Distribution of NADPH-diaphorase-positive neurons in the prefrontal cortex of the Cebus monkey

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Distribution of NADPH-diaphorase-positive neurons in the prefrontal cortex of the Cebus monkey Roelf J. Cruz-Rizzolo a,⁎, José de Anchieta C. Horta-Júnior b , Jackson C. Bittencourt c , Edilson Ervolino a , José Américo de Oliveira a , Cláudio A. Casatti a a

Department of Basic Sciences, Araçatuba Campus, UNESP - São Paulo State University, São Paulo, Brazil Department of Anatomy, Institute of Biosciences, Botucatu Campus, UNESP - São Paulo State University, São Paulo, Brazil c Laboratory of Chemical Neuroanatomy, Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil b

A R T I C LE I N FO

AB S T R A C T

Article history:

We studied the distribution of NADPH-diaphorase (NADPH-d) activity in the prefrontal

Accepted 25 January 2006

cortex of normal adult Cebus apella monkeys using NADPH-d histochemical protocols. The

Available online 13 March 2006

following regions were studied: granular areas 46 and 12, dysgranular areas 9 and 13, and agranular areas 32 and Oap. NADPH-d-positive neurons were divided into two distinct types,

Keywords:

both non-pyramidal. Type I neurons had a large soma diameter (17.24 ± 1.73 μm) and were

Nitric oxide

densely stained. More than 90% of these neurons were located in the subcortical white

Frontal lobe

matter and infragranular layers. The remaining type I neurons were distributed in the

Cerebral cortex

supragranular layers. Type II neurons had a small, round or oval soma (9.83 ± 1.03 μm), and

Cebus apella

their staining pattern varied markedly. Type II neurons were distributed throughout the

New World monkeys

cortex, with their greatest numerical density being observed in layers II and III. In granular areas, the number of type II neurons was up to 20 times that of type I neurons, but this proportion was smaller in agranular areas. Areal density of type II neurons was maximum in the supragranular layers of granular areas and minimum in agranular areas. Statistical analysis revealed that these areal differences were significant when comparing some specific areas. In conclusion, our results indicate a predominance of NADPH-d-positive cells in supragranular layers of granular areas in the Cebus prefrontal cortex. These findings support previous observations on the role of type II neurons as a new cortical nitric oxide source in supragranular cortical layers in primates, and their potential contribution to cortical neuronal activation in advanced mammals. © 2006 Elsevier B.V. All rights reserved.

1.

Introduction

The primate prefrontal cortex (PfC) is a structurally and functionally heterogeneous region, which consists of a series of areas with different architectonic and connective characteristics (Figs. 1B and 2). In addition, this structural heterogeneity

has also been observed in its chemical anatomy, with neuroactive substances showing different laminar and areal distributions. This heterogeneity may explain the variety of behavioral alterations and the diversity and specificity of cognitive deficits observed in human and non-human primates after lesion or reversible suppression of restricted areas

⁎ Corresponding author. Department of Basic Sciences, FOA-UNESP, José Bonifácio 1193, Araçatuba, SP 16015-100, Brazil. Fax: +55 18 36363332. E-mail address: [email protected] (R.J. Cruz-Rizzolo). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.01.098

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of the PfC (Rosenkilde, 1979; Goldman-Rakic, 1987; Fuster, 1991; Tremblay and Schultz, 1999; Davidson et al., 2000; Rolls, 2000). Among the neuroactive substances described in the primate PfC, nitric oxide (NO) has received considerable attention due to its role in numerous aspects of the physiology of the central nervous system, including cerebrovascular activity, neurotransmission, neuronal death, and synaptic plasticity (Moncada et al., 1991; Snyder, 1992; Akbarian et al., 1993; Bredt and Snyder, 1994; Dawson and Snyder, 1994; Schuman and Madison, 1994; Vincent, 1995; Wallace et al., 1996; Hölscher, 1997; Luth et al., 2001, 2002). NO-producing neurons in the primate cerebral cortex have been studied by immunohistochemical detection of nitric oxide synthase (NOS), the enzyme responsible for NO synthesis from L-arginine, and by NADPH-diaphorase (NADPH-d) histochemistry. The latter approach is based on the fact that NOS is able to selectively catalyze a histochemically detectable NADPH-d reaction in aldehyde-fixed tissue (Vincent, 1995). Biochemical and immunohistochemical evidence supports a one-to-one correspondence between neurons expressing NOS immunoreactivity and NADPH-d (Hope et al., 1991; Dawson et al., 1991; Bredt et al., 1991; Aoki et al., 1993; Hashikawa et al., 1994; Luth et al., 1994; Estrada and DeFelipe, 1998). In addition, complete loss of NADPH-d staining in the nervous system has been demonstrated in transgenic mice lacking a functional neuronal NOS gene, providing additional support to the nearly complete co-localization of NOS and NADPH-d (Huang et al., 1993). However, despite this evidence, some authors were unable to demonstrate this correspondence under certain experimental conditions (Matsumoto et al., 1993; Egberongbe et al., 1994; Vincent, 1995). This observation and the fact that

