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Neuroscience 184 (2011) 1–15

COMPARATIVE ANALYSIS OF THE NUCLEUS BASALIS OF MEYNERT

AMONG PRIMATES

M. A. RAGHANTI,a,b* G. SIMIC,c S. WATSON,a

C. D. STIMPSON,d P. R. HOFe AND C. C. SHERWOODd

aDepartment of Anthropology, Kent State University, Kent, OH 44242,SA

bSchool of Biomedical Sciences, Kent State University, Kent, OH4242, USA

cDepartment of Neuroscience, University of Zagreb Medical School,roatian Institute for Brain Research, Zagreb, 10000, Croatia

dDepartment of Anthropology, The George Washington University,ashington, DC 20052, USA

eFishberg Department of Neuroscience and Friedman Brain Institute,ount Sinai School of Medicine, New York, NY 10029, USA

Abstract—Long projection axons from the Ch4 cell groupof the nucleus basalis of Meynert (nbM) provide cholin-ergic innervation to the neurons of the cerebral cortex.This cortical cholinergic innervation has been implicatedin behavioral and cognitive functions, including learningand memory. Recent evidence revealed differences amongprimate species in the pattern of cholinergic innervationspecific to the prefrontal cortex. While macaques dis-played denser cholinergic innervation in layers I and IIrelative to layers V and VI, in chimpanzees and humans,layers V and VI were as heavily innervated as the supra-granular layers. Furthermore, clusters of cholinergic axonswere observed within the prefrontal cortex of both humansand chimpanzees to the exclusion of macaque monkeys,and were most commonly seen in humans. The aim of thepresent study was to determine whether the Ch4 cell groupwas modified during evolution of anthropoid primates as apossible correlate of these changes in cortical cholinergicinnervation. We used stereologic methods to estimate thetotal number of choline acetyltransferase-immunoreactivemagnocellular neurons within the nbM of New World mon-keys, Old World monkeys, apes, and humans. Linear re-gression analyses were used to examine the relationshipof the Ch4 cell group with neocortical volume and brainmass. Results showed that total nbM neuron numbers hy-poscale relative to both neocortical volume and brainmass. Notably, the total number of nbM neurons in humanswere included within the 95% confidence intervals for theprediction generated from nonhuman data. In conclusion,while differences in the cholinergic system exist amongprimate species, such changes appear to involve mostlyaxon collateral terminations within the neocortex and, withthe exception of the relatively small group of cholinergiccells of the subputaminal subdivision of the nbM at theanterointermediate and rostrolateral levels, are not accom-panied by a significant extra-allometric increase in the

*Correspondence to: M. A. Raghanti, Department of Anthropology,750 Hilltop Drive, 226 Lowry Hall, Kent State University, Kent, OH44242, USA. Tel: �1-330-672-9354; fax: �1-330-672-2999.E-mail address: mraghant@kent.edu (M. A. Raghanti).Abbreviations: Ach, acetylcholine; ChAT, choline acetyltransferase;

ChAT-ir, ChAT-immunoreactive; nbM, nucleus basalis of Meynert;NSP, nucleus subputaminalis; PBS, phosphate-buffered saline.

0306-4522/11 $ - see front matter. Published by Elsevier Ltd on behalf of IBRO.doi:10.1016/j.neuroscience.2011.04.008

1

overall number of subcortical neurons that provide thatinnervation. Published by Elsevier Ltd on behalf of IBRO.

Key words: acetylcholine, choline acetyltransferase, Ch4,substantia innominata, nucleus subputaminalis.

It was Theodor Meynert who first performed histologicalanalyses of the human basal forebrain in 1872. In his work,he described grey matter situated within the paleocorticalpart of the telencephalon and named this group of nucleithat “extends from the level of olfactory tubercle to the levelof lateral geniculate body” as the nucleus ansae lenticu-laris (Meynert, 1872). The well-known eponym “nucleusbasalis of Meynert” (nbM) was given by Kölliker (1896),although Meynert’s work does not exactly show this cellgroup (as later shown by Mettler, 1968). Along with themore detailed analysis of magnocellular groups of nucleibetween the anterior commissure and optic tract, Kölliker(1896) introduced the term “basal telencephalon” and alsosuggested cytological criteria to differentiate these neu-rons from others within this region. These criteria requirethat neurons be clearly larger than others by four to sixtimes when measuring their longer axis; neurons be hy-perchromatic (i.e. a more pronounced “reaction” uponNissl stain than for other neurons); the staining be morepronounced at the periphery of the perikaryon; and thatnuclei of these cells be pale and the nucleolus easily seen.By using these criteria, one group of such neurons situatedmostly below the lateral part of the putamen was namedand described in the human and chimpanzee brains as thenucleus subputaminalis (NSP) by Giuseppe Ayala (Ayala,1915, 1924; Simic et al., 1999) (see Fig. 3L, M at all levels),whereas Foix and Nicolesco (1925) were the first to de-scribe these magnocellular neurons within the internal andexternal medullary laminae and the internal capsule (seeFig. 3M at the intermediate level). Finally, Brockhaus real-ized that the magnocellular neurons in Meynert’s nucleusare only one component of the whole complex of the basalforebrain magnocellular cell groups. Therefore, he includednuclei of the diagonal band of Broca and the olfactory tuber-cle into the complex of the basal telencephalon nuclei—the“Basalkernkomplex” (Brockhaus, 1942). By describing notonly magno- but also microcellular nuclei, Andy andStephan provided the most precise description of the basalforebrain nuclei as delineated by classic Nissl-staining(Andy and Stephan, 1968).

The development of new histochemical and immuno-cytochemical methods that allowed detection of cholinergicneurons in the late 1970s and early 1980s provided evi-

dence that the nbM is a major source of long projection

O

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*

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–152

neurons that provide cholinergic innervation to the neuronsof the cerebral cortex and the amygdala (Hong and Jang,2010; Johnston et al., 1979; Mesulam et al., 1983, 1986;Mesulam and Geula, 1988b; Mesulam and Van Hoesen,1976; Pearson et al., 1983). Specifically, it was demon-strated that more than 90% of the neurons in the nbM arecholinergic in that their perikarya and axons contain cho-line acetyltransferase (ChAT) (German et al., 1985; Mesu-lam and Geula, 1988a; Mesulam et al., 1983; Nagai et al.,1983; Pearson et al., 1983; Saper and Chelimsky, 1984).Based on the topographical distribution of ChAT-immuno-reactive (ChAT-ir) cell bodies in the rhesus macaque brain,a new nomenclature proposed by Mesulam and col-leagues (1983) was established. This has become themost widely accepted terminology for the primate magno-cellular basal forebrain. Although the human nbM is rela-tively larger and more complex, the same terminology hasbeen adopted for the human brain. According to this no-menclature, the main part of the human magnocellularforebrain, nbM, is designated as the Ch4 cell group and isfurther divided into six sectors: the anterior part (Ch4a) isdivided by vasculature into the anteromedial (Ch4am) andthe anterolateral (Ch4al) sectors; the anterointermediatedivision (Ch4ai) that spans the anterior and intermediateparts (and is not well-developed in nonhuman primates);the intermediate part (Ch4i) is divided by the ansa pedun-cularis into the intermediodorsal (Ch4id) and the interme-dioventral (Ch4iv) sectors; the posterior division occupiesa sector designated as Ch4p. Comparing the terminologyof Mesulam and collaborators (1983, based on ChAT im-munostaining) with the classification provided by Brock-

