Butyrylcholinesterase and the cholinergic system - Semantic Scholar

Neuroscience 234 (2013) 53–68

BUTYRYLCHOLINESTERASE AND THE CHOLINERGIC SYSTEM G. A. REID, a N. CHILUKURI b AND S. DARVESH a,c*

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 the CNS. BuChE staining with histochemistry and immunohistochemistry produced the same distribution of labeling throughout the brain and spinal cord. Immunofluorescent double labeling demonstrated that many nuclei in the medulla oblongata, as well as regions of the spinal cord, had neurons that contained both BuChE and ChAT. BuChE-positive neurons without ChAT were found in close proximity with ChAT-positive neuropil in areas such as the thalamus 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 evidence of a role for BuChE in the regulation of acetylcholine levels in the CNS. Ó 2013 IBRO. Published by Elsevier Ltd. Open access under CC

a

Department of Medical Neuroscience Dalhousie University, Halifax, Nova Scotia, Canada

b

Research Division, Physiology & Immunology Branch, US Army Medical Research, Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA

c

Department of Medicine (Neurology and Geriatric Medicine), Dalhousie University, Halifax, Nova Scotia, Canada

Abstract—The cholinergic system plays important roles in neurotransmission in both the peripheral and central nervous systems. The cholinergic neurotransmitter acetylcholine is synthesized by choline acetyltransferase (ChAT) and its action terminated by acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The predominance of AChE has focused much attention on understanding the relationship of this enzyme to ChAT-positive cholinergic neurons. However, there is ample evidence that BuChE also plays an important 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

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Key words: cholinesterases, acetylcholinesterase, pseudocholinesterase, choline acetyltransferase, 129S1/SvImJ mice, acetylcholine.

