distribution of butyrylcholinesterase in the human amygdala and hippocampal formation

Distribution of Butyrylcholinesterasein the Human Amygdala

and Hippocampal Formation

S. DARVESH,1,2* D. L. GRANTHAM,2 AND D. A. HOPKINS2

1Department of Medicine, Divisions of Neurology and Geriatric Medicine,Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada

2Department of Anatomy and Neurobiology, Dalhousie University,Halifax, Nova Scotia B3H 4H7, Canada

ABSTRACTThe distribution of the major cholinergic regulatory enzyme acetylcholinesterase (AChE,

EC 3.1.1.7) has been extensively studied in the human brain, but the distribution of the closelyrelated enzyme butyrylcholinesterase (BuChE, EC 3.1.1.8) is largely unknown. Because of theimportance of BuChE and AChE in Alzheimer’s disease, we have studied the distribution ofBuChE in the normal human amygdala and hippocampal formation and compared it with thatof AChE by using histochemical techniques.

In the amygdala, the distribution of BuChE differed significantly from that of AChE inthat BuChE was found primarily in neurons and their dendritic processes, whereas AChE wasfound predominantly in the neuropil. BuChE-positive neurons were present in up to 10% ofthe neuronal profiles in lateral, basolateral (basal), basomedial (accessory basal), central,cortical, and medial amygdaloid nuclei. AChE was found primarily in the neuropil in thesenuclei with only a few AChE-positive neurons.

In the hippocampal formation, BuChE was also found in neurons and not in the neuropil,whereas AChE was found in both neurons and in the neuropil. BuChE and AChE neuronswere present in the polymorphic layer of the dentate gyrus, as well as the stratum oriens andstratum pyramidale of the hippocampus proper. There was considerable overlap in shapes,sizes, and numbers of BuChE- and AChE-positive neurons, suggesting that the enzymes werecolocalized in neurons of the hippocampal formation.

The distinct distribution of BuChE suggests that it may have specific functions includingcoregulation of cholinergic and noncholinergic neurotransmission in human amygdala andhippocampal formation. J. Comp. Neurol. 393:374–390, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: cholinesterases; pseudocholinesterase; central nervous system; Alzheime’s

diseaser

Cholinergic neurotransmission plays an important rolein the normal human central nervous system and has beenimplicated in cognitive functions (Bartus et al., 1982;Gallagher and Colombo, 1995; Lawrence and Sahakian,1995). One of the major enzymes regulating cholinergicneurotransmission is acetylcholinesterase (AChE, EC3.1.1.7), which catalyzes the hydrolysis of the neurotrans-mitter acetylcholine (Silver, 1974). AChE also has nonclas-sic actions (Greenfield, 1991; Appleyard, 1992; Balasubra-manian and Bhanumathy, 1993; Layer, 1995; Small et al.,1996), suggesting that it has a wide spectrum of activitiesin the nervous system. Interestingly, evidence suggeststhat the related enzyme butyrylcholinesterase (BuChE,EC 3.1.1.8) also plays an important role in the nervoussystem as a coregulator of the action of acetylcholine and

as an enzyme with functions independent from AChE(Desmedt and LaGrutta, 1957; Vigny et al., 1978; Giaco-bini et al., 1996).

Grant sponsor: Medical Research Council of Canada; Grant number:MT-7369; Grant Sponsors: The Scottish Rite Charitable Foundation ofCanada, University Internal Medicine Research Foundation, Departmentof Medicine, Dalhousie University, Halifax, Nova Scotia, Canada, CampHill Medical Centre Research Fund, and Queen Elizabeth II HealthSciences Centre, Halifax, Nova Scotia, Canada.

*Correspondence to: S. Darvesh, M.D., Ph.D., FRCPC, Department ofAnatomy & Neurobiology, Faculty of Medicine, Dalhousie University,Halifax, Nova Scotia, B3H 4H7 Canada. E-mail: [email protected]

Received 7 October 1997; Revised 24 November 1997; Accepted 26November 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 393:374–390 (1998)

r 1998 WILEY-LISS, INC.

Neuroanatomical maps of the distribution of BuChE inthe nervous system in numerous species reveal distinctpatterns of distribution in neurons and neuropil (Cava-nagh et al., 1954; Okinaka et al., 1961; Foldes et al., 1962;Friede, 1967; Silver, 1974; Bhatt and Tewari, 1978; Gray-biel and Ragsdale, 1982; Sethi and Tanwar, 1984; Robert-son et al., 1986; Hartz-Shutt and Mai, 1991; Darvesh et al.,1992; Tago et al., 1992). In the rat brain, BuChE is found incertain populations of cholinergic and noncholinergic neu-rons as well as in some neurons that do not have AChE(Robertson et al., 1986; Darvesh et al., 1992; Tago et al.,1992). In the monkey brain, BuChE is differentially distrib-uted in specific layers of the lateral geniculate nucleus,and cytoarchitectonic boundaries are respected in thedistribution of BuChE in the striate cortex (Graybiel andRagsdale, 1982). In the human brain, BuChE has beenreported in restricted populations of neurons but morepredominantly in subcortical white matter, glia, and endo-thelial cells (Okinaka et al., 1961; Foldes et al., 1962;Friede, 1967; Hartz-Shutt and Mai, 1991). In terms of itsneuronal distribution, BuChE has been found in wide-spread areas of the cerebral cortex, including primarysomatosensory, cingulate, and visual cortices as well as thehippocampal formation, but comprehensive maps of thedistribution of BuChE in the human brain are not avail-able in the literature.

Both BuChE and AChE are found in high levels duringdevelopment of the nervous system, suggesting a role inneurogenesis (Kostovic and Goldman-Rakic, 1983; Robert-son and Mostamand, 1988; Layer, 1991). Although BuChEis found in high levels during development of the centralnervous system and low levels in mature brain, theneuronal systems that retain the capacity to synthesizeBuChE in the human brain have not been mapped indetail. Mapping of BuChE in mature brain has particularsignificance when one considers that in neurodegenerativedisorders such as Alzheimer’s disease, there are higherthan normal levels of BuChE in the brain (Perry et al.,1978; Arendt et al., 1984, 1992). The emergence of highlevels of BuChE has been suggested to play a significantrole in the formation and maturation of neuritic plaquesand neurofibrillary tangles, the hallmarks of Alzheimer’sdisease (Geula and Mesulam, 1995).

The possible importance of BuChE in the normal humancentral nervous system and in Alzheimer’s disease hasprompted us to study the distribution of BuChE-positiveneural elements in the normal human brain with the viewthat knowledge gained would aid in elucidating possibleroles for BuChE in the central nervous system. In thisreport, we describe the distribution of BuChE in thenormal human amygdala and hippocampal formation,structures, which are consistently affected in Alzheimer’sdisease (Brockhaus, 1938; Ball, 1977; Herzog and Kemper,1980; Kemper, 1983; Hyman et al., 1984; Van Hoesen and

Damasio, 1987; Kromer Vogt et al., 1990; Ulrich et al.,1990; Mirra et al., 1993), and in which we have founddistinct populations of BuChE-positive neurons. We alsoprovide comparisons with the distribution of the relatedenzyme AChE in these structures. Portions of this workhave been presented in abstract form (Darvesh et al.,1996).

