Local circuit neurons in the medial prefrontal cortex (areas 24a,b,c, 25 and 32) in the monkey: I. Cell morphology and morphometrics

THE JOURNAL OF COMPARATIVE NEUROLOGY 364567-608 ( 1996)

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

in the Monkey: I. Cell Morphology and Morphometrics

PAUL L.A. GABBOTT AND SARAH .J. HACON University Department of Pharmacology, Oxford, England

ABSTRACT This paper provides a comprehensive morphological description of local circuit neurons in

the medial prefrontal cortex (mPFC: areas 24a, 24b, 24c, 25 and 32) of the monkey. Cortical interneurons were identified immunocytochemically by the expression of the

calcium binding proteins calretinin (CR), parvalbumin IPV) and calbindin D-28k (CB). Interneurons were also identified using GABA immunocytochemistry. The areal and laminar distributions of CR, PV, and CB cells were consistent across mPFC; their morphological characteristics identified them as local circuit neurons. Throughout layers 2-6: CR immunoreactivity labelled double bouquet and bipolar neurons, PV was localised in large and small basket neurons and in chandelier (axoaxonic) cells, while CB immunoreactivity was present in double bouquet, Martinotti, and neurogliaform neurons. In addition, some cells in layer 1 (including Cajal-Retzius neurons) were CR immunoreactive. Calbindin immunoreactivity also labelled a population of large nonpyramidal neurons deep in the cortex. Other types of CR, PV and CB cells were also immunolabelled. A small population of layer 3 pyramidal cells was weakly CB immunoreactive. Peak cell densities occurred in layer 2iupper layer 3 for CR+ neurons and in upper to midlayer 3 for CB+ cells. PV+ neuron density peaked in midcortex. These observations support and extend a similar study of monkey prefrontal cortex (Conde et al. 119941 J. Comp. Neurol. 341:95-116). The morphologies and combined cortical depth distributions of CR+, PV+, and CB+ neurons were similar to GABA-immunolabelled cells.

Local circuit neurons in mPFC displaying NADPH diaphorase activity composed less than 0.25% of the total neuron population, and were distributed in two horizontal strata, in mid- to lower layer 3 and in lower layer 5iupper layer 6. CR, PV and CB immunoreactivity was colocalised in NADPH diaphorase-reactive neurons.

The interrelationships between CR+, PV+ and CB+ neurons were investigated using dual immunocytochemistry. CR+ puncta were found to be closely associated with the cell bodies and proximal processes of PV+ neurons, whereas CR+ puncta were located more distally over processes from CB+ cells. Additionally, PV+ puncta were found closely apposed to PV+ somata and processes and CR+ puncta abutted against CR+ cell bodies.

The companion paper (Gabbott and Bacon [ 19961 J. Comp. Neurol.) presents quantitative data regarding the areal and laminar distributions of the identified cell classes in mPFC. Such data provide a realistic structural framework with which to investigate neuronal operations in monkey mPFC. ' IXK Wile!.-Liss. Ine.

Indexing terms: limbic system, cingulate cortex, GABA, calcium binding proteins, NADPH diaphorase

The prefrontal cortex of primates is involved with complex behaviours that require the integration of higher brain functions. These associative functions include motivation, attention, cognition, learning and memory and the spatio- temporal organisation of goal-directed behaviours (Fuster, 1989; Goldman-Rakic, 1990).

orbital and insular surfaces of the brain are not only

involved in these higher brain functions (Vogt and Gabriel, 19931, but also act together with the limbic system to provide part of the complex physiologcal background to a wide range of emotional reactions and expressions (Smith

Accepted J u n e 19, 1995. Address reprint rcqucsts t.o Paul L.A. Gahhott, University Departmcnt of Parts of the prefrontal cortex, located Over the

Pharmacology, Mansfield Road, Oxford. OX1 3QT. United Kingdom.

( 1996 WILEY-LISS, INC.

568

and DeVito, 1984). The medial prefrontal cortex (mPFC) subserves a variety of autonomic (somatovisceral) activities that underlie emotional behaviour, for example, respiration and cardiovascular functions (breathing, heart rate and blood pressure), gastrointestinal mobility and secretion, and pupillary dilation (Neafsey et al., 1993; Buchanan and Powell, 1993; Powell et al. 1994).

On the basis of cytoarchitecture, mPFC is composed of Brodmann areas 24a, 24b, and 24c (anterior cingulate cortex or anterior limbic cortex), area 32 (prelimbic cortex) and area 25 (infralimbic cortex). This structural definition of mPFC is concordant across a number of mammalian species, including humans (rat, Van Eden et al., 1991; rabbit, Buchanan et al., 1994; cat, Room et al., 1985; Musil and Olson, 1993; monkey, Carmichael and Price, 1994; see also Figs. 1A,B of this study; and humans, Uylings and Van Eden, 1990; Vogt et al. 1995).

The functional activity of each cortical area is governed by the dynamic interplay between cortical afferents, intrinsic circuitry, and their relationship with the corticoefferent pathways. Although the afferent and efferent connectivities of the cortical areas composing the mPFC are beginning to be unravelled in detail (Van Hoesen et al., 1993; Carmichael and Price, 19941, comparatively little is known about the internal architecture and synaptic connectivities of these areas in the monkey. However, detailed wiring diagrams of their internal construction would identify the routes via which afferent information (derived from other cortical areas and subcortical sources) is integrated and processed within defined neural networks and subsequently relayed to other cortical fields and brain regions.

Neural networks in the cortex are composed of interconnected populations of pyramidal cells and local circuit or nonpyramidal neurons (Douglas and Martin, 1990). Pyrami- dal neurons are excitatory in function, and, in addition to having local intracortical axon arbors, they project to other cortical areas and/or subcortically. Golgi and immunocytochemical studies show that local circuit neurons in the monkey PFC are heterogeneous in axon and dendritic morphologies and laminar location (Lewis and Lund, 1990; Lund and Lewis, 1993). Moreover, the vast majority of cortical interneurons use GABA to mediate their inhibitory intracortical operations (see Mize et al., 1992; Jones et al., 1994). Specific types of inhibitory cortical local circuit neurons (e.g., axoaxonic, basket, and double bouquet neurons; see review by Jones et al., 1994) have defined patterns of synaptic connectivity with specific parts of pyramidal cells (e.g., axon initial segments, somata, and dendrites).

ABP CB CBPs CR DAB FITC GABA GAD mPFC PNADPH

NBT NO NOS P B PBS PV RT TRIS

Abbreviations

avidin-biotin-peroxidase calbindin D-28k calcium binding proteins calretinin 3,3’-diaminobenzidine Auorcscein isothiocyanate gamma aminobutyric acid glutamic acid decarboxylase medial prefrontal cortex reduced form of nictotinamide adenine dinucleotide phos-

phate nitroblue tetrazolium nitric oxide nitric oxide synthase phosphate buffer phosphate-buffered saline parvalbumin room temperature Tris (hydroxymethy1)-methylamine

P.L.A. GABBOTT AND SJ. BACON

Furthermore, extensive connections also exist between cortical interneurons (Jones et al., 1994). In concert, local circuit neurons play crucial and specific functional roles in shaping the physiological response properties of pyramidal cells, thereby strongly influencing neural activity in the cortex (Douglas and Martin, 1992; Wilson et al., 1994).

Recent studies have shown that the separate types of GABAergic local circuit neuron in monkey cerebral cortex can be readily identified using immunocytochemistry for the calcium binding proteins, calretinin, parvalbumin and calbindin D-28k (DeFelipe et al., 1989a,b; Demeulemeester et al., 1989, 1991; Blumcke et al., 1990; Van Brederode et al., 1990; Lewis and Lund, 1990; Huntley and Jones, 1990; DeFelipe and Jones, 1991, 1992; Morino-Wannier et al., 1992; Hof et al., 1993; Conde et al., 1994). The current investigation was undertaken with the specific aim of expanding on the studies of Hof et al. (1993) and Conde et al. (1994) by investigating both the morphology and quantitative distribution of the structural components involved in the local neuronal circuitry of the monkey mPFC.

The present study was performed at two related levels. Firstly, a detailed and comprehensive morphological account of the areal and laminar distribution of local circuit neurons in mPFC that show immunoreactivities for the calcium binding proteins, calretinin, parvalbumin and calbindin D-28k. In addition, the structural relationships between neurons containing different calcium binding proteins was investigated using double immunocytochemistry. A further aim was a thorough documentation of interneurons in monkey mPFC displaying NADPH diaphorase activity (Sandell, 1986). Since diaphorase activity is colocalised with nitric oxide synthase (the biosynthetic enzyme for nitric oxide; Hope et al., 1991), these interneurons have the potential to affect cortical operations, not only through GABA-mediated mechanisms, but also via the actions of the neuromodulator nitric oxide (NO; see reviews by Vincent, 1994, 1995). Indeed, NADPH diaphorase activity has been colocalised with specific calcium-binding proteins within interneurons (Vincent, 1995).

The second part of the study was a quantitative analysis of the areal and laminar distributions of the immunocytochemically and histochemically identified classes of local circuit neuron in the separate areas of the monkey mPFC. These data are presented in the companion paper (Gabbott and Bacon, 1996).

The overall rationale behind this light microscopic investigation is that detailed qualitative and quantitative information concerning the morphology and distribution of local circuit neurons in the adult monkey mPFC is of prime importance, firstly, towards understanding the involvement of interneurons in cortical circuitry during normal and abnormal development, and secondly, by providing a framework with which to interpret alterations in the functional architecture of local circuit neurons in the human mPFC that are considered to underlie psychiatric disorders such as schizophrenia, Pick’s disease, Wernicke-Korsakoff s syn- drome, and Alzheimer’s disease (Benes et al., 1991; and reviews by Benes, 1993a,b, 1995; Braak and Braak, 1993).

MATERIALS AND METHODS Animals and tissue preparation

Material used in this study was obtained from five adult female cynomologus monkeys (Macaca fasicularis; 6.0-

LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I 569

10.0 kg). The animals had been previously used in experiments studying the primate visual system. At the end of these experiments the animals had been given a lethal overdose (180 mg) of sodium pentobarbitone (Baker and May. UK). When sufficiently anaesthetised the animals were perfused transcardially with either 4% paraformaldehyde (animal M5-AC 302), or a mixture of aldehyde fixa- tives containing 3% paraformaldehyde with either 0.3% (animal M2-KACM 4/94), 0.5% (animals M1-KACM 1/94 and M4-KACM) or 0.74 (animal M3-KACM 13/93) glutar- aldehyde in 0.1 M phosphate (PB) buffer (pH 7.4). The brains had been removed, postfixed in fixative and stored in PB buffer (pH 7.4) at 4°C.

For the studies presented here, blocks of tissue containing the anterior medial wall of the cortex that included cortical areas 24a, 24b, 24c, 25 and 32 were carefully excised from the brains (Fig. 1A,B). In three of the animals tissue was taken from both hemispheres. These tissue blocks were sectioned serially using a Vibratome in either the coronal plane (animals M1-3 and M5; Fig. 1C) or parasagitally (animal M4). Sections were collected as sets; each set contained 5-8 serial sections cut at one of several thicknesses (50, 80 or 100 pm). After sectioning, the tissue was rinsed thoroughly in 50 mM TRIS-HCl pH 7.4 buffer (TRIS).

NADPH diaphorase histochemistry and immunocytochemistry

Overview. In each set of sections, individual sections were reacted for either: ( i ) diaphorase enzyme activity alone; ( i i ) for diaphorase activity in combination with GABA immunocytochemistry or immunocytochemistry using an antiserum directed against one of the calcium binding proteins (CBPs): calretinin (CR), parvalbumin (PV), or calbindin (CB); or (iii) treated with osmium tetroxide, dehydrated, and embedded in resin (see below). The last group of sections was used in morphometric studies (Gab- bott and Bacon, 1996). In addition, selected sets of sections were Nissl-counterstained and some sections were processed solely for NOS immunocytochemistry.

The technical procedure used to demonstrate the immunocytochemical localisation of a marker substance in material previously reacted to reveal NADPH diaphorase activity has been described in detail previously (Gabbott and Bacon, 1994). The methodological procedures are therefore described in brief.

NADPH diaphorase histochemistry. After several washes in TRIS, tissue sections were reacted histochemically for NADPH diaphorase activity in a solution containing 0.25 mgiml of Nitro Blue Tetrazolium (NBT) and 1.0 mgiml of reduced nitotinamide adenine phosphate (,NADPH). Tissue sections were incubated at 37°C for 45-120 minutes and the reaction terminated by thorough rinses in cold (4°C) TRIS buffer. (NBT and ,,NADPH were purchased from Sigma Chemicals Ltd).

Preembedding immunocytochemistry. Selected sections were then rinsed for an extended period in TRIS buffer containing 0.5% Triton X-100 for 1-2 hours or were freeze-thawed as described previously (Bolam, 1992). Sec- tions were then washed in either 20% normal goat serum or 20% normal swine serum (30-60 minutes) and then incubated in a primary antiserum against either GABA, CR, PV, CB or nitric oxide synthase (NOS, brain and endothelial forms; Vincent, 1994,1995). The source of the antisera, the range of working dilutions, incubation times, and sera specificity are given in Table 1. The use of these antisera in

immunocytochemical studies has been given previously (Table 1). All sera were made up in TRIS (pH 7.4) buffer containing 0.01% sodium azide.

Immuolabelling was visualised using one of the following methods:

(i) The standard immunoperoxidase procedures using appropriate species matched Vectastain ABC kits (Vector Laboratories) and developed with 3,3’-diaminobenzidine (DAB) as the chromagen (incubating sections in TRIS (pH 7.4) with 0.05% DAB and 0.01% HzOz for 3-10 minutes at room temperature).

(ii) Immunolabelling procedures using appropriate species-specific secondary antibodies tagged with alkaline phosphatase. Prior to labelling with secondary antibody, tissue sections were incubated in TRIS (pH 7.4) buffer containing 0.015% levamisol for 3 x 10 minutes (to abolish endog- enous phosphatase activity). Fast red TRinapthol AS-MX (Kit F4523, Sigma Chemicals Ltd.) was then used as the chromagen to reveal immunospecific alkaline phosphatase activity. The colouration of fast redinapthol-alkaline phosphatase labelling is bright red.

(iii) An indirect immunofluorescence method. After the primary antisera incubations, selected sections were thoroughly washed in phosphate-buffered saline (PBS) and then incubated in the appropriate species-specific secondary IgG serum conjugated with fluorescein isothiocyanate (FITC, Sigma Chemicals Ltd).

In the above immunocytochemical procedures, sections were washed thoroughly in TRIS buffer pH 7.4 (3 x 15 minutes) between each of the incubation steps.

In control sections incubated without ,NADPH, no specific diaphorase enzyme reactivity was present. Specific immunolabelling was absent in control sections that had been incubated without primary antiserum. However, in these latter sections a uniform and weak colouration was present throughout the tissue due to nonspecific labelling.

Double preembedding immunocytochemical labelling. Following diaphorase enzyme histochemistry, certain sections were reacted using a double preembedding immunocytochemical labelling procedure described recently by Bevan et al. (1994). Essentially, sections were incubated either simultaneously or sequentially with two primary antisera. (Combinations of CRIPV, CRICB, CBIPV antisera were used during simultaneous incubations. During sequential incubations the same antisera combinations were used but the order of the antisera was varied to achieve optimal results.) Antisera dilutions and incubation times were the same as those for single antiserum incubations (Table 1). The primary antisera were sequentially tagged with avidin- biotin-peroxidase ( ABP) complex using appropriate Vec- tastain ABC kits (Vector Laboratories). The first ABP complex was revealed using DAB as the chromagen and the second ABP complex visualised with Vector SG (Vector Laboratories). Excess peroxidase activity was quenched after the first visualisation reaction by washing sections in 0.05%, H202 in TRIS buffer (pH 7.4) x 10 minutes. Sections were then washed thoroughly in TRIS buffer (pH7.4). In addition, an avidinibiotin blocking step was included between first and second labelling sequences. Control incubations were undertaken to ensure method specificity (Bevan et al., 1994).

