Calretinin neurons in human medial prefrontal cortex (areas 24a,b,c, 32?, and 25)

Calretinin Neurons in HumanMedial Prefrontal Cortex(Areas 24a,b,c, 328, and 25)

PAUL L.A. GABBOTT,* PAUL R.L. JAYS, AND SARAH J. BACON

University Department of Pharmacology, Oxford University,Oxford OX1 3QT, United Kingdom

ABSTRACTThe calcium-binding protein calretinin (CR) is present in a subpopulation of local-circuit

neurons in the mammalian cerebral cortex containing g-aminobutyric acid. This lightmicroscopic investigation provides a detailed qualitative and quantitative morphologicalanalysis of CR-immunoreactive (CR1) neurons in the medial prefrontal cortex (mPFC; areas24a,b,c, 328, and 25) of the normal adult human.

The morphology of CR1 neurons and their areal and laminar distributions wereconsistent across human mPFC. The principal organisational features of CR1 labelling werethe marked laminar distribution of immunoreactive somata and the predominantly verticalorientation of labelled axon-like and dendritic processes. Several types of CR1 neurons werepresent in layer 1, including horizontally aligned Cajal-Retzius cells. In layers 2–6, CR1

neurons displayed a variety of morphologies: bipolar cells (49% of CR1 population), verticallybitufted cells (35%), and horizontally bitufted cells (3.5%). These neuron types were mainlylocated in layer 2/upper layer 3, and their dendritic processes were commonly aspiny andsometimes highly beaded. Aspiny (8%) and sparsely spiny multipolar (5%) CR1 neurons werealso found. The mean somatic profile diameter of CR1 cells was 11.6 6 0.3 µm (mean 6 S.D).

CA1 puncta formed pericellular baskets around unlabelled circular somatic profiles inlayers 2/3 and around unlabelled pyramidal-shaped somata in layers 5/6. The somatic sizes ofthese unlabelled cell populations were significantly different. Immunolabelled puncta werealso found in close contact with CR1 somata.

Cortical depth distribution histograms and laminar thickness measurements defined theproportions of the overall CR1 cell population in each layer: 7% in layer 1, 78% in layers 2/3,14% in layers 5/6, and 1% in the white matter. In the tangential plane, CR1 neurons weredistributed uniformly at all levels of the cortex. By using stereological counting procedures onimmunoreacted Nissl-stained sections, CR1 neurons were estimated to constitute a mean8.0% (7.2–8.7%) of the total neuron population in each cortical area. These data are comparedwith similar information obtained for the mPFC in monkey and rat (Gabbott and Bacon[1996b] J. Comp. Neurol. 364:657–608; Gabbott et al., [1997] J. Comp. Neurol. 377:465-499).This study provides important morphological insights into a neurochemically distinctsubclass of local-circuit inhibitory neurons in the human mPFC. J. Comp. Neurol. 381:389–410, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: cingulate and limbic cortex; calcium-binding proteins; g-aminobutyric acid; corticalmodules; vertical inhibition

Neural networks in the mammalian cerebral cortex areprimarily composed of interconnected populations of exci-tatory pyramidal projection cells and of nonpyramidallocal-circuit neurons that contain the inhibitory neurotrans-mitter g-aminobutyric acid (GABA). Cortical interneuronsnot only provide strategic synaptic innervation to specificcellular compartments (e.g., axon-initial segments, so-

Contract grant sponsor: MRC; Contract grant number: G9312936N;Contract grant sponsor: Royal Society; Contract grant number: RSRG15788.*Correspondence to: Paul L.A. Gabbott, University Department of Phar-

macology, Oxford University, Mansfield Road, Oxford OX1 3QT, UnitedKingdom. E-mail: [email protected] 3 September 1996; Revised 2 December 1996; Accepted 10

December 1996

THE JOURNAL OF COMPARATIVE NEUROLOGY 381:389–410 (1997)

r 1997 WILEY-LISS, INC.

mata, and dendrites) of pyramidal neurons, but they alsoinnervate other interneurons (Jones et al., 1994). Subpopu-lations of electrophysiologically characterised andmorpho-logically identified local-circuit neurons can be distin-guished by the presence of the calcium-binding proteinsparvalbumin (PV) and calbindin D-28K (CB; Kawaguchiand Kubota, 1993, 1996). Evidence exists demonstratingthat PV is localised in large basket neurons and inchandelier (axoaxonic) cells, whereas CB immunoreactiv-ity is expressed by double-bouquet, Martinotti, and neuro-gliaform neurons (for references, see Table 3 in Gabbottand Bacon, 1996a; see also Del Rio and DeFelipe, 1996).By contrast, the calcium-binding protein calretinin (CR;

Rogers, 1987) is present in bipolar cells, neurons resem-bling double-bouquet cells, multipolar cells, a type of smallbasket neuron, and a distinct population of cells in layer 1that includes Cajal-Retzius neurons (Glezer et al., 1992;Conde et al., 1994; Belichenko et al., 1995; Fonseca andSoriano, 1995; Gabbott and Bacon, 1996a; Martin andMeskenaite, 1997; Gabbott et al., 1997). Of functionalsignificance is the finding that, unlike CB- and PV-containing neurons and their synaptic connectivities, local-circuit neurons in human prefrontal cortex that containCR are comparatively resistant to the degenerativemecha-nisms underlying specific neurological conditions, such asAlzheimer’s disease and schizophrenia (Arai et al., 1987;Ichimiya et al., 1988; Iwamoto and Emson, 1991; Hof andMorrison, 1991; Hof et al., 1991, 1993b; Fonseca et al.,1993; Daviss and Lewis, 1995; Fonseca and Soriano, 1995).In a series of recent papers, we have investigated the

morphology and distribution of local-circuit neurons in themedial prefrontal cortex (mPFC) of the monkey and ratcontaining either PV, CB, or CR (Gabbott and Bacon,1996a,b; Gabbott et al., 1997). In this light microscopicstudy, we extend our observations and provide a specificaccount of the anatomy and quantitative distribution ofthe subtypes of CR1 neurons and processes found in areas24a,b,c, posterior 32, and 25 of the normal adult human(Vogt et al., 1995). By using a limited stereological sam-pling procedure, estimates are presented of the proportionthat CR neurons constitute of the total neuron populationin specific regions of the human mPFC. The investigationalso includes a comparative review of the CR1 neuronpopulations in the mPFC of the human, monkey, and rat.In addition, the results are discussed in relation to local-circuit neurons in the cortex containing vasoactive intesti-nal peptide (VIP), because evidence shows that CR andVIP are colocalised in similar populations of GABA-containing interneurons in themammalian cerebral cortex(Rogers, 1992; Kubota et al., 1994; Kawaguchi andKubota,1996; Gabbott and Bacon, 1997).

MATERIALS AND METHODS

Human subjects

Material from the brains of three elderly adult humansubjects (.60 years of age; cases 1–3) were used in thisstudy. The material was obtained from the Department ofHuman Anatomy, Oxford. All three subjects had died fromnatural causes, and there was no evidence of neuropathol-ogy. Following postmortem examinations, brain tissue hadbeen placed in a solution of buffered 10% formalin. Thepostmortem delay was not longer than 24 hours. The timebetween tissue fixation and further histological processingranged between 0.5 and 2.0 years.

Tissue preparation and immunocytochemicalprocedures

Large blocks of tissue containing the cingulate, paracin-gulate, and subcallosal cortical fields (Fig. 1; see also Vogtet al., 1995; Paus et al., 1996) were dissected from themedial surfaces of the left and right cerebral hemispheresfrom cases 1 and 3, respectively. Brain tissue from case 2was received as serial coronal slabs (5 mm thick) in whichboth hemispheres were present (Fig. 1B). Serial Vibratomesections (100 µm thick) were subsequently cut in thecoronal plane through Brodmann (1909) areas 24a,b,c,posterior 32, and 25 (Fig. 1B–E; posterior area 32 isdenoted by 328 and lies superior to areas 24a,b,c; see alsoVogt et al., 1995).Additional sets of serial sections were cut parallel to the

pial surface from selected parts of areas 24b and 328 in case3. Due to the curvature of the cortex, care was taken tooptimise the plane of section to be as parallel as possiblewith the pial surface.The tissue sections were thoroughly washed in 50 mM

Tris-HCl buffer, pH 7.4 (Tris), before being permeabilisedwith 0.3% Triton X-100 for 30 minutes or freeze thawed(Cuello, 1993). Sections were then washed in 20% normalgoat serum (60 minutes) and subsequently incubated in aprimary antiserum against CR (rabbit polyclonal, code7696; SWant, Switzerland; Schwaller et al., 1993). Incuba-tion conditions were 1:1,000–1:8,000 dilution in Tris buffercontaining 0.01% sodium azide for 2–3 days at 4°C orovernight at room temperature.Immuolabelling was localised by using standard immu-

noperoxidase procedures (Cuello, 1993) with a species-matchedVectastainABCkit (Vector Laboratories, Peterbor-ough, United Kingdom). Peroxidase activity was visualisedby incubating the sections in a solution of 0.05% 3,38-diaminobenzidine (DAB) and 0.01%H2O2 for 3–10minutesor by using the SG peroxidase substrate kit (Vector Labora-tories).Control incubations. Specific immunoreactivity was

absent in control sections of human tissue incubatedwithout primary antiserum or secondary link antibody.Tissue sections from the frontal cortices of monkey and ratwere used as positive controls, because they were pro-cessed by using the same incubation conditions as those forthe human material. CR immunoreactivity in sectionsfrom monkey and rat labelled a specific subset of corticalneurons whose morphologies have been described in detailpreviously (Gabbott and Bacon, 1996a; Gabbott et al.,1997).Tissue processing and examination. Individual sec-

tions were mounted onto gelatin-coated slides and airdried. Several CR1 immunolabelled sections were alsoNissl stained. These sections were spaced at regularintervals through the rostrocaudal extent of the cingulateand paracingulate regions. The tissue sections were dehy-drated in an ascending series of alcohols, passed throughxylene, and finally embedded in DPX mountant. Thehistological material was observed in a photomicroscopeequipped with a drawing tube. Objects of interest wererecorded through drawings and/or series of photomicro-graphs. The terms immunoreactive or immunopositive(CR1) refer to specific CR immunolabelling in cell bodiesand processes.

390 P.L.A. GABBOTT ET AL.

Quantitative methods

Parameters. Tissue sections were analysed quantita-tively to determine 1) the depth of laminar and sublaminarboundaries in each of the cortical areas studied, 2) size-frequency distributions of somatic profiles (Nissl-stainedneurons, CR1 neurons, and unlabelled somatic profilessurrounded by CR1 pericellular baskets; see below), 3)spine density measurements of CR1 dendrites, 4) thecortical depth distribution of CR1 neurons, 5) the percent-age that CR1 neurons constituted of the total neuronpopulations in each layer or sublayer (P%), and 6) thepercentage that CR1 neurons constituted of the totalneuron populations in each area (C%).Planar measurements. Planar measurements were

calculated and recorded by using a computerised planim-eter (Apple PowerMac 8500/MacStereology digitising sys-tem interfaced to a Kurta high-resolution graphics tablet).Measuring and statistical analysis programs were scaledappropriately to accommodate tissue shrinkage (15%) anddifferent magnification factors.

