Local circuit neurons in the medial prefrontal cortex (areas 24a,b,c, 25 and 32) in the monkey: II. Quantitative areal and laminar distributions

THE JOURNAL OF COMPARATIVE NEUROLOGY :364:609-636 ( 1996)

Local Circuit Neurons in the Medial Prefrontal Cortex (Areas 24a,b,c, 25 and 32)

in the Monkey: 11. Quantitative Areal and Laminar Distributions

PAUL L.A. GABBOTT ANI) SARAH J. BACON University Department of Pharmacology, Oxford, England

ABSTRACT The companion paper (Gabbott and Bacon 119961 J. Comp. Neurol.) describes the

morphology of calretinin (CR)-, parvalbumin (PW, calbindin (CB)-, and GABA-immunoreactive neurons, and NADPH diaphorase-reactive cells, in the medial prefrontal cortex (mPFC; areas 24a, 24b, 24c, 25 and 32) of the adult monkey. Since these local circuit neurons play crucial functional roles, the aim of this study was to provide supportive quantitative data defining their areal and laminar distribution in mPFC.

The numerical densities of neurons (Nv, number of cells per mm:’) in each area and layer were calculated stereologically. The mean total neuronal Nv estimates across mPFC was 55,727 % 3,319 per mm3 (mean % S.D.; n = 3); values ranged from 50,489 -t- 8,374 per mm3 (area 24a) to 59,938 % 7,214 per mm3 (area 24c). Interareal differences were not significant. Cortical depth measurements and neuronal Nv estimates for each area allowed the absolute number of neurons in a column of cortex under 1 mm2 of surface to be calculated; values varied between 86,457 % 15,063 (area 24a) and 128,464 t 24,050 (area 24c).

Using immunolabelled Nissl-stained sections of mPFC, CR+ neurons constituted 11.2%, PV+ neurons 5.9%, and CB+ neurons 5.0%) of the total neuron population. GABA+ neurons represented an overall 24.9%, (23.5-27.3% ) of neurons in the mPFC. Differences between areas were not significant.

The cortical depth distribution histograms of CR+, PV+, CB+, and GABA+ cell populations in each area were derived and the percentage of a given cell population in each layer subsequently calculated. Peaks in the cortical depth distributions of CR+ and CB+ neurons occurred in layer 2 and upper layer 3, respectively; the peak distribution of PV+ neurons occurred between lower layer 3 and upper layer 5. The depth distribution of GABA+ cells reflected the combined distributions of CR+, PV+ and CR+ neurons. In all areas, the majority (74.4-84.0%) of the GABA cell population was located in layers 213. The depth distributions for each cell type were similar between areas.

Diaphorase-reactive neurons accounted for 0.25%’ (0.2-0.32% ) of‘ all cortical neurons in mPFC and were distributed in two horizontal strata, in midlayer 3 and in midiupper layer 6. A large population of diaphorase-reactive cells was present in the white matter.

The absolute numbers of CR+, PV+, CB+ and GABA+ neurons within individual layers in a column of cortex under 1 mm2 and 50 x 50 pm of cortical surface have been derived.

The data presented provide the basis for a quantitative definition of cortical circuits in monkey mPFC. , I:)%; ~ i ~ r y - ~ i s s , Inc.

Indexing terms: cortical modules, GAllA, calcium binding proteins, NADI’H diaphorase, quantitation

The medial prefrontal cortex (mPFC, Brodmann areas 24a, 24b, 24c, 25, and 32) of primates, and especially of humans, is involved in a wide range of complex higher order brain functions, such as learning and memory, cognition, association and the sequencing of tasks that are responsible for integrated patterns of behaviour (Fuster, 1989, 1995;

Goldman-Rakic, 1990; Vogt and Gabriel, 1993). The mPFC is also involved in autonomic functions in the body that

AcceptedJune ,9, ,995, Address reprint requests to L.A. ~ ~ b h ~ t t . Cniversity Dcpartment of

Pharmacology, Mansfieid Kwd, Oxford. OX1 3Q1‘. United Kmgdom.

< 1996 WILEY-LISS. INC.

P.L.A. GABBOTT AND S.J. BACON

modules, which are considered to be fundamental structural units in the cortex (Mountcastle, 1978; Peters and Sethares, 1991; Peters, 19931, in terms of neurochemically identified neurons and circuits.

Combined with information detailing the afferent and efferent synaptic connectivities, neurochemical characteristics and physiological properties of local circuit neurons, the present study will enable the functional anatomy of the primate mPFC to be quantitatively defined and realistic models of neural circuits in this region of the cerebral cortex to be constructed.

underlie the expression of emotional states (Smith and DeVito, 1984; Neafsey et al., 1993; Buchanan and Powell, 1993).

Inhibitory local circuit neurons critically influence the physiological activity of neurons within the cerebral cortex (Douglas and Martin, 1990, 1992; Jones et al., 1994). In an attempt to more fully understand the contribution of these neurons to the overall functional architecture of the mPFC, a detailed and comprehensive morphological description of local circuit neurons in the monkey mPFC has been given in the companion paper (Gabbott and Bacon, 1996). The approach involved the immunocytochemical identification of mPFC neurons expressing the calcium binding proteins calretinin (CR), parvalbumin (PV), and calbindin (CB; Andressen et al., 1993), markers for defined subclasses of inhibitory local circuit neurons in the cortex (DeFelipe et al., 1989; DeFelipe and Jones, 1992; Van Brederode et al., 1990; Demeulemeester et al., 1991; Hendrickson et al., 1991; Ferrer et al., 1992; Hof and Nimchinsky, 1992; Williams et al., 1992; Lewis and Lund, 1993; Hof et al., 1993; Conde et al., 1994). Cortical interneurons were also identified using gamma aminobutryric acid (GABA) immunocytochemistry (Jones et al., 1994). In addition, local circuit neurons displaying nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity were localised histochemically (Sandell, 1986).

The current paper complements and extends the morphological descriptions by providing quantitative data concerning the distribution of identified local circuit neurons influencing mPFC function. Using morphometric procedures, the absolute numbers and the relative proportions of CR-, PV-, CB-, GABA-, and diaphorase-reactive neurons in a gven cortical area and layer under defined unit areas of cortical surface have been estimated. Such data are important direct measures of the anatomical and physiological significance of each cell type. Furthermore, it becomes possible to define the cellular organisation of cortical

C%

CB CBPs CK D DAB D.circ D.nuc GABA GAD IN mPFC N, N,

NI

N\ pNADPH

N

NO NOS N N P%

PV

Abhr-euiations

cortical percentage-number of neurochemically identified neurons as a percentage of the total number of neurons throughout whole depth of cortex

calbindin D-28k Calcium binding proteins calretinin NADPH diaphorasc reactive 3,3'-diaminobenzidine diameter of an area equivalent circle diameter of a spherical nucleus gamma aminobutyric acid glutamic acid decarboxylase number of immunocytochemically defined test neurons medial prefrontal cortex number of neuronal nuclei per unit area of quadrat absolute number of neurons in a cortical column (i.e. under

1 mm2 of cortical surface and extending through whole depth of cortex)

absolute number of neurons in a given layer under 1 mm' of cortical surface

numerical density (number of neurons per mm.' of tissue) reduced form of nictotinamide adenine dinucleotide phos-

number of Nissl stained test neurons in the upper and lower

nitric oxide nitric oxide synthase Nissl stained 'test' neurons population percentage-number of neurochemically idcnti-

phate

immunoreacted regions of the tissue

fied neurons in a given lamina as a percentage of the total number of such cells throughout the whole depth ofcortex

parvalbumin

MATERIALS AND METHODS Preparation of material

Details concerning animals, tissue fixation and section- ing, as well as the subsequent enzyme histochemical and immunocytochemical staining procedures are described in the preceding paper (Gabbott and Bacon, 1996).

Briefly, serial sets of coronal Vibratome sections through Brodmann areas 24a, 24b, 24c, 25, and 32 of the adult monkey mPFC were stained histochemically for NADPH diaphorase enzyme activity in combination with either GABA, CR, PV, or CB immunocytochemistry. Each set of sections were cut at a different thickness (50, 80, or 100 pm). Selected sets from each cortical area were Nissl- stained to clearly identify cortical areas and reveal the pattern of cellular lamination. Material from five hemi- spheres taken from three animals was analysed.

In addition, a set of coronal sections from each area was processed with osmium tetroxide, dehydrated in an ascend- ing series of alcohols, and embedded in resin (Bolam, 1992). Semithin resin sections (0.5-pm-thick) were cut perpendicular to the cortical surface through each layer in each cortical area. These sections were then lightly counterstained with an aqueous solution of 0.294 Toluidine blue (containing 0.5% borax).

Areal definition Areas 24a, 24b, 24c, 25 and 32 of monkey mPFC were

identified in the Nissl-stained sections using defined cytoar- chitectural criteria (see Fig. 1A-E; see alsoVogt et al., 1987; Dum and Strick, 1993; Carmichael and Price, 1994).

Identification of cell types The immunocytochemically defined cell populations stud-

ied in the quantitative analyses described below were taken from material reacted via the immunoperoxidase-DAB method (see Gabbott and Bacon, 1996). In this material, specific immunostaining of neurons resulted in dark brown diaminobenzidine (DAB) labelling of cell bodies and processes in the immunoreacted regions of the tissue. Immuno- reactive neurons were clearly defined against the nonspe- cific light brown colouration of the background material.

Nissl staining of such material served to identify and separate unlabelled neurons and glial cells from neurons labelled immunocytochemically and/or histochemically (Fig. 2). lmmunoreactive neurons were clearly identified and on occasions their morphologes well-defined (Fig. 2B,C,J, and K). Counterstained neurons and glial cells (astroglia, oligo- dendroglia and microglia) in both the Nissl sections and the semithin sections stained with Toluidine blue, were distin- guished on the basis of cell size and nuclear staining characteristics (Fig. 2A, C-H; see also Gabbott and Stew- art, 1987).

Fig. 1. A-E: Photographs of Nissl-stained sections from areas 24a, by intervening bands of neuropil (arrow). Capillary (c). H: Photomicro- 24b, 24c, 25 and 32 showing the position of laminar boundaries within graph of layer 5 in cingulate area 24b. J Enlargement of region boxed each cortical region. WM, white matter. F: Photomicrograph showing in H showing the vertical alignment of cells (arrows) characteristic for horizontal tiers of cells in layer 5 of area 32. Capillary (c). G this part of the cortex. Capillaries (c). Calibration bars: A-E = 500 bm; Enlargement of the boxed region in F illustrating the horizontal F,H = 200 km; G,J = 50 km. clustering of layer 5 pyramidal neurons. The tiers of cells are separated

Figure 2

LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I1 613

In material processed for immunocytochemistry and subsequently reacted histochemically for NADPH diaphorase activity, neurons that were double-labelled had brown immunolabelling of their nuclei and the characteristic dark blueipurple formazan end-product of the diaphorase enzyme histochemical reaction in their cellular cytoplas (see Fig. 4c in Gabbott & Bacon, 1996).

Terminology and symbols The terms immunoreactive or immunopositive (either

CR+, PV+, CB+ or GABA+) refer tospecificimmunolabel- ling in cell bodies. Unless otherwise stated, the term diaphorase-reactive (D+ ) refers to strong enzyme activity in neuronal somata (Gabbott and Bacon, 1995a). The terms immunonegative (either CR-, PV-, CB- or GABA-) and diaphorase-nonreactive (D-) refer to the absence of specific immunolabellingistaining in cells. Finally, cells displaying both specific immunoreactivity and NADPH diaphorase activity are termed immunopositive/diaphorase-reactive neurons (e.g., CR+/D+, PV+/D+, CB+/D+ or GABA+/ D+) .

Quantitative methods Overuiew to quantitative data. The following quantita-

tive parameters were derived for each cortical area: ( i ) cortical thickness, depth below the pial surface of lower laminar boundaries, and absolute laminar thicknesses; (ii) cortical depth distribution histograms for individual populations of CR+, PV+, CB+ or GABA+ neurons; (iii) neuronal numerical densities (Nv, number of neurons per mm:’ of tissue); mean neuronal Nv estimates were calculated for each cortical layer and subsequently for each cortical area; (iv) cortical percentage (C% ) that either CR+, PV+, CB+ or GABA+ neurons constituted of the total

Fig. 2. Material reacted immunocytochemically for either calretinin ICRI, parvalbumin (PV), calbindin ICB) or GABA and subsequently ~iss l~counters ta ined. A: Nissl-counterstained section of layer 3 in area 24c with the somatic profiles of several neurons indicated (n) . One neuron lies near to the surface of the section with its soma bisected; the remaining nuclear profile (double-headed arrow) does not contain a nucleolus. Several glial cells are indicated by single white arrows. Capillaries ( c ) . B: Low power photomicrograph showing a parvalbumin- immunopositive neuron (white arrow) in layer 5 ofarea 24c. C: Higher power micropaph of neuron shown in B (rotated by approximately 45” clockwise: white arrow). Neighbouring neurons (nl and glial cells ( g ) are indicated. D: Layer 3, area 24a. A calretinin-immunoreactive neuron (CR+ i and processes (small white arrows) located amidst Nissl-stained neuronal cn i and glial cells (g). E: Calbindin-immunoreactive neuron I C R + I with neighbouringcounterstainedglial cells ( g ) and the profile of a neuronal somata ( n ) . A CB-immunoreactive beaded fibre is indicated Ismall white arrow). F: A calretinin-immunopositive neuron (CR+ I in layer 4 of area 32 with labelled processes emergmg from opposite somatic poles (white arrowsl. A fine process also arises from the cell body and descends obliquely through the cortex (three white arrows). Counterstained neuron (n l and glial cells (gl. G: GABA-immunoreactive neurons (GI with labelled processes lwhitearrowsl. Counterstained somata of glial cells (gl and the soma of a neuron (n l are indicated. H: Pavalbumin-immunopositive neuron IPV+ 1 with labelled processes ismall whitearrowsl in the white matter beneath area 24b. The somata of counterstained neuroglial cells (gl are indicated. J: Drawing of the identified PV immunoreactive neuron larrowed) and the surrounding counterstained neurons seen in B and C. K Drawing of the PV immunoreactive neuron shown in B and C. An immunolabelled “axon- like” process laxi emerged from the soma of this cell and gave rise to collateral processes that ascended and descended through the cortex (arrows). Calibration bars: K = 100 pm; A,B,J = 50 pm; C = 25 pm; D.E.F,H = 20 pm; G = 10 pm.

number of cortical neurons; (v) population percentage (P% ) that neurochemically identified neurons in a given lamina constituted of the total number present through the whole depth of cortex; (vi) absolute number of neurons (Nc) in a column of cortex occupying 1 mm2 of cortical surface and extending through the whole depth of cortex, determined from cortical depth data and cortical Nv estimates (above); (vii) absolute number of neurons (NL) in a given layer under 1 mm2 of cortical surface, determined from laminar thickness data and laminar Nv estimates (above); (viii) absolute number of CR+, PV+, CB+ and GABA+ neurons in a column of cortex under 1 mm2 (and under 50 x 50 km) of cortical surface area, determined from Nc and Cc/r data (above); and (ix) absolute numbers of CR+, PV+, CB+, and GABA+ neurons in each layer under 1 mm2 (and under 50 x 50 km) of cortical surface area, determined from Pc/r data and the estimates obtained in viii (above).

