Neuroscience 234 (2013) 53–68

BUTYRYLCHOLINESTERASE AND THE SYSTEM

G. A. REID, a N. CHILUKURI b AND S. DARVESH a,c* 129S1/SvImJ mice were stained for BuChE and ChAT using a Department of Medical Neuroscience Dalhousie University, Halifax, histochemical, immunohistochemical and immunofluorescent Nova Scotia, Canada techniques. Both BuChE and ChAT were found in neural elements throughout the CNS. BuChE staining with histochem- b Research Division, Physiology & Immunology Branch, US istry and immunohistochemistry produced the same distribu- Army Medical Research, Institute of Chemical Defense, tion of labeling throughout the brain and spinal cord. Aberdeen Proving Ground, MD 21010, USA Immunofluorescent double labeling demonstrated that many c Department of Medicine (Neurology and Geriatric Medicine), nuclei in the medulla oblongata, as well as regions of the spinal Dalhousie University, Halifax, Nova Scotia, Canada 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 thal- Abstract—The cholinergic system plays important roles in amus and amygdala. BuChE-positive neuropil was also found neurotransmission in both the peripheral and central nervous closely associated with ChAT-positive neurons, particularly systems. The cholinergic is in tegmental nuclei of the pons. These observations synthesized by acetyltransferase (ChAT) and its provide further neuroanatomical evidence of a role for BuChE action terminated by (AChE) and butyr- in the regulation of acetylcholine levels in the CNS. ylcholinesterase (BuChE). The predominance of AChE has Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. focused much attention on understanding the relationship of this to ChAT-positive cholinergic neurons. How- ever, there is ample evidence that BuChE also plays an impor- Key words: , acetylcholinesterase, pseudo- tant role in cholinergic regulation. To elucidate the , choline acetyltransferase, 129S1/SvImJ mice, relationship of BuChE to neural elements that are producing acetylcholine. acetylcholine, the distribution of this enzyme was compared to that of ChAT in the mouse CNS. Brain tissues from

*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 for mouse ; 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, 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.

0306-4522/12 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.12.054 53 54 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

INTRODUCTION Materials

Cholinergic neurotransmission in the mammalian CNS is Unless otherwise stated, all reagents were purchased from regulated predominantly by the enzyme Sigma–Aldrich (St. Louis, MO). acetylcholinesterase (AChE, EC 3.1.1.7) by catalyzing the hydrolysis of the cholinergic neurotransmitter Preparation of brain tissue acetylcholine (Silver, 1974). Improved histochemical techniques (Koelle and Friedenwald, 1949) for detecting Mice (between 10 and 18 weeks old) were deeply anesthetized this enzyme led to the first map of the distribution of with an intra-peritoneal injection of sodium pentobarbital AChE in the rodent brain (Shute and Lewis, 1963). (200 mg/kg) and perfused with approximately 25 ml of 0.9% Subsequently, antibodies were developed to detect saline solution containing 0.1% sodium nitrite followed by 50 ml of 0.1 M phosphate buffer (PB, pH 7.4) containing 4% choline acetyltransferase (ChAT, EC 2.3.1.6), the paraformaldehyde. Brains were removed and post-fixed in PB enzyme that catalyzes the synthesis of acetylcholine with 4% paraformaldehyde for 1–2 h, cryoprotected and stored (Eng et al., 1974). This permitted elucidating the in PB with 30% sucrose and 0.05% sodium azide. Brains were organization of cholinergic neurons (Kimura et al., 1980; cut in 40-lm serial sections in a coronal plane on a Leica Armstrong et al., 1983). Combined AChE histochemical SM2000R microtome with Physitemp freezing stage and BFS- and ChAT immunohistochemical staining studies 30TC controller. Sections were stained for BuChE or AChE by demonstrated that most ChAT-positive neurons were histochemical (HC) technique, and for BuChE and ChAT using immunohistochemical (IHC) methods. Double labeling for also AChE-positive. (Eckenstein and Sofroniew, 1983; BuChE and ChAT was performed using immunofluorescence Levey et al., 1983, 1984). (IF). In addition to AChE, the enzyme butyrylcholinesterase (BuChE, EC 3.1.1.8) is important in the regulation of the Cholinesterase histochemistry cholinergic system (Darvesh et al., 1998, 2003; Mesulam et al., 2002b; Giacobini, 2003; Duysen et al., Cholinesterase histochemical staining was performed using a 2007). Like AChE, BuChE is able to efficiently catalyze modified (Darvesh et al., 2012a) Karnovsky–Roots method the hydrolysis of acetylcholine (Silver, 1974). BuChE is (Karnovsky and Roots, 1964). Briefly, tissue sections were expressed in distinct populations of neurons, some of rinsed in 0.1 M maleate buffer (pH 7.4) for 30 min and which also contain AChE (Friede, 1967; Tago et al., 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, 1992; Darvesh et al., 1998; Darvesh and Hopkins, 2003; 0.05 mM potassium ferricyanide, 0.8 mM butyrylthiocholine Geula and Nagykery, 2007). The importance of BuChE iodide and 0.01 mM BW 284 C 51 (to inhibit AChE). All in cholinergic neurotransmission is further supported by sections were then rinsed with gentle agitation for 30 min in the observation that AChE-knockout mice survive to dH2O and placed in 0.1% cobalt chloride in water for 10 min. adulthood. This indicates BuChE is able to compensate After further rinsing in dH2O, sections were placed in PB 0 for the lack of AChE, allowing the continued regulation containing 1.39 mM 3,3 -diaminobenzidine tetrahydrochloride (DAB). After 5 min in the DAB solution, 50 ll of 0.15% H O in of cholinergic neurotransmission (Li et al., 2000; Xie 2 2 dH2O was added per ml of DAB solution, and the reaction was et al., 2000; Mesulam et al., 2002a). carried out for approximately 3 min. Sections were then washed To date, study of the colocalization of BuChE and in 0.01 M acetate buffer (pH 3.3), mounted on slides, cleared in ChAT in the mammalian CNS has been limited to the xylene and cover-slipped. Control experiments to demonstrate spinal cord in the rat (Mis, 2005). Because of the earlier specificity of BuChE staining were performed as described indications of BuChE co-regulation of cholinergic previously (Darvesh et al., 1998) and indicated the staining neurotransmission (Xie et al., 2000; Mesulam et al., pattern observed was specific for BuChE activity. The procedure for the visualization of AChE activity was 2002a,b), the present work was undertaken to examine similar to that for BuChE except that the reaction solution was the organization of BuChE-expressing neural elements 24.99 mM sodium citrate, 14.72 mM cupric sulfate, 2.43 mM as they relate to the ChAT-defined cholinergic system in potassium ferricyanide, 2.46 mM acetylthiocholine iodide and the mouse CNS. 0.13 mM ethopropazine (to inhibit BuChE), at room temperature This work has not been presented elsewhere except in 0.1 M maleate buffer (pH 6.0) for 15 min with gentle agitation. in abstract form (Darvesh et al., 2012b). Generation and characterization of polyclonal antibodies to BuChE

