The gyral surface of a Nissl-stained rhesus monkey case was evaluated to determine if the characteristics of transition from area 24 to area 23 observed in human studies are applicable to the monkey. Thirty of the 240 sections through the left cingulate gyrus were photographed at the “b” subdivisions to assess cytoarchitectural transition along the full length of the gyrus. An equally spaced sample series of 10 sections are shown in where magnifications were selected to emphasize mid-cortical layers, where the transition from agranular to granular cortex occurs, to identify the location of the first layer IV neurons. The star at section #159 in refers to the original border between areas 24 and 23 (Vogt et al., 1987
) and this is 3 mm caudal to the VCA. The first evidence of a layer IV is in section #191 (arrow to layer IVd). At this level there is a bridge of neurons between layers IIIc and Va and this lack of a complete layer IV is characteristic of a dysgranular layer IV. Since the photographs are aligned at the superficial border of layer Va, it can be seen in section #159 that layer Va has a neuron dense and granular appearance. This feature is characteristic of pMCC and may have contributed over the past century to mis-localization of the border between areas 24 and 23 when viewed with Nissl stains. No such neuron density occurs in layer Va of areas 24b or a24b′. In addition, claims that area 24 is dysgranular seem to be incorrect as can be seen in the high magnification photographs of ; layer IIIc and Va directly abut in areas p24b′, a24b′, and 24b and there is no dysgranular layer.
Thus, the monkey has a MCC with areas a24′ and p24′ as in human and there is a complex transition between areas 24 and 23; i.e., there is not a simple and progressive increase in the number of layer IV neurons. Rather, there is an increase in small-neuron density of layer Va of p24′ when compared to a24′, a dysgranular layer IV in area 23d, and a granular layer IV in area 23 in conjunction with a dense layer Va.
Cynomolgus Monkey: Surface Features & Flat Map
The medial surface is oriented parallel to the anterior commissure/posterior commissure plane in and the VCA drawn perpendicular thereto. The plane of coronal sections for this case is shown with arrowheads at the face of each of the three blocks. The corpus callosum was drawn at its dorsal border, placed above the medial surface photograph, and flattening produced in one dimension by extension from the corpus callosum. The retrosplenial areas were drawn by extension below the dorsal part of the corpus callosum. The splenial sulcus (spls) in this case is not directly attached to the cingulate sulcus (cgs), although there is a vascular indentation, and RSC (shaded) is placed ventral to the dorsal border of the callosum so that the overlying area 23 of the caudomedial lobule can be represented. The knee of the cgs at the inflection of the mr is noted with an arrow in because this is an important point of area confluence; just ventral to the knee area 23d on the gyral surface and area 23c in the cgs disappear. Finally, the caudomedial lobule forms the terminal part of the posterior cingulate gyrus and lies just dorsal to the ac-pc line.
Flat maps were generated like that shown in with reference to the genu of the corpus callosum, VCA, knee of the cgs, and caudal edge of the splenium of the corpus callosum. To verify that the brains were a similar size, the corpora callosi were measured from the rostral tip of the genu to the caudal tip of the splenium. This distance was 2.55±0.217 cm and there was no correlation between body weight and the length of the corpus callosum. Thus, coordinates in the “y” and “z” planes for any area in this case are similar for all adult cynomolgus monkeys with less than a 10% measurement error.
Although the basic layout of cynomolgus cingulate cortex is similar to that reported for rhesus monkey, there have been a number of modifications based on rhesus monkey (Vogt, 1993
; Vogt et al., 1997
) and human observations (Vogt et al., 2003
) and these are reported herein. First, MCC is identified with a prime following area 24 to refer to its posterior placement and agranular form and it is comprised of 7 unique cytoarchitectures and three fundal extensions. Second, transition to the posterior cingulate region occurs via a dysgranular area 23d between areas 23 and p24′. Third, PCC is comprised of dorsal and ventral divisions.
Anterior cingulate cortex
The ACC includes areas 32 and 24a rostral to the genu, area 24b dorsal to and 25 ventral to the genu, and area 24c with its fundal extension (f 24c) in the cgs. shows the most rostral part of ACC including areas 24 and 32; ventral area 9 (9v) is described below because it is more fully elaborated dorsal to the genu. Area 32 is a dysgranular cortex with a thin layer IV. Only layer Va neurons express NFPs, with very few such cells in layers IIIc and Vb (). In contrast, area 24c has a broad layer of NFP-expressing neurons in layer V, but activity in layer IIIc is still weak. Generally, the large neurons in both divisions of layer V are larger in area 24c than in area 32 (, NeuN at high magnification). Layer VI in area 24c is also much more dense and differentiated than it is in area 32.
Figure 3 Location and composition of areas 32 and 24c in the rostral cgs. Higher magnification shows the dysgranular layer IV in area 32 with NeuN (N), the lack of one in area 24c, and the much higher level of SMI32 immunoreactivity (S) in area 24c than area 32. (more ...)
