Forebrain connectivity of the prefrontal cortex in the marmoset monkey (Callithrix jacchus): An...

Forebrain Connectivity of the PrefrontalCortex in the Marmoset Monkey

(Callithrix jacchus): An Anterogradeand Retrograde Tract-Tracing Study

ANGELA C. ROBERTS,1,2* DAVORKA L. TOMIC,2 CAROLINE H. PARKINSON,3

TOM A. ROELING,3 DAVID J. CUTTER,2 TREVOR W. ROBBINS,1,3

AND BARRY J. EVERITT1,3

1Behavioural and Clinical Neuroscience Institute, University of Cambridge,Cambridge, CB2 3EB, UK

2Department of Physiology, Development and Neuroscience, University of Cambridge,Cambridge, CB2 3DY, UK

3Department of Experimental Psychology, University of Cambridge,Cambridge, CB2 3EB, UK

ABSTRACTThe cortical and subcortical forebrain connections of the marmoset prefrontal cortex

(PFC) were examined by injecting the retrograde tracer, choleratoxin, and the anterogradetracer, biotin dextran amine, into four sites within the PFC. Two of the sites, the lateral andorbital regions, had previously been shown to provide functionally dissociable contributionsto distinct forms of behavioral flexibility, attentional set-shifting and discrimination reversallearning, respectively. The dysgranular and agranular regions lying on the orbital and medialsurfaces of the frontal lobes were most closely connected with limbic structures includingcingulate cortex, amygdala, parahippocampal cortex, subiculum, hippocampus, hypothala-mus, medial caudate nucleus, and nucleus accumbens as well as the magnocellular divisionof the mediodorsal nucleus of the thalamus and midline thalamic nuclei, consistent withfindings in the rhesus monkey. In contrast, the granular region on the dorsal surface closelyresembled area 8Ad in macaques and had connections restricted to posterior parietal cortexprimarily associated with visuospatial functions. However, it also had connections withlimbic cortex, including retrosplenial and caudal cingulate cortex as well as auditory pro-cessing regions in the superior temporal cortex. The granular region on the lateral convexityhad the most extensive connections. Based on its architectonics and functionality, it resem-bled areas 12/45 in macaques. It had connections with high-order visual processing regions inthe inferotemporal cortex and posterior parietal cortex, higher-order auditory and polymodalprocessing regions in the superior temporal cortex. In addition it had extensive connectionswith limbic regions including the amygdala, parahippocampal cortex, cingulate, and retro-splenial cortex. J. Comp. Neurol. 502:86–112, 2007. © 2007 Wiley-Liss, Inc.

Indexing terms: lateral prefrontal cortex; orbitofrontal cortex; medial prefrontal cortex;

mediodorsal thalamus; striatum; nucleus accumbens

Grant sponsor: Wellcome Trust; Grant number: 19407/1.27 (to T.W.R.,B.J.E., A.C.R., and Barbara Sahakian); Grant sponsor: Medical ResearchCouncil (Career Establishment grant to A.C.R.); Grant sponsor: from theWellcome Trust (travel fellowship to D.L.T.).

Current address for Dr. Tom Roeling: Rudolf Magnus Institute of Neu-roscience, Dept. of Pharmacology and Anatomy, University Medical Cen-tre, Utrecht, The Netherlands.

*Correspondence to: Angela C. Roberts, Department of Physiology, De-velopment and Neuroscience, University of Cambridge, Downing St., Cam-bridge, CB2 3DY, UK. E-mail: [email protected]

Received 8 May 2006; Revised 20 September 2006; Accepted 21 Decem-ber 2006

DOI 10.1002/cne.21300Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 502:86–112 (2007)

© 2007 WILEY-LISS, INC.

The common marmoset (Callithrix jacchus), a NewWorld monkey, is proving to be a valuable primate speciesin which to study the neural and neurochemical basis ofhigher-order cognitive and emotional functions. Theirsmall size makes them well suited to behavioral testing ina laboratory setting and their relatively invariable brainsize, compared to that of larger primates, is ideal forhighly specific physical and neurochemical interventionsin target subcortical brain structures. In addition, theirability to perform touch-screen-based cognitive tests, di-rectly analogous to those undertaken by human subjects,increases the ease with which experimental results can beextrapolated into the clinic. As a consequence, marmosetsare also becoming a popular primate species in which tostudy the pharmacological modulation of cognition andemotion for the purpose of developing novel drug treat-ments for a range of neuropsychiatric disorders.

One specific area of investigation in the marmoset hasfocused on the functional organization of the prefrontalcortex (PFC) and its modulation by the monoamines. Adouble dissociation has been revealed between the effectsof excitotoxic lesions of the lateral and orbital regions ofthe marmoset PFC on performance of a series of multidi-mensional visual discriminations designed to measure dif-ferent forms of behavioral flexibility, including attentionalset-shifting and discrimination reversal (Dias et al., 1996,1997). The results support a role for the orbitofrontalregion in changing behavior in the face of alterations inthe reward value of specific stimuli in the environment

and for the lateral region in the shifting of higher-orderattentional sets. These functions are not only dependenton distinct regions of the PFC but are also modulateddifferentially by the catecholamines. Thus, higher-orderattentional selection is disrupted by dopaminergic (Croftset al., 2001), but not serotonergic lesions, of the PFC(Clarke et al., 2005), while the ability to reverse respond-ing following changes in reward contingencies is im-paired by serotonergic (Clarke et al., 2004) but notdopaminergic lesions (Roberts et al., 1994, Clarke et al.,2007). A similar dissociation between impairments indiscrimination reversal and attentional set-shifting isevident in the pattern of behavioral impairments re-ported in patients with Parkinson’s disease (Downes etal., 1989), Huntington’s disease (Lange et al., 1995), anddementia of the frontal lobe type (Rahman et al., 1999).In addition, in human functional neuroimaging studiesattentional set-shifting and discrimination reversal in-duce distinct patterns of task-dependent activationswithin the prefrontal cortex (Konishi et al., 1998; Na-kahara et al., 2002; Kringelback and Rolls, 2003;O’Doherty et al., 2003; Hampshire and Owen, 2006).While these findings highlight the apparent homologiesbetween the functioning of marmoset and human pre-frontal cortex, our understanding of the prefrontal cor-tex of marmosets is relatively poor.

A cytoarchitectonic classification of marmoset prefron-tal cortex was originally performed by Brodmann (1909)and Pedin and Von Bonin (1947) and subsequently a num-ber of studies examined its connections. First, Leichnetz

Abbreviations

Abmg Accessory basal nucleus of the amygdala, magnocellulardivision

Abpc Accessory basal nucleus of the amygdala, parvocellulardivision

ac Anterior commissureAM Anteromedial nucleus of the thalamusAV Anteroventral nucleus of the thalamusBi Basal nucleus of the amygdala, intermediate divisionBmg Basal nucleus of the amygdala, magnocellular divisionBpc Basal nucleus of the amygdala, parvocellular divisionC Cortical nucleus of the amygdalaCA1 Sector of the hippocampus properCe Central nucleus of the amygdalaCeM Centromedial nucleus of the thalamusCl ClaustrumDA Dorsoanterior extrastriate areaDM Dorsomedial nucleus of the hypothalamusEnt Entorhinal cortexgpe Globus pallidus, external segmentgpi Globus pallidus, internal segmentG Gustatory cortexHDB Horizontal limb of the diagonal bandHF Hippocampal formationIam Interanteromedial nucleus of the thalamusIn InsulaITG Inferotemporal gyrusL Lateral nucleus of the amygdalaLHA Lateral hypothalamic areaLPOA Lateral preoptic area of the hypothalamusLV Lateral ventricleM Medial nucleus of the amygdalamc Mediodorsal nucleus of the thalamus, magnocellular divi-

sionMD Mediodorsal nucleus of the thalamusmg Magnocellular division of the ventroanterior nucleus of

the thalamus

MP Medial pulvinar nucleusMPOA Medial preoptic area of the hypothalamusMT Middle temporal visual areaMTr Mamillary tractMTc Middle temporal crescent visual areaNA Nucleus accumbensNBM Nucleus basalisOT Optic tractpc Mediodorsal nucleus of the thalamus, parvocellular divi-

sionPer Periamygdaloid cortexPFC Prefrontal cortexPH Periventricular hypothalamusPL Paralamellar nucleus of the amygdalaPOC Paraolfactory cortexPoS ProsubiculumPP Posterior parietal cortexPrCO Precentral orbital areaPrS PresubiculumPVN Paraventricular nucleus of the hypothalamusRe Nucleus reuniens of the thalamusS SubiculumSCP Superior cerebral pedunculeSS II Secondary somatosensory areaSTG Superior temporal gyrusSTS Superior temporal sulcusTP Temporal polar cortexV2 Second visual areaV III Third ventricleVA Ventroanterior nucleus of the thalamusVLA Ventrolateral anterior extrastriate areaVLP Ventrolateral posterior extrastriate areaVP Ventroposterior nucleus of the thalamus

The Journal of Comparative Neurology. DOI 10.1002/cne

87CONNECTIVITY OF MARMOSET PREFRONTAL CORTEX

and Astruc (1975) described the pattern of fiber degener-ation following partial ablations of the orbitofrontal cortexand later, Schwerdtfeger (1979) and Brysch et al. (1990)described labeling within the subiculum and thalamus,respectively, following an injection of horseradish peroxi-dase into the dorsal prefrontal cortex. More recently, Bur-man et al. (2006) described the organization of the dorso-lateral frontal areas using a combination of architecturalmethods and injections of fluorescent tracers in dorsalvisual areas. However, the overall pattern of forebrainconnectivity within marmoset prefrontal cortex is stilllargely unknown and so the present study used the an-terograde tracer, biotin dextran amine (BDA), and theretrograde tracer, choleratoxin, to compare the pattern ofconnections within four cytoarchitectonically distinct re-gions of prefrontal cortex. Two of the injections sites tar-geted the lateral and orbital regions that had been inves-tigated in the earlier functional studies (Dias et al., 1996,1997) and the other two included cortex lying on the dorsaland medial surfaces, respectively. These sites differed inthe overall extent of their granularity but based on ourown unpublished findings each site was composed of anumber of cyto- and myelo-architectonically distinct re-gions.

MATERIALS AND METHODS

Twelve common marmosets (Callithrix jacchus) (sevenmales and five females) obtained from Porton Down, Salis-bury, UK (n � 5) and bred on site at the Medical ResearchCouncil colony (n � 7), with a mean age of 26 months,were used in this study. All procedures were conducted inaccordance with the project and personal licenses held bythe authors under the UK Animals (Scientific Procedures)Act of 1986.