EXPERIMENTAL PROCEDURES Rabbit polyclonal antibodies to recombinant mouse BuChE (rmoBuChE) were generated for this study. Production of rmoBuChE (immunogen) was accomplished in human Animals embryonic kidney epithelial cells (293A cells) using an adenovirus containing the gene for mouse BuChE (Ad- Twenty male, wild-type (129S1/SvImJ) mice were purchased moBuChE) (Parikh et al., 2011). The mouse BuChE from The Jackson Laboratory (USA). This mouse strain was (moBuChE) gene contained a 6x tag at its carboxyl chosen because it has been utilized to examine components of terminus suggesting that the rmoBuChE used was a fusion the cholinergic system in other studies (Li et al., 2000; protein. Large-scale expression of rmoBuChE was Mesulam et al., 2002a; Duysen et al., 2007). Animals were accomplished by overnight culturing of 293A cells (10 106)in cared for according to the guidelines set by the Canadian 150-cm2 tissue culture dishes and infecting them for 1 h with Council on Animal Care. Formal approval to conduct the 10 ll of 4th cycle crude viral lysate (high titer CVL) of Ad- experiments was obtained from the Dalhousie University moBuChE in 10 ml of infection medium (DMEM containing 2% Committee on Laboratory Animals. 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 were detected using Infrared Imager (Licor Inc. NE). The (DMEM containing 10% FBS, 50 lg/ml penicillin and reactivity of anti-moBuChE IgG toward recombinant moBuChE streptomycin, and 50 lg/ml sodium pyruvate) was then added or hBuChE was also determined by direct enzyme-linked and plates were returned to the incubator for 5–7 days. immunosorbent assay (ELISA). One hundred ll of recombinant Seventy to one hundred culture dishes were infected in a single moBuChE or hBuChE in PBS (5 lg/ml) was added to each well experiment. When BuChE activity in the culture medium of a 96-well plate and incubated overnight at 4 °C. After reached between 3–4 units/ml, it was collected and cleared of washing the wells with PBS containing 0.05% Tween 20 cells and debris by centrifugation at 2500 rpm for 15 min, 4 °C. (washing buffer), they were blocked with 3% BSA in PBS for Culture media containing 60,000 units (or 70 mg) of the 2 h at 24 °C. After washing the plate three times with washing recombinant enzyme was used for purification of rmoBuChE. buffer, 100 ll each of five or fourfold serial dilutions (ranging Ellman assay (Ellman et al., 1961), using butyrylthiocholine as from 1:1000 to 1:4,000,000) of anti-moBChE IgG in PBS substrate was employed to monitor moBuChE activity during containing 0.1% BSA was added and incubated for 2 h at various steps of the enzyme purification. 24 °C, followed by five washes with washing buffer. One The purification scheme for rmoBuChE involved ammonium hundred ll of goat anti-rabbit IgG (H + L) conjugated with sulfate fractionation followed by affinity chromatography using horseradish peroxidase diluted 1:10,000 in PBS containing procainamide and nickel-affinity resins. The culture media was 0.1% BSA was then added to each well and incubated for 1 h fractionated with ammonium sulfate (0–50% saturation). The at 24 °C. After eight to 10 washes with washing buffer, 100 ll precipitate was collected by centrifugation at 10,000 rpm for of substrate solution containing 3,30,5,50-tetramethylbenzidine 15 min and discarded as the supernatant contained all the (TMB) was added and incubated for 15 min in the dark at enzyme activity. Ammonium sulfate was added to the 24 °C. The reaction was stopped with the addition of 100 llof supernatant to 80% saturation and the precipitate containing 2 N sulfuric acid and end point absorbance of each well was the entire recombinant enzyme was collected as described measured in a plate reader at 450 nm. BSA blocked wells were above. The enzyme was dissolved in 100 ml of 50 mM PB (pH used as reagent controls. 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 Butyrylcholinesterase immunohistochemistry 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 For BuChE immunohistochemistry (IHC), sections were rinsed in 500 mM NaCl and 2.5 mM imidazole and loaded onto a 10-ml PB for 30 min and placed in 0.3% H2O2 in PB for 30 min to column of nickel Sepharose resin equilibrated against the same quench endogenous peroxidase activity. Sections were rinsed buffer. The bound enzyme was eluted with 25 mM PB (pH 8.0) again in PB for 30 min and then incubated in PB containing containing 500 mM NaCl and 500 mM imidazole. The enzyme 0.1% Triton X-100, normal goat serum (NGS, 1:100) and rabbit fractions were combined and dialyzed against 25 mM PB (pH anti-BuChE primary antibody (1:10,000) for approximately 16 h 8.0). For long-term storage of the enzyme, glycerol was added at room temperature. After rinsing, sections were incubated in (50%) and stored in 20 °C freezer. PB with 0.1% Triton X-100, NGS (1:1000) and biotinylated goat Polyclonal antibodies to rmoBuChE were produced in two 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- New Zealand white rabbits according to standard polyclonal Ò antibody production protocols (Washington Biotechnology Inc., 100 and Vectastain Elite ABC kit (Vector) according to the 6200 Seaforth Street, Baltimore, MD 21224). Three mg of the manufacturer’s instructions. After rinsing the sections in PB, purified rmoBuChE was supplied to Washington Biotechnology they were placed in a solution of PB containing 1.39 mM DAB. as lyophilized powder for antibody development. The protocol After 5 min, 50 ll of 0.3% aqueous H2O2 per mL of DAB was approved by the Institutional Animal Care and Use staining solution was added and incubated for 5 min. The Committee of the Johns Hopkins University, Baltimore, reaction was stopped by rinsing the sections in 0.01 M acetate Maryland. Sera collected after the booster injections were used buffer (pH 3.3). Sections were mounted on slides, cleared in for purification of anti-moBuChE immunoglobulin G (IgG) by xylene and cover-slipped. 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. Choline acetyltransferase immunohistochemistry The bound IgG were eluted with 50 mM glycine–HCl (pH 2.7) and fractions containing IgG were neutralized using 1 M dibasic For ChAT-IHC, sections were rinsed in 0.05 M tris-buffered saline PB (pH 7.5). Later, sodium azide and glycerol were added to (TBS; pH 7.6) for 30 min and placed in 0.3% H2O2 in TBS for final concentrations of 0.05% and 10%, respectively for long- 30 min to quench endogenous peroxidase activity. Sections term storage of the antibody. were rinsed again for 30 min and incubated in TBS containing The reactivity of anti-moBuChE IgG toward rmoBuChE or 0.3% Triton X-100, normal rabbit serum (NRS, 1:100) and goat human BuChE (hBuChE) was determined by Western blotting. anti-ChAT primary antibody (1:1000; Millipore, AB144P) for Sodium dodecyl sulfate–polyacrylamide gel electrophoresis about 16 h at room temperature. After rinsing, sections were (SDS–PAGE) was carried out for 100 ng of each enzyme using incubated in TBS containing 0.3% Triton X-100, normal rabbit precast 10% Tris–HCl gels. One microgram of bovine serum serum (1:1000) and biotinylated rabbit anti-goat secondary albumin (BSA) was included in the sample to prevent non- antibody (1:500; Vector) for 1 h. After another rinse, sections specific loss of the enzyme. After electrophoresis, proteins were placed in TBS containing 0.3% Triton X-100 and were transferred to nitrocellulose membrane using IBlot gel VectastainÒ Elite ABC kit according to the manufacturer’s transfer apparatus (InVitrogen, CA). The membrane was instructions. After rinsing, sections were placed in a solution of blocked in blocking buffer (Licor Inc., NE) for 2 h at 24 °C, TBS (pH 8.0) containing 1.39 mM DAB and 0.6% nickel washed once with 50 mM phosphate buffer containing 0.05% ammonium sulfate. After 5 min, 50 ll of 0.3% H2O2 in dH2O Tween 20 (washing buffer) and kept overnight in blocking buffer was added per mL of DAB staining solution and the mixture containing anti-moBChE antibody (1:20,000 dilution). The was incubated for 2–5 min. The reaction was stopped by membrane was then washed five times with intermittent rinsing the sections in TBS. Sections were mounted on slides, shaking for 5 min and incubated with secondary antibody cleared in xylene and cover-slipped. Control experiments were conjugated with infra red dye 680 (Licor Inc., NE, 1:10,000 performed by omitting the primary antibody and no staining was dilution) made in blocking buffer for 1 h, and protein bands observed. 56 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