Table 1. Specimens used in this study

Group Species Commo

Humans Homo sapiens HumanHomo sapiens Human

Apes Pan troglodytes ChimpaPan troglodytes ChimpaSymphalangus syndactylus Siamang

ld World monkeys Macaca nemestrina PigtailedMacaca nemestrina PigtailedMacaca nemestrina PigtailedMacaca nemestrina PigtailedCercopithecus kandti GoldenCercocebus agilis Golden

ew World Monkeys Saguinus oedipus CottontoSaguinus oedipus CottontoAotus trivirgatus Owl moAotus vociferans Owl moAotus vociferans Owl moAlouatta caraya HowlerAlouatta caraya HowlerSaimiri boliviensis SquirrelSaimiri boliviensis SquirrelPithecia pithecia White-faCebus apella Brown cCebus apella Brown cAteles fusciceps Spider mAteles belzebuth Spider m

Duration of time in fixation is given in days.

haus (1942, based on Nissl staining), the following conclu-sions can be derived: the Ch4am group mainly correspondsto the pars diffusa, and the Ch4i to the pars compacta ofBrockhaus. The posterior group (Ch4p) of Mesulam and col-leagues corresponds to the cell clusters that were designatedby Kostovic (1986) as the pars aggregata.

The nbM has been implicated in numerous behavioraland cognitive functions, including attention, learning andmemory, and cortical plasticity (Cabrera et al., 2006; Con-ner et al., 2010; Gold, 2003; Hasselmo, 1999; Ramana-than et al., 2009; Sarter and Bruno, 2000; Sarter andParikh, 2005). Specifically, neurons within the Ch4 cellgroup respond to novel stimuli (Santos-Benitez et al.,1995; Wilson and Rolls, 1990), triggering the release ofacetylcholine (ACh) within the cerebral cortex. ACh pro-motes long-term potentiation and synaptic plasticity via aninhibition of potassium conductance that enhances re-sponsiveness to further excitatory inputs. These effectsare mediated mainly by the muscarinic M1 receptor sub-types that are preferentially expressed within the cerebralcortex (von Bohlen und Halbach and Dermietzel, 2006).

The Ch4 cell group is a target for human-specific neu-ropathological processes and several reports indicate thatit is affected early and progressively with cognitive impair-ment (e.g. Grothe et al., 2010; Iraizoz et al., 1999; Mesu-lam et al., 2004; Mufson et al., 1989b; Zaborszky et al.,2009), although other reports indicate that the cholinergicsystem is not affected until cognitive impairment is estab-lished (Davis et al., 1999; DeKosky et al., 2002; Gilmor etal., 1999). A decrease in nbM total neuron number hasbeen associated with a number of neurodegenerative dis-

Sex Age Fixation* Total neurons in nbM

M 24 5 231,214F 56 5 201,901M 25 �60 161,978F 45 �60 153,407M 33 14 151,987

e F 9 7 96,051e F 6 7 97,854e F 15 7 121,067e M 3 7 91,730

M Adult �60 104,808y F 19 7 81,285

n M 11 18 18,955n F 10 45 20,563

M �18 �60 28,308M 18 �60 41,536F 5 7 29,614M 21 �60 24,378M 3 15 20,712F 9 35 40,262F 9 8 95,074F 1.5 �60 60,221M 16 7 108,760F 12 7 141,514F 16 45 117,868F 36 14 134,835

n name

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macaqumacaqumacaqumacaqu

guenonmangabep tamarip tamari

nkeynkeynkeymonkeymonkeymonkeymonkeyced sakiapuchinapuchinonkeyonkey

pCtiCtaaa

WsWWd11psapeG1taaeh2cIahl(cccape

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–15 3

eases, such as Alzheimer’s disease and Parkinson’s dis-ease (Arendt et al., 1983; Cullen and Halliday, 1998; Kleinet al., 2010; Lehéricy et al., 1993; Mufson et al., 1991;Teipel et al., 2005). Furthermore, progressive decline incholinergic neuron numbers correlates with the worseningof dementia in Alzheimer’s disease (Iraizoz et al., 1999)with total numbers of neurons decreasing with duration ofdisease (Mufson et al., 1989b).

Gorry (1963) compared the nbM among a variety ofhylogenetic groups, reporting an absence of a definableh4 nucleus in the taxonomic orders Marsupialia, Insec-

ivora, and Chiroptera. Among the other taxonomic ordersnvestigated (including Primates, Lagomorpha, Rodentia,etacea, Carnivora, Sirenia, Perissodactyla, and Artiodac-

yla), Gorry reported phylogenetic variation in the extentnd differentiation of the nbM that correlated with the over-ll size of the cerebral cortex, with the greatest differenti-tion of the nbM in Primates and Cetacea.

Among primates, the nbM has been described in Oldorld monkeys (e.g. Ghashghaei and Barbas, 2001; Me-

ulam et al., 1983, 1986; Smiley and Mesulam, 1999), Neworld monkeys (Everitt et al., 1988; Kordower et al., 1989;u et al., 2000), apes (Gorry, 1963), and humans (Halli-

ay et al., 1993; Kostovic, 1986; e.g. Mesulam and Geula,988a; Mesulam, 2004; Perry et al., 1984; Selden et al.,998). The anatomical descriptions of the nbM amongrimates are in general agreement with one another (Me-ulam and Geula, 1988a; Mufson et al., 1989a; Selden etl., 1998), although the distribution of cholinergic cortico-etal cells in the human nbM has been described as morextensive relative to that of macaques (Mesulam andeula, 1988a; Mesulam et al., 1983, 1986; Mufson et al.,989a; Simic et al., 1999). Species-specific differences inhe neurochemical phenotype of the Ch4 neurons havelso been observed (Melander and Staines, 1986; Wu etl., 2000). In addition, differences in the pattern of cholin-rgic innervation within the prefrontal cortex occur amonguman and nonhuman primate species (Raghanti et al.,008a). In areas 9 and 32 of the prefrontal cortex, ma-aques have denser cholinergic innervation in layers I andI relative to layers V and VI. In contrast, layers V and VIre as heavily innervated as the supragranular layers inumans and chimpanzees. Furthermore, clusters of cho-

inergic axons that may be involved in cortical plasticityMesulam et al., 1992) were observed within the prefrontalortex of both humans and chimpanzees but not in ma-aque monkeys, and this morphology appears to be moreommon in humans. The goal of the present study was tossess the evolution of the Ch4 cell group among anthro-oid primates in the context of the role of cortical cholin-rgic innervation in cognition.