Surgery and tracer injections

Anesthesia was induced by administering 0.4 mL saffan(Schering-Plough, Welwyn Garden City, UK) intramuscu-larly (i.m.) and maintained with supplementary doses of0.3 mL saffan for the duration of surgery. Each monkeywas placed in a stereotaxic frame using a head holder withincisor and zygoma bars specially modified for the mar-moset. Burr holes were made in the skull, the dura in-cised, and the cortex exposed.

Injections of tracers (the anterograde tracer, BDA,D1956; Molecular Probes, Cambridge Bioscience, Cambs,UK; and the retrograde tracer choleratoxin; Cholera toxinB subunit, C-7771, Sigma, Dorset, UK) were placed in thedorsal, lateral, medial, and orbital regions of PFC (Fig.1A–C) of 12 marmosets. Prefrontal injection sites werechosen according to Brodmann’s description of the cytoar-chitectural properties of subdivisions of the frontal cortexin the marmoset (Fig. 1D,E) and our own observations(Fig. 2). Since it is possible to administer multiple tracersin one animal, an injection of BDA was placed into oneregion of one hemisphere, while choleratoxin was placedinto another region of the other hemisphere of four mar-mosets. The remaining eight marmosets received a singleinjection into one or other hemisphere. The stereotaxiccoordinates that were used for each injection site are pre-sented in Table 1. Pressure injections were made througha glass micropipette attached to a syringe at a rate of 0.02�L / 20 seconds that was then left in place for about 25minutes after the injection in order to prevent the spread

of tracer upwards along the cannula tract. The volumes oftracers, as well as the number of injections per site, variedbetween the different regions (see Table 1). The injectionswere deliberately large, since the purpose of this studywas to reveal the general pattern of connectivity of thegranular, dysgranular, and agranular regions of PFC inthe marmoset.

Due to the inherent individual variability in brain size,infusion coordinates were individually determined foreach marmoset. To achieve this the depth of the brain atthe stereotaxic coordinate of AP 17.5, LM �1.5 (standard-ization coordinate) was measured for each marmoset. Thiswas calculated by taking a stereotaxic reading as the tip ofa fine dental burr (0.05 mm diameter) pierced the surfaceof the brain and then again as the burr touched the baseof the skull. (For the latter, a small lateralized movementof the burr at the surface of the brain indicated that the tiphad touched the base of the skull.) If the depth of the brainbetween these two readings was 5.8–6.5 mm no adjust-ments were made to the injection coordinates. However, ifthe depth of tissue fell outside of this range then thisindicated that the absolute position of the coordinatewithin the frontal lobes was either too rostral or too cau-dal. Consequently, the standardization coordinate was ad-justed accordingly along the anterior–posterior plane un-til the depth measurement fell within this range. If thestandardization coordinate had to be moved posteriorly by0.5 mm to obtain a thickness of tissue 5.8–6.5 mm, thenaccordingly all injection coordinates for that particularmarmoset would then be adjusted by the same amount,i.e., an AP injection site of 16.5 would be adjusted to AP16.0.

Histological procedures

After 10 days of survival animals were given an over-dose of pentobarbitone (“Sagatal,” Genusxpress, York,UK) and perfused transcardially with 500 mL of 0.1 Mphosphate-buffered saline (PBS), pH 7.4, followed by 1.5 Lof fixative solution containing 4% paraformaldehyde in 0.1M phosphate buffer. Brains were removed and postfixedovernight in the same fixative, then transferred to a 30%sucrose solution for 48 hours. Coronal sections (40 �m)were cut on a freezing sledge microtome and placed intocold PB in 10 series, so sections of one series were equallyspaced by 400 �m. The first and fifth series were stainedfor Nissl and adjacent series reacted for BDA and/or pro-cessed for choleratoxin-immunocytochemistry.

The BDA reaction product was visualized by washingthe free-floating sections in 0.05 M Tris-NaCl with 0.5%Triton X-100 then incubating them in 0.05 M Tris-NaClbuffer (pH 7.4) containing 0.5% Triton X-100 and avidin-biotin complex (ABC, Vectorstain elite, Vector, Peterbor-ough, UK) for 1 hour at room temperature. The sectionswere then reacted in a solution of 0.1 M PB containing 0.5mg/mL 3,3�-diaminobenzidine-tetrahydrochloride (DAB,Sigma, Dorset, UK), 0.015% H2O2, and intensifying agentnickel-ammonium sulfate for 20 minutes. Sections forcholeratoxin processing were washed in 0.1 M Tris-NaClbuffer (pH 7.4) for 45 minutes, then in order to removeendogenous peroxidase, sections were placed in a solutionof 10% methanol and 10% H2O2 (1:1) for 5 minutes; theywere then washed again, as above, then placed in 0.1 MTris-NaCl buffer, 0.2% Triton-X and 10% normal swineserum for 1 hour. This was followed by incubation inTris-NaCl buffer with 0.2% Triton X-100, 1% normal


88 A.C. ROBERTS ET AL.

swine serum, and goat anti-choleragenoid primary anti-body (Quadratech, Surrey, UK) at a dilution of 1:2,000 for24 hours at room temperature. Next, sections werewashed and then incubated in Tris-NaCl buffer containing0.2% Triton X-100 and biotinylated donkey antigoat sec-ondary antibody (Stratech, Bedford, UK) 1:200 for 2 hoursat room temperature. Sections were then transferred intoTris-NaCl buffer containing ABC for 2 hours. Finally, thesections were washed prior to visualization with DAB,mounted on gelatin-coated slides, dried overnight at roomtemperature, and then dehydrated and coverslipped withDPX.

One of the remaining series of sections in four of themonkeys was processed for calbindin immunocytochemis-try in order to visualize subdivisions of the nucleus accum-bens in the marmoset, previously described by Meredith et

al. (1996). Sections were washed in 0.1 M Tris-NaCl buffer(pH 7.4) for 45 minutes, then incubated in 0.1 M Tris-NaClbuffer (pH 7.4) containing 0.3% Triton X-100, 1% normalgoat serum, and anti-calbindin D-28K 1:500 for 24 hoursat room temperature. The monoclonal anti-calbindinD-28K (Sigma Biosciences, St. Louis, MO; Lot number:095H4814) was a mouse IgG derived from the hybridomaproduced by fusion of mouse myeloma cells and spleno-cytes from mice immunized with calbindin D-28k purifiedfrom chicken gut. The antibody specifically stains the45Ca-binding spot of calbindin D-28k (MW 28k) in a two-dimensional gel. Staining of sections through the nucleusaccumbens produced a pattern of calbindin immunoreac-tivity identical to previous descriptions in the marmoset(Meredith et al., 1996). The sections were washed andthen transferred to 0.1 M Tris-NaCl buffer (pH 7.4) con-

Fig. 1. A–C: Lateral, medial, and orbital views of the surface of themarmoset brain illustrating the location of the various injection sites.For each region, i.e., dorsal (D), lateral (L), orbital (O), and medial(M), the black and gray lines represent injections of retrograde (chol-eratoxin; ctx) and anterograde (BDA) tracers, respectively, while thesolid and dotted lines represent individual cases of each tracer.D,E: Lateral and medial views of the surface of the marmoset brain,showing Brodmann’s parcellation of the marmoset cortex and indicat-ing the approximate anteroposterior coordinates based on the inter-aural line (Stephan et al., 1980). F: Lateral view of the marmosetbrain illustrating the parcellation of dorsolateral prefrontal cortexand adjacent regions according to Burman et al. (2006). It has beenredrawn, with permission, from Burman et al. (2006), copyright JohnWiley & Sons. For each of the anterograde and retrograde cases thatare illustrated, the full extent of the tracer injections is depicted

across a series of coronal sections in (G). The middle section repre-sents the approximate center of the injection site and the sections oneither side represent the most rostral (left) and caudal (right) extentof the injection site. Dorsal injection site depicted in A: Black solidline, case D.ctx-1; gray solid line, case D.bda-1; black dotted line, caseD.ctx-2; gray dotted line, case D.bda-2. Dorsal aspect of lateral injec-tion site depicted in A and ventral aspect depicted in C: Black solidline, case L.ctx-1; gray solid line, case L.bda-1; black dotted line, caseL.ctx-2; gray dotted line, case L.bda-2. Orbital injection site depictedin C: black solid line, case O.ctx-1; gray solid line, case O.bda-1; blackdotted line, case O.ctx-2; gray dotted line, case O.bda-2. Medial PFCinjection site depicted in B. Black solid line, case M.ctx-1; gray solidline, case M.bda-1; black dotted line, case M.ctx-2; gray dotted line,case M.bda-2. Scale bars � 4 mm in A-F; 5 mm in G.



Fig. 2. A: A low-power photomicrograph of a Nissl-stained coronalsection taken from an intermediate level of the marmoset frontal lobeson which all four injection sites impinge. The approximate boundariesof all four injection sites at this level are indicated by the dotted lines.High-power photomicrographs (B–E) illustrate the cytoarchitecturalproperties of the dorsal, lateral, orbital, and medial injection sites.Dorsal (B) and lateral (C) injections are located in regions that aregranular, with a particularly well-developed layer IV. The expansionof layer III within the dorsal injection site can be seen in A. Cillustrates the region at the lateral apex which is the cytoarchitec-tonically distinct region that predominates within the lateral injection

site. In contrast, the orbital injection site (D) is located in a regionwith a far less-developed layer IV, considered dysgranular, while inthe medial injection site (E) layer IV is absent. The arrows in D andE highlight layers V and VI in the medial aspect of orbitofrontal cortexand the ventral aspect of medial PFC in which are found prominentalignments of cells into horizontal rows running parallel to the sur-face. The asterisks mark the same locations in the high-power pho-tomicrographs of B–E. as in the low-power photomicrograph of A. s,cortical surface; w, white matter. Scale bars � 1 mm in A; 500 �m inB–E.


taining 0.3% Triton X-100, 1% normal goat serum, andbiotinylated goat antimouse secondary antibody (Dako,Carpinteria, CA), for 2 hours. After washing, the sectionswere incubated in Tris-NaCl buffer containing ABC for 1hour. Finally, the sections were washed prior to visualiza-tion with DAB. Sections were then mounted and cover-slipped as described above.