After the histochemical reactions, tissue sections were processed in one of two ways. The majority of sections were mounted in order onto gelatin-

Control incubations.

Tissue processing.

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

A c+R

V

cc

C

Fig. 1. A: Photograph of the anterior medial wall of the left cerebral hemisphere of monkey M3. Calibration bar = 0.5 cm. B: Diagram showing the positions of the cytoarchitectonic areas 24a,b,c, 25 and 32 over the surface of the medial wall. Coronal sections (1-6) are shown in C. (Note the proximity of sections 3 and 4 to accommodate the highly curved aspect, the genu, of the corpus callosum.) C: Coronal sections

taken at the rostrocaudal sampling levels in C. The rostrocaudal extents of cortical areas 24a, 24b, 24c, 25 and 32 are indicated. Abbreviations: CS, cingulate sulcus; LOS, lateral orbital sulcus; MOS, medial orbital sulcus; PS, principal sulcus: RS, rostral sulcus: and CC, corpus callosum. Orientation markers: R, rostral; C, caudal; M, medial; L, lateral; V, ventral; and D, dorsal.

coated slides and air-dried. Of these, specific sets of sections were lightly counterstained with cresyl violet. The slides were finally passed through an ascending series of alcohols,

passed swiftly through xylene and the sections embedded in DPX mountant. Other sections were treated with 0.5% Os04 in 0.1 M PB for 0.5-1.0 hour, dehydrated through an


TABLE 1. Primary Antisera: Suppliers, Incubation Dilutions and Times

Working Incubation dilutions t imes Serum Abbreviation Source/ Ref'. Code Host animal

I ' 1.000-5.000

I 200-.500 1 500-2.500

1~20(1-500 l.200-1.000

1.200-250 I . 100-200 1 :500-1.500

1. 1.000-2.000

ascending series of alcohols (including 1% uranyl acetate in 70% alcohol for 1 hour), flat-embedded in resin (ACM Durcupan) and cured for two days at 56°C.

Tissue sections reacted using immunofluorescence were thoroughly washed (3 x 10 minutes) in PBS and then mounted in 100% glycerol on glass slides.

Sections were observed in a light microscope and objects of interest recorded with photomicrographs or drawings. Immunofluorescence was detected using a Leitz Lz filter system attached to a photomicroscope.

Postembedding GABA immunocytochemistry. Semi- thin (0.5-2.0 pm) resin-embedded sections were obtained from selected cortical areas and lamina and reacted using the postembedding GABA immunocytochemistry procedure described by Somogyi and Hodgson (1985).

Tissue examination.

Cell morphometry, statistical analysis and spine density counts

At high magnification (using a 1 0 0 ~ oil immersion objective lens and 10x eyepieces) the cytoplasmic and nuclear profiles of both strongly and weakly reactive diaphorase-positive cells as well as for CR+, PV+, CB+, and GABA+ immunoreactive neurons were traced onto paper using a drawing tube attached to a photomicroscope. The samples of somatic and nuclear profiles were derived from neurons situated in all layers of the cortex and from the different cortical areas investigated in this study. Only cells in which the nuclear profiles could be discerned were included in the analysis. The cell populations were taken from material reacted using the DABiimmunoperoxidase procedure (see above).

Somatic profile areas were measured and then calculated using a computerised planimeter (Apple QuadraiKurta digtising tablet system operated using MacStereology). Measuring programs incorporated a linear and isotropic tissue shrinkage factor of 15% (see Gabbott and Bacon, 1995). The diameters of area equivalent circles (D.Circle) were subsequently calculated for the individual somatic profile populations (Weibel, 1980). Somatic profile data were then analysed using a statistical software package (InStat. GraphPad Software, CAI. Data were initially sub- jected to Bartlett homogeneity tests before the population means were compared using multiple t-tests (Fry, 1993). Significant differences between means were considered to occur when a P value of <0.05 was obtained (Fry, 1993). Five animals were used to gather somatic profile data for CR, PV, CB, and GABA-immunopositive cell populations, while four animals were analysed to provide data concerning the diaphorase-reactive neuron populations.

Spine density estimates were determined for selected dendritic segments from NADPH diaphorase-reactive neurons and immunoreactive cells using the procedure of Feldman and Peters (1979).

Terminology and symbols. The terms diaphorase- reactive ( D + 1 and immunoreactive or immunopositive (either CR+, PV+, CB+, GABA+ or NOS+) refer to specific stainingiimmunolabelling in cells and cellular processes. The terms diaphorase-nonreactive (D - ) and immunonegative (either CR-, PV-, CB-, GABA- or NOS-) refer to the absence of specific stainingiimmunolabelling in cells. Finally, cells and processes displaying both specific immunoreactivity and NADPH diaphorase activity are termed immunopositiveidiaphorase-reactive neurons (e.g., CR+i D+, P V + / I ) + , CB+/D+ or GABA+iD+).

RESULTS Technical considerations

The following variables: ( i ) quality of tissue fixation (fixative composition, postfixation processing, storage), (ii) subsequent treatment with either Triton X-100 or freeze- thawing, (iii) characteristics of the primary antiserum, (iv) incubation times in sera, and (v) the type of chromagen used to visualise immunolabelled structures, all affected the depth and quality of immunoreactivity within the tissue. In contrast, NADPH diaphorase activity was present throughout the thickness of tissue sections and diaphorase- reactive cells were revealed with great morphologcal detail, rivalling that of cells seen in Golgi preparations (Vincent, 1995).

In this investigation, several chromagens were used separately to visualise immunoreactive neurons (see Mate- rials and Methods). Irrespective of the chromagen used, all the characteristic features of a cell class were present in immunolabelled neurons; variations were, however, found in the clarity with which fine morpholoGcal details were revealed.

As described below, diaphorase-reactive neurons in the monkey mPFC were present in two separate populations based on the intensity of somatic staining, either strongly or weakly labelled cells. The existence of these two separate populations was not related to methodological factors (e.g., incubation time) since both cell classes were consistently present in material cut at different thicknesses and incubated using substantially different reaction times (45 versus 120 minutes). Furthermore, the somatic profile-size distributions of each staining type did not represent two

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

Fig. 2. Unfolded surface map of areas 24a, 24b, 24c, 25 and 32. The line used to unfold the curvature of the cortex lay at the boundaries between layers 315 (area 24a,b,c and 25) and layers 314 (area 32). The unfolded segments were centred on the concavity of the cingulate sulcus. Orientation markers: R, rostral; C, caudal; V, ventral; and D, dorsal. Corpus callosum, cc. Calibration bar = 1.75 mm.

discrete parts of a continuum but overlapped markedly (Fig. 24).

Identification of cortical areas and laminae Areas 24a, 24b, 24c, 25 and 32 of monkey mPFC were

identified in the coronal and parasagittal Nissl-stained sections using previous cytoarchitectural criteria (Fig. 1A-C; see also Walker, 1940; Vogt et al., 1987; Barbas and Pandya, 1989; Matelli et al., 1991; Dum and Strick, 1991; Carmichael and Price, 1994; see also Fig. 1A-E in Gabbott and Bacon, 1996). Of note, is that area 24c was identified as extending onto the dorsal bank of the cingulate sulcus where it represented approximately half of the cortical territory (Fig. 1C). The anterior dorsal borders of area 24c were not well defined rostral to the genu of the corpus callosum (Fig. 1C).

Areas 24a, b, c, and 25 were typically agranular cortical fields with pyramidal shaped neuronal somata located throughout layers 2-6. Pyramidal cells were abundant in layers 2 and 3 and comparatively sparse in layer 6. The largest pyramidal neurons were located in the middle of layer 5. Area 32 was defined as a granular cortical field (layer 4) due to the presence of a distinct, numerically large population of small spherical neurons located above layer 5 (Yeterian and Pandya, 1994).

The distribution of cortical areas 24a,b,c 25 and 32 over the unfolded surface of the medial wall is shown in Figure 2. The definition of areal borders is in good agreement with previous studies (Dum and Strick, 1991; Carmichael and Price, 1994). The rostrodorsal boundary of area 24c was difficult to define with certainty (Figs. lC, 2).

Cellular morphology NADPH diaphorase-reactive neurons Characteristics of diaphorase labelling in nPFC neu-

Neurons in the cortex and white matter that were rons.

histochemically labelled for NADPH diaphorase activity contained the characteristic dark blueipurple formazan end-product of the diaphorase reaction. Diaphorase enzyme activity in the monkey mPFC was present in two separate types of cell that could be distinguished clearly as either weakly or strongly diaphorase-reactive on the basis of the intensity of formazan labelling (Fig. 4c).

In weakly labelled neurons, cytoplasm within the cell body and in the proximal regions of emerging processes was faintly discoloured by the formazan end-product. In addition, fine particles of the formazan precipitate were dis- persed throughout their somata (Fig. 4C). The staining of weakly diaphorase-reactive neurons varied considerably but never reached the intensity nor intracellular distribution found within strongly diaphorase-reactive cells. Conse- quently, the morphology of weakly diaphorase-reactive neurons was not investigated further. By contrast, the cytoplasm within the cell bodies and cellular processes of strongly diaphorase-reactive neurons was labelled intensely and their morphology revealed with great clarity and detail (Figs. 5-7).

Within the somata of both strongly and weakly diaphorase-reactive neurons, cell nuclei were invariably unlabelled and provided distinctive translucent regions within the somata of diaphorase-labelled cells. However, in neurons with comparatively large amounts of cytoplasm surrounding centrally placed nuclei, strong diaphorase labelling could totally obscure the unstained nuclei (Figs. 3A,B; 4B,D,M; 5E). Nevertheless, the absence of nuclear staining is a characteristic hallmark of diaphorase-reactive neurons in the mammalian nervous system that has been reported in previous studies (Sandell, 1986; Cipolloni and Pandya, 1991; Vincent, 1994).

Qualitatiue distribution. Diaphorase-reactive cells appeared as scattered solitary neurons (Thomas and Pearse, 1964) that were present in all the cortical layers and in the underlying white matter of the five areas investigated in this study (Figs. 6-8). The cortical depth distribution of the diaphorase-reactive neurons indicated that there were three peaks in cell density. These peaks occurred in all cortical areas and were located: (i) in the middle of layer 3, (ii) in a region extending from very deep layer 5 to upperimidlayer 6, and (iii) within the white matter beneath each cortical area at a depth of between 100 and 200 pm below layer 6. The distribution peaks in the cortex produced two tangentially distributed tiers of stained neurons. Quantitative aspects of the areal and laminar distribution of diaphorase

Fig. 3. A Diaphorase-reactive neurons (1-3) in the superficial layers of area 24a. Diaphorase reactivity in several smallimedium bore capillaries is shown (double-headed arrows). c, capillaries. B: Layer 2 diaphorase-reactive neuron (1 in A) with ascending dendrites (arrows) that enter layer 1. B: High magnification of the somatic region of cell 1 seen in B. A fine descending axon-like process (arrows) arises proximally from a reactive dendritic process. C: Radiate multipolar diaphorase-reactive neuron (n) in layer 3 of area 25. D: A diaphorase-reactive neuron in upper layer 3 of area 24c with a descending arcade (small arrows) of dendrites emerging from the basal pole of the cell body. One ascending dendritic process curves downwards (double arrow). c, capillary. E: Beaded dendrites (arrows) from a diaphorase-reactive neuron. F Diaphorase-reactive neuron (n) in layer 5 of area 24b. This neuron was situated in the centre of a tissue section. All the diaphorase- reactive processes from this neuron ascended through the cortex (small arrows). An axon-like process can be seen to emerge from the basal pole of the soma (double-headed arrow). G: Bitufted diaphorase-reactive neuron ( n ) in layer 4 of area 32. Calibration bars: A, B, C = 100 pm; D, F, G = 50 Fm; E = 5 Fm.

Figure 3

Figure 4

LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I 57.5

reactive cells are detailed in the companion paper (Gabbott and Bacon, 1996).

Diaphorase neurons i n monkey m.PFC. Diaphorase- reactive neurons composed between 0.20% and 0.32% of all cortical neurons in monkey mPFC. Within the population of diaphorase-reactive neurons, strongly reactive cells represented about 75% of the total cell count. Approximately 60% of weakly stained cells were located in superficial layers 1 , 2 and 3. (See Table 15 in Gabbott and Bacon, 1996).

Light microscopic evidence indicates that both weakly and strongly diaphorase- reactive cells were morphologically diverse types of smooth and sparsely spinous nonpyramidal cortical neurons (Feld- man and Peters, 1978; Vogt and Peters, 1981; Peters and Regidor, 1981; Sandell, 1986; Lewis and Lund, 1990; Cipolloni and Pandya, 1991; Lund and Lewis, 1993; Jones et al., 1994). Firstly, their somata did not give rise to true apical and basal dendrites nor were the cell bodies of the vast majority of labelled neurons unequivocally pyramidal in outline. Secondly, the distribution of their dendritic fields was characteristically nonpyramidal. Thirdly, the origm and initial course of axon-like processes from these neurons was highly varied and unlike cortical pyramids. Finally, spine density counts set diaphorase-reactive neurons apart from spiny stellate and pyramidal neurons (see below).

Nonpyramidal versus pyramidal.

Fig. 4. A. Diaphorase-reactive neuron ( D + I situated in layer 1 of area 24b. A descendingspray of dendritic processes (arrows) can be seen entering layer 2. Several of these processes descended for over 250 pm reaching the layer 213 border. B: Diaphorase-reacted and Nissl- counterstained section. A multipolar diapborase-reactive neuron ( D + I in layer 3 of area 24b gives rise to a descending axon-like process (ax); part of this process (boxed) is shown a t higher magnification in B’. Diaphorase-reactive axon fibres and fibre swellings are present in the neuropil. Some of these labelled axonal processes are aligned vertically (arrowheads). Several Nissl-stained neurons are indicated (n) . B’: High magnification of the boxed region in B. The identified diaphorase axon-like process bifurcates (arrowhead), with one segment possessing several varicosities (small arrows). C: Examples of strongly and weakly diaphorase-reactive neurons. rJote that there was no gradation in diaphorase reactivity between the two intensities of labelling. A fine axon-like process (double-headed arrow) can be seen arising from a labelled dendrite. c, capillary. Clumps of formazan precipitate (small thin arrows; end-products of the diaphorase reaction) can be seen in the soma of the weakly labelled neuron. D: Diaphorase-reactive dendritic process I D * ) with spine-like protrusions (arrows). One protrusion [double-headed arrow) shows the clear features of a dendritic spine seen in Gnlg preparations, a spine head and a slender spine neck. Note also, the occurrence of short stub-like protrusions. E: A labelled diaphorase- reactive swelling (D+I comes into close contact (encircled) with a diaphorase-reactive dendritic process (D+ 1. F: Two diaphorase-reactive axonal swellings ( D + , arrows) are closely associated with the somata of a Nissl-stained neuron ( n ) . G: Diaphorase-reactive dendrites (D+ 1 with numerous dendritic spines (arrows) on primary and secondary segments. Note the variation in spine density between segments. Also note the predominance of long thin spines. H: Diaphorase-reactive axonal fibres in layer 3 of area 24c. Labelled fibres surround the somata of unstained cell bodies (nl. J: Combined GABA immunocytochemistry and diaphorase enzyme histochemistry. A diaphorase-reactive axonal swelling iD+ i is associated with the cell body of a GABA-immunoreactive neuron tG+) . K: Diaphorase-reactive neuron ( D + ) in layer 5 of area 32 with a lightly labelled axon-like process emerging (arrow) from the lower part of the cell body. L: Diaphorase-reactive neuron in layer 6 of area 2.5. Labelled processes are indicated (arrows). The boxed re&<on is shown enlarged in M. M: A fine axon-like process emerges (arrows) from the diaphorase-reactive soma (D+ I and travels horizontally within layer ti. Along its length the labelled process gave rise to beaded segments (double-headed arrows). Calibration bars: A = 100 ym; B, H = 50 pm; C, L = 25; D, E, F, J, K, M = 10 ym; G = 5 ym.