Similar quantitative parameters were derived for the laminar and areal distributions of strongly NADPH diaphorase-reactive neurons in the mPFC. The relative proportion of strongly and weakly diaphorase-reactive neurons was calculated (Gabbott and Bacon, 1996). The degree ofcoexist- ence of NADPH diaphorase activity with either CR, PV, CB, or GABA immunoreactivities were also determined (Gab- bott and Bacon, 1996). In addition, the proportion of the diaphorase-reactive neuron population that colocalised either CR, PV, CB or GABA immunoreactivities was determined.

Sampling procedure and linear measurements To provide representative quantitative information con-

cerning the cortical areas comprising monkey mPFC, data were derived from sections spaced throughout the rostrocau- dal extent of each cortical area investigated (see Fig. 1 in Gabbott and Bacon, 1996). This served to reduce the confounding effects of local variations in neuron composition and density not associated with cellular lamination.

Linear measurements were obtained using a computerised planimeter (Apple QuadraiKurta digitising tablet system operated using MacStereology). Measuring and statistical analysis programs were scaled appropriately to accommodate magnification factors. A tissue shrinkage of 15% (Rockel et al., 1980; Beaulieu and Colonnier, 1983) was incorporated into measuring programs; shrinkage was considered to be linear and isotropic.

Cortical depth and laminar thickness Cortical depth and laminar thickness measurements

were made on Nissl-stained coronal sections. Using either 10 x or 25 x objectives and 10 x eyepieces, the pial surface and lower laminar boundaries were traced onto paper with the aid of a drawing tube. The cortical depth and laminar thickness measurements were then calculated along lines positioned perpendicularly to the pial surface. Ten sets of cortical and laminar measurements were made per area per animal. Mean values were subsequently derived.

Neuronal density (Nv) estimates The stereological procedures for estimating neuronal

numerical densities (Nv) in cortical tissue have been reported in detail previously (Gabbott and Somogyi, 1986; Gabbott and Stewart, 1987; see also Weibel, 1979).

The number of neurons per unit area of tissue (NA) were calculated for each cortical layer using an area-weighted random counting procedure (Weibel, 1979; Fry, 1993). This

614 P.L.A. GABBOTT AND S.J. BACON

procedure was performed on Toluidine blue-stained resin semithin sections (0.5-bm-thick) cut perpendicular to the cortical surface. The counting procedure required that each cortical layer was sampled in proportion to its absolute cortical thickness (Table 2) using a defined number of square sampling quadrats (100 x 100 pm). Between 10 and 20 test quadrats were used to sample an individual cortical layer. At low magnification, quadrats were superimposed onto defined regions of the tissue section through a drawing tube. Counting procedures were then carried out at a higher magnification ( 4 0 ~ or 100 x oil immersion). Quadrats were only analysed if the whole of its sampling area and its surrounding borders contained tissue from a given layer. Sampling quadrats containing large blood vessels were not analysed.

Cell nuclei were used to define test objects (Weibel, 1979). All test neuronal nuclei lying completely within the sampling area of each quadrat were recorded. In addition, neuronal nuclei that intersected the left and upper borders were included for morphometric analysis (Gundersen, 1977). The number of test neuronal nuclei per unit area of quadrat (NA ) was calculated and mean laminar values derived.

The area equivalent circle diameters (D.circ) of test nuclear profiles in each layer were measured. Nuclear size-frequency distributions were then constructed and corrected for missing nuclear profiles (Weibel, 1979). The mean nuclear profile diameter (d) was calculated, and the mean sphere diameter of nuclei (D.nuc) derived (D.nuc = (41 7 ~ ) x d; Weibel, 1979).

From corrected NA, D.nuc and section thickness (t) measurements, Nv estimates were then calculated for each layer and mean areal values derived subsequently (Nv = NA/ [D.nuc + tl; Weibel, 1979).

Cortical depth distribution of identified cell types

The cortical depth distributions of GABA+, CR+, PV+, CB+ and diaphorase-reactive neurons were determined for each cortical area. The outline of a large rectangular sampling grid (700-pm-wide and extending the whole depth of the cortex and into the underlying white matter) was superimposed over stained sections via a drawing tube and positioned perpendicular to the pial surface. Each grid was divided into 100-pm squares. Using a 1 0 ~ objective lens, the positions of identified test neurons occurring in each quadrat that were: (i) immunopositive alone (GABA+, CR+, PV+ or CB+), (ii) strongly diaphorase-reactive alone (D+ ), (iii) weakly diaphorase-reactive alone, or (iv) displayed both immunolabelling and diaphorase reactivity (GABA+/D+,CR+/D+,PV+/D+,orCB+/D+)werecode- marked onto the grid. Identified test neurons that intersected the right border were included on the grid while those crossing the left border were excluded from analysis (Gundersen, 1977). Laminar boundaries were also drawn onto each grid.

Regions relatively unaffected by the curvature of the cortex were primarily chosen for grid analysis. Regions near the gyrus of area 24b and in the sulcal region of area 24c were therefore selectively avoided.

The absolute number of identified neurons occurring along each horizontal tier of the grid was calculated. Cortical depth distribution histograms were later constructed expressing the number of neurons present within a given horizontal sampling tier as a percentage of the total number of such neurons present in the area of cortex sampled by the grid. From knowledge of laminar thicknesses, the percentage of an identified neuron population (GABA+,

CR+, PV+, CB+ or D+) within a given layer was defined. Three grids were used to sample each cortical area. Data were combined to g v e mean areal data for each animal.

Cortical percentage of GABA, CR, PV, CB, and diaphorase-reactive cells

The percentage composition (0%) that GABA-, CR-, PV-, CB- and diaphorase-reactive cell populations constituted of the total neuron population in each cortical area was calculated directly from thick sections that had been immunocytochemically and/or histochemically reacted and then Nissl-stained. The procedure was performed on sections 50-km-thick and the sampling method was similar to that described above for neuronal Nv estimates.

The penetration of immunoreagents into the tissue produced two regions of immunoreactivity at the upper and lower surfaces of the tissue. The depth of these immunoreactive zones varied between 5 and 20 pm and depended on the pretreatment of the tissue sections and the antisera used (Gabbott and Bacon, 1996). Immunocytochemically defined neurons (IN) at the upper and lower surfaces of the tissue with test nuclei inside each quadrat or crossing its surrounding inclusion borders were counted. Immunola- belled neurons in which nuclear profiles were not clearly defined were not counted. The number of Nissl-stained neurons that had test nuclei (n) in the two immunoreacted regions of the tissue were also counted. Since diaphorase- reactive neurons were present throughout the thickness of the section, all test diaphorase-reactive neurons (D) were counted, as were all nonreactive Nissl-stained test neurons (nn; see Fig. 4B in Gabbott and Bacon, 1996).

Each layer was sampled separately and data were combined across layers to give a mean areal value of the percentage composition that a given cell type (GABA+, CR+, PV+, CB+, or diaphorase-reactive) constituted of the total neuron population; C% = 100 x [IN/(IN + n)], or CLTC = 100 x LD/(D + nn)].

[Note: Statistical analyses of nuclear profile size distributions indicated no significant differences between GABA+ , CR+, PV+ and CB+ cell populations].

Colocalisation of NADPH diaphorase reactivity and either CR, PV, CB, or GABA

immunolabelling in neurons Material that had been immunostained and reacted for

NADPH diaphorase activity was used to calculate the degree of colocalisation of diaphorase reactivity with either CR, PV, CB or GABA immunolabelling in mPFC neurons. This was defined as the percentage that dual-labelled neurons represented of all immunoreactive neurons within a cortical sampling grid. Mean areal values were derived.

In addition, the proportion that diaphorase-reactive neurons immunolabelled for either CR, PV, CB or GABA composed of the total diaphorase population within the immunoreactive regions of the tissue was estimated.

Absolute numbers of neurons under a defined area of cortical surface

Using mean cortical neuronal Nv estimates together with cortical depth measurements (above), the absolute numbers of neurons (Nc) in a column of cortex occupying 1 mm2 of cortical surface and extending through the whole depth of cortex were calculated. Similarly, the mean absolute number of neurons (NL) in a given layer under a 1 mm2 unit of cortical surface was determined from mean laminar Nv estimates and laminar thickness data.

LOCAL CIRCUIT NEURONS IN MONKEY mI'FC: I1 615

From data defining the percentage composition (C%) that a gwen cell type (GABA+, CR+, PV+, CB+, or D + ) constituted of the total neuron population, the absolute numbers of GABA-, CR-, PV-, CB-, and diaphorase-reactive neurons in a cortical column could be calculated I=Nc x iC%/lOO)]. In addition, from knowledge of how each defined cell population was distributed between cortical layers (see Cortical depth distributions of cell types, above) it was possible to calculate the absolute number of GABA+, CR+, PV+, CB+, or D+ cells in a gwen layer under 1 mm2 or cortical surface.

The absolute numbers of GABA+, CR+, PV+, and CB+ neurons in a column of cortex under 50 x 50 pm of surface area were subsequently determined, dividing the value under 1 mm2 of cortical surface by 400. A cortical column 50 x 50 pm wide represents the approximate width of cortical modules in the cortex (see Peters, 1993). The issue is considered further in the Discussion.

(Comment: The term cortical column is used below to describe a vertical column of tissue, occupying a unit area of cortical surface, extending from pia to white matter.)

Ancilliary morphometric procedures Two additional morphometric procedures were per-

formed to provide comparative data with which to assess the validity of the principal measuring techniques.

Laminar Nv estimates of CR+ neurons in layers 2 and 3 of area 24c were calculated separately using the unbiased disector method (Sterio, 1984). In brief, serial pairs of semithin sections (0.5-pm-thick and spaced 3 pm apart 1 were collected from the immunoreacted regions of thick resin-embedded sections. The semithin sections were then lightly counterstained with 0.2% Toluidene blue to identify unlabelled neurons and to discriminate between neurons and glial cells (Gabbott and Stewart, 1987). Sam- pling quadrats i 100 x 100 pm) were superimposed within layer 2 or 3 on the first section and the corresponding regon located in the second section. The number of test neuronal nuclei occurring on the first section but not the second section were counted according to the disector principle incorporating inclusion and exclusion borders (Gundersen, 1977). The counting procedure was reversed i i.e., counting test neuronal nuclei occurring on the second section but not the first section). Twenty pairs of sampling quadrats were used to analyse material from two animals. Mean laminar Nv estimates of CR+ neurons in layer 3 of area 24c were subsequently calculated using the unbiased disector method (Sterio, 1984). The proportion that CR+ neurons constituted of the total neuron population was also determined.

Postem bedding GABA immunocgtochemistrg. Postem- bedding GABA immunocytochemistry was carried out on semithin (0.5-2.0 pml resin-embedded sections using the procedure described by Somogyi and Hodgson (1985; see also Gabbott and Bacon, 1995). Sections were counterstained with Toluidine blue. Sampling quadrats were superimposed over selected areas of tissue sections and test GABA+ and GABA- neurons counted. The proportion that GABA+ neurons represented of the total neuron composition was calculated for area 24b (Gabbott and Somogyi, 1986).

Statistics

Disector technique.

The results are presented as mean laminar and areal values. Results were analysed using a statistical software package iInStat, GraphPad Software, CA). Data were ini-

tially subjected to a Bartlett test for the homogeneity of variance. Animal mean values were then compared using a one-way ANOVA, before the individual means were compared using multiple t-tests (Fry, 1993). Significant differences between areal means were considered to occur when a P value of < 0.05 was obtained (Fry, 1993). Mean laminar and areal values and have been ranked in order and significant probability values given as either P < 0.05, P < 0.02. or P < 0.01.

RESULTS Definition of cortical areas and lamination The definition of cortical areas and laminar boundaries as

seen in Nissl-stained sections are shown in Figure 1A-E. Areas 24a,b,c and 25 are agranular fields without a distinct layer 4. Area 24b lay over the cingulate gyrus while area 24c extended over the greater portion of the ventral bank and onto the dorsal bank of the cingulate sulcus (see Figs. 1 and 2 in Gabbott and Bacon, 1996; also Dum and Strick, 1993). Area 32 was granular in appearance, possessing a distinct population of small sized neurons distributed as a thin band above layer 5 (Fig. 1E).

Area 32 was characterised by horizontal tiers of clustered pyramidal-shaped neurons in layer 5 (Fig. 1F.G).

In area 24b, bundles of vertically aligned pyramidal- shaped somata were found in layers 5 and 6 (Fig. 1H). The mean diameter of an individual somatic bundle was 43.2 2 3.7 pm (mean 2 SD; n = 26 columns) with a mean centre- to-centre distance between neighbouring bundles 69.1 ?

5.3 pm. The space between adjacent bundles (26 pm) contained vertically aligned fasicles of unstained fibres. This arrangement was similar to two concentric cylinders arranged vertically within the cortex, the inner cylinder containing the bundles of somata, the outer cylinder containing the fibre bundles. The vertical clustering of pyramidal- shaped somata i Fig. 1H,J) strongly resembled the arrangement of pyramidal cell bodies whose apical dendrites engaged in bundling (Peters and Kara, 1987; Peters and Sethares, 1991; Peters, 1993). i Comment: In Nissl-stained sections, unstained fibres and fibre bundles were rendered visible by increasing the contrast of the material by closing down the condenser diaphragm of the microscope.)

The laminar boundaries between layers 112, 213 and 315 were well defined in Nissl material (Fig. 1 ). Attention to cell shape and size, as well as relative cell packing densities, were necessary to define a laminar boundary between layers 516. The placement of a boundary between layer 6 and the underlying white matter was more difficult (especially for area 24b which lies at the top of the cingulate gyrusl since there was no sharp gradation in neuronal density.

