chemical organization of the macaque monkey olfactory bulb: iii. … · 2017. 9. 6. · 1986). the...

Chemical Organization of the MacaqueMonkey Olfactory Bulb: III. Distribution

of Cholinergic Markers

ANGEL PORTEROS, CARMELA GOMEZ, JORGE VALERO,

FERNANDO CALVO-BALTANAS, AND JOSE RAMON ALONSO*

Dpto. Biologıa Celular y Patologıa, Instituto de Neurociencias de Castilla y Leon,Universidad de Salamanca, E-37007 Salamanca, Spain

ABSTRACTThe distribution patterns of choline acetyltransferase (ChAT) and acetylcholinesterase

(AChE) were studied in the olfactory bulb (OB) of three species of macaque. AChE wasdetected by a histochemical method and ChAT immunoreactivity by immunocytochemistry.Similar results were observed in all species analyzed. With the exception of the olfactorynerve layer, all layers of the macaque monkey OB demonstrated a dense innervation ofAChE- and ChAT-positive fibers. The distribution patterns of AChE- and ChAT-labeled fiberswere similar for both cholinergic markers, although the number of AChE-labeled fibers wasclearly higher than the number of ChAT-immunoreactive fibers. The highest density of AChEand ChAT-stained fibers was observed in the interface between the glomerular layer and theexternal plexiform layer and in the internal plexiform layer. Dense bundles of labeled fiberswere observed in the caudal OB, coursing from the olfactory peduncle. All ChAT-immunopositive elements were identified as centrifugal fibers, derived from neurons caudalto the OB. Neither olfactory fibers nor intrinsic neurons were observed after ChAT immu-nocytochemistry. However, a few AChE-positive cells were observed in the glomerular layerand in both external and internal plexiform layers. These neurons were presumably identi-fied as periglomerular cells, superficial short-axon cells, and/or external tufted cells and deepshort-axon cells. Contrary to other neurotransmitters and neuroactive substances, the dis-tribution patterns of ChAT and AChE activities in the macaque monkey OB closely resem-bled the patterns described in macrosmatic mammals and showed laminar differences withthe distribution pattern observed in humans. J. Comp. Neurol. 501:854–865, 2007.© 2007 Wiley-Liss, Inc.

Indexing terms: acetylcholinesterase; choline acetyltransferase; olfaction; primate

Most primates are anatomically classified as microsmaticanimals due to the lower relative size and less distinct lam-ination of the olfactory structures, mainly the olfactory bulb(OB) and the anterior olfactory nucleus, in comparison withother telencephalic areas of the same animals and with theolfactory centers of other groups of mammals (Crosby andHumphrey, 1939; Stephan, 1975; Halasz, 1990). The OBaccounts for 17.6% of the volume of the telencephalon inbasal insectivores, where olfaction provides important infor-mation about the external conditions, whereas this ratio isreduced to 2.9% in prosimians and 0.2% in simians, typicalmicrosmatic animals (Stephan and Andy, 1970). Allometricstudies demonstrate that there is a positive correlation be-tween the reduction in the OB size and the decrease in thefunctional significance of the olfactory system (Stephan andAndy, 1970).

Although the OB cytoarchitecture is relatively constantamong vertebrates (Allison, 1953), there are significant vari-ations on its anatomical and functional organization be-tween different groups of vertebrates, and especially be-tween macrosmatic and microsmatic animals (Takagi, 1981,

Grant sponsor: Ministerio de Educacion y Ciencia; Grant number:SAF2006-05705; Grant sponsor: Junta de Castilla y Leon.

*Correspondence to: Dr. J.R. Alonso, Dpto. Biologıa Celular y Patologıa,Facultad de Medicina, Campus Miguel de Unamuno, Universidad deSalamanca, E-37007 Salamanca, Spain. E-mail: [email protected]

Received 8 March 2006; Revised 16 October 2006; Accepted 1 December2006

DOI 10.1002/cne.21280Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 501:854–865 (2007)

© 2007 WILEY-LISS, INC.

1986). The chemoarchitecture of the OB demonstrates forsome neurotransmitters and neuroactive substances (e.g.,neurotensin, NADPH-diaphorase, aromatic-L amino acid de-carboxylase, and calcium-binding proteins) a considerableinterspecies variability (Baker, 1986; Matsutani et al., 1989;Baker et al., 1991; Davis, 1991; Alonso et al., 1995, 1998,2001; Porteros et al., 1996), whereas other neurochemicallyidentified neuronal populations are remarkably constant be-tween microsmatic and macrosmatic mammals (Sanides-Kohlrausch and Wahle, 1991; Bassett et al., 1992).

The cholinergic and cholinoceptive elements of the OBhave been identified using both acetylcholinesterase(AChE: EC 3.1.1.7) histochemistry and choline acetyl-transferase (ChAT: EC 2.3.1.6) immunohistochemistry.Available studies on the distribution of cholinergic andcholinoceptive elements in the OB have been carried outin rat (Godfrey et al., 1980; Nickell and Shipley, 1988; LeJeune and Jourdan, 1991, 1993, 1994; Crespo et al., 1995;Kasa et al., 1996; Gomez et al., 2005), mouse (Carson andBurd, 1980; Weruaga et al., 2001), hamster (Macrides etal., 1981), rabbit (Chao et al., 1982), dog (Nadi et al.,1980), cat (Kimura et al., 1981), opossum (Brunjes et al.,1992), and hedgehog (Crespo et al., 1999), all of themmacrosmatic animals. In these studies a relatively com-mon distribution pattern is observed: the fiber stainingdemonstrates a high density of extrinsic fibers terminat-ing predominantly in the glomerular and internal plexi-form layers, whereas the somal staining demonstrates afew AChE-active neurons and no ChAT-immunoreactiveintrinsic neurons.

In contrast to the extensive information available on thedistribution of cholinergic and cholinoceptive elements inthe OB of macrosmatic animals, much less is known aboutthe differences or similarities with the nonhuman primateOB. The organization of the cholinergic system has beendescribed in the human OB, both in nondemented personsand in patients affected by Alzheimer’s disease (Kasa etal., 1997; Kovacs et al., 1998), but no data are available inthe OB of primate models such as the macaque monkey.The aim of this study was to carry out a detailed descrip-tion of the location and morphological characteristics of

AChE-active and ChAT-immunoreactive elements in themacaque monkey OB and to compare these observationswith previous data on macrosmatic animals and humans.