Cortical depth and laminar thickness

Cortical depth and laminar thickness measurements foreach area were made on the Nissl-stained coronal sections.Using either a 310 or a 325 objective lens and 310eyepieces, the pial surface and lower laminar boundarieswere traced onto paper with the aid of a drawing tube. Thecortical depth and laminar thickness measurements werethen calculated along lines positioned (as perpendicularlyas possible) between the pial surface and the underlyingwhite matter (Fig. 2). Twenty sets of cortical and laminarmeasurements were made per area per subject. Meanintersubject values were subsequently derived.

Cell size measurements and dendriticspine counts

At high magnification (365 oil-immersion objective lensand 310 eyepieces), the somatic profiles of CR1 neuronswere traced via a drawing tube fixed to a microscope. CRneurons in all layers of the cortex and from the different

Fig. 1. Location of cytoarchitectural areas 24a,b,c, 328, and 25 inhuman medial prefrontal cortex (mPFC). A: Drawing of the medialsurface of the left cerebral hemisphere from case 2. The anterior-posterior (p&a) extents of the cingulate, paracingulate, and subcallo-sal areas investigated in the three subjects were from regions extend-ing between the two thin vertical black lines. The coronal slab of tissueindicated by the thick black line is shown in B. B: Coronal slice (5 mmthick) taken at the level indicated in A. The boxed regions are shownenlarged in C and E, respectively. Note the distinct gray appearance ofthe outlying cerebral cortex (note also that the circular marks on thetissue slab shown here resulted from the rotary action of the tissueslicer). C: Enlargement of the region outlined in B. The distribution of

cingulate and paracingulate areas is illustrated in D. cc, corpuscallosum, cc. D: Drawing showing the distribution and location ofcytoarchitectural areas over the superior aspect of themPFC. Note thedeep invagination of the cingulate sulcus (CS). Note also that paracin-gulate area 328 extends to the inferior lip of the superior cingulatesulcus (CS8). Superior, sup; inferior, inf. E: Enlarged photomicrographof the subcallosal region indicated in B. The definition of cortical areasin the region indicated is illustrated in F. F: Drawing showing thecytoarchitectural position of subcallosal areas 25 and 12 (note that theboundaries between areas 24c/328 and areas 25/12 are difficult todefine). Scale bars 5 2.5 cm in B, C, D; 1.0 cm in E, F.

CR NEURONS IN HUMAN mPFC 391

cortical areas investigated in this study were sampled—only cells containing a nuclear profile were included foranalysis. The same condition was applied to neuronssurrounded by CR1 pericellular baskets. Samples of so-matic profile areas of each neuron type (i.e., solely Nissl-stained neurons, CR1 neurons, and unlabelled somatasurrounded by CR1 baskets) were measured, and thediameters of area equivalent circles (Dcirc) obtained.Representative samples of spine-bearing CR1 dendrites

were drawn at high magnification (3100 oil immersion).These drawings were then enlarged to a known finalmagnification, and spine density estimates were subse-quently calculated by using the method of Feldman andPeters (1979).

Cortical depth distribution of CR1 neurons

The depth distributions of CR1 neurons in areas 24a,b,c,328, and 25 of the human mPFC were determined by usingrectangular sampling grids. Each grid was 1,000 µm wideand 4,500 µm deep, with the long axis positioned perpen-dicularly to the pial surface. The grids were divided into acontinuous series of 45 tangential tiers (each 100 µm deep)that extended from the pial surface into the white matterunderlying each area.Sampling grids were superimposed through a drawing

tube onto each tissue section to be analysed. By using a310 or a 325 objective lens, the positions of identified‘‘test’’ CR1 neurons occurring in each sampling tier weremarked onto the grid. The identities of some neurons wereconfirmed at high magnification. CR1 neurons in theunderlying white matter were also included (Figs. 10, 11).Identified test CR1 neurons whose nuclear profiles inter-sected the right and upper border of each tier wereincluded for analysis, whereas those crossing the left andlower border were excluded (Gundersen, 1977). Wheresufficient dendritic morphology was present, individualtest CR1 cells weremarked as being either bipolar, bitufted,horizontal, or multipolar in appearance (Feldman andPeters, 1978; also see Results). From neighbouring Nissl-stained sections, laminar boundaries were also drawn ontoeach grid (Figs. 2, 10).The absolute numbers of identified neurons occurring

along each horizontal tier of the grid were calculated andrecorded. Cortical depth distribution histograms werelater constructed expressing the number of CR1 cellspresent within each 100 µm horizontal sampling tier as apercentage of the total number of CR1 neurons present inthe area of cortex sampled by the grid. From the placementof laminar boundaries, the percentages of the CR1 neuronpopulation that occurred within each layer/sublayer of thecortex and subjacent white matter (to a depth of 200–250µm below the layer 6B/white matter boundary) were alsodefined.Three grids were used to sample each cortical area in

each subject. Results were combined to give mean arealdata for individual cases. Mean intersubject values foreach area were subsequently derived.

Fig. 2. Low-power photomicrograph showing the depth distribu-tion of calretinin (CR)-immunoreactive (CR1) neurons in area 328 ofhumanmPFC. The somata of CR-immunolabelled neurons are seen asblack dots. Several immunoreactive somata at various levels of thecortex have been indicated by arrows. By reference to an adjacentNissl-stained section, the lamination of the cortex (layers 1–6B) hasbeen defined. Pial surface, pia; white matter, wm. Scale bar 5 500 µm.


Percentage of CR1 neurons in human mPFC

The percentage compositions that CR1 neurons consti-tuted of the total neuron population in each cortical layerand sublayer (P%) in each area were determined directlyfrom the 100-µm-thick sections that had been reactedimmunocytochemically and then Nissl stained (see Fig. 2in Gabbott and Bacon, 1996b). The quantitative methodwas based on the unbiased optical-dissector countingprocedure described previously (Sterio, 1984; West, 1993;Coggeshall and Lekan, 1996). It is important to state thatthe approach employed here was used principally to collectquantitative data regarding the percentage composition ofCR1 neurons rather than to rigorously derive the absolutenumbers and densities of Nissl-stained and of CR-immunolabelled neurons in each area and layer of humanmPFCusing rigid stereological sampling procedures (West,1993; Coggeshall and Lekan, 1996).For each section analysed, a large rectangular grid (750

µm wide 3 1,250 µm long) made up of a regular lattice ofunbiased counting squares (50 3 50 µm) was randomlypositioned within each cortical layer/sublayer at low mag-nification. By using a randomly selected start square (1 to3), a 1-in-3 series of squares was sampled across eachhorizontal row within each cortical layer. At high magnifi-cation (3100 oil immersion), the block of tissue under eachcounting square was, in effect, optically sectioned with acontinuous series of focal planes over a calibrated z-axisdistance of 30.4 µm (Gabbott et al., 1997). The volumes oftissue sampled were taken from the central/upper regionsof the Vibratome sections to preferentially avoid regions ofphysical distortion at the cut surfaces. Of note was theobservation that Nissl staining and CR immunoreactivityextended through the depth of each section.Nuclei were used to define test neurons (West, 1993).

Counterstained neurons, glial cells, and endothelial cellswere distinguished by their Nissl-staining characteristics(see Gabbott and Stewart, 1987; also see Fig. 3A,C–H inGabbott and Bacon, 1996b). All test profiles of neuronalnuclei lying completely within the sampling volume ofeach frame that conformed to ‘‘unbiased x, y, and z axisinclusion and exclusion rules’’ were counted (Gundersen,1977; Sterio, 1984; West, 1993). The number of test nucleiof CR1 neurons (A) and of other neurons stained solelywith Nissl (B) that were contained within each block oftissue were recorded on the sampling grid. Countingframes were scanned several times to ensure that all testnuclei had been correctly recorded. Between 15 and 30counting squares were used to sample individual layers/sublayers in a single section from a given area per subject.The number of test CR1 neuronal nuclei (A) per countingblock varied between zero and six (the highest counts perblock were present in layer 2, and the lowest numbers,including zero counts, were found frequently in the deeplayers of the cortex; see Table 4). The number of testneuronal nuclei per counting block stained solely withNissl (B) ranged between zero in layer 1 to 16 in layers 2and 3. In total, five to eight nonadjacent sections weresampled to provide data for a given area in an individualsubject. Mean layer/sublayer counts of test CR1 neuronalnuclei (A) per subject had coefficients of variation (COVs)of 16.2–21.9% (the lower values were obtained from layer2, and the higher estimates were obtained from layer 1 andlayers 5A-6B).The percentages that CR1 neurons composed of the total

neuron population in a given layer/sublayer (P%) werecalculated for each area and subject (P% 5 [(A/A 1 B) 3

100]). Mean intersubject laminar values were derived.These mean estimates had COV values of between 6.8%and 43.1%. The mean percentages that CR1 neuronsconstituted of the total neuron populations in each area(C%) were subsequently calculated for each subject. Thelatter estimates had COV values between 10.8% and16.8%.

Interspecies comparison of CR1 neurondistributions in rat, monkey, and human

The cortical depth distributions of CR1 neurons inhuman mPFC can be compared with similar distributionsin the mPFCs of the monkey and rat (Gabbott and Bacon,1996b; Gabbott et al., 1997). Data from the cited studieshave been transformed to facilitate interspecies compari-sons. A single representative field of mPFC (area 32) hasbeen used for comparative purposes. The transformationrequired dividing the full depth of cortex (100%) in eachspecies into 5% depth bins (including two 5% bins in thewhite matter underlying each area; 100–105% and 105–110%) and subsequently calculating the percentage of thetotal CR1 neuron population occurring in each 5% tier (seeFig. 11). The percentage distribution of CR1 neurons perlayer/sublayer (P%) in area 32 has also been calculatedpreviously for rat andmonkey and allows a direct compari-son with the data of the present study in human area 328.

Statistical analyses

The results derived from an individual case were consid-ered as a single sample for the statistical analyses. Thequantitative results are presented as mean group layer/sublayer and areal values. Results were analysed by usinga statistical software package (InStat, GraphPad Soft-ware, CA). Data were initially tested by using a one-wayanalysis of variance (ANOVA); then, mean values werecompared by using multiple t tests (Fry, 1993). Thepercentage laminar distributions of CR1 neurons in area32 of rat and monkey and human area 328 were analysedby using x2 tests to determine whether the distributionswere uniformly distributed through the cortex or whetherthere were significant nonuniform distributions. Meanlayer values were then compared by using multiple inter-species t tests. In all statistical tests, significant differ-ences between mean values were considered to occur whena probability (P) value of ,0.05 was obtained (Fry, 1993).