Subgenual cingulate cortex is comprised of areas 24a, 25, and a frontal cortex extension termed area 10m (Carmichael and Price, 1994
) as shown in . Area 24a surrounds the genu and has a broad and undifferentiated layer V with substantial expression of NFPs. It also has a dense layer VI (NeuN) with many parvalbumin-ir neurons and dendrites (Parv). In contrast, area 25 has much weaker NFP expression in layer V, poorly differentiated layers V and VI, and fewer parvalbumin-expressing neurons in layer VI. Finally, area 10m has a dense layer V, relatively few NFP and parvalbumin expressing neurons and axonal plexuses, and a dysgranular layer IV. Although there are some parvalbumin-ir neurons in layer IV, no appreciable expression of NFPs occurs.
Figure 4 The ventral and rostral areas are differentiated with NeuN, SMI32, and parvalbumin (Parv). A Parv plexus is particularly notable in layer VI of area 24a and differentiation of areas 25 and 10m is clear in all preparations. The composition of area 24a (more ...)
Cortex in sulcal ACC is shown in . Pivotal to identifying individual layers in each cingulate area is using designations that are consistent with those for neocortex. Since cortical layers are differentially distorted in the fundus with deep layers being proportionately more stretched and superficial layers compressed, a level adjacent to area 4 was selected so that Betz neurons in layer Vb could be identified in SMI32 of a medioventral division of area 4 (4mv; ). The Betz neuron closest to fundal area 24d (f 24d) is circled and the layers identified in the fundus and on the ventral bank of the cgs in area 24d. The most dense plexus and somatic impregnation is layer Vb, while the neurofilament-sparse layer is layer Va.
Figure 5 Anterior cingulate sulcal cortex is comprised of area 24c, its fundal extension (f24c) and area 9 in the dorsal bank. Although there is a bilaminar pattern in parvalbumin expression in areas 9v, f 24c, and 24c, the level of staining in layer VI is much (more ...)
Area 24c has the thickest and least differentiated NFP-expressing deep pyramidal layers V and VI of any part of the cingulate gyrus. There are also very few layer IIIc neurons expressing NFPs, while f 24c does have an enhancement of them in layer IIIc. Layer Va is present (; NeuN), however it overlaps to some extent with the NFP-plexus and the differentiation between layers Va and Vb is not pronounced. Parvalbumin expression shows two prominent layers of neuron labeling; one in layer Va and another in layer VI; the latter of which is more dense.
Cortex on the dorsal bank of the cgs is not “cingulate” in structure, although it has transitional features. The ventral area 9 is labeled 9v in and it has a slender layer Va, although it is less prominent than in area 24c. At higher magnification the larger layer Va neurons are apparent in area 9v. The cingulate “signature” of a very neuron-dense layer Va, that is disproportionate in relation to the less neuron-dense layer IIIc, is not prominent in area 9v. Importantly, area 9v has very large layer IIIc pyramids (, NeuN and SMI32; high magnification photographs) which is not true for area 24c. Parvalbumin also distinguishes between these areas because the layer VI expression in area 24c is very weak in area 9v, while the number of labeled neuron and axons in superficial layers is much greater than in area 24c. Thus, although area 9v has transitional features characteristic for both areas 24c and 9, it appears to share more features with area 9, in particular, the fully developed and NFP+ layer IIIc pyramidal neurons and very large pyramids in both divisions of layer V.
Cortex on the dorsal and ventral banks of the cingulate sulcus
Most of the cingulate gyrus abuts frontal cortex, except for part of the PCC. In addition to defining the specific organization of each cingulate area, an important question is to what extent cortex on the dorsal bank of the sulcus is “cingulate” or “frontal” as partially considered above. This question is best resolved with low magnification macrophotographs of the entire sulcus as in and higher magnifications to detail particular neuron structures and protein expression patterns. shows three rostrocaudal levels of the cingulate sulcus and shows that the dorsal bank is quite different in architecture than the ventral bank. The fundal part of area a24c′ (f a24c′) abuts a ventral part of area 6aβ (v6aβ), the fundal division of area p24c′ (f p24c′) abuts the ventral division of area 6aa (v6aa), while a medioventral division of area 4 (4mv) abuts area 24d. Area 4mv is selected in to demonstrate how significantly different the dorsal and ventral banks are at a higher magnification, although the low magnification at all levels is also quite compelling. Area 4mv is also useful in determining the location of layer Vb in SMI32 preparations (). Some differences between areas 4mv and 24d include the formers extensive numbers of NFP-expressing neurons in layer III and much larger Betz neurons in layer Vb (arrows in ). Differences in superficial layer SMI32-ir are obvious at all levels of magnification. Area 24d has a very dense layer Va with both large and small neurons as is characteristic of most cingulate areas and this is not true of area 4mv.