Butyrylcholinesterase and choline acetyltransferase its binding to hBuChE (Fig. 1B). ELISA results also immunofluorescence double labeling showed a similar trend. Certain dilutions of the antibody (1:25,000, 1:125,000, and 1:500,000) produced a Double labeling for BuChE and ChAT was done on the same fourfold higher absorbance values with moBuChE sections using an immunofluorescent method. Sections were compared to hBuChE (Fig. 1C). The specificity of rinsed in TBS for 30 min and incubated for approximately 16 h moBuChE antibody was also confirmed by conducting at room temperature in TBS containing 0.3% Triton X-100, normal donkey serum (1:100) and both primary antibodies, BuChE-IHC with the antibody following incubation with rabbit anti-BuChE (1:7000) and goat anti-ChAT (1:1000). After BuChE from mouse serum. No staining was observed rinsing in TBS, sections were incubated for 1 h in TBS indicating specificity of this antibody (Fig. 1D). All containing 0.3% Triton X-100, donkey anti-goat Alexa FluorÒ together, these results demonstrate the specificity of this 488 (1:350; Molecular Probes, A11055) and donkey anti-rabbit antibody toward moBuChE. Alexa FluorÒ 555 (1:350; Molecular probes, A31572). Sections were rinsed, mounted on slides, cleared in xylene and cover- slipped. Validation of BuChE immunohistochemical staining The mouse BuChE antibody was evaluated for its ability Data analysis to recapitulate the distribution of BuChE activity through established histochemical analysis. To this end, Sections stained by HC, IHC or IF were analyzed on a Zeiss sections, at representative levels throughout the brain, Axioplan 2 microscope and photographed with a Zeiss Axiocam were stained by BuChE-HC, BuChE-IHC or AChE-HC HRc digital camera and AxioVision 4.6 software. Photomicrographs of IF sections were also acquired on a Zeiss and compared. Fig. 2 provides two examples, the LSM 510 laser scanning-confocal microscope. Orthogonal piriform cortex (Pir; Fig. 2A–C) and the motor trigeminal images were rendered and edited with LSM imaging software nucleus (Mo5; Fig. 2D–F), that illustrate the (Zeiss). Photographs were assembled using AdobeÒ comparability of BuChE-HC (Fig. 2A, D) and BuChE- Ò Photoshop CS5. Images were contrast enhanced and the IHC (Fig. 2B, E) staining and emphasizes differences in brightness adjusted to match the background from different distribution of this enzyme relative to that of AChE-HC images. Images of IF labeling for BuChE and ChAT were merged to illustrate the relationship between these two staining (Fig. 2C, F). These areas were chosen as markers. BuChE-IF appeared green while ChAT appeared red. examples because of the distinct distribution of BuChE Neural elements with both BuChE and ChAT appeared yellow and AChE neural elements that occur therein. in merged images. Throughout the brain, the patterns of staining for BuChE Anatomical maps of the distribution of BuChE or ChAT were were comparable whether HC or IHC was used, except produced by plotting the neuronal distribution on a Wacom that neuropil staining by BuChE-HC was generally more drawing tablet with Microbrightfield Neuroleucida software linked to a Microfire digital camera on an Olympus BMAX diffuse than that revealed by BuChE-IHC. These microscope with Heidenhain linear potentiometers and Prior experiments confirmed the ability of the BuChE antibody Optiscan controller. The same sections were then to accurately visualize the location of BuChE that is photographed at low power and assembled into a single image conventionally obtained by HC analysis. 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 Distribution of butyrylcholinesterase and choline AdobeÒ IllustratorÒ CS5 by overlaying and aligning the acetyltransferase neuroleucida drawings on the low power photomicrographs. Within the mouse CNS, BuChE staining labeled distinct populations of neurons, neuropil, glia and white matter RESULTS while ChAT labeled neurons and neuropil (see Fig. 3 for The present investigation was undertaken to elucidate the examples). The glial perikarya were distinguished from relationships between BuChE-positive neural elements the neuronal perikarya based on their size and and ChAT in the mouse CNS. Using HC and IHC morphology (Kettenmann and Ransom, 2005). Staining methods, the distributions of BuChE and ChAT neural patterns for each marker were consistent in all brains elements were analyzed. Colocalization of these two examined. Comparative photomicrographs and markers was confirmed with immunofluorescent (IF) corresponding schematic maps (Fig. 4) of BuChE-HC double labeling. (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 Characterization of mouse BuChE antibodies maps, each neuron stained for BuChE or ChAT is Analysis of rmoBuChE by reducing SDS–PAGE revealed represented by a black circle. Stained neuropil and that it was a pure protein and the molecular weight of white matter, not dealt with in the maps, can be seen 85 kDa for its subunit was similar to that for purified directly for each marker in the corresponding native hBuChE (Fig. 1A). Polyclonal moBuChE antibody photomicrographs (Fig. 4). A summary of structures in was characterized for its binding and specificity to the mouse brain containing neuronal somata or neuropil recombinant moBuChE and hBuChE by Western stained for BuChE and ChAT is presented in Table 1 blotting (Fig. 1B) and direct ELISA (Fig. 1C). Both with an estimate of soma numbers and density of assays show that moBuChE antibody preferred binding neuropil. In this tables, boxes shaded in gray indicate to moBuChE than hBuChE. By Western blotting, nuclei that contain neurons stained for both BuChE and antibody binding to moBuChE was 10-fold greater than ChAT as demonstrated by IF double labeling. The G. A. Reid et al. / Neuroscience 234 (2013) 53–68 57