MATERIALS AND METHODS

Animals and tissue preparation

Four adult male cynomolgus monkeys (Macaca fascicu-laris), each weighing 2.7–3.8 kg, five adult male rhesusmonkeys (Macaca mulata), weighing 4.7–5.3 kg, and fiveadult male pig-tail monkeys (Macaca nemestrina), weigh-ing 4.5–6.5 kg, were used in the present study. The brainsof these animals were used in other neuroanatomical stud-ies, not related to the olfactory system. The OBs weredonated by different groups working in the neuroanatomyof other parts of the brain (see Acknowledgments). In allcases, animal manipulations were performed according toNIH guidelines and subjected to authorization from thepertinent ethical committees.

The cynomolgus monkeys were tranquilized withketamine-HCl (10 mg/kg body weight, i.m.) and anesthe-tized with sodium pentobarbital (50 mg/kg, i.v.). Thenthey were transcardially perfused with 500 mL of 0.9%saline and the following fixative mixtures: 4% paraformal-dehyde in 0.1 M sodium acetate buffer, pH 6.5 (250 mL/min for 5 minutes and 100 mL/min for 15 minutes), and4% paraformaldehyde in 0.1 M sodium borate buffer, pH9.5 (100 mL/min for 30 minutes). The OBs were dissectedout and postfixed in the last fixative for 6 hours at 4°C.

The rhesus and pig-tail monkeys were anesthetizedwith 10 mL/kg ketamine-HC1 (i.m.) and 50 mg/kg sodiumpentobarbital (i.v.). They were transcardially perfusedwith 0.8–1 of 0.9% saline followed by 4–5 1 of 4% para-formaldehyde in 0.1 M phosphate buffer, pH 7.3 (PB). TheOBs were dissected out and postfixed in the same fixativefor an additional 2 hours at 4°C.

The OBs were carefully washed in PB and cryoprotectedin either 30% sucrose or 20% glycerol with 2% dimethylsulfoxide (DMSO) in PB for 1 day. Then they were frozenwith cooled isopentane (Rosene et al., 1986) and stored at�70°C until further processing; 30-�m serial coronal orparasagittal sections were cut on a freezing, sliding mic-rotome.

ChAT immunohistochemistry

Material analyzed for this study was prepared withmonoclonal antibody AB8, kindly donated by Dr. BruceWainer. This antibody has been fully characterized (Arm-strong et al., 1983; Levey et al., 1983). It was raisedagainst partially purified ChAT from bovine caudate,emulsified in Freund’s adjuvant, and injected into Lewisrats. Rat spleen cells were fused with mouse myelomacells, obtaining a hybridoma cell line secreting antibodiesthat reacted equally well with ChAT from all mammalianspecies tested (bovine, human, sheep, cat, guinea pig, rat,and mouse) and also bound to avian ChAT. The AB8antibody was also obtained in high titer in ascites fluid ofnude mice. Specificity of the antibody was assessed byWestern blotting of crude extract of bovine brain or par-tially purified bovine ChAT, obtaining two immunostainedprotein bands of 68 and 70 kDa, as expected (Cozzari andHartman, 1980; Ryan and McClure, 1980). Staining ofsections through the monkey brain with AB8 ChAT anti-serum showed a clear and distinct cholinergic fibrillarpattern (Alonso and Amaral, 1995; Alonso et al., 1996).

ABBREVIATIONS

AB 0.1 M acetate buffer, pH 6.5AChE AcetylcholinesteraseBSA Bovine serum albuminChAT Choline acetyltransferaseDAB 3,3� DiaminobenzidineDMSO Dimethyl sulfoxideEPL External plexiform layerG GlomerulusGCL Granule cell layerGCLext Granule cell layer, external partGCLint Granule cell layer, internal partGL Glomerular layerGLext Glomerular layer, external partGLint Glomerular layer, internal partIPL Internal plexiform layerMCL Mitral cell layerOB Olfactory bulbONL Olfactory nerve layerOP Olfactory pedunclePAP Peroxidase antiperoxidasePB 0.1 M phosphate buffer, pH 7.3PBS 0.1 M phosphate-buffered saline, pH 7.3TBS 0.2 M Tris buffered saline, pH 7.6WM White matter

The Journal of Comparative Neurology. DOI 10.1002/cne

855CHOLINERGIC MARKERS IN MONKEY OLFACTORY BULB

The free-floating sections were sequentially incubatedin the following solutions: 1) AB8 primary antibody 1:500in 0.1 M phosphate-buffered saline, pH 7.3 (PBS), contain-ing 2% bovine serum albumin (BSA), 20% normal rabbitserum, and 0.5% Triton X-100, for 48 hours at 4°C; 2)rabbit antirat IgG secondary antiserum (Cappel Labora-tories, Durham, NC) diluted 1:50 in 0.2 M Tris bufferedsaline, pH 7.6 (TBS), containing 2% BSA, 20% normalrabbit serum, 10% normal monkey serum, and 0.2% Tri-ton X-100, for 1 hour; 3) rat peroxidase antiperoxidase(PAP) (Sternberger Monoclonals, Jarretsville, MD), di-luted 1:50 in the same solution as the secondary anti-serum, for 2 hours. The sections were processed followinga double-bridging procedure by returning them to thesecondary antiserum solution for 1 hour and then PAPcomplex for 90 minutes. Steps 2) and 3) were carried out atroom temperature. After incubation, the sections werecarefully washed and the tissue-bound peroxidase wasvisualized with 0.05% 3,3� diaminobenzidine (DAB) inTBS containing 0.015% hydrogen peroxide for 15 minutes.The sections were mounted onto gelatin-coated slides, air-dried overnight, defatted in a 1:1 solution of chloroformand absolute ethanol for 1 hour, and rehydrated. The DABreaction product was intensified by placing sections in0.005% aqueous osmium tetroxide for 30 minutes, runningwater for 30 minutes, 0.05% aqueous thiocarbohydrazidefor 15 minutes, running water for 30 minutes, and againin the original osmium solution for 30 minutes. Thereaf-ter, the slides were rinsed in running water for 30 min-utes. Controls for the immunohistochemical procedurewere carried out to determine the specificity of the immu-nolabeling: omission of the primary or secondary antibod-ies and incubation of sections exclusively in 0.05% DABand 0.015% H2O2 in TBS. No residual immunoreactivitywas observed.