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different NOS isoforms may express NADPH-d activity (Hilbig et al., 2001) indicate that a more careful interpretation of NADPH-d staining is needed (for a review, see Vincent, 1995). The study of the areal distribution of NADPH-d-positive neurons in the primate cerebral cortex has produced conflicting results. Some authors have concluded that there is an overall uniform areal distribution of NADPH-d-positive neurons in the primate cerebral cortex (Egberongbe et al., 1994; Hashikawa et al., 1994; Yan et al., 1996). However, in studies supported by quantitative data areal variations in the densities of these cell types have been reported (Luth et al., 1994; Barone and Kennedy, 2000; Garbossa et al., 2005). Specifically in the monkey PfC, NADPH-d-positive neurons were found to predominate in agranular (olfactory and limbic) areas, whereas isocortical granular areas showed the lowest density (Dombrowski and Barbas, 1996). There is also controversy regarding the morphological characteristics of cortical NADPH-d cells, as well as their laminar distribution. It has been reported that in the monkey's cerebral cortex NADPH-d-positive neurons can be divided into two distinct types (Gabbott and Bacon, 1996; Yan et al., 1996; Franca et al., 1997; Barone and Kennedy, 2000). Type I neurons have a relatively large soma, with an intense reaction product filling the soma and processes (Golgi-like reaction). These neurons are distributed throughout the cortex, but mainly in the subcortical white matter and infragranular cortical layers. Type II neurons are more numerous, smaller, with weak NADPH-d reactivity, and are distributed fundamentally in supragranular cortical layers. However, in a study carried out specifically in the monkey PfC, the existence of two different neuronal populations was not mentioned (Dombrowski and

Fig. 1 – A shows the widely cited cytoarchitectonic map of the Macaca prefrontal cortex proposed by Walker (1940). In B, surface view of the lateral, medial and orbital prefrontal cortex of Cebus apella, showing its anatomical organization. Gray rectangles represent the regions analyzed in this study. The numbers near the rectangles correspond to the areas according to Walker's parcellation, except for area 32 (from Vogt et al., 1987) and Oap. Abbreviations: ACgG, anterior cingulate gyrus; as, arcuate sulcus; cgs, cingulate sulcus; FOG, fronto-orbital gyrus; GRe, gyrus rectus; IFG, inferior frontal gyrus; iras, inferior ramus of arcuate sulcus; los, lateral orbital sulcus; LOrG, lateral orbital gyrus; MFG, medial frontal gyrus; MOrG, medial orbital gyrus; mos, medial orbital sulcus; Oap, periallocortical division of the agranular orbital cortex; PrG, precentral gyrus; prs, principal sulcus; ros, rostral sulcus; SFG, superior frontal gyrus; sras, superior ramus of arcuate sulcus.

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Fig. 2 – Brightfield photomicrographs of the prefrontal cortex areas analyzed in this study showing their cytoarchitectonic characteristics. In areas 46 and 12, note the large pyramidal neurons in layers III and V and the well-demarcated layer IV. In areas 9 and 13, a thin layer IV is still discernible. However, in areas 32 and Oap, layer IV is no longer visible. Scale bar, 1 mm.

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Barbas, 1996). Thus, it remains unclear whether there are significant variations in the areal distribution of NADPH-dpositive neurons across the primate cerebral cortex and in the PfC in particular, as well as whether different NADPH-d neuronal types exist in the primate PfC. In view of these discrepancies, the present study was undertaken to examine the laminar and areal distribution of NADPH-d-positive neurons in selected areas of the PfC of the capuchin monkey (Cebus apella) using NADPH-d histochemistry. This New World monkey was chosen for this study because of its similarity with the most intensively studied Macaca monkeys. In fact, the pattern of cortical fissuration is virtually identical in Cebus and Macaca, facilitating anatomical comparison. Some of the results reported here have been published in abstract form (Cruz-Rizzolo et al., 2003).

2.

Results

2.1.