However, despite the foregoing, a few cortical diaphorase- reactive neurons were encountered that had many (Fig. 3A), but not all, of the characteristic features commonly ascribed to true cortical pyramidal neurons. The most extreme example was of inverted pyramidal-like neurons (Fig. 6, cell IP). These cells closely resembled true cortical pyramidal neurons but possessed a thick primary process that descended through the cortex and a tuft of processes emergmg from the upper surface of the cell body. By considering the size and shape of somata, dendritic field structure (tree shape and spine density measurements) and the origm, course, and morphology of axonal arbors, these cells could be classified as nonpyramidal neurons represent- ing extremes of a morphological spectrum.

The shape of diaphorase-reactive neuronal somata commonly ranged from highly ellipti- cal (Figs. 3G; 6, cells a,dj,q; 7, cells b,d,c,h,m,q; 8, cells b,c) to nearly circular (Figs. 6, cells e,f,k; 7, cell k,i,p; 8, cell a ) in outline. However, unusual shapes were frequently encountered, such as lobulated profiles where cytoplasm appar- ently blistered from the cell body (Figs. 6, cells i,n,p; 7, cell j ) or somata in which large amounts of cellular cytoplasm appeared funnelled into exceptionally thick primary processes (Figs. 6, cell 0 ; 7, cells c,g,m). Inverted pyramidal- shaped cell bodies were also observed (Fig. 6, cell IP).

Somatic profile-size distributions of populations of strong and weakly diaphorase-reactive neurons located in either layers 213 or layers 516 are given in Figure 24 and data provided in Table 2. Statistical analyses established that whereas no somatic size differences occurred between intensely and weakly labelled cells in the superficial layers, strongly diaphorase-reactive somata were significantly larger than weakly labelled cell bodies in the deep layers of the cortex (Table 2) .

Two types of diaphorase-reactive neurons were found in layer 1. The first type lay deep in the molecular layer and gave rise to a spray of descending beaded processes that ramified in layer 2 and the superficial part of layer 3 (Fig. 4A). The second type of cell was comparatively rare, but when present was located immediately beneath the pial surface (Fig. 6, cell a) . This type of diaphorase-reactive neuron had an ovoid soma giving rise to long and relatively thick aspiny processes that were oriented horizontally.

Diaphorase-reactive neurons throughout layers 2-6 usually possessed multipolar, bipolar or bitufted somata (Figs. 3-71, Although diaphorase-reactive cells in these layers had diverse morphologies, several overlapping categories were identified: ( i) Diaphorase-reactive neurons with radiate dendritic arbors in layers 2-6 (Figs. 6, cells b,e,f; 7, cells k,i,o; 8, cell a ) , especially within the central territory of the cortex (lower layer 3, layer 4 in granular area 32, and upper layer 5; Figs. 3C,G; 5; 6, cells i,m-t; 7, cells b,c,e,f,m,n,o; 8, cell a ) ; (ii) cells located in layers 2 and upper layer 3 that had radiate tufts of apical processes and long basal processes aligned vertically (Figs. 3D; 6, cells c,d,gj). The ascending dendrites from these cells would freely enter layer 1; (iii) neurons in lower layer 5 and 6 with radiate basal processes and long processes ascending vertically through the cortex (Figs. 3F; 7, cells j,m,o,p); (iv) cells in lower layer 6 and the white matter with ovoid cell bodies and long horizontally aligned processes (Figs. 4L,M; 7, cell q; 8, cells b,c); and finally, (v) a comparatively small group of inverted pyramidal-like neurons (Fig. 6, cell IP).

Cell body shape and size.

Dendritic morpho1og.y.


F

B

Figure 5


Of note is that some diaphorase-reactive neurons had processes that travelled for comparatively wide lateral extents (see especially cell h in Fig. 6). However, the horizontal spread of diaphorase-reactive processes was not a primary feature of these cells, except for the minority of cells found in upper layer 1, lower layer 6 and the white matter.

The dendritic arbors of bitufted diaphorase neurons commonly arose as a spray of processes emerging from thick primary dendrites (see Figs. 6, cells d,g; 7, c). More distally, these dendritic segments would become wispy and sometimes highly beaded (Figs. 3D,E,F; 4A; and cell c in Fig. 7).

Pyramidal-like diaphorase-reactive neurons were located predominantly in superficial layers 2 and 3 (particularly layer 3 in caudal area 24c; Fig. 6, cell IP). They possessed an inverted pyramidal-shaped cell body with a thick descending process that penetrated into the deeper layers of the cortex before giving rise to terminal branches. Several dendrites emerged from the upper somatic pole of these cells in a fashion reminiscent of (but not totally similar to) basal dendrites arising from the lower surface of true cortical pyramidal cells.

Spines were present over the dendritic surfaces of diaphorase- reactive neurons. Spines varied in shape from short stub- like protrusions (Fig. 4D) to relatively long thin pedicles with bulbous heads (Fig. 4G; Peters and Kaiserman- Abramof, 1970).

Relative to Golgi-impregnated neurons (DeFelipe and Farinas, 1992) and cells intracellularly filled with HRP (Larkman, 1991), the dendritic processes of diaphorase- reactive neurons ranged from being virtually spine-free (smoothiaspiny, Figs. 3E, 4E) to possessing a lowimoder- ate density of spines (sparsely spiny, Figs. 4D,G; 5C,D). Dendritic spines predominantly occurred on second and higher order processes (Figs. 4D,G; 5C,D; 8, cell c), and spines were found over the dendrites of diaphorase neurons located throughout the cortex, even over processes in lower layer 6 that originated from diaphorase-reactive neurons situated in the white matter (Fig. 8, cell c).

Spine density measurements were calculated for samples of selected secondary and tertiary dendritic processes derived from neurons situated in layers 213 and 516. The samples were collected from all the studied cortical areas and the data are presented in Figure 9. No statistical differences were detected between diaphorase neurons situated in the superficial and deep layers (Fig. 9). The density of spines over the processes of inverted pyramidal-like neurons was not significantly different from other diaphorase-reactive neurons in the cortex.

Dendritic spines: shape and surface density.

Fig. 5. A A tufted bipolar diaphorase-reactive neuron ( n ) in lower layer 2 of area 24b. The neuron gave rise to the labelled proximal part of an axonal arbor (ax) that ramifies at the layer 2/3 border. Parts of the dendritic arbor (boxed) are shown enlarged in C and D. B: Drawing of the neuron seen in A. The axonal arbor is shown in more detail in F. C: Part of a distal dendritic segment showingdendritic spines (arrows). D: Dendritic spines (arrows) arising from a proximal dendritic segment. E: Soma of the diaphorase-reactive neuron shown in A. A diaphorase- reactive spicule emerges from the cell body (arrow). F Drawing of the labelled axon arbor (ax) from the cell shown in A. Numerous axonal swellings are present over the axon field. One segment of the axon arbor (boxed) is shown in G. G: Photomicrograph of the axonal swellings along the diaphorase-reactive axonal fibre indicated in F. Calibration bars: F = 100 pm; A, B = 50 pm; G = 20 pm; D, E = 10 pm; C = 5 pm.

Spine-like protrusions were found emerging from the somata of diaphorase-reactive neurons (Feldman and Pe- ters, 1978). These somatic spicules were not uncommon and occurred either singly or in pairs (Fig. 5E; 7, cell b).

Axon-like processes were observed to emerge from diaphorase-reactive neurons in layers 2-6. These processes were exceptionally fine and frequently emerged from labelled somata (Figs. 3F; 4B,B’,K,L,M) or from the proximal segments of primary or secondary dendrites (Fig. 3B’; 4C; 5A,B; 6; 7). Occasionally, two fine calibre axon-like processes were observed to come from a single diaphorase-reactive cell. Usually these processes would arise separately from the soma and a proximal primary process, and would either ascend or descend through the cortex. Such neurons were most commonly located in layers 213.

No axon-like processes were seen coming from stained neurons in layer 1. However, diaphorase-reactive varicose fibres were found coursing horizontally through layer 1 in cingulate areas 24a,b, and c.

The axonal processes from the inverted pyramidal-like diaphorase neurons would commonly emerge very near to the soma or from a primary dendrite and course vertically upwards before either recurving or giving rise to collaterals (Fig. 6, cell IP).

Only the initial parts of axonal arbors from identified diaphorase-reactive neurons could be traced (Figs. 3-7). One reason for this may be that more distal axon segments from diaphorase-reactive cells in the cortex contain specifically low or insufficient levels of enzyme activity for histochemical detection. The initial trajectories of identified axonal arbors were either vertically or tangentially oriented in the cortex (Figs. 3B’,F; 4B,B’,K,L,M; 5B,F; 6-8). On occasions, these arbors could be traced into relatively diffuse arrays of fine, beaded processes ramifying close to their parent somata (Figs. 4B‘; 5F,G).

Strongly diaphorase-reactive puncta were present uniformly throughout the cortex (Fig. 4B,B’). These puncta were commonly located along interconnected webs of labelled processes (Fig. 4B). Diaphorase-reactive puncta were found closely associated with the processes of diaphorase-reactive neurons (Fig. 4E) and also the somata of counterstained neurons (Fig. 4F). Indeed diaphorase-reactive puncta in lower layer 3 could be seen to form pericellular arrays, or baskets, partially or completely encircling the somata of unlabelled cells (Fig. 4H). These basket-like structures appeared to be produced by 1-3 varicose fibres. They were present in all areas of mPFC investigated.

Diaphorase-reactive puncta were not observed to form the short vertically aligned strings of beads (axon cartridges) described below for PV-immunoreactive puncta.

Finally, few diaphorase-reactive axon-like processes and labelled puncta were observed in the white matter under- neath the cortical areas investigated.

NOS-immunoreactiue neurons. Cells in the monkey mPFC were immunolabelled using an antisera against b-NOS (Fig. 10). Although the morphology of these neurons was not revealed in great detail, many cellular characteristics of these cells were similar to neurons stained histochemically for NADPH diaphorase enzyme activity (Fig. 10. cf Figs. 3-81. In addition, the cortical depth distribution of bNOS-immunoreactive neurons was essentially similar to that observed for diaphorase-reactive neurons (see above).

Axonal morphology.

Diaphorase-reactive puncta in the cortex.


Fig. 6. Composite drawing of diaphorase-reactive neurons in layers 1-314 of areas 24a,b,c, 25 and 32. Note the morphology and distribution of cellular processes as well as the shape and orientation of labelled somata. Axon-like processes are indicated by arrows. Layout of figure: The calibration bar applies to all neurons. In order to present neurons from different cortical areas, where laminar boundaries occur at different depths, cells have been placed in their corresponding position

within each lamina although the exact depth of each lamina is not to scale. Laminar location ofcells (layer/area): a, 1/32; b, 2124a; c, 2/25; d, 212413; e, 2124~; f, 2124a; g, 2124b; h, 3/24a; i, 3124~; j , 3/32; k, 3124b; m, 3/32; n, 3124~; o, 3124b; p, 3124a; q, 3/25; r, 3/32; s, 3/25; t, 4/32. Inverted pyramidal-like neuron, cell IP from layer 3 in caudal area 24c. The thick descending process from cell IP is indicated by the curved open arrow. Calibration bar: 100 km.

LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I

\ Diaphorase t \

,579

Fig. 7. Composite drawing illustrating the cellular morphologies of into superficial cortical layers; the processes from cells j and p both diaphorase-reactive neurons in layers 5 and 6 of areas 24a,b,c, 25 and entered upper layer 3. Laminar location ofcells (1ayer:area): a, 5/24a; b. 32. Axon-like processes are indicated byarrows. The layout of the figure 5124b; c, 5/32; d, 5/25; ( I , 5 1 2 4 ~ ; f, 5 1 2 4 ~ ; g, 5124b; h, 5/24a; i, 6/32; j , is described in the legend to Figure 6. The soma of neuron b is shown 6124b; k, 6/25; m, 6/24b; n , 6 1 2 4 ~ ; 0, 6124a; p. 6/32; q, 6/25. enlarged to illustrate a somatic spicule (arrow). The arrows adjacent to Calibration bar: 100 kin. processes a t the top of the figure indicate that these processes continued

580

Diaphorase

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

f J I b

,/ /

f Fig. 8. Composite drawing of the cellular morphologies of diaphorase-

reactive neurons at the border between layer 6 and the underlying white matter (wmj of areas 24a,b,c, 25 and 32. Such cells display three clear morphologies: ( i ) lower layer 6 neurons with processes that enter the white matter (cell a) (ii) cells in the white matter that are oriented along the fibre bundles that constitute the white matter (cell b), and (iii)

TABLE 2. Morphometric Data'

D. circ Range (pm) (pm)

Calcium binding proteins' ICR. PV and CB populations combined) 11.6 2 1.2 4.9-27.8

Calretinin iCRl** 9.0 2 0.8 5.1-13.4 Parvdbumin 1PVI 12 9 2 1.1 6.1-33.2 Calbindin ICRI 11 9 i 1.1 5.2-25 3 GABA 10.8 ? 1.2 5.7-26.3 Diaphorase-reactive

Whole population 10.2 ? 0.9 5.617.3

Strongly reactive 12.7 L 1.2 8.1-16.2 Weakly reactive 1 0 5 ? 1.0 6.0-17.1

Strongly reactive 13.9? 1.1 8.5-19.8 Weakly reactive 9.8 ? 0.9 5.0-14.7

Layers 2:3

Layers 5:6***

'Mean area equivalent circle diameters !D. circle) of the cell populations investigated in this study- a1 CBPs icombined CR, PV and CB populations), CR+, PV+, CB+, and GABA+ immunoreactive neurons. Populations were sampled from all layers of the cortical arras investigated. bl Strongly and weakly reactive diaphorase-positive cells in superficial and deep layers within the cortex. These cell populations were composed of neurons taken from layers 213 and 516, respectively, of the corticd areas studied. !Mean values ? S D.; n = 3 three data sets. I Statistical analysis of somatic profile data: 'CBPs vs. GABA: t = 0.47, df = 4, P = 0.07. Difference not sipificant. "CR vs PV: t = 3 9. df = 4, P < 0.05. Difference significant. ***L5;6 strong vs. weakdiaphorase: t = 2.88, df = 4, P < 0.05. Difference significant. Comparisons between other cell populations were not significant !P > 0.05).

The b-NOS antisera used in this study produced a high level of background nonspecific staining at all serum dilutions. However, no b-NOS immunostaining was observed in endothelial or glial cells. Conversely, no specific labelling of cortical neurons was found with an antiserum directed against eNOS.

cells in the white matter that have ascending processes that enter the cortex (cell b). Cell b has two processes that are beaded (arrows). Part of one process from cell c (boxed) is shown enlarged to illustrate how dendritic processes become sparsely spiny (arrow) on entering the cortex. Laminar location of cells (laymiarea). a, 6/32; b, wmi24c; c, wmi25. Calibration bar: 100 wm.

Morphology o f calcium binding proteins in mPFC. Neu- rons immunoreactive for the calcium binding proteins (either CR, PV, or CB) investigated in this study displayed strong immunolabelling of their somata and nuclei as well as their cellular processes. Neurons immunoreactive for a given CBP displayed consistent morphologies across the five cortical areas 24a, 24b, 24c, 25, and 32 of the monkey mPFC. However, within each population of CBP-immunoreactive cell, several morphologically distinct subtypes were identified. Quantitative aspects of cell densities, cortical and laminar distributions are detailed in the companion paper (Gabbott and Bacon, 1996).