AChE histochemistry

An adjacent series to that prepared immunohistochemi-cally for ChAT was processed for the demonstration ofAChE using a modification (Hedreen et al., 1985) of theKoelle method (Geneser-Jensen and Blackstad, 1971) asdescribed elsewhere (Bakst and Amaral, 1984; Amaraland Bassett, 1989; Crespo et al., 1999). Briefly, afterwashing in 0.1 M sodium acetate buffer (AB) pH 6.0, thesections were incubated with agitation at room tempera-ture for 30 minutes. The incubation medium was made upof 1.7 mM acetylthiocholine iodide (Sigma, St. Louis, MO,#A5751), 0.49 mM sodium citrate (Sigma, #S4641), 2.9mM cooper sulfate (Sigma, #C7631), 1.25 mM potassiumferricyanide (Sigma, #P8131), and 0.2 mM ethopropazine(Sigma, #E2880) in AB. Ethopropazine was used as inhib-itor of nonspecific cholinesterases. Acetylcholinesteraseactivity was visualized using two different techniques: Inthe first technique the sections were rinsed after incuba-tion in AB, treated for 1 minute with 2% ammoniumsulfide, and washed (3 � 10 minutes) in 0.1% sodiumnitrate. The free-floating sections were then stained with0.05% silver nitrate for 1 minute and washed in 0.1%sodium nitrate (3 � 5 minutes) and PB (2 � 5 minutes).The second group of sections was rinsed after incubationin 0.2 M Tris-HCl buffer, pH 7.6 (3 � 10 minutes) and thereaction was visualized using 0.0125% DAB, 0.5% nickelammonium sulfide, and 0.001% hydrogen peroxide in thesame Tris-HCl buffer. The reaction was stopped by rinsingthe sections with 0.2 M Tris-HCl buffer, pH 7.6 (3 � 5

minutes) and distilled water (2 � 5 minutes). Controls forthe specificity of the histochemical staining were per-formed as previously described (Crespo et al., 1999). Re-sidual activity was not detected in any control.

A complete third series of sections from each OB wasstained with 0.25% thionin for the demonstration of cellbodies.

All sections were serially mounted on gelatin-coatedslides, dehydrated in ethanol graded series, cleared withxylene, and coverslipped with Entellan (Merck, Darm-stadt, Germany).

Analysis

Sections were analyzed using brightfield and darkfieldoptical systems. Digital images were obtained with anApogee KX digital camera (Apogee Instruments, Tucson,AZ) coupled to an Olympus Provis AX70 photomicroscope.The capture software was connected to a trichromaticsequential filter (Cambridge Research & Instrumentation,Boston, MA). Original pictures were processed digitallywith Adobe Photoshop 7.0 software (San Jose, CA). Thesharpness, contrast, and brightness were adjusted to re-flect the appearance seen through the microscope. Cellsizes were measured using a Neurolucida image analysissystem (MicroBrightField, Burlington, VT). For each iden-tified neuronal type the maximum diameters of 140AChE-positive neurons were measured. Measured cellswere selected at random in the OB of all animals used inthe study (10 cells of each neuronal type for each animal).Only cells with their focusing plane in the middle of the30-�m sections were measured. The sizes are presented asmean � SEM of the maximum diameter.

In order to gain a better understanding of the results, asemiquantitative analysis of the fiber density was per-formed using ImageJ software (Scion, Bethesda, MD) aspreviously described (Gomez et al., 2005). Measures werecarried out delimiting the different areas of each layerfrom randomly selected digital pictures obtained with a10� objective (UPlanFl 10.30 NA). The digital imageswere treated to balance the signal-to-noise ratio in such away that positive elements were clearly distinguishablefrom the background. Following this, they were manuallytransformed into binary images in which only stainedelements appeared as white pixels. Then the surface ofanalysis was delimited, using the original image as refer-ence and fiber density was calculated as the white pixelratio in the entire estimated area. The densities are pre-sented in percentage as mean � SEM. Statistical differ-ences were assessed by one-way analysis of variance(ANOVA) and Student-Newman-Keuls post-hoc analysis.The level of significance was set at P � 0.01.

RESULTS

As a general feature, the morphologic characteristicsand distribution of ChAT- and AChE-labeled elements inthe different layers of the OB were consistent in all threespecies studied (Macaca fascicularis, Macaca mulatta,and Macaca nemestrina). Therefore, the following descrip-tion of the distribution of both markers refers to all spe-cies.

As described previously, two different techniqueswere used to visualize AChE-active elements. Therewere no differences in the distribution and number ofstained structures after using both methods and only


856 A. PORTEROS ET AL.

minor variations in the quality of the labeling could benoted. Thus, using the silver nitrate technique, the fiberstaining was excellent but the precipitate in the cellbodies was weak, and the visualization of labeled cellswas difficult. When revealing AChE-active elements byusing DAB, the neuronal staining improved notably,although the profiles of labeled fibers appeared lessclearly delineated. Brightfield Figures 1a and 2b showthe general overview of AChE staining in coronal sec-tions of the macaque monkey OB. In Figure 2a compar-ison with a Nissl-staining section is presented. Thedensity of AChE labeling is higher in the glomerularlayer (GL) and the inner plexiform layer (IPL) than inany other region of the macaque monkey OB.

The distribution of labeled elements was relatively sim-ilar for both markers, ChAT and AChE. However, twomain differences can be noted. Whereas after ChAT-immunohistochemistry only stained fibers were observed,the AChE histochemical technique labeled, in addition tofibers, some neuronal bodies. AChE-labeled neuronsshowed a dark reaction product in the cell body, whereastheir processes, if stained, were only visible in their prox-imal part, and therefore classifying neuronal types wasdifficult. The second main difference between the ChATand AChE labelings was that, although the distribution ofChAT-immunoreactive and AChE-active structuresshowed a similar distribution through the layers of theOB, the density of AChE-positive fibers was clearly higherthan that of the ChAT-immunoreactive ones (see Figs. 1,3). Data from the semiquantitative analysis (Table 1, Fig.3) corroborate these findings, and also pointed out differ-ences in the relative density between the internal part ofthe glomerular layer (GLint) and external part of theglomerular layer (GLext) and between the internal part ofthe granule cell layer (GCLint) and the external part ofthe granule cell layer (GCLext).

All layers of the OB, with the only exception of theolfactory nerve layer (ONL), demonstrated abundantAChE- and ChAT-positive fibers. The most superficiallayer of the OB, the ONL, was AChE- and ChAT-negativesince all olfactory fibers were negative for both markers(Figs. 1, 2b).