Morphological features of NADPH-d-positive neurons

Two morphologically different types of NADPH-d-positive neurons were identified in the Cebus PfC, irrespective of the

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area studied. Type I neurons had a darkly stained soma and process with a Golgi-like appearance, and were of medium size (average diameter: 17.24 ± 1.73 μm, n = 390). The morphology of the type I neurons was fundamentally nonpyramidal, with a predominance of the stellate and bipolar forms which were especially abundant in the subcortical white matter (Fig. 3). Here, type I neurons showed tangential processes between the cortex and white matter, but some of them, as well as those located in the cerebral cortex itself, also exhibited perpendicular processes reaching the supragranular layers including layer I. A variable amount of dendritic spines could be observed in many of these neurons (Fig. 3F). A small number of type I neurons also showed a characteristic pyramidal shape, appearing to be inverted pyramidal-like neurons (Fig. 3A). Another much more numerous neuronal population was observed in the Cebus PfC. These neurons (type II; Fig. 4) had a round or oval soma and were smaller than type I neurons (average diameter: 9.83 ± 1.03 μm, n = 1100). Some of these type II neurons exhibited a very pale staining, while others had an intense coloration. In control sections incubated without NADPH or NBT, no specific diaphorase enzyme reactivity was present.

Fig. 3 – Photomicrographs of different cytological types of NADPH-d type I neurons shown in the center of the image. In A, an inverted pyramidal-like neuron; in B, C and E, stellate neurons; in D, a bipolar type I neuron. F, High power photomicrograph showing thick dendrites with abundant spines (arrows) of a type I neuron. Arrows in A to E, type II neurons. Scale bar in E is the same for all photomicrographs, except for F.

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Fig. 4 – In A and B, type II neurons. In C, a rare type I neuron in the supragranular layers. In C and D, note the clearly visible morphological differences between type I (arrows) and type II NADPH-d-positive neurons. Scale bar (in C) is the same for all photomicrographs.

2.2.

Laminar distribution of NADPH-d-positive neurons

We observed clear differences in the vertical distribution of the two neuronal populations. Type I neurons were located fundamentally in a band that included layer VI and the white matter immediately below the cerebral cortex (Figs. 5 and 6). A very small number, which did not exceed 4% of the total number of cortical type I neurons, was located in the supragranular compartment, and a slightly larger number was located in deeper regions of the white matter. Most type II neurons were found in the supragranular compartment, particularly in layer II and upper layer III. It was in this band that we observed the more densely stained type II neurons. The remaining type II neurons were distributed within the other cortical layers of the infragranular compartment. Although rare, type II neurons could still be observed in the white matter (Figs. 5 and 7, Table 1). The density of type II neurons in the supragranular compartment was up to 2.8 times greater than in the infragranular compartment (Table 1). This proportion was observed fundamentally in granular areas (12 and 46). In agranular areas (32 and Oap), the density of type II neurons in the supragranular compartment was similar to that observed in the infragranular compartment, fundamentally in the Oap area.

2.3.

Type I vs. type II neurons

When analyzing the total number of type I and II neurons in the different cortical areas, including all compartments, we observed that the population of type II neurons in granular and dysgranular areas was in some cases more than 20

times that of type I neurons in the same region (Table 2). In contrast, in the agranular areas this difference was markedly reduced.

2.4.

Areal distribution of NADPH-d-positive neurons

The neuronal density of type II neurons was found to vary depending on the areal architectonic characteristics (Figs. 8– 11). Agranular areas (32 and Oap) exhibited the lowest cellular densities of this neuronal type. These differences were more evident when we compared the supragranular compartments of the different areas due to the larger cellular density of type II neurons in layers II and III (Figs. 8 and 9). In all experimental cases, area Oap showed a decrease in neuronal density in relation to granular areas 12 and 46. Area 32 also exhibited a smaller neuronal density in relation to areas 12 and 46 but these differences were not always statistically significant. These differences became more evident when we grouped the areas according to their architectonic characteristics, with the granular (12 and 46), dysgranular (9 and 13), and agranular areas (32 and Oap; Figs. 9 and 11) being included in different groups. In this case, the group of agranular areas of all animals studied presented a significantly smaller density of type II neurons in the supragranular compartment as well as in the cortex as a whole (layers I–VI) compared to the group of granular areas. The dysgranular areas 9d and 13l usually presented an intermediate cellular density of type II neurons compared to granular and agranular areas. In some cases, these areas exhibited differences that were statistically significant compared to granular or agranular areas, but these differences were not consistent in all the animals studied.

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Fig. 5 – Distribution of NADPH-d-positive neurons in representative sections of areas selected in this study. Dotted lines indicate limits between the supra- and infragranular compartment, and between the cortex and white matter. Large black circles, type I neurons; small gray circles, type II neurons.