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 ventromedial corner of the Pir. There was very little in specific regions of the mouse brain is described below BuChE-positive neuropil in the cortex with the exception in more detail. of the cingulate cortex (Cg), which had distinct processes localized to layers 2–3. There was also Cerebral cortex. BuChE-positive neuronal somata diffuse neuropil staining in the retrosplenial cortex (RS), were scattered throughout the mouse cerebral cortex predominantly in layer 4. As reported previously (Table 1; Fig. 4A, C, E, G), as reported previously (Mesulam et al., 2002a), there were numerous ChAT- (Mesulam et al., 2002a). Labeled neurons were more positive neurons evident in the cerebral cortex with the numerous rostrally but generally were few in number exception of the Cg and MEnt (Table 1; Fig. 4B, D, F, and located in layer 6 with an occasional neuron in H, J). ChAT-positive cells were concentrated in layers layers 1–2. On the other hand, the medial entorhinal 2–3, but were also scattered throughout layers 4–6. cortex (MEnt) had numerous BuChE-positive neurons Also, as previously reported (Kitt et al., 1994), there was primarily located in layer 2 (Fig. 4I). There was also a laminar distribution of ChAT-positive neuropil throughout group of BuChE-positive neurons located between the the cerebral cortex. Experiments using IF double Pir, the ventral portion of the tenia tecta, the anterior labeling for BuChE and ChAT demonstrated that these olfactory cortex (AO) and the olfactory tubercle (Tu). two markers labeled different populations of neurons in Caudally, these neurons continued adjacent to the the cerebral cortex. 58 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

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 positive neurons (Fig. 4C), as in the anterior cortical sparse within the basal ganglia with only a few scattered (ACo), basolateral (BL), basomedial (BM) and lateral neurons in the caudate putamen nucleus (CPu) and the (La) amygdaloid nuclei or were not positive for BuChE, ventral pallidum (VP) (Table 1; Fig. 4A, C). In contrast, as in the central amygdala (Ce). ChAT staining within there were numerous ChAT-positive neurons and the amygdala was limited to neuropil (Fig. 4D), as neuropil labeling in these regions of the basal ganglia reported previously (Kitt, 1994). There were no BuChE- (Fig. 4B, D), as reported previously (Kitt et al., 1994; positive neurons stained for ChAT in this region. Mesulam et al., 2002a). There were no BuChE/ChAT double-labeled neurons in this region. Hypothalamus. A number of nuclei of the hypothalamus contained BuChE-positive neuronal Basal forebrain. Within the basal forebrain there were somata (Table 1; Fig. 4A, C, E) including the medial scattered BuChE-positive neuronal somata (Table 1, supramammillary (SuMM), ventral premammillary Fig. 4A) in the medial septal nucleus (MS), substantia (PMV), lateroanterior hypothalamic (LA), posterior part innominata (SI) and vertical (VDB) and horizontal (HDB) of the anterior hypothalamic (AHP), dorsal limb nuclei of the diagonal band of Broca. There was premammillary (PMD), lateral hypothalamic (LH), generally sparse BuChE neuropil staining in these magnocellular preoptic (MCPO), terete hypothalamic, regions except for islands of Calleja (ICj), which had submammillothalamic (SMT), lateral preoptic, intense labeling. In contrast, ChAT staining within the paraventricular hypothalamic (Pa), perifornical and basal forebrain was much more prominent (Table 1; posterior hypothalamic (PH) nuclei (Fig. 4A, C, E). Fig. 4B), as shown previously (Kitt et al., 1994). Distinct BuChE-positive neuropil was labeled in the Immunofluorescent double labeling showed that BuChE SuMM, SMT, LH, PH with sparse staining in the and ChAT were located in different populations of mammillary nucleus. As observed previously in the neuronal somata in the basal forebrain. mouse (Bobkova et al., 2001), the MCPO had ChAT- positive perikarya (Fig. 4B). In addition, other Amygdala. The amygdala had several nuclei with hypothalamic nuclei had ChAT-positive neuronal somata BuChE-positive neurons (see Table 1). There were and neuropil, including the LH and SuMM (Fig. 4D, F). numerous BuChE-positive neuronal somata in the There were no neurons within the hypothalamus that anterior amygdaloid area (AA), medial anterior were positive for both BuChE and ChAT labeling. amygdala (MeA; Fig. 4C) and in the most caudal tip of the posteromedial cortical amygdala (PMCo). The Thalamus. There was extensive BuChE staining remaining nuclei either had a few scattered BuChE- associated with neuronal somata and neuropil within the G. A. Reid et al. / Neuroscience 234 (2013) 53–68 59