MATERIALS AND METHODS

The brains from three men, age 64, 68, and 74 years, andtwo women, age 88 and 89 years, were obtained at autopsyand were used in this study (Table 1). None of theindividuals had a history of neurologic or psychiatricdisorder, and the primary clinical diagnosis was nonneuro-logic disease.

The brains were removed within 26 hours (range, 5–26hours) of death and fixed by immersion in 10% formalin inphosphate buffer at pH 7.4 for 36 hours at 40C. The brainswere bisected in the midline. One half of the brain wasused for neuropathologic examination and the other halffor cholinesterase histochemistry.

The half of the brain used for neuropathologic examina-tion was immersion fixed in 10% formalin in phosphatebuffer at pH 7.4 for 2 weeks, and then cut in 1- to 2-cmcoronal slabs. Areas of the brain blocked from these slabsincluded several representative areas of neocortex, basalforebrain, amygdala, hippocampal formation, entorhinalcortex, basal ganglia, thalamus, and the midbrain. Blockedareas of the brain were paraffin-embedded and 8-µm serialsections were cut and mounted on glass microscope slides.One series was stained with hematoxylin and eosin, andanother series was stained by using a modified Bielschow-sky method (Mirra et al., 1993). The slides were cover-slipped and examined by means of brightfield microscopyfor the presence of neuropathology, especially neuriticplaques and neurofibrillary tangles as seen in Alzheimer’sdisease. The two older brains (Table 1) showed someneuritic plaques and neurofibrillary tangles in severalareas of the brain, including the hippocampal formationand amygdala but there were too few neuritic plaques tomeet the criteria for neuropathologic diagnosis of Alzhei-mer’s disease (Khachaturian, 1985; Mirra et al., 1991,1993). In addition, these two patients had been examinedby geriatricians with experience in dementias and werejudged cognitively intact before death. Thus, on clinicaland neuropathologic grounds, all the brains used in thepresent study were normal for their age.

The half of the brain used for cholinesterase histochem-istry was cut in 1- to 2-cm thick coronal slabs after theinitial 36 hours of fixation, and fixed for a further 12–24hours (Moran and Gomez-Ramos, 1992). After this, blockscontaining the amygdala and hippocampal formation werecut and their outlines and identifying boundaries were

TABLE 1. Clinical Profile of Subjects

NumberAge

(years) Gender Cause of deathTime1

(hours)Brain weight

(g)Clinical neurologic

diagnosisNeuropathologic

diagnosis

96-A-10 64 Male Cancer 23 1450 Normal Normal97-A-7 68 Male Myocardial infarction 26 1553 Normal Normal95-A-109 74 Male Pneumonia 6 1525 Normal Normal94-A-146 88 Female Cardiomyopathy 8 1343 Normal Normal2

96-A-124 89 Female Congestive heart failure 5 1066 Normal Normal2

1Time to autopsy.2Some neurofibrillary tangles and neuritic plaques.

BUTYRYLCHOLINESTERASE IN HUMAN FOREBRAIN 375

drawn by water-proof ink tracing on 40 3 75 mm micro-scope slides for future orientation. After cryoprotection byimmersion in 30% sucrose in phosphate buffer at pH 7.4 for36 hours, each block was cut in three series of 40-µm-thickserial sections. One series was stained for Nissl substancewith thionin and the other two were processed for thedemonstration of BuChE and AChE.

Histochemical procedures

A modified Karnovsky-Roots method (Karnovsky andRoots, 1964; Tago et al., 1986; Geula and Mesulam, 1989)was used to demonstrate the presence of cholinesterases.This method identifies cholinesterases in normal neuralelements and cholinesterases found in pathologic struc-tures such as neuritic plaques and neurofibrillary tanglesin the human brain (Geula and Mesulam, 1989). It hasbeen reported that the optimum pH for demonstration ofcholinesterases in the normal human brain is 8.0, whereasat lower pH (6.8), cholinesterases in pathologic structuressuch as neuritic plaques and neurofibrillary tangles arepreferentially detected (Geula and Mesulam, 1989). There-fore, to demonstrate cholinesterase-containing structuresin normal brains, all the reactions were carried out at pH8.0. Because treatment of the sections with hydrogenperoxide to remove endogenous peroxidase activity in redblood cells leads to loss of BuChE and AChE staining, thispretreatment was not done. Residual red blood cell stain-ing did not interfere with cholinesterase staining.

The substrate used for demonstration of BuChE wasbutyrylthiocholine with AChE activity inhibited by BW284 C 51 (1,5-bis [4-allyl dimethylammonium phenyl]pentan-3-one dibromide; Sigma Chemical Co., St. Louis,MO). The sections were rinsed in 0.1 M maleate buffer, pH7.4, for 1 hour and then placed in a reaction mediumcontaining 0.5 mM sodium citrate, 0.47 mM cupric sulfate,0.05 mM potassium ferricyanide, 0.36 mM butyrylthiocho-line iodide, and 0.19 mM BW 284 C 51 (AChE inhibitor) in0.1 M maleate buffer, pH 7.4, and incubated for 15 hours atroom temperature. Sections were then rinsed with gentleagitation for 10 minutes in distilled water and placed in0.1% cobalt chloride in water for 10 minutes. After afurther rinse in distilled water, sections were placed in asolution containing 1.39 mM 3,3’-diaminobenzidine tetra-hydrochloride (DAB), 7.48 mM ammonium chloride and11.1 mM D(1)-glucose in 100 ml of 0.1M phosphate buffer(pH 7.4). After 1 minute, a solution of 6 mg of glucoseoxidase in 100 ml of distilled water was added at a ratio of10:1 (DAB solution: glucose oxidase solution) and thereaction was carried out for approximately 30 minutes.Sections were then washed in 0.1 M acetate buffer, pH 3.3,mounted on slides, cover-slipped, and examined withbrightfield and darkfield microscopy.

The procedure for the demonstration of AChE wassimilar to that for BuChE except the substrate was 0.37mM acetylthiocholine. In addition, BuChE activity wasinhibited by either 0.06 mM ethopropazine (Sigma) or 0.05mM tetraisopropyl pyrophosphoramide (iso-OMPA)(Sigma), and the sections were incubated for 3 hours atroom temperature.

Control experiments

Several control experiments were performed to establishthe specificity of BuChE and AChE staining in humanbrain. (1) Histochemical procedures were done in theabsence of the substrates acetylthiocholine or butyrylthio-choline to determine whether the reagents used stained

endogenous pigments. (2) Butyrylthiocholine was used asthe substrate without BuChE or AChE inhibitor to deter-mine the extent of hydrolysis of butyrylthiocholine by thetwo enzymes. (3) Butyrylthiocholine was used as thesubstrate with the BuChE inhibitors ethopropazine oriso-OMPA to determine whether ethopropazine or iso-OMPA specifically inhibits BuChE in these experimentalconditions. (4) Acetylthiocholine was used as the substratewithout AChE or BuChE inhibitor to determine the extentof hydrolysis of acetylthiocholine by the two enzymes. (5)Acetylthiocholine was used as the substrate in the pres-ence of AChE inhibitor BW 284 C 51 to determine whetherit inhibits AChE specifically.