In relation to type I neurons, we were not able to determine statistical differences in their distribution across the areas studied.

3.

Discussion

3.1.

Methodological considerations

In the present study, the quantitative data were obtained by counting neuronal profiles and not neurons in semi-

serial sections from the PfC. This counting method does not ensure a correct calculation of the total number or real density of neurons per area since we are actually counting neuronal profiles (Coggeshall and Lekan, 1996). In addition, because the number of neuronal profiles (and neurons themselves) can vary depending on sex, disease, experimental conditions, fixative, etc., we also did not use our results to make comparisons among animals, although we used apparently healthy animals of the same sex and the same approximate age. Our purpose was to compare areas

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Fig. 6 – Laminar distribution of type I neurons. “n” corresponds to the total number of type I neurons counted in each animal. Observe that in several experimental cases no type I neurons were found in the supragranular compartment. Layer IV was included in the infragranular compartment.

inside the same animal and, within this context, our counting method seems appropriate. There are no reasons to suppose that, for example, experimental conditions could have modified the neuronal density in certain areas of the frontal lobe at the expense of others in the same brain. Because this might have happened in the case of edema or inadequate fixation, Nissl-stained sections were carefully examined and no signs of these alterations were observed. Our final results reveal that the regional distribution of NADPH-d-positive neurons was similar among cases, although the overall density, when comparing the same area in various animals, differed to some extent. The results shown in Figs. 8–11 refer to the population of type II neurons, the most abundant NADPH-d population in the primate cerebral cortex. The number of type I neurons was insufficient to determine any statistical tendency. However, it is important to point out that, due to the relatively small number of type I neurons, when including type I and type II neurons as a single population of NADPH-d-positive cells in the analysis, the final statistical results did not change the areal distribution (unpublished data).

3.2.

Types of NADPH-d-positive neurons

Based on the present findings we conclude that there are two different types of NADPH-d-positive neurons in the

Cebus PfC, and that these neurons show differences in their areal and laminar distribution. The existence of two NADPH-d-positive neuronal types in the monkey cerebral cortex has been reported by several authors (Sandell, 1986; Hashikawa et al., 1994; Luth et al., 1994; Gabbott and Bacon, 1996; Yan et al., 1996; Franca et al., 1997; Barone and Kennedy, 2000). These cells have also been found in the primate hippocampal formation (Mufson et al., 1990; Sobreviela and Mufson, 1995) and amygdala (Pitkanen and Amaral, 1991; Brady et al., 1992), indicating the widespread nature of these neurons in the primate telencephalon. However, in a previous study analyzing specifically the distribution of NADPH-d neurons in the rhesus PfC the existence of two neuronal populations was not shown (Dombrowski and Barbas, 1996). According to species-specific studies (Yan and Garey, 1997), the population of cortical type II neurons is especially abundant in primates and is not observed in rodents. Type II neurons may form a group of NADPH-d-positive neurons newly differentiated in higher mammals during evolution from a subpopulation of calbindin-GABAergic interneurons (Yan et al., 1996). Our results seem to confirm this possibility. Indeed, in Cebus, type II neurons are especially abundant in granular areas of the PfC, including area 46 and, according to comparative studies, these areas of the dorsolateral PfC have emerged more recently in the primate evolutionary history (Preuss and Goldman-Rakic, 1991).

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Fig. 7 – Laminar distribution of type II neurons. “n” corresponds to the total number of type II neurons counted in each area. Observe that the disproportion between the number of type II neurons in the supragranular and infragranular compartment decreases in agranular areas 32 and Oap. Layer IV was included in the infragranular compartment. Type II neurons in the white matter are not shown because of their very small number.

The different laminar distribution of type I and type II neurons observed here in Cebus confirms previous studies on Old World monkeys (Luth et al., 1994; Gabbott and Bacon, 1996; Yan et al., 1996; Barone and Kennedy, 2000), and may reflect different ontogenetic characteristics between the two neuronal populations (Rakic, 1988; Barone and Kennedy, 2000). An interesting aspect in our results is the small number of type I neurons in the Cebus supragranular compartment. In Macaca, some studies indicate that the proportion of type I neurons in the supragranular layers can reach up to 20% of all cortical type I neurons (Gabbott and Bacon, 1996; Yan et al., 1996; Barone and Kennedy, 2000). This number is not higher than 4% in Cebus. To some extent, this difference can be related to methodological variations. In our study, the supragranular compartment did not include layer IV, in contrast to other quantitative investigations (Barone and Kennedy, 2000). However, this fact does not seem to explain completely these differences, mainly because very few type I neurons were observed by us and others in layer IV. According to some studies (Yan and Garey, 1997), there exists a tendency in phylogeny toward a reduction in the number of type I neurons and an increase in the number of