Preparation of illustrations

All tone and line figures presented in this paper aredigitised images that were derived from black-and-whitephotomicrographs and line drawings. These photographsor drawings were initially scanned by using a high-resolution, flat-bed scanner (ScanJet IIIc, Hewlett-Pack-ard). The digitised images were thenmanipulated by usinganApple PowerMac 8500/120 computer and image process-ing software (Adobe Photoshop, version 3.0; Adobe Sys-tems Inc.). The images were subsequently composed,masked, tone standardised, and labelled. Files of graphicaldata were also prepared with commercially available dataanalysis/graphics application software (Kaleidagraph, ver-sion 3.0; Abelbeck Software, Reading, CA; Powerpoint,version 4.0, Microsoft Corp. Redmond, WA).The final digital images were then transferred to a

Digital VAX 6000-620, and bromide prints were obtainedfrom a Monotype Prism PS-plus Post Script Imagesetter.During composition and standardisation, only the layoutand photographic quality of the images were modified to


achieve optimum print resolution. It is important to statethat the scientific content of the illustrations presented inthis article have not been altered by these procedures inany way.

RESULTS

Tissue preservation

Due to the nature of tissue fixation and storage (i.e.,postmortem delay, fixation method, fixative composition,and postfixation storage interval), the preservation of thematerial used in this study was not optimal. Nevertheless,compared with the immunoreacted control sections fromthe frontal cortices of the rat and monkey, the brainsections from the three subjects produced consistent andspecific immunocytochemical labelling of cortical and sub-cortical neuronal structures. However, the preservation ofthe material used in this study precluded an ultrastruc-tural analysis of the synaptic connectivity of CR1 neuronsin human mPFC.

Surface features and areal cytoarchitecture

There was variation in the number of gyri betweensubjects. Cases 1 and 2 had one cingulate gyrus, whereascase 3 had two adjacent gyri. Such variation in thepatterns of cingulate and paracingulate gyri and sulci arewell known (Vogt et al., 1995; Paus et al., 1996).The anterior-posterior extents of cingulate areas 24a,b,c,

the paracingulate area 328, and subcallosal cortical area25 in the mPFC of the human that were sampled areshown in Figure 1A. The cytoarchitectures of these corticalareas (see Fig. 1) were defined from the coronal Nissl-stained sections. The morphological criteria (neuronalsize, shape, packing density, and patterns of neuronalclustering) used to distinguish between these corticalfields in human mPFC were similar to those reportedpreviously (Vogt et al., 1995). The descriptions provided byVogt et al. (1995) were also used as guidelines to determinethe boundaries between cortical layers. Measurements ofthe depths of lower laminar boundaries for areas of mPFCstudied in the three subjects are presented in Table 1 (seealso Fig. 2). In this study, no attempt wasmade to partitionlayer 3 into a,b, and c subdivisions (cf. Vogt et al., 1995).

Characteristics of CR immunolabelling

Specific CR immunoreactivity was present throughoutthe mPFC of the three subjects investigated. CR-immuno-labelled somata were found unevenly distributed through-out all layers (1–6) of the areas investigated and were alsopresent in the underlying white matter (Figs. 2–7, 10). Noglial cells were found to be CR1. The overall appearance of

immunolabelling in the neuropil of the five cortical areasclearly showed that, within layers 2–5A, CR1 processeswere tightly bundled and preferentially aligned along thevertical axis of the cortex (Figs. 3F, 6, 7). A characteristic ofCR immunoreactivity in human mPFC was the prominentbackground staining of layer 1 neuropil, particularly inareas 24a,b,c.

Morphology of CR1 cells in human mPFC

In the areas of human mPFC examined, CR immunore-activity labelled a distinct subpopulation of cortical neu-rons. CR1 cells bore all the typical hallmarks of nonpyrami-dal local-circuit neurons in the cortex (Jones et al., 1994)and could be subdivided into several distinct types on the

Fig. 3. Morphology of CR1 neurons in areas of human mPFCindicating cortical lamination (layers 1–6A) and boundaries betweenlayers (dotted lines). A: A CR1 neuron (n) situated immediatelybeneath the pial surface (pia) in layer 1 of area 24b. Processes emergefrom the cell and travel for long horizontal distances (arrows). B: CR1

neurons in layers 2 and 3 of area 24b. A bitufted CR1 neuron (n) inlayer 3 has a spray of vertically oriented varicose dendrites (arrows). Acluster of three immunopositive somata (arrowheads) is seen in lowerlayer 2. C:Abitufted CR1 neuron in layer 3 in area 328. The ascendingand descending vertical dendritic arbors (arrows) of this cell are morerestricted than those seen in B. D: Bipolar CR1 neuron in upper layer3 in area 24a. Immunoreactive processes are indicated by arrows. E:CR1 stained neuron (n) in upper layer 6A of area 24b. A long process(arrows) from the labelled neuron ascends into layer 5A. The boxedregion is enlarged in J. F: A string of vertically aligned varicose CR1

processes (thick arrow) is seen in layer 3 of area 328. Two unlabelledneurons (n) in upper layer 3 are indicated. Of note, several CR1

boutons (thin arrows) are seen in close contact with a weaklyimmunoreactive cell body (CR1). G: A multipolar CR1 neuron (n) inlayer 5A of area 24b. Labelled processes (arrows) radiate from the cellbody and course along the radial axis of the cortex. H: In layer 3, area328, processes arise from a multipolar CR1 neuron (n). One processesis beaded (thick arrow) and possesses a long, thin dendritic spineswith two separate heads (thin arrow) as well as a stubby type of spine(double-headed arrow). J: Enlargement of the region indicated in E. Athin axon-like process (thick arrow) is seen descending from thelabelled neuron (n); a varicose segment of this process is indicated(thin arrows). g, Glial cells. K: A pyramidal-like CR1 neuron in lowerlayer 3 of area 24b. Note the triangular-shaped cell body and a thickascending process (long arrow) that gives rise to two daughterbranches (short arrows). L: CR1 neuron (n) in layer 5 of area 24b. Theboxed region is shown at higher magnification in M.M: Enlargementof the boxed region indicated in L. An axon-like process is seen toemerge from the CR1 neuron. Of particular interest is that a fineaxon-like process displaying numerous varicosities (double-headedarrow) descends over the initial segment of the axon-like process(thick arrow). One varicosity is shown in close apposition withaxon-like process (thin arrow). Scale bars 5 25 µm in A–E,G,K,L, 20µm in F, 10 µm in H,J, 5 µm in M.

TABLE 1. Quantitative Definition of Cortical Lamination in Areas 24a, b, c, 328, and 25 of Human Medial Prefrontal Cortex1

Layer

Area

24a 24b 24c 328 25

1 432 6 69 (11.4)2 393 6 82 (11.0) 532 6 63 (14.5) 292 6 31 (8.5) 367 6 42 (10.9)2 587 6 73 (15.5) 614 6 78 (17.2) 712 6 104 (19.4) 609 6 72 (17.7) 659 6 88 (19.6)3 1,783 6 193 (47.1) 1,491 6 152 (41.7) 1,873 6 202 (51.0) 1,472 6 165 (42.8) 1,465 6 181 (43.5)5A 2,252 6 203 (59.5) 1,895 6 216 (53.0) 2,372 6 270 (64.6) 1,861 6 244 (54.1) 1,882 6 212 (55.9)5B 2,673 6 351 (70.6) 2,642 6 319 (73.9) 2,883 6 291 (78.5) 2,094 6 249 (60.9) 2,510 6 286 (74.6)6A 3,089 6 336 (81.6) 3,146 6 380 (88.0) 3,198 6 310 (87.1) 2,596 6 286 (75.5) 2,980 6 292 (88.6)6B 3,785 6 392 (100.0) 3,575 6 444 (100.0) 3,672 6 432 (100.0) 3,439 6 357 (100.0) 3,365 6 473 (100.0)Total depth 3,785 6 392 3,575 6 444 3,672 6 432 3,439 6 357 3,365 6 473

1Data represent depth µm from the pial surface to the lower laminar/sublaminar boundaries in the areas examined (mean values 6 S.D.).2Percentage of the depth that each lower laminar/sublaminar boundary represents of the total depth.


basis of their laminar locations and the orientation andextent of their dendritic arbors, their somatic features,and, where present, their axonal morphologies (see Fig. 8).

Dendritic morphology

Layer 1. Three types of CR1 neuron were present inlayer 1. The first type of neuron was commonly encoun-

Figure 3


tered immediately below the surface of the cortex andpossessed a large bipolar/tripolar ovoid cell body with longprocesses that ramified horizontally in the upper reaches

of the molecular layer (Fig. 3A; Fig. 5, cells a,b). Figure 5shows that these cells sometimes had dendritic processesthat coursed obliquely or perpendicularly into the tissue

Fig. 4. Morphology of CR1 neurons in areas of human mPFC. A:Lower layer 3 in area 328. CR1 boutons (short arrows) seen in closecontact with the somata of a pyramidal shaped cell body (P) fromwhich appears to arise the apical dendrite (ad; long arrow). Twoimmunonegative neurons (n) and an immunolabelled cell (thickarrow) are shown. Note that immunonegative cell n8 is not surroundedby CR1 puncta—the nucleolus in cell n8 is indicated by the thin arrow.B: An unlabelled pyramidal shaped soma (P) in lower layer 3 in area24a encircled by a cluster of CR1 boutons (thin arrows).An immunoposi-

tive cell is also indicated (thick arrow). C,D: Clusters of CR1 boutons(arrows) surrounding the somata (n) of two immunonegative cellbodies. In D, the cell nucleolus is visible. E: Upper layer 3, area 25.CR1 boutons in close contact (small arrows) with the soma of animmunonegative pyramidal shaped cell body (P). Other immunonega-tive neurons are indicated (n). A neighbouring CR1 cell is indicated(large arrow). F,G: Two examples of CR1 boutons (arrows) in closeapposition with the somata of two weakly immunoreactive neurons(CR1). Scale bars 5 10 µm inA,B,E–G, 5 µm in C,D.

Fig. 5. Composite drawing of CR1 neurons (a–e) in layer 1 of areas24b,c, 25, and 328. Cells a and b were located immediately beneath thepial surface (pia) and had processes that coursed tangentially for longdistances in upper layer 1. Other dendrites from both of these cellsemerged perpendicular to the axis of viewing and extended deep intothe tissue sections. The latter processes also lay below the pial surfaceand are shown in the regions indicated by the curved arrows. The

dendrites of these cells were aspiny and highly beaded. Other CR1

neurons (c–e) occurred at different depths through layer 1. Anaxon-like process from cell c (straight arrow) descended into layer 2.Cell e ramified parallel to the cortical surface at the layer 1/2 border.Areal location of cells: a, 24b; b, 328; c, 25; d, 24c; e, 24b. Scale bar 550 µm.


