bobkova et al., 2001), the MCPO had ChAT-

positive perikarya (Fig. 4B). In addition, other

hypothalamic nuclei had ChAT-positive neuronal somata

and neuropil, including the LH and SuMM (Fig. 4D, F).

There were no neurons within the hypothalamus that

were positive for both BuChE and ChAT labeling.

Thalamus. There was extensive BuChE staining

associated with neuronal somata and neuropil within the

Fig. 3. Overview of butyrylcholinesterase (BuChE; A–C) and choline acetyltransferase (ChAT; D) staining of neural elements in the mouse brain.

BuChE neuronal (asterisk) and glial (arrows) labeling (A). The inset in A shows glia at higher magnification. BuChE-positive neuropil (B) and BuChE-

positive white matter (C). BuChE labeling was obtained through histochemistry (A, C) and immunohistochemistry (B). ChAT-positive neuronal and

neuropil staining (D). Scale bars = 25 lm and 10 lm (inset).

G. A. Reid et al. / Neuroscience 234 (2013) 53–68 59

thalamus (Table 1; Fig. 4C, E). The anterodorsal (AD),

anteromedial (AM), anteroventral (AV), dorsal lateral

geniculate (DLG), laterodorsal (LD), medial habenula

(MHb), paratenial (PT) and reuniens (Re) thalamic

nuclei were all densely populated with BuChE-positive

neurons. BuChE-positive neurons within the MHb were

generally located more caudally, along the dorsal edge

of the nucleus and were closely associated with the stria

medullaris (sm) and the habenular commissure. The

marginal zone of the medial geniculate, interanterior

dorsal and lateral posterior (LP) thalamic nuclei had

fewer BuChE-stained neurons. There were also a few

scattered BuChE-positive cells in the ventral posterior

lateral thalamus along the lateral edge, closely

associated with the external medullary lamina (eml).

Generally, there was distinct BuChE-positive neuropil

throughout the thalamus. However, a few areas with

relatively more intense BuChE neuropil staining included

lateroventral portion of the AV, the habenula (Hb) and

mediodorsal (MD) nuclei. ChAT staining labeled neuropil

throughout the thalamus (Table 1; Fig. 4D, F), as

observed previously (Kitt, 1994). The only ChAT-positive

soma was located within the MHb (Table 1; Fig. 4D) as

noted elsewhere (Mesulam et al., 2002a). IF double

labeling analysis demonstrated that BuChE and ChAT

labeled different populations of cells within the MHb.

Hippocampal formation. The hippocampal formation

(H) of the mouse brain had few BuChE-positive

neuronal somata found in the caudal portion of the

polymorphic cell layer of the dentate gyrus (PoDG) and

the stratum oriens (Or) of hippocampus proper (Fig. 4E,

G). There was BuChE neuropil staining at the most

rostral level of the hippocampus proper in the CA3

region. ChAT staining within the hippocampal formation

labeled neuropil, as described previously (Kitt et al.,

1994). There were no neurons double labeled for

BuChE and ChAT in the hippocampal formation.

Midbrain. Within the midbrain, there were several

nuclei with numerous BuChE-positive neuronal somata

including the interstitial nucleus of Cajal (InC), medial

terminal nucleus of the accessory optic tract (MT),

dorsal raphe nucleus (DR) and red nucleus (R)

(Table 1; Fig. 4G, I). Other nuclei in this region had

relatively fewer BuChE-positive cells (see Table 1).

There was dense BuChE-positive neuropil in the

intermediate white layer of the superior colliculus

(InWh), anterior pretectal nucleus (APT), DR and the

retroparafascicular nucleus, with less dense BuChE-

positive neuropil throughout other midbrain regions

(Fig. 4G, I). As reported previously (Mesulam et al.,

2002a; Vanderhost et al., 2006), a number of nuclei in

Fig. 4. Butyrylcholinesterase histochemistry (left) and choline acetyltransferase immunohistochemistry (right) in the mouse brain. For each marker,

the section photomicrograph is accompanied by a neuroleucida drawing of the same section with neurons represented by black circles. Scale

bar = 1 mm.

60 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

Fig. 4. (continued)

G. A. Reid et al. / Neuroscience 234 (2013) 53–68 61

Table 1. Butyrylcholinesterase (BuChE) and choline acetyltransferase (ChAT) neuronal somata and neuropil distribution in the mouse brain. For

neuronal density, + = <10, ++ = 10–20, +++ = 20–30 and ++++ = >30 neurons in each nucleus/section. For the neuropil, x is an

estimate of the density of neuropil staining. Examples of the different neuropil densities for BuChE staining can be seen in (Fig. 4. Low density = x

(e.g. HDB), moderate density = xx (e.g. LH); dense = xxx (e.g. APT); very dense = xxxx (e.g. ICj). Nuclei with neuronal somata colocalization of

BuChE and ChAT are shaded in gray.