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).

thalamus (Table 1; Fig. 4C, E). The anterodorsal (AD), Hippocampal formation. The hippocampal formation anteromedial (AM), anteroventral (AV), dorsal lateral (H) of the mouse brain had few BuChE-positive geniculate (DLG), laterodorsal (LD), medial habenula neuronal somata found in the caudal portion of the (MHb), paratenial (PT) and reuniens (Re) thalamic polymorphic cell layer of the dentate gyrus (PoDG) and nuclei were all densely populated with BuChE-positive the stratum oriens (Or) of hippocampus proper (Fig. 4E, neurons. BuChE-positive neurons within the MHb were G). There was BuChE neuropil staining at the most generally located more caudally, along the dorsal edge rostral level of the hippocampus proper in the CA3 of the nucleus and were closely associated with the stria region. ChAT staining within the hippocampal formation medullaris (sm) and the habenular commissure. The labeled neuropil, as described previously (Kitt et al., marginal zone of the medial geniculate, interanterior 1994). There were no neurons double labeled for dorsal and lateral posterior (LP) thalamic nuclei had BuChE and ChAT in the hippocampal formation. fewer BuChE-stained neurons. There were also a few scattered BuChE-positive cells in the ventral posterior Midbrain. Within the midbrain, there were several lateral thalamus along the lateral edge, closely nuclei with numerous BuChE-positive neuronal somata associated with the external medullary lamina (eml). including the interstitial nucleus of Cajal (InC), medial Generally, there was distinct BuChE-positive neuropil terminal nucleus of the accessory optic tract (MT), throughout the thalamus. However, a few areas with dorsal raphe nucleus (DR) and red nucleus (R) relatively more intense BuChE neuropil staining included (Table 1; Fig. 4G, I). Other nuclei in this region had lateroventral portion of the AV, the habenula (Hb) and relatively fewer BuChE-positive cells (see Table 1). mediodorsal (MD) nuclei. ChAT staining labeled neuropil There was dense BuChE-positive neuropil in the throughout the thalamus (Table 1; Fig. 4D, F), as intermediate white layer of the superior colliculus observed previously (Kitt, 1994). The only ChAT-positive (InWh), anterior pretectal nucleus (APT), DR and the soma was located within the MHb (Table 1; Fig. 4D) as retroparafascicular nucleus, with less dense BuChE- noted elsewhere (Mesulam et al., 2002a). IF double positive neuropil throughout other midbrain regions labeling analysis demonstrated that BuChE and ChAT (Fig. 4G, I). As reported previously (Mesulam et al., labeled different populations of cells within the MHb. 2002a; Vanderhost et al., 2006), a number of nuclei in 60 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

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

Fig. 4. (continued) 62 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, + = <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. G. A. Reid et al. / Neuroscience 234 (2013) 53–68 63

Table 1 (continued)

the midbrain had ChAT-positive neurons and neuropil detected with IF double labeling, a close association of (Table 1; Fig. 4H, J). The only nucleus that had both BuChE-positive neuropil and ChAT-positive somata was ChAT- and BuChE-positive neurons was the R but IF evident within the LDTg and PPTg. For example, in the double labeling indicated that BuChE and ChAT were LDTg, confocal microscopy indicated that BuChE- not colocalized within the same neurons. positive neuropil surrounded ChAT-positive somata (Fig. 5).

Pons. The pons had numerous BuChE-positive neuronal somata in the locus coeruleus (LC) and Medulla oblongata. Within the medulla oblongata, subcoeruleus nucleus (SubC; Fig. 4I, K). Other nuclei numerous BuChE-positive neurons were found in the had scattered BuChE-positive cells (Table 1; Fig. 4I, K). facial (7N), dorsal motor nucleus of the vagus (10N), There was extensive BuChE-positive neuropil in the inferior olivary (IO), inferior salivatory (IS) and solitary pedunculopontine (PPTg), laterodorsal (LDTg) and tract (Sol) nuclei as well as the A5 noradrenalin cell microcellular (MiTg) tegmental nuclei. Other areas had group (see Table 1; Fig. 4M, O). There were also moderately dense staining for BuChE-positive neuropil numerous BuChE-positive neurons located in the (see Table 1). As has been shown previously (Mesulam caudoventral portion of the hypoglossal nucleus (12N) et al., 2002a; Vanderhost et al., 2006), numerous and scattered neurons in the ambiguus nucleus (Amb), regions in the pons had ChAT-positive neurons and intermediate reticular nucleus (IRt) and the interpolaris neuropil (Table 1; Fig. 4J, L). Although there was no (Sp5I) and ventrolateral oralis (Sp5OVL) divisions of the neuronal colocalization of BuChE and ChAT in the pons spinal trigeminal nucleus among several others (see 64 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