Cortical depth and laminar thickness measurements

Mean cortical depth measurements for each area are given in Table 1. The total depths of area 24a,b, and c are due, respectively, to changes in cortical thickness over the cingulate gyrus and sulcus. Mean laminar thickness and percentage depth measurements are given in Table 2. Of significance is that although layer 2 in area 24b was of similar thickness to other areas, it represented a substan- tially reduced percentage depth. This was due to marked increases in the absolute thicknesses of layers 5 and 6 over the course of the c inpla te gyrus.


TABLE 1. Depths of Lower Laminar Boundaries (pm) of Areas in mPFC' TABLE 2. Absolute Laminar Thickness (and percentage value of total cortical depth), Laminar and Cortical Neuron Densities (Nv per mm'),

Absolute Number of Neurons in Each Layer (N,.,, and Total Number of Neurons in a Cortical Column Under 1 mm' of Surface Area tN<. = XNl.)','

Area

Layers 24a 24b 24c 25 32 Area 24a I 139 i 7

2 346 I 13

3 967 ? 27

4

i 1.246 i 19

9.4%

23.6%

65.9% -

84.9'%

218 I 8 10 4'4

260 i 7 13 1%

195 t 8 11.6%

1x0 i 10 10.3%

372 t 9 21.3'1

1,013 i 18 52.2%

1,146 i 15 65.5%

1,400 i 10 80.0%

1,750 t 19

1,750 i 19 100%

Laminar thickness N,,, no. of neurons Laver (wm) N v under 1 mm'

506 i 6 25.5%

412 ? 21 24.6%

1.001 t 52 47.6'k -

1.420 2 63 67.5(k

2,104 i 72

2,104 i 72 1 00'Z

1,203 ? 13 60 6% -

1.532 2 29

1,046 i 14 62.3'Z -

1,407 i 23 83.8%

1,678 t 21 100'k

1,678 * 21

1 139 i 27 19.5%) 2,501 f 321 348 i 54 I 0 4'3 )

2 207 i 39 114.1'%1 72.521 f 8.127 15,012 2 6,116 !17.4'%! 3 621 I 18 (42.3'4 1 70,065 i 9,692 43.510 ? 4,673 150 3%1 5 279 z 33 119.0'%! 66.572 i 6,017 18,574 2 1,942 t21.5'kl 6 221 I 25 (15.1% I 40.784 i 10,602 9,013 i 876 110.4% I

Mean Nv 50.489 i 8,374 N< 86,457 2 15,063 i 100'k I 77.2%

1,985 i 25 100';;

6 1,467 ? 34

Total depth 1,467 I 34 l0OD Area 24b

1,985 ? 25

Laminar thickness N,, no. of neurons Layer ( p m ) NV under 1 mm2

1 218 i 18 110.4W 2,434 t 501 531 t 31 10.4'%! 2 187 i 48 (8 9'4) 76.346 ? 8,661 14,277 i 2.971 i l l 8%1

419 I 38 119.9(k! 66,134 t 13.490 27,291 t 2.536 122.6'2 ! 6 684 i 54 i32.5Ul 50,928 ? 15.375 94,835 2 3.567 128 R'k!

Mean Nv 53,696 i 10,139 N,. 120,821 t 16,104 199.9%)

3 596 ? 50 128.35! 73,636 i 8.108 43,887 i 4,112 136.3'k I >

'Depths also expressed a s percentage ofoverall cortical thickness. Mean 2 S.E.M ; n = 3 .

Neuronal density (Nv) estimates Mean total neuronal density (Nv) estimates for each

cortical area and layer are given in Table 2 and presented graphically in Figure 3. A mean Nv estimate for all areas of the mPFC of 55,727 2 3,319 neurons per mm3 was obtained. Individual areal Nv estimates ranged from 50,489 to 59,938 neurons per mm3. No significant differences in mean Nv estimates were detected between cortical areas (Table 2). The high coefficients of variation for the mean group data reflect wide interanimal variation in NA and Nv estimates related to limited sample size (Table 2).

The visual impression gained from the Nissl-stained sections is confirmed by the depth variation in neuron densities between layers (Fig. 3). Within each cortical area, layer 1 had very low neuronal Nv-estimates. In agranular areas 24a,b,c and 25 the highest neuronal densities were located in layer 2; layer 4 in granular area 32. A decline in neuronal density was present in layers 5 and 6 for all areas. A significant number of neurons were located in the underlying white matter (Fig. 3).

Area 24c

Laminar thickness N,., no. of neurons Layer ( k m ) NV under 1 mm"

1 260 i 17 113.1',$! 3,262 t 562 848 i 79 10.7%! 2 246 A 41 112.4(k I 83.677 i 5.823 20,585 ? 4,017 (16.0'4)

> 329 i 42 I 16.6Q 1 73,516 i 5,179 24,187k 3,416 11H.H%! 6 453 i 31 122.8'Z 1 58,205 i 10,008 26,367 i 2,254 i20.5%1

3 697 L 45 135.1'4 I 81,029 i 10,442 56.477 ? 3,396 144.0% 1

Mean Nv 59,938 2 7.214 N,. 128,464 i 24.050 1 100%)

Area 25

Laminar thickness N!,, no. of neurons Layer ( p m ) NV under 1 mm2

1 195 L 18 (11 6%) 2.749 i 549 536 2 61 10.5%! 2 217 t 41 l12.9!t) 88,206 i 4.230 19,141 ? 3,630 117.9%1 3 634 t 36 137.8Cfi 80.206 i 7,312 50,853 ? 2,917 147.6% I

6 271 t 19 i16.2LIi 43.125 i 6,070 11.687 i 1,079 !10.9'%1 5 361 ? 38 121.Sd) 68,265 i 6,683 24,644 i 2,764 t23.l'/rl

MeanN, 56,511 i 7,993 N,- 106,861 2 20.443 I100'kl

Area 32

Laminar thickness N,,, no. of neurons Layer (pm) Nv under 1 mm'

Total number of neurons under 1 mm2 of cortical surface

From cortical depth measurements and mean cortical neuron densities (Nv), the absolute number of neurons (Nc) that were present in a column of cortex were calculated (Table 2). The mean number of neurons in a cortical column under 1 mm2 of surface varied significantly between cortical fields and followed the trend, area 24c > 24b > 25 > 32 > 24a; the number (Nc) in a column of cortex in area 24c was 50% greater than in area 24a (Table 2).

Similarly, from mean laminar thickness measurements and laminar Nv densities, the absolute total number of neurons in a given layer (NL) under 1 mm2 of cortical surface were calculated (Table 2). The highest absolute numbers of neurons (43,500-56,500) were found in layer 3. In all areas the absolute number of neurons in layer 1 was approximately 0.45% of the total number of neurons in a cortical column (Table 2). In areas 24a, 24c, and 25, there was a significantly higher proportion of cortical column neurons in layers 213 (60%-68%) compared with layers 516 (40%-32%; Table 2). In area 24b a similar proportion of neurons was present in layers 213 compared with 516. In granular area 32,65% of cortical column neurons resided in layers 21314 with 34% in layers 516.

1 180 i 24 110.32) 3,114 2 492 561 i 74 (0.5'Z ! 2 192 L 31 111.0%) 74,721 i 9,371 14,346 i 3,196 (13.6%1 3 641 L 29 136.6%) 67,996 i 8.886 43,585 i 4,821 (41.4'%! 4 133i 20 176'irl 80,539 i 11,113 10,712 i 1,591 (10.2'11 5 254 i 34 i14.S',i! 65,916 t 5,842 16,479 2 1,532 115.7%! 6 350 2 28 (2Il.O'i, I 55,736 t 5,046 19,508 ? 1,601 Il8.6%!

Mean NL 58.004 i 7.397 NI. 105.191 2 16.863 t100'k)

IN,, values are g v e n as percentages o f N < . Mean values ? S D.: n = 3 . 'Statistical Analyses. 1) Comparison of mean neuronal densities tNt.1 between cortical areas.

Rank order: A24c > A32 ,, A25 > A24b > A24d One-way ANOVA: F = 0.88, df = 14; P = 0.51. Differences not significant.

2 ) Comparison between cortical areas of the absolute numbers of neurons in a cortical colurnntNl.i under 1 mm'ofsurface.

Rank order- A24c > A24h > A25 > A32 > A24a. 1 way-ANOVA: F = 3.50: df = 14; P = 0.05. Differences siggificant.

A24b A24c A25 A32 A24a t = 2.94, df = 4 , P < 0.05 t = 3 .49 ,d f= 4,P < 0.05 n s ns A24b - ns ns ns

ns ns A24c - ns A25 -

- - -

Cortical depth distributions of GABA+, CR+, PV+, and CB+ neurons

The cortical distributions of immunolabelled CR+, PV+, CB+ and GABA+ somata in tissue sections from area 24b are shown in Figure 4.

Area 24a

w 0

2000

0

500 - u f 4 a iooo 3

m 3

k

U

I 1500

w 0

2000

: wrn

....... ... ..... -~ ........

0 20 40 60 80 100

NEURONAL N, ( x103) . rnd

Area 24c

Area 24b - 1

500 - u E, 5 a 1000

3

m 3 W

I 1500 b- h w n

2000

2

3

5

6

wrn . _

0 20 40 60 80 100

NEURONAL N, ( x103) mm3

500 h

v E, Q: a iooo 3

m

n w

3 w I 1500 I-

-. ._. .........

P J wrn

0 20 40 60 80 100

NEURONAL N, ( x 1 0 3 ) . r n d 0 20 40 60 80 100

NEURONAL N, ( x103) . rnrK3

Area 32 0

500 - u % a a 1000 3

m 3

k

w I 1500

w cl

2000

L. I. .- ..'.u --!.~-->- !Li ~:--

0 20 40 60 80 100

~ - !

NEURONAL N, ( xl O3 ) . rnm3

Fig. 3. Cortical depth distributions of laminar neuronal Nv estimates. Mean values ? S.D.; n = 3.

618

' 2

3 .

5.'

P.L.A. GABBOTT AND S.J. BACON

.......... - - - . . , . ** .*: .-

c . 0 . . .. *.

.. a . .. 9 .

0..

.. * . '

' 0 % 0. :... *.. :*.

*- .. . -0.: :. .. J . ! 0 . ' .so. ...'.. .. .* -. . . ' . . . .. . * -. 8

+ - - *- ...... .*- . * .. 0. :. . **.

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e

WIT

CR CB .- - - -- - - * - --..

9..:.-:. ~ :...... .. 2 .. ......... #.*

0 : .......... : 3; *.: 0.'

. . .. .. * + :,e.>*+*. -* -* " :*+

*. .- . *

. . 0 ~. .. .. .*.*.- .. .. .*. *. 0 .

. . . . . + '= 8 ..............

t

* . . - . ." ....... -0. ..:-*-.

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GABA .

Fig. 4. Composite drawings of the cortical depth distribution of calretinin (CR)-, calbindin (CBI-, parvalbumin (PV)-, and GABA-immunoreactive neuronal somata in area 24b (Monkey M3). Stars indicate the positions of strongly NADPH diaphorase reactive neuronal somata. Drawings obtained from several sections. Calibration bar = 500 um

Figure 5 presents histograms of the cortical depth distribution of CR+, PV+, CB+ and GABA+ somata in the five areas investigated. These distributions were similar across all areas of the mPFC. The different cell types were not uniformly distributed throughout the cortex since the majority of each population was resident in layers 2 and 3 (Fig. 5). The peak distribution of calretinin neurons occurred in middle and upper layer 2 with a marked decline through lower layers of the cortex. The distribution for calbindin neurons revealed two peaks, a major peak occurring in lower layer Ziupper layer 3 with a minor peak situated in layer 5. By contrast, the cortical depth distribution of parvalbumin neurons displayed a peak centred on lower layer Siupper layer 5. In granular area 32, there was a marked reduction in the number of PV+ neurons in layers 5 and 6 (Fig. 5). The depth distributions of GABA+ neurons in all areas mirrored the combined distributions of CR+, PV+, and CB+ neurons (Fig. 5).

From these distributions it was possible to define the population percentage (P%), the percentage distribution of CR+, PV+, CB+ and GABA+ cell populations in a given cortical layer. Results were obtained for each animal and mean laminar data are given for each area (Tables 4-8).

Cortical depth distribution of diaphorase-reactive neurons

Figure 6 shows the distribution of diaphorase-reactive neurons in area 24b. Cortical depth distribution histograms are given for each area in Figure 7. Apparent from these histograms are the three peaks in the distribution of D+ neurons; the distributions were similar for all cortical areas investigated. Within the cortex, diaphorase-reactive neurons were distributed in two horizontal strata, midlayer 3

and midiupper layer 6 (Fig. 7). A distinct trough in D+ neurons occurred in the region centred on lower layer 3 to upper layer 5. The major peak in diaphorase-reactive cell distributions occurred in the white matter underlying each area (Fig. 7 ) . Diaphorase-reactive neurons in the white matter represented a large proportion of the total number of D+ neurons in each area, 42.0% in area 24a; 27.5% in area 24b; 41.7% in area 24c; 40.1% in area 25; and 37.7% in area 32 (Fig. 8). (Comment: The comparatively low figure for area 24b may relate to the difficulty in defining the layer 6iwhite matter boundary in this region.)

Cortical percentage of CR+, PV+, CB+, GABA+ and diaphorase-reactive neurons

The percentage composition ((2%) that each population of identified neurons (CR+, PV+, CB+, and GABA+) constituted of the total number of neurons in a cortical area are given in Table 3. Mean data for the mPFC indicate that CR+ neurons constituted 11.2 2 1.9% of all neurons, with PV+ neurons representing 5.9 i 1.4% and CB+ neurons 5.0 2 1.4% (Table 3). Differences between cortical areas were not significant (Table 3).

For the mPFC as a whole, GABA+ neurons represented 24.9 5 2.1% (mean 2 S.D.; n = 3) of all cortical neurons. On average, 1 in 4 neurons in monkey mPFC were GABA+. The relative percentage that GABA+ neurons constituted of the total neuron population in each area varied with respect to layer (Table 3 cf Table 2). In layer 1, the majority of neurons (87%) were GABA+. Across the mPFC, GABA+ neurons represented 36% of the total number of neurons in layers 2 and 3; this value declined to 11.1% for layers 5 and 6. In layer 4 of area 32, 12.9% of neurons were GABA immunopositive.

LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I1

Diaphorase-reactive neurons accounted for 0.25 2 0.05% of all neurons in mPFC (i.e., about one D+ neuron per 400 D- cells). Values for individual areas ranged from 0.20% (area 32) to 0.32% (area 25; Table 14).

Absolute numbers of CR+, PV+, CB+ and GABA+ neurons

The total numbers of CB+, CR+, PV+ and GABA+ neurons in a column under 1 mm2 of cortical surface for each area investigated are given in Tables 4-8. (These data are presented graphically in Fig. 8.) From population percentages (P%, the percentage laminar distributions of CR+, PV+, CB+ and GABA+ cell populations) the absolute numbers of CR+, PV+, CB+ and GABA+ cells in a given cortical layer under 1 mm2 (and under 50 x 50 pm) of surface area have been calculated for each cortical field (Tables 4-8).

Tables 9-13 provide statistical comparisons of laminar data (i.e., absolute numbers of GABA+, CR+, PV+ and CB+ neurons in defined layers under 1 mm2 of surface) between the various cortical areas. One major finding to emerge from this statistical data is the significantly greater number of cell types (CR+, PV+, CB+ and GABA+) in layers 2 and 3 of area 24c compared with other areas.

The mean ratio of CR:PV:CB neurons in them PFC was 2.2:1.2:1 (Table 14). This value was consistent between all areas investigated (Table 14).

Table 15 gives a statistical comparison of the absolute numbers of diaphorase-reactive neurons under 1 mm2 of cortical surface between the various cortical areas. The highest number of diaphorase-reactive neurons in a cortical column was present in area 24c (n = 361) with the lowest number in area 24a (n = 202).

Nv estimates of CR+, PV+, CB+ and GABA+ neurons

From cortical thickness measurements and the absolute numbers of CR+, PV+, CB+ and GABA+ neurons under 1 mm2 of surface (Table 2 and Tables 4-8), it is possible to define Nv estimates for each cell populations by post hoc calculations. Average Nv estimates across all areas of mPFC [mean, 2 S.D. (range)J: CR+, 6,832 ? 746 per mm3 (6,029-8143 per mm"); PV+, 3,615 2 318 per mm3 (3,216- 3,949 per mm"); CB+, 3,042 2 212 per mm3 (2,814-3,388 per rnm,''); and GABA+, 15,205 2 1,077 per mm3 (13,495- 16,091 per mm") neurons.

GABA cell population versus CR, PV and CB cell populations

In each area, the combined number of CR+, PV+ and CB+ neurons in a cortical column was less than the total number of GABA+ cells (Tables 4-8 and Fig. 9). The CR, PV and CB neuron population represented the following combined percentages: 83.5%, area 24a; 89.4%, area 24b; 95.1%, area 24c; 88.5%, area 25; and 87.0%, area 32 of the total number of GABA+ cells in each area (Fig. 9). (Com- ment: Caution should be exercised in interpreting these data since it was not established whether all the neurons in each cell population were GABA+.)

Layer 4 in area 32 Layer 4 in area 32 represented 7.6% of the total cortical

depth and due to its high neuronal density contained approximately 10% of total number of neurons in a cortical

619

column (Table 2 ) . Of note is the relatively large number of PV+ cells in this layer compared with the distribution of neurons throughout the rest of area 32 (Table 8 and Fig. 8 ) . The trough in the cortical distribution of diaphorase reactive neurons in area 32 was centred on layer 4 (Fig. 7).

Colocalisation of diaphorase activity with CR, PV, CB, and GABA immunoreactivities

Diaphorase activity was found to be colocalised with either CR, PV, CB, or GABA immunoreactivities in neurons throughout the areas of the mPFC investigated (Table 16A). Mean values for the mPFC as a whole indicated that 0.3%; of CR+ neurons, 0.4% of PV+ neurons, and 0.3% of CB+ neurons colocalised NADPH diaphorase reactivity. Of GABA+ neurons in mPFC, approximately 0 .59 were diaphorase-reactive. The degree of colocalisation varied markedly across mPFC areas (Table 16A). Dual-labelled neurons were located throughout the cortex and showed no preferen- tial depth distribution.

The proportion that diaphorase-reactive neurons containing immunoreactivity for either CR, PV, CB or GABA composed of the total diaphorase population was calculated for each area, and a mean value for mPFC derived (Table 16B). (Technical comment: The ability to identify diaphorase-reactive neurons in the immunoreactive regions of the tissue was a methodological problem that affected this quantitative parameter. Cells colocalising diaphorase activity and immunoreactivity were easily identified; however, the identification of neurons in the immunoreactive zones of the tissue that were diaphorase-reactive but immunonegative was problematic [see Figs. 25A,C in Gabbott and Bacon, 19761. The problem of false negatives could not be ruled out; therefore, the data presented in Table 16B may be overestimates.)

Within the NADPH diaphorase-reactive cell population in mPFC (areas 24a,b,c, 25 and 32), CR immunoreactivity was separately colocalised in 11.5% of diaphorase-reactive neurons, PV immunolabelling separately colocalised in 4.9%' of diaphorase-positive cells, and CB immunoreactivity colocalised separately in 10.1% of diaphorase neurons (Table 16B). Assuming nonoverlapping colocalisation of calcium binding proteins (CBP) in diaphorase-reactive neurons (i.e., no multiple coexistence of, for example, CR and CB immunoreactivities in individual diaphorase-reactive cells), then CBP were colocalised in a total 26.6% (range 17.4-37.2% ) of diaphorase-reactive neurons in layers 1-6 of monkey mPFC (Table 16B). A gradient in combined CBPidiaphorase reactivity colocalisation was observed in anterior cingulate areas: Area 24a (19.7%,) + area 24b (29.2%) + area 24c (29.5%;). (Comment: The actual number of neurons in a cortical column under 1 mm2 of surface that colocalise diaphorase activity and immunoreactivity for a calcium binding protein is very low. For instance, a cortical column in area 25 contains 106,861 neurons; of these, approximately 22 neurons colocalise CB immunoreactivity and NADPH diaphorase activity.)

Finally, GABA immunoreactivity was colocalised in 81 3%. (range 62-90%') of diaphorase-reactive neurons in monkey mPFC (Table 16B). Conversely, 18.1% of diaphorase- reactive neurons in monkey mPFC were GABA immunonegative.

Nv estimates using the disector technique Using the disector technique (Sterio, 1984) Nv estimates

for CR+ neurons in layers 2 and 3 of area 24c were calcu-

620 P.L.A. GABBOTT AND SJ. BACON

AREA 24a Calretlnln M1 Parvalbumin M i

0 TTrs--T7'-,7 1

2

- 500

4 n.

3 v E, -

5

6

3 1000 3 w I m

111 ................. 1500

t U

t n = 4 6 2 j

0 5 10 15 20 0 5 10 15 20 % OF POPULATION % OF POPULATION

Calbindin M I 4 ....

. . . . .

n = 599 ~

0 5 10 15 20 % OF POPULATION

2LLI .LL .L L.l.A-1 ..-,.L.L.& r 0 5 10 15 20

% OF POPULATION

AREA 24b Calreti n I n Parvalbumin MI Calblndln ~1 GABA M1

1

2 .. . . .

3

5

.- ...... - ..........

6

. . . . . . . . . . . . . . . . . . . . . . . - .!.L.t . .LLL.L1.&. d.L -2.

wm 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20

% OF POPULATION % OF POPULATION % OF POPULATION % OF POPULATION

AREA 24c M l Parvalbumin MI Calbindin MI GABA M1 Calretinln

......... ~~~~ -- .........

. . .

0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 % OF POPULATION % OF POPULATION % OF POPULATION % OF POPULATION

Figure 5

LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I1 62 1

AREA 25 Calretinin M I

U

500 v 5 4 a 3 1000 3 w I m

1500 D

I

2

3

~~ 5

AREA 32

0

- 500

4 u 5. a 3 1000 3 w m r

1500 0

2000

Yo OF POPULATION

Calretlnin M I

Parvalbumin M I 7 , rr r r rr, 7' 7 - 1 7 - 7 '7

Parvalbumln M I Calbindin M1

i n = 373 l w m l n=365 1 1 n = 403 u 1 d 1 L L l l 1 u I LJ A-, I 1 j , L L I i d L 1 2 L L l l L LILL_ L l _ l I L 1 1 L I 1

0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 % O F POPULATION % OF POPULATION % OF POPULATION

1 0 5 10 15 20

% OF POPULATION

Fig. 5. Cortical depth distributions of CR+, PV+, CB+ and GABA+ neurons in each area. The depth distributions are taken from one representative animal (M1). (Laminar boundaries are indicated).

lated as: layer 2 (mean ? S.D.; n = 2), 17,602 % 3,198 per mm,'; layer 3, 13,168 i 1,166 per mmR. The procedure used in this investigation gave CR+ Nv estimates of 18,526 ? 2,286 per mm3 in layer 2 and 13,985 2 1,267 per mm3 in layer 3. Estimates using the latter procedure differed by +5.2% for layer 2 and by +6.28 for layer 3 compared with the disector technique. The differences were not significant (layer 2: t = 0.386, n = 3, P = 0.725; layer 3: t = 0.725, n = 3, P = 0.521).

Postembedding GABA immunocytochemistry Stereologxal counting procedures performed on GABA-

immunoreacted semithin sections (Gabbott and Somogyi, 1986) indicated that GABA neurons constituted 25.2 +- 1.8% (mean ? S.D.) of the total number of neurons in area 24b. This compares with a value of 23.5 2 2.6% determined using the direct counting method employed in this study

(Table 5 ) . These values are not significantly different ( t = 0.79, df = 3, P = 0.49).

DISCUSSION This paper forms part of an immunocytochemical study

investigating the morphology and quantitative distribution of neurons in the medial prefrontal cortex (mPFC) of the adult macaque monkey expressing the calcium binding proteins, calretinin (CR), parvalbumin (PV), and calbindin (CB; Andressen et al., 1993). Substantial anatomical and immunocytochemical evidence indicates that these identified cell populations represent distinct subclasses of local circuit GABAergic neurons in the monkey cerebral cortex (DeFelipe et al., 1989; Hendry et al., 1989; Huntley and Jones, 1990; Van Brederode et al., 1990; Hendry and Jones, 1991; Hendrickson et al., 1991; Ferrer et al., 1992; Hof and


Diaphorase

Fig. 6. Composite drawing illustrating the morphology and distrihu- tion of diaphorase-reactive neurons in area 24h. (Laminar position of cells: layer 1, a; layer 2, h,c; layer 3, d-h; layer 5, i-k,m; layer 6, n-q; white matter, s,r.) The strip to the right of the diagram shows the cortical depth distribution of a population of 122 diaphorase-reactive neuronal somata (taken from a sample of 28 sections). The handing of the somata into two hands (superficial and deep) within the cortex is apparent. Calibration bar = 300 pm.

Nimchinsky, 1992; Williams et al., 1992; Lund and Lewis, 1993; Hof et al., 1993; Conde et al., 1994; see also Table 3 in Gabbott and Bacon, 1996). In addition, the study has used enzyme histochemistry to identify cortical neurons containing NADPH diaphorase enzyme activity, which colocalises with the activity of nitric oxide synthase (NOS), the biosyn- thetic enzyme of nitric oxide (NO; Vincent, 1994, 1995). Diaphorase-reactive (D) cells in the mammalian cortex are also local circuit neurons (Thomas and Pearse, 1964; Sandel, 1986; Valtschanoff et al., 1993; Liith et al., 1994; Vincent, 1994, 1995).

The companion paper (Gabbott and Bacon, 1996) provides a detailed morphological description of the morphology of CR+, PV+, CB+ and D+ local circuit neurons in areas 24a, 24b, 24c, 25 and 32 of the adult monkey mPFC. The present paper extends the morphological description by quantitatively defining the cortical and laminar distributions of these cell types. In addition, GABA immunocytochemistry was used to identify the complete population of local circuit neurons in the mPFC (Jones et al., 1994). Quantitative data concerning the cortical and laminar distribution of GABA+ neurons provide direct measures with which to gauge the contribution and significance of the individual subclasses of local circuit neuron towards the overall cellular composition and synaptic circuitry of monkey mPFC.

Methodological considerations The rationale behind the quantitative methods employed

in this paper was straightforward. Cortical depth measurements and cortical neuronal Nv data allowed the absolute numbers of neurons in a cortical column to be readily calculated for each area. Laminar estimates were also calculated. Using immunocytochemical and/or histochemical data, the percentage of neurons in a cortical column belonging to an identified neuronal class (CR+, PV+, CB+, GABA+ or D+) were then determined ('2%). Finally, from knowledge of how the identified cell populations within each area were distributed between individual layers (P%,), the absolute numbers of identified neurons in a given cortical lamina were derived.

Several aspects of the methodology deserve discussion. Firstly, the assumption that the observed cortical distribution of identified test neurons (for example see Figs. 4 and 5) represents the true distribution of a given cell population. The central issue here is the definition and identification of test neurons. The presence of immunocytochemically defined test neurons is directly related to: ( i) morphometric parameters of the cell population (cell size, density and distribution), (ii) fixation, (iii) the immunocytochemical procedure, (iv) antiserum specificity and penetration, and (v) the quantitative sampling procedures. Sections used for the quantitative analysis were prepared with conventional immunoperoxidase techniques. These techniques gave specific labelling within the tissue that was well defined (Fig. 2; see Gabbott and Bacon, 1996). Although postembedding immunocytochemistry provides optimal con- ditions for cell counting (see Gabbott and Somogyi, 1986) most of the antisera could only be used with preembedding procedures. Indeed, quantitative results derived from GABA- immunoreacted semithin resin sections did not vary significantly with data obtained from material immunoreacted using the preembedding procedure (see Results).

Secondly, the sampling and quantitative methods employed in the present study are standard procedures for estimating the volume numerical density of particles (Nv)

Area 24b 0

Area 24a 0

1 2

500 3 -

v E, 4 loo0

2

3 500 - - E,

2 1000

3 3 wm 1500

,... LL

I I- w n

a 2000

25W

3 5 10 15 20

O h DIAPHORASE NEURONS

2500

0 5 10 15 20 25

Yo DIAPHORASE NEURONS

Area 24c 0 8- -

1 1 - _ . _ .