Data analysis

Brain sections processed according to the methods ex-plained above were viewed under darkfield illumination(sections reacted for BDA) or brightfield illumination (sec-tions reacted for choleratoxin). Examples of the injectionsites for BDA and choleratoxin are illustrated in Figure 3,as are examples of choleratoxin-labeled cell bodies andBDA-positive terminals. It was found that the histologicalprocedure for labeling choleratoxin containing cell bodiescrossreacted, to a small degree, with BDA- positive neu-rons within the BDA injection site of those monkeys thathad received a choleratoxin injection in the contralateralhemisphere. However, such crossreactivity could easily beidentified since a BDA-containing neuron labeled by thecholeratoxin procedure showed uniform staining through-out the entire neuron (cytoplasm and nucleus) in compar-ison to a choleratoxin containing neuron in which, as canbe seen clearly in Figure 3D, labeling was restricted to thecytoplasm.

All coronal sections reacted for choleratoxin weremapped at low power using a Zeiss-Jena Microfilm Reader(DL2, Zeiss, Welwyn Garden City, UK), equipped with a30-cm wide screen. The outlines of sections, location ofcholeratoxin-positive neurons, and the location of the in-jection sites were mapped by drawing over the screen. Asimilar procedure of analysis was used for those sectionsreacted for BDA except that sections were drawn underlow-power darkfield microscopy using a camera lucidaattachment in order to enhance the details of stainedfibers. Injection sites for choleratoxin were defined as thatpart of each coronal section that included densely stainedregions adjacent to the cannula tract (Fig. 3B), but not theneighboring region with moderate to light backgroundstaining where individual labeled neurons could be seen.For BDA, injection sites were determined as part of adensely stained cortex immediately around the cannulatract (Fig. 3A). Drawings were made of each of the injec-tion sites (Fig. 1A–C) as well as the distribution of cellbodies in the case of choleratoxin or terminals of the

projections arising from each injection site, in the case ofBDA within a range of cortical and subcortical forebrainstructures. The drawings were scanned into a computerimage processing package, Adobe Photoshop (San Jose,CA), and drawings of corresponding sections were thensuperimposed, separately for anterograde and retrogradetracers, in order to create composite maps showing thelocation of the injection sites as well as the terminationpattern of projections from the injection sites in the stri-atum, amygdala, and thalamus. In addition, bright- anddarkfield photomicrographs were taken using a Leitz Di-alux 20 microscope (Leica, Milton Keynes, UK) in order toillustrate injection sites and the distribution of BDA-positive terminals or choleratoxin-labeled neurons inother structures, such as the nucleus accumbens, hypo-thalamus, hippocampus, and basal forebrain. Photomicro-graphs were then scanned into a computer image process-ing package and adjusted for contrast and brightnessusing Adobe Photoshop. Photomicrographs and drawingswere finally combined into plates using the Adobe Inde-sign desktop publishing program.

RESULTS

The nomenclature used to describe dorsolateral regionsof the PFC (see Fig. 1F) and visual extrastriate regions inmarmosets is that of Burman et al. (2006). The nomencla-ture for superior temporal cortical regions is that of de laMothe (2006). For all other cortical regions the nomencla-ture is that of Brodmann’s unless otherwise specified.

BDA and choleratoxin injection sites withinmarmoset PFC

The injection sites are depicted on the lateral, medial,and ventral views of the brain of the marmoset in Fig.1A–C, respectively. The dorsal PFC injection site was in amoderately granular region with a relatively well-developed layer IV containing loosely packed granule cells(Figs. 1A, 2A,B). According to Burman et al. (2006) itcorresponds to area 8Av following the nomenclature ofPetrides and Pandya (1999). The site may have impingedon area 9 rostrally. The lateral PFC injection site (Figs.1A,C, 2A,C, 3B) was in a highly granular region with adensely packed layer IV. It most likely corresponds to area12/45 described by Burman et al. (2006). In contrast, theorbital PFC injection site lay in a dysgranular region (Fig.

TABLE 1. Sterotaxic Coordinates Used to Inject Tracer into Dorsal, Lateral, Orbital, and Medial Prefrontal Cortex (PFC)

InjectionSite

AP(mm)

LM(mm)

Angle(°)

Depth1

(mm)Volume of Tracer

(�L) BDA2 CT2

Dorsal PFC �16.00�17.25

3.803.50

——

0.7s0.7s

0.200.20

2 2

Lateral PFC �16.00 6.20 10 0.9b 0.20 2 2�16.75 5.90 8 1.5b 0.20�17.50 5.60 8 1.0b 0.20�18.25 5.30 8 1.0b 0.20�19.00 4.60 8 0.7b 0.20

Medial PFC �16.75 1.25 — 3.1s 0.20 2 2�17.75 1.00 — 3.1s 0.20�18.50 0.75 — 2.5s 0.15

Orbital PFC �16.00 4.00 — 0.7b 0.25 2 2�16.75 3.00 — 0.7b 0.25�17.75 2.00 — 0.8b 0.25�18.50 2.00 — 0.8b 0.25

1Position of glass micropipette above the base (b) of the skull or below the brain’s surface (s).2Number of monkeys injected for each site.



Fig. 3. A: A low-power brightfield photomicrograph of a BDA in-jection site within orbital PFC (case O.bda-1). The injection encom-passes all cortical layers. B: A low-power brightfield photomicrographof the injection site of the retrograde tracer choleratoxin into thelateral PFC (case L.ctx-1). The white arrowhead indicates the locationof the cannula tract representing an orientation point for the place-ment of the injection site. The boundaries of the lateral injection siteare highlighted by black arrows. Low- (C) and high-power (D) pho-tomicrographs of neurons containing choleratoxin labeling after an

injection of tracer into lateral PFC (injection site shown in B). Areabetween arrowheads in C is magnified in D. Note that choleratoxinproduces a dark reaction product in the cytoplasm and proximal partsof the dendrites of the neuron, while the nucleus is not stained.Low-power (E) and high-power (F) darkfield photomicrographs of theBDA reaction product in the striatum following an injection intoorbital PFC (depicted in A). Scale bars � 1 mm in A,B; 150 �m in C,E;100 �m in D,F.


1C, 2A,D, 3A) composed of two cytoarchitectonically dis-tinguishable areas. In both, layers II and IV were poorlydefined and the supragranular layers were either widerthan, or equivalent in width to, the infragranular layers.In the more medial of the two regions the cells in layer VIformed orderly rows running parallel to the surface. Fi-nally, the medial PFC injection site lay in an agranularregion within the central portion of the medial frontalsurface (Figs. 1B, 2A,E), encompassing two distinct cyto-architectonic areas. Both areas lacked granular layers IIand IV and had a high cell density in layers III and V. Thecells in layer VI of the more ventral region formed distinc-tive rows that ran parallel to the surface. As in the orbitalsite, supragranular layers were considerably thicker thaninfragranular layers. These two areas most likely corre-spond to area 32 dorsally and area 25 ventrally based ontheir architecture (Preuss and Goldman-Rakic, 1991a;Carmichael and Price, 1994).

In all but one case, where the BDA injection into thedorsal PFC was restricted to layers I–V, tracer injectionsinvolved all cortical layers. In the case of one of the medialinjections of BDA (case M.bda-2) and choleratoxin (caseM.ct-2), the tracer extended downward, occupying a smallpart of the orbital cortex at the medioventral convexity(see Fig. 1B). In the case of one of the orbital injections ofBDA (case O.bda-2), two adjacent injections of the tracerremained separated in the tissue, rather than mergingtogether. There was no evidence that the tracer spreadalong the cannula tracts to the white matter.

Neuronal and terminal labeling insubcortical structures of the forebrain

Striatum. High-power microscopic analysis of termi-nal labeling showed that in the caudate nucleus, putamen,and nucleus accumbens, terminal fields consisted of adense network of beaded fibers (Fig. 3E,F). The predomi-nant round shape of these beaded structures suggests thatthey represent synaptic terminals. All PFC areas pro-jected to the striatum, predominantly to the caudate nu-cleus, while far fewer terminals were found in the puta-men (Fig. 4). In addition, after medial and orbitalinjections projections were also found in the nucleus ac-cumbens (Fig. 4A,B). As shown in Figure 4, each region ofthe PFC projected to a restricted portion of the caudatenucleus, topographically maintaining the position of theirterminal fields throughout the longitudinal extent of thehead and body of the caudate nucleus. In the case of themedial and the lateral injections, terminals were alsofound to extend all the way back into the tail of thecaudate nucleus (Fig. 4G,H).

Caudate nucleus and putamen. At the rostral levelwithin the head of the caudate nucleus the terminal fieldsof the dorsal and lateral injection sites overlapped (Fig.4A); at more caudal levels the terminal fields of each siteseemed to be clearly segregated (Fig. 4B–G). After aninjection into the dorsal PFC, terminals were found in thedorsal caudate (Fig. 4A,B). More caudally, they appearedmore ventromedial, still maintaining, however, their moredorsal position compared to the terminal fields of the otherinjection sites (Fig. 4C–E). In contrast, the terminals ofthe lateral prefrontal neurons occupied the ventrolateralaspect of the head and body of the caudate nucleus. Adja-cent to the lateral terminal field, positioned in the ventro-medial region of the caudate nucleus were the terminals oforbital prefrontal fibers. At rostral levels these were

heavily intermingled with terminals from the medial PFC(Fig. 4A) but further caudally, orbital and medial termi-nals were more clearly segregated, with the medial termi-nal field bordering the lateral wall of the lateral ventricle(Fig. 4C,D). In the case of all four injection sites, terminalfibers were seen to extend laterally into the putamen (Fig.4A–D). In contrast to the lateral PFC terminal field, whichwas most abundant at mid-rostral levels of the caudate (AP11.5 and 10.5), medial and orbital PFC projections were seento be most dense at more rostral levels, AP 12.5 and 11.5.

Nucleus accumbens. As in the rat and rhesus mon-key, there was a differential distribution of CaB immuno-reactivity in the nucleus accumbens of the marmoset. Ingeneral, the rich CaB immunoreactivity of the caudatenucleus and putamen became less intense ventrally at thelevel of the nucleus accumbens but the border between thecaudate and putamen and the nucleus accumbens couldnot be discerned. In the nucleus accumbens of the ratthere is an inner core and an outer crescent-shaped shelllocated medial and ventral to the core, which is easilyidentified by its poor CaB immunostaining (Zahm andBrog, 1992). In the marmoset this region was also CaB-poor at the most rostral level (Fig. 5A) but at mid-rostrocaudal levels a relatively high density of CaB immu-nostaining was found in the ventral region (Fig. 5B),which was maintained at more caudal levels (Fig. 5C,D).Indeed, at more caudal levels both the medial and ventralregions of the nucleus accumbens were richer in CaBimmunostaining than parts of the lateral core region (Fig.5C,D), consistent with the findings of Meredith et al.(1996) in the marmoset.