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 with both were examined following IF double prepositus (Pr), solitary (Sol) and gracile nuclei. Many labeling to determine if neural elements contained both nuclei within the medulla oblongata contained ChAT- enzymes. From these experiments, a number of positive neurons, as described previously (Vanderhost relationships between BuChE and ChAT labeling could et al., 2006). In addition, numerous ChAT-positive be identified. First, there were BuChE-positive neuronal neurons were detected in the medial vestibular (MVe), somata that were colocalized with ChAT. Second, in spinal vestibular (SVe), and IS nuclei as well as the other areas BuChE-positive neuronal somata did not noradrenaline cell group A5 (Table 1, Fig. 4N). IF contain ChAT but were in close proximity to ChAT- double labeling demonstrated that BuChE and ChAT positive neuropil. Third, elsewhere, there were ChAT- were colocalized in neurons in the 7N (Fig. 6A–C), 10N positive neurons that did not contain BuChE but were (Fig. 6D–F), 12N, Amb (Fig. 6G–I), IS (Fig. 6J–L), A5 closely associated with BuChE-positive neuropil. (Fig. 6M–O), LPGi, and IRt. Confocal imaging with Within the mouse CNS, colocalization of BuChE and orthogonal view of these regions provided further ChAT labeling within neuronal somata was found in the evidence of colocalization of BuChE and ChAT within medulla oblongata and the spinal cord (Table 1, shaded the same neurons (see Fig. 7 for example). boxes). Nuclei showing neuronal colocalization included the 7N, 10N and 12N (Figs. 6 and 7). Confocal imaging Spinal cord. Within the cervical spinal cord BuChE demonstrated that BuChE was located within the cell staining was observed in scattered neuronal somata in body of the neuron and was not confined to the cell laminae 5, 7 and 10 and in distinct, varicose neuropil in surface. The distribution of these BuChE-positive lamina 4. As reported previously, ChAT staining labeled cholinergic neurons within cranial nerve nuclei suggests neurons in laminae 3 (Mesnage et al., 2011), 7, 9 and 10 a role for BuChE in motor and visceromotor control. (VanderHorst and Ulfhake, 2006). In addition, ChAT-positive BuChE-positive neuronal somata in most areas were neurons were located in laminas 5 and 8. ChAT-positive ChAT-negative but coincided with ChAT-positive neuropil was found throughout this level of spinal cord but neuropil (Table 1, Fig. 4). Due to the extensive was particularly dense in lamina 2. IF double labeling, for distribution of ChAT-positive neuropil and the BuChE and ChAT demonstrated colocalization of these widespread use of acetylcholine as a neurotransmitter, it markers in neurons of laminae 7 and 10. 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 DISCUSSION neuronal somata as well as ChAT-positive neuropil. This Cholinergic neurotransmission plays a significant role in a area receives cholinergic input from the MS and VDB wide variety of functions in the nervous system including (Heys et al., 2012) and primarily projects to the dentate memory, behavior, and motor and visceromotor control gyrus and CA3 hippocampal region via the medial (Giacobini and Pepeu, 2006). Regulation of the perforant pathway (Kerr et al., 2007). Therefore, cholinergic system, through acetylcholine hydrolysis, BuChE-positive neurons in this region could affect has been largely attributed to AChE. However, it has cholinergic modulation of functions subserved by this been demonstrated that BuChE can also play a region, such as spatial novelty detection and path fundamental role in cholinergic neurotransmission in integration (Van Cauter et al., 2013). Similarly, many view of the fact that AChE knock-out mice are viable thalamic nuclei had ChAT-positive neuropil and large (Xie et al., 2000). The present work describes the populations of BuChE-positive neurons. These BuChE- association of BuChE with ChAT-positive cholinergic positive cells could be receiving cholinergic innervation elements in the mouse CNS and provides detailed maps from a wide variety of sources including the basal of neuronal somata positive for each enzyme. Areas forebrain and tegmental nuclei (Mesulam et al., 1983; G. A. Reid et al. / Neuroscience 234 (2013) 53–68 65

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 of BuChE in this area which, in turn, may regulate strong cholinergic input to the thalamus is most likely thalamic functions including relaying sensory and motor regulated, at least in part, by the extensive distribution signals to the cerebral cortex and regulation of 66 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