Calretinin (CR)-immunoreactiue neurons. CR-immunoreactive neurons were found in all layers (1-6) of the areas investigated and were also present in the underlying white matter. Several classes of CR+ neurons were evident in each cortical area investigated (Fig. 14). The numerical depth distribution of CR+ neurons indicated that peak density occurred in layer 2 and upper layer 3, with a marked decrease in cell numbers through layer 5 and into layer 6. The somata and processes of CR+ neurons were strongly immunoreactive in all cortical layers. CR-immunoreactive neurons were generally small (9.0 pm in diameter; Fig. 24; Table 2) with ovoid bipolar somata that had either: (i) two long vertically oriented processes arising from opposite somatic poles (Figs. 1Oc; 14, cells k, n, q, t, u, x, y), or (ii) two vertical tufts of processes (Fig. 14, cells j, m, 0, p, s, v, w, z). Although these cells were found throughout all layers of the mPFC they were most prevalent in middle to upper layer 3.

LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I 58 1

Diaphorase Reactive Neurons - L 2 / 3

Mean: 0.60 spines / pm

SD: 0.26 .d 2 20 Counts 48 C W a &I 15 0 & W P 10 Ei 1 C o i 5 P 4

Range 0 09 - 1 56 spines I pm

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

Spine density (spines / pm)

Diaphorase Reactive Neurons - L 5 / 6

Mean: 0.49 spines / pm 25 r SD 019

v)

W r

$J 20 Counts 51

5 a ru ' 5 0 k W P 10 Ei 1 C o i 5 P d

.r(

Range 0 08 - 0 98 spines / pm

n I 0 0.2 0 4 0 6 0 8 1 0 1.2 1 4 1 6 1 8

Spine density (spines / pm)

Fig. 9. Distributions of spine densities over the processes of diaphorase-reactive dcndritic processes in superficial (I, 2 / 3 ) and deep ( L 5/61 layers of monkey mPFC. Data derived from pooled samples from cortical areas 24a,b,c, 25 and 32 in 4 animals. (Arrows indicate mean values,. Statistical analysis: L213 versus L5/6: t = 0.68, n = 6, P = 0.52. Difference not significant.

A common structural feature of these CR+ neurons in layers 2-4, and upper layer 5 was the tight bundling and vertical orientation of their dendritic and axon-like processes.

CR+ neurons were also found in layer 1 where they were commonly encountered at the junction with layer 2. A numerically smaller population of CR+ cells was located superficially within layer 1 typically beneath the pial surface (Figs. 12A,B; 14, cells b,c). The cell bodies of these latter neurons were spindle-shaped and oriented parallel with the pia (i.e., horizontally). Thick processes emerged from opposite poles of their somata and were aligned horizontally within layer 1. Although rare, occasional side

branches would arise from the thick parent stems and descend for short distances within layer 1.

The dendritic fields of CR+ neurons in layer 2 were more radiate than for other CR+ cells. These dendritic fields arose from multipolar somata and radiated freely into layer 1 (Fig. 14, cells d-g, i). Occasionally the cell bodies of CR+ neurons in layers 3 and 5 were oriented horizontally, gwing rise to processes that initially coursed obliquely but then adopted vertical orientations within the cortex (Fig. 14, cell r).

A separate class of bipolaribitufted CR+ neuron in the deeper layers of the cortex had triangular or ovoid somata with long thick ascending processes (Fig. 14, cells z, a ' ) that passed through superficial layers, sometimes rising into upper layer 2 where they tapered gradually (Fig. 14, cell b'). Tufts of basal processes would emerge from these neurons and ramify horizontally in the deep layers of the cortex. The lateral spread of the basal processes from these deep lying CR+ neurons increased as the position of the parent cell body approached the white matter. Layer 6 CR+ neurons lying near the white matter did not have ascending processes and were aligned horizontally (Fig. 14, cell c'). The processes from these latter cells extended for considerable tangential distances ( < 300 pm) deep within the cortex. None of the processes from these cells ramified outside the grey matter.

Beaded dendrites were a characteristic structural feature of all classes of CR+ neurons in the cortex (Figs. 11D, 14, inset). In addition, many of the CR-immunoreactive dendritic processes bore a sparse number of dendritic spines. The qualitative appearance of low spine density over the processes of CR+ neurons was confirmed quantitatively, with spine density calculated as 0.89 i 0.09 spines per micron (mean 2 S.E.M.: sample ( n ) of 30 primaryisecond- ary processes. Each process was > 30 pm in length).

Axon-like processes arose from CR+ neurons located throughout the cortex. For CR+ neurons in layers 2-6 these process would emerge either from upper or lower somatic poles or more commonly from primary or proximal secondary dendrites (or even the intervening dendritic branch point). They frequently adopted an initial descending course but would give rise to ascending branches. Axonal fields were predominantly oriented vertically within the cortex and frequently confined within the lateral extent of the dendritic arbor (Fig. 14, cells j, s ) . Although uncommon, notable exceptions were found where the vertically aligned dendritic and axonal fields were slightly displaced laterally (Fig. 13). The axon arbors of CR+ neurons gave rise to swellings or varicosities (Fig. 13B).

Fine axon-like CR+ processes were also observed to come from the somata of labelled layer 1 neurons; however, these processes would course horizontally within layer 1 (Fig. 12A). No collateral branches were observed from these latter processes.

The overall appearance of immunolabelling in the neuro- pi1 of the five cortical areas clearly showed that within layers 2-4, CR+ immunoreactive processes were tightly bundled and preferentially aligned along the vertical axis of the cortex (Figs. 11C,D,H; 13; 14). Within layer 5 and particularly layer 6, CR+ labelled processes were less strictly oriented vertically.

CR-immunoreactive puncta were present throughout all cortical layers. Of particular note is that CR+ puncta were found to be closely associated with the somata and initial dendritic processes of other CR+ neurons. Indeed, on occasions strings of CR+ puncta could be found draped


Fig. 10. NOS immunocytochemistry. A NOS-immunoreactive neuron in) in layer 3 of area 24a with labelled processes (arrowed). B: Neuron ( n ) in layer 4 of area 32 immunoreactive for NOS. Immunola- belled processes extend laterally (arrows) and become beaded (encircled). The cell body of another NOS-immunopositive cell is indicated (double arrow). c, capillary. C: NOS-immunopositive neuron in) in layer 5 of area 25 with labelled processes (arrows). One fine immunore-

closely over CR-immunoreactive cell bodies (Fig. 26R,S) in a fashion reminiscent of pericellular baskets (see below).

Parvalbumin (PV)-immunoreactiue neurons. Strongly immunoreactive PV+ neurons were present in layers 2-6 and in the subjacent white matter of all the cortical areas investigated (Fig. 15A-F). In these layers immunoreactive neurons were strongly labelled. Cells in the deep region of layer 1 were also PV-immunoreactive. However, the somata of these neurons were only weakly labelled and their dendritic morphology not visible.

The depth distribution of PV+ neurons indicated that peak density occurred midcortex, in a region centered on middle to lower layer 3 and upper layer 5 (or layer 4 in the case of area 32). A decline in PV cell numbers was present in lower layer 5 and maintained in layer 6.

In general, PV + neurons in layers 2-6 could be divided into three broad classes on the basis of the size and shape of their somata and the distribution of their processes (Figs. 15-17). The first class had small bipolaribitufted fusiform somata (6.1-12.5 km in diameter; Table 2; Fig. 24) with either vertically aligned processes (Figs. 15C; 16, cell a) or with small radiate dendritic fields (Fig. 16, cells b-0. This latter type of PV+ neuron was most common in upper layer 2, but could also be found throughout layer 3. The second type of PV+ cell had a medium sized fusiform soma (10-16.5 pm in diameter; Table 2; Fig. 23) that was either vertically or horizontally disposed (Figs. 15c; 16, cells h, q, r, s). The processes from these cells radiate outwards along the axis of their parent somata. Neurons in this second category were common in lower layer 3, (layer 41, and layers 5 and 6.

The third type of PV+ neuron found in this study of the mPFC had exceptionally large round multipolar somata (18.0-30+ km in diameter; Table 2; Fig. 24) with wide

active process (double arrowhead) emerges from the labelled soma. (Note the weak immunolabelling of capillaries [cj and compare with Fig. 3A.) D: Neuron ( n ) and processes (arrows) in layer 3 area 24c immunoreactive for NOS. E: Multipolar neuron ( n ) with processes (arrows) immunoreactive for NOS lying in layer 6 of area 24b. Calibration bars: A-D = 50 pm; E = 25 pm.

radiate dendritic arbors (Figs. 15B, 16). The dendritic fields were commonly composed of between 3 and 8 thick primary dendrites that bifurcated proximally with secondary branches coursing radially outwards from the soma for considerable distances (Fig. 16, cells g, i, j , p). This third type of PV+ neuron was less frequent than the former two types of PV+ cells. However, when set amidst other PV-immunoreactive neurons, these cells were conspicuous by size alone (Fig. 15B,E). Indeed, there was a distinct cortical depth distribution of PV+ somatic profile size, with a strong tendency for large somatic profiles to occur in the deeper layers of the cortex, particularly lower layer 5 (Fig. 16).

Fig. 11. Calretinin (CR) immunoreactivity. A Superficial laminae (1-3) of area 24c showing the distribution of CR-immunoreactive neurons (arrows). Note presence of labelled neurons in layer 1 tdouble- headed arrow). B: CR-immunopositive neurons (n) in layer 2 of area 25. A strongly labelled neuron 1 thick arrow) gives rise to numerous radiate processes (small arrows). C: Lower layer 2 of area 24b. Elongated CR-immunolabelled neuron (n), oriented vertically in the cortex (arrowheads), with labelled processes (arrows). D: CR-immunopositive neuron ( n ) in layer 3 of area 24c. The neuron gives rise to two vertically oriented tufts of beaded processes (arrows). E: CR-immunopositive neuron (n) in layer 3 of area 24a. The labelled processes bear spines; examples are shown in F and G. F: Enlargement of boxed region shown in E. The immunolabelled process gives rise to many lightly CR- immunoreactive dendritic spines (arrows). G: Further example of CR-immunoreactive spines (arrows) arising from the cellular process of the neuron seen in E. H: CR-immunoreactive neuron in layer 3 of area 32. Note the laterally directed processes of this neuron (arrowheads) and the oriented immunoreactive processes (arrows) coursing vertically through the cortex. Calibration bars: A = 100 pm; B, C, H = 50 pm; D = 25 pm; E = 20 pm; F = 10 pm (G same magnification as F).

LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I 5x3

Figure 11


The dendritic processes of PV+ neurons were, as far as could be ascertained, aspiny, and the more distal processes were frequently beaded in appearance (Figs. 16, cell h; 17). Dendritic arbors of all types of PV+ cells ramified without regard for laminar boundaries, type 3 PV+ neurons situated at the midlevel of the cortex were found to have processes that ascended into layer 2 and descended through layer 5 towards layer 6 (e.g., cell j in Fig. 16 and cell m in layer 4 of area 32) . Although rare, PV+ cells were encountered in the white matter (Fig. 15F). These cells had processes that coursed horizontally for long distances beneath the grey matter with occasional side branches that entered into layer 6 where they bifurcated (Fig. 15F).

PV+ immunoreactive axon-like fibres were seen to emerge from the somata of labelled cells or from proximal processes (Fig. 16). In the majority of cases it was impossible to trace these processes for long distances (Fig. 15). However, on occasions the initial part of axonal arbors were detailed (Fig. 17). The principal trunk of these processes (which were particularly thin over their proximal lengths) would either ascend or descend vertically through the cortex (Fig. 16). Collateral shoots would then branch and run for short distances horizontally (Fig. 17); the terminal arbor of axon collaterals were not identified with certainty.

PV+ immunoreactive processes and puncta were distributed throughout all layers of the cortex (Fig. 18) but were most dense in layers 2 and 3. Layer 1, however, contained a very low density of PV+ puncta and the processes in this layer could frequently be traced back to P V + neurons in layer 2. PV+ puncta were also present within the underlying white matter (Fig. 25).

Of particular importance is that some PV+ puncta formed two morphologically distinct structures, termed axon cartridges and pericellular clusters (see Akil and Lewis, 1992). These two structures were most frequently found within prelimbic area 32 and cingulate areas 24b and 24c.

Axon cartridges were composed of numerous strongly immunoreactive PV+ varicosities (akin to strings of dark beads) that were vertically aligned within the cortex and situated at variable distances (4-15 Fm) beneath the somata of unlabelled neurons (Fig. 18A-C,GJ). On occasions the unlabelled somatic profiles were clearly seen to be pyramidal in shape (Fig. 18C). Axon cartridges were highly variable in both length and complexity (Fig. 18A-C,G and H). Their length ranged from 10 pm or so to over 30 Fm; as a rough first estimate (leaving methodological considerations aside) each cartridge was composed of between 8 and 45 individual PV+ puncta (for example compare Figs. 18A and 18H). These puncta were either closely apposed to each other or interconnected by fine immunoreactive strings (cyto/axoplasmic bridges; DeFelipe et al., 1985; Williams et al., 1992; see Fig. 18A-C and G J ) .

Axon cartridges were predominantly situated in superficial cortical layers 213 but were also present (albeit to a

Fig. 12. Calretinin (CRJ immunoreactivity. A: CR-immunopositive neuron lying very near the pial surface (piaJ of area 24c. A thick labelled process (thick arrows) emerges from one pole of the soma and courses horizontally within layer 1. In contrast, a fine immunolabelled process arose from the opposite pole of the soma and travelled close to the pial surface for over 200 pm. B: Layer 1 neuron in area 32 displaying CR immunoreactivity. The neuron lies close to the pia and has processes (arrows) oriented horizontally beneath the cortical surface. Calibration bars: A, B = 50 pm.

LOCAL CIRCUIT NEURONS IN MONKEY mI’FC: I 585

A CR

3

much lesser degree) in layer 5 and occasionally in layer 6. They were frequently encountered in the upper layers of areas 24b and 24c (Fig. 18A,B,G). Quantitative estimates indicated that the number of PV+ puncta composing an individual axon cartridge were more numerous in upper layers 213 (10-45) than in lower layers 516 (12-33).

Pericellular clusters were identified as numerous PV+ immunolabelled puncta fully or partially encircling the somata of unlabelled neurons (Fig. 18C-H,J). Similar to axon cartridges, the unlabelled cells were commonly pyramidal in shape, although circular somatic profiles were also encountered (Fig. 1 8 - H ) . PV+ puncta were additionally found around the descending presumed axon hillocks of pyramidal cells (Fig. 18C,H). Immunoreactive puncta surrounded not only the profiles of unlabelled pyramidal somata but also surrounded the outlines of their proximal apical (and sometimes basal) dendrites (Fig. 18C).

Of note is the observation that the pericellular clusters and axon cartridges formed by PV+ puncta could be present in three types of arrangements around the somata and presumed descending axonal profiles of unlabelled neurons: ( 1) PV+ puncta forming pericellular clusters alone, (2) PV+ puncta forming axon cartridges alone, or (3) PV+ puncta forming both a pericellular cluster and an axon cartridge around the same unlabelled cellular profile (Fig. 18A,B,C,H; see also Fig. 27). Figure 18H illustrates a type 3 PV+ puncta formation surrounding the profile of an unlabelled cell body together with its presumed descending axon hillock and axon initial segment.

All three types of PV+ puncta arrangements were present throughout layers 2-6. However, type 1 arrangements were predominant in layers 516, whereas arrangements of types 2 and 3 were most common in layers 213 (Fig. 27). The occurrence of this tripartite PV+ immunolabelling surrounding cortical pyramidal neurons occurred most frequently in areas 24b,c and 32.

I t should be pointed out that methodological constraints may be responsible for these observations and data reported above, for example, whether both the pericellular cluster and axon cartridge around a given pyramidal cell were equally available for immunocytochemical identification. The depth of the immunoreactive zone and the orientation of pyramidal cell somata within the tissue section could select which parts of the cell, and therefore the number of puncta, were immunolabelled. However, the observations were too consistent across sections and areas for such labelling to be related exclusively to methodology.