Fig. 6. Composite drawing of CR1 neurons in layers 1–3 of areas24a,b,c, 25, and 328. Note the morphology and distribution of cellularprocesses as well as the shape and orientation of labelled somata.Axon-like processes are indicated by arrows. Note that the processes ofsome labelled neurons lying deep in layer 3 extend into layer 5.Layout: The scale bar applies to all neurons. To present neurons fromdifferent cortical areas, where laminar boundaries occur at differentdepths, cells have been placed in their corresponding position within

each lamina, although the exact depth of each lamina is not to scale.Laminar location of cells (layer/area): a, 2/24a; b, 2/328; c, 2/24b; d,2/24c; e, 2/24a; f, 2/328; g, 2/25; h, 2/328; i, 2/24c; j, 2/25; k, 2/25; m,2/24a; n, 2/24b; o, 2/328; p, 2/24a; q, 2/24b; r, 3/328; s, 3/24b; t, 3/24a; u,3/328; v, 3/24c; w, 3/25; x, 3/328; y, 3/24b; z, 3/24c; a8, 3/24b; b8, 3/328; c8,3/24b; d8, 3/24c; e8, 3/25; f8, 3/24b; g8, 3/328; h8, 3/24b; i8, 3/25; j8, 3/24a;k8, 3/328; m8, 3/24b; n8, 3/24c; o8, 3/24b; p8, 3/25; q8, 3/328; r8, 3/328; s8,3/25; t8, 3/24a; u8, 3/24a; v8, 3/24b; w8, 3/24c. Scale bar 5 100 µm.

Fig. 7. Composite drawing of CR1 neurons in layers 5A–6B ofareas 24a,b,c, 25, and 32. Note the morphology and distribution ofcellular processes as well as the shape and orientation of labelledsomata. Axon-like processes are indicated by arrows. Note that theprocesses of some labelled neurons lying in upper layer 5A extend intolayer 3. The scale bar applies to all neurons. The layout of the figure isdescribed in the legend to Figure 6. Laminar location of cells (layer/

area): a, 5A/24a; b, 5A/25; c, 5A/328; d, 5A/24a; e, 5A/24c; f, 5A/24b; g,5A/328; h, 5A/24a; i, 5A/24c; j, 5A/24b; k, 5A/24b; m, 5A/24c; n, 5A/328;o, 5A/5B border/25; p, 5B/24a; q, 5B/24b; r, 5B/328; s, 5B/24b; t, 5B/24a;u, 5B/24c; v, 6A/25; w, 6A/24a; x, 6A/328; y, 6A/24b; z, 6B/24c; a8, 6B/25;b8, 6B/328; c8, 6B/24a;; d8, 6B/24b; e8, 6B/24a; f8, 6A/24c; g8, 6B/24b; h8,6B/328; i8, 6B/25. Scale bar 5 100 µm.

sections, remaining close beneath the pial surface. Al-though the dendrites of such CR1 neurons were highlybeaded and virtually aspiny (Fig. 3A), they sometimesgave rise to the initial segments of descending dendritic

‘‘branchlets’’ (Marin-Padilla, 1984). These cells repre-sented about 0.5% of the CR1 cell population in humanmPFC (Table 2).The second type of CR1 neuron was also aligned horizon-

tally, but these cells were generally much smaller in bothsoma size and lateral extent of their dendritic arbors andwere located at varying depths within in layer 1 (Fig. 5,cells d,e). The dendritic processes of this second type of‘‘horizontal cell’’ did not traverse the layer 1/2 border (Fig.5, cell e). The third type of CR1 cell in layer 1 was amedium-sized neuron situated in the lower two-thirds ofthis lamina with dendrites that radiated upward (Fig. 5,cell c). A characteristic feature of this neuron type was thata varicose axon-like process would emerge from a verti-cally oriented cell body and descend into upper layer 2.Together, these three cell types constituted approximately1% of the cortical population of CR1 neurons (Table 2).Layers 2/3. Bipolar, bitufted, and multipolar CR1 neu-

rons and horizontally aligned CR1 cells with bitufteddendritic arbors were found in layers 2 and 3. The firsttype of CR1 neurons were small bipolar cells with ovoid/fusiform somata (meanminor andmajor soma diameters c.7 3 13 µm; Fig. 8, Table 3). Neurons of this type werecommonly located in layer 2 and in upper layer 3 and hadvertically oriented dendritic arbors. These arbors werecomposed of one primary ascending and one primarydescending dendrite that arose from opposite somaticpoles. The majority of these dendritic processes did notcourse for long distances and tapered gradually (Fig. 3D;Fig. 6, cells e,f,k,m,n; Fig. 8). Other bipolar cells hadmarkedly longer dendritic processes (Fig. 6, cells a,c,i, j,s,t,v,x,y,a8–c8,e8, j8,m8,n8,u8). Occasionally, a third parentdendritic process would emerge from the somata of theseneurons (Fig. 6, cells d,q). Most frequently, this additionaldendrite arose close to the main ascending or descendingprocesses, giving a tripolar appearance. Bipolar/tripolarneurons were the most frequent encountered neuron typein layers 2/3 and represented 52% of CR1 cells in theselayers (Table 2).The second type of CR1 neurons were bitufted cells (Fig.

8). These neurons had predominantly long tufts of verti-cally oriented dendrites arising either directly from oppo-

Fig. 8. Diagram illustrating the characteristic morphological fea-tures of the three principal types of CR1 neurons in human mPFC.Cell a: a bitufted neuron from layer 3 in area 25. Cell b: A multipolarneuron from layer 3 in area 24b. Cell c:Abipolar neuron from layer 2 inarea 328. The initial portions of axonal arbors are indicated by arrows.Note that the bipolar neuron (cell c) has ascending and descendingaxonal processes. Scale bar 5 50 µm.

TABLE 2. Different Types of Calretinin Immunopositive Neuronsin Layers 1–6 of Areas 24a, b, c, 328, and 25 of Human Medial

Prefrontal Cortex1

Cell typeLayers1/2/3 (%)

Layers5/6 (%)

Corticalmean (%)

Layer 1 cellsCajal-Retzius2 0.5 — 0.25Other types 1.5 — 0.75

Bipolar/tripolar3,8 52 45 48.5Vertical bitufted4,8 27 42 34.5Horizontal bitufted5 4 3 3.5Multipolar6,8Aspiny 8 7 7.5Sparsely spiny 6 3 4.5

Pyramidal like7 1 — 0.5

1Cell types expressed as a percentage of classifiable calretinin-immunopositive (CR1)neurons (e.g., classifiable 5 1,134; unclassifiable 5 203).2For examples of Cajal-Retzius neurons, see Figures 3A and 5, cells a, b.3For examples of bipolar neurons, see Figures 6, cells i, e8 and 7, cells o, f8. For examplesof tripolar neurons, see Figure 6, cells d, q.4For examples of vertical bitufted neurons, see Figures 6, cells z, f8 and 7, cells m, r.5For examples of horizontal bitufted neurons, see Figure 6, cells r, i8.6For examples of aspiny multipolar neurons, see Figures 6, cells s8 and 7, cell p. For anexample of a spiny multipolar neuron, see Figure 3H.7For an example of a pyramidal-like neuron, see Figure 3K.8See also Figure 8 for examples of the three principal types of CR1 neurons in the humanmedial prefrontal cortex.


site somatic poles or from short dendritic processes.Bitufted cells represented 27% of the CR1 cells in layers2/3 (Table 2). Two subtypes could be found. The firstsubtype had ascending and descending dendrites re-stricted to long, narrow columns of cortex (Fig. 3L,M; Fig.6, cells z,f8,g8; Fig. 8), and the second subtype of cell had amore radiate distribution of dendritic arbors (Fig. 3B,C;Fig. 6, cells g,o,p,u,w,d8,h8,q8,o8,r8,t8,v8,w8). Another type ofbitufted neuron situated in layers 2/3 had horizontallyoriented, spindle-shaped cell bodies (Fig. 6, cells b,h,r,i8).Cells in the latter category represented 4% of layer 2/3CR1 neurons (Table 2) and were readily apparent by theorientation of their long and widely ramifying dendriticprocesses (Fig. 6, cells r,i8).Neurons belonging to the last major category were

multipolar CR1 cells located throughout layers 2/3 withsymmetrically radiate dendritic arbors (Fig. 6, cellsk8,p8,q8,s8; Fig. 8). These cells were most frequently situ-ated in layer 3. Two subtypes could be found: the firstsubtype possessed aspiny processes, whereas the secondsubtype had spine-bearing secondary and tertiary den-drites (see below). The sparsely spinous and aspiny multi-polar neurons accounted for 14% of CR1 cells in layers 2/3(Table 2).Pyramidal-shaped CR1 neurons were also present in

layer 3 (Fig. 3K, Table 2). These pyramidal-like neuronswere rare, representing less than 1.0% of layer 2/3 CR1

cells (Table 2). They possessed a stout dendrite arisingfrom the apex of a triangular-shaped cell body and anumber of thinner dendritic processes radiating from thebasal aspects of the soma. Unlike true pyramidal neurons,the apical process soon bifurcated into daughter segments(Fig. 3K).Layers 5/6. Similar to their counterparts in layers 2/3,

CR1 cells in layers 5 and 6 could be divided into bipolarvertically and horizontally oriented bitufted cells andmultipolar neurons (Fig. 8). In general, cells in the deeplayers of mPFC had larger somata and longer processesthan their more superficial counterparts; however, con-spicuous exceptions could be found. CR1 bipolar neuronshad long processes that ascended and descended throughthe cortex without respecting laminar boundaries (Fig. 7,cells a,c–f,n,t,u,v,b8,c8,f8,h8,i8; see especially cell o). Bipolarneurons at the base of the cortex had long ascending

processes and a descending dendrite that branched intosmall basal arbors within lower layer 6 (Fig. 7, cell z).Bitufted CR1 neurons ranged between cells with disperseddendritic arbors (Fig. 7, cells b,i,k,m,s,y,d8) and cells with along, narrow, columnar arrangement of their ascendingand descending dendrites (Fig. 7, cells a8,j, and especiallycell r). Some horizontally bitufted CR1 neurons in layers5/6 gave rise to processes that initially coursed obliquelybut then adopted vertical orientations within the cortex(Fig. 7, cells d8,e8). Multipolar cells in layers 5 and 6 variedbetween small-sized cells with radiate dendritic trees (Fig.7, cells g,h,p,q,w,x,e8,g8) to a comparatively rare type oflarge aspiny neuron with exceptionally long dendriticprocesses commonly found in layer 5Aof areas 24a,b,c (Fig.7, cells p,q). The latter type of cell was never stronglyimmunoreactive for CR. No pyramidal-like CR1 cells werefound in the deep layers.Table 2 presents data defining the percentage composi-

tion of bipolar, bitufted, horizontal, and multipolar cells inlayers 5/6. Figure 8 presents the typical morphology of thethree principal classes of CR1 neuron in layers 2–6B ofhuman mPFC.White matter. CR1 neurons in the white matter below

each area were bipolar or bitufted cells with their long axesaligned along the common paths of fibre bundles (notillustrated). The processes of these cells were highlybeaded and sometimes bore dendritic spines. Labelleddendrites from CR1 neurons in the white matter rarelyentered the cortex. A few CR1 fibres were observed cours-ing through the white matter.Cell body morphology and immunoreactivity. The

cell bodies of CR1 neurons displayed a variety of shapesand sizes. The shapes ranged from circular to highlyelongated in outline. Ovoid- and fusiform-shaped somatahad long axes perpendicular to the pial surface (e.g., Fig.3D). The mean size of CR1 somatic profiles in the humanmPFC was 11.6 µm compared with the statistically signifi-cant larger mean somatic size for immunonegative, Nissl-stained neurons of 16.1 µm (Table 3). Size-frequencydistributions of these two cell populations are shown inFigure 9. In CR1 immunolabelled neurons, nuclei werefrequently more intensely stained than the surroundingsomatic cytoplasm (Figs. 3A–C,H,K, 4A,B,E).