Data analysis

The distribution of neurons was plotted by using an X-Yplotter linked to the stage of a Leitz Orthoplan microscopeby a linear potentiometer. Neuronal profiles positive forBuChE, AChE, and Nissl-substance were counted in adja-cent sections by using a Leitz Orthoplan microscope togain some insight into the percentage of BuChE-positiveneurons relative to AChE-positive neurons. The percent-age of BuChE- or AChE-positive neuronal profiles in aparticular amygdaloid nucleus was calculated by dividingthe total number of BuChE- or AChE-positive neuronalprofiles by the total number of thionin-stained neuronalprofiles. BuChE- or AChE-positive neuronal profiles in anucleus were counted in three areas on the same sectionand at three different rostrocaudal levels of the nucleus ina field at 4003 magnification. The total number of neuro-nal profiles was counted in these areas on adjacent thionin-stained sections. In the hippocampal formation, the totalnumber of BuChE- andAChE-positive neurons was countedin the polymorphic cell layer at two rostrocaudal levels,and the numbers were averaged. In the stratum oriens andstratum pyramidale, the total number of BuChE- andAChE-positive neurons were counted at two rostrocaudallevels at 1003 magnification, and the numbers wereaveraged.

Sections were photographed by using a Leitz Orthoplancompound microscope and an Olympus 5240 dissectingmicroscope. Images were captured with a JVC framecapture digital camera. The photographic plates wereassembled by using Adobe Photoshop 4.0 on a PowerMacintosh 7100/80 computer. The images were color bal-anced, contrast enhanced, and the brightness adjusted sothat the background from different images matched.

RESULTS

The histochemical procedures for demonstrating BuChEand AChE resulted in different patterns of staining for thetwo enzymes in the amygdala and the hippocampal forma-tion. BuChE-positive elements included neuronal somata,processes, and glia. BuChE showed granular staining inthe neuronal somata and processes. The distribution ofAChE-positive elements in these structures was compa-rable to that reported in the literature (Nitecka and Nar-kiewicz, 1976; Mellgren et al., 1977; Svendsen and Bird,1985; Green and Mesulam, 1988; Amaral and Insausti,1990; de Olmos, 1990; Sims and Williams, 1990) withstrong and widespread staining of the neuropil and onlysmall numbers of neuronal somata and their dendrites inselected regions. In the neuronal somata and dendrites,AChE showed granular staining, but in contrast to BuChE,there was lighter staining of the nuclei. The distribution of

376 S. DARVESH ET AL.

BuChE in the amygdala and hippocampal formation willbe described and compared with the distribution of AChEin adjacent sections.

Technical considerations

Fixation. It has been observed that prolonged fixationof the tissue in formalin leads to loss of cholinesterasestaining (Moran and Gomez-Ramos, 1992). We observedthat when brains are fixed in formalin for more than 60hours, there is loss of cholinesterase histochemical stain-ing. In brains processed after less than 60 hours of fixation,there was some variability in intensity of staining, but theoverall patterns were consistent and robust across allbrains.

Controls. Control experiments (summarized in Table2) were performed to determine the degree of specificity orcross-reactivity between BuChE and AChE staining ofhuman forebrain. The basolateral (basal) nucleus of theamygdala was used as a reference area because it exhibitsstrong, characteristic staining for both of the enzymes(Fig. 1). When sections were incubated in a reactionmedium without either of the substrates butyrylthiocho-line or acetylthiocholine, no staining was observed. Withdarkfield microscopy, endogenous pigment was readilyobserved, and this pigmentation did not interfere withcholinesterase staining (see below).

The inhibitors of BuChE and AChE were compared todetermine their selectivity and whether the inhibitor ofone enzyme affected staining for the other enzyme (Table2). When sections were incubated in the presence ofbutyrylthiocholine without either a BuChE orAChE inhibi-tor, there was a light staining of the neuropil, as shown inthe basolateral (basal) amygdaloid nucleus (Fig. 1A). Thepattern of staining of this neuropil corresponded to that ofAChE staining as reported in the literature (Nitecka andNarkiewicz, 1976; Svendsen and Bird, 1985; de Olmos,1990; Sims and Williams, 1990). In addition, there werenumerous cholinesterase-positive neurons. This findingindicated that butyrylthiocholine was being hydrolyzed byboth the cholinesterases. However, because the staining ofthe neuropil was weak, this observation demonstratedthat butyrylthiocholine was being hydrolyzed by AChEconsiderably less efficiently than by BuChE as has beenconfirmed in enzyme kinetic studies (Darvesh and Martin,unpublished results).

When the sections were incubated in the presence ofbutyrylthiocholine and the BuChE inhibitors ethopro-pazine or iso-OMPA, there was positively, albeit weakly,stained neuropil, as shown in the basolateral amygdaloidnucleus (Fig. 1B). The intensity of staining was similar tothat seen when sections were incubated with butyrylthio-choline without any inhibitor. The most significant conclu-

sion to be drawn from this control experiment was that theneurons did not show any staining indicating that BuChEwas being completely inhibited by ethopropazine andiso-OMPA and that butyrylthiocholine was being hydro-lyzed by AChE but less efficiently. That is, cells in thebasolateral nucleus were not positive because BuChE wasbeing inhibited by its inhibitors, and they do not containAChE. In addition, AChE staining was positive in theneuropil because butyrylthiocholine was being hydrolyzedby AChE and AChE was not being inhibited by ethopro-pazine or iso-OMPA.

When the sections were incubated with acetylthiocho-line without AChE or BuChE inhibitors, there was heavilystained AChE-positive neuropil, and there were manycholinesterase-positive cells (Fig. 1C). This finding indi-cated that both the cholinesterases hydrolyzed acetylthio-choline.

When the sections were incubated in the presence ofacetylthiocholine and BW 284 C 51 to inhibit AChE, therewas no staining of the neuropil, but there was strongstaining of some neurons (Fig. 1D). This finding indicatedthat the hydrolysis of acetylthiocholine by AChE wasinhibited by BW 284 C 51, and the hydrolysis of acetylthio-choline by BuChE was not being inhibited.

When the sections were incubated in the presence ofbutyrylthiocholine and BW 284 C 51 to inhibit AChE,there was no AChE-positive neuropil, and there werenumerous cholinesterase-positive cells (Fig. 4). This find-ing indicated that the AChE was being inhibited and thatBuChE was being detected specifically. This method wasthe procedure used to detect BuChE-positive neural ele-ments.

When the sections were incubated with acetylthiocho-line in the presence of ethopropazine to inhibit BuChE,there was a heavily labeled neuropil as described for AChE(Fig. 2B,D,F), and there were only isolated labeled cells.This finding indicated that AChE was being detectedspecifically. This method was the procedure used to detectAChE-positive neural elements.

Taken together, these control experiments show that useof butyrylthiocholine as the substrate and BW 284 C 51 asthe inhibitor of AChE during histochemical procedureresults in specific staining for BuChE. Similarly, the use ofacetylthiocholine as the substrate and ethopropazine oriso-OMPA as the inhibitors of BuChE results in specificstaining for AChE.