PV+ puncta were also found closely abutting onto the somata and processes of PV+ immunoreactive neurons at midlevels within the cortex.

Thick calibre PV-immunoreactive fibres were found in the neuropil of lower layer 3 and layer 4 (Fig. 25H). Similarly large PV-immunoreactive fibres were also present in the white matter underlying the cortical areas investigated here. These labelled processes coursed for considerable distances along the common path of unlabelled fibres in the white matter (Fig. 245).

Fig. 13. A Drawing of a CR-immunoreactive neuron in layer 3 of area 24c. The cell gves rise to a vertically oriented axonal arbor (ax1 part of which (boxed region) is shown in B. B: Photomicrograph of a segment of the axonal region boxed in A. Numerous swellings (arrows) are present along the CR-immunolabelled axonal fibre. Calibration bars: A = 100 pm; B = 20 pm.

586

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

Fig. 14. Composite drawing of calretinin (CR)-immunoreactive neurons in layers 1-6 ofareas 24a,b,c, 25 and 32. Note the morphology and distribution of cellular processes as well as the shape and orientation of labelled somata. Axon-like processes are indicated by arrows. The boxed inset in the centre of the figure shows the typical beaded appearance of a CR-immunoreactive dendrite (see also Fig. 12A). wm,

white matter. The layout of figure is described in the legend to Figure 6. Laminar location ofcells tlayeriarea): a, 1125; b, 1/32; c, 1124~; d, 2/32; e, 2124a; f, 2124b: g, 2132; h, 2i25; i, 2/24a; j, 3124~; k, 3124b; m, 2124~; n, 2/25: 0,3132: P, 2124~; q, 3124a: r, 3i24b; s, 3125; t, 5124~: U , 4/32; V, 3i24b; w, 5i24c; x, 5124b; y, 5124a; z, 6124a; a ' , 6124c; b', 6i24b; c' , 6/32. Calibration bar = 100 pm.


Fig. 15. Parvalbumin t PV) immunoreactivity. A: Low power photomicrograph showing darkly labelled PV immunopositive neurons in layer 3 in area 24b. B: A large multipolar PV-immunoreactive neuron i n ) in layer 4 of area 32. The cell gives rise to numerous labelled processes (arrows) radiating outwards for long distances. A fine axon- like process arises from the cell body (ax). Several other immunoreactive neuron cell bodies are indicated (arrowheads). Compare the sizes of immunolabelled cells. C: Layer 3 area 25. PV-immunoreactive neuron i n ) oriented vertically with a spray of labelled processes (arrows) above the cell body. D: PV-immunolabelled neuron ( n ) in layer 5 of area 24c.

Finally, in all the cortical areas investigated, PV+ immunoreactivity defined a band of cortex that extended from lower layer 3 through to upper layer 5 (encompassing layer 4 in area 32). This band was particularly evident due to the increased frequency of PV+ somata as well as the presence of immunoreactive puncta.

In the cortical areas studied, CB immunoreactivity was found in pyramidal and non-pyramidal neurons (Fig 19G). These

Calbindin (CB)-immunoreactiue neurons.

two subpopulations-were morphologicallydistinct (Figs. 19G, 20).

Labelled process iarrow). E: Layer 3, area 32. PV-immunolabelled soma o f a large multipolar neuron (double-headed arrow); compare the size of this neuron with the somatic sizes of other neighbouring immunolabelled cells (arrows). F: PV-immunoreactive neuron ( n ) lying in the white matter (wm) below layer 6 of area 24aib. Note: immunolabelled processes elongated along fibres coursing in the white matter (arrows); process ascending into layer 6 (double-headed arrow); beaded process in layer 6 (arrowheads). The layer 6iwhite matter boundary is indicated (dotted line). Calibration bars: A = 100 km: B = 75 pn: F = SO pm: C. D = 25 km; E = 20 kin.

The majority of CB + immunoreactive pyramidal neurons were located in lower layer 3 and also present although to a much lesser extent in layer 5. Both tiers of cells were very weakly to faintly immunolabelled and possessed characteristic pyramidal somata with thick processes emerging from the apical pole of their somata (Figs. 19, 20) . This staining pattern was present in all the cortical areas examined.

By comparison, neurons within the nonpyramidal cell population were strongly immunolabelled for calbindin (Fig. 19G) and several subtypes were evident (Figs. 19-21). The cortical depth distribution of the nonpyramidal CB-


Fig. 16. Composite drawing of parvalbumin (PV)-immunoreactive neurons in layers 2-6 of areas 24a,b,c, 25 and 32. Note the morphology and distribution of cellular processes and size of labelled somata. Axon-like processes are indicated by arrows. PV-immunoreactive neurons in layer 1 have not been included since only their somata were lightly stained and no morphological detail of their processes revealed.

1 ' . . . . . . . _ .

wm, white matter. The layout of this figure is described in the legend to Figure 6. Laminar location of cells (layerlarea): a, 2124b; b, 2124a; c, 2132; d, 2125; e, 2124~; f, 2124b; g, 3132; h, 3125; i, 3124b; j , 3124b; k, 5132; m, 4132; n, 512413; 0, 5124~; p, 5/32; q, 5124b; r, 5124c; s, 6124a. Calibration bar = 100 Fm.

589 LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I

\

PV

1 :: I+ i-

i Fig. 17. Drawing of PV-immunoreactive multipolar neuron in layer

4 of area 32. An axonal process emerged (small arrow) from the proximal part of a dendritic process and gave rise to a vertically and horizontally disposed axonal field (large arrows) with several identified collaterals. Calibration bar = 100 pm.

immunoreactive cell population was essentially similar to that of CR+ neurons, but peak density occurred comparatively lower in the cortex, within upper to middle layer 3. Thereafter CB+ cell density gradually diminished with increasing cortical depth.

The first two types of CB+ immunoreactive nonpyramidal neurons were located primarily within layers 2 and 3 (Fig. 20). These cell types comprised vertically oriented bipolaribitufted and radiate multipolar neurons, respectively (Fig. 20, cells a-k). The processes from the bitufted neurons were confined within narrow, vertically oriented columns. The somatic profiles of both cell types were small to medium in size and ranged from 5.2 to 15 pm in diameter (Fig. 24). Beaded dendrites were a common characteristic of these CB+ cells (Fig. 19B,F); fine varicose axon-like fibres were seen to arise from proximal dendrites (Fig. 19F). Spine-like protrusions were also present over the surfaces of CB+ neurons in these two classes (Fig. 19E). Mean spine density for these cell types was calculated as 0.71 2 0.08 spines per micron (mean t S.E.M.: Sample (n ) of 8 processes. Process length ranged from 24 to 46 pm).

The third type of CB+ nonpyramidal neuron was very distinct and found in layers 2-6 (Fig. 21). Neurons characteristic of this subtype possessed very small round multipolar somata (6-10 Fm in diameter; Fig. 24). The processes that emerged from these neurons bifurcated readily and ramified extensively in the vicinity of the cell body. Rarely did the dendritic territory exceed 200 Frn in diameter. No axonal processes were seen arising from neurons in this category of CB+ cell.

Neurons belonging to the fourth category of CB+ neuron had exceptionally large somata and were regularly located in the deeper layers of the cortex (Figs. 19D,J; 20). Somatic measurements ranged from 16 to 25.3 pm in diameter. Such neurons were multipolar cells with expansive smooth dendritic arbors that radiated outwards for over 350 km from the parent somata. The initial segments of fine axonal processes were seen to emerge from the lower surface of the cell body (Fig. 19J').

CB+ neurons in the fifth category were located predominantly in layers 5 and 6 (Fig. 22). Similar to cells in the previous category, these neurons also had large multipolar cells (somatic diameter range 15-25 pm) with radiate dendritic fields. However, a characteristic feature of these neurons was a well defined single axon-like process that merged either from the soma or a proximal dendrite and coursed vertically upwards into superficial layers. Terminal segments of these axon arbors could not be identified.

The last type of CB+ neuron was located in the white matter. Like PV+ neurons, the somata and processes of these cells were aligned horizontally beneath the grey matter. Occasional side branches would travel obliquely or perpendicularly into layer 6; these side branches were not beaded in appearance.

No CB+ cells were found in layer 1 of the cortical areas investigated. However, CB+ processes were frequent components in layer 1 and were commonly derived from CB+ neurons in layers 2 and 3. On occasion, horizontally oriented CB+ processes were found coursing near the pial surface. Within the superficial layers 2 and 3, CB+ immunoreactive processes were frequently encountered as vertically oriented bundles (fascicles) of labelled processes (similar to CR+ processes).

CB+ puncta were distributed throughout the cortex, particularly within layers 2 and 3. In the superficial layers


Figure 18

LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I .591

strings of immunolabelled puncta were frequently distributed radially.

The range of mor- phologes of GABA+ neurons in the monkey mPFC (Fig. 23) overlapped the morphologies of CR+, PV+ and CB+ immunoreactive neurons described in detail above. One exception, however, was that GABA immunoreactivity was never found in the somata of pyramidal neurons in layer 3 (cf calbindin immunoreactivity).

The cortical depth distribution of the GABA-immunoreactive cell populations mirrored the combined cortical depth distribution of CR+, PV+, and CB+ neurons. Peak cell density for GABA-immunoreactive neurons in monkey mPFC occurred in layer 2 and diminished gradually towards the white matter without secondary peaks occurring in lower cortical layers (for quantitative details see Gabbott and Bacon, 1995).

GABA+ puncta were found throughout the layers of the mPFC and predominantly in layers 2 and 3. In both layers 213 and 516 GABA+ puncta formed pericellular clusters (Fig. 18K,I,,M) and axon cartridges (Fig. 18K) similar to those described above for PV-immunoreactive puncta. How- ever, GABA-immunoreactive puncta were not present in the white matter.

The somatic profile-size distribution of GABA+ neurons was not significantly different from that of the combined population of CR+, PV+, and CB+ neurons (calcium binding proteins, CBPs; Fig. 24). Interestingly, both somatic profile-size distributions (GABA+ versus CBPs+ )

CABA-immunoreactiue neurons.

Fig. 18. CoinparisonofPV (A-HI and GABA (K-M) immunoreactivities. A: 1,aye.r 3 , area 24h. Rows of vertically aligned PV-immunorcac-

trings" (arrows) lying beneath unlabelled cell bodies (n; presumably the unlabelled somata of cortical pyramidal neurons). B: Layer 4, area 32. I n this typical example, a PV-immunopositive string (thick arrow1 can be seen to be composed of numerous immunoreactive puncta ismall arrows). C: Layer 3 , area 24c. PV-immunoreactive puncta are not only clustered around the unlabelled soma and proximal apical dendritic shaft (ad) of a pyramidal neuron (P; small arrows) but are also clustered around (thick arrow) the basal pole of the neuron, presumably encasing the axon initial segment. D: PV-immunoreactive neuron tPV+ I in layer 3 of area 24c. Dark PV-iinmunoreactive puncta surround the cell bodies of unlabelled neurons ( n ) . E, F: Numerous PV-immunoreactive puncta (small arrows) encircle the unlabelled somatic profiles of two cells ( n ) . The nuclei and nucleoli of both neurons are visible. Cell E is located in layer 3 of area 25, while cell F is located in layer 5 of area 24b. G: Layer 3 , area 24c. The soma of an unlabelled neuron ( n ) is surrounded by PV-immunoreactive puncta (example indicated by thin arrow). PV-immunoreactive puncta also encase (thick arrows) a thin unlabelled process that emerges from the lower surface of the cell. A neighbouring immunoreactive neuron (PV+) is indicated. !The arrows identify structures shown in H and J.) H: Higher magnification photomicrograph of unlabelled neuron ( n ) and the perisoinatic immunoreactive puncta (small arrows). The descending immunoncsgative process remains surrounded by PV-immunoreactive puncta for ovcr 30 pm (thick arrows). Inset J: Drawing of the PV-immunoreactive puncta associated with the unlabelled neuron In) and the fine process descending from its soma. K GABA immunoreactivity in layer 3 of area 25. The somata of two GABA-immunonegative pyramidal neurons (PI are covered by GABA-immunopositive puncta (thin arrows I . The proximal part of the apical dendrite (ad) of one of the cells is siirrounded by immunoreactive puncta. In addition a descending string of GABA-immunoreactive puncta is present a t the basal pole of the same pyramidal neuron (thick arrows). L Layer 4, area 32. GABA-immunoreactive puncta encircle the soma of an unlabelled neuron !nl. M: Soma and proximal apical dendrite (ad) of an unlabelled pyramidal neuron (PI in layer 3 of area 24c is surrounded by GABA- immunoreactive puncta (arrows). Calibration bars: A, C, D, K, M = 20 pm; H = 15 pm: J = 12.5 pm; B, E, F, G. L.

showed minor outlying peaks in the range of 19-23 Fm (Fig. 24), derived from large PV+ and CB+ cells.

NAIIPHdiaphorase histochemistry in comhination with (peroxidaselllAB) inimunoc.ytochemistry

Cells displaying both N N P H diuphorase activity and im.munoreactivity. Neurons displaying combined diaphorase activity and specific immunoreactivity (for either CR, PV, CB, or GABA) were readily identified by brown nuclear labelling contrasted against a blueipurple cytoplasm containing the formazan end-product of the NADPH diaphorase reaction (Fig. 25A). Such double-labelled neurons were uncommon and only encountered within the immunoreactive regions of the tissue. No direct correlation could be established relating the coexistence of NADPH diaphorase activity and immunoreactivity (either for CR, PV, or CB) with staining intensities nor size of labelled somata.

The relative infrequence of diaphorase-reactiveiimmuno- labelled cells resulted from two confounding factors, the limited penetration of antisera into tissue sections coupled to the comparative rarity of diaphorase reactive neurons in the mPFC. (Comment: diaphorase-reactive neurons constitute 0.20%-0.32% of the cortical neuron population in monkey mPFC; see Table 15B in Gabbott and Bacon, 1996.)

However, CR, PV, CB and GABA immunoreactivities were found to be separately colocalised within a proportion of diaphorase-reactive neurons in layers 2-6 and the underlying white matter (Fig. 25A-K; quantitative data are gwen in Gabbott and Bacon, 1996). Neither the CBPs nor GABA immunoreactivities were colocalised in layer 1 diaphorase- reactive cells. The rarity of these neurons together with the above mentioned technical considerations may have been responsible for this finding. Furthermore, within the cortex proper, it was not possible to differentiate between CR+, PV+, or CB+ containing NADPH diaphorase-reactive neurons solely on the basis of either dendritic tree morphology or specific features (e.g., size and shape) of the cell body (Fig. 25).

In addition to the distribution of diaphorase-reactive puncta described above, some puncta were also found closely apposed to the somata and both proximal and distal processes from CR-, PV-, CB-, and GABA-immunoreactive neurons (Figs. 45; 26N,O,P). Conversely, CR+ and CB+ immunoreactive puncta were found in close proximity with cell bodies and processes of diaphorase reactive neurons.

Dual immunocytochemical labelling: CRIPV, CBIPV, and CRiCE Using a double immunolabelling technique (Bevan et al., 1994) the structural interrelationships of CR-, PV-, and CB-immunoreactive structures were investigated in the monkey mPFC.

Following specific dual immunostaining, DAB-labelled structures appeared brown and SG-labelled structures grey. No cells were found double- labelled using this technique. Indeed, immunolabelled neurons and processes were morphologically similar to labelled structures found in corresponding tissue reacted with only one antiserum. It was therefore considered that the chromagens SG and DAB were localised in neurochemically distinct types of cells and cellular processes.