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 Taking into account these close relationships between addition, the hypothalamus contained several nuclei with ChAT- and BuChE-positive neural elements, there are a BuChE- and ChAT-positive neuronal somata and number of ways BuChE could participate in the neuropil (Table 1; Fig. 4A–F) such as the MCPO, SuMM regulation of cholinergic neurotransmission. BuChE and LH. However, BuChE and ChAT were not could be expressed in the synapse of neurons with colocalized within the same neurons in these regions. cholinergic receptors and be involved in modulation of Although ChAT-positive neurons have been previously direct cholinergic neurotransmission. On the other hand, described in the mouse MCPO (Bobkova et al., 2001) BuChE-positive neural elements may also be involved in the present study indicated that the SuMM and LH also regulation of tonic levels of volume acetylcholine. Tonic contained ChAT-positive perikarya. ChAT-positive neurotransmission could occur when acetylcholine neurons in these areas have been described previously diffuses out of the synaptic cleft or when acetylcholine is in the rat (Choi et al., 2012) and cat (Vincent and released from non-synaptic varicosities (Descarries Reiner, 1987), respectively. The distribution of BuChE et al., 1997; Sarter et al., 2009; Ren et al., 2011). These within the hypothalamus suggests its involvement in considerations are in keeping with previous observations hypothalamic cholinergic functions such as modulating demonstrating that highly specific BuChE inhibitors, stress responses, feeding behavior, sleep-wake cycles such as (Greig et al., 2005) and ()- and homeostasis in general. N1phenethyl-norcymserine (Cerbai et al., 2007), A close relationship was also observed between increased acetylcholine levels in the rat cerebral cortex, BuChE-positive neuropil and ChAT-positive cholinergic leading to improved cognition in these animals. In neurons within the LDTg and PPTg in the pons. This addition, in AChE-deficient mice, the selective BuChE caudal cholinergic cell column innervates a number of inhibitors bambuterol and bisnorcymserine, significantly areas of the brain, including the thalamus (Mesulam raised acetylcholine in the brain (Hartmann et al., 2007), et al., 1983), pontomedullary reticular formation and indicating the importance of BuChE in the regulation of mesolimbic regions and is involved in control of cholinergic neurotransmission. locomotion and regulation of sleep-wake cycle, arousal, Thus, based on neuroanatomical evidence presented stimulus and reward learning, visual orienting and herein, BuChE may be involved, along with AChE, in sensory motor systems (VanderHorst and Ulfhake, maintaining appropriate levels of acetylcholine in the 2006). As in the rat (Tago et al., 1992), BuChE-HC brain. The level of physiologically relevant acetylcholine staining demonstrated that this enzyme was associated may be maintained by each enzyme in a different way. with neuronal perikarya. However, BuChE-IHC and That is, although BuChE and AChE both hydrolyze confocal microscopy in double labeled sections acetylcholine efficiently, at high levels of acetylcholine, indicated that this staining was, in fact, BuChE-positive AChE activity is inhibited while that of BuChE is neuropil surrounding ChAT-positive neurons forming a activated (Silver, 1974). This complementary BuChE-containing pericellular basket. This suggests that relationship between these two enzymes may be the apparent difference observed in the mouse in the important in maintaining appropriate cholinergic tone present study was most likely a result of the finer detail within the CNS. enabled through BuChE-IHC, rather than species differences. Given this close association of BuChE neuropil to ChAT-positive neurons in the LDTg and CONCLUSIONS PPTg, BuChE may be involved in maintaining This study indicates a possible cholinergic regulatory acetylcholine levels in the arousal systems of the CNS. 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 Eckenstein F, Sofroniew MV (1983) Identification of central observed for the distribution of AChE in the brain, not all cholinergic neurons containing both choline acetyltransferase BuChE-positive neuronal somata contain ChAT. BuChE and acetylcholinesterase and of central neurons containing only acetylcholinesterase. J Neurosci 3(11):2286–2291. and ChAT colocalization within neurons was found to be Ellman GL, Courtney KD, Andres Jr V, Feather-Stone RM (1961) A limited to particular nuclei within the medulla oblongata and new and rapid colorimetric determination of acetylcholinesterase spinal cord. However, there were other relationships activity. Biochem Pharmacol 7:88–95. evident between these two enzymes, including BuChE- Eng LF, Uyeda CT, Chao LP, Wolfgram F (1974) Antibody to bovine positive neuronal somata in areas of ChAT neuropil and choline acetyltransferase and immunofluorescent localisation of BuChE neuropil that formed pericellular baskets around the enzyme in neurons. Nature 250(463):243–245. Friede RL (1967) A comparative histochemical mapping of the ChAT-positive, cholinergic neurons. distribution of butyryl cholinesterase in the brains of four species This work provides further neuroanatomical evidence of mammals, including man. Acta Anat (Basel) 66(2):161–177. that BuChE plays a role in the modulation of cholinergic Geula C, Nagykery N (2007) Butyrylcholinesterase activity in the rat neurotransmission. forebrain and upper brainstem: postnatal development and adult distribution. Exp Neurol 204(2):640–657. Giacobini E (2003) Butyrylcholinesterase: its function and Acknowledgments—This work was supported in part by Cana- inhibitors. United Kingdom: Martin Dunitz. dian Institutes of Health Research, Capital District Health Author- Giacobini E, Pepeu G (2006) The brain cholinergic system: in health ity Research Fund, Nova Scotia Health Research Foundation, and disease. United Kingdom: Informa Healthcare. Faculty of Medicine (Clinician-Scientist Program), Dalhousie Uni- Greig NH, Utsuki T, Ingram DK, Wang Y, Pepeu G, Scali C, Yu QS, versity Department of Medicine (University Internal Medicine Re- Mamczarz J, Holloway HW, Giordano T, Chen D, Furukawa K, search Fund) and Dalhousie Medical Research Foundation Sambamurti K, Brossi A, Lahiri DK (2005) Selective (S.D.) and Defense Threat Reduction Agency (DTRA), Joint Sci- butyrylcholinesterase inhibition elevates brain acetylcholine, ence and Technology Office, Medical S&T Division (N.C.). We augments learning and lowers Alzheimer beta-amyloid peptide would like to thank Professor Earl Martin for critically reviewing in rodent. Proc Natl Acad Sci USA 102(47):17213–17218. this manuscript. 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. REFERENCES Heckers S, Geula C, Mesulam MM (1992) Cholinergic innervation of the human thalamus: dual origin and differential nuclear Armstrong DM, Saper CB, Levey AI, Wainer BH, Terry RD (1983) distribution. J Comp Neurol 325(1):68–82. Distribution of cholinergic neurons in rat brain: demonstrated by Heys JG, Schultheiss NW, Shay CF, Tsuno Y, Hasselmo ME (2012) the immunocytochemical localization of choline acetyltransferase. Effects of acetylcholine on neuronal properties in entorhinal J Comp Neurol 216(1):53–68. cortex. Front Behav Neurosci 6(32) (Epub Jul 24). Bobkova NV, Nesterova IV, Nesterov VV (2001) The state of Jones E (2007) The thalamus. second ed. Cambridge, United cholinergic structures in forebrain of bulbectomized mice. Bull Kingdom: Cambridge University Press. Exp Biol Med 131(5):427–431. Karnovsky MJ, Roots L (1964) A direct-coloring thiocholine method Cerbai F, Giovannini MG, Melani C, Enz A, Pepeu G (2007) for cholinesterases. J Histochem Cytochem 12:219–221. N1phenethyl-norcymserine, a selective butyrylcholinesterase Kerr KM, Agster KL, Furtak SC, Burwell RD (2007) Functional inhibitor, increases acetylcholine release in rat cerebral cortex: a neuroanatomy of the parahippocampal region: the lateral and comparison with and . Eur J Pharmacol medial entorhinal areas. Hippocampus 17(9):697–708. 572(2–3):142–150. Kettenmann H, Ransom BR (2005) Neuroglia. New York: Oxford Choi WK, Wirtshafter D, Park HJ, Lee MS, Her S, Shim I (2012) The University Press. characteristics of supramammillary cells projecting to the Kimura H, McGeer PL, Peng F, McGeer EG (1980) Choline hippocampus in stress response in the rat. Korean J Physiol acetyltransferase-containing neurons in rodent brain demonstrated by Pharmacol 16(1):17–24. immunohistochemistry. Science 208(4447):1057–1059. Darvesh S, Hopkins DA (2003) Differential distribution of Kitt CA, Ho¨hmann C, Coyle JT, Price DL (1994) Cholinergic butyrylcholinesterase and acetylcholinesterase in the human innervation of mouse forebrain structures. J Comp Neurol thalamus. J Comp Neurol 463:25–43. 341(1):117–129. Darvesh S, Grantham DL, Hopkins DA (1998) Distribution of Koelle GB, Friedenwald JA (1949) A histochemical method for butyrylcholinesterase in the human amygdala and hippocampal localizing cholinesterase activity. Proc Soc Exp Biol Med formation. J Comp Neurol 393:374–390. 70(4):617–622. Darvesh S, Hopkins DA, Geula C (2003) Neurobiology of Levey AI, Wainer BH, Mufson EJ, Mesulam MM (1983) Co- butyrylcholinesterase. Nat Rev Neurosci 4(2):131–138. localization of acetylcholinesterase and choline Darvesh S, Cash MK, Reid GA, Martin E, Mitnitski A, Geula C acetyltransferase in the rat cerebrum. Neuroscience 9(1):9–22. (2012a) Butyrylcholinesterase is associated with b-amyloid Levey AI, Wainer BH, Rye DB, Mufson EJ, Mesulam MM (1984) plaques in the transgenic APPSWE/PSEN1dE9 mouse model of Choline acetyltransferase-immunoreactive neurons intrinsic to Alzheimer disease. J Neuropathol Exp Neurol 71(1):2–14. rodent cortex and distinction from acetylcholinesterase-positive Darvesh S, Reid GA, Chilukuri N (2012b) Butyrylcholinesterase and neurons. Neuroscience 13(2):341–353. cholinergic neural elements in the mouse brain. Soc Neurosci Li B, Stribley JA, Ticu A, Xie W, Schopfer LM, Hammond P, Brimijoin Abstr. Program#/Poster#:840.15/B5. S, Hinrichs SH, Lockridge O (2000) Abundant tissue Descarries L, Gisiger V, Steriade M (1997) Diffuse transmission by butyrylcholinesterase and its possible function in the acetylcholine in the CNS. Prog Neurobiol 53(5):603–625. acetylcholinesterase knockout mouse. J Neurochem Duysen EG, Li B, Darvesh S, Lockridge O (2007) Sensitivity of 75(3):1320–1331. butyrylcholinesterase knockout mice to ()- and Mesnage B, Gaillard S, Godin AG, Rodeau JL, Hammer M, Von donepezil suggests humans with butyrylcholinesterase Engelhardt J, Wiseman PW, De Koninck Y, Schlichter R, deficiency may not tolerate these Alzheimer’s disease drugs Cordero-Erausquin M (2011) Morphological and functional and indicates butyrylcholinesterase function in characterization of cholinergic interneurons in the dorsal horn of neurotransmission. Toxicology 233(1–3):60–69. the mouse spinal cord. J Comp Neurol 519(16):3139–3158. 68 G. A. Reid et al. / Neuroscience 234 (2013) 53–68