EXPERIMENTAL PROCEDURES

Specimens

This study included brain specimens from 23 individuals repre-senting 12 anthropoid species. Postmortem nonhuman brainswere obtained from zoological or research institutions where an-imals were housed according to each institution’s guidelines. All

institutions were either American Zoo and Aquairium (AZA) or the

Association for Assessment and Accreditation of Laboratory Ani-mal Care (AAALAC)-accredited. The animals died of naturalcauses or were humanely euthanized for reasons independent ofthis research. The golden guenon brain was provided by the OfficeRwandais du Tourisme et des Parcs Nationaux and the MountainGorilla Veterinary Project in compliance with CITES regulations.Brain specimens from adult, nongeriatric humans were providedby the Cuyahoga County Coroner’s office. All human brains weredevoid of gross and microscopic neuropathologic lesions. Table 1provides details on the individuals included in this analysis.

All samples for this study were derived from the left hemi-sphere. In many cases, the right hemisphere was not available foranalysis and previous studies reported no differences in neurondensity or volume of the nbM between hemispheres or sexes inhumans (Halliday et al., 1993; Zaborszky et al., 2008). The samplewas limited to specimens that were sectioned continuouslythroughout the nbM and for which the entirety of the nbM wasavailable.

Tissue processing

All brains were collected postmortem and were immersion-fixed in10% buffered formalin. Brain mass was recorded for each speci-men prior to histological processing. Once fixed, the brains weretransferred to a 0.1 M phosphate-buffered saline (PBS, pH 7.4)solution containing 0.1% sodium azide and stored at 4 °C toprevent bacterial growth and further tissue shrinkage and antigenblockade. Prior to sectioning, samples were cryoprotected byimmersion in a series of sucrose solutions (10%, 20%, and 30%).Samples were frozen on dry ice and cut to 40 �m-thick sectionsusing a Leica SM2000R sliding microtome (Leica, Bannockburn,IL, USA). Individual sections were placed into eppendorf tubescontaining freezer storage solution (30% each distilled water,ethylene glycol, and glycerol and 10% 0.244 M PBS) and num-bered sequentially. Sections were stored at �20 °C until immu-nohistochemical or histological processing.

Fig. 1. Primate phylogeny used for independent contrasts analyses.

1ntm

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–154

A one-in-10 series for all samples was stained for Nissl sub-stance with a 0.5% solution of Cresyl Violet to reveal cell somata.Nissl-stained sections were used to identify the cytoarchitecturalboundaries of the magnocellular neurons within the basal fore-brain. Once delineated, a series of equidistant sections spanningthe nbM was selected for immunohistochemical processing. Eachseries included sections that were rostral and caudal to the nbM toensure full representation of the Ch4 cell group.

The Ch4 cell group has been described in humans (Hallidayet al., 1993; Mesulam and Geula, 1988a; Perry et al., 1984; Saperand Chelimsky, 1984; Zaborszky et al., 2008), macaque monkeys(Mesulam et al., 1983, 1986; Smiley and Mesulam, 1999), capu-chins (Kordower et al., 1989), owl monkeys (Melander andStaines, 1986), and marmosets (Everitt et al., 1988; Wu et al.,2000). These descriptions were used to inform the boundaries ofthe nbM within the species studied here.

Immunohistochemistry

Free-floating tissue sections were immunostained for cholineacetyltransferase (ChAT) using the avidin-biotin-peroxidasemethod, as described previously (Raghanti et al., 2008a). Briefly,sections were pretreated for antigen retrieval by incubating in a 10mM citrate buffer (pH 8.5) at 86 °C for 30 min. Sections wererinsed and endogenous peroxidases were quenched using a so-lution of 75% methanol, 2.5% hydrogen peroxide (30%), and22.5% distilled water for 20 min at room temperature. Sectionswere preblocked in a solution of PBS with 4% normal serum, 0.6%

Fig. 2. The extent and distribution of the NSP in human (A–C) and c

levels, respectively. Black arrows point to neurons that constitute the nucleus

Triton X-100, and 5% bovine serum albumin. Sections were thenincubated in primary antibody (goat anti-ChAT polyclonal anti-body, AB144, Millipore, Billerica, MA, USA) at a dilution of 1:500in PBS for 48 h at 4 °C. Following this, sections were incubated ina biotinylated secondary antibody (1:200) in a solution of PBS and2% normal serum. The sections were then incubated in an avidin-peroxidase complex (PK-6100, Vector Laboratories, Burlingame,CA, USA) followed by a 3,3’-diaminobenzidine-peroxidase sub-strate with nickel enhancement (SK-4100, Vector Laboratories).Omission of the primary or secondary antibodies resulted in acomplete absence of staining.

Data collection and analysis

Quantitative data were collected using an Olympus BX-51 photo-microscope equipped with a Ludl XY motorized stage, Heidenhainz-axis encoder, StereoInvestigator software (MBF Bioscience,Williston, VT, USA, version 8), and a digital camera that projectsimages onto a 24-inch LCD flat panel monitor. The Ch4 cell groupwas outlined in each section at low magnification (4�). The Ch2group was readily distinguishable from Ch4 by the more verticalorientation of neurons. The outline included the entirety of the Ch4group, including interstitial elements and the outline was reducedto a line between clusters of cells. The optical fractionator (West,999) was used to determine total neuron number at high mag-ification (60�). A guard zone of at least 2 �m was employed at

he top and bottom of the sections and section thickness waseasured at every fifth sampling site. Stereology parameters

ee (D–F). The panels represent anterior, intermediate, and posterior

himpanz subputaminalis. Scale bars�500 �m.

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–15 5

were variable depending on brain size. Counting frames were setat 100�100 �m2 with an optical disector height of 10 �m. Thesampling grid area ranged from 40,000 to 360,000 �m2. Therewas an average of 253�155.21 sampling sites per individual(range 81–608) and an average of 229�144.41 neurons countedper individual (range 46–629).