The GL, by contrast, demonstrated together with theIPL the highest density of labeled fibers for both mark-ers (Figs. 1, 2b). These ChAT-immunoreactive andAChE-positive fibers entered the deep side of the glo-meruli and outlined them, extending superficially up tothe external boundary with the ONL (Figs. 1, 2b). Mostpositive fibers and terminations were located in theperiphery of the glomeruli limiting with the externalplexiform layer (EPL), but in all glomeruli a few poorlyramified fibers entered within the glomerular core(Fig. 2b).

The highest density of AChE-positive cell bodies wasalso found in the GL, where two types of AChE-containingneurons were distinguished. The most abundant typeshowed an ovoid shape, small size (6.78 � 0.76 �m), andwere normally located surrounding the glomeruli: typicalmorphological characteristics of periglomerular cells. Oc-casionally, they were found inside the glomeruli (Fig. 4a).The second population of AChE-active neurons located inGL was less numerous, and they were always locatedsurrounding the glomeruli mainly on its superficial part,close to the border with the ONL (Fig. 4b). They hadpredominantly ovoid or fusiform shape and their size was

larger (11.23 � 1.12 �m). According to these characteris-tics they were presumably classified as superficial short-axon cells or external tufted cells.

The superficial portion of the EPL close to the GLshowed a very dense network of fibers (Figs. 1, 2b),which was reduced in the inner part. The distributionsof the labeling and the morphology of stained elementswere very similar for both ChAT and AChE; however,the density of AChE-active fibers was clearly higherthan the density of ChAT-immunoreactive ones (Figs.1, 3).

AChE-labeled neurons were also detected in the EPL.These neurons were observed throughout the whole extentof this layer, although most of them were located in itssuperficial one-third (Fig. 4c,d), where fibers were alsomore abundant. They had ovoid, piriform (Fig. 4c), orround (Fig. 4d) shapes and medium size (12.83 � 1.65�m). They were classified as superficial short-axon cellsand/or external tufted cells.

The mitral cell layer (MCL) showed very scarce AChE-and ChAT-stained elements, with only some labeled fiberscrossing with radial or oblique trajectories between theunstained profiles of the mitral cells (Fig. 2b). In this layerneither mitral cells nor any other neuronal type was ob-served after AChE staining.

The density of positive fibers for both markers increasedin the thin IPL, which showed abundant horizontal axonslocated just below the mitral cells (Fig. 2b). The AChE andChAT stainings in this layer were similar to those ob-served in the superficial portion of the EPL for both mark-ers and only one difference could be noted: in the IPL therewas a relative abundance of thicker fibers. Most labeledfibers coursed parallel to the OB lamination. In this layerand at the border between the IPL and the granule celllayer (GCL), only a few AChE-labeled cell bodies weredetected (Fig. 4e,f). They were medium-sized (13.06 � 1.23�m) and showed piriform, round, or ovoid shapes. Thesecells could belong to some type of oriented interneurons ofthe deep layers such as horizontal cells and/or verticalcells of Cajal due to their location and size, notably largerthan the granule cells.

The staining for ChAT and AChE decreased again inthe GCL, where the density of stained fibers was similarfor both markers. This layer was the zone of the OBwhere most variability was observed among the activeelements. The fibers showed varying thicknesses (Fig.5e), did not show any definite orientation, and were seenfrequently branching throughout this layer (Fig. 5e).Moreover, the density varies notably from one zone toanother (Fig. 5a– c). In addition, there were some clus-ters of densely packed stained fibers sparsely distrib-uted throughout the GCL (Figs. 5d, 6a). The GCL waspractically devoid of AChE-reactive neurons with theexception of those described in the superficial region,close to the IPL.

The density of the staining decreased slightly at thedeeper portions of the GCL and in the white matter (WM).In the WM, as well as in the proximal region of theolfactory peduncle (OP), only sparsely distributed fiberswere seen. Among these fibers a distinct population ofAChE-positive neurons was observed (Fig. 4g,h). Theseneurons were large (25.78 � 2.01 �m) and they normallyhad a fusiform shape, although some had ovoid or irregu-lar morphologies. The dendrites of these neurons werefrequently stained and it was possible to follow them for



Fig. 1. Brightfield overview of AChE- and ChAT-stained sectionsof the Macaca fascicularis monkey olfactory bulb (OB). Note that thehighest density of fibers is observed in the glomerular layer (GL) andthe internal plexiform layer (IPL). The staining laminar pattern is

similar for both markers. ONL, olfactory nerve layer; EPL, externalplexiform layer; IPL, internal plexiform layer; MCL, mitral cell layer;GCL, granule cell layer; GL, glomerular layer; WM, white matter.Scale bar � 1 mm.



long distances coursing parallel to the OB lamination. Asin the case of the neurons located in the EPL, the negativecell nucleus could be observed (Fig. 4g,h).

Finally, it was possible to observe abundant labeledfibers entering the GCL from the olfactory tract throughthe OP (Fig. 6b). These labeled fibers demonstrated alongitudinal orientation (Fig. 6b,d) and there was a de-crease in the number of AChE- or ChAT-labeled fibersfrom the border to the central part, where the density wasclearly lower (Fig. 6c). The junction zone between the GCLand the OP was clearly distinguishable because of thedifferent arrangement of positive fibers in both regions(Fig. 6b). Similar AChE-labeled neurons to those de-scribed in the WM were observed in the OP.

DISCUSSION

The results of this study constitute the first detailedreport of the localization of AChE and ChAT in the ma-caque monkey OB. The histochemical distribution ofAChE was very similar to the immunohistochemical dis-tribution pattern of ChAT, although the density of ChAT-labeled fibers was clearly lower and no ChAT-positive cellswere observed. The staining features for both markerswere constant and no significant differences were ob-served in the results among the specimens used, whichsuggests the existence of a common pattern in the cholin-ergic innervation of the monkey OB, at least in theseclosely related species.

Fig. 2. Photographic composition of coronal Nissl-stained section (a) and AChE stained section (b) ofthe Macaca fascicularis monkey olfactory bulb (OB) to show the laminar distribution of labeled AChEfibers. ONL, olfactory nerve layer; EPL, external plexiform layer; IPL, internal plexiform layer; MCL,mitral cell layer; GCL, granule cell layer; GL, glomerular layer; WM, white matter. Scale bar � 250 �m.