62 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

Table 1 (continued)

G. A. Reid et al. / Neuroscience 234 (2013) 53–68 63

the midbrain had ChAT-positive neurons and neuropil

(Table 1; Fig. 4H, J). The only nucleus that had both

ChAT- and BuChE-positive neurons was the R but IF

double labeling indicated that BuChE and ChAT were

not colocalized within the same neurons.

Pons. The pons had numerous BuChE-positive

neuronal somata in the locus coeruleus (LC) and

subcoeruleus nucleus (SubC; Fig. 4I, K). Other nuclei

had scattered BuChE-positive cells (Table 1; Fig. 4I, K).

There was extensive BuChE-positive neuropil in the

pedunculopontine (PPTg), laterodorsal (LDTg) and

microcellular (MiTg) tegmental nuclei. Other areas had

moderately dense staining for BuChE-positive neuropil

(see Table 1). As has been shown previously (Mesulam

et al., 2002a; Vanderhost et al., 2006), numerous

regions in the pons had ChAT-positive neurons and

neuropil (Table 1; Fig. 4J, L). Although there was no

neuronal colocalization of BuChE and ChAT in the pons

detected with IF double labeling, a close association of

BuChE-positive neuropil and ChAT-positive somata was

evident within the LDTg and PPTg. For example, in the

LDTg, confocal microscopy indicated that BuChE-

positive neuropil surrounded ChAT-positive somata

(Fig. 5).

Medulla oblongata. Within the medulla oblongata,

numerous BuChE-positive neurons were found in the

facial (7N), dorsal motor nucleus of the vagus (10N),

inferior olivary (IO), inferior salivatory (IS) and solitary

tract (Sol) nuclei as well as the A5 noradrenalin cell

group (see Table 1; Fig. 4M, O). There were also

numerous BuChE-positive neurons located in the

caudoventral portion of the hypoglossal nucleus (12N)

and scattered neurons in the ambiguus nucleus (Amb),

intermediate reticular nucleus (IRt) and the interpolaris

(Sp5I) and ventrolateral oralis (Sp5OVL) divisions of the

spinal trigeminal nucleus among several others (see

Fig. 5. Confocal laser scanning photomicrographs illustrating immunofluorescent (IF) labeling of the laterodorsal tegmental nucleus (LDTg) for

butyrylcholinesterase (BuChE; A) and choline acetyltransferase (ChAT) (B). BuChE and ChAT overlaid images with orthogonal view on the

periphery (C). Note, BuChE-IF labeling forms a pericellular, neuropil basket surrounding ChAT-positive neurons. Scale bar = 50 lm.

64 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

Table 1). BuChE-positive neuropil was observed in the

prepositus (Pr), solitary (Sol) and gracile nuclei. Many

nuclei within the medulla oblongata contained ChAT-

positive neurons, as described previously (Vanderhost

et al., 2006). In addition, numerous ChAT-positive

neurons were detected in the medial vestibular (MVe),

spinal vestibular (SVe), and IS nuclei as well as the

noradrenaline cell group A5 (Table 1, Fig. 4N). IF

double labeling demonstrated that BuChE and ChAT

were colocalized in neurons in the 7N (Fig. 6A–C), 10N

(Fig. 6D–F), 12N, Amb (Fig. 6G–I), IS (Fig. 6J–L), A5

(Fig. 6M–O), LPGi, and IRt. Confocal imaging with

orthogonal view of these regions provided further

evidence of colocalization of BuChE and ChAT within

the same neurons (see Fig. 7 for example).

Spinal cord. Within the cervical spinal cord BuChE

staining was observed in scattered neuronal somata in

laminae 5, 7 and 10 and in distinct, varicose neuropil in

lamina 4. As reported previously, ChAT staining labeled

neurons in laminae 3 (Mesnage et al., 2011), 7, 9 and 10

(VanderHorst and Ulfhake, 2006). In addition, ChAT-positive

neurons were located in laminas 5 and 8. ChAT-positive

neuropil was found throughout this level of spinal cord but

was particularly dense in lamina 2. IF double labeling, for

BuChE and ChAT demonstrated colocalization of these

markers in neurons of laminae 7 and 10.