type II neurons in the mammalian neocortex. For example, in rodents, no NADPH-d type II neurons are observed and the population of type I neurons occupies all cortical layers (Yan et al., 1994). In macaques, besides the existence of a numerous population of type II neurons, a drastic decrease of type I neurons is observed in supragranular layers. In Cebus, this decrease seems to be still more pronounced, probably indicating that this substitution process of type I with type II neurons might be in a more advanced stage in Cebus than in Macaca.

3.3.

Areal distribution of NADPH-d-positive neurons

We observed areal differences in the distribution of NADPH-d-positive neurons according to the areal laminar characteristics. Agranular areas of the Cebus PfC always exhibited lower cellular densities of type II neurons than observed in granular areas. In fact, the neuronal density of area Oap was statistically lower in seven of eight possible comparisons when compared, for example, with granular area 12 in the supragranular compartment and in the cerebral cortex as a whole (Figs. 8 and 10). These differences were more evident when areas 12 and 46, 9

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Table 1 – Cellular densities of type II neurons Case M1 Area Supragranular compartment Cortex b SG/IG c

b

46 (n = 2960) a

12 (n = 4153)

9 (n = 4932)

13 (n = 4364)

32 (n = 2141)

Oap (n = 1838)

181.30 (±29.29) 148.5 (+23.72) 1.8

199.87 (±49.51) 152.2 (±47.48) 1.76

155.91 (±28.47) 117.8 (±30.31) 1.8

118.91 (±3.90) 88.63 (±11.84) 1.7

83.04 (±11.41) 73.3 (±5.53) 1.25

92.59 (±5.90) 80.72 (±12.87) 1.58

46 (n = 1894)

12 (n = 1986)

9 (n = 1877)

13 (n = 2428)

32 (n = 976)

Oap (n = 1164)

82.03 (±13.23) 66.78 (±10.23) 1.56

67.1 (±9.12) 60.89 (±9.72) 1.2

51.05 (±11.76) 47.30 (±8.69) 1.03

13 (n = 2618)

32 (n = 1015)

Oap (n = 1010)

122.65 (±9.13) 94.99 (±6.12) 1.93

81.48 (±4.39) 61.38 (±4.49) 2

56.91 (±7.82) 53.79 (±5.31) 1.12

13 (n = 2383)

32 (n = 1156)

Oap (n = 1126)

122.63 (±9.76) 93.76 (±6.82) 2.06

86.04 (±6.33) 65.00 (±8.05) 1.86

68.22 (±19.12) 62.77 (±11.94) 1.2

Case M2 Area Supragranular compartment Cortex b SG/IG c

b

93.56 (±14.46) 83.51 (±12.9) 1.43

108.4 (±14.37) 81.16 (±6.14) 1.92

75.29 (±10.89) 60.20 (±5.46) 1.59

Case M3 Area

46 (n = 2212)

12 (n = 2009)

138.62 (±8.55) 95.88 (±3.25) 2.8

134.4 (±11.13) 90.6 (±11.82) 2.53

Area

46 (n = 2034)

12 (n = 2162)

Supragranular compartment b Cortex b SG/IG c

128.44 (±15.51) 92.11 (±15.66) 2.48

142.13 (±10.46) 98.19 (±8.85) 2.37

Supragranular compartment Cortex b SG/IG c

b

9 (n = 2453) 88.72 (±8.46) 70.00 (±8.90) 1.75

Case M4

a b c

90.22 (±13.41) 69.80 (±8.99) 1.85

Total number of type II neurons counted in the area in all compartments. Mean and standard deviation of the densities in the supragranular compartment and in the cortex as a whole (neurons/mm2). Proportion between the densities of type II neurons in the supragranular and infragranular compartment.

and 13, and 32 and Oap were grouped according to their architectonic characteristics into granular, dysgranular and agranular groups, respectively (Figs. 9 and 11). In this situation, the neuronal density of NADPH-d-positive neurons was significantly greater (P < 0.001) in the “granular” group than in the “agranular” one in all animals studied (these differences did not differ when type I and type II neurons were counted together, even when including noncortical type I neurons; see the methodological considerations above). These results disagree with those reported by Dombrowski and Barbas (1996), who observed that the cellular densities of NADPH-d-positive neurons (there is no distinction between neuronal types in their study) is always greater in the “limbic” agranular areas of the orbital and medial PfC of rhesus monkeys. These areas correspond to areas Oap and

Table 2 – Proportion between type II and type I neurons (type II/type I) a Case M1 M2 M3 M4 a

9 (n = 2531)

46

12

9

13

32

Oap

36.31 19.56 14.69 15.45

18.80 15.05 13.06 15.18

47.75 24.89 17.49 20.96

21.87 23.29 28.97 25.7

14.33 9.06 8.66 10.60

18.55 13.99 10.10 10.30

Calculated from the sum of all type II and type I neurons in each area and in all compartments, including white matter.