The discrete laminar pattern of distribution of ChATand AChE activities in the macaque monkey OB is in goodagreement with previous works in rodents (Carson andBurd, 1980; Godfrey et al., 1980; Macrides et al., 1981;Nickell and Shipley, 1988; Le Jeune and Jourdan, 1991,1993, 1994; Crespo et al., 1995; Kasa et al., 1996, Weruagaet al., 2001; Gomez et al., 2005), rabbit (Chao et al., 1982),dog (Nadi et al., 1980), cat (Kimura et al., 1981), opossum(Brunjes et al., 1992), and hedgehog (Crespo et al., 1999).The cholinergic innervation observed in the human OBshows a similar laminar pattern in the deep layers, al-though no labeling has been described in the GL or EPL(Kasa et al., 1997; Kovacs et al., 1998).

The macaque monkey OB presents reciprocal connectionswith areas of the primary olfactory cortex, including theanterior olfactory nucleus, piriform cortex, anterior corticalnucleus of the amygdala, periamygdaloid cortex, and ento-rhinal cortex, and also with the nucleus of the horizontallimb of the diagonal band of Broca (Carmichael et al., 1994).In the monkey, ChAT-immunopositive and AChE-activeneurons can be observed in the Ch3 region of the basaltelencephalon, equivalent to the horizontal limb of the diag-onal band of Broca following Mesulam’s nomenclature (Me-sulam et al., 1983a,b; Mesulam and Geula, 1988). This cho-linergic population may constitute the main source ofcholinergic centrifugal fibers to the macaque monkey OB.

Although the general distribution of cholinergic fibers inthe macaque monkey OB is generally coincident with previ-ous reports in macrosmatic mammals, there are some minordifferences. Thus, the prominent AChE staining in the MCLof the rat OB reported by Shute and Lewis (1967) is notobserved in the monkey OB, where the low density of fibersin the MCL is comparable to that in the deepest region of theEPL. Godfrey et al. (1980) reported in mice that cholinergicelements in the GL were as plentiful within as outside theglomeruli. However, in other reports in mouse (Carson andBurd, 1980) and rat (Ojima et al., 1988; Phelps et al., 1992)as well as in our sections, most ChAT-immunoreactive andAChE-active fibers were located surrounding the glomeruliand only a few fibers and terminals entered within them.The presence of cholinergic activity in the ONL is controver-sial. Some data support an involvement of acetylcholine inthis layer by the presence of a low AChE enzymatic activityin the olfactory receptors (Baradi and Bourne, 1953), theexistence of muscarinic acetylcholine receptor binding sitesin the dog ONL (Nadi et al., 1980) and in the olfactoryreceptors of the salamander (Hedlund and Shepherd, 1983),and the presence of ChAT and AChE activities in thesalamander ONL. Our results in the macaque monkey con-firm previous observations in rodents (Carson and Burd,1980; Ojima et al., 1988; Phelps et al., 1992), where noactivity was detected in the ONL after either ChAT-immunocytochemistry or AChE-histochemistry.

The use of AChE, ChAT, and other cholinergic markersin the rat olfactory bulb defines two different populationsof olfactory glomeruli (Zheng et al., 1987; Ojima et al.,1988; Zheng and Jourdan, 1988; Le Jeune and Jourdan,1991, 1993, 1994; Le Jeune et al., 1995, 1996; Crespo etal., 1995, 1997; Weruaga et al., 2001; Gomez et al., 2005).In the caudalmost OB, a subset of atypical glomerulishows a dense innervation from centrifugal fibers as wellas high levels of nicotinic receptors. This glomerular het-erogeneity has been related to a topographically definedspecificity of the olfactory glomeruli involved in the pro-cessing of particular signals (Zheng et al., 1987; Zhengand Jourdan, 1988; Le Jeune and Jourdan, 1991, 1993).By contrast, in the macaque monkey OB the density of

TABLE 1. Density of AChE and ChAT Staining in the Macaque MonkeyOB

Layer AChE ChAT

GLext 24.53 � 3.15 29.83 � 3.75GLint 73.16 � 1.48 51.11 � 3.22EPL 35.12 � 1.15 26.27 � 2.96MCL 6.30 � 2.80 6.92 � 2.55IPL 70.18 � 5.07 38.09 � 2.49GCLext 27.56 � 2.74 7.89 � 4.03GCLint 7.00 � 0.79 5.31 � 4.63WM 2.73 � 0.98 2.13 � 0.89

Data represent the mean � SEM of percentage of stained pixels in digital images.OB, olfactory bulb; EPL, external plexiform layer; GCLext, granule cell layer, externalpart; GCLint, granule cell layer, internal part; GLext, glomerular layer, external part;GLint, glomerular layer, internal part; IPL, internal plexiform layer; MCL, mitral celllayer; WM, white matter.

Fig. 3. Comparative graph showing AChE and ChAT staining densities in the different layers of themacaque monkey olfactory bulb (OB). Data represent the mean � SEM of percentage of stained pixels indigital images. Significant differences are observed in the GLint, the IPL, and the GCLext. GLint, granule celllayer, internal part; IPL, internal plexiform layer; GCLext, granule cell layer, external part. **P � 0.01.



Fig. 4. Brightfield photomicrographs of AChE-active neurons inthe Macaca fascicularis monkey olfactory bulb (OB). a: Small (5–8�m) AChE-positive neurons (arrows) within a glomerulus (G) sur-rounded by numerous AChE-active fibers. b: Medium-sized neuron(10–12 �m) located in the external zone of a glomerulus (G). Note thepresence of some positive fibers inside the glomerulus and the lack ofAChE-activity in the ONL (asterisks). c,d: Medium-sized neurons(11–14 �m) in the superficial region of EPL showing piriform (c)

(arrow) and ovoid (d) shapes. e: AChE-active neurons displayingdifferent morphologies (arrows) in the deep region of IPL. f: PiriformAChE-active neuronal body located at the IPL–GCL border (arrow).g,h: Large AChE-positive neurons (25–30 �m) located in the WM andin the proximal region of the OP. These neurons had fusiform (g) orirregular (h) shapes with long dendrites (arrows). IPL, internal plex-iform layer; GCL, granule cell layer; OP, olfactory peduncle. Scalebars � 50 �m.



AChE-active and ChAT-immunoreactive fibers was rela-tively homogeneous throughout the whole rostrocaudalextent of the GL. Atypical glomeruli in the rat OB alsoshow lower numbers of NADPH-diaphorase active/NOS(nitric oxide synthase) immunoreactive-periglomerularcells (Crespo et al., 1996) and neurocalcin-containingtufted cells (Crespo et al., 1997), but this sort of heteroge-neity has not been found in the macaque monkey OB(Alonso et al., 1998, 2001).