*Correspondence to: S. Darvesh, Department of Medical Neuroscience, Dalhousie University, Room 1308, Camp Hill Veterans’ Memorial, 5955 Veterans’ Memorial Lane, Halifax, Nova Scotia, Canada B3H 2E1. Tel: +1-902-473-2490; fax: +1-902-473-7133. E-mail address: [email protected] (S. Darvesh). Abbreviations: AChE, acetylcholinesterase; Ad-moBuChE, adenovirus containing the gene for mouse butyrylcholinesterase; Amb, ambiguus nucleus; AA, anterior amygdaloid area; ac, anterior commissure; ACo, anterior cortical amygdala; AD, anterior dorsal thalamus; AO, anterior olfactory cortex; AHP, anterior posterior hypothalamic nucleus; APT, anterior pretectal nucleus; ATg, anterior tegmental nucleus; AV, anterior ventral thalamus; Au, auditory cortex; BL, basolateral amygdala; BM, basomedial amygdala; BST, bed nucleus of stria terminalis; BSA, bovine serum albumin; BuChE, butyrylcholinesterase; CPu, caudate putamen nucleus; Ce, central amygdala; cp, cerebral peduncle; ChAT, choline acetyltransferase; Cg, cingulate cortex; Cl, claustrum; cc, corpus collosum; DpMe, deep mesencephalic nucleus; DAB, 3,30 -diaminobenzidine tetrahydrochloride; DLG, dorsal lateral geniculate nucleus; 10N, dorsal motor nucleus of the vagus; DR, dorsal raphe nucleus; DTg, dorsal tegmental nucleus; ELISA, enzyme-linked immunosorbent assay; En, endopiriform cortex; ec, external capsule; ECu, external cuneate; eml, external medullary lamina; 7N, facial nucleus; fr, fasciculus retroflexus; FBS, fetal bovine serum; fi, fimbria; f, fornix; g7, genu of the facial nucleus; Gi, gigantocellular reticular nucleus; GP, globus pallidus; H, hippocampal formation; HC, histochemical; HDB, horizontal limb nuclei of the diagonal band of Broca; hBuChE, human butyrylcholinesterase; 12N, hypoglossal nucleus; IgG, immunoglobulin G; icp, inferior cerebellar peduncle; IC, inferior colliculus; IO, inferior olivary nucleus; IS, inferior salivatory nucleus; I, insular cortex; InG, intermediate gray layer of superior colliculus; IRt, intermediate reticular nucleus; InWh, intermediate white layer of the superior colliculus; ic, internal capsule; IP, interpeduncular nucleus; InC, interstitial nucleus of Cajal; ICj, islands of Calleja; La, lateral amygdala; LDTg, lateral dorsal tegmental nucleus; LD, lateral dorsal thalamus; LEnt, lateral entorhinal cortex; LH, lateral hypothalamus; LPGi, lateral paragigantocellular nucleus; LP, lateral posterior thalamus; LRt, lateral reticular nucleus; LS, lateral septal nucleus; LVe, lateral vestibular nucleus; LC, locus coeruleus; MCPO, magnocellular preoptic nucleus; mt, mammillothalamic tract; MeA, medial anterior amygdala; MD, medial dorsal thalamus; MEnt, medial entorhinal cortex; MG, medial geniculate nucleus; MHb, medial habenula; ml, medial lemniscus; mlf, medial longitudinal fasciculus; MS, medial septal nucleus; MT, medial terminal nucleus of the accessory tract; MVe, medial vestibular nucleus; MnR, median raphe nucleus; MiTg, microcellular tegmental nucleus; M, motor cortex; Mo5, motor trigeminal nucleus; moBuChE, mouse butyrylcholinesterase; ns, nigrostriatal bundle; A5, noradrenaline cell group A5; LL, nucleus of lateral lemniscus; Tu, olfactory tubercle; opt, optic tract; Pa, paraventricular hypothalamic nucleus; PPTg, pedunculopontine tegmental nucleus; PAG, periaqueductal gray; PB, phosphate buffer; PBS, phosphate-buffered saline; Pir, piriform cortex; PoDG, polymorphic cell layer of the dentate gyrus; Pn, pontine nuclei; PnO, pontine reticular nucleus, oral part; pc, posterior commissure; PH, posterior hypothalamic nucleus; PMCo, posteromedial cortical amygdala; PMD, premammillary nucleus, dorsal; PMV, premammillary nucleus, ventral; PO, preoptic nucleus; Pr, prepositus nucleus; Pr5, principal sensory trigeminal nucleus; Po, pulvinar thalamic nucleus; py, pyramidal tract; rmoBuChE, recombinant mouse BuChE; R, red nucleus; Rt, reticular nucleus; RtTg, reticulotegmental nucleus of the pons; RS, retrosplenial cortex; Re, reuniens; RI, rostral interstitial nucleus of mlf; RPO, rostral paraolivary nucleus; rs, rubral spinal tract; S, sensory cortex; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; sol, solitary tract; Sol, solitary tract nucleus; Sp5I, spinal trigeminal nucleus, interpolaris; Sp5OVL, spinal trigeminal nucleus, ventrolateral oralis; sp5, spinal trigeminal tract; SVe, spinal vestibular nucleus; Or, stratum oriens; sm, stria medullaris; st, stria terminalis; SubC, subcoeruleus nucleus; SMT, submammillothalamic nucleus; SI, substantia innominata; SN, substantia nigra; scp, superior cerebellar peduncle; str, superior thalamic radiation; SuVe, superior vestibular nucleus; SuMM, supramammillary, medial nucleus; ts, tectospinal tract; TeA, temporal association cortex; TMB, 3,30 ,5,50 -tetramethylbenzidine; m5, trigeminal nerve root; TBS, tris-buffered saline; VA, ventral anterior thalamic nucleus; VLG, ventral lateral geniculate; VP, ventral pallidum; VTA, ventral tegmental area; VDB, vertical limb nuclei of the diagonal band of Broca; V, visual cortex; ZI, zona incerta. 0306-4522 Ó 2013 IBRO. Published by Elsevier Ltd. Open access under CC BY-NC-ND license. http://dx.doi.org/10.1016/j.neuroscience.2012.12.054 53

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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).

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 BFS30TC 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 polyclonal antibodies to BuChE

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.