Amygdala

The parcellation and nomenclature used to define thenuclei of the amygdala is based on that of Hilpert (1928),Johnston (1923), Brockhaus (1940), Crosby and Humphrey(1941), Lauer (1945), Price et al. (1987), and Amaral et al.(1992), as adapted and extended by de Olmos (1990) for thehuman amygdala. The synthesis by de Olmos (1990) waschosen because it permits generalizations to primatesother than the human and will ultimately facilitate under-standing of human amygdala connections.Alternate parcel-lation and nomenclature used in the literature to definesome of the nuclei of the amygdala is indicated in the textbelow as appropriate.

The nuclei of the amygdala were readily distinguishedin low-magnification photomicrographs of sections stainedfor BuChE and AChE by virtue of differential stainingpatterns (Fig. 2). BuChE-positive neurons were present inmany amygdaloid nuclei, whereas only rarely were AChE-positive neurons identified (Fig. 3). Fiber tracts were

TABLE 2. Control Experiments to Determine Specificityof Butyrylcholinesterase and Acetylcholinesterase Inhibitors

Inhibitor

Substrate

Butyrylthiocholine Acetylthiocholine

Neuropil1 Neurons2 Neuropil1 Neurons2

None 1 11 11 11Ethopropazine or Iso-OMPA 1 0 11 1BW 284 C 51 0 11 0 11

1Neuropil: 0 5 no stained neuropil; 1 5 lightly stained neuropil; 11 5 heavily stainedneuropil.2Neurons: 0 5 no stained neurons; 1 5 few stained neurons; 11 5 many stainedneurons.


stained darkly after BuChE histochemistry, although indi-vidual axons were not distinct (Fig. 2A,C,E). AChE showeda distinct pattern of staining of the neuropil in whichaxons could be discerned in amygdaloid nuclei (Fig.2B,D,F). The BuChE-positive neurons were found in thelateral, basolateral (basal), basomedial (accessory basal),central, cortical, and medial amygdaloid nuclei.

Lateral amygdaloid nucleus. The lateral nucleusoccupies about three-quarters of rostrocaudal extent of theamygdala (Figs. 2, 3). It contains predominantly large-sized neurons dorsally with a gradual decrease in neuronalsize ventrad (de Olmos, 1990).

The lateral amygdaloid nucleus contained numerousBuChE-positive neurons (Fig. 3). Most of these BuChE-

positive neurons were medium-sized to small, had round,fusiform, or triangular profiles (Fig. 4A), and were foundthroughout its rostrocaudal and dorsoventral extent. Someof these neurons had multiple branching processes thatcould be followed for a distance of up to 150 µm from thecell body. Relative to the total number of Nissl-stainedneurons, this nucleus contained approximately 8% BuChE-positive neurons.

In keeping with the observations of others (Nitecka andNarkiewicz, 1976; Svendsen and Bird, 1985; de Olmos,1990; Sims and Williams, 1990), the lateral nucleus had anAChE-positive neuropil that was more intense rostrally(Fig. 2). There were isolated small and medium-sizedAChE-positive neurons with fusiform or triangular profiles.

Fig. 1. Photomicrographs of sections from control experimentsshowing the effect of inhibitors on butyrylcholinesterase (BuChE) andacetylcholinesterase (AChE) histochemical staining in the lateral (La)and basolateral (BL) amygdaloid nuclei. A: Histochemical stainingwith butyrylthiocholine as the substrate without an inhibitor. Thereare many stained neurons, and there is light staining of the neuropil.B: Histochemical staining with butyrylthiocholine as the substrate

and ethopropazine (BuChE inhibitor). There is staining of the neuro-pil, but the neurons are not stained. C: Histochemical staining withacetylthiocholine as the substrate without inhibitor. There is heavystaining of the neuropil and some neurons. D: Histochemical stainingwith acetylthiocholine as the substrate and BW 284 C 51 (AChEinhibitor). There is no neuropil staining, but there are numerousstained neurons. Scale bar 5 250 µm (applies to A–D).


Fig. 2. Photomicrographs showing histochemical staining for butyr-ylcholinesterase (BuChE) (left panels) and acetylcholinesterase (AChE)(right panels) in adjacent sections of the human amygdala. A,B: BuChEand AChE staining in the rostral amygdala. C,D: BuChE and AChEstaining at a middle level of the amygdala. Boxes identify the highmagnification photomicrographs in Figure 7E,F. E,F: BuChE and

AChE staining in the caudal amygdala. AC, anterior commissure; BL,basolateral amygdaloid nucleus; BM, basomedial amygdaloid nucleus;Ce, central amygdaloid nucleus; Co, cortical amygdaloid nucleus; La,lateral amygdaloid nucleus; Me, medial amygdaloid nucleus; PL,paralaminar amygdaloid nucleus. Scale bar 5 5 mm (applies to A–F).

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Fig. 3. Composite drawings showing the distribution of butyrylcho-linesterase (BuChE)- and acetylcholinesterase (AChE)-positive neu-rons in the human amygdala, from a rostral (top) to a caudal level(bottom). Black dots, BuChE-positive neurons; red triangles, AChE-positive neurons. The sections used for these drawings are the same asthose in the photomicrographs of Figure 2. BL, basolateral amygdaloid

nucleus; BM, basomedial amygdaloid nucleus; Ce, central amygdaloidnucleus; Co, cortical amygdaloid nucleus; GP, globus pallidus; H,hippocampus; La, lateral amygdaloid nucleus; Me, medial amygdaloidnucleus; OT, optic tract; P, putamen; PL, paralaminar amygdaloidnucleus; V, ventricle. Scale bar 5 5 mm.


Fig. 4. Photomicrographs of representative butyrylcholinesterase(BuChE)-positive neurons in human amygdaloid nuclei. A: Lateralamygdaloid nucleus. B: Basolateral amygdaloid nucleus. C: Basolat-eral amygdaloid nucleus. D: Basolateral amygdaloid nucleus. Dark-

field photomicrograph of the same neurons as in C showing BuChE-positive neurons and neurons containing lipofuscin (arrowheads).E: Basomedial amygdaloid nucleus. F: Cortical amygdaloid nucleus.Scale bar 5 50 µm (applies to A–F).

Basolateral amygdaloid nucleus. The basolateralnucleus is situated immediately medial to the lateralamygdaloid nucleus (Figs. 2, 3). Following the nomencla-ture and parcellation of the primate basolateral amygda-loid nucleus promulgated by de Olmos (1990), the nucleusis divided into a dorsal large-celled division, and ventral tothis is a medium-celled division, followed ventrally by asmall-celled division, and the most ventral division is theparalaminar division (de Olmos, 1990). The nomenclatureand parcellation of the primate basolateral amygdaloidnucleus has varied in the literature (Johnston, 1923;Hilpert, 1928; Brockhaus, 1940; Crosby and Humphrey,1941; Lauer, 1945; Price et al., 1987; de Olmos, 1990;Amaral et al., 1992). In the monkey amygdala, the basolat-eral nucleus has been called the basal nucleus, and large-(magnocellular) and small-celled (parvicellular) divisionshave been recognized (Lauer, 1945, Price et al., 1987,Amaral et al., 1992). In the human amygdala, the dorsallarge-celled and the medium-celled divisions have alsobeen called collectively the laterobasal nucleus, and thesmall-celled and the paralaminar divisions have beencalled collectively the mediobasal nucleus (Crosby andHumphrey, 1941).