32 in our study. The authors conclude that, “… as the degree of laminar definition increases from agranular limbic to dysgranular limbic and then to eulaminate areas, the density of diaphorase-positive neurons decreased.” According to their results, allocortical (agranular) areas exhibit larger cellular densities of NADPH-d-positive neurons than isocortical areas (for example, PfC granular areas 46 and 12 are among those showing the lowest cellular densities of NADPH-d-positive neurons). The discrepancies between our results and those of Dombrowski and Barbas (1996) might be attributed to species-specific differences. However, the well-documented similarity between Cebus and Macaca brains with regard to their macroscopic, architectonic and connective characteristics and phylogenetic origins (Le Gross Clark, 1959; Chiarelli, 1980; Falk, 1980, 1982; Rosa et al., 2000) does not support the possibility of an independent emergence of a new and so numerous neuronal population in the supragranular compartment of the Cebus PfC. In addition, other studies analyzing different cortical areas of Old and New World monkeys, including some areas of the PfC (Gabbott and Bacon, 1996; Yan et al., 1996; Franca et al., 1997; Yan and Garey, 1997; Barone and Kennedy, 2000), are in agreement with our results and also indicate the existence of two NADPH-d-positive neuronal populations, with densities and laminar distributions similar to those observed in the Cebus PfC. Probably, the discrepancies between the results reported by Dombrowski and Barbas (1996) and the findings

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Fig. 8 – Histograms showing the densities (mean ± SEM) of NADPH-d-positive type II neuronal profiles in the supragranular compartment (layers I–III) in different prefrontal cortex areas. The Kruskal–Wallis statistical value (H) and the probability (P) are provided in each case. Dunn's post test was used for individual comparisons. M1, M2, M3, M4, experimental cases (animals). *P < 0.05; **P < 0.01; ***P < 0.001.

obtained in our study and other experiments reflect methodological differences. When describing the NADPHd-positive neurons, Dombrowski and Barbas (1996) pointed out the intense staining of cellular bodies and processes, and the fact that in most areas the relative distribution of NADPH-d-positive neurons in the cortex was higher in deep layers than in the upper layers. However, according to our observations, these are exclusive characteristics of type I neurons. The low neuronal density reported by these authors, lower than 13 neurons/mm2, seems to reinforce the possibility that, for some reason, the population of type II neurons was not completely included in their study. In this case, because type II neurons are especially abundant in granular areas (and supragranular layers), the final results regarding the vertical (laminar) and horizontal (areal) distribution might show numerical distortions and lead to erroneous interpretations.

3.4.

Functional implications

The relatively high density of type II neurons in the superficial layers of the cerebral cortex seems to be strategic in order to control the vascular irrigation of the cerebral cortex as a whole, fundamentally because cerebral blood vessels invade the cortex from the pia mater. It has been reported that greater availability of NO in superficial layers of the developing and mature cortex may adjust

the blood supply of specific cortical areas by controlling the diameter of the blood vessels through release of NO and concomitant vasodilatation (Adachi et al., 1992; Iadecola et al., 1993; Yan et al., 1994; Estrada and DeFelipe, 1998; Wiencken and Casagrande, 2000). Thus, NO, in addition to its direct action on neuronal physiology, may modulate local cortical activity by regulating blood flow. In addition, our observation of a larger cellular density of NADPH-d-positive neurons in granular areas may have other functional consequences. Previous studies indicate that, from a phylogenetic perspective, these granular PfC areas have emerged more recently. Preuss and GoldmanRakic (1991) concluded that Walker's area 46 does not exist in prosimians such as galago, having differentiated in later phases of primate evolution. This area 46 is associated with the execution of extremely complex cognitive tasks, fundamental in processes of temporary planning of behavior (Goldman-Rakic, 1987; Fuster, 1989, 1991). These tasks depend on an elaborated set of connections and complex intra-areal neuronal circuits. It is valid to assume that the emergence of a NADPH-d neuronal population able to supply the cortical microenvironment with NO, developing in parallel with the appearance of new cortical areas, has been an important step in the evolutionary process. The relevance of this possibility is reinforced because, according to some studies, NO seems to participate in neuronal