Mesulam MM, Mufson EJ, Wainer BH, Levey AI (1983) Central Sarter M, Parikh V, Howe WM (2009) Phasic acetylcholine release cholinergic pathways in the rat: an overview based on an and the volume transmission hypothesis: time to move on. Nat alternative nomenclature (Ch1–Ch6). Neuroscience Rev Neurosci 10(5):383–390. 10(4):1185–1201. Shute CC, Lewis PR (1963) Cholinesterase-containing systems of Mesulam MM, Guillozet A, Shaw P, Levey A, Duysen EG, Lockridge the brain of the rat. Nature 199:1160–1164. O (2002a) Acetylcholinesterase knockouts establish central Silver A (1974) The biology of cholinesterases. Amsterdam: Elsevier. cholinergic pathways and can use butyrylcholinesterase to Tago H, Maeda T, McGeer PL, Kimura H (1992) hydrolyze acetylcholine. Neuroscience 110(4):627–639. Butyrylcholinesterase-rich neurons in rat brain demonstrated by Mesulam M, Guillozet A, Shaw P, Quinn B (2002b) Widely spread a sensitive histochemical method. J Comp Neurol butyrylcholinesterase can hydrolyze acetylcholine in the normal 325(2):301–312. and Alzheimer brain. Neurobiol Dis 9(1):88–93. Van Cauter T, Camon J, Alvernhe A, Elduayen C, Sargolini F, Save E Mis K (2005) Colocalization of acetylcholinesterase, (2013) Distinct roles of medial and lateral entorhinal cortex in butyrylcholinesterase and choline acetyltransferase in rat spinal spatial cognition. Cereb Cortex 23(2):451–459. cord. Hum Exp Toxicol 24(10):543–545. VanderHorst VG, Ulfhake BJ (2006) The organization of the Parikh K, Duysen EG, Snow B, Jensen NS, Manne V, Lockridge O, brainstem and spinal cord of the mouse: relationships between Chilukuri N (2011) Gene-delivered BuChE Is Prophylactic against monoaminergic, cholinergic, and spinal projection systems. Chem the toxicity of chemical warfare nerve agents and Neuroanat 31(1):2–36. organophosphorus compounds. J Pharmacol Exp Ther Vincent SR, Reiner PB (1987) The immunohistochemical localization 337:92–101. of choline acetyltransferase in the cat brain. Brain Res Bull Paxinos G, Franklin KBJ (2001) The mouse brain in stereotaxic 18(3):371–415. coordinates. second ed. San Diego: Academic Press. Xie W, Stribley JA, Chatonnet A, Wilder PJ, Rizzino A, McComb RD, Ren J, Qin C, Hu F, Tan J, Qiu L, Zhao S, Feng G, Luo M (2011) Taylor P, Hinrichs SH, Lockridge O (2000) Postnatal Habenula ‘‘cholinergic’’ neurons co-release glutamate and developmental delay and supersensitivity to in acetylcholine and activate postsynaptic neurons via distinct gene-targeted mice lacking acetylcholinesterase. J Pharmacol transmission modes. Neuron 69(3):445–452. Exp Ther 293(3):896–902.

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