The basolateral amygdaloid nucleus contained manyBuChE-positive neurons (Fig. 3). The BuChE-positiveneurons were more numerous in the dorsal large- andmedium-celled subdivisions of the nucleus. These neuronswere round, fusiform, or triangular in profile, with manybranching processes (Fig. 4B). Approximately 10% of theneurons in these two subdivisions of the nucleus containedBuChE. The ventrally located small-celled and the para-laminar subdivisions of the basolateral amygdaloid nucleuscontained BuChE-positive neurons that were morphologi-cally similar to the neurons in the dorsal part of thebasolateral nucleus (Fig. 4C,D). Pigment-containing neu-rons that were not stained with BuChE were easilydistinguished from those with BuChE by using brightfieldand darkfield microscopy (Fig. 4D). Approximately 6% ofthe neurons in the small-celled and the paralaminarsubdivisions contained BuChE.

The basolateral amygdaloid nucleus contained the mostintensely stained AChE-positive neuropil in the amygdala(Fig. 2B,D,F), and the staining was lighter ventrallytoward the paralaminar subnucleus (Nitecka and Narkie-wicz, 1976; Svendsen and Bird, 1985; de Olmos, 1990;Sims and Williams, 1990). There were only isolated AChE-positive neurons in this nucleus (Fig. 3), with fusiform ortriangular profiles. The percentage of AChE-positive neu-rons in this nucleus was less than 1%.

Basomedial amygdaloid nucleus. The basomedialnucleus is situated medial to the basolateral nucleus (Figs.2, 3). The basomedial amygdaloid nucleus has also beencalled the accessory basal nucleus (Crosby and Humphrey,1941; Lauer, 1945; Price et al., 1987; Amaral et al., 1992).The cytoarchitecture of this nucleus follows the generalschema found in most amygdaloid nuclei in that the largecells are located in the dorsal portion of the nucleus, andthe size gradually becomes smaller ventrally (Crosby andHumphrey, 1941; Lauer, 1945; Price et al., 1987; de Olmos,1990; Amaral et al., 1992).

The basomedial amygdaloid nucleus contained numer-ous BuChE-positive neurons (Fig. 3). These neurons weresmall to medium in size and had round, fusiform, ortriangular profiles. Some had several branching processes(Fig. 4E). The size and shape of BuChE-positive neuronswere similar in the dorsal and the ventral parts of the

basomedial amygdaloid nucleus. Approximately 9% of theneurons in this nucleus contained BuChE.

In keeping with the observations of others (Nitecka andNarkiewicz, 1976; Svendsen and Bird, 1985; de Olmos,1990; Sims and Williams, 1990), this nucleus containedmoderately stained AChE-positive neuropil (Fig. 2B,D,F).There were only a few AChE-positive fusiform or triangu-lar neuronal profiles in this nucleus. Approximately 1% ofthe neurons in this nucleus contained AChE.

Central amygdaloid nucleus. The central nucleus ismost well defined in the caudal levels of amygdala (Figs. 2,3). The central amygdaloid nucleus contained a few BuChE-positive neurons. These neurons were small to medium insize with fusiform profiles (Fig. 3). Approximately 1% ofthe neurons in this nucleus contained BuChE. The neuro-pil of the central nucleus was lightly positive for AChE(Fig. 2B,D,F). There were no AChE-positive neurons inthis nucleus in four of the five brains studied. In the fifthbrain, a few AChE-positive neurons could be discerned.

Cortical amygdaloid nucleus. The cortical nucleusis medial to the basomedial amygdaloid nucleus (Figs. 2,3). This nucleus contained many BuChE-positive neurons(Fig. 3). These neurons were small to medium in size withround, fusiform, or triangular profiles, some with multiplebranching processes (Fig. 4F). The approximate percent-ages of BuChE-positive neurons in this nucleus were 14%in the rostral level, 2% in the middle level, and 7% in thecaudal level.

There was a lightly stained AChE-positive neuropil, theintensity of which increased from rostral to caudal levels(Fig. 2B,D,F). There were AChE-positive neurons thatwere small to medium in size and had triangular profiles.Approximately 5% of the neurons in the rostral level hadAChE, whereas there were only isolated AChE-positiveneurons in the middle and caudal levels.

Medial amygdaloid nucleus. The medial nucleus iswell defined in middle levels of amygdala (Figs. 2, 3). Themedial amygdaloid nucleus contained a few small tomedium-sized triangular BuChE-positive neurons (Fig. 3).Processes of some of these neurons were also stained.Approximately 5% of the neurons were BuChE-positiveand were found predominantly in the medial portion of thenucleus. There was a lightly stained AChE-positive neuro-pil in this nucleus (Fig. 2D), with only isolated AChE-positive neurons.

Hippocampal formation

The nomenclature used for the subdivisions and layersof the hippocampal formation is based on that of Ramon yCajal (1968) and Lorente de No (1934), as adapted andextended by Amaral and Insausti (1990). The hippocampalformation is composed of the dentate gyrus, hippocampusproper (cornu Ammonis), subicular complex, and the ento-rhinal cortex. BuChE and AChE were differentially distrib-uted in the hippocampal formation (Fig. 5).

BuChE was found in neurons and processes in thepolymorphic cell layer in the hilus of the dentate gyrus, inthe stratum oriens and in the stratum pyramidale of thehippocampus proper. In addition, there were a few BuChE-positive cells in the deeper layers of the subicular complexand the entorhinal cortex. There was no BuChE-positiveneuropil. AChE was found in neurons and neuropil in thehippocampal formation, and its distribution paralleledthat reported in the literature (Mellgren et al., 1977;Green and Mesulam, 1988; Amaral and Insausti, 1990; DeLacalle et al., 1994).


Dentate gyrus. The dentate gyrus has three layers(Fig. 5) (Amaral and Insausti, 1990). The granule cell layercharacteristically stains darkly with Nissl substance. The

hilus of the dentate gyrus is defined here as the areaenclosed within the end blades of the granule cell layer. Inthe hilus of the dentate gyrus, and immediately adjacent to

Fig. 5. Photomicrographs showing Nissl-staining (A,B) and histo-chemical staining for butyrylcholinesterase (BuChE) (C,D) and acetyl-cholinesterase (AChE) (E,F) in the human hippocampal formation.A,C,E: Nissl-, BuChE-, and AChE-staining in adjacent sections in arostral level of the hippocampal formation. B,D,F: Nissl-, BuChE-,and AChE-staining in adjacent sections in a middle level of the

hippocampal formation. Boxes in D identify areas from which high-magnification photomicrographs in Figure 7 were taken. DG, dentategyrus; CA1, CA1 field of stratum pyramidale; P, polymorphic cell layerof the dentate gyrus; S, subiculum; SO, stratum oriens. Scale bar 5 5mm (applies to A–F).


the granule cell layer, is the polymorphic cell layer of thedentate gyrus. The layer overlying the granule cell layer isthe molecular layer of the dentate gyrus.