All subsequent analyses used species mean values, withthe exception of the genera Aotus and Ateles in which individ-uals from different species were grouped because of limitedsample sizes. To examine the scaling of nbM neurons within

Fig. 3. Distribution of ChAT-ir corticopetal cells within the anterior (leftCh4 cell group in tamarin (A), owl monkey (B), howler monkey (C),macaque (H), golden guenon (I), golden mangabey (J), siamang (K),corpus callosum; C, caudate; CP, cerebral peduncle; ic, internal caps

pallidus; oc, optic chiasm; ot, optic tract; P, putamen; Th, thalamus. Arrows poin

the brain, the logarithm (base 10) of total neuron number wasregressed on the logarithm of brain mass. The brain mass fromeach specimen used in this study was measured prior to his-tological sectioning. We also analyzed the relationship betweentotal neuron number and neocortical volume (data taken fromStephan et al., 1981) to explore the scaling relationship be-tween the Ch4 cell group and, specifically, the major region thatit innervates. Because not all the same species are representedin the Stephan et al. (1981) dataset and the current study, weused neocortical volumes from the closest relatives that were

ntermediate (middle panel), and posterior (right panel) divisions of themonkey (D), white-faced saki (E), capuchin (F), spider monkey (G),zee (L), and human (M). Abbreviations: ac, anterior commissure; cc,globus pallidus; EGP, external globus pallidus; IGP, internal globus

panel), isquirrelchimpanule; GP,

t to neurons of the nucleus subputaminalis in chimpanzee and human.

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–156

available. Although this is not ideal, it is notable that our scalinganalyses against brain mass were similar to those obtained forneocortical volume. Because both brain mass and neocorticalmass have been shown to scale linearly with the total numberof neurons that they contain in primates (Gabi et al., 2010), thescaling exponents derived from analyses of nbM neuron num-ber against these variables are expected to reflect proportionalchanges in the population of neurons in each structure relativeto one another.

Fig. 3. (Contin

Regression coefficients and associated standard errors werealso generated using phylogenetic independent contrasts (Gar-land et al., 1992). We used Mesquite version 2.74 (mesquitepro-ject.org) to perform these analyses, based on a Bayesian estimateof phylogeny and associated branch lengths downloaded from the10k Trees website (http://10ktrees.fas.harvard.edu/; see phylog-eny in Fig. 1). A least-squares linear prediction of human valuesbased on nonhuman data was performed to determine if humannbM neuron number deviates significantly from allometric expec-

ued).

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–15 7

tations for neocortex volume and brain mass. Predictions basedon independent contrasts were calculated according to themethod described in Garland and Ives (2000). An � level of 0.05was set for all statistical tests.

RESULTS

The nbM was identifiable in all species examined, with theanterior, intermediate, and posterior divisions readily ap-parent. The magnocellular neurons within the Ch4 cellgroup were large and multipolar, with variable orientationand dendritic arborization patterns throughout the extent ofthe nucleus in all species analyzed. The most rostral por-

Fig. 3. (Contin

tion of the nbM was ventral and anterior to the anteriorcommissure. The nucleus extended caudally until it grad-ually disappeared lateral to the optic tract, approximately atthe level of the lateral geniculate nucleus. Overall, thedistribution of ChAT-ir magnocellular neurons within thenbM was relatively similar among species. A notable ex-ception, however, was found in the New World howlermonkey (Alouatta). While the nbM occupied similar ana-tomical levels as in the other primates, the neurons weremarkedly limited in dispersion and in numbers (see Fig.3c). Somewhat comparably, the tamarin and owl monkey(both small, New World primates) displayed relatively lim-

ued).

at

eesc

actptibpps

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–158

ited dispersion of the anterior division of the Ch4 cellgroup, but not to the extent observed in Alouatta. Interest-ingly, Alouatta is the only folivorous species in this samplend it possesses one of the lowest encephalization quo-

ients among primates (Jerison, 1973).The most notable qualitative difference in the cholin-

rgic groups of cells in the basal forebrain was the pres-nce of the rostrolateral and anterointermediate part of theubputaminal nucleus of Ayala (NSP) in the human andhimpanzee brains (Fig. 2). This group of neurons was

Fig. 3. (Contin

bsent in all other species and although present in thehimpanzee, it contained fewer neurons that were rela-ively dispersed compared to the dense clusters that com-rise the human NSP, a difference that was pronounced athe anterior level (see Fig. 2A, D). At the anterior, antero-ntermediate, and intermediate levels, the NSP is definedy the presence of very large (cholinergic) cells below theutamen (i.e. between the ventral caudate and the anteriorerforated substance) and lateral to the anterior commis-ure. At the posterior part, the NSP is again situated below

ued).

tsR

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tpRs

p

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–15 9

the putamen, but medial to the anterior commissure. Con-sidering the absence of a distinct NSP in the siamang, alesser ape, it will be interesting to ascertain the status ofthe NSP in other hominid species, such as gorillas andorangutans.

As expected for a sample of species spanning a 131-fold range in total brain size (minimum—Saguinus, maxi-mum—Homo), phylogenetic variation was evident in thetotal number of cholinergic neurons within the Ch4 cellgroup (see Table 1). The total number of Ch4 neuronsvaried by 11-fold, with humans having the largest numberof neurons. The total neuron estimates generated for thenbM in humans were consistent with the value of 200,000previously reported by Arendt et al. (1985). The distributionof ChAT-ir cells in the anterior, intermediate, and posteriordivisions of the Ch4 cell group is illustrated in Fig. 3A–M.For Fig. 3, photo montages of the basal forebrain wereobtained at 4� and markers were placed over ChAT-irneurons using Adobe Photoshop. Photomicrographs ofChAT staining for all species are presented in Fig. 4.Additional images of the tamarin, howler monkey, andsiamang are provided in Fig. 5.

There was a hypometric scaling relationship betweenotal nbM neuron numbers and brain mass (contemporarypecies data: F�20.9, P�0.001, RMA slope�0.593,2�0.655; independent contrasts: F�9.368, P�0.011,

RMA slope�0.674, R2�0.460), indicating that the numberf neurons in the nbM increase at a slower rate than

ncreases in brain mass across species (Fig. 6). The 95%rediction interval for total neuron number within the nbMenerated from nonhuman data included the human mean,owever, the observed mean total neuron number in hu-ans was lower than expected (contemporary speciesata–log observed human point: 5.34, log predicted point:.66, 95% prediction interval: 4.95�6.03; observed totaleuron number: 216,558, predicted total neuron number:54,106; independent contrasts–log predicted point: 5.46,5% prediction interval: 4.61�6.31; predicted total neuron

Fig. 3

umber: 289,953).

A hypometric scaling relationship was also observed be-ween total neuron numbers and neocortex volume (contem-orary species data: F�21.2, P�0.001, RMA slope�0.557,2�0.658; independent contrasts: F�9.158, P�0.012, RMAlope�0.654, R2�0.454; Fig. 7). The 95% prediction interval

generated using the nonhuman data included the humanmean, but the observed human value was lower than thepoint estimate derived from the nonhuman least-squares re-gression (contemporary species data–log observed humanpoint: 5.34; log predicted point: 5.64, 95% prediction interval:4.95�6.03; observed total neuron number: 216,558, pre-dicted total neuron number: 441,124; independent contrasts–log predicted point: 5.49, 95% prediction interval: 4.62�6.36;redicted total neuron number: 309,430).