Area 25 0 8 -~

1 -

. -

. A

2

3 '

2 . 500

h

v E, 3 ,

3 3

k 1500

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w 2ow

4 5 w 1500 m I 6

ill k - n

2000

- ._ . - - 2500

0 3 l o 15 20

% DIAPHORASE NEURONS

. . i _ . . 2500

6 - 5 10 15 20

% DIAPHORASE NEURONS

Area 32 0s 1

2

3

4

5

6

wm

CL W n

2000 Fig. 7. Cortical depth distributions of strongly diaphorase-reactive neurons in each area. The distributions define the percentage of the total number of cells throughout the cortex and underlying white matter that occur in a given horizontal sampling tier. (Comment: Diaphorase-reactive neurons in the white matter have been incorporated in the distributions sinccr they represent a substantial proportion of the ovcrdll cell population.)

2500

0 5 10 15

% DIAPHORASE NEURONS 20


CALRETININ

IF- F 2 2

2 3

8 5

U

3 0

6

0 2 4 6 8 1 0 0 2 4 6 8 1

AREA 24c AREA 25 AREA 32

1 I I I

1 2 4 6 8 1 0 0 2 4 6 8 1 0

Absolute number of neurons ( xi03 ) In each lamlna under immz of cortlcal surface

PARVALBUMIN

AREA 24a

4- 2 2

5 2 3

ki 8 5

0

6

0 1 2 3 4

AREA 24b

I--

L 0 1 2 3 4

AREA 24c r--

0 1 2 3 4 L- Absolute number of neurons ( xi03 ) In each lamlna under lmm2 of cortlcal surface

CALBlNDlN

AREA 248 I

2 2 5

2 3

8 5 0 U

6

AREA 24b r---

0%-

AREA 24c

I_____

0 1 2 3 4

Absolute number of neurons ( xi03 ) In each lamlna under lmmz of cortlcal surface

GABA

0 5 10 15 20 0 5 10 15 20 0 5 10 15

AREA 25 AREA 32

Absolure number of neurons ( xlO3 ) In each lamlna under Imm2 of cortical surface

Fig. 8. Histograms showing the absolute number of calretinin-, calbindin-, parvalbumin-, and GABA- immunoreactive neurons in each lamina under 1 mm2 of cortical surface area. Mean values; n = 3. (Error estimates are gwen in Tables 4-81.


T.ABLE 3 Mean Percentage That Calretinin-, Parvalbumin-, Calbindin-, and GABA-Immunopositive Neurons and Diaphorase-Reactive Neurons Compose of the Total Neuron Population in Each Cortical Area'

Area Areal

24a 24b 24c 25 32 mean

<4 CR 10.9 i 2.1 10.5 i 1.9 12.5 2 2.4 11.2 f 1 7 10.7 1 1.3 11.2 I 1.9 PV 6.7 z 2.8 5.6 i 1.9 5.8 I 2.6 6.1 i 1.5 5.4 2 1.7 5.9 t 1.4 C B 5 . 2 t 1.6 4.9 t 1.5 5.2 t 2.1 4.9 t 1.3 4.7 2 1.4 5.0 k 1.4

(4 CBPS Total 22.8 1 3 . 0 21.0 1 2.4 23.5 2 1.7 22.2 2 2.5 20.8 f 1.6 22.1 2 1 . 3

/4 GARA 27.3 i 4.1 23.5 i 2.6 24.7 k 2.3 26 1 2 :%.(I 23.9 i 2 0 24.9 2 2 1

'Mean va lu~s i S.I).. n = 3 'Statistical Analyses. '4 CK Rank order. A24c > A25 > A24a > A32 > A24b.

'k PV Rank order. A24a > A25 > A24c > 24b > A32.

'4 CB Rankorder. A24ciA24a > A24biA25 > A32.

'4 GARA Rank order. A248 > A25 > A24c > A32 > A24b.

Ont.~wayANOVA: F = 0.51 ,df = 14. P < 0 7 3 Differenc~snots i~~if icant

One-way ANOVA: F = (1.1.5, df = 14. P < 0.96. DifTerPnces not significant.

One-way ANOVA. F = 0.06, df = 14. P < 0.99. Differences not s i p i f c a n t .

On?-way ANOVA: F = 0.79. df = 14. P < 0.56. Differences not sikmilicant.

TABLE 4. Quantitative Data for Area 24a'

Calretinin Parvalbumin Calbindin GABA

27.3 2 4.1 C%: 10.9 ? 2.1 6.7 2 2.8 5.2 2 1.6 ~

No of neurons under 9,424 ? 1,479 I mm'

Nl d I ( ' r I ~ J O J

5,793 ? 1,038 4,496 2 753 23,603 2 4,190

Neurons under Neurons under Neurons under Neurons under

Layer P% l m m L 50 x 5 0 p m P'k I m m ' 50 x 5 0 p m P'Z I m m ' 50 x 5 0 ~ m P% I m m ' 50 x 50pm

1 0 5 2 0 . 1 4 7 2 5 0.1220.04 0 .1010 .01 6 .0 -1 .2 0 .01 t0 .004 - - - 1 2 z 0 I 283 2 33 0 71 k 0.12 2 : 3 2 0 i 5 . 9 3 , 0 1 6 2 7 5 6 7 . 5 i 1 . 3 1 7 . I i 2 . 8 9912159 2 . 5 1 0 . 3 2 7 . 5 2 4 1 1 , 2 3 6 i 2 0 6 3.120.6 21.8z3.1 5.145t1.008 1 2 9 k 2 . 7 1 56.9 i 2 . 3 5.362 t 1,103 13.4 i 2.14 57.2 i 3.2 3,313 5 634 8.3 1.4 61.2 i 1.3 2,751 k 436 6.9 2 1.1 62.2 f 2.9 14.681 t 2,681 36.7 1 5.5 5 4.7 2 2.4 443 2 61 1.1 i 0.2 18.9 2 3.3 1,095 i 209 2.7 i 0.4 7.6 2 0.9 342 I 49 0.9 2 0.2 9.6 f 1.8 2 . 2 6 6 ~ 388 5.7 1 1.13 6 5 . 9 i 0 . 8 5 5 6 2 7 3 1 4 2 0 3 6 . 7 2 1 . 2 3 8 8 2 5 2 1 . 0 1 0 . 2 3 . 7 1 0 . 6 1 6 6 1 2 9 0 . 4 k O . l 5 . 2 k O X 1 . 2 2 7 1 2 1 1 :3.1+0.5 Total 100.0 9,424 23.5 1000 5,793 14.5 100.0 4.495 11 2 100.0 23,602 59 0

IN, 'To ta l number ofneurons in atortical column under 1 mm'ofcortex = 86,457 k 15.063 (mean -I S.D.: n = 31

from sections of finite thickness using stereological techniques (Weibel, 1979). However, the methods are based on the assumptions that neurons in mPFC represent a hetero- geneously distributed population of unequally sized cells and that each cell possesses a single spherical nucleus placed concentrically within the soma. Neuronal nuclei closely adhere to these basic assumptions concerning par- ticle size, shape and distribution. The methods have been previously used to derive neuronal NV estimates in the visual cortices of several species (O'Kusky and Colonnier, 1982; Beaulieu and Colonnier, 1983; Gabbott and Somogyi, 1986; Gabbott and Stewart, 1987; see also Peters, 1987). Comparing the quantitative methods employed in this study with the unbiased disector technique (Sterio, 1984) produced NV estimates for CR+ neurons in layers 2 and 3 of area 24c that differed by +5.2% and +6.2% (respectively); these differences were not significant (see Results). Never- theless, it is recognised that the disector technique is the more efficient method to obtain neuronal NV estimates (Gundersen et al., 1988a,b; Brsendgaard et al., 1990).

Finally, comparisons between cell populations are facili- tated using percentage values (P%) to describe the cortical depth distribution of a given cell population. Unlike absolute numbers, percentage values overcome some of the problems associated with the penetration of different antisera into tissue sections. It is emphasised that the potential lack of immunolabelling within neurons does not indicate

the lack of the antigen. Cell physiology may cause significant differences in antigen concentration that are below the level of immunocytochemical demonstrability and therefore underlie false negative results. Consequently, the numbers of immunostained neurons counted in this study may be underestimates.

Total neuron populations Using stereological measuring procedures carried out on

semithin resin-embedded sections, O'Kusky and Colonnier (1982) estimated that in area 17 of the monkey (Macaca mulatta and Macaca fascicularis) there are approximately 119,100 neurons per mm3 of cortical tissue. For the areas of mPFC investigated here, neuronal NV estimates ranged from 50,500 to 60,000 neurons per mm3. In monkey, the density of neurons in area 17 is therefore more than double (x2.2) the average NV estimate found in mPFC.

Total neuronal Nv estimates have also been calculated for area 17 in other species (Peters, 1987): cat, 48-50,000 and 54,210 per mm3 (respectively, Beaulieu and Colonnier, 1983; Gabbott and Somogyi, 1986); rabbit, 38,000 per mm3 (Vrensen et al., 1977); rat, 60,000 and 80,537 per mm3 (respectively, Gabbott and Stewart, 1987; Peters and Kara, 1985). For areas 6,8 and 17 in the mouse cortex, Schuz and Palm (1989) obtained a mean estimate of 92,000 neurons per mm3. Recent quantitative studies have calculated the


TABLE 5. Quantitative Data for Area 24b'


23.5 2 2.6 C V : 10.5 z 1.9 5.6 2 1.9 4.9 5 1.5

No. of neurons

1 mm' XI # I " ' , lll(l8

under 12,686 z 2,811 6,766 z 2,209 5,920 2 1,501 28.393 ? 5,311


Layer Pr4 I m m ' 50 x 5 0 p m P% I m m 2 50 x 5 0 p m P% l m m L 50 x 5 0 p m P% I m m ' 50 x 5 0 p m

I 0.40 2 0 . 1 51 i 11 0.13 1 0 . 0 4 - - - - - - I . % ? 0.1 341 i 65 0.85 2 34.4 I 3.1 4,364 i 798 10.9 t 1.8 9.4 ? 6.5 6 3 6 t 137 1.6? 0.3 33.0 I 11.6 1,954 2 348 4.9 2 0.9 2 5 3 i 9.5 7,183 i 1.207 18.0 3 51 7 L 1 4 6.559 I 1.082 16 7 i 2.5 53.4 i 7.4 :i,(iI:l i 438 9.0 I 1.8 45.6 L 4.2 2,700 I 505 6 7 I 1.4 48.8 ? 6.5 13.856 2 2,607 34 7 1 6 6 2 0 8 8 3 7 2 1 4 7 2 . 1 1 0 . 3 25 .814 .8 1,7462295 4 .4L0 .9 9 . 8 1 2 . 4 5HOi I18 1 S i 0 . 2 14 .824 .1 4,2022980 1 0 1

'l'litd 100.0 12.686 31 7 100.0 6,766 16.9 100.0 5.921 14.8 100.0 28,393 71 1 6 6 9 ? l l H75<15:3 2 . 2 2 0 . 4 11.412.4 7711149 1.9?O.S 1 1 . 6 1 2 . 9 6H7?108 1 7 i 0 . 3 9 . 9 2 2 . 1 2 ,811 i497 7.0

IN, ' T o t a l n u m h r r o l n e u r o n s i n a a ~ r t ~ c d l c o i u m n under 1 mm'ofairtex = llO.X21 5 16,104 (mean ? S.D.; n = 3 1

TABLE 6. Quantitative Data for Area 24c'


CSt : 12.5 2.7 5.8 2 1.0 5.2 2 1.1 24.7 2 4.4

No. of neurons under 1 mm2

s, / 1 1 " / I11111

16,164 2 3,059 7,500 2 1.391 6,725 ? 1.418 31,940 2 6,279

Neurons under Neruons under Neurons under Neurons under

Layer P% I m m 2 50 x 5 0 p m P'X I m m 2 50 x 5 0 p m P I m m 2 50 x 5 0 p m P'% l m m ' 50 x 5 0 p m

1 I I .- 0.2 178 + 27 0.45 i O.OR 0.10 I o.02 8.0 z I .'j o w z 001 - - - 2.5 2 0.2 678 z 56 1 7 0.2 2 290 t 13.5 4,688 A 921 I 1 7 i 1 9 19.6 i 4.0 1.470 i 246 9.7 2 0.6 31.0 i 6 8 2,085 I 463 5.2 I 0.7 26.5 i 10.2 8,464 2 2,619 21 2 3 60 .7210 .59 ,812 i1 ,486 2 4 . 5 t 5 . 4 61.9iH.3 4,6422,578 1 1 . 6 + 2 8 4 7 , 9 1 4 8 3 , 2 2 1 1 4 7 7 8 . 1 i 1 . 3 5 6 . 6 i 4 . 3 I8.107?2,335 4 5 3 2 7 5 > 7.2 2 1.1 1.164 z 217 2 . 9 ~ 0.5 16.1 1 2.2 1 , 2 0 7 ~ I68 3 0 ? 0 6 10 1 I 1 2 679 t 116 1 7 2 0.3 10.7 + 1 6 3,446 I 583 8 6 ? 1 H 6 2.0 ? 0.2 32:l i 61 0.8 i 0.1 2 3 i 0.3 1 7 S ? 31 0.4 2 0.1 11 0 i 2.1 740 i 104 1.8 z 0.4 3 9 i 0.6 1,246? 212 3 1 i 0.5 'Iota1 100.IJ 16.165 40.4 100.0 7.500 18.8 100.0 6,725 16.8 100.0 31,941 79 9

density of neurons in the rat somatosensory cortex (54,900 per mm:'; Ren et al., 1992) and in the primary auditory cortex ( A l ) of the rabbit (55,000 cells per mm"; McMullen et al., 1994). Taking into consideration species differences and variations in areal cytoarchitecture (Vogt, 1993), as well as quantitative methodologies, the Nv data obtained for monkey mPFC (this study) are consistent with estimates derived in other investigations.

Of importance for the present study is that the monkey visual cortex contains approximately 2.5 times more neurons per column than other cortical areas in the same animal (Peters, 1987). O'Kusky and Colonnier (1982) calculated that there were about 202,000 neurons in a column of cortex under 1 mm2 of surface. This compares with the range of values for monkey mPFC of between 86,500 and 120,800 neurons in a similar column of cortex. Hence the mean absolute number of neurons in a cortical column from area 17 is 1.7-2.3 times greater than in a similar column from areas of the mPFC. The two sets of data are thus in good general agreement. Furthermore, data given by Rockel et al. (1980) indicate that in primate species, including humans, the number of neurons in area 17 is similar to that in macaque. Whether this applies to monkey and human medial prefrontal cortex remains to be determined. If so, then the data presented in this study could be used directly for comparative investigations in humans (Vogt et al., 1995).