DISCUSSION

Cholinergic neurotransmission plays a significant role in a

wide variety of functions in the nervous system including

memory, behavior, and motor and visceromotor control

(Giacobini and Pepeu, 2006). Regulation of the

cholinergic system, through acetylcholine hydrolysis,

has been largely attributed to AChE. However, it has

been demonstrated that BuChE can also play a

fundamental role in cholinergic neurotransmission in

view of the fact that AChE knock-out mice are viable

(Xie et al., 2000). The present work describes the

association of BuChE with ChAT-positive cholinergic

elements in the mouse CNS and provides detailed maps

of neuronal somata positive for each enzyme. Areas

with both enzymes were examined following IF double

labeling to determine if neural elements contained both

enzymes. From these experiments, a number of

relationships between BuChE and ChAT labeling could

be identified. First, there were BuChE-positive neuronal

somata that were colocalized with ChAT. Second, in

other areas BuChE-positive neuronal somata did not

contain ChAT but were in close proximity to ChAT-

positive neuropil. Third, elsewhere, there were ChAT-

positive neurons that did not contain BuChE but were

closely associated with BuChE-positive neuropil.

Within the mouse CNS, colocalization of BuChE and

ChAT labeling within neuronal somata was found in the

medulla oblongata and the spinal cord (Table 1, shaded

boxes). Nuclei showing neuronal colocalization included

the 7N, 10N and 12N (Figs. 6 and 7). Confocal imaging

demonstrated that BuChE was located within the cell

body of the neuron and was not confined to the cell

surface. The distribution of these BuChE-positive

cholinergic neurons within cranial nerve nuclei suggests

a role for BuChE in motor and visceromotor control.

BuChE-positive neuronal somata in most areas were

ChAT-negative but coincided with ChAT-positive

neuropil (Table 1, Fig. 4). Due to the extensive

distribution of ChAT-positive neuropil and the

widespread use of acetylcholine as a neurotransmitter, it

is likely that BuChE-positive neurons in such regions are

involved in acetylcholine regulation. For example, layer

2 of the MEnt has a large population of BuChE-positive

neuronal somata as well as ChAT-positive neuropil. This

area receives cholinergic input from the MS and VDB

(Heys et al., 2012) and primarily projects to the dentate

gyrus and CA3 hippocampal region via the medial

perforant pathway (Kerr et al., 2007). Therefore,

BuChE-positive neurons in this region could affect

cholinergic modulation of functions subserved by this

region, such as spatial novelty detection and path

integration (Van Cauter et al., 2013). Similarly, many

thalamic nuclei had ChAT-positive neuropil and large

populations of BuChE-positive neurons. These BuChE-

positive cells could be receiving cholinergic innervation

from a wide variety of sources including the basal

forebrain and tegmental nuclei (Mesulam et al., 1983;

Fig. 6. Staining in the facial nucleus (7N; A–C), dorsal motor nucleus of the vagus (10N; D–F), ambiguus nucleus (Amb; G–I), inferior salivatory

nucleus (IS; J–L) and the noradrenaline group A5 (M–O) with butyrylcholinesterase (BuChE) immunofluorescence (IF; A, D, G, J, M), choline

acetyltransferase (ChAT) IF (B, E, H, K, N) and BuChE and ChAT double labeling IF (C, F, I, L, O) in the same nuclei. Scale bar = 100 lm.

G. A. Reid et al. / Neuroscience 234 (2013) 53–68 65

Heckers et al., 1992; Darvesh and Hopkins, 2003). The

strong cholinergic input to the thalamus is most likely

regulated, at least in part, by the extensive distribution

of BuChE in this area which, in turn, may regulate

thalamic functions including relaying sensory and motor

signals to the cerebral cortex and regulation of

Fig. 7. Confocal laser scanning photomicrographs illustrating immunofluorescent (IF) labeling of the hypoglossal nucleus for butyrylcholinesterase

(BuChE; A), choline acetyltransferase (ChAT; B) and overlaid images of BuChE and ChAT showing colocalization with orthogonal view on the

periphery (C). Note BuChE and ChAT IF colocalization throughout the cell body of numerous neurons within the hypoglossal nucleus. Scale

bar = 50 lm.

66 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

consciousness, sleep, and alertness (Jones, 2007). In

addition, the hypothalamus contained several nuclei with

BuChE- and ChAT-positive neuronal somata and

neuropil (Table 1; Fig. 4A–F) such as the MCPO, SuMM

and LH. However, BuChE and ChAT were not

colocalized within the same neurons in these regions.

Although ChAT-positive neurons have been previously

described in the mouse MCPO (Bobkova et al., 2001)

the present study indicated that the SuMM and LH also

contained ChAT-positive perikarya. ChAT-positive

neurons in these areas have been described previously

in the rat (Choi et al., 2012) and cat (Vincent and

Reiner, 1987), respectively. The distribution of BuChE

within the hypothalamus suggests its involvement in

hypothalamic cholinergic functions such as modulating

stress responses, feeding behavior, sleep-wake cycles

and homeostasis in general.