Only orbital and medial PFC were found to project tothe nucleus accumbens in the marmoset (Fig. 4A,B). Alarge number of fibers from both regions terminated in thelateral region of the nucleus accumbens, while far fewerterminated in the ventral and medial regions. The ventralregion received fibers arising from both the medial andorbital PFC, while the medial region tended to receivefibers from the medial PFC only (Fig. 6A,B).

Thalamus. The thalamus was a site of very denselabeling following all four injections. The projections fromthe different prefrontal injection sites were distributedthroughout several thalamic nuclei: the mediodorsal(MD), ventral, anterior, intralaminar, mesial, and medialpulvinar nuclei. The most abundant, reciprocal projec-tions from all injection sites were found in the mediodorsalnucleus (Fig. 7E,F). In Nissl-stained sections the MD nu-cleus can be defined as the group of cells positioned be-tween the internal medullary laminae and the periventralgray in the middle third of the thalamus (Fig. 7D). Inmarmosets, two major parts of the MD can be recognized:a magnocellular division located rostrally and medially,containing large, intensely stained multipolar neurons,and a parvicellular division that extends more caudallywithin the nucleus and consists of smaller and paler neu-rons (Fig. 7D). The pars paralamellaris as well as a parsdensocellularis seen in the macaque could not be dis-cerned in the marmoset.

Terminals from all four injection sites were topograph-ically organized within the MD nucleus, with terminallabeling arising from the orbital and medial injection sitesconcentrated in the magnocellular part, while terminallabeling arising from the lateral and dorsal injectionsoccurred almost exclusively in the parvicellular portion(Fig. 7F), although there was some labeling from the lat-



Fig. 4. A–H: A composite map of the camera lucida drawings ofterminals in the striatum following an injection of BDA into the dorsal(case D.bda-2), lateral (case L.bda-1), orbital (case O.bda-2), or medial(case M.bda-2) PFC. The projections from each region of the PFCoccupy a distinct, well-defined position within the caudate nucleus,maintaining this position throughout the longitudinal extent of the

nucleus. Only lateral and medial PFC were found to project longitu-dinally all the way back to the tail of the nucleus (G,H). Note that onlythe medial and orbital PFC project to the nucleus accumbens, orbitalPFC projecting mainly to the central region (c) while medial PFCprojects to the central, as well as the medial (m) and ventral regions(v; A,B). Scale bar � 1 mm.

eral site in the most ventral part of the magnocellularregion. In addition, while terminals from the dorsal andmedial PFC were distributed dorsally in the MD, termi-nals from lateral and orbital PFC were distributed primarilyin the ventral part of the MD. An almost identical distribu-tion pattern was seen with respect to choleratoxin-labeledcells, highlighting the strong reciprocity in the connec-tions between the PFC and MD thalamus in marmosets(compare Fig. 7E,F).

There were also reciprocal connections between all fourprefrontal injection sites and the ventral anterior nucleus(Fig. 7A) with cells and fibers present throughout therostrocaudal extent of the nucleus (Fig. 7B,C). In contrast,connections with the anteromedial and anteroventral nu-cleus were not reciprocal. While labeled neurons werefound in both nuclei after lateral and orbital injections,labeled terminals were only found after a medial injection(Fig. 7C). Scattered neurons, but not terminals, were also

found in intralaminar centrodorsal nucleus following in-jections into the medial site, as well as in the mesialnucleus (paratenial, centromedial, interanteromedial, andreuniens) after injections into all sites (Fig. 7B). In addi-tion to the ipsilateral connections, in most cases scatteredcells and fibers were found occupying the same part of thethalamic nuclei contralaterally.

Finally, a distinctive topographic arrangement of termi-nals was found in the medial pulvinar (Fig. 7G), withprojections from the medial PFC terminating in the dor-somedial part, terminals from the dorsal PFC terminatingmore ventrally in the ventromedial region, and terminalsfrom the lateral PFC being found most laterally, in thecentromedial zone of the pulvinar nucleus (Fig. 7I). Noterminals were found in the medial pulvinar after orbitalinjections of BDA and choleratoxin-positive cells werefound in the medial part of the medial pulvinar only afterthe lateral injection (Fig. 7H).

Fig. 5. Low-power photomicrographs illustrating the distributionof CaB immunostaining throughout the rostrocaudal extent of thenucleus accumbens (A–D). Rostrally (A), both the ventral (v) andmedial (m) aspects of the nucleus accumbens are relatively poor inCaB immunostaining compared to the central region (c) of the nucleusaccumbens, which in turn is stained more lightly than that of theneighboring caudate and putamen. At mid-rostrocaudal levels of the

nucleus accumbens (B), however, there is dense CaB immunostainingin the ventral region (arrow) that is maintained at the most caudallevels (C,D). At these more caudal levels, patches of dense CaB im-munostaining can also be seen in the medial region (arrow in C), whilethe central region at these more caudal levels has patches of quitelight staining. Scale bar � 500 �m.



Amygdala. Since the cytoarchitecture of the amygda-loid nuclei in the marmoset has not been reported exten-sively, we adopted the nomenclature of subdivisions de-scribed in the atlas of the marmoset brain by Stephan etal. (1980) along with additional subdivisions, based on ourown delineation of the nuclei, stained with cresyl violet,

acetylcholinesterase, and immunostained for parvalbu-min and calbindin. These additional nuclei that were notincluded in the atlas by Stephan et al. were describedrecently in the rat (McDonald, 1997) and macaque mon-key (Amaral and Basset, 1989). We identified 12 subdivi-sions of the amygdala in the marmoset, the majority of

Fig. 6. Darkfield photomicrographs of the pattern of fiber termi-nations in the nucleus accumbens (NA) following an injection of BDAinto the orbital (A; case O.bda-1) and medial PFC (B; case M.bda-1).While there is moderately dense labeling in the central region (c) of

the NA following an injection into the orbital PFC (A), there is verylittle labeling in the neighboring medial (m) and ventral (v) regions. Incontrast, there are BDA-positive fibers throughout the NA after aninjection of BDA into the medial PFC (B). Scale bar � 200 �m.



Fig. 7. Low-power photomicrographs of Nissl-stained sectionsthrough the thalamus illustrate the location of the anterior nuclearcomplex (A), mediodorsal nucleus (D), and pulvinar (G). Compositemaps of terminals (C,F,I) (case D.bda-2; case L.bda-1; case O.bda-2;case M.bda-2) and cell bodies (B,E,H) (case D.ctx-2; case L.ctx-1; caseO.ctx-2; case M.ctx-1) within the thalamus following BDA and CTinjections into the PFC show that projections between the PFC and

individual thalamic nuclei are topographically organized, such thateach part of the PFC is connected to a specific part of the thalamus.Note that choleratoxin-labeled neurons are found in the medial pulv-inar nucleus only after the lateral injection (H; case L.ctx-1), whileBDA-positive fibers are found following lateral (case L.bda-1), dorsal(case D.bda-2), and medial (case M.bda-2) injections (I). Scale bars �1 mm in A,D,G–I; 1.5 mm in B,C,E,F.



them illustrated in Figure 8A: lateral, basal magnocellu-lar, basal parvocellular, basal intermedial, accessory basalmagnocellular, accessory basal parvocellular, cortical, me-dial, central, paralaminar, anterior, and periamygdaloidcortex.

Both anterograde and retrograde tracers injected intothe PFC produced labeling in the amygdala. As illustratedin Figure 8B–G, lateral, orbital, and medial PFC, but notdorsal PFC, all had extensive reciprocal connections withthe basal magnocellular nucleus of amygdala, easily rec-ognized by its large, hyperchromatic cells forming atriangular-shaped territory in the central part of theamygdaloid complex. The projections from the PFC wereorganized topographically. The densest projection camefrom the orbital injection sites, with fibers terminating inthe basal magnocellular nucleus as well as in the basalintermediate nucleus (Fig. 8C). Fibers from the medialPFC were more sparse, targeting mainly the ventral partof the basal magnocellular nucleus (Fig. 8D). In contrast,lateral PFC gave rise to labeled terminals in the dorsalpart of the basal magnocellular nucleus (Fig. 8B). In allcases incoming fibers were seen emerging from the whitematter of the external capsule, coursing downward andmedially into the temporal pole then ramifying into theneighboring parts of the basal magnocellular nucleus. Inthe case of the orbital injection site (Fig. 8C) some of thefibers passed all the way through the basal and lateralnucleus, finally forming vertical columns of terminal fi-bers in the underlying entorhinal cortex. In addition to thebasal nucleus, lateral, orbital, and medial PFC injectionsgave rise to labeled fibers and terminals in the lateralnucleus, although these were far fewer in number. Termi-nal labeling in the central nucleus was only seen followingmedial and orbital injections and the cortical nucleus wasonly labeled following orbital injections.

In contrast to the widespread pattern of incoming fibersfrom the PFC, choleratoxin-positive neurons were primar-ily seen in the basal magnocellular nucleus and unlike theterminal labeling, the neurons did not show any apparenttopographical preference within the basal magnocellularnucleus (Fig. 8E–G). The majority of labeled basal neu-rons, from lateral, orbital, and medial injection sites, werefound in the middle sector of the amygdala, within thegroup of magnocellular neurons that form a distinctivetriangular shape. Only following injections of choleratoxininto the medial PFC were neurons also labeled more ros-trally in the medial region of the basal magnocellularnucleus, often referred to as the accessory basal magno-cellular nucleus (see Fig. 8A). In addition, a few scatteredlabeled neurons were visible in the cortical nucleus follow-ing the medial injection.

Basal forebrain. Retrogradely labeled neurons in thebasal forebrain could be seen after injecting choleratoxininto all regions of the PFC. The neurons were large, mul-tipolar, and hyperchromatic and showed the characteristicmorphology of the magnocellular cholinergic neurons ofthe Ch4 cell region in the marmoset (see fig. 9A–C inEveritt et al., 1988). All prefrontal injection sites labeledneurons rostrally, anterior to the decussation of the ante-rior commissure, in the Ch4am cell group (Fig. 9A). Inaddition, labeled cells were present more caudally, at thelevel of, and caudal to, the anterior commissure decussa-tion (Fig. 9B), within Ch4al, Ch4iv, and Ch4id cell groups(see fig. 9C–H in Everitt et al., 1988). In addition, follow-ing the lateral and dorsal injections labeled magnocellular

neurons were also found in the internal pallidal lamina(Fig. 9C) and following all injections, a few labeled cellswere seen in the horizontal limb nucleus of the diagonalband of Broca (HDB) (Fig. 9A). Overall, cell labeling withinthe Ch4 cell groups appeared most numerous and most wide-spread following choleratoxin injections into the lateral PFCand most sparse following injections into dorsal PFC.