Local circuit neurons GABA cell population. Previous studies have estimated

the proportion that GABA+ neurons constitute of the total neuron population in various regions of the monkey cerebral cortex. Hendry et al. (1987) provide extensive data concerning the proportions of GABA-immunoreactive neurons in the precentral motor, somatic sensory, parietal, visual and other areas of monkey cortex. GABA+ neurons were found to compose 19.0% of all neurons in area 17 and 22.7%) of neurons in area 3b. The value for area 17 compares with estimates of 15% and 20% derived, respectively, by Fitzpatrick et al. (1987) and Beaulieu et al. (1992). In other cortical areas investigated by Hendry et al. (1987) the overall proportion of GABA+ neurons was 24.7%. Of significance is that GABA+ neurons were found to constitute 24.2%, and 24.5%, of the total number of neurons within the orbital and lateral frontal cortices, respectively. These values are in direct agreement with the mean estimate of 24.9% (range 23.5-27.3%) derived for areas of the medial prefrontal cortex investigated in the present study.

The proportions that GABA-immunoreactive neurons represent in the cortex of other species have also been determined. In the cat, GABA+ neurons represent approximately 20.6% (1 in 5) of neurons in area 17 (Gabbott and


TABLE 7. Quantitative Data f i r Area 25'

Calretinin Parvalbumin Calhindin GABA ' T C'( : 11.2 f 1.7 6.1 % 1.5 4.9 % 1 . 3 2.1 1 5 3 0

No. of neurons under 11,969 ? 2,144 1 mm'

v, . I " , NN,,

6,519 f 912 5,236 L 930 26,822 ? 4.292


La.ycxr I"% lmrn' 50 x 50 prn P% I m m 2 50 x 50bm P% l m m ' 50 x 50 prn P% 1 rnrn' 50 x 50 urn I - - - - - 1.9 I 0.2 504 2 47.4 1.3 2 6.2 1.010 I 1,59 2 5 I 0.4 lH.li 4 I 142 2 4 I 0.5 25.7 10.X 6$93 I 1.289 17.2 1 1 I 6 4 i :1.0 49.0 I 5.2 3,194 I :I79 8 0 I 1.3 6 2 3 2 ! 551 x.2 I 1 5 r,7 1 4 7 15,181 I 2.H7ti : lX . I l > 4.3 1.747 i 293 4 4 2 0.7 IX5 707 4 1.X I 8 4 0 :I 11.6 1.9 , ' l .ll2 . 484 7.9 I 1 4 6 I 2 567 1 8 9 1.4 + 0 2 ,5.6 2% .! 56 07 L 0.1 4.1 I1 6 1.100 2 1x3 2.7 !. 0 4 'I.0iill I00 0 I 1.969 29.9 1000 6.518 16.3 100.0 5.236 I d . I 100.0 26.X22 67.1

IS, 'l'ot:il nuuihrr of'nruronh in a c ~ r t i ~ a l column undrr I mini ofcortex = 106.H6I I 20,443 linean I %I).; n = :]I.

TABLE 8. Quantitative Data for Area 32)

Calretinin Parvalbumin Calhindin GABA

CCC : 10.7 2 1.3 5.4 2 1.7 4.7 % 1.4 23.9 ? 2.0

No. of neurons under 11,255 2 1,791 5.680 ? 913 4,944 ? 735 25,141 f 4,041 1 mnil

u, , I 'I ill,^


Layer F'% 1 rnrni 50 x 5 0 p m P% 1 mmL .iO x 5 0 p m P% 1 mrn' 50 x 50pm P'C I m m ' 50 x 50pm

1 L 22 1 1H. .5 2.487 1 4 1 0 6.2 I 0 9 11 9 1 4 . 7 676 2 121 1.7 i 0.9 25.4 2 X.2 1.25 :I H c t i 4 6.7:ll 2 1.345 16.H 1.8 :1.:100 I 415 X X I 1.3 51 7 1 4 . 2 2.55 4 6 4 0.9 6SOI 112 1.6 .H (i93 2 127 1.7 1 0.3 5 6 I O . 8 27 I O . 8 l.:18:1 2 231 35 ! 0.6 > x * 1.5 991 + 165 2.5 .4 7 1 6 1 1 1 4 l N i 0 . 2 l 2 . l ? 2 . : 3 59 ti 2 9 I 0 5 :ati 2 ti2 0 8 0 1 .5.2 I 0.9 295 1 45 0 7 1 (1.1 5 2 i 0 9 25 '"0t;tl 100 0 I I.ZS.5 28.1 100 0 5,BXO 14.2 100.l1 4.944 909.0

- 0.8 10.1 90 i 16 0.22 i 0 05 - - -

Somogyi, 1986) and 24.6% (1 in 4) of cells in primary auditory cortex (Prieto et al., 1990). GABA+ neurons account for approximately 15% (1 in 7.5) of neurons in rat visual cortex (Meinecke and Peters, 1987; Beaulieu, 1993).

Using stereological techniques, Hendry et al. ( 1987) also derived Nv estimates for GABA+ neurons in the monkey cortex. These authors obtained a mean Nv estimate of about 23,400 GABA+ neurons per mm3 of area 17, giving approximately 35,400 GABA+ neurons in a cortical column beneath 1 mm2 of surface. This corresponds to 89 GABA+ neurons per column under 50 x 50 pm of cortical surface. In cortical areas 1-2, 3b, 4 and 5, the Nv estimates for GABA+ neurons obtained by Hendry et al. (1987) were lower and ranged from 10,400 to 17,200 per mm:l providing between 50 and 61 GABA+ neurons in a 50 x 50 pm column of cortex.

These data compare with the results of this study in the following manner. The densities of GABA+ neurons in monkey mPFC were calculated as: 16,089 per mm" in area 24a; 13,495 per mm:] in area 24b; 16,091 per mm3 in area 24c; 15,985 per mm:' in area 25; and 14,366 per mm:j in area 32 (derived from Tables 4-8), which are in good accord with the data of Hendry et al. (1987). However, the absolute numbers of GABA+ neurons in 50 x 50 bm columns of mPFC ranged between 59 and 80 cells. This range lies midway between the estimates derived by Hendry et al.

(1987) for areas 1-2, 3b, 4 and 5, and area 17. Variations in cortical depth measurements together with GABA+ Nv estimates for different areas of the cortex are likely to underlie such variations. Indeed, as mentioned above, the number of neurons in area 17 is greater than for other cortical areas while the overall percentage of GABA+ neurons is lower (20% in area 17 cf 25% in mPFC).

In the monkey mPFC, 74-84% of the GABA+ cell populations were located in layers 2 and 3, with the highest number of GABA+ cells in layer 3 (Fig. 8). By contrast, layers 5 and 6 had 14.8-23.95% of the GABA+ cell population. In area 32, layer 4 contained 5.5% of GABA+ neurons. These data indicate the significant distribution of GABAer- gic neurons between the superficial and deep laminae of mPFC areas. Since afferents to mPFC from the thalamus, amygdala, and hippocampus have heavy terminal fields within the superficial layers of the cortex (Van Hoesen et al., 1993), the distribution of GABA+ neurons possibly reflects the prominent role of GABAergic inhibition in the early stages of information processing within mPFC (see also Discussion in Gabbott and Bacon, 1996).

Finally, Schwartz et al. (1988) have demonstrated a bimodal periodicity in the tangential distribution of GABA+ neurons in the prefrontal cortex of monkeys. Over area 46 of the dorsolateral prefrontal cortex GABA+ neurons were distributed in two overlapping density bands with repeat


TABLE 9. Layer 1. Statistical Comparison of Absolute Numbers of Neurons Occurring in Layer 1 of Each Area Under 1 mm2 of Cortical Surface

1) Calretinin: Rank order: A24c > A25 > A32 > A24b > A24a. Significant differences. One-way ANOVA: F = 32.34; df = 14; P < 0.01. A24b A24c A25 A32

A24a ns' 424h - A24c - A25

2) Parvalbumin: Rank order: A24c > A24a. [A24b, A25, and A321. No significant differences. Few neurons in areas A24a and A24c (see Results).

3) Calbindin: Rank order: [A24a, A24b, A24c, A25, and A321. No significant differences. No neurons.

4) GABA: Rank order: A24c > A25 > A32 > A24b > A24a. Significant differences. One-way ANOVA: F = 26.31; df = 14; P < 0.01

t = 8.34, df = 4, P < 0.01 t = 7.68, df = 4, P < 0.01

t = 6.80, df = 4, P < 0.01 t = 5.48, df = 4, P < 0.01 t = 4.01, df = 4, P < 0.02

t = 4.46, df = 4, P < 0.02 t = 3.57, df = 4, P < 0.05 t = 4.92, df = 4, P < 0.01 ns

- - - -

A24b A24c A25 A32

A24d A24h A24c A25

t = 10.52, df = 4, P < 0.01 t = 6.80, df = 4, P < 0.01

- -

t = 6.57, df = 4, P < 0.01 t = 3.49. df = 4. P < 0 05 t = 4.08. df = 4, P < 0.02

-

t = 5.91, df = 4 , P < 0.01 t = 3.27, df = 4, P < 0.05 t = 3.88, df = 4, P < 0.02 ns

TABLE 10. Layer 2. Statistical Comparison of Absolute Numbers of Neurons Occurring in Layer 2 of Each Area Under 1 mm2 of Cortical Surface

1) Calretinin: Rank order: A24c > A24b > A25 > A24a > A32. Significant differences. One-way ANOVA: F = 6.14; df = 14; P < 0.01. A24b A24c A25 A32

A24a A24b A24c A25

t = 2.80, df = 4, P < 0.05 t = 2.82, df = 4, P < 0.05 ns' -

- - - -

ns ns ns ns

t = 3.78, df = 4, P < 0.02 t = 4.02. df = 4, P < 0.02 ns -

2) Parvalbumin: Rank order: A24c > A25 > A24a > A32 > A24b. Significant differences. One-way ANOVA: F = 11.66; df = 14; P < 0.01. A24b A24c A25 A32

A24a ns A24h - t = 5.13, df = 4 , P < 0.01 t = 3.09, df = 4, P < 0.05 ns A24c - - ns t = 5.02, df = 4. P < 0.01 A25 - - - t = 2.90, df = 4, P < 0.05

t = 2.93. df = 4, P < 0.05 t = 2.83, df = 4. P < 0.05 ns

3) Calbindin: Rank order: A24c > A24b > A32 > A24a > A25. Significant differences. One-way ANOVA: F = 17.52; df = 14; P << 0.01. A24b A24c A25 A32

A24a t = 3.07, df = 4, P < 0.05 t = 5.19,df = 4 , P < 0.01 ns ns A24h - ns A24c - A25

4) GABA: Rank order: A24c > A24b > A25 > A32 > A24a. Significant differences. One-way ANOVA: F = 10.07; df = 14; P < 0.01.

t = 4.52, df = 4, P < 0.02 t = 6.37, df = 4. P < 0.01

t = 2.91, df = 4, P < 0.05 t = 5.04, df = 4, P < 0.01 ns

- - - -

A24b A24c A25 A32

A24a A24b A24c A25

ns -

t = 4 52, df = 4, P < 0.02 t = 3.07, df = 4, P < 0.05

- - - -

ns ns t = 3.21, df = 4, P < 0.05

-

ns ns t = 3.89, df = 4. P < 0 02 ns

Ins, not significant

distances of 125-250 pm and 1,000-1,275 pm. Data from the present study did not allow periodicities in the tangential distribution of identified neurons to be examined. Nevertheless, they may also be a structural feature of the functional organisation of GABA+ neurons in the mPFC.

Across mPFC in the monkey, CR+ neurons constituted 11.2%, PV+ neurons 5.9%, and CB+ neurons 5.0% of the total cell populations. CR+ and CB+ neurons were predominantly distributed in layer 2 and upper layer 3, where 86% and 80% of the each cell type was, respectively, located. The peak distribution of PV+ cells occurred in lower layer 3 to upper layer 5; however, 71% of cortical PV+ neurons were positioned in layers 2 and 3.

Cortical depth and laminar distributions of CR+, PV+ and CB+ neurons in monkey prefrontal and cingulate cortices have been obtained by Hof et al. (1993) and Conde et al. (1994). Hof et al. (1993, Table 5.1) show an asymmetri- cal distribution of CR+, PV+, and CB+ neurons between layers 213 and 516 in area 24c of the monkey cortex.

CR, PV, and CB cell populations.

Approximately 77% of CR+, 62% of PV+, and 66% of CB+ neurons were present in the superficial layers compared with the deep layers. In contrast, lower distribution estimates were obtained for areas 9, 11, and 46 of the monkey prefrontal cortex. Conde et al. (1994) calculated that of the total population for each cell type, 63% of CR+, 52% of PV+ and 64% of CB+ neurons were present in layers 2 and 3. Differences reflect the fact that areas 9, 11 and 46 are granular cortical areas possessing a distinct layer 4. Signifi- cant proportions of each cell type were located in layer 4 of each area, especially in area 46, which contained 30% of cortical PV+ neurons (see Table 3 in Conde et al., 1994). The studies of Hof et al. (1993) and Conde et al. (1994) do not provide quantitative data concerning the percentage that each cell type represents of the total neuron population.

In the visual cortices of primates, Bliimcke et al. (1990) report that the distributions of PV+ cells in monkeys and humans were similar but not identical. The distribution of PV+ neurons was highest in cortical layers receiving input from the dorsal lateral geniculate nucleus (see below). PV+


TABLE 11. Layer 3. Statistical Comparison of Absolute Numbers of Neurons Occurring in Layer 3 of Each Area Under 1 mmp of Cortical Surface

11 Calretinin: Rank order: A24c > A32 > A24bIA25 > A24a. Significant differences. One-way ANOVA: F = 32.34; df = 14; P < 0.01. A24b A24c A25 A32

K24a ns' t = 4.30. df = 4, P < 0.02 ns ! = 3.15. df = 4, P < 0.05 A24h - t = 3.07. df = 4. P < 0.05 ns "S

A24c - t = C3.31, df = 4 , P < 0 05 ns A25

2) Parvalburnin: Rank order: A25 > A24c > A24a > A24b > A32. Significant differences. One-way ANOVA: F = 4.74; df = 14; P < 0.05.