DISCUSSION

Comparative neuroanatomical studies of the mammalianbasal forebrain have demonstrated phylogenetic variationin the size and cytoarchitectonic complexity of the magno-cellular basal complex (Divac, 1975; Gorry, 1963). Forexample, humans and apes (gibbon, chimpanzees, andgorillas) possess a different localization of galanin-ir withinthe basal forebrain relative to monkeys (brown capuchinsand rhesus macaques). Specifically, monkeys display co-localization of galanin in basal forebrain magnocellularneurons whereas apes and humans do not (Benzing et al.,1993). Rather than colocalization, apes and humans havea dense plexus of galanin-ir fibers that appeared to inner-vate the magnocellular neurons. In addition, carnivoreshave a less extensive nbM with more elaboration of itsmedial part (Kimura et al., 1981; St-Jacques et al., 1996;Steriade et al., 1987), while rodents have only medial,subpallidal, and peripallidal equivalents of the nbM (Arm-strong et al., 1983; Bigl et al., 1982; Cuello and Sofroniew,1984; Fibiger, 1982; Johnston et al., 1979; Rye et al.,1984). By comparison with primates, it could be concludedthat the lineage-specific specializations of the magnocel-

ued).

. (Contin

lular chain of nuclei within the basal forebrain has been

b

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–1510

“directed” mostly towards the expansion of its rostrolateralpart. The prominence of the nbM in the primate brain maybe therefore explained by the remarkable expansion of thecerebral cortex that represents its main innervation target(Bigl et al., 1982; Divac, 1975; Everitt et al., 1988; Gorry,1963; Johnston et al., 1979; Jones et al., 1976; Kievet andKuypers, 1975; Mesulam and Geula, 1988a; Mesulam etal., 1983; Mesulam and Van Hoesen, 1976; Pearson et al.,1983; Saper, 1987, 1990; Struble et al., 1986). Our earlierresearch identified differences in the patterning of corticalcholinergic innervation among humans, chimpanzees, andmacaque monkeys (Raghanti et al., 2008a). Due to therole that cortical cholinergic innervation plays in learning,

Fig. 4. ChAT-ir neurons and axons within the nbM of each species inthe level of the posterior anterior commissure (corresponding to thecommissure is visible in the upper left corner. Pictured are (A) tamarin,(F) capuchin, (G) spider monkey, (H) golden guenon, (I) golden mangabars�250 �m for panels (A–M). High magnification photomicrographar�50 �m.

memory, and cognitive flexibility, the current study tested s

the hypothesis that the evolution of cognitive functions inthe primate lineage might have entailed a significant alter-ation of this neurochemical system.

When the basal forebrain cholinergic system is com-pared among a larger range of mammals, the expansion ofits rostrolateral subdivision is remarkable in humans andsome hominoids, considering that these species are theonly ones noted to have all rostrolateral groups of the nbMcells. Moreover, at the most rostral and anterointermediatelevels of the nbM complex, only humans and chimpanzeespossess a subputaminal subdivision, with the human sub-putaminal division being more cell dense and extensive(see Fig. 2) (Ayala, 1915; Simic et al., 1999). The larger

this study. Images were taken ventral to the anterior commissure, atdiate division of the Ch4 cell group). In panels (A–L), the anterioronkey, (C) squirrel monkey, (D) white-faced saki, (E) howler monkey,acaque monkey, (K) siamang, (L) chimpanzee, and (M) human. ScalehAT-ir neurons and axons in chimpanzee (N) and human (O), scale

cluded ininterme

(B) owl mbey, (J) ms show C

ize of the subputaminal subdivision of the nbM within the

t1m(ts1miaa

tl(cnmF

wrrwaStptd2

saeHebdwm

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–15 11

left hemisphere of the human brain (Halliday et al., 1993;Simic et al., 1999), the ascension of subputaminal cholin-ergic fibres through the external capsule (Kostovic, 1986)owards the perisylvian language areas (Simic et al.,999), and its most protracted development among all theagnocellular aggregations within the basal forebrain

Kracun and Rosner, 1986), suggest that, although rela-ively small in size, this part of the nbM may have human-pecific contributions to cognitive functions (Simic et al.,999). Therefore, the pathological changes of these hu-an-specific parts of basal forebrain may be particularly

mportant in the context of human-specific diseases, suchs schizophrenia (Heimer, 2000), Alzheimer’s disease,nd primary progressive aphasia (Boban et al., 2006).

Interestingly, the quantitative allometric scaling pat-erns reported here are similar to what was found for theocus coeruleus in humans and nonhuman primatesSharma et al., 2010). Long projection neurons of the locusoeruleus provide the noradrenergic innervation to theeocortex and have also been implicated in learning andemory processes (Gaspar et al., 1989; Morrison and

Fig. 4

oote, 1986). Loss of these neurons is also associated s

ith neurodegenerative conditions. Sharma et al. (2010)eported that the number of tyrosine hydroxylase-immuno-eactive neurons within the human locus coeruleus fallsithin predicted ranges based on nonhuman primate data,nd that the human total is lower than the predicted value.urprisingly, while this subcortical neuron population re-

ains a scaling relationship with the areas it innervates, theattern and density of tyrosine hydroxylase-immunoreac-ive axon collaterals within the cerebral cortex itself alsoisplays significant variation among species (Hof et al.,000; Raghanti et al., 2008b).

We predicted that the human nbM would possess aignificantly higher total number of neurons to supportlterations in the patterning of prefrontal cortical cholin-rgic innervation and associated cognitive specializations.owever, humans actually had fewer nbM neurons thanxpected based on nonhuman data predictions for bothrain mass and neocortical volume. In conclusion, whileifferences in prefrontal cortical cholinergic innervationere reported among humans and other catarrhine pri-ates (Raghanti et al., 2008a), these changes were not

ued).

. (Contin

upported by significant alterations in the subcortical cell

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–1512

population that supplies that innervation. Primates pos-sess a disproportionately large neocortex relative to otherspecies and it is possible that there are significant devel-opmental constraints on basal forebrain systems relative tothe neocortex. However, as evidenced by phylogenetic

Fig. 5. Low power (4�) image montages in tamarin (A, B)

Fig. 6. Total neuron number within the nbM regressed on brain mass.Data points for New World monkeys are dark grey; Old World monkey

data points are white; lesser and great ape data points are light grey.

diversity of neurotransmitter-immunoreactive axon densi-ties and distributions, there is demonstrable plasticity in thepatterning of axon collaterals within the cortical mantle,which is independent of variation in the number of neurons

monkey (C, D), and siamang (E, F). Scale bars�500 �m.

Fig. 7. Total neuron number within the nbM regressed on neocorticalvolume. Data points for New World monkeys are dark grey; Old Worldmonkey data points are white; lesser and great ape data points are

, howler

light grey.

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–15 13

that give rise to these projections. Thus, evolutionary mod-ifications in the anatomy and function of such diffuse neu-romodulatory systems in the brain may involve decouplingof terminal axon patterns and the distribution of neurons inthe basal forebrain nuclei themselves.