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Fig. 9 – Histograms showing the densities (mean ± SEM) of NADPH-d-positive type II neuronal profiles in the supragranular compartment (layers I–III), with the different prefrontal cortex areas being grouped according to their cytoarchitectonic characteristics. The Kruskal–Wallis statistical value (H) and the probability (P) are provided in each case. Dunn's post test was used for individual comparisons. G, granular group (areas 46 and 12); D, dysgranular group (areas 9 and 13); A, agranular group (areas 32 and Oap); M1, M2, M3, M4, experimental cases (animals). *P < 0.05; **P < 0.01; ***P < 0.001.

selection processes during periods of cortical formation and maturation through neurotoxic mechanisms (Yan et al., 1994; Kiss and Vizi, 2001) and this final shaping occurs mainly in layers VI and II/III during the first postnatal days. Probably, it is not a coincidence that these layers are the ones with the denser distribution of NADPH-d-positive cells in the primate isocortex. Thus, these new and complex cortical areas might have used this phylogenetically recent population of type II neurons to generate their definitive phenotypic pattern.

4.

Experimental procedures

For this study we used four young adult male Cebus apella obtained from the Primate Center of the “Júlio de Mesquita Filho” São Paulo State University, São Paulo, Brazil. Experimental procedures were conducted according to the Guidelines for the care and use of mammals in neuroscience and behavioral research (2003) and were approved by the local laboratory animal care and use committee. All efforts were made to reduce the number of animals and to minimize suffering. The animals were anesthetized with sodium pentobarbital (30 mg/kg, i.p.) and transcardially perfused with 0.9%

saline (800 ml) followed by 1500 ml of 4% paraformaldehyde in 0.1 M acetate buffer, pH 6.5, and subsequently by 1500 ml of 4% paraformaldehyde in 0.1 M borate buffer, pH 9.0. The brains were exposed and blocked with the aid of a stereotaxic apparatus. The blocks were then removed from the skull and placed in a cryoprotective solution containing 10% glycerol and 2% dimethyl sulfoxide in 0.1 M borate buffer, pH 9.0, at 4 °C. After 3 days, the blocks were transferred to a similar solution but with an increased concentration of glycerol (20%) for four additional days, according to the method described by Rosene et al. (1986). The blocks were then frozen, sectioned at 40 μm on the coronal plane, and collected in a solution of 0.1 M phosphate buffer, pH 7.3. NADPH-d histochemistry was performed according to the protocol of Weinberg et al. (1995), with minor modifications. Briefly, free-floating sections were rinsed in phosphate buffer and incubated at 37 °C for 140 to 180 min in Tris buffer, pH 8.0, containing 0.08% β-NADPH (Sigma 1630), 0.06% NBT (Sigma 6876) and 0.1% Triton-X under constant shaking. Adjacent sections were counterstained with thionin (Nissl stain) and used as cytoarchitectonic references. All tissue sections from each animal were reacted at the same time, and all animals were submitted to the same histological treatment. In control sections for histochemistry, NADPH or the electron acceptor

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Fig. 10 – Histograms showing the densities (mean ± SEM) of NADPH-d-positive type II neuronal profiles in the cortex (layers I–VI) in different prefrontal cortex areas. The Kruskal–Wallis statistical value (H) and the probability (P) are provided in each case. Dunn's post test was used for individual comparisons. M1, M2, M3, M4, experimental cases (animals). *P < 0.05; **P < 0.01; ***P < 0.001.

(NBT) was omitted but all other steps followed the same protocol.

4.1.

Identification and selection of cortical areas

In order to correlate the density of NADPH-d-positive neurons with the architectonic characteristics of some PfC areas, we selected six different areas: two granular, two dysgranular, and two agranular areas. In the context of this study, we use the term granular cortex to designate five or six layered regions where layers II and IV are both granular and clearly demarcated from adjacent laminae. The agranular cortex is characterized by the lack of a discernible layer IV and the remaining layers show a simpler organization, limited to three to four cellular strata. The dysgranular cortex represents a transition between the granular and agranular cortex. In this type of cortex layer IV is rudimentary, showing no full laminar demarcation. The areas selected in this study are shown in Figs. 1B and 2. Due to their topographical and architectonic similarity between Cebus and Macaca brains, we denominated these areas according to the parcellation proposed by Walker (1940) for the macaque monkey (Fig. 1A), with some modifications. We adopted this terminology not to establish homologies but only to permit a rapid topographic representation in view of the