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 (AdmoBuChE) (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 AdmoBuChE 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 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 longterm 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 nonspecific 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

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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 ll of 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 of 2 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 X100 and VectastainÒ 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 VectastainÒ 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.

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Butyrylcholinesterase and choline acetyltransferase immunofluorescence 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 FluorÒ 488 (1:350; Molecular Probes, A11055) and donkey anti-rabbit Alexa FluorÒ 555 (1:350; Molecular probes, A31572). Sections were rinsed, mounted on slides, cleared in xylene and coverslipped.

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 AdobeÒ PhotoshopÒ 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 AdobeÒ PhotoshopÒ CS5. The anatomical parcellation, based on the mouse brain atlas by Paxinos and Franklin (2001), and neuronal distributions were then redrawn using AdobeÒ IllustratorÒ 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 BuChEIHC (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 choline acetyltransferase 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


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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.

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 ChATpositive 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.

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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 lm and 25 lm (inset).

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 BuChEpositive 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 ChATpositive 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


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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 BuChEpositive 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).

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 BuChEpositive neuropil throughout other midbrain regions (Fig. 4G, I). As reported previously (Mesulam et al., 2002a; Vanderhost et al., 2006), a number of nuclei in

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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.


Fig. 4. (continued)

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G. A. Reid et al. / Neuroscience 234 (2013) 53–68 Table 1. Butyrylcholinesterase (BuChE) and choline acetyltransferase (ChAT) neuronal somata and neuropil distribution in the mouse brain. For neuronal density, + = 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.


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Table 1 (continued)

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 BuChEpositive 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

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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.

Table 1). BuChE-positive neuropil was observed in the prepositus (Pr), solitary (Sol) and gracile nuclei. Many nuclei within the medulla oblongata contained ChATpositive 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 ChATpositive neuropil. Third, elsewhere, there were ChATpositive 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 BuChEpositive cells could be receiving cholinergic innervation from a wide variety of sources including the basal forebrain and tegmental nuclei (Mesulam et al., 1983;


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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.

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

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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.

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,


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 BuChEpositive 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 Canadian Institutes of Health Research, Capital District Health Authority Research Fund, Nova Scotia Health Research Foundation, Faculty of Medicine (Clinician-Scientist Program), Dalhousie University Department of Medicine (University Internal Medicine Research Fund) and Dalhousie Medical Research Foundation (S.D.) and Defense Threat Reduction Agency (DTRA), Joint Science and Technology Office, Medical S&T Division (N.C.). We would like to thank Professor Earl Martin for critically reviewing this manuscript.

REFERENCES Armstrong DM, Saper CB, Levey AI, Wainer BH, Terry RD (1983) Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase. J Comp Neurol 216(1):53–68. Bobkova NV, Nesterova IV, Nesterov VV (2001) The state of cholinergic structures in forebrain of bulbectomized mice. Bull Exp Biol Med 131(5):427–431. Cerbai F, Giovannini MG, Melani C, Enz A, Pepeu G (2007) N1phenethyl-norcymserine, a selective butyrylcholinesterase inhibitor, increases acetylcholine release in rat cerebral cortex: a comparison with donepezil and rivastigmine. Eur J Pharmacol 572(2–3):142–150. Choi WK, Wirtshafter D, Park HJ, Lee MS, Her S, Shim I (2012) The characteristics of supramammillary cells projecting to the hippocampus in stress response in the rat. Korean J Physiol Pharmacol 16(1):17–24. Darvesh S, Hopkins DA (2003) Differential distribution of butyrylcholinesterase and acetylcholinesterase in the human thalamus. J Comp Neurol 463:25–43. Darvesh S, Grantham DL, Hopkins DA (1998) Distribution of butyrylcholinesterase in the human amygdala and hippocampal formation. J Comp Neurol 393:374–390. Darvesh S, Hopkins DA, Geula C (2003) Neurobiology of butyrylcholinesterase. Nat Rev Neurosci 4(2):131–138. Darvesh S, Cash MK, Reid GA, Martin E, Mitnitski A, Geula C (2012a) Butyrylcholinesterase is associated with b-amyloid plaques in the transgenic APPSWE/PSEN1dE9 mouse model of Alzheimer disease. J Neuropathol Exp Neurol 71(1):2–14. Darvesh S, Reid GA, Chilukuri N (2012b) Butyrylcholinesterase and cholinergic neural elements in the mouse brain. Soc Neurosci Abstr. Program#/Poster#:840.15/B5. Descarries L, Gisiger V, Steriade M (1997) Diffuse transmission by acetylcholine in the CNS. Prog Neurobiol 53(5):603–625. Duysen EG, Li B, Darvesh S, Lockridge O (2007) Sensitivity of butyrylcholinesterase knockout mice to ()-huperzine A and donepezil suggests humans with butyrylcholinesterase deficiency may not tolerate these Alzheimer’s disease drugs and indicates butyrylcholinesterase function in neurotransmission. Toxicology 233(1–3):60–69.