Double CRIPV immunolabelling. Calretinin-immunoreactive fibres were frequently found lying near proximal processes and cell bodies of identified PV-immunoreactive neurons situated in layers 213 of the mPFC (Fig. 26A-E, H-L). When such interactions occurred, fibre swellings would be closely apposed to the proximal processes and somata of PV-immunoreactive neurons (Fig. 26A-E, H-L).

Comment on methodology.

Figure 19


The somata of PV neurons were the common targets of CR+ puncta; between 1 and 8 CR-immunoreactive fibre swellings were associated with a given PV+ cell body (Fig. 26E,J,L; see also Fig. 28).

Similar to CRIPV interactions described above, CR+ immunoreactive fibres and fibre swellings were found to be associated with calbindin-immunoreactive cell bodies, particularly CB + somata located in layer 3. However, unlike CRiPV interactions there was a distinct tendency for CR+ axonal swellings to be predominantly located away from labelled PV+ somata and preferentially onto primary and secondary dendritic processes (Fig. 26M,Q; see also Fig. 28).

(Overview. It could not be determined whether the CR+ fibres and puncta associated with PV+ neurons were derived from the same fibres and puncta contacting CB+ cells. The possibility therefore remains of two distinct populations of CR+ cell, each preferentially contacting the somata and processes of either PV+ or CB+ neurons [see Fig. 281.1

No definitive characteristic interactions were observed between CB+ and PV+ immunoreactive structures.

Double CRICB immunolabelling.

Double CBIPV immunolabelling.

DISCUSSION The present paper provides a detailed and comprehensive

qualitative description of the distribution and morphology of neurons in the monkey medial prefrontal cortex (mPFC) displaying immunoreactivity for the calcium binding proteins calretinin (CR), parvalbumin (PV) and calbindin (CB). The study also describes the morphology and cortical distribution of NADPH diaphorase-reactive neurons in the same region of the monkey cerebral cortex.

The investigation concentrated on the mPFC, a region located in the anterior medial wall that includes Brodmann areas 24a, 24b, and 24c (anterior cingulate cortex or anterior limbic cortex), area 32 (prelimbic cortex) and area 25 (infralimbic cortex; Carmichael and Price, 1994; and see Fig. 1). This paper directly supports several recent investi-

Fig. 19. Calbindin (CB) immunoreactivity. A Multipolar CB- immunoreactive neuron (n) in lower layer 3 of area 24a. Numerous labelled processes (arrows) radiate from the soma. B: CB neuron in layer 2 of area 32. Small arrows indicate beaded dendrites. A fine varicose process arises from the descending process proximal to the soma (thick arrow). C: Immunoreactive neuron ( n ) with labelled processes (arrowheads) in upper layer 3 of area 24b. A fine side process (arrows) emerges from the principal ascending dendrite. D: Neighbour- ing CB-imrnunoreactive cells (arrows) in layer 5 of area 24c. Note size differences. E: Part of a CB-immunopositive dendritic process showing spine-like protrusions (small arrows). F: A descending beaded (small arrows) process from a CB-immunolabelled neuron gives rise to an exceptionally fine axon-like process (thick arrow). G: Bipolar CB- immunoreactive neuron (n) in superficial layer 5 of area 24c. Labelled processes (thin arrows) ascend into layer 3 and descend through layer 5. A faintly CB-immunoreactive layer 3 pyramidal neuron is indicated (thick arrow). c, capillaries. H: Bipolar CB-immunoreactive neuron ( n ) in layer 6 of area 24b with ascending and descending sprays of immunolabelled processes (arrows). J: Giant CB-immunoreactive neuron (n i in layer 5 of area 25. Segments of the dendritic processes of this cell are beaded (arrowheads). One labelled process (thick arrow) ascends to superficial layers. An axon-like process (small arrow) emerges from the lower surface of the cell. Inset J' is an enlargement of the boxed region in J and shows the axon-like process coursing away from the labelled soma (arrows). Calibration bars: B, C, H = 50 pm; A, G = 25 pm; J .= 20 pm; D = 15 pm; F, J' = 10 pm; E = 5 pm.

gations studying the regional distributions and morphologies of neurons displaying CR, PV and CB immunoreactivities in the monkey prefrontal and cingulate cortices (Hof and Nimchinsky, 1992; Hofet al., 1993; Conde et al., 1994).

The study of Conde et al. (1994) qualitatively studied Brodmann areas 9-13, 32 and 46 of the prefrontal cortex and also provided a quantitative assessment of the relative densities of CR-, PV-, CB-immunoreactive neurons in areas 9, 11 and 46. The current investigation supplements these observations by examining pre- and infralimbic cortical areas as well as the anterior cingulate cortex. Furthermore, the current paper also extends the cyto- and chemoarchitec- tural observations of Hof and Nimchinsky (1992) and Hof et al. (1993) by providing a detailed qualitative and quantitative morphological survey of these neuron populations in the anterior cingulate cortex (areas 24a, b, and c). In addition, the present study has provided an extensive account of NADPH diaphorase-reactive neurons in the mPFC.

Taken together, this collection of studies gives a comprehensive description of the areal and laminar distributions and cellular morphologies of neurons expressing CR, PV and CB immunoreactivities and diaphorase enzyme activities in the prefrontal and cingulate cortices of the adult macaque monkey.

Qualitative areal and laminar distribution of CBPs in mPFC

The pattern and intensity of labelling within each cortical area examined here did not vary significantly across areas. However, in area 32 there was a distinct band of PV+ immunopositive neurons situated in lower layer 3, layer 4, and upper layer 5 (see below).

The cortical depth distributions of CR-, PV- and CB- immunoreactive neurons were similar across the mPFC. Peak cell densities occurred in layer 2Iupper layer 3 for CR+ neurons and in upper to middle layer 3 for CB+ cells, whereas maximum density for PV+ neurons was located in lower layer 3 to upper layer 5. These findings corroborate the observations of Hof and Nimchinsky (19921, Hof et al. (1993) and Conde et al. (1994).

Ferrer et al. (1992) have described the distribution and morphology of CB+ neurons in the temporal neocortex of normal humans. Similar to the monkey mPFC, CB+ neurons were predominantly distributed within superficial layers 2 and 3.

CR-, PV-, CB-immunoreactiue cells: local circuit GABAergic neurons Convergent evidence derived from a large number of studies investigating the morphology, distribution and synaptic connectivity of cortical neurons displaying immunoreactivities for CBPs, indicate that CR, PV and CB are expressed in well defined populations of local circuit GABAergic neurons in the monkey cerebral cortex. The anatomical evidence stems from the cortical and laminar distributions, as well as the characteristic morphologies (somatic size and shape, dendritic and, where possible, axonal morpholojges) of each immunoreactive cell class compared with the structure of defined local circuit neurons seen in Golgi specimens and in GABA (and related) immunocytochemical studies. The morphological evidence is summarised in Table 3, and discussed in detail by Lund and Lewis (1993) and Conde et al. (1994).

Although Table 3 provides strong evidence for the similarity between neurons displaying CBPs and specific types of interneurons in the monkey cortex, it is emphasised that

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

Fig. 20. Composite drawing of calbindin (CB)-immunoreactive neurons in layers 2-6 of areas 24a,b,c, 25 and 32. Note the morphology and distribution of cellular processes as well as the size of labelled somata. Axon-like processes are indicated by arrows. A population of weakly immunolabelled layer 3 pyramidal neurons (p) is shown. Note also CB-immunoredctive neuron in the white matter (wm). The layout of

the figure is described in the legend to Figure 6. Laminar location of cells (layer/area): a, 2124~; b, 2124b; c, 2/25; d, 2124a; e, 2/32; f, 2124c; g, 3/32; h, 3124b; i, 3124~; j, 3124a; k, 3/25; m, 4124~; n, 5124a; 0,5/32; p, weakly CB+ pyramidal-shaped neurons; q, 5124b; r, 6/24a; s, 6/32; t, wmi24c; u, wm/25; v, wm132. Calibration bar = 100 pm.

LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I .595

Fig. 21. Calhindin (CB)-immunoreactive neurons (A-F) throughout the cortex (inset). An initial segment of an “axon-like” process is seen emerb.ng from cell b (small arrow). Note the compact dendritic arbors

ramifying near to the cell bodies. Areal location of cells (layeriareal: A, 32; B, 32,; C, 24c; D, 25; E, 24c; F, 24b. Calibration bar = 100 Fm.

Fig. 22. Large calhindin (CBI-immunoreactive neurons distributed in the deeper layers (insets) of areas 24a,c, 25, and 32. Identified immunolahelled “axon-like” processes (small arrows) emerged from these cells and ascended through the cortex. The somatic profiles of

not all CBP-immunoreactive neurons fit the schemata of classification. Indeed, several classes of CR+, PV+ and CB+ neurons were present in the mPFC whose identities could not be catalogued unequivocally, for example, CR+ neurons with radiate dendritic fields in layer 2 or bipolar PV neurons in layer 3 (see Fig. 15C; see also Table 2 in Lund and Lewis, 1993). Without clear descriptions of the afferent and efferent synaptic connectivity of such neurons (see WilIiams et al., 1992) further classification of these

neighhouring CB-immunoreactive neurons are shown. Laminar location of cells (layeriareai: A, 5124a; B, 5/32; C, upper 6124~; D, 5/25; E, lower 5/32; F, 6124~. Calibration bar = 100 um.

cells is problematic. Nevertheless, spine density measurements along representative CR- and CB-immunoreactive processes indicate that together with the battery of other morphological criteria, CR+ and CB+ neurons belong to aspiny or sparsely spiny cell categories, with PV+ neurons being predominantly smooth cells (Stichel et al. 1987; Blumcke et al., 1990; Conde et al., 1994).

Immunoreactivity for CB was also expressed in a population of layer 3 pyramidal neurons; the somata of these cells


1994). However, the functional significance of this labelling pattern is unclear but may signify chemically distinct populations of pyramidal neurons in layer 3. Such neurons may receive qualitatively and quantitatively similar sources of synaptic input and themselves innervate a similar spectrum of intracortical targets and subcortical target regions.

The localisation of CB and CR immunoreactivities in neurons that resemble double bouquet cells (Somogyi and Cowey, 1981, 1984) suggests that either two separate populations of these cells are present in monkey mPFC or that the two calcium binding proteins are differentially expressed within the same class of local circuit neuron (see Rogers, 1992; Rogers and Resbois, 1992 ). Immunoreactive neurons with double bouquet morphologies were much less frequently encountered in the CB+ cell population than in the CR+ neuron population (Figs. 14 and 20). Indeed, CB+ neurons with double bouquet morpholoses were located mainly in layer 2, whereas such CR+ cells were predominantly present in layer 3 (Figs. 14 and 20).

Although CR and CB antisera may exhibit some cross- reactivity (since an 86% amino acid sequence homology in their molecular structures has been identified; Winsky et al., 1989), Conde et al. (1994) do not report the colocalisation of CR and CB in cortical neurons in layers 2-6. Furthermore, while Conde et al. (1994) report C R + double bouquet cells, they do not specifically mention CB immunoreactivity in double bouquet neurons in prefrontal cortex. Importantly, DeFelipe et al. (198913, 1990) and DeFelipe and Jones (1992) provide extensive evidence of CB- immunopositive double bouquet cells in other areas of monkey cortex, and Ferrer et al. (1992) also report large CB+ double bouquet neurons in human temporal cortex. It therefore seems likely that CR and CB are expressed in two neurochemically distinct populations of double bouquet cell in monkey mPFC.

With regard to the postsynaptic targets of double bouquet cells, DeFelipe and Jones ( 1992) have demonstrated ultra- structurally that the long, narrow, vertically aligned strings of CB+ puncta, derived from CB+ double bouquet neurons in monkey somatosensory cortex, formed symmetric synaptic contacts on unlabelled dendritic shafts (62% ) and onto dendritic spines (38%)). However, despite the density and close proximity of boutons within an individual CB+ bouquet, relatively few immunolabelled boutons coverged onto the same postsynaptic target. Such targets included the side branches of pyramidal cell apical and basal dendrites. DeFelipe and Jones (1992) report that apical den-

were lightly CB-immunoreactive. This observation has also been reported previously (DeFelipe et al., 1989b; Hof and Morrison, 1991; Hof and Nimchinsky, 1992; Conde et al.,

Fig. 23. GABA immunoreactivity. A GABA immunopositive neurons (G+I in layer 3 of area 24h. Processes (thin arrows) can be seen radiating from one of the labelled neurons. B: GABA-immunoreactive neurons (G+) in layer 2 of area 24b. Beaded processes (arrows) can be seen arising from one neuron. Immunoreactive puncta are present in the neuropil (encircled). GABA immunonegative neuron ( G - 1. C: GABA-immunoreactive puncta (arrows) surrounding two GABA- irnmunonegative somata (G- 1. GABA-immunolahelled neurons are indicated (G+) and immunopositive puncta are shown in the neuropil (encircled). D Postemhedding GABA-immunoreacted section from layer 3 of area 24c showing immunopositive (G+ 1 and immunonegative (G- ) neuronal profiles. Note GABA-immunoreactive puncta closely apposed (arrows) to the somata of the immunolabelled neuron and the unlahelled cell. (The structural arrangement of GABA-immunopositive puncta around GABA-immunoreactive soma correlates with known synaptic interconnections between GABAergic neurons that mediates disinhibition within cortical circuits: see Mize et al., 1992; Jones et al.. 1994.) Calibration bars: A, B, C = 25 pm; D = 10 pm

CBPs +ve 150

m Mean 11 6 pn

x 2% c Counts 1800

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tK)

z STRONG Mean 1 2 7 p SD 155 Counts 400 Range 8 1 - 16 2 pn

WEAK Mean l 0 5 p SD 268 Cowls 400 Range 6 0 17 1 prn

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Diaphorase Layers 516 liy)

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Fig. 24. 1)il;trihutions of' somatic profile size iD. Circle, p m ) . tions for diaphorasc-rc;ictive neurons have been divided into cells located in superficial t2it'3) and deep 1516) layers, as well as into strongly and weakly labelled somata. tAbs. Frequency is absolute number ofcells occurring within each sizc bin.)