All the prefrontal areas except dorsal area 8 sent pro-jections back to the basal forebrain region. The densestprojections were from the orbital PFC innervating the Ch4cell region as well as the region containing the large cho-linergic neurons of the HDB (Fig. 10A). This innervationwas apparent over the full rostrocaudal extent of bothregions. In contrast, terminal labeling after injections intothe medial and lateral PFC was far weaker to that seenfollowing orbital PFC injections and was only seen in theventral Ch4 cell region and in the region of the HDB (Fig.10B,C).

Hypothalamus. The distinctive subdivisions of thehypothalamus are illustrated in Figure 11A–C. Injectionof BDA into the orbital and medial PFC resulted in abun-dant labeling throughout the hypothalamus with a cleardistinction between the terminal fibers of the two injectionsites. The medial PFC injection resulted in dense axonallabeling in much of the hypothalamus throughout its ros-trocaudal extent. Laterally, terminals were visible in thelateral preoptic area, rostrally (Fig. 11D) and throughoutthe lateral hypothalamic area, more caudally (Fig. 11E,F).Medially, there were many labeled axons and terminals inthe medial preoptic area (Fig. 11D), anterior hypothalamicarea (Fig. 11E), and the dorsomedial nucleus (Fig. 11F). Aparticularly dense plexus of fibers and terminals sur-rounded the dorsal aspects of the third ventricle morecaudally and ramified into the dorsomedial nucleus (Fig.11F). No label was present in the supraoptic nucleus, thesuprachiasmatic nucleus, the paraventricular nucleus,the ventromedial nucleus, and the mammillary nuclei al-though axons were observed to lie adjacent to their bor-ders. In contrast to the medial PFC, the orbital PFCtended to project exclusively into the lateral parts of thehypothalamus, forming dense terminals in the lateral hy-pothalamic area (Fig. 10A) through its entire rostrocaudalextent. Labeling was not seen in more medial regions. More-over, no terminals were seen within the hypothalamus fol-lowing BDA injections into the lateral or dorsal PFC.

In contrast to BDA terminals, choleratoxin-positive hy-pothalamic neurons were far more restricted. The mostdistinct labeling was present in the tuberal division of thelateral hypothalamic area extending into the rostral partof the posterior division (Fig. 12A). These were clusters oflarge neurons stained for CT following injections into lat-eral, orbital, and medial PFC sites.

Claustrum. Large numbers of choleratoxin-labeledcells were found in the claustrum after injections into allsites (Fig. 14A–D). Labeling was observed in both parts ofthe nucleus, the ventral part (the nucleus endopiriformis)and the dorsal part, depending on the injection site. Themost abundant labeling in both parts of the nucleus wasfound after the lateral injection, with positive cells presentthroughout the whole rostrocaudal extent. In contrast, thedorsal injection produced labeled cells that were restrictedto the dorsal part while the orbital injection producedlabeling confined to the ventral part. After the medialinjection labeling was present in the dorsal part morerostrally and the ventral part caudally. BDA-positive fi-



Fig. 8. A: A low-power photomicrograph of a coronal sectionstained for Nissl showing cytoarchitectural subdivisions of the amyg-dala in the marmoset. Note the distinctive triangular shape of themagnocellular basal nucleus (Bmg), the major nucleus of the amyg-dala, that has strong connections with the lateral, orbital, and medialPFC injection sites in the marmoset (B–G). Camera lucida drawingsof BDA terminal labeling in the amygdala following injections intolateral (B; case L.bda-1), orbital (C; case O.bda-1), and medial (D; caseM.bda-1) PFC show that in all cases the main site of termination wasin the basal magnocellular nucleus (Bmg), although many fibers of

passage can also be seen in basal, lateral, and central nuclei. Whilelateral PFC projected to the dorsal part of the Bmg (B), medial PFCprojected to more ventral parts of the Bmg (D). C,D also reveals BDAterminal labeling within the neighboring hypothalamus. Semisch-ematic drawings representing choleratoxin labeled neurons in theamygdala in E–G reveal that labeling was restricted to the basalmagnocellular nucleus (Bmg) following lateral (E; case L.ctx-1), or-bital (F; case O.ctx-2), and medial (G; case M.ctx-1) PFC injections.Scale bars � 1 mm in A; 2 mm in B–G.



bers were found throughout the claustrum after all injec-tions, indicating that connections between the PFC andclaustrum were highly reciprocal.

Neuronal and terminal labeling in thecortex

Prefrontal areas. A choleratoxin injection into any ofthe four prefrontal sites labeled neurons throughout thePFC. These connections were generally reciprocal. The mostwidespread intrinsic prefrontal connections were found after

Fig. 9. A–C: High-power photomicrographs of coronal sectionsthrough the basal forebrain illustrating labeled magnocellular neu-rons following an injection of choleratoxin into the lateral PFC (caseL.ctx-1). At the most rostral level, labeled magnocellular neuronswere found both in the horizontal limb of the diagonal band (HDB)and in the nucleus basalis (NBM) (A). In addition, magnocellularneurons stained for choleratoxin were scattered around the caudalextension of the anterior commissure (B) and in glial sheets lyingbetween the internal and external segments of the globus pallidus(arrowheads) (C). Scale bars � 400 �m.

Fig. 10. Camera lucida drawings of coronal sections at the level ofthe basal forebrain representing the pattern of termination of BDAfibers within the basal forebrain after an injection of BDA into orbital(A; case O.bda-1), medial (B; case M.bda-1), and lateral (C; caseL.bda-1) PFC. Scale bar � 2 mm.



choleratoxin and BDA injections into the lateral PFC (area12/45) that resulted in extensive labeling of both terminalsand neurons throughout most dorsal and ventral areas. Ros-trally, this included area 10 in the frontal pole, areas 8b, 8ad,and 8av and area 46 on the dorsal surface, area 12 on thelateral convexity and extending medial to lateral on theorbital surface. Caudally, labeling was seen in 6dr, 8av, and12/45, as well as on the orbital surface including rostralinsula and olfactory and gustatory cortex. Areas 24 and 32were labeled on the medial surface (Fig. 13B). Contralaterallabeling followed a similar pattern but was not so extensivein the orbital and medial prefrontal areas. Injections into themedial sector of the orbital cortex, the most restricted of allthe injection sites, produced labeling in the rostral pole, area10, adjacent to the injection site, areas 24 and 32 in medialPFC, area 13 in orbital PFC and caudal insula (Fig. 13C).Injections into the dorsal region produced labeling, rostrally,

in caudal area 9 and in a restricted region of the lateralconvexity, area 12/45. Caudally, labeling was found in pre-motor areas 6m and 6dr and medial PFC, area 32 (Fig. 13A).Injections into the medial PFC produced the heaviest label-ing in the frontal pole of all injections. It also producedlabeling in area 9 and 8ad and more caudally, in areas 6mand 6dr. Discrete pockets of labeling were also seen on theorbital surface including area 12/45 as well as more medialregions (Fig. 13D).

Temporal cortex. After a lateral injection retro-gradely labeled neurons were found in the auditory cortex,corresponding to the secondary auditory regions (AII), asdefined by Hackett and colleagues in the marmoset (Lisaet al., 2006). No labeled BDA fibers were found in thatarea. Positive neurons extended ventrally along the supe-rior temporal gyrus into polysensory cortex in and aroundthe superior temporal sulcus (STS). In this area, as well as

Fig. 11. High-power photomicrographs of cresyl-violet-stainedcoronal sections (A–C) and camera lucida drawings representing thepattern of termination of BDA fibers of adjacent sections (D–F) atthree different levels within the hypothalamus, rostral, (A,D), mid(B,E), and caudal (C,F) after an injection of BDA in medial PFC (case

M.bda-1). Labeling can be seen within the lateral (LPOA) and medial(MPOA) preoptic area, rostrally (A,D), in the lateral hypothalamicarea (LHA) and anterior hypothalamic area (AHA) at mid levels (B,E)and in the LHA and dorsomedial nucleus (DM) caudally (C,F). Scalebar � 500 �m.



in the ventrally adjacent inferotemporal/higher order vi-sual association cortex, numerous labeled neurons werefound following the lateral injection (Fig. 14B), through-out the entire rostrocaudal extent of the region. Antero-grade labeling in the inferior and superior temporal cor-tices after the lateral injection confirmed the reciprocity ofthe connections. There were also numerous labeled cells inthe temporal pole. In contrast, there was very little neu-ronal and terminal labeling of the temporal cortex follow-ing the dorsal injection (Fig. 14A) except for a restrictedset of neurons around the polysensory region of the supe-rior temporal sulcus, caudally, and another restrictedgroup of neurons in the secondary auditory region (AII).Small clusters of labeled neurons and terminals werefound in the inferior temporal cortex, rostrally, andaround the superior temporal sulcus, caudally, after theorbital injection (Fig. 14C). There was also considerablelabeling in the temporal pole and both the inferior tempo-ral cortex and superior temporal sulcus following the me-dial injection (Fig. 14D). Only after a lateral injection wasthere labeling in the visual areas of the caudal temporallobe including the middle temporal area (MT) and themiddle temporal crescent (MTc), as defined by Rosa andTweedale (1998).

Parietal cortex. Labeled neurons were found withinthe parietal cortex including somatosensory area SII andthe posterior parietal cortex following the lateral and dor-sal injections only (Fig. 14A,B). The lateral area receivedthe larger and more widespread projection from posteriorparietal cortex including both rostral (Fig. 14B, AP7.5)and caudal sectors (Fig. 14B, AP4). In contrast, the dorsalsite received from a more restricted region, caudally, inand around the shallow groove lying on the cortical sur-face (see Fig. 14A). Terminal labeling mirrored this gen-eral pattern, with lateral injections resulting in the pres-ence of BDA labeling more rostrally than that followingdorsal injections. Unlike the neuronal labeling, terminallabeling was always restricted to narrow columns of fiberswithin posterior parietal cortex.

Occipital cortex. Labeled cells were found within anumber of rostral extrastriate regions of the occipital cor-tex following injections of choleratoxin into the lateralsite. These regions in the marmoset have been definedfunctionally by Rosa and colleagues (Rosa and Elston,1998; Rosa and Tweedale, 2000; Rosa et al., 2005) andtheir nomenclature is used here in defining the areas oflabeling. Scattered neurons were present in the ventralvisual areas (Fig. 14B, AP1,1.5) including V2, as well asadjacent regions probably corresponding to VLP and VLAof Rosa and Tweedale (2000). In addition, labeling wasalso present in the dorsal convexity (Fig. 14B, AP2) in aregion that likely corresponds to area DA, as defined byRosa et al. (2005). Labeling was not seen following injec-tions into the other sites except for some discrete labelingof neurons following the medial injections, in a regionadjacent to area V2 (Fig. 14D, AP0) that may correspondto a prostriate region as defined by Rosa and Tweedale(2000). Complementing the distribution of cells from thelateral and medial sites were BDA-positive fibers from thesame sites.