67

Eckenstein F, Sofroniew MV (1983) Identification of central cholinergic neurons containing both choline acetyltransferase and acetylcholinesterase and of central neurons containing only acetylcholinesterase. J Neurosci 3(11):2286–2291. Ellman GL, Courtney KD, Andres Jr V, Feather-Stone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95. Eng LF, Uyeda CT, Chao LP, Wolfgram F (1974) Antibody to bovine choline acetyltransferase and immunofluorescent localisation of the enzyme in neurons. Nature 250(463):243–245. Friede RL (1967) A comparative histochemical mapping of the distribution of butyryl cholinesterase in the brains of four species of mammals, including man. Acta Anat (Basel) 66(2):161–177. Geula C, Nagykery N (2007) Butyrylcholinesterase activity in the rat forebrain and upper brainstem: postnatal development and adult distribution. Exp Neurol 204(2):640–657. Giacobini E (2003) Butyrylcholinesterase: its function and inhibitors. United Kingdom: Martin Dunitz. Giacobini E, Pepeu G (2006) The brain cholinergic system: in health and disease. United Kingdom: Informa Healthcare. Greig NH, Utsuki T, Ingram DK, Wang Y, Pepeu G, Scali C, Yu QS, Mamczarz J, Holloway HW, Giordano T, Chen D, Furukawa K, Sambamurti K, Brossi A, Lahiri DK (2005) Selective butyrylcholinesterase inhibition elevates brain acetylcholine, augments learning and lowers Alzheimer beta-amyloid peptide in rodent. Proc Natl Acad Sci USA 102(47):17213–17218. Hartmann J, Kiewert C, Duysen EG, Lockridge O, Greig NH, Klein J (2007) Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity. J Neurochem 100(5):1421–1429. Heckers S, Geula C, Mesulam MM (1992) Cholinergic innervation of the human thalamus: dual origin and differential nuclear distribution. J Comp Neurol 325(1):68–82. Heys JG, Schultheiss NW, Shay CF, Tsuno Y, Hasselmo ME (2012) Effects of acetylcholine on neuronal properties in entorhinal cortex. Front Behav Neurosci 6(32) (Epub Jul 24). Jones E (2007) The thalamus. second ed. Cambridge, United Kingdom: Cambridge University Press. Karnovsky MJ, Roots L (1964) A direct-coloring thiocholine method for cholinesterases. J Histochem Cytochem 12:219–221. Kerr KM, Agster KL, Furtak SC, Burwell RD (2007) Functional neuroanatomy of the parahippocampal region: the lateral and medial entorhinal areas. Hippocampus 17(9):697–708. Kettenmann H, Ransom BR (2005) Neuroglia. New York: Oxford University Press. Kimura H, McGeer PL, Peng F, McGeer EG (1980) Choline acetyltransferase-containing neurons in rodent brain demonstrated by immunohistochemistry. Science 208(4447):1057–1059. Kitt CA, Ho¨hmann C, Coyle JT, Price DL (1994) Cholinergic innervation of mouse forebrain structures. J Comp Neurol 341(1):117–129. Koelle GB, Friedenwald JA (1949) A histochemical method for localizing cholinesterase activity. Proc Soc Exp Biol Med 70(4):617–622. Levey AI, Wainer BH, Mufson EJ, Mesulam MM (1983) Colocalization of acetylcholinesterase and choline acetyltransferase in the rat cerebrum. Neuroscience 9(1):9–22. Levey AI, Wainer BH, Rye DB, Mufson EJ, Mesulam MM (1984) Choline acetyltransferase-immunoreactive neurons intrinsic to rodent cortex and distinction from acetylcholinesterase-positive neurons. Neuroscience 13(2):341–353. Li B, Stribley JA, Ticu A, Xie W, Schopfer LM, Hammond P, Brimijoin S, Hinrichs SH, Lockridge O (2000) Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse. J Neurochem 75(3):1320–1331. Mesnage B, Gaillard S, Godin AG, Rodeau JL, Hammer M, Von Engelhardt J, Wiseman PW, De Koninck Y, Schlichter R, Cordero-Erausquin M (2011) Morphological and functional characterization of cholinergic interneurons in the dorsal horn of the mouse spinal cord. J Comp Neurol 519(16):3139–3158.