TABLE 3. Mean Somatic Profile Sizes (Diameter of the area-equivalent circles) of Nissl-Stained Neurons1 and Calretinin-Immunopositive (CR1)Neurons in Areas 24a, b, c of the Human Cingulate Cortex2

Nissl-stainedneurons

CR1

neurons

Unlabelled somata with CR1 baskets3

Layers 2/3 Layers 5/6

Mean 6 S.D. (µm) 16.1 6 0.6 11.6 6 0.3 13.4 6 0.4 15.8 6 0.4Minimum 6.3 5.7 5.9 9.0Maximum 32.2 17.1 16.8 20.1Number of profiles 1,500 1,500 250 250

Statistical analysis: Mean somatic profile sizes (t tests).Nissl-stained neurons vs. CR1 neurons vs. unlabelled somata with CR1 baskets.

Unlabelled somata with CR1 baskets

CR1 neurons Layers 2/3 Layers 5/6

Nissl-stained neurons t 5 11.62, df 5 4, P 5 0.0003 t 5 6.48, df 5 4, P 5 0.003 nsCR1 neurons - t 5 6.23, df 5 4, P 5 0.0034 t 5 12.48, df 5 4, P , 0.001

Rank order: Nissl-stained neurons/unlabelled somata with CR1 baskets in layers 5/6 . unlabelled somata with CR1 baskets in layers 2/3 . CR1 neurons.

Unlabelled somata with CR1 baskets in layers 2/3 versus 5/6. Significant difference: t 5 6.49, df 5 4, P , 0.003. Rank order: 5/6 . 2/3.

1Note that Nissl-stained neurons refers to neurons in CR-immunoreacted material that were immunonegative and only stained for Nissl.2The samples were taken from across the areas 24a, b, c and from all cortical layers. Mean values 6 S.D. (n 5 3 subjects).3The mean somatic profile size of unlabelled somata in layers 2/3 and 5/6 surrounded by more than ten CR1 boutons (CR1 baskets).


Morphology of dendritic processes. The dendrites ofCR1 neurons displayed a variety of surface morphologies(Figs. 3, 6–8). Dendrites ranged from being aspiny to beingsparsely spine bearing (Feldman and Peters, 1978; also seebelow) and from having a relatively constant diameter tohaving highly beaded processes (Figs. 3, 6–8). Beadeddendrites appeared as large ovoid or circular structuresinterconnected by immunoreactive strands (Fig. 3B,C).

Dendritic spine characteristics. Several morphologi-cally distinct types of dendritic spines were found over theprocesses of the sparsely spiny subtype of CR1 multipolarneuron. The structure of the dendritic spines ranged fromshort, stubby protrusions to long, thin stalks with singlecircular spine heads (Fig. 3H). Dendritic spines with twospine heads were also found (Fig. 3H). Several of thesedendritic spine types could be found over an individualspine-bearing CR1 process (Fig. 3H).Spine density estimates. The more distal dendrites

(second-/third-order processes) of some multipolar CR1

neurons bore a sparse number of dendritic spines, asdescribed above. Quantitative estimates indicated a verylow spine density over these dendritic segments [0.29 60.03 spines/µm (mean 6 S.E.M.); sample (n) of 20 primaryand secondary processes; each process was .15 µm inlength].Axon-like processes. Axon-like processes arose from

CR1 neurons located throughout the cortex (Figs. 3E,J,M,5–8). These fine processes would commonly emerge eitherfrom the upper or lower poles of labelled somata or fromprimary or proximal secondary dendrites (or the interven-ing dendritic branch point). The proximal segments ofsome axon arbors from CR1 neurons displayed numeroussmall varicosities ,1 µm in diameter (Fig. 3J). Althoughthe most frequent initial trajectory of axon-like processesarising from CR1 neurons was radially within the cortex(Fig. 3E; Fig. 6, cells a–c,k8,p8,v8; Fig. 7, cells b,m,s,g8; Fig.8, cells b,c), examples could be found of axon-like processesthat were initially aligned tangentially (Fig. 6, cells r,w,d8;Fig. 7, cell e8; Fig. 8, cell a). Fine axon-like processesarising from the somata of CR1 in layer 1 were alignedhorizontally.CR1 puncta. CR-immunolabelled puncta were present

in all cortical layers, but the greatest density was presentin layers 2–5. Strings of numerous CR1 puncta were foundbundled vertically in the cortex and were commonly lo-cated in layers 2/3 and in upper layer 5 (Fig. 3F). On anindividual string, CR1 puncta were joined by fine cytoplas-mic strands (Fig. 3F). Measurements established that themean centre-to-centre distance between these bundles wasabout 53 6 25 µm (mean 6 S.D.; n 5 102; range 26–121µm).Of particular note is that numerous CR1 puncta were

found to be closely associated with the somatic profiles ofsome unlabelled neurons (Fig. 4A–E). Numerous (up to 40)CR1 puncta could partially or completely encircle thesomatic profiles of these cells. When more than ten CR1

puncta were associated with an individual unlabelledsomatic profile, they were considered to form a pericellularbasket (Kisvarday, 1992). The number of CR1 punctainvolved in such pericellular baskets varied widely. CR1

pericellular baskets were most commonly found in layers2–5 of the cortical areas examined. No baskets were foundin layer 6B.A significant feature was that the unlabelled somata

surrounded by CR1 pericellular baskets in layers 5 and 6Awere mainly pyramidal in shape (Fig. 4A,B,E). On occa-sion, puncta forming these CR1 pericellular baskets werealso closely associated with the presumed apical dendritearising from the immunonegative pyramidal somata (Fig.4A,B,E). Importantly, immunoreactive puncta selectivelyformed pericellular baskets around some pyramidal neu-rons while avoiding neighbouring somatic profiles (see, inparticular, Fig. 4A). By contrast, CR1 pericellular baskets

Fig. 9. Top: Size-frequency histograms of CR1 somatic profilescompared with Nissl-stained immunonegative somatic profiles. Datapresented from area 24b in case 2. Mean 6 S.D. values for the subjectgroup (cases 1–3) are given in Table 3. Both sample populations werecomposed of the same numbers of profiles (n). Note that populationswere taken across all cortical layers. Bottom: Size-frequency histo-grams of immunonegative somatic profiles surrounded by pericellularbaskets of CR1 puncta. In layers 2/3, pericellular baskets mainlyencircled circular profiles (see Fig. 3C,D), whereas, in layers 5/6, theencircled somata were predominantly pyramidal in shape (see Fig.3A,B,E). Each pericellular basket was composed of more than tenimmunoreactive puncta. The data presented are from area 24b in case1. Mean6 S.D. values for each distribution are given. Note the greaterfrequency of larger sized profiles (D. Circle 17–25 µm) surroundedby pericellular baskets in layers 5/6 compared with layers 2/3. Themean 6 S.D. values for the subject group (cases 1–3) are given inTable 3.


in layers 2 and 3 mainly surrounded unlabelled circularsomatic profiles (Fig. 4C,D). CR1 puncta were not observedto form structures similar to the ‘‘axon-cartridges’’ thathave been described previously for PV1 puncta in monkeymPFC (see Fig. 18A–C,G,H in Gabbott and Bacon, 1996a).The mean somatic profile size of unlabelled cells that

were surrounded by CR-immunolabelled pericellular bas-kets was 15.8 6 0.4 µm for pyramidal shaped somata inlayers 5/6 and 13.4 6 0.4 µm for circular somata in layers2/3 (Fig. 4, Table 3). These mean somatic profiles sizes(pyramidal vs. circular) are significantly different fromeach other (Table 3).CR1 puncta were also found closely associated with the

somata of other CR1 neurons situated in layers 2/3 (Fig.4C,D). Although these arrangements were similar to peri-cellular baskets, the number of visible CR1 puncta in-volved in such arrangements around an individual CR1

soma was comparatively low (frequently less than eight);consequently, these structures were considered not torepresent ‘‘true’’ pericellular clusters (Fig. 4F,G) similar tothose formed by basket neurons (Kisvarday, 1992). Immu-nopositive puncta were found in close contact with theinitial dendritic processes arising from CR1 neurons (notillustrated). Finally, CR1 puncta were observed in closeapposition with the initial segment of axon-like processesarising from CR1 cells bodies (Fig. 3L,M).

Distribution of CR1 neurons in human mPFC

The cortical depth distributions of CR1 neurons weresimilar in all areas of humanmPFC examined (Figs. 2, 10).The majority of CR1 somata were concentrated in theupper one-third of the cortex at distances of 350–1,250 µmbelow the pial surface (Figs. 2, 10).From depth measurements of lower layer and sublayer

boundaries (Table 1), the cortical depth distributions ofCR1 neurons can be defined with respect to individuallayers and sublayers (Figs. 2, 10). The proportion of theoverall CR1 population in a given layer has been calcu-lated subsequently (Table 4). These data indicate that thepeak density of CR1 neurons occurred in layer 2 in all ofthe mPFC areas examined (Figs. 2, 10). By accounting forthe actual depth of each layer, it was determined that33–41% and 39–43% of the CR1 population in the cortexwere located in layers 2 and 3, respectively (Figs. 2, 10,Table 4). Indeed, over 78% of the CR1 cell population wasdistributed in layers 2 and 3 (Figs. 2, 10, Table 4). Bycomparison, layer 1 contained 7.5%, and layers 5A-6Bcontained 13.6% of the CR1 cell population (Figs. 2, 10,Table 4). Serial sections cut parallel with the pial surfacethroughout the depth of areas 24b and 328 indicated thatCR1 neuronal somata were arranged uniformly in thetangential plane and did not reveal any significant cluster-ing of labelled perikarya (not illustrated).