Insular cortex. Restricted connections with the in-sula were seen after the lateral, orbital, and medial injec-tions of choleratoxin. Labeled neurons were found in themore caudal, lateral orbital region, extending posteriorlyinto the caudal dysgranular and agranular areas under-lying the claustrum (B–D in Figs. 13, 14). BDA-positivefibers were present in the same areas as choleratoxin-positive neurons, suggesting reciprocity of connections be-tween the insula and PFC.

Cingulate cortex. Areas of the cingulate cortex werelabeled after an injection of choleratoxin or BDA into eachof the prefrontal sites, highlighting the reciprocity of pro-jections and the widespread connectivity between cingu-late cortex and prefrontal cortex. The lateral injectionsproduced cell body and terminal labeling throughout therostrocaudal extent of areas 24 and 23 as well as theretrosplenial cortex (Fig. 14B). Similarly, the dorsal injec-tions also produced extensive labeling in area 23 andretrosplenial cortex, but unlike the lateral injections therewas almost no labeling in area 24. In contrast, after or-bital injections cell body and terminal labeling was limitedmainly to area 24, with a few labeled cells in rostralretrosplenial cortex but little labeling in area 23 (Fig.14C). Medial injections also resulted in little labeling inarea 23, although there was considerable cell body andterminal labeling in rostral area 24 and throughout theretrosplenial cortex (Fig. 14D).

Parahippocampal cortex. The cytoarchitectonicallydistinct regions of the parahippocampus are illustrated in

Fig. 12. A: A high-power photomicrograph of a coronal sectionthrough the hypothalamus at AP7.5 stained with choleratoxin.Choleratoxin-labeled neurons can be seen in the lateral hypothalamus(LHA) following an injection of choleratoxin into the lateral PFC (caseL.ctx-1). The position of these choleratoxin-labeled neurons is illus-trated in the low-power photomicrograph of an adjacent coronal sec-tion stained with Giemsa (B). Scale bars � 400 �m in A; 2 mm in B.



Fig. 13. Outline drawings of representative coronal sections atfour different AP levels through the PFC, illustrating the distributionof labeled neurons following injections of choleratoxin into the dorsal(case D.ctx-2), lateral (case L.ctx-1), orbital (case O.ctx-1), and medial(case M.ctx-1) PFC. The dark shaded area illustrates the location ofthe injection site. Numbers within dorsolateral prefrontal regions

represent cytoarchitectonic subdivisions based on parcellation of thecerebral cortex of the marmoset by Burman et al. (2006). Numberswithin medial and orbital regions are based on Brodmann’s parcella-tion of the marmoset prefrontal cortex and our own unpublishedobservations. Scale bar � 5 mm.

Fig. 14. Outline drawings of representative coronal sections atdifferent AP levels (A0-A11.5) through the cerebral cortex, illustrat-ing the distribution of labeled neurons following injections of cholera-toxin into the dorsal (case D.ctx-2), lateral (case L.ctx-1), orbital (caseO.ctx-1), and medial (case M.ctx-1) PFC (A–D). Numbers represent

cytoarchitectonic subdivisions in the marmoset based on the parcel-lation of extrastriate visual areas by Burman et al. (2006), the par-cellation of auditory areas by de la Mothe et al. (2006), and theparcellation of the remaining regions by Brodmann (1909) and ourown unpublished findings. Scale bar � 5 mm.

Figure 14 (Continued)


Figure 15A. Some positive neurons were found in entorhi-nal and perirhinal regions of the anterior parahippocam-pal cortex as well as the presubiculum and parasubiculum

following injections into the lateral (Fig. 15B), orbital, andmedial, but not dorsal sites. However, only after medialand lateral injections were neurons observed in the pos-terior parahippocampal region, area TH. A very sparsenumber of positive BDA fibers were observed in the ante-rior parahippocampal region but only after an injectioninto the orbital site.

Hippocampal formation. Retrograde labeling in thehippocampal formation was found only after orbital andmedial injections. After the orbital injection pyramidalneurons were labeled in the CA1 field, prosubiculum, andsubiculum throughout the rostrocaudal extent of the hip-pocampus, extending into the retrocommissural part.After the medial injection, labeled neurons were alsofound in the CA1 field, the subiculum, and in the tran-sitional zone between the two, the prosubiculum (Fig.15C), but they were limited to the more rostral parts ofthese structures. No labeling was found in any territoryof the hippocampal formation following injections ofcholeratoxin into the dorsal or lateral PFC, nor follow-ing injection of the anterograde tracer BDA into anyprefrontal area.

DISCUSSION

This is the first study to compare the cortical and sub-cortical forebrain connections of distinct granular, dys-granular, and agranular regions within the dorsal, lateral,orbital, and medial PFC of the marmoset. The injectionsites were relatively large, spanning more than one cyto-architectonically distinct region, so as to provide an over-all impression of the differences in connections betweenthe granular, agranular, and dysgranular regions of thisNew World species of primate.

Pattern of connections of marmosetdorsal PFC

The dorsal injection site covered an area of six-layeredfrontal cortex with a well-developed granular layer IV inan area defined by Brodmann as area 8. It correspondsprimarily to area 8Ad, impinging on the more medialaspects of area 8Av, as defined by Burman et al. (2006)and following the division proposed by Petrides and Pan-dya (1999) in macaques. Very little tracer extended intoneighboring area 8B, dorsal area 9, anteriorly, or area 6dr,posteriorly. Within the frontal lobes this region was con-nected primarily to areas on the lateral surface. Caudally,it had reciprocal connections with adjacent premotor re-gions, areas 6m and 6dr, and rostrally it had restrictedconnections with the lateral region (area 12/45) and thedorsomedial region (area 9). Also, caudally it had recipro-cal connections with area 32, just anterior to the genu ofthe corpus callosum. Other cortical areas connected recip-rocally with this region included area 23 and retrosplenialcortex, posterior parietal area 7, polysensory superiortemporal cortex around the STS, and secondary somato-sensory cortex (Krubitzer and Kaas, 1990). With respect tothe posterior parietal cortex, the dorsal PFC in the mar-moset received projections from a particular subdivision ofarea 7, situated around the shallow cortical impressionwhich may be equivalent to the intraparietal sulcus ofhigher anthropoids (see Fig. 14A). Areas VIP and LIP(Preuss and Goldman-Rakic, 1991b) lie within the in-traparietal sulcus in macaque and these same areas have

Fig. 15. A low-power photomicrograph of a coronal section stainedfor Nissl is presented in A (Case M.ctx-1), illustrating the approxi-mate location of the boundaries of the various subdivisions of thehippocampal formation and parahippocampal gyrus as indicated byarrowheads. B,C: High-power photomicrographs of coronal sectionsthrough the anterior hippocampus stained for choleratoxin, illustrat-ing labeled neurons within the parasubiculum and entorhinal cortexfollowing an injection of choleratoxin into the lateral PFC (caseL.ctx-1) and the prosubiculum (PoS), subiculum (S), and CA1 fieldfollowing an injection of choleratoxin into the medial PFC (case M.ctx-1), respectively. Scale bars � 2 mm in A; 500 �m in B,C.



been recognized in the relatively smaller brain, strep-sirhine primate, galago, occupying the area around theshallow groove that divides the surface of the parietalcortex longitudinally into a dorsal and ventral part(Preuss and Goldman-Rakic, 1991b). Since in both ma-caque and galago the territory around the intraparietalsulcus is occupied by homologous areas, it is possible thatthese same areas are represented in and around the shal-low groove in the marmoset.

This pattern of cortical connectivity is consistent withthat described by Petrides and Pandya (1999) for area 8Adin the macaque. In this species, too, area 8Ad receivesprojections originating mainly from higher-order cingu-late regions, lateral parietal cortex, and the superior tem-poral cortex, including auditory areas. This pattern is incontrast to the extensive connectivity that neighboringarea 8Av has with extrastriate regions in the macaqueand is consistent with a more integrative polymodal rolefor 8Ad.

In addition to its cortical connections, the dorsal injec-tion site sent extensive projections to the caudate nucleuswhich occupied a central position in the head of the nu-cleus, both dorsal, and often more lateral to terminallabeling following a tracer injection into the lateral PFC.However, there was extensive overlap between the dorsaland lateral injections sites, particularly in the rostralhead of the caudate nucleus. These findings are, in gen-eral, consistent with those reported in macaque in whichtracer injections into dorsolateral prefrontal cortex, whichvariably include regions of areas 8, 9, and 46 produceextensive labeling in the central regions of the caudatenucleus (Selemon and Goldman-Rakic, 1985; Yeterian andPandya, 1991). This pattern of frontostriatal projectionfrom area 8 is in contrast to that from the neighboringpremotor areas (area 6), which innervate primarily theputamen and extreme lateral caudate (McFarland andHaber, 2000). It was this differential projection patternbetween premotor and the more rostral prefrontal associ-ation regions that led to a scheme that recognized a cen-tral associational zone along with a limbic-related ventro-medial zone and a sensorimotor dorsolateral zone (Parent,1990; Haber et al., 1995).

The only other forebrain structure that was intercon-nected extensively with this dorsal injection site was thethalamus. Like all the other regions of PFC examined, thedorsal site had extensive reciprocal connections with theMD nucleus of the thalamus. However, the multiformregion of the MD, in the ventrolateral corner of MD, whichhas been shown to project to dorsal area 8 in macaques(Giguere and Goldman-Rakic, 1988; Barbas et al., 1991),could not be identified in the marmoset. Instead, labelingwas found in the dorsolateral sector of the parvocellularregion, overlapping, but in general more dorsal and lat-eral, to the projection to lateral PFC. Besides the MD, thedorsal PFC site was reciprocally connected to a ventrome-dial and a dorsolateral sector of the VA and receivedprojections from a number of thalamic midline nuclei.Along with the other three injection sites, it also sentprojections into the medial pulvinar, but unlike the otherthree injection sites it was the only site to receive inputback from the medial pulvinar. Unlike adjacent premotorregions (Brysch et al., 1990) dorsal PFC did not receive aprojection from the thalamic motor nuclei, e.g., ventrolat-eral or ventroposterior nuclei. This pattern of thalamicconnections between the dorsal PFC and the thalamus in

the marmoset is similar to that reported between dorsalprefrontal areas, including areas 8, 9, and 46, and thethalamus in macaque (Barbas et al., 1991; Romanski etal., 1997) except that in the latter these more cytoarchi-tectonically differentiated dorsal regions of PFC did notreceive from midline nuclei (Barbas et al., 1991; Barbas,1995). Consistent with the macaque, this dorsal region inthe marmoset was not connected with the anterior tha-lamic nuclei.