A close relationship was also observed between

BuChE-positive neuropil and ChAT-positive cholinergic

neurons within the LDTg and PPTg in the pons. This

caudal cholinergic cell column innervates a number of

areas of the brain, including the thalamus (Mesulam

et al., 1983), pontomedullary reticular formation and

mesolimbic regions and is involved in control of

locomotion and regulation of sleep-wake cycle, arousal,

stimulus and reward learning, visual orienting and

sensory motor systems (VanderHorst and Ulfhake,

2006). As in the rat (Tago et al., 1992), BuChE-HC

staining demonstrated that this enzyme was associated

with neuronal perikarya. However, BuChE-IHC and

confocal microscopy in double labeled sections

indicated that this staining was, in fact, BuChE-positive

neuropil surrounding ChAT-positive neurons forming a

BuChE-containing pericellular basket. This suggests that

the apparent difference observed in the mouse in the

present study was most likely a result of the finer detail

enabled through BuChE-IHC, rather than species

differences. Given this close association of BuChE

neuropil to ChAT-positive neurons in the LDTg and

PPTg, BuChE may be involved in maintaining

acetylcholine levels in the arousal systems of the CNS.

Taking into account these close relationships between

ChAT- and BuChE-positive neural elements, there are a

number of ways BuChE could participate in the

regulation of cholinergic neurotransmission. BuChE

could be expressed in the synapse of neurons with

cholinergic receptors and be involved in modulation of

direct cholinergic neurotransmission. On the other hand,

BuChE-positive neural elements may also be involved in

regulation of tonic levels of volume acetylcholine. Tonic

neurotransmission could occur when acetylcholine

diffuses out of the synaptic cleft or when acetylcholine is

released from non-synaptic varicosities (Descarries

et al., 1997; Sarter et al., 2009; Ren et al., 2011). These

considerations are in keeping with previous observations

demonstrating that highly specific BuChE inhibitors,

such as cymserine (Greig et al., 2005) and (�)-N1phenethyl-norcymserine (Cerbai et al., 2007),

increased acetylcholine levels in the rat cerebral cortex,

leading to improved cognition in these animals. In

addition, in AChE-deficient mice, the selective BuChE

inhibitors bambuterol and bisnorcymserine, significantly

raised acetylcholine in the brain (Hartmann et al., 2007),

indicating the importance of BuChE in the regulation of

cholinergic neurotransmission.

Thus, based on neuroanatomical evidence presented

herein, BuChE may be involved, along with AChE, in

maintaining appropriate levels of acetylcholine in the

brain. The level of physiologically relevant acetylcholine

may be maintained by each enzyme in a different way.

That is, although BuChE and AChE both hydrolyze

acetylcholine efficiently, at high levels of acetylcholine,

AChE activity is inhibited while that of BuChE is

activated (Silver, 1974). This complementary

relationship between these two enzymes may be

important in maintaining appropriate cholinergic tone

within the CNS.

CONCLUSIONS

This study indicates a possible cholinergic regulatory

function for BuChE in modulation of motor control,

G. A. Reid et al. / Neuroscience 234 (2013) 53–68 67

awareness, cognition and behavior. Similar to what has been

observed for the distribution of AChE in the brain, not all

BuChE-positive neuronal somata contain ChAT. BuChE

and ChAT colocalization within neurons was found to be

limited to particular nuclei within the medulla oblongata and

spinal cord. However, there were other relationships

evident between these two enzymes, including BuChE-

positive neuronal somata in areas of ChAT neuropil and

BuChE neuropil that formed pericellular baskets around

ChAT-positive, cholinergic neurons.

This work provides further neuroanatomical evidence

that BuChE plays a role in the modulation of cholinergic

neurotransmission.

Acknowledgments—This work was supported in part by Cana-

dian Institutes of Health Research, Capital District Health Author-

ity Research Fund, Nova Scotia Health Research Foundation,

Faculty of Medicine (Clinician-Scientist Program), Dalhousie Uni-

versity Department of Medicine (University Internal Medicine Re-

search Fund) and Dalhousie Medical Research Foundation

(S.D.) and Defense Threat Reduction Agency (DTRA), Joint Sci-

ence and Technology Office, Medical S&T Division (N.C.). We

would like to thank Professor Earl Martin for critically reviewing

this manuscript.

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(Accepted 21 December 2012)(Available online 7 January 2013)