Laminar and areal proportionof CR1 neurons

The proportions that CR1 neurons constituted of theneuronal populations in each layer and sublayer (P%) ofhumanmPFCwere calculated from immunolabelled/Nissl-stained sections (Table 5). The proportion that CR1 neu-rons composed of the total neuron populations in eachcortical area (C%) was also determined (Table 5). Thesedata indicated that, across the mPFC areas studied, CR1

neurons composed 9.0% of layer 1 neurons, 16% of layer2/3 neurons, and 4% of neurons in layers 5 and 6. CR

neurons constituted an overall mean 8% of the totalneuronal populations in the cortical areas investigated(Table 5). There were no significant mean interarealdifferences (Table 5). The COV values for the C% dataindicate marked interindividual variability and empha-sise the small number of subjects examined (n 5 3).

Comparative data—Human, monkey, and rat

Figure 11 shows the depth distributions of CR1 neuronpopulations in layers 1–6B/white matter in area 32 of therat and monkey and for area 328 in the human. Table 6presents data of the percentage laminar distribution ofCR1 neurons throughout the cortex for each species. Thehuman data have been expressed in terms of the CR1 cellpopulation within the cortex; therefore, the data excludeCR1 neurons in the white matter.Statistical x2 tests confirmed that the overall CR1 cell

populations in the three species were not distributeduniformly across cortical layers (Table 6). Furthermore, ttests indicated that there were no significant differences inthe percentage distributions of CR1 neurons in corticallayers 1–3 between humans (87.4%) and monkeys (84.6%).However, the proportions of CR1 cell populations in layers2 and 3 of the monkey and human were significantlygreater (131%) than in the rat. In layers 5-6, the situationwas, reversed with the proportion of CR1 neurons in ratarea 32 significantly higher (1171%) than for the corre-sponding layers in either the monkey or human (Table 6).Table 7 presents data detailing the percentage that CR1

neurons composed of the overall neuron composition inspecific areas of rat, monkey, and human mPFCs. Themean areal percentage of CR1 neurons (C%) were signifi-cantly different between the three species: monkey(11%) . human (8%) . rat (4%; Table 7).

DISCUSSION

The major findings of this light microscopic study arethat 1) CR1 neurons in areas 24a,b,c, 25, and 328 of humanmPFC possess small/medium-sized somata and display avariety of morphologies, 2) the dendritic and axon-likeprocesses of CR1 neurons are predominantly aligned verti-cally within the cortex, 3) the great majority (79%) of CR1

somata are located in layers 2/3 with the peak distributionoccurring in layer 2, 4) CR1 neurons constitute 8% of thetotal neuron population in each area, and 5) CR1 punctaform pericellular baskets around unlabelled cell bodies—principally circular somatic profiles in layers 2/3 andpyramidal-shaped somata in layers 5/6.

Morphology of CR1 local-circuit neurons

In human mPFC, the CR1 cell population was princi-pally composed of bipolar (48.5%), vertically bitufted(34.5%), horizontally bitufted (3.5%), both aspiny (7.5%)and sparsely spiny (4.5%) multipolar neurons, and layer 1cells (1%) that included Cajal-Retzius neurons. These CR1

neuronal types are essentially similar to those described inother areas of human cortex (Glezer et al., 1992; Hof et al.,1993a,b), in the prefrontal and visual cortices of themonkey (Conde et al., 1994; Gabbott and Bacon, 1996a;Martin and Meskenaite, 1997), and in areas of the ratcortex (visual: Rogers, 1992; mPFC: Gabbott et al., 1997).On the basis of their somatic and dendritic characteristics,laminar and regional distributions, synaptic connectivi-ties, and neurochemical content, CR1 cells in the cerebral


cortex of a variety of mammalian species, including hu-mans, have been identified as a subpopulation of GABA-containing nonpyramidal local-circuit neurons (Rogers,1992; Kubota et al., 1994; Fonseca et al., 1995; Del Rio andDeFelipe, 1996; Martin and Meskenaite, 1997).

CR1 neurons in layers 2–6

Bipolar cells. Bipolar CR1 cells in human mPFCstrongly resemble bipolar neurons that have been shownto contain VIP in a number of mammalian species (mon-key: Gabbott and Bacon, 1997; cat: Wahle and Meyer,1989; rat: Rogers, 1992; Kubota et al., 1994; Kawaguchiand Kubota, 1996). Indeed, the majority (69–87%) of CR1

interneurons in the cortex colocalise VIP (Rogers, 1992;

Kubota et al., 1994; Kawaguchi and Kubota, 1996; Gabbottand Bacon, 1997). Importantly, VIP is not present in CB1

and PV1 neurons (Rogers, 1992; Kubota et al., 1994;Kawaguchi and Kubota, 1996). Consequently, importantinsights into the possible connectivity and intracorticalfunction of CR1 bipolar cells in human cortex emerge froma consideration of VIP1 bipolar neurons in the cortices ofother species (McDonald et al., 1982; Connor and Peters,1984; Morrison et al., 1984; Peters et al., 1987; Peters andHarriman, 1988; Peters, 1990; Kawaguchi and Kubota,1996).In area 17 of the rat, 85–90% of VIP1 neurons are

bipolar in form (Connors and Peters, 1984). A slightlylower estimate (78%) is found for VIP1 cells in monkey

Fig. 10. Cortical depth distribution histograms of CR1 neurons inareas 24a,b,c, 328, and 25 of human mPFC (case 2). The data arepresented as the percentage of the total CR1 cell population occurringat each horizontal sampling level. The number of immunoreactive

neurons (n) composing each histogram are given for each distribution.Cortical lamination is given to the right of each histogram. Note thesimilarity in CR1 cell distribution across cortical areas.


mPFC (Gabbott and Bacon, 1997). Similar to bipolar CR1

neurons, bipolar VIP1 neurons are commonly located insuperficial layers 2/3 and display characteristic verticallyaligned dendritic arbors (Morrison et al., 1984; Peters,1984, 1990; Peters et al., 1987; Peters and Harriman,1988; Gabbott and Bacon, 1997). In rat, cat, and monkey

cortices, bipolar VIP1 cells possess a long, vertically ori-ented main axon that gives rise to collaterals in thevicinity of the parent somata and then descends, withoutfurther collateralisation, into the deeper layers beforebranching into a narrow plexus of axon terminals (Petersand Harriman, 1988; Wahle andMeyer, 1989; Gabbott and

TABLE 4. Distributions of Calretinin-Immunoreactive Neurons in Cortical Areas 24a, b, c, 328, and 25 in the Human1

Layer

AreaLayermean24a 24b 24c 328 25

1 7.5 6 1.2 7.6 6 1.5 8.2 6 1.9 8.0 6 1.9 6.3 6 1.1 7.5 6 0.72 33.4 6 6.6 35.7 6 5.8 41.0 6 7.2 37.1 6 9.5 40.0 6 6.8 37.4 6 2.83 43.1 6 8.1 41.0 6 6.6 39.9 6 6.8 40.2 6 7.2 38.6 6 7.6 40.6 6 1.55A 5.0 6 0.9 4.9 6 0.8 4.6 6 0.9 5.3 6 1.1 4.4 6 3.2 4.9 6 0.35B 4.4 6 0.7 5.5 6 1.1 2.1 6 0.3 4.3 6 0.8 3.4 6 0.6 3.9 6 1.16A 3.4 6 0.6 2.4 6 0.4 2.5 6 0.4 1.7 6 0.3 2.9 6 0.5 2.6 6 0.66B 2.8 6 0.6 2.2 6 0.4 1.6 6 0.3 1.4 6 0.3 3.1 6 0.5 2.2 6 0.7Underlying wm2 0.3 6 0.1 0.7 6 0.2 0.1 6 0.03 1.9 6 0.4 1.4 6 0.3 0.9 6 0.7Total 99.9 100 100 99.9 100.1 100.0

1Mean values 6 S.D. (%). Data presented as the percentage that CR1 cell population present in each layer or sublayer constituted of the overall CR1 cell population (including CR1

neurons in the white matter).2Percentage of CR-immunoreactive neuronal population located between the layer 6B/white matter (wm) border and a distance of 200–250 µm into the underlying white matter.

TABLE 5. Percentages That Calretinin-Immunoreactive Neurons Compose of the Total Neuron Population in Areas 24a, b, c, 328,and 25 in the Human Cortex1

Layer

AreaLayermean24a 24b 24c 328 25

1 9.5 6 4.1 10.2 6 2.1 7.4 6 1.1 8.5 6 1.9 8.4 6 1.6 8.8 6 1.02 16.0 6 3.2 13.6 6 2.2 11.2 6 2.0 15.1 6 3.1 15.3 6 2.3 14.2 6 1.73 18.9 6 2.7 17.3 6 1.5 16.0 6 2.1 17.8 6 1.8 18.2 6 2.5 17.6 6 1.05A 7.0 6 1.0 4.7 6 0.7 7.3 6 1.2 6.0 6 0.9 6.2 6 1.1 6.2 6 0.95B 3.1 6 0.4 2.9 6 0.2 3.4 6 0.3 3.5 6 0.6 2.6 6 0.4 3.1 6 0.36A 3.5 6 0.5 3.3 6 0.6 2.9 6 0.3 3.2 6 0.4 3.9 6 0.5 3.4 6 0.16B 3.0 6 0.4 2.5 6 0.3 2.0 6 0.2 2.8 6 0.2 3.5 6 0.3 2.8 6 0.3Areal means (C%) 8.7 6 1.4 7.8 6 0.9 7.2 6 1.2 8.1 6 1.3 8.3 6 0.9 8.0 6 0.5

1Mean values 6 S.D. (%). Multiple t tests did not reveal statistically significant differences between mean areal values (C%).

Fig. 11. Comparison of the cortical depth distribution of CR1

neurons in areas 32 and 328 of the rat, monkey, and human, respec-tively. The actual depth of the cortex in each species has been dividedinto 5% depth sampling bins. Cortical lamination is indicated on theright in each histogram. Note that, because CR1 neurons are presentin the subjacent white matter (wm), two additional sampling bins(100–105% and 105–110%) are included for each depth distribution.

The number of neurons occurring in each 5% sampling tier has beenexpressed as a percentage of the total number of neurons (n) compos-ing each histogram. Comparative data: rat, Gabbott et al. (1997);monkey, Gabbott and Bacon (1996b). The data have been taken fromrepresentative subjects in each species (see Table 6 for a statisticalanalysis of the laminar distributions).