Although some authors have described a small popula-tion of cholinergic neurons in the mammalian OB (Carsonand Burd, 1980; Ojima et al., 1988; Lysakowski et al.,1989; Phelps et al., 1992), it is generally accepted that therodent OB is devoid of intrinsic cholinergic neurons, andonly a few cholinoceptive neurons displaying AChE activ-ity are present (Nickell and Shipley, 1988; Le Jeune andJourdan, 1991, 1993; Crespo et al., 1995; Kasa et al.,1996). These patterns of somal staining are coincident

Fig. 5. Darkfield photomicrographs of the granule cell layer (GCL)of the macaque monkey olfactory bulb (OB) after ChAT immunocyto-chemistry. a–c: Local differences in the density of ChAT-immunoreactive fibers within the granule cell layer (GCL). Someareas of this layer showed a high density of ChAT-positive fibers (a).

In other areas the density was moderate (b) whereas in other zonesthe density was low (c). Macaca mulatta. d: Dense cluster of ChAT-positive fibers in the GCL. Macaca fascicularis. e: Higher magnifica-tion of a ramified ChAT-immunoreactive fiber in the GCL of themacaque monkey OB. Macaca nemestrina. Scale bars � 250 �m.



with our observations in the macaque monkey OB, whereno ChAT-immunopositive cells and only a few AChE-active neurons were detected. Since ChAT is a more spe-cific marker for cholinergic elements, AChE-labeled neu-rons are probably noncholinergic in nature and AChEcould be involved, in these neurons, in the metabolism of

acetylcholine and/or other substances. In fact, AChE hy-drolyses met-enkephalin precursor (Millar and Chubb,1985), leu- and met-enkephalin (Chubb et al., 1983), andsubstance P (Chubb et al., 1980), all of them present in theOB. Despite these data, due to the similar patterns ofAChE with other cholinergic markers, it seems that most

Fig. 6. Dark-field photomicrographs of the deep layers of the ma-caque monkey olfactory bulb (OB) after AChE histochemistry. a:Cluster of densely packed AChE-positive fibers in the GCL. Macacamulatta. b: Boundary zone between the OP and the internal region ofthe GCL. Note the clear limit between both zones due to the differentdisposition of the centrifugal fibers. Macaca fascicularis. c: Caudal

region of the OB where an AChE-positive bundle courses close to thepial surface, whereas isolated fibers (arrows) penetrate obliquely inthe caudal GCL. Macaca nemestrina. d: AChE-positive fibers in theinternal region of the OP. In addition to the abundant longitudinalfibers, some perpendicular thin fibers can be observed (arrows). Ma-caca fascicularis. Scale bars � 250 �m.



AChE in the OB is involved in the hydrolysis of acetylcho-line (Nickell and Shipley, 1988).

AChE-labeled neurons in the macaque monkey OB werelocated in the GL, in the EPL, at the IPL–GCL border, andin the WM and OP. As in the macaque, a subpopulation ofperiglomerular cells has been identified displaying AChE-activity in the rat (Lysakowski et al., 1989; Phelps et al.,1992; Le Jeune and Jourdan, 1993, 1994) and in themouse (Carson and Burd, 1980), and the larger AChE-positive neurons of the GL have been identified as super-ficial short-axon cells (Nickell and Shipley, 1988). Ourresults in monkey agree with previous observations inrodents where AChE-positive cells in the EPL have beendescribed as superficial short-axon cells (Lysakowski etal., 1989; Carson and Burd, 1980) or as tufted cells (Car-son and Burd, 1980; Kasa et al., 1996). Since in the mon-key, as in the rat (Nickell and Shipley, 1988; Le Jeune andJourdan, 1994), AChE-positive neurons in the IPL–GCLborder were substantially larger than granule cells and,due to their location, they could correspond to some typesof deep short-axon cells (horizontal neurons and verticalcells of Cajal, among others).

The deepest population of AChE-positive neurons wasfound in the WM and in the OP. These cells have not beenpreviously described in other species. Large neurons re-sembling their morphological characteristics and their lo-cation have been observed in the OB of the rat (Celio,1990; Brinon et al., 1992) and human (Ohm et al., 1991)after calbindin D-28k immunostaining; however, theselatter cells had oriented and strongly varicose dendrites,whereas the AChE-labeled neurons demonstrated differ-ent dendritic morphologies. It has been proposed thatAChE-labeled deep neurons in the OP might belong to theterminal nerve (Wirsig and Leonard, 1986).

ACKNOWLEDGMENTS

The authors thank Dr. David G. Amaral (University ofCalifornia, Davis), Dr. Wendy A. Suzuki (New York Uni-versity), and Drs. Carmen Cavada and Estrella Rausell(Universidad Autonoma de Madrid) for help in obtainingthe macaque monkey olfactory bulbs, and Dr. BruceWainer for contributing the ChAT antibody used in thisstudy.

LITERATURE CITED

Allison AC. 1953. The morphology of the olfactory system in the verte-brates. Biol Rev 28:195–244.

Alonso JR, Amaral DG. 1995. Cholinergic innervation of the primate hip-pocampal formation. I. Distribution of choline acetyltransferase immu-noreactivity in the Macaca fascicularis and Macaca mulatta monkeys.J Comp Neurol 355:135–170.

Alonso JR, Arevalo R, Garcıa-Ojeda E, Porteros A, Brinon JG, Aijon J.1995. NADPH-diaphorase active and calbindin D-28k-immunoreactiveneurons and fibers in the olfactory bulb of the hedgehog (Erinaceuseuropaeus). J Comp Neurol 351:307–327.

Alonso JR, U HS, Amaral DG. 1996. Cholinergic innervation of the primatehippocampal formation: II. Effects of fimbria/fornix transection.J Comp Neurol 375:527–551.

Alonso JR, Porteros A, Crespo C, Arevalo R, Brinon JG, Weruaga E, AijonJ. 1998. Chemical anatomy of the macaque monkey olfactory bulb:NADPH-diaphorase/nitric oxide synthase activity. J Comp Neurol 402:419–434.

Alonso JR, Brinon JG, Crespo C, Bravo IG, Arevalo R, Aijon J. 2001.Chemical organization of the macaque monkey olfactory bulb: II. Cal-retinin, calbindin D-28k, parvalbumin, and neurocalcin immunoreac-tivity. J Comp Neurol 432:389–407.

Amaral DG, Bassett JL. 1989. Cholinergic innervation of the monkeyamygdala: an immunohistochemical analyses with antisera to cholineacetyltransferase. J Comp Neurol 281:337–361.