Acknowledgments—This work was supported by the National Sci-ence Foundation (BCS-0921079, BCS-0827531, BCS-0639180)and the James S. McDonnell Foundation (22002078). G.S. wassupported by the Croatian Ministry of Science, Education, andSports grant no. 108-1081870-1942. Brain materials used in thisstudy were loaned by the Great Ape Aging Project (NIH grantAG14308, “A Comparative Neurobiology of Aging Resource,” Dr.Joseph Erwin, PI), the Cleveland Metroparks Zoo, the Michale E.Keeling Center for Comparative Medicine and Research at theUniversity of Texas M.D. Anderson Cancer Center, the NationalPrimate Research Center at the University of Washington (NIHRR00166), the Comparative Pathology Department at the NewEngland Regional Primate Research Center (Division of ResearchResources, grant RR00168), the Foundaton for Comparative andConservation Biology, Office Rwandais du Tourisme et des ParcsNationaux (Dr. Antoine Mudakikwa), and the Cuyahoga CountyCoroner’s Office (Dr. Andrea McCollum).

REFERENCES

Andy OJ, Stephan H (1968) The septum in the human brain. J CompNeurol 133:383–410.

Arendt T, Bigl V, Arendt A, Tennstedt A (1983) Loss of neurons in thenucleus basalis of Meynert in Alzheimer’s disease, paralysis agi-tans and Korsakoff’s disease. Acta Neuropathol 61:101–108.

Arendt T, Bigl V, Tennstedt A, Arendt A (1985) Neuronal loss indifferent parts of the nucleus basalis is related to neuritic plaqueformation in cortical target areas in Alzheimer’s disease. Neurosci-ence 14:1–14.

Armstrong DM, Saper CB, Levey AI, Wainer BH, Terry RD (1983)Distribution of cholinergic neurons in rat brain demonstrated by theimmunocytochemical localization of choline acetyltransferase.J Comp Neurol 216:53–68.

Ayala G (1915) A hitherto undifferentiated nucleus in the forebrain(nucleus subputaminalis). Brain 37:433–438.

Ayala G (1924) Weitere untersuchungen über den Nucleus subputami-nalis. J Psychol Neurol 30:285–299.

Benzing WC, Kordower JH, Mufson EJ (1993) Galanin immunoreac-tivity within the primate basal forebrain: evolutionary change be-tween monkeys and apes. J Comp Neurol 336:31–39.

Bigl V, Woolf NJ, Butcher LL (1982) Cholinergic projections from thebasal forebrain to frontal, parietal, temporal, occipital and cingulatecortices: a combined fluorescent tracer and acetylcholinesteraseanalysis. Brain Res Bull 8:727–739.

Boban M, Kostovic I, Simic G (2006) Nucleus subputaminalis: ne-glected part of the basal nucleus of Meynert. Brain 129:E42.

Brockhaus H (1942) Vergleichend-anatomische untersuchungen überden Basalkernkomplex. J Psychol Neurol 51:57–95.

Cabrera S, Chavez C, Corley S, Kitto M, Butt A (2006) Selectivelesions of the nucleus baslais magnocellularis impair cognitiveflexibility. Behav Neurosci 120:298–306.

Conner JM, Kulczycki M, Tuszynski MH (2010) Unique contributions ofdistinct cholinergic projections to motor cortical plasticity and learn-ing. Cereb Cortex 20:2739 doi:10.1093/cercor/bhq022.

Cuello AC, Sofroniew MV (1984) The anatomy of the CNS cholinergicneurons. Trends Neurosci 7:74–78.

Cullen K, Halliday G (1998) Neurofibrillary degeneration and cell lossin the nucleus basalis in comparison to cortical Alzheimer pathol-

ogy. Neurobiol Aging 19:297–306.

Davis KL, Mohs RC, Marin D, Purohit DP, Perl DP, Lantz M, Austin G,Haroutunian V (1999) Cholinergic markers in elderly patients withearly signs of Alzheimer’s disease. JAMA 281:1401–1406.

DeKosky ST, Ikonomovic MD, Styren SD, Beckett L, Wisniewski S,Bennett DA, Cochran EJ, Kordower JH, Mufson EJ (2002) Upregu-lation of choline acetyltransferase activity in hippocampus andfrontal cortex of elderly subjects with mild cognitive impairment.Ann Neurol 51:145–155.

Divac I (1975) Magnocellular nuclei of the basal forebrain project toneocortex, brainstem and olfactory bulb. Review of some func-tional correlates. Brain Res 93:385–398.

Everitt B, Sirkia T, Roberts A, Jones G, Robbins T (1988) Distributionand some projections of cholinergic neurons in the brain of thecommon marmoset, Callithrix jacchus. J Comp Neurol 271:533–558.

Fibiger HC (1982) The organization and some projections of cholin-ergic neurons in the mammalian forebrain. Brain Res Rev 4:327–388.

Foix C, Nicolesco J (1925) Les noyaux gris centraux et la régionmésencéphalique optique, suivi d’un appendice sur l’anatomiepathologique de la maladie de parkinson. Anatomie cérébrale.Paris: Masson & Cie.

Gabi M, Collins CE, Wong P, Torres LB, Kaas JH, Herculano-HouzelS (2010) Cellular scaling rules for the brains of an exxtendednumber of primate species. Brain Behav Evol 76:32–44.

Garland T, Harvey PH, Ives AR (1992) Procedures for the analysis ofcomparative data using phylogenetically independent contrasts.Syst Biol 41:18–32.

Garland T, Ives AR (2000) Using the past to predict the present:confidence intervals for regression equations in phylogenetic com-parative methods. Am Nat 155:346–364.

Gaspar P, Berger B, Febvret A, Vigny A, Henry JP (1989) Catechol-amine innervation of the human cerebral cortex as revealed bycomparative immunohistochemistry of tyrosine hydroxylase anddopamine-beta-hydroxylase. J Comp Neurol 279:249–271.

German DC, Bruce G, Hersh L (1985) Immunohistochemical stainingof cholinergic neurons in the human brain using a polyclonal anti-body to human choline acetyltranferase. Neurosci Lett 61:1–5.

Ghashghaei HT, Barbas H (2001) Neural interaction between thebasal forebrain and functionally distinct prefrontal cortices in therhesus monkey. Neuroscience 3:593–614.

Gilmor ML, Erickson JD, Varoqui H, Hersh LB, Bennett DA, CochranEJ, Mufson EJ, Levey AI (1999) Preservation of nucleus basalisneurons containing choline actyltransferase and the vesicular ace-tylcholine transporter in elderly with mild cognitive impairment andearly Alzheimer’s disease. J Comp Neurol 411:693–704.

Gold PE (2003) Acetylcholine modulation of neural systems involvedin learning and memory. Neurobiol Learn Mem 80:194–210.