Pattern of connections of marmoset lateralPFC

The lateral prefrontal injection site included cortex onthe lateral convexity of the anterior PFC, within an areadefined by Brodmann as area 9. However, according toBurman et al. (2006) the cytoarchitectonics of this regionin the marmoset resemble those of the macaque’s area12/45 (Walker, 1940; area 12/47 of Petrides and Pandya,2002). Of all four frontal sites examined, this area ap-peared to have the most extensive connections within thefrontal lobes, although it must be borne in mind that thiswas also the largest of the injection sites. It received largebilateral projections from area 8 (8B, 8Ad, and 8Av) andpremotor area 6 (m and dr). In addition, it received fromrostral dorsolateral regions, including areas 46, 9, and 10.The rostral and caudal regions of orbitofrontal cortex alsosent dense projections to this lateral region, including theagranula insula and olfactory cortex. All these connectionswere reciprocal. There were no connections with the ros-tral and ventral regions on the medial surface of theprefrontal cortex, apart from a few in the caudal aspect ofarea 32, just at the level of the genu of the corpus callo-sum. This pattern of connectivity is relatively similar tothat shown for area 12/47 in macaques (Carmichael andPrice, 1996; Petrides and Pandya, 1999). In particular, thelack of connectivity with medial prefrontal regions is con-sistent with the proposal that much of area 12 in ma-caques is part of an orbital prefrontal network, distinctfrom an adjoining medial prefrontal network. Only area12o in macaques has connections with both networks(Carmichael and Price, 1996).

Besides having the most extensive connections withinthe frontal lobes, this lateral prefrontal region also hadthe most widespread cortical connections outside of thefrontal lobes of all four sites. This again is consistent withthe pattern of connectivity reported for area 12/47 in therhesus monkey by Petrides and Pandya (1999) and Car-michael and Price (1995a,1995b). Of particular signifi-cance were the extensive reciprocal connections with sen-sory association regions. These included the visualassociation areas of the inferotemporal cortex, the pol-ysensory region, in and around the superior temporalsulcus and auditory secondary and association regions.Moreover, the lateral region also received and sent con-nections to the visual extrastriate regions including MTand MTc, a dorsal visual area (DA), and ventral visualareas V2, VLP, and VLA. The latter results are consistentwith the study of Burman et al. (2006) in which tracer wasplaced into many of these extrastriate regions of the mar-moset and shown to result in neuronal labeling withinarea 12/45. Petrides and Pandya (1999) have establishedthat a major difference in the cortical inputs to area 12/47and 45 in the macaque is the far greater input into area 45from auditory cortex, compared to area 12/47. The findingthat this lateral prefrontal region in the marmoset had



quite extensive connections with both visual and auditoryassociation areas may suggest therefore that this region inthe marmoset includes areas comparable to that of both 12and 45 in the macaque, as has been proposed by Burmanet al. (2006), based on their cytoarchitectonic analysis.Extensive neuronal and terminal labeling was also evi-dent in posterior parietal cortex, not only within the re-stricted region of posterior parietal cortex (area 7) aroundthe shallow groove, as was the case for dorsal PFC, butalso in territory above and below this region. The lateralregion was also connected reciprocally with limbic associ-ation regions including the rostral and caudal cingulateareas and retrosplenial cortex. Moreover, while appar-ently having no direct connections with the hippocampusor subiculum, lateral PFC did receive projections from theposterior parahippocampal cortex, although projectionsfrom this lateral prefrontal region back to the parahip-pocampus were very scarce. In contrast to the marmoset,it has been reported in macaques that perirhinal, but notparahippocampal, cortex is reciprocally connected witharea 12r and other orbital areas related to the ‘orbital’network (Kondo et al., 2005). However, the posterior para-hippocampal cortex was reported to be connected to area12/47 by Petrides and Pandya (2001). The absence of di-rect connections from the hippocampal formation includ-ing the CA cell fields, the dentate gyrus, subiculum andprosubiculum to lateral granular PFC in marmosets isconsistent with the findings of Barbas and Blatt (1995) ofa paucity of projections from the hippocampal formation tosix-layered (eulaminate) dorsolateral PFC in rhesus mon-keys.

Subcortically, the lateral PFC sent projections to thedorsal head of the caudate nucleus, sandwiched betweenthe dorsal projections dorsolaterally and the orbital andmedial projections medially, occupying the central posi-tion, in agreement with tracing studies of area 12/47 in themacaque (Selemon and Goldman-Rakic, 1985; Yeterianand Pandya, 1991; Ferry et al., 2000). As with the otherinjection sites these projections ran longitudinally alongthe full length of the head and body of the caudate nucleusas well as entering the tail. Unlike the dorsal injectionsite, however, but like the orbital and medial sites, thelateral PFC had reciprocal connections with the amyg-dala. Our unpublished findings suggest that inputs fromthe amygdala are particularly dense within the more ven-tral aspects of this region that lie on the orbital surface,consistent with studies in macaque that report a markedreduction in the density of amygdala projections as theyextend from the ventrolateral surface onto the dorsolat-eral surface of the prefrontal cortex (Amaral et al., 1992;Carmichael and Price, 1995a). In contrast to the orbitaland medial sites, however, this lateral region did not haveextensive connections with the magnocellular division ofthe MD nucleus of the thalamus. In macaque monkeys athree-way interaction exists between the prefrontal cor-tex, MD thalamus, and amygdala such that those prefron-tal regions that receive the densest innervation from theamygdala, i.e., orbitofrontal and medial prefrontal cortex,are also interconnected entirely with the magnocellulardivision of the MD nucleus of the thalamus, the sector ofMD thalamus that receives a major input from the amyg-dala. Although the lateral prefrontal regions 12/47 in themacaque are recipients of amygdala input they differ fromthe medial orbitofrontal and medial prefrontal regions inthat they have connections with both the magnocellular

and parvocellular divisions of the MD nucleus (Barbas etal., 1991; Amaral et al., 1992; Ray and Price, 2000). In themarmoset, lateral prefrontal regions appear to be con-nected entirely with the parvicellular division of the MDnucleus of the thalamus. However, as it will be seen below,the orbital and medial injection sites in the marmoset areconnected almost exclusively with the magnocellular divi-sion of MD, although whether the latter is the site of thestrongest amygdala projections in the marmoset remainsto be determined.

In addition to the MD nucleus, the lateral region wasalso connected reciprocally with the VA nucleus of thethalamus and the medial pulvinar and received afferentinnervation from the anteromedial and anteroventral nu-clei of the thalamus. As the latter receive input from thehippocampus, this could provide the lateral PFC withindirect hippocampal input. Overall, the pattern of tha-lamic connections with the lateral injection site was sim-ilar to that described in a previous study on thalamocor-tical projections in the marmoset (Brysch et al., 1990),although the precise positioning of the injection site dif-fered somewhat from the present study.

The lateral PFC also received input from the hypothal-amus, specifically, the tuberal division of the lateral hy-pothalamic area. The former was composed of large mag-nocellular neurons that may correspond to thehistaminergic neurons that send diffuse projections to thecerebral cortex and that have been shown to be affected inAlzheimer’s disease and schizophrenia (Fernandez-Novoaand Cacabelos, 2001). Consistent with anatomical studiesin the macaque examining dorsolateral isocortical regions,no projections were found from the lateral PFC back to thehypothalamus. Only orbital and medial PFC sent projec-tions to the hypothalamus (Ongur et al., 1998, Rempel-Clower and Barbas, 1998).

Finally, tracer injection into the lateral PFC resulted incell labeling in the basal forebrain region including thehorizontal limb of the diagonal band and the nucleusbasalis of Meynert. In all cases the structure of the cellsand their topographical arrangement suggested that theywere the magnocellular cholinergic neurons (see Everitt etal., 1988).

Pattern of connections of marmoset orbitalPFC

The injection site on the orbital surface was confined tothe medial, dysgranular regions of cortex. More lateralregions of orbital PFC, including a rostral granular areaand a more caudal dysgranular area, were not included.From our own observations, this medial orbital region wascomposed of two cytoarchitectonically distinguishable ar-eas in both of which layers II and IV were poorly definedand the supragranular layers were either wider than, orequivalent in width to, the infragranular layers. Based ondescriptions of cytoarchitectonics of the orbitofrontal cor-tex in macaque (Brodmann, 1909; Walker, 1940; Car-michael and Price, 1994) and our own analysis of thisregion in marmosets this region in the marmoset is prob-ably most similar to areas 11 and 13 of macaque. It nei-ther received nor sent projections to the dorsolateral re-gions of prefrontal cortex. It was exclusively connected,reciprocally, with caudal regions of the orbitofrontal cor-tex and ventral regions of the medial prefrontal cortex. Italso had connections with the agranular insula cortex andolfactory cortex, as well as dysgranular insula. Outside of



the frontal lobes this orbital region had reciprocal connec-tions with the inferior and superior temporal cortex in-cluding visual association and polysensory cortex. Com-pared with Old World monkeys, the extent of orbitalconnections with sensory association cortices appearedrather limited (Carmichael and Price, 1995a); however,this could be explained by the rather restricted spread ofthe tracer, which, as mentioned above, did not extend verylaterally. Like medial PFC, but unlike lateral and dorsalPFC, it had no obvious connections with either posterior oranterior parietal cortex. However, it had reciprocal con-nections with limbic cortices including caudal cingulate,parahippocampal cortex, as well as the hippocampal for-mation, although connections with retrosplenial cortexwere sparse. It also had connections with subcortical lim-bic structures including reciprocal connections with theamygdala and efferent projections to the hypothalamusand ventral striatum. Its hypothalamic projections werefound more laterally to those from the medial injectionsite, primarily innervating the lateral hypothalamic areathrough its entire rostral-caudal extent, consistent withfindings in the macaque (Rempel-Clower and Barbas,1998; Ongur et al., 1998). Like the medial PFC, it too didnot appear to receive projections from the hypothalamus,in contrast to that of the macaque (Ongur et al., 1998).