- t = :3 Of,. df = 4. P < 0 Of, - - -

A24b A24c A25 A32

A24a A2417 A24c .\25

ns t = 2.92, df = 4. P < 0.05 n s ns n s ns 111 -

- ~ t = :3.63. df = 4, P < 0.05 t = :3.27. df = 4. P < 0 O R ~ "S - -

31 Calbindin: Rank order: A25 > A24c > A24a > A24b > A32. No significant differences. One-way ANOVA: F = 1.11; df = 14

41 GABA Rank order: A24c > A25 > A24a > A32 > 24b. No significant differences. One-way ANOVA: F = 0.15; df = 14.

Ins. not significant

TABLE 12. Layer 5. Statistical Comparison of Absolute Numbers of Neurons Occurring in Layer 5 of Each Area Under 1 mrnL of Cortical Surface

1) Calretinin Rank order A25 > A24c > A32 > A24b > A24a Significant differences One way ANOVA F = 13 79, df = 14, P < 0 01 A24b A24c A25 A32

A24a t = 7 1 1 . d f = 4 . r < o . o i A24h - ns ' t = :I 91, df = 4. P < n nz ns

n s ns A24c - - A25

2 ) Parvdlburnin: Rank order: A25 > A24b > A24c > A24a > A32. Significant differences. One-way ANOVA: F = 11.73; df = 14; P < 0.01

t = 4.24. df = 4, P < 0 0 2 t = 5.46. df = 4, P < 0.01 t = 5.42. df = 4. P < 0 01

! = 2.83. df = 4. P < 0.05 - - -

A24b A24c A25 A32

A24a t = 2.83. df = 4. P < 0.05 A24h - t = 2.72, dr = 4, P < n 05 n s t = 5 63. df = 4, P < 0.01 A24c - t = 4 10. df = 4. I' < 0.02 A2S - - - t = 5.80, df = 4. I' < 0.01

t = mfi. df = 4, P < 0.05 "S t = 3.19. df :: 4. P ': 0.05

t = 2 93. d r =I 4. P <. o 05 -

3) Calbindin: Rank order: A25 > A24c > A32 > A24b > A24a. Significant differences. One-way ANOVA: F = 5.56; df = 14; P < 0.02 A24b A24c A25 A32

A24a A24h - ns ns ns A24c - - ns ns

ns A25

41 GABA: Rank order: A24b > A24c > A25 > A32 > 24a. Significant differences. One-way ANOVA: F = 4.96; df = 14; P < 0.02.

t = 3.19. df = 4, P < 0.05 t = 4.52, df = 4,P < 0.02 t = 4.54. df = 4, f' < 0 02 t = 4 19. df = 4, P < 0.02

- - -

A24b A24c A25 A32

A24a A24b A24c A25

t = 3 01. df = 4. P < 0.05 t = 4.123, df = 4, f' < 0 02 ns ns

ns ns - ns " S t = 2.99. df = 4. P < 0.05 - -

"S - - -


neurons represented 11.5% of all cells in monkey area 17 and 6.8% of the all neurons in human visual cortex (Blumcke et al., 1990). Compared with the present study, several factors may account for the differences: (i) area 17 contains more then double the number of neurons than other areas and this may affect the relative proportion of different types of neurons (see above); (ii) cellular composition is likely to differ between areas with different cytoarchi- tectonic constructions (proisocortex versus neocortex; Vogt, 1993), (iii), the distribution of thalamocortical afferents is different between primary visual cortex and areas of mPFC in monkey (Douglas and Martin, 1990 cf Vogt, 19931, and (iv) functional differences between cortical area (visual versus prefrontal processing). McMullen et al. (1994) defined that PV+ neurons constitute 7.0% of neurons in the primary auditory cortex of the rabbit.

The study of Stichel et al. (1987) reported the concentration of CB+ neurons in layers 213 of area 17 in the cat. Similarly, Ferrer et al. (1992) demonstrated that 90% of CB+ neurons in the temporal neocortex of humans were

located in layers 1-3. In human visual cortex, C R + neurons were most prevalent in layers 2, 3 and 4abc with markedly reduced cell numbers in the infragranular layers (Glezer et al., 1992). These data and observations support the findings of the present study.

Diaphorase-reactive neurons As a population, diaphorase-reactive neurons were sparse

and widely dispersed accounting for only 0.25% (range 0.20-0.32%) of cortical neurons in mPFC (Table 15). An average of 275 diaphorase-reactive neurons (range 202- 361) were present in a cortical column under 1 mm2 of mPFC (Table 15). These populations of diaphorase-reactive neurons were principally distributed unequally into two horizontal strata within each area of mPFC, one-third of the cortical population in midlayer 3 and two-thirds in midiupper layer 6 (Fig. 7, Table 15). A similar cortical depth distribution has also been reported in monkey visual cortex (Sandel, 1986), in the visual cortex of humans (Liith


TABLE 13. Layer 6. Statistical Comparison ofAbsolute Numbers of Neurons Occurring in Layer 6 of Each Area Under 1 mrn2 of Cortical Surface

1) Calretinin: Rank order: A24b > A24a > A25 > A32 > A24c. Significant differences. One-way ANOVA: F = 11.07; df = 14; P < 0.01. A24b A24c A25 A32

A2 4 il n s l t = 4.40, df = 4, P < 0.02 ns A24h - A24c - A25

2) Pawalbumin: Rank order: A24b > A25 > A24a > A32 > A24c. Significant differences. One-way ANOVA: F = 27.89; df = 14; P c 0.01.

t = 4.24. df = 4, P < I1 02 t = 4.10. df = 4. P < 0 0 2

ns

t = 4.16, df = 4, P < 0.02 t = 2.96, df = 4. P < 0.05 " S 115

- - -

A24b A24c A25 A32 ~ ~ ~ ~ ~ ~ ~~~~~

A24a A24h - A24c - A25

3 ) Calbindin: Rank order: A24c > A24b > A25 > A32 > A24a. Significant differences. One-way ANOVA. F = 26.84; df = 14; P < 0.01.

t = 4.72, df = 4. P i 0.01 t = 6.45. df = 4, P < 0.01 t = 7.87. df = 4, P i 0.01

t = 3 04. df = 4. P < 0 05 ns t = 7 45, rif = 4, I' c 0.01

ns t = 6.04. df = 4. P c 0.01 t = 4.13. df = 4. P < 0 02 t = 4.83. dS = 4. P < 0 01

- - - -

A24b A24c A25 A32

A24a A24b - ns A24o - A2S

4) GABA: Rank order: A24b > A24c > A24a > A25 > A32. Significant differences. One-way ANOVA: F = 15.40; df = 14; P << 0.01.

t = 6.02. df = 4. P < 0 01 t = 9.02, df = 4, P << 0.01 t = C3.59, dl '= 4. P < 0.05 t = 4.32. df = 4. P < 0.02 t = 6.51. dl '= 4. P < 0.01

t = 3.27, df = 4. P i 0 05 t = 4.73. df = 4. P < 0.01 t = 7 20. df = 4. P < I1 01 ns

- - - -

A24b A24c A25 A32

AZ4a t = 3.85. df = 4, P < 0.02 ns 11s ns A24h t = 5.36. df = 4, P < 0 01 A24c - - 11s t = 3 8 7 . d f = 4 . P < 0 0 2 A25

- t = 3.78. df = 4, P < 0.02 t = 4.45. df = 4. P < 0 0%

115 - - -


TABLE 14 Ratio Between CR-, PV-. and CB-Immunoreactive Cells in Areas of the mPFC'

CR PV CB Total

ArYd 24a Abs n o column

CR:PV:CR ratio

Abs no column

CR:PV.CK ratio

Ahs. no columii

Area 24h

Area 24c

CR-PV CR rdtlll Area 25

Abs. nwcolumn

CR:PV CR rat111

Abs. nOcolurnii

CR-PV.CR ratio Mean mPFCnred

Area :I2

CRPV-CI% ratio

9.424 48'h 2.1

12.686 50% 2.2

16.164 53% 2.4

11,969 50% 2.3

11.255 51% 2.2

2.2

5.793 29% 1.3

6,766 27% 1.2

7,500 25% 1.1

6,519 28% 1.S

5,680 26% 1.1

1.2

4,496 23% 1

5.920 23% 1

6,726 22% 1

5.236 22% 1

4,944 2 : n 1

1

19,913 100%

25,372 100'1

:30,3x9 100%

23,724 100s

21,879 l00U

- 'The ahsolute number of CR. PV, and CB neurons per cortical cnlumn is given (Ahs. ni,,cr,lulnn = number dcells in a column extending throughout t h r whole depth of the cortex under 1 mm' ifcortical surface areal. The percentage tha t each cell type composed of the total cell count per column ICR + PV + CRI IS also given. The relationship between cell types 1s m v m 1CR:PV:CR ratioi.

et al., 1994), and is also present, albeit less pronounced, in the mPFC of the rat (Gabbott and Bacon, unpublished observations). The significance of this cell population and its distinct intracortical distribution remains to be determined. One interesting observation is that in the monkey the two strata of diaphorase-reactive neuronal somata straddle the main thalamorecipient territory of mPFC (Vogt et al., 1987; Vogt and Gabriel, 1993).

Finally, Hashikawa et al. (1994) report a uniformity in the distribution of diaphorase reactive neurons across several cortical areas in the Japanese macaque-macaca

fuscata. However, within the mPFC of macaca fasicularis although the qualitative distribution of strongly diaphorase reactive neurons appeared relatively uniform, when the absolute numbers of labelled cells under 1 mm2 of cortical surface were derived for each area, significant interareal differences were detected (this study - Table 15A).

Colocalisation of CR, PV, CB in GABA cell populations

The degree of colocalisation of CR, PV and CB immunoreactivities in mPFC neurons was not investigated in this paper. However, the subject is of considerable interest and has been reviewed in detail elsewhere (see Rogers and Resibois, 1992; Rogers, 1992; Andressen et al., 1993; Kubota et al., 1994).

I t is important to clarify that the quantitative data of the present study has been derived for separate populations of CR+, PV+, CB+ and GABA+ neurons. Assuming that there is little (or no) overlap in the expression of CBPs markers (cf Kubota et al., 1994), the data indicate that the combined CR+, PV+, and CB+ cell populations represent between 84% and 95% of the GABA cell population in mPFC (Fig. 9). This is in general accord with other studies which have shown that each of these calcium binding proteins represents an unique marker for distinct populations of cortical neurons in adult animals (DeFelipe et al., 1989; Hendry et al., 1989; Huntley and Jones, 1990; Van Brederode et al., 1990; Hendry and Jones, 1991; Hendrick- son et al., 1991; Demeulemeester et al., 1991; Ferrer et al., 1992; Hof and Nimchinsky, 1992; Williams et al., 1992; Rogers, 1992; Lund and Lewis, 1993; Hof et al., 1993; Conde et al., 1994; Kubota et al., 1994; see also Table 3 in Gabbott and Bacon, 1996). However, whether all neurons in a given class of interneuron contain the respective marker calcium binding protein (or combination of CBPs) is not known, for example, the difference in PV immunoreactivity of chandelier cells in the superficial and deep layers of


'TABLE 15.4. Percentage Composition ((3% 1 That Strongly Diaphorase- reactive, Neurons tlayers 1-6) Constituted of the Total Neuron Composition

in Areas 24a. 24b. 24c, 25, and 32'

Cortical area

24a 24b 24c 25 32

I", 0.2:1 I 0.09 0.21 I 0 08 (1 28 i 0 1 I 0 :I2 i 0 05 ll.20 I 0 0 4

as no <nr d i a p h m x w nwroti,. under 111,111~ 202 I :19 2,55 I 4 4 361 c 69 :345 L 59 212 i :iZ

Statistical Analysis. Rank order: A24c > h 2 5 > A24h > A32 > A24a. One-way ANOVA: F = 32.34; df = 14; P i 0.01.

A24b A24c A25 A32

A242 n.1 t = 3 47. dl' = 4. t = X.01. d i = 4, I>!:

A24h - ns "S "8

A%4c - - "a t = 3 37. d i = 4. I' < 0.05 M 5 - - - t = 2 80. d i = 4, P < 0.05

P < 0.05 P < 0.05

:From dater in 'l'ahle 2, the absolute number of strongly diaph(,r;tsu-rPactivc' nrurons under I mn.' o f cortical surface throughout the depth o i thr cortex has been calculated. .V.2t.;in v,iI~it.s '~ S.1) . n = :1 hi. not sipnliicmit

TIU~LE 15B. Strongly Diaphorase-Reactive Cells in mPFC

Combined areas 24a.b.c. 25 and 32

TABLE 15C. Mean Percentage of Strong and Weak Diaphorase-Reactive Neurons in Superficial and Deep Layers of the i lganular Cortical Areas

ComprisinE Monkey mPFC'

Diaphorese-reactive cells

Strong Weak

25 7'1 1%61 ,511 4 % 14641 76 1'4 17001

the cortex (Gabbott and Bacon, 1996; see also Kubota et al., 1994 1. Furthermore, a numerically small population of neurons in layer 1 represents another exception, since during development they are reported to colocalise specific combinations of calcium binding proteins (Huntley and Jones, 1990; Conde et al., 1994; Vogt-Weisenhorn et al., 1994 1. The shortfall in percentage compositions may be explained by the presence of unidentified populations of GABA neurons containing other calcium binding proteins or neuroactive substances. Inclusion of diaphorase-reactive neurons in the morphometric considerations would not significantly affect the findings because these neurons represent a numerically very small population of cortical neurons (Table 16). The discrepancies could also be due to methodological limitations. Moreover, a very low proportion of cortical neurons expressing CR, PV, or CB immunoreactivities do not contain GABA (Demeulemeester et al., 1991; Andressen, 1994; Kubota et al., 1994).

In addition to calcium binding proteins, local circuit GABAergc neurons in the cerebral cortex contain specific neuropeptides and at least one specific enzyme activity, NOS/ NADPH diaphorase activity (Hendry et al., 1984; Somogyi et al., 1984; Demeulemeester et al., 1991; Jones et

30 I t -

@ Calret : Paw & Calbin

GABA 25

20

% 15

10

5

n - 24a 24b 24c 25 32

AREA

Fig. 9. Summary diagram showing the percentage composition that the combined CR-, PV-, and CB-immunoreactive cell populations constituted of the total neuron population in each cortical area (dotted and cross-hatched histograms). For comparison, the percentage tha t GABA neurons constituted of the total neuron population in each area is also shown (black histograms). Mean values; n = 3. Error estimates Dven in Table 3.

al., 1994; Kubota et al., 1994; Gabbott and Bacon, 1996). The neuropeptide markers also define distinct populations of interneurons and the degree of coexistence of CR, PV, and CB with combinations of somatostatin, cholecystoki- nin, neuropeptide Y and vasointestinal polypeptide in GABA- ergic local circuit neurons remains to be fully unravelled in primate cortex (Hendry et al., 1984; Demeulemeester et al., 1991; Rogers, 1992; Miller and Robertson, 1993).