In the polymorphic cell layer in the hilus of the dentategyrus, there were many BuChE-positive neurons (Fig. 6).These neurons were medium-sized to large, with fusiformor triangular profiles, and most had many branchingprocesses (Fig. 7A,B). There were also many medium-sizedto large AChE-positive neurons in the polymorphic celllayer in the hilus of the dentate gyrus (Fig. 6) withfusiform or triangular profiles and multiple branchingprocesses. There was also a fine AChE-positive neuropil inthe polymorphic cell layer. To estimate relative numbers ofBuChE- and AChE-positive neurons, all the BuChE-positive and AChE-positive neurons were counted at twolevels in three brains. The average number of BuChE-positive neurons was 86 (range, 58–120), whereas that ofAChE-positive neurons was 102 (range, 47–130). Theoverlap in numbers of BuChE- and AChE-positive neu-rons, as well as overlap in their distribution and morphol-ogy suggest that in the polymorphic cell layer these twoenzymes may be found in the same populations of neurons.

The granule cell layer and the molecular layer wereBuChE-negative (Fig. 5). There was a fine AChE-positiveneuropil in the granule cell layer (Fig. 5B,D), which is inkeeping with the observation of others (Mellgren et al.,1977; Green and Mesulam, 1988; Amaral and Insausti,1990; De Lacalle et al., 1994).

Hippocampus proper (cornu Ammonis). The hippo-campus proper, which lies in the floor of the temporal hornof the lateral ventricle, can be divided into a head rostrally,body in the middle, and tail caudally. Because of thefoldings in the laminae of the head and the tail, they arestructurally relatively more complex than those in thebody (Fig. 5A). Therefore, it is easier to appreciate thelayers of the hippocampus in the body (Fig. 5B). In coronalsections, from the ventricular surface toward the pialsurface, the layers are the alveus, stratum oriens, stratumpyramidale, stratum lucidum, stratum radiatum, andstratum lacunosum-moleculare (Fig. 5). The stratum py-ramidale was originally divided into four fields designatedCA1–CA4 (Lorente de No, 1934). Within the hilus of thedentate gyrus, the assignment of the pyramidal neurons ofstratum pyramidale to either CA3 or CA4 has beencontroversial because of the absence of connectional orcytoarchitectonic criteria to support a separate designa-tion (Amaral and Insausti, 1990). Thus, all the pyramidalneurons of the stratum pyramidale in the hilus of thedentate gyrus are designated as belonging to the CA3 field(Amaral and Insausti, 1990). As shown in Figure 5B, theCA3 field is followed by the CA2 field, but the borderbetween these two fields is not sharp with Nissl staining.The CA2 field is followed by the CA1 field. Whereas theborder between these two fields also is not sharp, the CA1field becomes gradually broader than the CA2 field. TheCA1 field terminates by overlapping with the subiculum(Amaral and Insausti, 1990).

The alveus had a distinct BuChE-positive staining,although, as in other fiber tracts, individual axons couldnot be discerned (Fig. 5A,B). There were many clearlydiscernible AChE-positive axons in the alveus (Fig. 5B,D).

The stratum oriens contained many BuChE-positiveneurons (Fig. 6). These neurons were small to medium insize and were fusiform or triangular in profile (Fig. 7C,D).The long axis of some of these cells was oriented parallel tothe alveus. The number of BuChE-positive cells was

greater in the region subjacent to the CA1 field of stratumpyramidale. Some of these BuChE-positive cells had mul-tiple processes that penetrated deep into the stratumpyramidale (Fig. 7C,D). There were also many AChE-positive fusiform and triangular neurons in this layer, andthey became more numerous toward the region subjacentto the CA1 field. The relative numbers of BuChE- andAChE-positive neurons were estimated by counts at tworostrocaudal levels in three brains. The average number ofBuChE-positive neurons was 14 (range, 13–18), whereasthat of AChE-positive neurons was 12 (range, 7–19). Basedon the distribution pattern, morphology, and numbers ofBuChE- and AChE-positive neurons, BuChE and AChEappear to be present in the same populations of neurons(Fig. 7E,F).

In the stratum pyramidale there were scattered small tomedium-sized BuChE-positive neurons. These neuronswere fusiform or triangular in profile (Fig. 7C,D). TheBuChE-positive neurons were more numerous in the CA1region compared with other regions of the stratum pyrami-dale. There were also AChE-positive fusiform or triangularneurons in this layer, especially toward the CA1 region.There was an AChE-positive neuropil in the stratumpyramidale that was most intense in the CA4 and CA3regions and less intense toward the CA1 region (Fig.5B,D). The average number of BuChE-positive neurons instratum pyramidale was 20 (range, 11–25), whereas theaverage number of AChE-positive neurons was 16 (range,4–22).

The stratum lucidum, which contains mossy fibers thatare projections from the dentate granule cells to the CA3region and lies subjacent to the CA3 region, was BuChEnegative. This layer could be identified clearly with AChEbecause of its light staining compared with the heavystaining of adjacent layers. The stratum radiatum wasBuChE-negative but did have a distinct AChE-positiveneuropil (Fig. 5B,D). The stratum lacunosum-molecularehad BuChE staining similar to that in the alveus in thatindividual axons could not be discerned (Fig. 5A,C).

DISCUSSION

The present investigation demonstrates that BuChE ispresent in many populations of neurons in the humanamygdala and hippocampal formation. In the amygdala,the distribution of BuChE is different from that of AChE inthat BuChE is found predominantly in the neurons andtheir processes, whereas AChE is found predominantly inthe neuropil. In the hippocampal formation, BuChE andAChE are found in neurons and their processes with anoverlap in their distribution, shapes, and relative num-bers, but only AChE is found in the neuropil.

Technical considerations

Fixation. We have also observed that prolonged delayto fixation and prolonged time in formalin leads to loss ofenzymatic activity required for histochemical demonstra-tion of cholinesterases, BuChE in particular (Moran andGomez-Ramos, 1992). Animal neural tissues are also sensi-tive to postmortem conditions (Navaratnam and Lewis,1975; Graybiel and Ragsdale, 1982; Nance et al., 1987).Because delays in postmortem removal of the brain andprolonged fixation are not suitable for optimal demonstra-tion of BuChE, brains were removed within 26 hours ofdeath and were fixed for not more than 60 hours informalin.


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Fig. 6. Composite drawings showing the distribution of butyrylcho-linesterase (BuChE)- and acetylcholinesterase (AChE)-positive neu-rons in the human hippocampal formation, from a rostral (top) to acaudal level (bottom). Black dots, BuChE-positive neurons. Red

triangles, AChE-positive neurons. The sections used for these draw-ings are the same as those in the photomicrographs of Figure 5. CA1,CA1 field of the stratum pyramidale; DG, dentate gyrus; F, fimbria; LGB,lateral geniculate body; S, subiculum; V, ventricle. Scale bar 5 5 mm.

Fig. 7. Photomicrographs of representative butyrylcholinesterase(BuChE)- and acetylcholinesterase (AChE)-positive neurons in vari-ous regions of the hippocampal formation. A: Brightfield photomicro-graph of neurons in the polymorphic cell layer of the dentate gyrustaken from an area indicated by an inset with a solid arrow in Figure5D. B: Darkfield photomicrograph of the same field as in A. Note thepresence of pigmented neurons some of which are not stained withBuChE. C: Brightfield photomicrograph of neurons in the stratumoriens and stratum pyramidale taken from an area indicated by aninset with a solid arrow in Figure 5D. Note that processes of some

neurons penetrate the stratum pyramidale (arrows). D: Darkfieldphotomicrograph of the same region as C. Note the presence ofpigmented neurons some of which are not stained with BuChE.E: Brightfield photomicrograph of the BuChE-positive neurons in therostral stratum oriens and stratum pyramidale taken from an areaindicated by an inset in Figure 2C. F: Brightfield photomicrograph ofAChE-positive neurons in a section adjacent to the one in E taken froman area indicated by an inset in Figure 2D. SO, stratum oriens; SP,stratum pyramidale. Scale bar 5 50 µm (applies to A–F).