Gorry JD (1963) Studies on the comparative anatomy of the ganglionbasale of Meynert. Acta Anat 55:51–104.

Grothe M, Zaborszky L, Atienza M, Gil-Neciga E, Rodriguez-RomeroR, Teipel SJ, Amunts K, Suarez-Gonzalez A, Cantero JL (2010)Reduction of basal forebrain cholinergic system parallels cognitiveimpairment in patients at high risk of developing Alzheimer’s dis-ease. Cereb Cortex 7:1685–1695.

Halliday G, Cullen K, Cairns M (1993) Quantitation and three-dimen-sional reconstruction of Ch4 nucleus in the human basal forebrain.Synapse 15:1–16.

Hasselmo ME (1999) Neuromodulation: acetylcholine and memoryconsolidation. Trends Cogn Sci 3:351–359.

Heimer L (2000) Basal forebrain in the context of schizophrenia. BrainRes Rev 31:205–235.

Hof PR, Glezer II, Nimchinsky EA, Erwin JM (2000) Neurochemicaland cellular specializations in the mammalian neocortex reflectphylogenetic relationships: evidence from primates, cetaceans,

and artiodactyls. Brain Behav Evol 55(6):300–310.

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–1514

Hong JH, Jang SH (2010) Neural pathway from the nucleus basalis ofMeynert passing through the cingulum in the human brain. BrainRes 1346:190–194.

Iraizoz I, Guijarro JL, Gonzalo LM, de Lacalle S (1999) Neuropatho-logical changes in the nucleus basalis correlate with clinical mea-sures of dementia. Acta Neuropathol 98:186–196.

Jerison HJ (1973) Evolution of the brain and intelligence. New York:Academic Press.

Johnston MV, McKinney M, Coyle JT (1979) Evidence for a cholinergicprojection to neocortex from neurons in basal forebrain. Proc NatlAcad Sci U S A 76:5392–5396.

Jones EG, Burton H, Saper CB, Swanson LW (1976) Midbrain, dien-cephalic and cortical relationship of the basal nucleus of Meynertand associated structures in primates. J Comp Neurol 167:385–420.

Kievet J, Kuypers HGJM (1975) Basal forebrain and hypothalamusconnections to frontal and parietal cortex in the rhesus monkey.Science 187:660–662.

Kimura H, McGeer PL, Peng JH, McGeer EG (1981) The centralcholinergic system studied by choline acetyltransferase immuno-cytochemistry in the cat. J Comp Neurol 200:151–201.

Klein JC, Eggers C, Kalbe E, Weisenbach S, Hohmann C, Vollmar S,Baudrexel S, Diederich NJ, Heiss WD, Hilker R (2010) Neurotrans-mitter changes in dementia with lewy bodies and parkinson dis-ease dementia in vivo. Neurology 74:885–892.

Kölliker A (1896) Handbuch der Gewebelehre des menschen. Fürärzte und studirende. Nervensystem des menschen und der thiere.Leipzig: Engelmann.

Kordower J, Bartus R, Marciano F, Gash D (1989) Telencephaliccholinergic system of the New World monkey (Cebus apella):morphological and cytoarchitectonic assessment and analysis ofthe projection to the amygdala. J Comp Neurol 279:528–545.

Kostovic I (1986) Prenatal development of nucleus basalis complexand related fiber systems in man: a histochemical study. Neuro-science 17:1047–1077.

Kracun I, Rosner H (1986) Early cytoarchitectonic development of theanlage of the basal nucleus of Meynert in the human fetus. Int JDev Neurosci 4:143–149.

Lehéricy S, Hirsch E, Cervera-Pierot P, Hersh L, Bakchine S, Piette F,Duyckaerts C, Hauw J, Javoy-Agid F, Agid Y (1993) Heterogeneityand selectivity of the degeneration of cholinergic neurons in thebasal forebrain of patients with Alzheimer’s disease. J Comp Neu-rol 330:15–31.

Melander T, Staines WA (1986) A galanin-like peptide coexists inputative cholinergic somata of the septum-basal forebrain complexand in acetylcholinesterase-containing fibers and varicositieswithin the hippocampus in the owl monkey (Aotus trivirgatus).Neurosci Lett 68:17–22.

Mesulam M, Geula C (1988a) Nucleus basalis (Ch4) and corticalcholinergic innervation in the human brain: observations based onthe distribution of acetylcholinesterase and choline acetyltrans-ferase. J Comp Neurol 275:216–240.

Mesulam M, Mufson E, Levey A, Wainer B (1983) Cholinergic inner-vation of cortex by the basal forebrain: cytochemistry and corticalconnections of the septal area, diagonal band nuclei, nucleusbasalis (substantia innominata) and hypothalamus in the rhesusmonkey. J Comp Neurol 214:170–197.

Mesulam M, Mufson E, Wainer B (1986) Three-dimensional represen-tation and cortical projection topography of the nucleus basalis(Ch4) in the macaque: concurrent demonstration of choline acetyl-transferase and retrograde transport with a stabilized tetramethyl-benzidine method for horseradish peroxidase. Brain Res 367:301–308.

Mesulam M, Shaw P, Mash D, Weintraub S (2004) Cholinergic nu-cleus basalis taupathy emerges early in the aging-MCI-AD contin-uum. Ann Neurol 55:815–828.

Mesulam MM (2004) The cholinergic innervation of the human cere-

bral cortex. Prog Brain Res 145:67–78.

Mesulam MM, Geula C (1988b) Acetylcholinesterase-rich pyramidalneurons in the human neocortex and hippocampus: absence atbirth, development during the life span, and dissolution in Alzhei-mer’s disease. Ann Neurol 24:765–773.

Mesulam MM, Hersh LB, Mash DC, Geula C (1992) Differential cho-linergic innervation within functional subdivisions of the humancerebral cortex: a choline acetyltransferase study. J Comp Neurol318:316–328.

Mesulam MM, Van Hoesen GW (1976) Acetylcholinesterase projec-tion from basal forebrain of the rhesus monkey to neocortex. BrainRes 109:152–157.

Mettler FA (1968) Anatomy of the basal ganglia. In: Handbook ofclinical neurology (Vinken PJ, Bruyn GW, eds), pp 1–55. Amster-dam: North-Holland Publiching Company.

Meynert T (1872) Vom Gehirn der Saugetiere. In: Handbuch der lehrevon den geweben des menschen und tiere (Stricker S, ed), pp694–808. Leipzig: Engelmann.

Morrison JH, Foote SL (1986) Noradrenergic and serotonergic inner-vation of cortical, thalamic, and tectal visual structures in old andnew world monkeys. J Comp Neurol 243:117–138.

Mufson EJ, Bothwell M, Hersh L, Kordower J (1989a) Nerve growthfactor receptor immunoreactive profiles in the normal, aged, hu-man basal forebrain: colocalization with cholinergic neurons.J Comp Neurol 285:196–217.