Calcitiin hindiiig protein iCRPsl profile distribution derived from combined pitpulations ccqually sized; n = 600) ofCR+, PV+, and CR+ immunowacttvt~ cell profiles. Kotc the minor outlying peaks in the ratigt, 19 -2:i p.rn for CRPs+ and CABA+ neurons tarrowed). Distribu-


Fig. 25. Photomicrographs of tissue sections reacted for diaphorase enzyme activity (Dia.) in combination with immunocytochemical labelling for either calretinin (CR), parvalbumin (PV), calbindin (CB) or GABA. A Diaphorase and calretinin, layer 516 area 24b. Three distinct types of cellular labelling are shown: (i) a cell that is diaphorase-reactive alone (D+ alone); (ii) a neuron that is CRimmunoreactive (CR+ alone); and (iii) a cell that displays both diaphorase activity and CR immunoreactivity ID+CR+). Since the nuclei of diaphorase neurons are not labelled with formazan reaction end-product, the primary feature identifying the specific immunolabelling of diaphorase-reactive neurons is the presence of brown peroxidase labelling of nuclei in diaphorase- reactive neurons. Note that specific peroxidaseiDAB immunolabelling in the nuclei of cells that are either solely immunoreactive (CR+) or diaphorase reactiveiimmunopositive (D+CR+) is clearly a darker brown than the general nonspecific DAB peroxidase staining (brown colouration) of the tissue. Note also that the three neurons shown here were within the zone of immunolabelling. c, capillaries. B: Diaphorase and calretinin, layer 3 area 24a. Neuron displaying CR immunoreactivity (CR+ 1 neighbouringa neuron that displays both diaphorase enzyme activity and CR immunoreactivity (D+CR+ ). C: Diaphorase and calbindin, layer 3 area 24c. A neuron that is solely diaphorase-reactive (D+ 1 lacks brown peroxidase immunolabelling of its nucleus. Compare staining pattern with local CB-immunopositive neuron (CB+ ). Both

cells lay within the immunoreactive regions of the tissue section. C': Enlargement of the boxed region in C showing a fine axon-like process (arrowhead) emerging from the lower somatic pole of the diaphorase- reactive neuron. D: Diaphorase reactivity and GABA immunoreactivity in layer 3 of area 24b. A cell that is solely GABA-immunoreactive (G+) lies near to a diaphorase-reactiveIGABA immunopositive neuron (D+G+ ). Note the brown DAB peroxidase reaction product defining the nucleus of the double labelled cell. E: Colocalisation of diaphorase and calbindin within a neuron (D+CB+ 1 in layer 6 of area 32. c, capillary. F: Neuron lying in the white matter beneath area 24c showing both diaphorase activity and CB immunoreactivity (D+CB+ 1. G: Diapho- rase reactivity and calhindin immunolabelling colocalised tD+CB+ 1 in a neuron in layer 3 of area 25. H Large calibre PV-immunoreactive fibres (small white arrows) present in the neuropil of layer 4 in area 32. Diaphorase activity and PV immunolabelling are colocalised in a neighbouring cell (D+PV+ 1. J: Neuron lying horizontally in the white matter (wm) immediately below area 32. The cell displays both diaphorase activity and PV immunoreactivity (D+PV+ ). Note the large calibre PV-immunoreactive fibres (small white arrows) coursing in the white matter. K Weak GABA immunoreactivity colocalised with diaphorase activity in the cell body of a neuron deep in the white matter beneath areas 24a,b and c. Calibration bars: A, B = 100 pm; D, E, F, G, H, J = 20 pm; C, K = 10 pm.


dritic shafts did not receive input from the CB+ boutons associated with CB+ double bouquet cells. The above observations on the targets of CB+ double bouquet cells have been substantiated in a recent report in the human temporal neocortex (del Rio and DeFelipe, 1995). The above immuno-cytochemical data support ultrastructural studies investigating the postsynaptic targets of Golgi-impregnated double bouquet cells (Somogyi and Cowey, 1981, 1984). Whether the postsynaptic targets of CR+ double bouquet cells in monkey mPFC are similar to those of CB+ double bouquet is presently unknown (see Fig. 29). If the spectrum of postsynaptic targets is significantly different between the CB+ and the CR+ double bouquet cell populations, then the possibility exists for two separate, vertically oriented and overlapping intracortical circuits. These vertically aligned inhibitory local circuits could differentially and significantly influence neuronal operations in a radial cylin- der of cortex extending over several cortical laminae (see below).

PV+ immunoreactive pericellular clusters and axon cartridges

Cortical basket and chandelier neurons innervate specific parts of cortical pyramidal cells (see references in Table 3; Douglas and Martin, 1990); basket neurons preferentially innervate the soma and axon hillock, whereas chandelier cells provide highly selective and exclusive synaptic input to the axon-initial segment. Immunocytochemical evidence indicates that although both cell types are considered to be GABAergic, subtypes exist containing either neuropeptides or PV reactivities (Williams et al., 1992; Andressen et al., 1993).

The distribution, morphology and synaptic connectivity of PV-irnmunoreactive neurons in area 46 of monkey prefrontal cortex have been studied by Williams et al. (1992). These authors described PV+ boutons as heavily innervating pyramidal neurons in layers 2 and 3 of area 46. PV+ boutons formed pericellular clusters and axon cartridges resembling those described in mPFC (this study). Williams et al. (1992) report that PV+ boutons established symmetrical (Gray type 2) synapses and that there was no mixture of PV+ or PV- boutons composing a single axon cartridge. A more recent study by Wouterlood et al. (1995) has investigated PV+ cells in the enthorinal cortex of the rat. Here again, PV+ neurons were most prevalent in layers 213, as were immunopositive processes and puncta. In the rat cortex, PV+ boutons were also found to construct pericellular baskets and axon cartridges in the superficial layers, similar to monkey prefrontal cortex.

The overall structural complexity of the PV+ labelled pericellular baskets and axon cartridges resembled those described in previous GolgiiEM investigations and immunocytochemical studies in the monkey cortex (Somogyi et al., 1982; DeFelipe et al., 1985; Lund and Lewis, 1993; see also Table 3). Although GolgiiEM studies have provided estimates for the convergence of basket and axoaxonic cells onto a single postsynaptic cortical pyramidal cell (Somogyi et al., 1982; DeFelipe et al., 1985), such data are not presently available for neurochemically distinct subtypes within these interneuron classes.

Of particular interest is that PV+ immunoreactive pericellular clusters and axon cartridges in mPFC were found either together or separately around unlabelled somata (Fig. 27), many of which were pyramidal in shape (Fig. 18;

see also Figs. 6 and 8a in Williams et al., 1992). Based on two related assumptions: (i) that all cortical pyramids in mPFC have pericellular baskets and axon cartridges, and (ii) that the observed PV+ punctate structures (baskets and cartridges) were derived exclusively from PV+ basket and PV+ chandelier neurons, respectively (Williams et al., 1992), then the results of this investigation indicate the presence of two subpopulations of basket and chandelier neurons in monkey mPFC (see Fig. 27). This does not necessarily mean that two of these populations of basket and chandelier cells were PV-immunonegative (Fig. 27), since the somata of such cells may be immunopositive but the level of PV in their axon terminals below the level of immunocytochemical demonstrability (Fig. 29).

The study of Lewis and Lund (1990) identified two neurochemical types of axon cartridge in the frontal and occipital cortices of the monkey; these axon cartridges were either PV+ or immunopositive for corticotrophin releasing factor (CRF). Indeed, the concept of distinct populations of axoaxonic cells and of basket neurons is further supported by Akil and Lewis (1992). These authors report that PV+ axon cartridges may target small pyramidal neurons in the superficial layers that project corticocortically, whereas PV+ pericellular clusters are located around large pyramidal neurons deep in the cortex that project subcortically. Thus, there may be neurochemically distinct populations of axoaxonic neurons and basket cells that specifically innervate pyramidal neurons associated with given output pathways from mPFC.

A variability in the termination pattern of axoaxonic cells in monkey cortex was described in the GolgiiEM study of DeFelipe et al. (1985). Pyramidal neurons in layers 213 and 516 of the sensory-motor cortices were found to have different numbers of axoaxonic synaptic contacts; on aver- age, layer 213 pyramids had 2-52 synaptic contacts on their axon initial segments whereas layer 5 pyramidal neurons had lower numbers (2-26). These values correspond with the data for presumptive PV+ axoaxonic puncta in monkey mPFC, which showed marked differences in the number of PV+ puncta associated with presumed pyramidal neurons in the superficial and deep layers of the cortex (see Results).

Taken together, the above evidence suggests that PV+ basket and chandelier local circuit neurons preferentially innervate pyramidal cells in layers 213 of monkey mPFC compared with pyramidal cells in layers 516. Given the tactical siting of such input, PV+ pericellular baskets and axon cartridges could differentially influence the activity and response characteristics of defined pyramidal cell subpopulations in the superficial and deep layers of mPFC.

Neurons in layer I A characteristic type of neuron in the superficial part of

layer 1 was labelled by both diaphorase histochemistry and by CR immunocytochemistry (see Figs. 6 and 12, 14). Although infrequent, this type of neuron was present in all the cortical areas examined and possessed typically ovoid somata with long horizontally aligned processes. In appearance and location, these cells resemble the Cajal-Retzius neurons seen in Golg impregnation studies (Marin-Padilla, 1984). Marin-Padilla ( 1990) also describes these neurons in Golgi preparations of human cortex (see also Meyer and Gonzalez Hernandez, 1993).

Similar to the present study, Conde et al. (1994) found cells in layer 1 of the monkey mPFC that were CR- immunopositive. Conde et al. (1994) also report that these

Figure 26

LOC.41, CIRCUIT SEURONS IN MONKEY mPFC: I ti01

CR+ cells colocalised CB immunoreactivity. Aside of this, Huntley and Jones (1990) describe CB, PV, and acetyl cholinesterase immunoreactivities as being useful markers for Cajal-Retzius neurons in developing monkey neocortex. Although this latter study did not investigate whether these layer 1 neurons also displayed CR+ immunoreactivity. the issue has been addressed by Vogt-Weisenhorn et al. (1994) in the developing cortex of the rat. These latter authors concluded tha t calretinin is a specific marker protein for Cajal-Retzius cells throughout the whole period of corticogenesis into adulthood. Hence several lines of evidence suggest layer 1 CR+ neurons found in this study are Cajal-Retzius cells. Indeed, Cajal-Retzius neurons may express specific combinations of all three calcium binding proteins a t different functional stages during cortical development. Furthermore, the morphologcal similarity of these cells with the diaphorase-reactive cells found superficially in laver 1 indicates that they probably also have the potential to synthesise nitric oxide (see below).

Labelled neurons and processes in the white matter

In addition to a small number of calbindin- and parvalbumin-immunoreactive neurons in the white matter, a numerically large population of diaphorase-reactive neurons was located below each of the cortical areas investigated. Lund and Lewis (1993) report the presence of somatostatin

Fig 26. 1,ight microscope evidence for thc interrelationships of calrvtinin i(:R], parvalbumin-t PVJ-, and calbindin (CUI-immunoreactive structurcs in areas 24a,b,c, 25 and 32. A: Area 24a, layer 3 . (:R-inimunort.activc axonal swellings tCR+; brown] in proximity with the soma of a PV-immunorcactive neuronal soma tPV+: g e y l . A process from the P V t neuron is indicated (double-headed arrow). B: Enlargement of the boxed area in A. One of the C R t axonal swellings is close1.v apposed with the soma of the PV-immunorcactive neuron i PV t I The PV labelled process arising from the cell body is indicated (arrow). C: Area 32. layer 4. Calretinin-immunctreactive axonal swellings i C R i 1 abutting the soma (arrows) of a PV immunoreactive neuron I P V C 1. D, E: Area 24c. upper layer 2. Corresponding colour and black white photomicrographs of calretinin immunoreactive puncta I C R + I in closc contact with the soma (single arrows) and the proximal dendritic shaft cdouble-headed arrow) of a PV immunolabelled neuron iPV+ I . F: 'I'wn calretinin~immunoreactive puncta larrowst lying close to a prcicess coming from a CR-immunolabelled neuron tCR+I. A PV-imniunolahelled neuron is indicated. G: Area 24b, layer 2. Calretinin- iminunortwtive puncta (arrow) in close proximity to a calrctinin- immunolahellcd cell. H: Low power photomicroLTaph of PV-immunoreactive. neurons and CR-immunolabelled axons. Lower layer 3iarea 25. J: Drawing of the boxed regon in H showing a CR-immunoreactive fibre with axon swellings ICR-) in close apposition with the soma ofa PV-imniunnreactivc iPV+ J cell. The CR+ axon climbs over the processes iarrowi and cell body of the PV+ neuron. K, L Corresponding colour and blackiwhite photomicrog-aphs of calretinin-immunoreactive puncta ICR+ 1 in close contact with the soma and the proximal dendritic shaft of the PV immunolabelled neuron (PV+l seen in H and d. (The two chromagens can be readily differentiated Icfcolour and B/W f i p r w I . ) M and Q: Layer 3, area 24c. Calretinin-immunoreactivc axonal swellings t C R + . arrows) engage with the processes of calbindin- immunoreactive neuron tCR+l. N: Lower layer 6, area 24h. A diaphorase-reactive fibre tD+ 1 gives rise to a fibre swelling (arrow D + 1 that is closely associated with thc soma of a calbindin immunolabelled cell. 0 and P: Diaphorase-reactive puncta tD+ I in close contact (white arrows) with thc somata of PV- and CR-immunoreactive cell bodies tPV+, CR+, respc~tivt.1~-i. R Calretinin-immunoreactive neuron ( C R + ) in layer 5 of area 24c. S: Enlargement of the boxed reb'lon in R showing calretinin- immunoreactive puncta ICR+, arrows) closely apposed to the soma of thecalretinin-immunolabelledcell. Calibration bars: H = 50 pm; A = 25 pm. hl. N. Q =: 20 pm; B-F, K, L, 0. P, R = 10 pm; G , S = 5 pm.

I -:i

i li

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( 281-12 )-immunoreactive neurons positioned horizontally in the white matter beneath area 46. The strongly beaded dendrites of these cells set them apart from the neurons encountered in this study. Interestingly, Shering and Lowen- stein (1994) have recently shown in young kittens and adult cats, tha t corticoefferent axons emergng from primary visual, somatosensory and suprasylvian cortices provide synaptic input to the shafts and dendritic spines of interstitial neurons located in the white matter beneath each area. The function of neuron populations in the white matter is currently unknown, but may relate to cortical development (Nieuwenhuys, 1994 ) .

Thick calibre PV-immunoreactive fibres were common within the white matter and probably represent cortical


A 1 2 3

ais 9 4,9 B

Fig. 27. A Diagram illustrating the three types of PV immunolabelling observed around the somata and axon initial segments (aisl of pyramidal neurons (PI in the monkey prefrontal cortex: type 1, PV+ puncta located around both somata and axon initial segments; type 2, PV+ puncta located around both soma and axon initial segments; and type 3 , PV+ puncta located around axon initial segments. Type 1 arrangements were most common in layers 5/6, while types 2 and 3 were frequently located in layers 2 and 3. (Note: The study of Williams et al. 119921 has shown that there is no mixture of PV+ and PV- boutons composing a single axon cartridge.) B: A schematic illustration showing how the observed pattern of PV+ puncta surrounding the somata of pyramidal neurons (PI and/or their axon initial segments (ais) could be derived from two neurochemically different populations o f basket cell (Ba) and chandelier (axoaxonic) cell (Chl. These cell populations would be either PV+ (Ba', Ch' I or PV- (Ba', Ch21).

afferents derived from PV+ projection neurons in subcortical nuclei (Jones and Hendry, 1989).

Diaphorase-reactive cells The somatic and dendritic morphologies of strongly

diaphorase-reactive neurons in the mPFC indicate that these cells are also derived from a morphologically diverse population of smooth and sparsely spiny cortical interneurons. This conclusion is substantiated by other histochemical studies investigating diaphorase-reactive neurons in the

Fig. 28. Diagram illustrating the characteristic structural relationships observed between some CR+ puncta and the processes and somata of CB+ and PV+ cells in monkey mPFC: (1) CR+ puncta are predominantly distributed over more distal processes (particularly at branch points; see Fig. 26M) but also over the soma o f CB+ neurons, and (21 CR+ puncta were more frequently associated with the somata of PV+ neurons but also contacted proximal processes. Whether separate populations of CR+ neurons were responsible for these characteristic structural arrangements is not known.

visual and auditory cortices of the monkey (Sandell, 1986; Cipolloni and Pandya, 1991) and in the visual and temporal cortices of humans (Liith et al., 1994; DeFelipe, 1993). However, absolute evidence for their status as local circuit neurons in the mPFC would come from details concerning their axonal morphologies and spectrum of postsynaptic target structures.

The studies of Valtschanoff et al. (1993; rat), Hashikawa et al. (1994; monkey) and Luth et al. (1994; human) have demonstrated that cortical neurons displaying NADPH diaphorase activity invariably contain immunoreactivity for NOS, the biosynthetic enzyme of nitric oxide (NO; see Vincent, 1995). Importantly, Valtschanoff and her col- leagues have further shown GABA immunoreactivity to be localised in NOS-containing, NADPH diaphorase-reactive neurons in rat neocortex.

In monkey mPFC, diaphorase-reactive neurons are not all aspiny (c.f., Vincent, 1995). The processes of these neurons display a gradient of spine density, ranging from being completely aspiny to bearing moderate numbers of dendritic spines (Fig. 9). Spine-bearing diaphorase-reactive neurons have been reported in the visual cortex of humans (Liith et al., 1994) and are also present in rat mPFC (Gabbott et al. 1995). It is therefore likely that, similar to their diverse dendritic morphologies, cortical diaphorase- reactive neurons also have a range of spine densities over their dendritic processes. Whether distinct subclasses of diaphorase-reactive cells can be defined quantitatively on the basis of spine density remains to be determined.