Substrates and inhibitors. Histochemically, BuChEand AChE are capable of hydrolyzing both of the sub-strates acetylthiocholine and butyrylthiocholine. Based onour control experiments, butyrylthiocholine is hydrolyzedless efficiently by AChE as evidenced by light staining ofthe neuropil when using this substrate in the absence ofAChE inhibitor. In contrast, acetylthiocholine is hydro-lyzed efficiently by both the enzymes as evidenced byheavy labeling of the neuropil and the neurons in theabsence of inhibitors. Even though the efficiency of hydro-lysis of the substrates differs, these substrates do give riseto some cross-reactivity between these two enzymes dur-ing histochemical procedures. However, the control experi-ments show that ethopropazine or iso-OMPAinhibit BuChEspecifically and that this inhibition does not interfere withAChE staining. Similarly, use of BW 284 C 51 inhibitsAChE and does not interfere with BuChE staining. Thus,use of the appropriate inhibitor permits selective andspecific demonstration of BuChE or AChE in the humanbrain.

Butyrylcholinesterase in the amygdalaand the hippocampal formation

Amygdala. The human amygdala has been reportedto contain neurons positive for BuChE, but details of thedistribution of the enzyme in various nuclei and neuronalmorphology have not been shown (Hartz-Shutt and Mai,1991). We have demonstrated BuChE-positive neurons inlateral, basolateral, basomedial, central, cortical, and me-dial amygdaloid nuclei. There is no BuChE-positive neuro-pil, but fiber bundles in the amygdala stain strongly butdiffusely with BuChE.

The basolateral nuclear group, composed of lateral,basolateral, and basomedial amygdaloid nuclei, is thelargest nuclear group in the human amygdala (de Olmos,1990). Neurons containing BuChE are found in all thedivisions of the basolateral nuclear group. These neuronsrange in size from small to medium and are fusiform ortriangular in profile. Pigment architectonic analysis of thebasolateral nuclear group suggests that it consists of threetypes of neuronal populations (Braak and Braak, 1983).Class I neurons are spindle or pyramidal in shape withdendrites that are covered with spines and are pigmentladen. According to Braak and Braak (1983), these cellsappear to resemble cortical pyramidal cells. Class IIneurons are pigment-laden cells that are polygonal orspindle in shape with dendrites that are aspinous or havesparse spines, and range in size from small to large. Thesecells appear to resemble cortical stellate cells, a class ofinterneurons. Class III neurons are devoid of pigment,their dendrites are devoid of spines, and their shape andsize are variable. Our results show that both pigmentedand nonpigmented neurons are BuChE positive, but it isnot possible on the basis of BuChE staining to determinewhether these cells are spiny or aspinous.

In addition to the nuclei of the basolateral nucleargroup, BuChE-positive neurons were also found in thecentral, cortical, and the medial amygdaloid nuclei. Themorphologies and sizes of BuChE-positive neurons inthese nuclei were consistent with the predominant celltypes described in these nuclei (de Olmos, 1990).

The distribution of AChE in the amygdala in our mate-rial paralleled that reported in the literature (Nitecka andNarkiewicz, 1976; Svendsen and Bird, 1985; de Olmos,1990; Sims and Williams, 1990) with AChE found predomi-nantly in the neuropil. The most intense and characteristic

staining is in the dorsal part of the basolateral amygdaloidnucleus with variable intensity of staining of the otheramygdaloid nuclei.

The differences in the patterns of staining betweenBuChE and AChE in the amygdala are striking in thatBuChE is found in the neurons, whereas AChE is found inthe neuropil and there are only fewAChE-positive neurons.

Hippocampal formation. BuChE-positive neuronsare found in the polymorphic cell layer of the dentategyrus, the stratum pyramidale and the stratum oriens ofthe hippocampus proper as well as the deeper layers of thesubicular complex and entorhinal cortex.

In the polymorphic cell layer of the dentate gyrusBuChE-positive neurons are medium-sized and fusiformor triangular in profile. There is no BuChE-positive neuro-pil.

Golgi and pigment architectonic analysis of the hippo-campus proper suggests there are two main types ofneurons in the stratum pyramidale, namely, pyramidalcells and stellate cells (Braak, 1974). The morphology ofsome of the BuChE-positive neurons in the stratum pyrami-dale, particularly in the CA1 region, corresponds to that ofpyramidal cells, whereas the morphology of others iscomparable to stellate cells. Golgi studies of the stratumoriens suggest that this layer contains neurons with a widerange of shapes, including fusiform, spindle, or triangular,ranging in size from small to large (Braak, 1974). In thislayer, BuChE-positive neurons also range from small tolarge, are predominantly fusiform in profile, and aremainly found subjacent to the CA1 region.

The distribution of AChE in the hippocampal formationin our material paralleled that reported in the literature(Mellgren et al., 1977; Green and Mesulam, 1988; Amaraland Insausti, 1990; De Lacalle et al., 1994). The polymor-phic cell layer of the dentate gyrus contains AChE-positiveneurons similar in morphology to BuChE-positive neu-rons. The distributions, morphology, and numbers ofBuChE and AChE-positive neurons in this layer suggeststhat these two enzymes may be staining the same popula-tion of neurons.

There is a distinct pattern of AChE staining in thehippocampus proper in that there is neuropil labeling thatis heaviest in the stratum pyramidale within the endblades of the hilus of the dentate gyrus (CA3), whichbecomes lighter toward the CA1 region and the subicularcomplex and the entorhinal cortex. In addition, there are asome AChE-positive neurons in the stratum pyramidaleand stratum oriens (Mellgren et al., 1977; Green andMesulam, 1988; Amaral and Insausti, 1990; De Lacalle etal., 1994). The patterns of neuronal staining by BuChEand AChE in the hippocampal formation suggest thatthere may be many neurons in the hippocampal formationthat stain for both BuChE and AChE.