Armstrong DM, Saper CB, Levey AI, Wainer BH, Terry RD. 1983. Distri-bution of cholinergic neurons in rat brain: demonstrated by the immu-nocytochemical localization of choline acetyltransferase. J Comp Neu-rol 216:53–68.

Baker H. 1986. Species differences in the distribution of substance P andtyrosine hydroxylase immunoreactivity in the olfactory bulb. J CompNeurol 252:206–226.

Baker H, Abate C, Szabo A, Joh TH. 1991. Species-specific distribution ofaromatic-L-amino acid decarboxylase in the rodent adrenal gland, cer-ebellum and olfactory bulb. J Comp Neurol 305:119–129.

Bakst I, Amaral DG. 1984. The distribution of acetylcholinesterase in thehippocampal formation of the monkey. J Comp Neurol 225:344–371.

Baradi AF, Bourne GH. 1953. Gustatory and olfactory epithelia. Int RevCytol 2:289–330.

Bassett JL, Shipley MT, Foote SL. 1992. Localization of corticotrophin-releasing factor-like immunoreactivity in monkey olfactory bulb andsecondary olfactory areas. J Comp Neurol 316:348–362.

Brinon JG, Alonso JR, Arevalo R, Garcıa-Ojeda E, Lara J, Aijon J. 1992.Calbindin D-28k-positive neurons in the rat olfactory bulb. An immu-nohistochemical study. Cell Tissue Res 269:289–297.

Brunjes PC, Jazaeri A, Sutherland MJ. 1992. Olfactory bulb organizationand development in Monodelphis domestica (grey short-tailed opos-sum). J Comp Neurol 320:544–554.

Carmichael ST, Clugnet MC, Price, JL. 1994. Central olfactory connectionsin the macaque monkey. J Comp Neurol 346:403–434.

Carson KA, Burd GD. 1980. Localization of acetylcholinesterase in themain and accessory olfactory bulbs of the mouse by light and electronmicroscopic histochemistry. J Comp Neurol 191:353–371.

Celio MR. 1990. Calbindin D-28k and parvalbumin in the rat nervoussystem. Neuroscience 35:375–475.

Cozzari C, Hartman BK. 1980. Preparation of antibodies specific to cholineacetyltransferase from bovine caudate nucleus and immunohistochem-ical localization of the enzyme. Proc Natl Acad Sci U S A 77:7453–7457.

Crespo C, Arevalo R, Brinon JG, Porteros A, Bravo IG, Aijon J, Alonso JR.1995. Colocalization of NADPH-diaphorase and acetylcholinesterase inthe rat olfactory bulb. J Chem Neuroanat 9:207–216.

Crespo C, Porteros A, Arevalo R, Brinon JG, Aijon J, Alonso JR. 1996.Segregated distribution of nitric oxide synthase-positive cells in theperiglomerular region of typical and atypical olfactory glomeruli. Neu-rosci Lett 205:149–152.

Crespo C, Alonso JR, Brinon JG, Weruaga E, Porteros A, Arevalo R, AijonJ. 1997. Calcium-binding proteins in the periglomerular region of typ-ical and atypical olfactory glomeruli. Brain Res 745:293–302.

Crespo C, Brinon JG, Porteros A, Arevalo R, Rico B, Aijon J, Alonso JR.1999. Distribution of acetylcholinesterase and choline acetyltrans-ferase in the main and accessory olfactory bulbs of the hedgehog (Eri-naceus europaeus). J Comp Neurol 403:53–67.

Chao L, Kan KSK, Hung FM. 1982. Immunohistochemical localization ofcholine acetyltransferase in rabbit forebrain. Brain Res 235:65–82.

Chubb IW, Hodgson AJ, White GH. 1980. Acetylcholinesterase hydrolyzessubstance P. Neuroscience 5:2065–2072.

Chubb IW, Ranieri E, White GH, Hodgson AJ. 1983. The enkephalins areamongst the peptides hydrolyzed by purified acetylcholinesterase. Neu-roscience 10:1369–1377.

Crosby EC, Humphrey T. 1939. Studies of the vertebrate telencephalon. 1.The nuclear configuration of the olfactory and accessory olfactory for-mations and of the nucleus olfactorius anterior of certain reptiles,birds, and mammals. J Comp Neurol 71:121–213.

Davis BJ. 1991. NADPH-diaphorase activity in the olfactory system of thehamster and rat. J Comp Neurol 314:493–511.

Geneser-Jensen FA, Blackstad TW. 1971. Distribution of acetylcholinest-erase in the hippocampal region of the guinea pig. I. Entorhinal area,parasubiculum and presubiculum. Z Zellforsch 114:460–481.

Godfrey DA, Ross CD, Herrmann AD, Matschinsky FM. 1980. Distributionand derivation of cholinergic elements in the rat olfactory bulb. Neu-roscience 5:273–292.

Gomez C, Brinon JG, Barbado MV, Weruaga E, Valero J, Alonso JR. 2005.Heterogeneous targeting of centrifugal inputs to the glomerular layerof the main olfactory bulb. J Chem Neuroanat 29:238–254.

Halasz N. 1990. The vertebrate olfactory system. Chemical neuroanatomy,function and development. Budapest: Akademiai Kiado.



Hedlund B, Shepherd GM. 1983. Biochemical studies on muscarinic recep-tors in the salamander olfactory epithelium. FEBS Lett 162:428–431.

Hedreen JC, Bacon SJ, Price DL. 1985. A modified histochemical techniqueto visualize acetylcholinesterase-containing axons. J Histochem Cyto-chem 33:134–140.

Kasa P, Karcsu S, Kovacs I, Wolff JR. 1996. Cholinoceptive neurons with-out acetylcholinesterease activity and enzyme-positive neurons with-out cholinergic synaptic innervation are present in the main olfactorybulb of adult rat. Neuroscience 73:831–844.

Kasa P, Rakonczay Z, Gulya K. 1997. The cholinergic system in Alzhei-mer’s disease. Prog Neurobiol 52:511–535.

Kimura H, McGeer PL, Peng JH, McGeer EG. 1981. The central cholinergicsystem studied by choline acetyltransferase immunohistochemistry inthe cat. J Comp Neurol 200:151–201.

Kovacs I, Torok I, Zombori J, Kasa P. 1998. Cholinergic structures andneuropathologic alterations in the olfactory bulb of Alzheimer’s diseasebrain samples. Brain Res 789:167–170.