The orbitofrontal injection site produced a very specificpattern of terminal labeling in the striatum. Terminalswere found in the medial caudate nucleus extending downinto the medial region of the nucleus accumbens. In ratsthe nucleus accumbens has been divided into a shell andcore region that differ on morphological (Meredith et al.,1992), electrophysiological (Pennartz et al., 1992), neuro-chemical (Jongen-Relo et al., 1994), and behavioral (Par-kinson et al., 1999, 2000) grounds. Although calbindinstaining has been used to identify a core and shell regionin the nucleus accumbens of the rat (Zahn and Brog, 1992;Zahn and Heimer, 1993), and to a lesser extent the ma-caque (Meredith et al., 1996), calbindin staining appearedmore patchy in the marmoset, and consistent with theobservations by Meredith et al. (1996) the ventral andmedial limits of the nucleus accumbens were not consis-tently poor in calbindin staining throughout the rostro-caudal extent of the nucleus. However, terminal labelingin the ventral and medial limits of the nucleus accumbenswas very sparse following tracer injection into the orbito-frontal region, in contrast to that of the medial prefrontalinjection site, and overall the pattern of striatal connec-tivity was very similar to that described for the macaquemedial and orbital prefrontal cortex (Haber et al., 1995;Ferry et al., 2000).

Like the medial PFC, the marmoset orbital PFC hadextensive reciprocal connections with the magnocellulardivision of the MD nucleus of the thalamus, occupying aposition within the MD nucleus remarkably similar tothat seen for medial orbitofrontal connections in the ma-caque (Ray and Price, 1993). It also had reciprocal connec-tions with the VA nucleus and, to a lesser extent, it re-ceived input from the anteromedial and anteroventralnuclei as well as midline thalamic nuclei.

Pattern of connections of marmoset medialPFC

The medial PFC injection site comprised an agranularregion of cortex on the medial surface of the anteriorfrontal lobes including areas that Brodmann defined as 32

and 24. From our own observations it encompassed twodistinct cytoarchitectonic areas, both of which lackedgranular layers 2 and 4 and had a high cell density inlayers III and V. In addition, supragranular layers wereconsiderably thicker than infragranular layers. These re-gions may correspond to prelimbic and infralimbic re-gions, areas 32 and 25, respectively, lying in the caudalaspects of the medial cortical wall in macaques (Preussand Goldman-Rakic, 1991a; Preuss, 1995). This medialregion had connections with medial prefrontal areas bothrostral and caudal to the injection site. The rostral label-ing extended into the frontal pole, into an area reported byBurman et al. (2006) to be comparable to area 10 inmacaques. It had restricted connections with regions onthe orbital surface including the medial dysgranular re-gion rostrally and the agranular regions caudally. It alsohad discrete connections with the ventral aspects of thelateral convexity (area 12/45). This latter observation sug-gests that this ventral region of area 12 in marmosetscorresponds to area 12o in macaque, given that area 12o isthe only sector of area 12 to have projections with themedial PFC (Carmichael and Price, 1996). Dorsal granu-lar PFC was the only other prefrontal region to havereciprocal connections with the medial PFC, a region thatincluded association areas 9 and 8Ad and premotor re-gions 6m and 6dr.

Outside of the frontal lobes this medial prefrontal re-gion had prominent reciprocal connections with the tem-poral pole, consistent with the macaque (Kondo et al.,2003). However, unlike the macaque these were not re-stricted to the more dorsal regions of the temporal pole,regions that in the macaque are associated with auditoryconnectivity (Moran et al., 1987). Instead, they extendedinto ventral regions, too, regions that in macaque havebeen associated with visual input (Moran et al., 1987). Themedial PFC also had reciprocal connections with rostraland caudal inferior and superior temporal cortex includ-ing visual association cortex and polysensory cortex in andaround the STS. Although a similar pattern of connectiv-ity between the medial prefrontal region and polymodalsensory areas in the STS has been reported in the ma-caque, medial prefrontal connectivity with visual associa-tion areas in the inferotemporal cortex have not beendescribed in the macaque (Carmichael and Price, 1995b).However, medial prefrontal connectivity with visual asso-ciation areas in the marmoset may be seen as being con-sistent with its connectivity with ventral regions of thetemporal pole that, as mentioned above, are connectedwith visual regions in the macaque, although this remainsto be shown in the marmoset. In addition, marmoset me-dial prefrontal cortex had relatively dense reciprocal con-nections with limbic cortical structures including caudalcingulate cortex, retrosplenial cortex, and parahippocam-pal cortex, again relatively consistent with that shown formacaque (Carmichael and Price, 1995b; Kondo et al.,2005). Moreover, unlike lateral PFC, medial PFC had adirect input from the CA1 fields of the hippocampus andthe subiculum, also consistent with that seen in macaques(Barbas and Blatt, 1995), although unlike macaques therewas no evidence for a corresponding reciprocal projection.

Subcortically, the medial PFC received afferents fromthe amygdala, primarily from more caudal and ventralparts of the magnocellular division of the basal nucleuscompared to the orbital and lateral PFC, which receivedinputs from more rostral parts of the nucleus. Unlike the



lateral PFC it sent projections to the hypothalamus, spe-cifically the medial hypothalamic territory where termi-nals were found in the medial preoptic area, anteriorhypothalamic area, dorsomedial and posterior nuclei, ar-eas that have autonomic and endocrine function, as wellas the lateral preoptic and lateral hypothalamic areas.This pattern of hypothalamic terminations is consistentwith findings in the macaque in which medial prefrontalareas including 32 and 25 were shown to send projectionsinto these medial hypothalamic regions (Ongur et al.,1998; Rempel-Clower and Barbas, 1998). In contrast, itappears to be distinct from findings in rats, in whichmedial prefrontal regions are reported to send projectionsinto the lateral hypothalamic area only (Ongur et al.,1998). However, unlike studies in macaque, there ap-peared to be no projections from the hypothalamus intothe medial prefrontal area of the marmoset, although thiswill need to be confirmed in future studies.

There was a large projection from medial PFC to themedial edge of the caudate nucleus, the ventromedial pu-tamen, and the nucleus accumbens. Anteriorly, in thecaudate nucleus and putamen this projection overlappedconsiderably with that from the orbital PFC. However,more posteriorly the projections remained relatively seg-regated in the caudate nucleus, with the medial projectionlying most medially, adjacent to the wall of the lateralventricle. The medial PFC also projected into the nucleusaccumbens and, unlike the orbital PFC, it innervated allregions of the nucleus accumbens, central, medial, andventral. This pattern is very similar to that shown for themacaque (Haber et al., 1995; Ferry et al., 2001).

Comparable to all other injection sites the medial PFChad pronounced reciprocal connections with the MD nu-cleus of the thalamus. However, unlike the dorsal andlateral PFC that were connected to the parvocellular di-vision, the medial, like the orbital site, was connected tothe magnocellular division, occupying a position dorsal tothat of the orbital PFC, comparable to that of macaques(Ray and Price, 1993). In addition, medial PFC had recip-rocal connections with the VA nucleus and midline nuclei,consistent with findings in rhesus monkeys (Barbas et al.,1991). While the medial PFC also sent projections into themedial pulvinar, there did not appear to be a projectionfrom the pulvinar to the medial PFC, in contrast to thereciprocal projections with medial regions of the PFC de-scribed in rhesus monkeys (Barbas et al., 1991; Romanskiet al., 1997).

Functional considerations

The dorsal injection site, corresponding to area 8Ad, asdefined by Burman et al. (2006), had the most restrictedset of connections, being associated primarily with regionsinvolved in visuospatial functions within the posteriorparietal cortex. However, its connections with theauditory-related areas of the superior temporal gyrus sug-gest that this region is also involved in auditory process-ing. Moreover, it may also have some involvement in emo-tional processing, or at least it may be regulated by theemotions, given its significant connections with the caudalcingulate regions and, in particular, the retrosplenial cor-tex (Maddock, 1999). This pattern of connectivity is re-markably similar to that described for area 8Ad in themacaque (Petrides and Pandya, 1999) and distinguishes itfrom neighboring 8Av, which is hypothesized to corre-spond to the marmosets frontal eye field (Burman et al.,

2006). The only difference in this region connectivity inthe marmoset to that of the macaque is the existence ofsomatosensory connections. Whether this may be attrib-uted to the injection site impinging on neighboring regionsremains to be determined. Overall, though, the pattern ofconnectivity of area 8Av suggests it has integrative poly-modal functions.

The granular region on the lateral convexity, corre-sponding to area 12/45, as defined by Burman et al. (2006),had the most widespread connections both within andoutside of the frontal lobes. Not only did this region haveextensive connectivity with posterior parietal cortex, im-plicating this lateral prefrontal region in visuospatial andattentional functions, it also had extensive connectionswith temporal cortical structures involved in higher-orderprocessing of visual form and audition, as well as poly-modal processing. Its connectivity with limbic regions in-cluding the amygdala, rostral and caudal cingulate cortex,retrosplenial cortex, and parahippocampus also impli-cates this region in emotional processing. Thus, overall,this pattern of cortical connectivity is similar, not to area9, as originally proposed by Brodmann (1909), but to thatof area 12/47 and 45 in macaques and is thus more con-sistent with the recent cytoarchitectonic analysis of thisregion in marmosets by Burman et al. (2006). This ana-tomical consistency between species is complemented by afunctional consistency, in that lesions of this region inmarmosets has been shown to disrupt attentional set-shifting (Dias et al., 1996) and activation of the ventrolat-eral convexity region accompanies the shifting of atten-tional sets in humans and macaques (Nakahara et al.,2002). Together, these findings are consistent with thisregion’s proposed role in higher-order rule learning (Rob-erts, 2007).

As expected from studies in other species, the dysgranu-lar and agranular regions of the orbital and medial areasof the PFC were highly interconnected with each other aswell as with limbic structures, consistent with findings inthe rhesus monkey (Morecroft et al., 1992; Carmichaeland Price, 1995a,b). The orbitofrontal cortex had exten-sive connectivity with higher-order visual processing re-gions within the inferotemporal cortex and with the poly-modal processing region within the STS. It also hadconnections with the rostral and caudal cingulate regionsand the amygdala, the latter both directly and indirectlyvia the magnocellular division of the MD thalamus. Fi-nally, it had major projections to the nucleus accumbens.This pattern of connectivity is consistent with the involve-ment of the orbitofrontal cortex in processing the affectivesignificance of environmental stimuli and contributing tomotivated behavior. The medial PFC, on the other hand,had extensive connectivity with retrosplenial cortex aswell as rostral and caudal cingulate cortex, agranularinsula cortex, and the hypothalamus, consistent with itsrole in emotional regulation.

ACKNOWLEDGMENTS

We thank Dr. R.M. Ridley for supplying marmosets, J.Bashford for photographic assistance, and A. Newman, I.Bolton, and L. Hopewell for help with preparation of thetable and figures. This is a publication from the Universityof Cambridge Behavioural and Clinical Neuroscience In-stitute.



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Forebrain connectivity of the prefrontal cortex in the marmoset monkey (Callithrix jacchus): An...

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