Bacon, 1997). In areas of rat cortex, VIP1 axonal varicosi-ties are most dense in upper layers 2–4 (Morrison et al.,1984). Throughout the visual cortex of the rat, VIP1

boutons form symmetric synaptic junctions with smooth(19%) and spiny (25%) dendritic shafts as well as the cellbodies of pyramidal neurons (16%) and the somata ofbipolar (3%) and multipolar (.0.5%) nonpyramidal cells—small-calibre dendritic shafts represent 36% of the targets(Peters, 1990). A similar range of targets has been foundpostsynaptic to VIP1 boutons in cat visual cortex (axoden-dritic, 90%; pyramidal somata, 10%; Peters et al., 1987).Taking into consideration the relative proportion of pyrami-dal cells and GABA1 neurons, this evidence suggests thatVIP1 bipolar neurons principally target the somata andprocesses of pyramidal neurons as well as the smoothdendritic shafts of interneurons (Peters et al., 1987; Pe-ters, 1990). These structures may also be the primesynaptic targets innervated by CR1 bipolar neurons inhuman mPFC (see below). Whether individual bipolarneurons colocalising VIP and CR innervate different post-synaptic targets in the superficial layer compared with thedeep layer of the cortex has not been established.It is interesting to note that, in monkey mPFC, 78% of

VIP1 cells are bipolar neurons located in layers 2/3 andthat CR immunoreactivity was colocalised in 80.5% ofVIP1 neurons (Gabbott and Bacon, 1997). Because VIP isintimately involved in the regulation of energymetabolismand blood flow dynamics in the cortex (Magistretti, 1990),VIP-containing bipolar CR1 cells in human cortex may notonly participate directly in local inhibitory circuitry butmay also play a crucial part in the local regulation of tissue

physiology within a radial column of cortex (Magistretti etal., 1984; also see below).Bitufted cells. From their somatodendritic and axonal

characteristics, bitufted CR1 neurons in layers 2–5 ofhuman mPFC are likely to be from the double-bouquetclass of narrow-arbor cortical interneurons (Somogyi andCowey, 1984). In this context, it is important to note thatcells in monkey and human cortices with typical double-bouquet morphology and connectivity have been found tobe immunoreactive for CB (DeFelipe et al., 1989, 1990;DeFelipe and Jones, 1992; Del Rio and DeFelipe, 1995).Identified CB1 double-bouquet cells possessed a descend-ing ‘‘horse-tail’’ axonal cascade composed of fine varicoseprocesses that ramified within a confined radial column ofcortex (Somogyi and Cowey, 1981, 1984; Lund and Lewis,1993; Jones et al., 1994; Kawaguchi and Kubota, 1996).Previous ultrastructural studies have identified the pre-dominant postsynaptic targets of these narrow axonalarbors as the shafts (60%) and spines (40%) of obliqueapical and basal dendrites arising from pyramidal neurons(Somogyi and Cowey, 1984; DeFelipe et al., 1989, 1990).Because CR and CB immunoreactivities rarely coexist inthe same cell, it is likely that these calcium-bindingproteins are present in distinct subpopulations of double-bouquet cells [Lund and Lewis, 1993; Conde et al., 1994;Gabbott and Bacon, 1996a (especially their Fig. 28); DelRio and DeFelipe, 1996; Martin and Meskenaite, 1997].Multipolar cells. The third and least frequent type of

CR1 cell in human mPFC encompasses the aspiny andsparsely spiny multipolar neurons. Multipolar CR1 cellshave been described in human area 17 (Glezer et al., 1992;Fonseca and Soriano, 1995) as well as in areas of monkeycortex (Conde et al., 1994; Gabbott and Bacon, 1996a;Martin and Meskenaite, 1997). Multipolar neurons thatwere immunoreactive for VIP have also been reported inarea 17 of the rat and cat (Connor and Peters, 1984; Peterset al., 1987), in rat frontal cortex (Kawaguchi and Kubota,1996), and in the mPFC of the monkey (Gabbott andBacon, 1997). Indeed, Kawaguchi and Kubota describe amultipolar VIP1 basket neuron forming multiple contactswith the somata and proximal dendrites of target cells.Whether some multipolar CR1 neurons in human mPFCare basket neurons (Kisvarday, 1992) remains to be deter-mined ultrastructurally following labelling of their axonalarbors.The sparsely spinous subtype of multipolar CR1 neurons

in human cortex described here strongly resembles thesparsely spiny multipolar neurons described by previousGolgi-impregnation studies in a variety of cortical areasand species (human: Mrzljak et al., 1988, 1992; monkey:Lund, 1973; Jones, 1975; Valverde, 1978, 1985; Lund andLewis, 1993; cat: Peters and Regidor, 1981; rat: Feldmanand Peters, 1978). The division of multipolar CR1 neuronsinto aspiny and sparsely spiny cell types received recentsupport from our previous observations on monkey mPFC(Gabbott and Bacon, 1996a) and from the work of Gulyaset al. (1992) describing spine-bearing CR1 neurons in therat hippocampus. The significance of these two types ofcortical CR1 interneuron deserves further investigation.

Synaptic targets of CR1 neuronsin primate cortex

The efferent synaptic connectivity of identified CR1

neurons has recently been described in the primary visualcortex of the monkey (Martin and Meskenaite, 1997).

TABLE 6. Percentage Distribution of the OverallCalretinin-Immunoreactive Neuron Population in Layers 1–6B in Area 32

of the Rat and Monkey and Area 328 of the Human1

LayerRat

(n 5 8)Monkey(n 5 3)2

Human(n 5 3)

1 7.9 6 1.3 7.1 6 1.2 8.4 6 1.92 22.9 6 3.4 34.2 6 2.8 37.8 6 7.23 31.0 6 3.5 43.3 6 3.8 41.2 6 2.95 26.0 6 5.0 12.2 6 1.2 9.8 6 0.56A 9.9 6 1.7 2.6 6 0.5 1.6 6 0.26B 2.3 6 0.1 0.7 6 0.05 1.3 6 0.1Total (%) 100.0 100.1 100.1

Statistical analysis‘Uniform’ versus ‘nonuniform’ laminar distributions: Individual x2 tests identified

that the distributions were significantly different from a uniform distributionof calretinin neurons between cortical laminae in the three species: rat, x2 532.19, df 5 5, P , 0.001; monkey, x2 5 100.9, df 5 5, P , 0.001; human, x2 5122.1, df 5 5, P , 0.001.

Laminar differences between species using multiple t tests.Layer 1: Rat vs. monkey vs. human—not significant.Layer 2: Monkey HumanRat t 5 5.10, df 5 9, P , 0.001 t 5 5.68, df 5 9, P , 0.001Monkey — n.s.Rank order: Human, monkey . rat.

Layer 3: Monkey HumanRat t 5 5.09, df 5 9, P , 0.001 t 5 4.46, df 5 9, P , 0.002Monkey — n.s.Rank order: Monkey, human . rat.

Layer 5: Monkey HumanRat t 5 4.59, df 5 9, P , 0.001 t 5 5.42, df 5 9, P , 0.001Monkey — t 5 3.20, df 5 4, P 5 0.033Rank order: Rat : monkey . human.

Layer 6A: Monkey HumanRat t 5 7.11, df 5 9, P , 0.001 t 5 8.16, df 5 9, P , 0.001Monkey — t 5 3.22, df 5 4, P 5 0.032Rank order: Rat : monkey . human.

Layer 6B: Monkey HumanRat t 5 25.88, df 5 9, P , 0.001 t 5 14.77, df 5 9, P , 0.001Monkey — t 5 9.295, df 5 4, P 5 0.007Rank order: Rat . human . monkey.

1Mean values 6 S.D. (%).2Calculated from Gabbott and Bacon (1996b).


Although Martin and Meskenaite did not concentratespecifically on cell subtypes, they found that, as a popula-tion, CR1 neurons chiefly innervate GABA-containingneurons in the superficial layers, whereas they only inner-vate pyramidal cells in the infragranular layers of area 17.Although CR1 cells and VIP1 cells innervate broadlysimilar postsynaptic elements, both sets of targets aredissimilar from other cortical interneurons (e.g., basketand axoaxonic cells) whose principal (.80%) targets areprojection pyramidal cells (Jones et al., 1994). Martin andMeskenaite also reported that, in the deep layers, 11% ofthe CR1 synaptic boutons were onto the GABA-immu-nonegative somata of presumed deep-lying pyramidalprojection neurons. Presumably, these CR1 boutons formedpericellular baskets similar to those described in thisstudy (see Fig. 4A,B,E). Furthermore, a recent observationhas demonstrated that VIP1 neurons form pericellularbaskets around GABA-immunonegative profiles in themonkey mPFC and that such pericellular nests inter-mingled with GABA1 puncta (Gabbott and Bacon, 1997).In the mPFC of the human, monkey, and rat (Gabbott

and Bacon, 1996a; Gabbott et al., 1997; this study), CR1

puncta were also found closely apposed with the somata ofCR1 neurons in the superficial layers, indicating thepossibility that CR1 neurons were interconnected viaaxosomatic contacts. Martin and Meskenaite (1997) haveultrastructurally confirmed this observation in monkeycortex and report that CR1 cell bodies formed 8% of thepostsynaptic targets innervated by CR1 boutons in thesuperficial laminae of monkey area 17.

Layer 1 CR1 cells

One important subclass of CR1 neuron that is notmentioned above is located in layer 1 (Marin-Padilla, 1984,1990). Previous studies have described several types ofCR1 cells in layer 1 of different mammalian species,including Cajal-Retzius neurons. Some debate exists overwhether Cajal-Retzius cells endure into adulthood orundergo developmental changes in their morphology andneurochemical content (Derer and Derer, 1992). Verneyand Derer (1995) have described Cajal-Retzius neurons inthe prenatal human cerebral cortex that show immunore-activity for nonphosphorylated neurofilament protein andcalcium-binding proteins, including CR. The evidence ofthis study, similar to that of Glezer et al. (1992) and ofBelichenko et al. (1995), indicate that CR1 neurons withthe structural hallmarks of Cajal-Retzius cells are alsopresent in the mPFC of the adult human. Indeed, Fonseca

and Soriano (1995) reported CR1 Cajal-Retzius cells in thetemporal cortex of elderly humans with Alzheimer’s dis-ease. Cajal-Retzius cells containing CR1 immunoreactiv-ity have also been reported in the adult monkey cortex(Conde et al., 1994; Gabbott and Bacon, 1996a; Martin andMeskenaite, 1997) and in developing and mature rodentcortex (Vogt-Weisenhorn et al., 1994; del Rio et al., 1995;Gabbott et al., 1997). It is possible that only a functionallyand neurochemically distinct subpopulation of Cajal-Retzius cells persist into adulthood (Huntley and Jones,1990).Of functional importance is that, unlike other neurons

containing calcium-binding proteins, evidence suggeststhat Cajal-Retzius cells do not contain GABA (Yan et al.,1995) and are excitatory in function (del Rio et al., 1995;Kim et al., 1995). Finally, the full identities of the postsyn-aptic targets of Cajal-Retzius neurons in human cerebralcortex are unknown and deserve consideration because ofthe influence that such comparatively rare cells (Table 2)could exert over the large numbers of superficial and deeppyramidal cells with apical dendritic tufts in layer 1.