Mufson EJ, Bothwell M, Kordower JH (1989b) Loss of nerve growthfactor receptor-containing neurons in Alzheimer’s disease: a quan-titative analysis across subregions of the basal forebrain. ExpNeurol 105:221–232.

Mufson EJ, Presley L, Kordower J (1991) Nerve growth factor receptorimmunoreactivity within the nucleus basalis (Ch4) in Parkinson’sdisease: reduced cell numbers and co-localization with cholinergicneurons. Brain Res 539:19–30.

Nagai T, Pearson T, Peng G, McGeer EG, McGeer PL (1983) Immu-nocytochemical staining of the human forebrain with monoclonalantibody to human choline acetyltransferase. Brain Res 265:300–306.

Pearson RCA, Gatter KC, Brodal P, Powell TPS (1983) The projectionof the basal nucleus of Meynert upon the neocortex in the monkey.Brain Res 259:132–136.

Perry RH, Candy JM, Perry EK, Thompson J, Oakley AE (1984) Thesubstantia innominata and adjacent regions in the human brain:histochemical and biochemical observations. J Anat 138:713–732.

Raghanti MA, Stimpson CD, Marcinkiewicz JL, Erwin JM, Hof PR,Sherwood CC (2008a) Cholinergic innervation of the frontal cortex:differences among humans, chimpanzees, and macaque mon-keys. J Comp Neurol 506:409–424.

Raghanti MA, Stimpson CD, Marcinkiewicz JL, Erwin JM, Hof PR,Sherwood CC (2008b) Cortical dopaminergic innervation amonghumans, chimpanzees, and macaque monkeys: a comparativestudy. Neuroscience 155:203–220.

Ramanathan D, Tuszynski MH, Conner JM (2009) The basal forebraincholinergic system is required specifically for behaviorally medi-ated cortical map plasticity. J Neurosci 29:5992–6000.

Rye DB, Wainer BH, Mesulam MM, Mufson EJ, Saper CB (1984)Cortical projections arising from the immunohistochemical localiza-tion of choline acetyltransferase. Neuroscience 13:627–643.

Santos-Benitez H, Magariños-Ascone CM, Garcia-Austt E (1995) Nu-cleus basalis of Meynert cell response in awake monkeys. BrainRes Bull 37:507–511.

Saper CB (1987) Diffuse cortical projection systems: anatomical orga-nization and role in cortical function. In: Handbook of physiology—the nervous system (Plum F, ed), pp 169–210. Bethesda: Ameri-can Phyiological Society.

Saper CB (1990) Cholinergic system. In: The human nervous system(Paxinos G, ed), pp 1095–1114. San Francisco: Academic Press.

Saper CB, Chelimsky TC (1984) A cytoarchitectonic and histochemicalstudy of nucleus basalis and associated cell groups in the normal

human brain. Neuroscience 13:1023–1037.

M. A. Raghanti et al. / Neuroscience 184 (2011) 1–15 15

Sarter M, Bruno JP (2000) Cortical cholinergic inputs mediatingarousal, attentional processing and dreaming: differential afferentregulation of the basal forebrain by telencephalic and brainstemafferents. Neuroscience 95:933–952.

Sarter M, Parikh V (2005) Choline transporters, cholinergic transmis-sion and cognition. Nat Rev Neurosci 6:48–56.

Selden N, Gitelman D, Salamon-Murayama N, Parrish T, Mesulam M(1998) Trajectories of cholinergic pathways within the cerebralhemispheres of the human brain. Brain 121:2249–2257.

Sharma Y, Xu T, Graf WM, Fobbs A, Sherwood CC, Hof PR, Allman JM,Manaye KF (2010) Comparative anatomy of the locus coeruleus inhumans and nonhuman primates. J Comp Neurol 518:963–971.

Simic G, Mrzljak L, Fucic A, Winblad B, Lovric H, Kostovic I (1999)Nucleus subputaminalis (Ayala): the still disregarded magnocellu-lar component of the basal forebrain may be human specific andconnected with the cortical speech area. Neuroscience 89:73–89.

Smiley J, Mesulam M (1999) Cholinergic neurons of the nucleusbasalis of Meynert receive cholinergic, catecholaminergic andGabaergic synapses: an electron microscopic investigation in themonkey. Neuroscience 88:241–255.

St-Jacques R, Gorczyca W, Mohr G, Schipper HM (1996) Mapping ofthe basal forebrain cholinergic system of the dog: a choline acetyl-transferase immunohistochemical study. J Comp Neurol 366:717–725.

Stephan H, Frahm HD, Baron G (1981) New and revised data onvolumes of brain structures in insectivores and primates. FoliaPrimatol 35:1–29.

Steriade M, Parent A, Pare D, Smith Y (1987) Cholinergic and non-cholinergic neurons of cat basal forebrain project to reticular and

mediodorsal thalamic nuclei. Brain Res 408:372–376.

Struble RG, Lehmann J, Mitchell SJ, McKinney M, Price DL, Coyle JT,DeLong MR (1986) Basal forebrain neurons provide major cholin-ergic innervation of primate neocortex. Neurosci Lett 66:215–220.

Teipel SJ, Flatz WH, Heinsen H, Bokde ALW, Schoenberg SO,Stockel S, Dietrich O, Reiser MF, Moller H-J, Hampel H (2005)Measurement of basal forebrain atrophy in Alzheimer’s diseaseusing MRI. Brain 128:2626–2644.

von Bohlen und Halbach O, Dermietzel R (2006) Neurotransmittersand neuromodulators. Handbook of receptors and biological ef-fects, 2nd ed, p 386. Weinheim, Germany: Wiley-VCH VerlagGmbH & Co.

West M (1999) Stereological methods for estimating the total numberof neurons and synapses: issues of precision and bias. TrendsNeurosci 22:51–61.

Wilson FAW, Rolls ET (1990) Neuronal responses related to novelty-and familiarity of visual stimuli in the substantia innominata, diag-onal band of Broca, and periventricular region of the primate basalforebrain. Exp Brain Res 80:104–120.

Wu C, Hersh LB, Geula C (2000) Cyto- and chemoarchitecture ofbasal forebrain cholinergic neurons in the common marmoset (Cal-lithrix jacchus). Exp Neurol 165:306–326.

Zaborszky G, Atienza M, Gil-Neciga E, Rodriguez-Romero R, TeipelSJ, Amunts K, Suarez-Gonzalez A, Cantero JL (2009) Reductionof basal forebrain cholinergic system parallels cognitive impair-ment in patients at high risk of developing Alzheimer’s disease.Cereb Cortex 20:1685–1695.

Zaborszky L, Hoemke L, Mohlberg H, Schleicher A, Amunts K, Zilles K(2008) Stereotaxic probabilistic maps of the magnocellular cell

groups in human basal forebrain. Neuroimage 42:1127–1141.

(Accepted 6 April 2011)(Available online 13 April 2011)