widespread acceptance of the division proposed by Walker (1940) for the primate PfC. As examples of granular areas, we selected the ones located in the inferior bank of the principal sulcus (area 46 in Walker's parcellation), and in the inferior frontal gyrus (area 12), both in the dorsolateral surface of the PfC. As examples of dysgranular areas, we selected the ones in the caudal third of the superior frontal gyrus (area 9, in the dorsolateral PfC), and the central region of the orbitofrontal cortex in the lateral orbital gyrus (area 13). Finally, the cortex of the anterior cingulate gyrus in the medial PfC (area 32 according to the nomenclature of Vogt et al. (1987)) and the periallocortical division of the paraolfactory cortex (Oap), in the caudal orbitofrontal cortex, were selected as examples of agranular areas.

4.2.

Cell counting and statistical analysis

The quantification methods are shown in Fig. 12. Briefly, cells were counted in five sections for each cortical area, with a distance of 300 μm between each section in the same area. The laminar distribution of NADPH-d-positive neurons was determined by comparison of adjacent thionin-stained sections. Columnar sectors (1500–2000 μm wide), orthogonal to the pial surface (Fig. 12A), corresponding to the center of each area selected in this study, were drawn in each

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Fig. 11 – Histograms showing the densities (mean ± SEM) of NADPH-d-positive type II neuronal profiles in the cortex (layers I–VI), with the different prefrontal cortex areas being grouped according to their cytoarchitectonic characteristics. The Kruskal–Wallis statistical value (H) and the probability (P) are provided in each case. Dunn's post test was used for individual comparisons. G, granular areas 46 and 12; D, dysgranular areas 9 and 13; A, agranular areas 32 and Oap; M1, M2, M3, M4, experimental cases (animals). *P < 0.05; **P < 0.01; ***P < 0.001.

section. These “rectangles” included the cerebral cortex and subcortical white matter. Using adjacent Nissl-stained sections, the cerebral cortex of the histochemical preparations was divided into two compartments. The superficial compartment included layers I, II and III, and the deep one layers IV, V and VI in granular and dysgranular areas, and layers V and VI in agranular areas. The extension of white matter included in each sector corresponded to approximately the extension of the deep compartment (Fig. 12F). Sections were examined by brightfield microscopy (Fig. 12B). Images of the previously described columnar sectors were acquired using a digital camera coupled to the microscope (Fig. 12C). In the computer, photomontages of tissue sections were composed using drawing software (Figs. 12C and D). Profiles of NADPH-d-positive neurons were labeled with different symbols directly on the screen of the computer after careful examination of the corresponding section under the microscope using a 40× objective (Fig. 12E). Final images showing the contour of the columnar sectors, the limits between the different compartments and the labeled neuronal profiles (Fig. 12F) were used to determine the density of NADPH-d neurons (number of profiles/mm2) in the total cortex (superficial + deep com-

partment), superficial compartment, deep compartment and white matter, using morphometric software (Image ProPlus®, Media Cybernetics). The densities of NADPH-d-positive neuronal profiles in each area were compared by the nonparametric Kruskal– Wallis test. Dunn's post hoc test was used for individual comparisons among areas. In two monkeys, the diameter (average diameter) of approximately 1500 NADPH-d-positive neuronal profiles in the different PfC cortical areas analyzed in this study was measured using appropriate software (Image Pro-Plus®, Media Cybernetics). Because of the intense labeling pattern of most NADPH-d-positive neurons, fundamentally type I neurons, we could not specifically count only the neuronal profiles in which the nucleus or nucleolus was visible, but instead whole cell body counts (excluding dendrites) were obtained. Labeled cellular fragments, NADPH-d-positive neurons cut only through the level of a dendritic tree, or neurons with dubious labeling were not included. The counting methods and sampling strategies used in this study allowed us to count neuronal profiles and not the neurons themselves. Thus, our results cannot be used to establish the absolute number of NADPH-d-positive neurons, which could only be obtained by complete serial

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Fig. 12 – Schematic representation of the counting methods and sampling strategies used in this study. For details, see text.

reconstruction which, in the context of this work, would be impracticable. However, to facilitate reading, in the text we will use the terms neuronal profiles and neurons interchangeably.

Acknowledgments This work was partly supported by grants from FAPESP (9910236-2) and FUNDUNESP (557-01-2005). The authors are grateful to José Ari Gualberto Junqueira and Arnaldo César dos Santos for technical assistance.

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