CR1 pericellular baskets

The formation of CR1 pericellular baskets around twodifferent populations of unlabelled somata in humanmPFC(circular somata in layers 2/3 and pyramidal cell bodies inlayers 5/6) is preliminary evidence that CR1 neurons maybe targeting the somata of distinct types of neurons. This issimilar to the findings of Martin and Meskenaite (1997) inmonkey visual cortex (see above). However, one unan-swered question is the possible intermingling of CR1

pericellular baskets with those from PV1 basket neuronsaround common postsynaptic somata. Martin andMeskenaite (1997) report that CR1 boutons formed nearlyhalf of the symmetric synaptic junctions to target pyrami-dal somata in the deep layers of monkey area 17. Theneurochemical identity of the remaining axosomatic syn-apses was not determined.

Distribution of CR1 neurons in mPFCof human, monkey, and rat

In human mPFC, the highly asymmetrical distributionof CR1 somata in superficial layers 2/3 (79%) comparedwith deep layers 5/6 (13%) reported here agrees closelywith similar data presented previously by Hof et al.(1993a,b) for human area 24c. Indeed, the asymmetricaldistribution of CR1 cell bodies with a selective location in

TABLE 7. Percentage that Calretinin Immunoreactive Neurons Constituted of the Total Neuron Populations in the Medial Prefrontal Cortex of the Rat,Monkey, and Human1,2,3

Subjects

AreaAreal

mean (%)24a 24b 24c 328 25

Rat (n 5 8) nd4 4.3 6 1.4 nd4 3.9 6 1.1 3.8 6 0.9 4.0 6 0.22Monkey (n 5 3) 10.1 6 2.1 10.5 6 1.9 12.5 6 2.7 10.7 6 1.3 11.2 6 1.7 11.0 6 0.8Human (n 5 3) 8.7 6 1.4 7.8 6 0.9 7.2 6 1.2 8.1 6 1.3 8.3 6 0.9 8.0 6 0.5

Statistical analysisInterspecies comparison of areal mean differences using multiple t tests.

Monkey HumanRat t 5 24.38, df 5 9, P , 0.001 t 5 19.35, df 5 9, P , 0.001Monkey — t 5 5.50, df 5 4, P 5 0.005Rank order: Monkey . human . rat.

1Mean values 6 S.D. (%).2Data: rat, Gabbott et al. (1997): monkey, Gabbott and Bacon (1996b); human, this study.3Samples: rat, n 5 8; monkey, n 5 3; human, n 5 3.4nd, Not determined.


the superficial layers is seen in several species: CR1

somata represent 77% of the CR1 cell population in themPFC of the monkey (Gabbott and Bacon, 1996a) and 55%in the rat (Gabbott et al., 1997). Furthermore, the peakcortical depth distribution of CR1 somata in human,monkey, and rat mPFCs are all prominently located inlayer 2 (Gabbott and Bacon, 1996a,b; Gabbott et al., 1997;this study). The variations in the distributions and mor-phologies of CR1 neurons in the mPFC of different speciesmay relate to an increase in the functional specialisation ofthis class of inhibitory interneuron.Finally, in this study, CR1 neurons represented 8% of

the total neuron population of human mPFC comparedwith similar estimates of 11% in monkey and 4% in ratmPFC (Gabbott and Bacon, 1996a,b; Gabbott et al., 1997).The low value in human compared with monkey may bedue to reduced CR immunoreactivity and the methodologi-cal difficulties associated with examining postmortemhuman material.

FUNCTIONAL CONSIDERATIONS

CR1 neurons and vertical inhibitionin human mPFC

A columnar organisation of monkey mPFC has beenreported previously (Arikuni et al., 1983) and has beendescribed recently in the human cingulate cortex (Schlauget al., 1995). This columnar architecture relates to pyrami-dal cell groups in mPFC and the bundling of their ascend-ing apical dendrites (Sakai, 1985; Peters and Sethares,1991; Gabbott and Bacon, 1996c). The average width of theanatomically defined cell columns was estimated to beapproximately 40 µm in human area 24b, with columnsbeing separated by a similar distance of neuropil (Schlauget al., 1995). The role of the CR1 narrow-arbor neuronscould be to provide selective intralaminar inhibition radi-ally within a column of cortex. This idea receives strongsupport from the vertical and bundled organisation of CR1

dendrites and axons.Moreover, Fonseca and Soriano (1995)report that, in human temporal cortex, bundles of CR1

varicose fibres are spaced 25–75 µm apart, whereas, inhumanmPFC,CR1 axon-like fascicleswere spaced approxi-mately 53 µm apart, with a range of 26–121 µm (thisstudy). Given the postsynaptic targets of CR1 cells, suchnarrow radial inhibition could act in concert with otherinhibitory circuits to synchronise the activity and output ofprojection cells at different levels within individual cellcolumns.

Superficial vs. deep layers in ananatomical column

The highly skewed distribution of CR1 neurons inprimate cortex indicates the possible differential involve-ment of these interneurons in the cortical operationsperformed in the superficial versus deep layers. Althoughthe dendrites of CR1 cells freely cross laminar boundaries,the prime location of labelled somata in layers 2/3 suggeststhat the majority of CR1 neurons and their processes arepositioned to receive a specific array of afferent synapticinput. Data in nonhuman primates and other mammalsindicate that such superficial input could arise from localsources (axons of spiny stellate neurons, recurrent axoncollaterals of pyramidal neurons in layers 2/3 and 5A/6A,other cortical interneurons; Lund et al., 1994) as well as

from sets of corticocortical projections and from specificsubcortical pathways to mPFC, for example, from theamygdala, which, in the monkey, terminates principally inlayer 2, and from thalamic nuclei, which mainly innervatelayer 3 (Amaral and Price, 1984; Vogt et al., 1987; Vogt andPandya, 1987; Nimchinsky et al., 1996; for review, see Vogtand Gabriel, 1993).The observations of Martin and Meskenaite (1997) in

monkey striate cortex suggest that CR1 neurons withprojections to both the superficial and deep layers mediatea dual function. One interpretation of their ultrastructuraldata is that CR1 neurons in layers 2/3 simultaneouslydisinhibit specific cellular compartments of pyramidalcells in the upper cortical layers through the internuncialrole of other inhibitory interneurons (such as axoaxonic,basket, or double-bouquet cells) while monosynapticallyinhibiting deep layer pyramids. Such connections haverecently been investigated in the rat hippocampus byGulyas et al. (1996). If similar local circuits are present inother cortical areas and species, then the connectivity ofCR1 neurons in human mPFC could be one potent mecha-nism differentially affecting projection pathways from theupper and lower layers of anatomical columns. In thecingulate cortex of the monkey (and possibly also inhumans), pyramidal cells in the superficial layers projectpredominantly to other cortical areas (intracingulate, mo-tor, temporal, and frontal cortices), whereas projectionsout of the deep layers innervate not only a range of othercortical areas but also subcortical structures, such as thethalamus, striatum, red nucleus, pons, nucleus of thesolitary tract, and spinal cord (Dum and Strick, 1993; VanHoesen et al., 1993; Carmichael and Price, 1995a,b; He etal., 1995; Hof et al., 1995; Nimchinsky et al., 1996).Differential inhibition within these pathways could signifi-cantly alter information outflow from superficial and deepcortical layers, thereby producing consequent alterationsin behaviour.

CR1 neurons in neurodegenerative diseases

Functional imaging studies have shown that areas24a,b,c are involved in tasks requiring controlled atten-tion, motivation, and response selection (Pardo et al.,1990; Posner and Petersen, 1990), whereas area 32 pro-cesses information concerning declarative memory andword generation and is also involved in emotional reac-tions and expressions (Grasby et al., 1993; Raichle et al.,1994; George et al., 1995). The function of human sub-genual area 25 is comparatively unknown but may sub-serve aspects of affective disorders (Biver et al., 1995). Insum, humanmPFCmediates a wide range of motor, highercognitive, and associative processes as well as being in-volved in autonomic activities (Buchanan and Powell,1993; Neafsey et al., 1993). These functions are differen-tially compromised in psychiatric disorders (Devinsky andLuciano, 1993).Local-circuit neurons in the cingulate and prefrontal

cortices are affected during schizophrenia andAlzheimer’sdisease (Braak and Braak, 1993; Benes, 1995). Both thenumber and the morphology of CB1 neurons and, to someextent, of PV1 neurons are affected during these neurode-generative diseases (Arai et al., 1987; Ichimiya et al., 1988;Hof and Morrison, 1991; Iwamoto and Emson, 1991;Fonseca et al., 1993; Hof et al., 1993a,b; cf., Hof et al.,1991). It is of functional significance that CR neurons infrontal cortex and possibly also their synaptic connections


are comparatively unaffected (Daviss and Lewis, 1995;Fonseca and Soriano, 1995; cf., Brion and Resibois, 1994).Schizophrenia results in part from abnormalities of the

ventral tegmental mesocortical dopaminergic system,which heavily innervates frontal areas of the cortex,including the mPFC. In the primate, dopaminergic affer-ents innervate both GABA-containing interneurons andpyramidal cells (Goldman-Rakic et al., 1989; Sesack et al.,1995a). However, dopamine inputs selectively avoid CR1

neurons (Sesack et al., 1995b). This suggests that theirlikely postsynaptic targets are CB- and PV-containinginterneurons, especially because CR, PV, and CB neuronscomprise the vast majority (89%) of cortical interneuronsin monkey mPFC (Gabbott and Bacon, 1996b).This raises the intriguing possibility that the apparently

selective protection of the CR cell population could bepartly due to the specific neurochemical content of CRneurons, their Ca21 regulating capacity (Kostyuk andVerkhratsky, 1995), and the functional properties of theintracortical synaptic circuits in which they are embedded.Furthermore, it may be that the subpopulation of pyrami-dal neurons containing nonphosphorylated neurofilamentproteins, which have been shown to be selectively sparedin Alzheimer’s disease (Hof et al., 1990), preferentiallyreceives synaptic input to its somata and processes fromCR1 neurons. Of note here is the apparent selectivity forCR1 puncta in deep layers of human mPFC to formpericellular baskets around some pyramidal cell bodiesbut not around neighbouring somata (Fig. 4A; see also DelRio and DeFelipe, 1994). Such fundamental questionsconcerning the role of CR1 local-circuit neurons in humancortical function, both in health and in disease, deservefuture study.

ACKNOWLEDGMENTS

The technical help of Tracy-Ann Warner is acknowl-edged with gratitude. We also thank Kunle Adebowale forhelp with digital-imaging aspects of this project. PaulGabbott is a Beit Memorial Research Fellow.

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Calretinin neurons in human medial prefrontal cortex (areas 24a,b,c, 32?, and 25)