Le Jeune H, Jourdan F. 1991. Postnatal development of cholinergic mark-ers in the rat olfactory bulb: a histochemical and immunocytochemicalstudy. J Comp Neurol 314:383–395.

Le Jeune H, Jourdan F. 1993. Cholinergic innervation of olfactory glomer-uli in the rat: an ultrastructural immunocytochemical study. J CompNeurol 336:279–292.

Le Jeune H, Jourdan F. 1994. AChE containing intrinsic neurons in the ratmain olfactory bulb: cytological and neurochemical features. EurJ Neurosci 6:1432–1444.

Le Jeune H, Aubert I, Jourdan F, Quirion R. 1995. Comparative laminardistribution of various autoradiographic markers in adult rat mainolfactory bulb. J Chem Neuroanat 9:99–112.

Le Jeune H, Aubert I, Jourdan F, Quirion R. 1996. Developmental profilesof various cholinergic markers in the rat main olfactory bulb usingquantitative autoradiography. J Comp Neurol 373:433–450.

Levey AI, Armstrong DM, Atweh SF, Terry RD, Wainer BH. 1983. Mono-clonal antibodies to choline acetyltransferase: production, specificity,and immunohistochemistry. J Neurosci 3:1–9.

Lysakowski A, Wainer BH, Bruce G, Hersch LB. 1989. An atlas of theregional and laminar distribution of choline acetyltransferase immu-noreactivity in rat cerebral cortex. Neuroscience 28:291–336.

Macrides F, Davis BJ, Youngs WM, Nadi NS, Margolis FL. 1981. Cholin-ergic and catecholaminergic afferents to the olfactory bulb in the ham-ster: a neuroanatomical, biochemical, and histochemical investigation.J Comp Neurol 203:495–514.

Matsutani S, Senba E, Tohyama M. 1989. Distribution of neuropeptidelikeimmunoreactivities in the guinea pig olfactory bulb. J Comp Neurol280:577–586.

Mesulam MM, Geula C. 1988. Nucleus basalis (Ch4) and cortical cholin-ergic innervation in the human brain: observations based on the dis-tribution of acetylcholinesterase and choline acetyltransferase. J CompNeurol 275:216–240.

Mesulam MM, Mufson EJ, Levey AI, Wainer BH. 1983a. Cholinergicinnervation of cortex by the basal forebrain: cytochemistry and corticalconnections of the septal area, diagonal band nuclei, nucleus basalis(substantia innominata), and hypothalamus in the rhesus monkey.J Comp Neurol 214:170–197.

Mesulam MM, Mufson EJ, Wainer BH, Levey AI. 1983b. Central cholin-ergic pathways in the rat: an overview based on alternative nomencla-ture (Chl-Ch6). Neuroscience 10:1185–1201.

Millar TJ, Chubb IW. 1985. [Met]-enkephalin.Arg6.Phe7 can be convertedto [Met]-enkephalin immunoreactive material by acety1cholinesterase.Neurosci Lett Suppl 19:86.

Nadi NS, Hirsch JD, Margolis FL. 1980. Laminar distribution of putativeneurotransmitter amino acids and ligand binding sites in the dogolfactory bulb. J Neurochem 34:138–146.

Nickell WT, Shipley MT. 1988. Two anatomically specific classes of candi-date cholinoceptive neurons in the rat olfactory bulb. J Neurosci8:4482–4491.

Ohm TG, Muller H, Braak E. 1991. Calbindin-D-28k-like immunoreactivestructures in the olfactory bulb and anterior olfactory nucleus of thehuman adult: distribution and cell typology — partial complementaritywith parvalbumin. Neuroscience 42:823–840.

Ojima H, Yamasaki T, Kojima H, Akashi A. 1988. Cholinergic innervationof the main and the accessory olfactory bulbs of the rat as revealed bya monoclonal antibody against choline acetyltransferase. Anat Em-bryol 178:481–488.

Phelps PE, Houser CR, Vaughn JE. 1992. Small cholinergic neurons withinfield of cholinergic axons characterize olfactory-related regions of rattelencephalon. Neuroscience 48:121–136.

Porteros A, Arevalo R, Crespo C, Brinon JG, Weruaga E, Aijon J, AlonsoJR. 1996. Nitric oxide synthase activity in the olfactory bulb of anuranand urodele amphibians. Brain Res 724:67–72.

Rosene DL, Roy NJ, Davis BJ. 1986. A cryoprotection method that facili-tates cutting frozen sections of whole monkey brains for histologicaland histochemical processing without freezing artifact. J HistochemCytochem 34:1301–1315.

Ryan R, McClure WO. 1980. Physical and kinetic properties of cholineacetyltransferase from rat and bovine brain. J Neurochem 34:395–403.

Sanides-Kohlrausch C, Wahle P. 1991. Distribution and morphology ofsubstance P-immunoreactive structures in the olfactory bulb and olfac-tory peduncle of the common marmoset (Callithrix jacchus), a primate.Neurosci Lett 131:117–120.

Shute CC, Lewis PR. 1967. The ascending cholinergic reticular system:neocortical, olfactory and subcortical projections. Brain 90:497–520.

Stephan H. 1975. Allocortex. Berlin: Springer.Stephan H, Andy OJ. 1970. The allocortex in primates. In: Noback CR,

Montagna W, editors. The primate brain. New York: Appleton-CenturyCrofts. p 109–135.

Takagi SF. 1981. Multiple olfactory pathways in mammals. A review.Chem Senses 6:329–333.

Takagi SF. 1986. Studies on the olfactory nervous system of the Old Worldmonkey. Prog Neurobiol 27:195–250.

Weruaga E, Brinon JG, Porteros A, Arevalo R, Aijon J, Alonso JR. 2001. Asexually dimorphic group of atypical glomeruli in the mouse olfactorybulb. Chem Senses 26:7–15.

Wirsig CR, Leonard CM. 1986. Acetylcholinesterase and luteinizinghormone-releasing hormone distinguish separate populations of termi-nal nerve neurons. Neuroscience 19:719–740.

Zheng LM, Jourdan F. 1988. Atypical olfactory glomeruli contain originalolfactory axon terminals: an ultrastructural horseradish peroxidasestudy in the rat. Neuroscience 26:367–378.

Zheng LM, Ravel N, Jourdan F. 1987. Topography of centrifugalacetylcholinesterase-positive fibres in the olfactory bulb of the rat:evidence for original projections in atypical glomeruli. Neuroscience23:1083–1093.



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