Functional considerations

Cholinergic neurotransmission. Because BuChEcatalyzes the hydrolysis of a wide variety of esters, includ-ing acetylcholine, propionylcholine, butyrylcholine, succi-nylcholine, heroin, cocaine, and aspirin, it has been called‘nonspecific‘ or ‘pseudocholinesterase‘ (Silver, 1974; Lock-ridge et al., 1980; Gatley, 1991; Soreq and Zakut, 1993).However, it has been suggested that BuChE may beinvolved in coregulation of cholinergic neurotransmissionbecause it catalyses the hydrolysis of acetylcholine (Vignyet al., 1978; Giacobini et al., 1996). In terms of cholinergicneurotransmission, both amygdala and hippocampal for-


mation receive cholinergic projections as evidenced by thepresence of choline acetyltransferase (ChAT)-immunoreac-tive fibers in these two structures (Ransmayr et al., 1989;Benzing et al., 1993; De Lacalle et al., 1994). Neither theamygdala nor the hippocampal formation contain ChAT-positive neurons (Ransmayr et al., 1989; Mesulam andGeula, 1991; Mesulam et al., 1992; Benzing et al., 1993; DeLacalle et al., 1994), indicating that there are no intrinsiccholinergic neurons in these two structures. The presentanatomical studies suggest that in the amygdala and thehippocampal formation, BuChE-positive neurons in areasinnervated by ChAT-positive fibers may function as cho-linoceptive neurons because BuChE inhibitors increasecortical acetylcholine level (Giacobini et al., 1996). In thisregard, BuChE may function as a coregulator of choliner-gic neurotransmission. However, the degree of cholinergicinnervation as evidenced by the density of ChAT-immuno-reactive fiber staining does not appear to vary proportion-ately with the number of BuChE-positive neurons. Forexample, a high–ChAT-fiber density is found in the basolat-eral amygdaloid nucleus and a low-fiber density in thelateral amygdaloid nucleus, but the percentage of BuChE-positive neurons is similar, 10% and 8% respectively.Therefore, it is possible that BuChE may have different oradditional functions in these structures.

Noncholinergic neurotransmission. In addition toesterase activity, BuChE has other enzymatic activitiesthat parallel in part those of AChE. Both BuChE andAChE have amidase activity because they catalyze thehydrolysis of aryl acylamides (George and Balasubrama-nian, 1981; Balasubramanian and Bhanumathy, 1993;Checler et al., 1994). The function of this activity in thenervous system, and the natural substrate for this enzy-matic action is unknown. Both these enzymes have pepti-dase activity because they are capable of catalyzing thehydrolysis of peptides such as substance P (Lockridge,1982), proenkephalins (Chubb et al., 1983), and chromogra-nins (Small et al., 1996), although this capability is amatter of debate (Checler et al., 1994). It has been ob-served that the active sites for the esterase and peptidaseactivities are in separate parts of the BuChE enzyme (Raoand Balasubramanian, 1990). Because of the capacity ofBuChE to catalyze the hydrolysis of amides and peptides,it could be involved in regulation of neuropeptide neuro-transmission. Both amygdala and the hippocampal forma-tion contain a wide variety of neuropeptides in neuronsand axons. These include neurotensin-, neuropeptide Y-,somatostatin-, substance P-, corticotropin releasing fac-tor-, cholecystokinin-, enkephalin-, and vasoactive intesti-nal polypeptide-immunoreactive neurons and fibers (Zechand Bogerts, 1985; Chan-Palay et al., 1986; Michel et al.,1986; Zech et al., 1986; Bouras et al., 1987; Lotstra andVanderhaeghen, 1987; Mai et al., 1987; Sakamoto et al.,1987; Amaral and Insausti, 1988; Mufson et al., 1988;Unger et al., 1988; Benzing et al., 1993). The presence ofaxons and neurons with neuropeptides in the regions ofamygdala and hippocampal formation containing BuChE-positive neurons may reflect possible involvement of BuChEin their regulation. Further studies are needed to deter-mine the role of BuChE in neuropeptide metabolism in theamygdala and hippocampal formation.

It has been suggested that AChE may have functionsindependent of its enzymatic activity (Greenfield, 1991;Appleyard, 1992, 1995). For example, infusion of AChEinto guinea pig hippocampal slices induces long-termpotentiation in the neurons (Appleyard, 1995). This action

has been shown to be independent of its esterase activitybut dependent on direct action of AChE on metabotropicglutamate receptors. Comparable activity of BuChE wasnot observed (Appleyard, 1995). The distribution of BuChEin the central nervous system varies from species tospecies. For example, only scattered neurons have beenobserved in the mouse amygdala (Sethi and Tanwar, 1984)and none in the rat amygdala (Darvesh et al., 1992; Tago etal., 1992). Furthermore, the rat or mouse hippocampalformation have not been shown to have BuChE-positiveneurons (Sethi and Tanwar, 1984; Darvesh et al., 1992;Tago et al., 1992). It is not known to what degree theguinea pig hippocampal formation contains BuChE. How-ever, given the variation of the distribution of BuChE fromspecies to species, physiologic experiments need to becarefully correlated with anatomic distribution of BuChE.Whereas the distribution of BuChE in the monkey brainhas not been studied in detail, during histochemical map-ping of the monkey hippocampal formation (Bakst andAmaral, 1984), BuChE-positive neurons have been identi-fied, which potentially provides an animal model forphysiologic experiments. Such physiologic studies wouldhave particular relevance because of the presence ofBuChE in human amygdala and hippocampal formation,structures that are implicated in cognitive functions(Milner, 1970; Aggleton, 1992; Squire et al., 1992), andwhich are consistently affected in Alzheimer’s disease(Brockhaus, 1938; Ball, 1977; Herzog and Kemper, 1980;Kemper, 1983; Hyman et al., 1984; Van Hoesen andDamasio, 1987; Kromer Vogt et al., 1990; Ulrich et al.,1990; Mirra et al., 1993).

Alzheimer’s disease. In Alzheimer’s disease, there isa severe loss of basal forebrain cholinergic neurons (Perryet al., 1978; Coyle et al., 1983). This neuronal loss isaccompanied by a decrease in the levels of AChE and anincrease in the levels of BuChE (Op Den Velde and Stam,1976; Perry et al., 1978). Both enzymes are found in theneuritic plaques and neurofibrillary tangles, the neuro-pathologic hallmarks of Alzheimer’s disease (Friede, 1965;Moran et al., 1994; Geula and Mesulam, 1995). Further-more, the number of BuChE-containing plaques is muchhigher in Alzheimer’s disease than in normal age-matchedcontrols (Mesulam and Geula, 1994). It has been observedin histochemical studies that the properties of cholinester-ases in these pathologic structures are altered, based onthe fact that the optimum pH for histochemical staining isaltered (Geula and Mesulam, 1989). Furthermore, boththe cholinesterases are more susceptible to inhibition byindolamines and protease inhibitors than esterase inhibi-tors (Geula and Mesulam, 1989; Wright et al., 1993).Because the cerebral cortex contains very little BuChEand the biochemical properties of the cholinesterases inthe neuritic plaques and neurofibrillary tangles resemblethose found in glia, it has been postulated that the sourceof BuChE may be glia that contain high levels of thisenzyme (Wright et al., 1993). Furthermore, it has beensuggested that the increase in BuChE in Alzheimer’sdisease may play a role in the formation of pathologicneuritic plaques and neurofibrillary tangles (Moran et al.,1994; Geula and Mesulam, 1995). Taken together, thepresence of BuChE in distinct populations of neurons inthe amygdala and the hippocampal formation, and itspresence in neuritic plaques and neurofibrillary tanglesemphasize the importance of deciphering the roles ofBuChE in normal nervous system and in Alzheimer’sdisease.


ACKNOWLEDGMENTS

We thank Dalya Abdulla and Scott Pronych for theirexcellent technical assistance and Dr. V. Sangalang (De-partment of Pathology, Queen Elizabeth II Health Sci-ences Centre, Halifax, Nova Scotia) for neuropathologicdiagnoses. The brains used in this study were provided byThe Maritime Brain Tissue Bank, Halifax, Nova Scotia,Canada.

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distribution of butyrylcholinesterase in the human amygdala and hippocampal formation

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