Similar to the observations of the present study, Liith et al. (1994) report somatic spicules (spines) arising from the somata of diaphorase-reactive neurons in human visual cortex. Aside of species and areal differences, the occur-


pia

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2

3

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6

Fig. 29. Summary diagram illustrating the structural relationships between neurons expressing CR, PV and CB immunoreactivities and pyramidal neurons situated in layers 213 and 516 of monkey mPFC. Specific m o r p h o l o g d types of neuron are indicated: M, Martinotti cell; Ng, neurogliaform cell; DB, double bouquet cell; CR, Cajal-Retzius cell; and Ba. basket neuron. Of special note are chandelier cells in the deep layers of the cortex (Ch) . Results of several studies indicate that the terminal axon cartridges from these neurons are PV-; however,

rence of these structures appears to be much higher in humans than monkeys; compare Figure 3B in Luth et al. ( 1994) with Figure 5E in this study.

Sandell ( 1986) reports two tiers of diaphorase-reactive neurons in the primary visual cortex (area 17) of the rhesus monkey, a finding also reported here for mPFC (see Fig. 7 in Gabbott and Bacon, 1996). Labelled cells in area 17 were most common in layers 213 and in layer 6iwhite matter. The significance of such stratified positioning of diaphorase- reactive neurons in monkey cortex is unclear but could relate to the termination pattern of thalamic afferents which occurs in a region between the two tiers of diaphorase- reactive somata.

Although the axon-like processes of a few diaphorase- reactive neurons could be traced for some distances from their cell bodies, these processes frequently disappeared, possibly due to decreased levels of enzyme activity. This is, however, in marked contrast to the strongly diaphorase- reactive varicose fibres seen throughout the cortex (Fig. 4Bi. The origin of these fibres (intrinsic or extrinsic) remains to be determined.

The finding that diaphorase-reactive punctate fibres encircled the somata of unlabelled cells in monkey mPFC (Fig.

whether the cell bodies of such neurons are also PV- is unknown (?; see also Fig. 27). The postsynaptic targets of some neurons in mPFC are unknown ( ? I . Note the presence of CR+ and CB+ double bouquet cells in monkey mPFC. Note also that CR+ double bouquet cell input to pyramidal cell apical dendrites is currently unknown For clarity, afferent and efferent pathways have been omitted. I Drawing modified from Williams et al.. 1992; Andressen ct al., 1993: Nieuwenhuys, 1994; and Conde et al.. 1994.)

4H) has also been observed in layers 2-6 of the temporal cortex of humans (see Fig. 2B,C in DeFelipe, 1993). In addition, basket-like formations of diaphorase-reactive fibres have been reported in layers 4-6 of the human visual cortex (Luth et al., 1994). However, although these structures were closely apposed around cellular profiles, their relative simplicity is in marked contrast to the pericellular baskets derived from true basket neurons seen in Golgi or immunocytochemical studies (Table 3). As discussed by DeFelipe i 1993), the diaphorase-reactive basket-like formations are probably produced by ascending serotonergic afferents to the cortex rather than being derived from intrinsic sources.

FUNCTIONAL CONSIDERATIONS CBPs, nitric oxide, calcium, and neuron

function in mPFC Colocalisation studies revealed that diaphorase-reactive

neurons in the cortex and white matter also contained GABA immunolabelling and immunoreactivities for CR, PV and CB (see Results). One common feature of these cell


classes is t,he predominant involvement of Ca2+ in their functional roles (Andressen et al., 1993).

Although the precise functions of CBPs are poorly understood, they are intimately involved in the intracellular sequestering, buffering and transport of Ca2+ ions (see reviews by Baimbridge et al., 1992; Andressen et al., 1993). Neurons expressing CBPs have high metabolic rates and slow adaption properties. Changes in Ca2+ can affect neuron function by altering the time course of the action potential, via increasing the probability of bursting activity (as a result of inhibiting calcium-dependent K+ conduc- tances) and by allowing Ca2+ entry to contribute to the overall depolarization of cell membranes (as a result of inhibiting Ca2+ -dependent inactivation of voltage-operated Ca2+ channels; Baimbridge et al., 1992).

Kawaguchi and Kubota (1993) have recently demonstrated that PV+ and CB+ neurons represent two distinct physiological subgroupings of nonpyramidal neurons in layer 5 of rat frontal cortex. PV+ and CB+ cells can be divided, respectively, into fast spiking (FS) and low threshold spiking (LTS) local circuit neurons. FS cells fire repeti- tively at depolarised potentials in response to synaptic excitation with virtually no spike frequency adaption, whereas LTS nonpyramidal cells produce low threshold spikes at hyperpolarised potentials. These physiological properties allow for rapid signal propagation from PV and CB interneurons to postsynaptic targets in the cortex. The findings of Kawaguchi and Kubota (1993) may also relate to other cortical layers and areas as well as to other species. Further, upon binding Ca2+ PV releases Mg2+ which is a powerful activator involved in a variety of other intracellular physiological activities that may also influence the signalling properties of PV+ interneurons (Celio, 1990). The physiological characteristics of CR+ neurons in the cortex are comparatively unknown, but may functionally define this type of cortical interneuron. Finally, the presence of CBPs in local circuit neurons may protect cells against the damaging effects of excessive Ca2+ influx during prolonged periods of high activity (Andressen et al., 1993).

The observations that diaphorase-reactive neurons and CBPs are colocalised in local circuit neurons may underlie complementary aspects of their intracortical function. Di- aphorase activity colocalises with the activity of NOS, the biosynthetic enzyme for nitric oxide (Bredt et al., 1991; Hope et al., 1991; Vincent, 1995). Intracellular calcium is essential for the synthesis of NO. Following elevations in the local concentration of Ca2++, NO is synthesised inter- nally and released from NOS-containing neurons. Such rises in intracellular Ca2+ can occur via N-methyl-D- aspartate (NMDA) gated channels or by the release of Ca2+ from calcium binding stores within the cell. Nitric oxide is a freely diffusible signal molecule that can exert its neurophar- macological actions without the direct involvement of either synaptic interactions between neurons or specific membranes receptors (Vincent, 1994, 1995). From diffu- sion and decay constants, NO could influence the activity of target structures up to 100 pm from the site of production (Vincent, 1995); this may explain the relative paucity of diaphorase-reactive cells in the cortex. Within neurons, NO specifically regulates the activity of soluble guanylyl cyclase which synthesises the second messenger cyclic guanosine monophosphate (cGMP). Increased levels of cGMP can exert a variety of short- or long-term neuromodulatory effects, for example, long-term potentiation (LTP). These effects may underlie other specific aspects of neuronal

function, such as during neural development, plasticity, and in learning and memory, not only in the medial prefrontal cortex but elsewhere in the cerebral cortex (Vogt and Gabriel, 1993).

In addition to its actions as a neuromodulator (see above), NO may also act as a potent vasodilator within the mPFC (Iadecola, 1993; Vincent, 1995). Recent studies have shown NO to be the endothelium-derived relaxing factor released from endothelial cells lining blood vessels that produces strong vasodilation (Iadecola, 1993). Neurons synthesising and releasing NO may therefore be operating at two interrelated levels, firstly by directly affecting the function of local and projection circuits in the cortex, and secondly, by altering the biodynamics of blood flow in response to local neural activity within defined regions of the mPFC (Iadecola, 1993; Gabbott and Bacon, 1993).

Excitation and disinhibition of local circuits in mPFC

Thalamic afferents to the visual cortex monosynaptically innervate both pyramidal and spiny neurons, as well as GABA-immunoreactive neurons (Freund et al., 1985,1989). These afferents excite their cortical targets (see review by Douglas and Martin, 1990). This indicates that local circuit GABAergic neurons are involved at the earliest stage of information processing in the visual cortex. Whether thalamic afferents (Ray and Price, 1993) and possibly other inputs to the monkey mPFC (e.g., from other cortical areas, amygdala, hippocampus, ventral tegmental area, and brain- stem structures; see Van Hoesen et al., 1993; Finch, 1993) also innervate local circuit neurons, thereby directly influencing the functioning of intracortical inhibitory mechanisms, remains to be determined. It can be envisaged that local inhibitory circuits within the cortex could be strate&- cally primed by direct feedforward excitation (or inhibition, see below) following activation of, for example, thalamocortical or hippocampal projection cells (Finch, 1993). Indeed, the finding that the majority of PV+ cells (basket and axoaxonic neurons) were found in the principal thalamocortical territories of mPFC supports this idea (Vogt et al., 1987). Indirect evidence comes from the observations that afferents to areas 24a, b, and c from other cortical areas (e.g., areas 25 and 23b of posterior cingulate cortex, and from regions of the frontal and temporal lobes) terminate predominantly in layers 2 and 3, where peak cell densities in both CR+ and CB+ neurons are located.

Of direct significance are the recent observations of Gigg et al. (1994). These authors show that glutamatergic projections from the hippocampal formation to the mPFC in the rat are directly influenced by GABAergic inhibition and that such inputs converge with glutamatergic projections from the mediodorsal thalamic nucleus onto individual cortical neurons. Gigg et al. (1994) speculate that the source of the GABA-mediated inhibition could be local circuit neurons in mPFC (see also Finch, 1993).

Pyramidal cells are the predominant targets of local circuit neurons in the cortex (Douglas and Martin, 1990). However, synaptic interconnections do occur between inhibitory cortical interneurons (Jones et al., 1994). Such connections would allow for direct disinhibition within intracortical networks (see Fig. 23D; Mize et al., 1992). Of particular interest is the structural relationship between CR+ immunoreactive puncta and the somata and processes of PV+ or CB+ immunoreactive neurons (Fig. 28). In addition, CR+ puncta were found to be closely associated with CR-


immunolabelled cell bodies, and PV+ puncta with PV+ somata and processes. These structural relationships could underlie disinhibitory mechanisms in mPFC (Douglas and Martin, 1992). One powerful form of disinhibition would be the CR+ innervation of PV+ basket cells and PV+ axoaxonic neurons in the superficial layers. Furthermore, the preferential location of CR+ puncta, more distally on CB+ cellular processes compared with their proximal location on the somata and initial dendritic processes of PV+ neurons, indicates selective innervation aimed to differentially affect the electrotonic properties of the target structures (Douglas and Martin, 1992). The question arises whether two functionally and morphologically distinct populations of CR+ neurons are involved (see Fig. 28). Conclusive evidence for this must, however, come from future electrophysiological and ultrastructural studies.

Local inhibitory circuits within the cortex are directly innervated by inhibitory afferents ascending from subcortical sources. Recent studies combining tract-tracing and immunocytochemistry have shown that different populations of inhibitory cortical interneurons (identified as containing either somatostatin, parvalbumin or calbindin immunoreactivities) are monosynaptically innervated by afferents from GABA+ neurons in the basal forebrain (Freund and Gulyas, 1991; Freund and Meskenaite, 1992). Such innervation, albeit very sparse compared with other sources of afferentation to local circuit neurons in the cortex (Vogt and Gabriel, 1993), could underlie defined functional aspects of information processing within local cortical networks (see Discussion in Wilson et al., 1994). The innervation of selected PV+ axoaxonic and PV+ basket cells by ascending afferents to mPFC could influence widespread populations of pyramidal neurons that are involved in specific corticocortical and subcortical pathways. Of related interest, is that dopamine immunoreactive boutons, presumably origmating from the dopamine- containing projection neurons in the ventral tegmental area, form symmetrical synaptic junctions with the somata, dendritic shafts and spines of identified pyramidal cells in the prefrontal, cingulate and motor cortices (Goldman- Rakic et al., 1989). Goldman-Rakic et al. (1989) also report dopamine-immunopositive terminals innervating the somata of GABA-containing cortical interneurons. Since inhibition is the most commonly reported effect of dopamine on the spontaneous activity of neurons in the prefrontal cortex (Vogt and Gabriel, 19931, such innervation could represent a further example of ascending feedforward disinhibition of local circuit neurons in mPFC. The functional significance of these and other nonspecific afferents to mPFC is poorly understood (Crino et al., 1993).

As mentioned above, inhibitory mechanisms in the cortex act in concert to control the output pathways provided by the pyramidal cell populations in layers 213 and 516. Figure 29 is a summary schematic diagram illustrating the structural interactions between defined classes of CR+, PV+ and CB+ local circuit neurons and pyramidal cell populations in the mPFC of the monkey.

Human mPFC and neuropsychiatric disorders Changes in the functional architecture of local circuits

within the human prefrontal cortex and in the cingulate cortices (posterior and anterior divisions) have been identified as underlying neuropsychiatric disorders such as Pick’s disease (Arai et al., 1991), Alzheimers disease (Braak and

Braak, 1993), and in schizophrenia (Vogt and Gabriel, 1993; Akbarian et al., 1995; Benes, 1995).

Several clinical investigations have clearly indicated that the pathophysiology of schizophrenia may involve marked alterations of intrinsic circuits within the anterior cingulate cortex (areas 24a, b, and c; Benes et al., 1993). Indeed, a statistically significant reduction was found in the absolute number of local circuit cells in layer 2 of area 24 (Benes et al., 1991). Whether a particular type of local circuit neuron was affected was not determined. Interestingly, afferents from the basal nuclei of the amygdala to the areas 24a, b and c of the cingulate cortex terminate specifically within deep layer 1 and layer 2 (Vogt and Pandya, 1987), the cortical zone with peak numbers of CR+ local circuit neurons. Given the central importance of inhibitory mechanisms in cortical function (Mize et al., 1992; Douglas and Martin, 19921, future studies should therefore investigate the extent to which specific types of cortical interneurons, embedded in functionally strategc local circuits in the mPFC, are differentially affected by psychiatric disorders and long-term alterations in emotional behaviour (Neafsey et a]., 1993; Benes, 1993a,b, 1995; and Braak and Braak, 1993).

CONCLUSIONS This study has documented the form and distribution of

neurons in the mPFC (areas 24a, b, c, 25 and 3 2 ) of the monkey that express the calcium binding proteins CR, PV, and CB. Also investigated were cortical neurons displaying diaphorase enzyme activity. This enzyme activity colocalises with nitric oxide synthase, the biosynthetic enzyme of nitric oxide. Morphological evidence from a variety of sources indicates that these cell populations are local circuit neurons subserving GABA-mediated inhibitory mechanisms in the monkey mPFC.

Moreover, the neurochemically identified neurons investigated in this study strongly resemble several classes of local circuit neurons seen in Golgi preparations of human prefrontal cortex (Mrzljak et al., 1988, 1990, 1992). Hence this study, in combination with previous reports (Hof and Nimchinsky, 1992; Lund and Lewis, 1993; Hof et al., 1993; Conde et al., 1994; Vogt et al., 1995) provide realistic structural models with which to assess alterations in the morphology of local circuit neurons in the prefrontal and cingulate cortices as a result of psychiatric and emotional disorders in humans.

The companion paper (Gabbott and Bacon, 1996) provides data that will allow such structural alterations to be assessed quantitatively.

ACKNOWLEDGMENTS The excellent technical assistance of Paul Jays, Tracy-

Ann Warner, Angela Robinson, and Leslie Annetts is gratefully acknowledged. This work was supported in part by the MRC (UK). The material used in this study was kindly provided by Dr. Kevan A.C. Martin (MRC Anatomi- cal Neuropharmacology Unit, Oxford) and Professor Alan Cowey (Dept. of Experimental Psychology, Oxford). Paul Gabbott is a Beit Memorial Research Fellow and Sarah Bacon is a Bristol-Meyers Squibb Research Fellow.


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Local circuit neurons in the medial prefrontal cortex (areas 24a,b,c, 25 and 32) in the monkey: I. Cell morphology and morphometrics