68


Mesulam MM, Mufson EJ, Wainer BH, Levey AI (1983) Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Ch1–Ch6). Neuroscience 10(4):1185–1201. Mesulam MM, Guillozet A, Shaw P, Levey A, Duysen EG, Lockridge O (2002a) Acetylcholinesterase knockouts establish central cholinergic pathways and can use butyrylcholinesterase to hydrolyze acetylcholine. Neuroscience 110(4):627–639. Mesulam M, Guillozet A, Shaw P, Quinn B (2002b) Widely spread butyrylcholinesterase can hydrolyze acetylcholine in the normal and Alzheimer brain. Neurobiol Dis 9(1):88–93. Mis K (2005) Colocalization of acetylcholinesterase, butyrylcholinesterase and choline acetyltransferase in rat spinal cord. Hum Exp Toxicol 24(10):543–545. Parikh K, Duysen EG, Snow B, Jensen NS, Manne V, Lockridge O, Chilukuri N (2011) Gene-delivered BuChE Is Prophylactic against the toxicity of chemical warfare nerve agents and organophosphorus compounds. J Pharmacol Exp Ther 337:92–101. Paxinos G, Franklin KBJ (2001) The mouse brain in stereotaxic coordinates. second ed. San Diego: Academic Press. Ren J, Qin C, Hu F, Tan J, Qiu L, Zhao S, Feng G, Luo M (2011) Habenula ‘‘cholinergic’’ neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes. Neuron 69(3):445–452.

Sarter M, Parikh V, Howe WM (2009) Phasic acetylcholine release and the volume transmission hypothesis: time to move on. Nat Rev Neurosci 10(5):383–390. Shute CC, Lewis PR (1963) Cholinesterase-containing systems of the brain of the rat. Nature 199:1160–1164. Silver A (1974) The biology of cholinesterases. Amsterdam: Elsevier. Tago H, Maeda T, McGeer PL, Kimura H (1992) Butyrylcholinesterase-rich neurons in rat brain demonstrated by a sensitive histochemical method. J Comp Neurol 325(2):301–312. Van Cauter T, Camon J, Alvernhe A, Elduayen C, Sargolini F, Save E (2013) Distinct roles of medial and lateral entorhinal cortex in spatial cognition. Cereb Cortex 23(2):451–459. VanderHorst VG, Ulfhake BJ (2006) The organization of the brainstem and spinal cord of the mouse: relationships between monoaminergic, cholinergic, and spinal projection systems. Chem Neuroanat 31(1):2–36. Vincent SR, Reiner PB (1987) The immunohistochemical localization of choline acetyltransferase in the cat brain. Brain Res Bull 18(3):371–415. Xie W, Stribley JA, Chatonnet A, Wilder PJ, Rizzino A, McComb RD, Taylor P, Hinrichs SH, Lockridge O (2000) Postnatal developmental delay and supersensitivity to organophosphate in gene-targeted mice lacking acetylcholinesterase. J Pharmacol Exp Ther 293(3):896–902.

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