Kubota et al., (1994) provide details of the coexistence of calcium binding proteins, neuropeptides, and NOS in layers 213 and 516 of the frontal agranular cortex in the rat. One significant difference between the data obtained for the rat frontal agranular cortex and monkey mPFC concerns the proportion of CR+ cells. In monkey mPFC, CR+ neurons (as a single population) represented approximately 45% of GABA+ cells in mPFC, whereas in the rat they constituted about 20% of the GABA cell population. Calretinin cells were also found to be the most abundant CBP cell type (double that of CB+ and PV+ neurons) by Conde et al., (1994) in the PFC of monkey. Species and areal differences, together with methodological factors (e.g., fixation proto- cols and antibody specificities) may partially underlie the differences between rat and monkey. Alternatively, the differences may indicate an increase in the CK+ cell population directly related to the functional significance of the processing operations performed by CR+ cells in monkey mPFC compared with rat (Preuss, 1995).

Colocalisation of CR, PV, CB and GABA in diaphorase-reactive cell populations

The colocalisation of either CR, PV, CB and GABA immunoreactivity with NADPH diaphorase activity in neurons has been reported in several brain regions (see refer- ences cited in review by Vincent, 1995).

As demonstrated in Gabbott and Bacon (1996), diaphorase-reactive neurons in monkey mPFC are morphologically diverse and closely resemble local circuit neurons in the cortex. This was substantiated by the finding that GABA immunoreactivity was colocalised in a large propor-


TABLE 16A. Percentage Colocalisation of Diaphorase Reactivity With Either CR, PV, CB, or GABA Immunoreactivity in mPFC Neurons'

Cortical area

24a 24b 24c 25 32

CR 0.12 2 0.04'k 0.27 2 0.06% 0.31 r 0.09% 0.22 2 0.05V 0.33 2 0.07% PV 0.17 2 0.04'% 0.16 2 0.08'2 0.21 f 0.09'% 0.19 f 0.07% 0.29 2 0.05% CB 0.42 + 0.049 0.50 i 0.03% 0.62 2 0.05'Z 0.44 ? 0.04'4 0.51 ? 0.27'4 GABA 0 58 1 1.2% 0.47 + 0.06a 0.54 i 0.10'i 0.43 ? 0.07% 0.49 2 0.06%

'Results expressed as t h e percentage tha t dual-labelled neurons in the immunoreactive zones of the tissue constituted of the total number of immunoreactive neurons. Mean data ? S.D.: n = 3.

TABLE 16B. Proportion of the Diaphorse-Reactive Neuron Population (layers 1-6) in mPFC That Colocalise Either CR, PV, CB or GABA

Immunoreactivities'

Mean data for mPFC (Areas 24a, b, c , 25 and 32)

CR PV CB CBPs GABA

Percent ( i f

NADPH diaphorase 11.5 ? 4.40 4.9 2 1.4% 10.1 2 2 1% 26.6 -t 7.2% 81.8 2 10.1'Z neurnn DODU~atlOn

'Results expressed as the percentage tha t dual-labelled neurons constituted of the total number of diaphorase reactive neurons present in the jmmunoreactive- zones or t h e tissue. The total percent coexistence for the calcium binding proteins is also given (CBPs). Mean data 2 S.D.: n = 3.

tion of diaphorase-reactive neurons (82%; Table 16B). The precise neurochemical identity of the remaining 18% of the diaphorase-reactive neurons is of interest, since these neurons may represent a subpopulation of GABA-immunonegative diaphorase-reactive cells. Such a subset of diaphorase-reactive neuron is unlikely to comprise excitatory spiny stellate cells, as these nonpyramidal neurons are virtually nonexistent in monkey PFC (Wilson et al., 1994). Furthermore, numerical considerations indicate that this subset of diaphorase-reactive neurons would represent about 50 cells in a cortical column containing in the order of 110,000 neurons. Methodology is likely to be responsible for this result. Valtschanoff et al. (1993) report that using combined preembedding immunocytochemistryi histochemistry, most, but not all, diaphorase reactive cells in rat neocortex were GABA-positive. However with postembedding immunocytochemistry, all diaphorase-reactive neurons examined were found to contain immunolabelling for GABA. Indeed, using postembedding immunocytochemistry, the somata of eight morphologically diverse diaphorase- reactive neurons, situated in different lamina of rat mPFC (areas 25, 32 and 24b), were all GABA-immunopositive (Gabbott and Bacon, 1995).

The data given in Table 16A indicate that the percentage colocalisation of diaphorase activity with CBP immunoreactivity is highest within the CB-immunoreactive population than for either CR+ or PV+ neuron types. However, when absolute cell numbers are taken into consideration and the percentage colocalisation expressed with reference to the diaphorase-reactive neuron population, the situation is different; more diaphorase-reactive cells in monkey mPFC express CR, than CB or PV immunoreactivities (Table 16B).

The colocalisation of CB and NOS immunolabelling in cortical interneurons has been described recently in the rat frontal agranular cortex (Kubota et al., 1994). Dun et al. ( 1994) provide quantitative data that indicates the infre-

quent co-localisation of NOS immunoreactivity and CBP immunolabelling in rat neocortical neurons - findings cor- roborated by the data of the present investigation in monkey mPFC (Table 16A). Similar to the monkey, CB was the CBP most frequently co-localised with NOS immunolabelling in rat neocortex (Table 1 in Dun et al. 1994).

Finally, the data of the present study can be readily transformed should future quantitative studies further define the degree of colocalisation of CBPs, GABA, and NOS/diaphorase reactivities in local circuit neurons in the prefrontal cortex of primates.

Comparisons between areas of mPFC Statistical interareal and interlaminar analyses indicated

a general trend in the quantitative data across areas 24a, 24b and 24c of the anterior cingulate cortex (Tables 9-13). The absolute numbers of most cell types (CR, PV, CB, and GABA) were higher in area 24c compared with areas 24a and 24b. This trend was particularly pronounced for superficial layers 2 and 3 (Tables 10 and 11). Additionally, the absolute number of diaphorase-reactive neurons in a cortical column followed this trend (Table 15).

Together with a ventrodorsal gradient (24a + 24b + 24c) in the absolute total number of neurons in a cortical column (Table 2), there is a concomitant increase in the numbers of local circuit inhibitory neurons in each area (Tables 4-8). This quantitative trend in cellular composition (and therefore neural circuitry) may reflect the progressive ventrodorsal differentiation and functional specialisation of the anterior areas of the monkey cingulate cortex (see reviews by Vogt, 1993; Dum and Strick, 1993).

Functional architecture of monkey mPFC: Cortical modules

The present investigation has defined the number of neurons that are present in a column of cortex under a defined unit of surface area (Table 2 and Tables 4-81, Such data enable an initial quantitative definition to be made concerning the number of neurochemically identified local circuit neurons in a module of cortex processing information in monkey mPFC.

Cortical modules. It is important to define a cortical module. A cortical module represents a narrow column of vertically organised neurons that share common physiological properties (see review by Peters, 1993). Cell modules may represent the fundamental building blocks of cortical architecture (Mountcastle, 1978; see also Douglas and Martin, 1992). In monkey mPFC, it is likely that the neurons within a cortical module are simultaneously processing information from multiple overlapping functional streams (Goldman-Rakic, 1984, 1990; Vogt and Gabriel, 1993; Fuster, 1995).

Structurally, cortical modules are thought to be centred on the vertically aligned and clustered apical dendrites derived from clusters of layer 5 pyramidal cells (Peters, 1993). A similar structural organisation has been well described in the cerebral cortex of primates (von Bonin and Mehler, 1971; Sakai, 1985; Peters and Sethares, 1991) and in other mammalian species (Peters, 1993). In monkey, apical dendrites from layer 5 pyramidal neurons aggregate to form bundles which then ascend in close apposition through the upper layers of the cortex (Peters and Sethares, 1991). The vertically oriented bundles are joined en route by the apical dendrites from pyramids in layers 3 and 2. In

LOCAL CIRCUIT NEURONS IN MONKEY mYFC: I1 633

upper layer 2, the dendrites extensively bifurcate to form the apical dendritic tufts characteristic of layer 1. Separate dendritic clusters are also formed by pyramidal neurons in layer 6.

Peters and Sethares (1991) have found that the centre-to- centre spacing between dendritic bundles in the visual cortex of the monkey is about 31 pm and that each bundle is composed of 142 cortical neurons. In the cortices of other species vertical modules of pyramidal neurons have significantly larger centre-to-centre distances of 40-70 pm (Pe- ters, 1993). Peters and Sethares (1991) consider that the comparatively small intercluster distance is related to the very high density of neurons in monkey area 17.

In a recent quantitative study on human cingulate cortex, a columnar arrangement of neurons was demonstrated via computerised image analysis (Schlaug et al., 1995; cf Fig. 1H,J of this study). These authors report that columns of cells, or modules, in area 24b were approximately 40 )*m in mean width and separated by a neuropil- rich fascicle of the same dimension. Further, the bundling of layer 6 pyramidal apical dendrites has been described in frontal association areas of the monkey cortex (Sakai, 1985). In the latter study, dendritic bundles were 50 pm wide with a 30 pm (cell free) space between adjacent bundles. In the posterior cingulate (granular retrosplenial) cortex of rats, the width of dendritic bundles derived from layer 2 pyramids was 30-100 pm with a similar range of centre-to-centre distances (Wyss et al., 1990). The above data are in strong agreement with the results of the present investigation for monkey area 24b (see Results).

Local circuit neurons and cortical modules in monkey mPFC. Given that the mean centre-to-centre distance between cortical modules in area 24b of the monkey mPFC is 69 )*m and assuming that modules in area 24b are arranged as a hexagonal honeycomb pattern parallel to the pial surface (Peters and Kara, 1987), then there are approximately 243 modules under 1 mm2 of surface (cf 1,041 modules under 1 mm2 of monkey area 17; from Peters and Sethares, 1991). Subsequent calculations define that an individual cortical module in area 24b contains 497 neurons, which includes 117 GABAergic neurons (Table 5). Of significance is that there are 96 layer 5 pyramids associated with an individual module. On morphometric grounds it is likely that the apical dendrites from these pyramids as- semble into several dendritic bundles (cf Peters and Sethares, 1991; Peters, 1993). Nevertheless, the comparatively high number of neurons within a cortical module in mPFC may reflect an increased capacity to parallel process neural information.

Pharmacologically identified subpopulations of local circuit neurons in a cortical module in monkey mPFC can be further defined. The 117 GABAergic neurons comprise 52 CR+, 28 PV+ and 24 CB+ neurons. These neurons are distributed between layers as defined in Table 5 (P%). Since the calcium binding proteins identify distinct classes of local circuit neuron in mPFC (Gabbott and Bacon, 1996) then it becomes possible to provide an initial guideline as to the absolute numbers of particular types of interneurons in a cortical module. Hence in layers 2 and 3 of a cortical module in area 24b there would be an average of 18 PV+ cells, composed of basket and chandelier neurons (Williams et al., 1992; Conde et al., 1994; Gabbott and Bacon, 1996). Similar calculations can be made for other lamina and areas of the mPFC. This is fundamental information concerning

the cellular composition of the cerebral cortex. Basket neurons and chandelier cells provide GABAergic input to selective parts of pyramidal neurons (soma and axon initial segment, respectively; Jones et al., 1994), thereby strongly influencing the response properties of these cells (Douglas and Martin, 1992; Jones et al., 1994). Although the relative numbers of cell types composing the classes of neurons defined by the calcium binding proteins are presently unknown, the data of the present study can be easily revised when this information becomes available.

Quantitative data concerning the spatial distribution and synaptic wiring diagram of different pharmacological classes of identified GABAergx local circuit neuron will enable the role of these cells in mPFC function to be evaluated (Kawaguchi and Kubota, 1993; see also Fig. 29 in Gabbott and Bacon, 1996). Of particular interest is the conver- gence of specific local inhibitory circuits onto specific regions of pyramidal neurons. This is important since pyramidal neurons represent the predominant source of excitatory output from the cortex to other cortical areas and subcorti- cal regons. Coupled with physiologcal evidence, this information could identify the structural location and neurochemical characteristics of strategic synaptic circuits both vertically within a defined module and horizontally across functionally related modules. Pharmacologcal manipula- tion of these synaptic circuits may have potential implications for the clinical treatment of abnormal cortical function (Hof and Morrison, 1991; DeFelipe et al., 1993). This is of profound significance since the prefrontal cortex and adjacent cingulate cortices are important sites of ravaging neurological disorders such as Alzheimer’s disease and schizophrenia in humans (see reviews by Benes, 1993, 1995; Braak and Braak, 1993).

Conclusion In conjunction with the companion paper (Gabbott and

Bacon, 19961, this study has provided a comprehensive account of local circuit neurons expressing the calcium binding proteins CR, PV and CB in the adult monkey mPFC. Cortical neurons displaying diaphorase reactivity were also investigated.

In this paper, quantitative descriptions were provided of these cell populations and the data used to estimate the number and type of local circuit neurons that may be present in a cortical module in the mPFC. Together with quantitative information concerning the synaptic maps of the individual cell types, it will eventually become possible to create realistic and simple structural models of mPFC in the monkey that subserve defined physiological functions. Such data could provide insights into the functional architecture of the medial prefrontal cortex in humans (Vogt et al. 1995).

ACKNOWLEDGMENTS The technical assistance of Paul Jays, Tracy-Ann Warner

and Angela Robinson is acknowledged with gratitude. The material used for this study was kindly provided by Dr K.A.C. Martin (MRC Anatomical Neuropharmacology Unit, Oxford) and Professor A. Cowey (Dept. of Experimental Psychology, Oxford). Paul Gabbott is a Beit Memorial Research Fellow and Sarah Bacon is a Bristol-Meyers Squibb Research Fellow. This work was supported in part by the MRC (UK).

634 Y.L.A. GABBOTT AND S.J. BACON

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Local circuit neurons in the medial prefrontal cortex (areas 24a,b,c, 25 and 32) in the monkey: II. Quantitative areal and laminar distributions