Figure 6 Architecture of three levels of midcingulate cortex including the dorsal bank of the cingulate sulcus. SMI32 shows that dorsal bank cortex does not share important similarities with cingulate cortex; the most profound differences being the very large (more ...)
shows a progressive, rostral-to-caudal build-up of small neurons in layer Va on the gyral surface of the rhesus monkey case and a similar trend occurs on the dorsal bank of the cingulate gyrus in the cynomolgus monkey. shows that neurons in layer Va of area a24c′ are larger and less dense than in area p24c′. Area 24d has a very neuron dense layer Va with many small neurons intermingled with the larger ones (). Photographs at the asterisks were enlarged and rephotographed for to show the size and density of neurons in all sulcal areas including area 24c. It appears that the overall largest neurons are in layer Va of area a24c′ when viewing NeuN preparations. Although this is also true for Nissl-stained tissue, NFP expression by the largest cingulate neurons shows that these latter are an infrequent part of cingulate architecture and are largest in the fundus adjacent to area 24d.
Figure 7 High magnifications of NeuN in cingulate sulcal areas to assess the composition of layer Va. The asterisks are positioned the same way as in . Neurons in this layer tend to be larger rostrally. At caudal levels the neurons can be somewhat smaller (more ...)
Cingulate sulcus fundal divisions
Curvature of cingulate cortex around the fundus of the cgs greatly attenuates architecture in this region. To the extent that these areas participate in skeletomotor regulation, these particular architectures can be considered the primary substrate for motor control as they are “stripped” of all but the largest neurons. The dorsal midcingulate gyral areas and their fundal extensions are shown in . In all instances, the largest neurons labeled with SMI32 are larger in the fundal extension. It appears that the largest neurons on the cingulate gyrus are in layer Vb of areas f p24c′ and 24d. Interestingly, the interneurons labeled with parvalbumin are also largest in layer Va of these same areas. These size differences are not as noticeable when viewing the total population of neurons with NeuN. Finally, the proportionate size differences also hold for layer IIIc neurons which are relatively larger in fundal areas. Thus, the fundal cytoarchitectures are not simply the product of laminar and neuronal stretching; they are also a consequence of pyramidal neuron enlargement and this may be necessary for these distorted areas to maintain their functional contribution to skeletomotor function.
Figure 8 The fundal extension of each cingulate gyrus area contains neurons that are generally larger both in SMI32 which emphasizes large, pyramidal-projection neurons and in parvalbumin which labels interneurons. “S” is SMI32 and “N” (more ...)
Differentiation of cingulate gyral surface
As a general rule, the ventral “a” divisions are relatively homogeneous when compared to their dorsal “b” counterparts. Since this homogeneity in the former is due to a less well differentiated layer Va, the “b” divisions also have a thicker and more dense layer Va and many more NFP-expressing neurons in this layer than is the case for the “a” divisions. provides an intermediate level of magnification of “a:b” pairs of areas at all levels of the cingulate gyrus. The increase in layer Va neuron density and SMI32-ir is present at the transition from area 24a to 24b and a24a′ to a24b′. This difference is not as pronounced in posterior cortex, however, differences in layer IIIc become more pronounced at caudal levels (i.e., areas d23a and d23b).
Figure 9 Architecture and cytology of cortex along the surface of the cingulate gyrus. Comparisons of the “a” and “b” divisions are enhanced with the vertical arrows to the border of layers IIIc and Va. Dysgranular area 23d has (more ...)
The level 4 in shows there is no a/b distinction at the dysgranular area 23d transition. A segment of this photograph was magnified X2 to show the dysgranular cortex with an intermingling of large pyramidal neurons in layers IIIc and Va. Also notice that SMI32-ir neurons are quite similar in all layers along the entire section photographed. Although the densities of NFP-expressing neurons in layer Va of both divisions of area d23 are similar, those in layer IIIc are much higher in the “b” than the “a” division of this area. Another important feature of cingulate architecture is discernable in where area 23 is not uniform in its dorsal and ventral parts. Area v23b has many more NFP-ir neurons in layers IIIc and Va and they are substantially larger than is the case for area d23b as discussed in more detail in the next section.
Parvalbumin is a sensitive marker of each anterior and midcingulate area as shown in . The following observations are notable: a) Homogeneity of architecture in the dorsal part of area 24a is reflected in parvalbumin-ir, layer VI has a prominent plexus with diffuse labeling in layer III of area 24b and these plexi are very prominent, particularly in layer Va in area 24c. b) Midcingulate areas a24′ and p24′ have very high layer Va activity but that in p24′ is more diffuse. c) Parvalbumin does not differentiate the posterior cingulate areas particularly well except for the retrosplenial areas as noted below. In this regard, areas 23d and both divisions of area 23 have two plexi in layers III and V that do not vary substantially in this region.
Posterior cingulate and retrosplenial cortices
Although the posterior cingulate gyrus has been considered above, the composition of areas 23c, f 23c, 31, and 7m have not been addressed nor have the dorsal and ventral divisions of PCC been considered in detail. Area 23c on the dorsal bank of the cingulate gyrus (; #1) has a broad external granular layer in relation to the deeper layers, dense layers II and IV, extensive NFP-expressing layer IIIc pyramids, and a poorly differentiated layer V; all of which distinguish it from area 24d rostrally. The fundal division of area 23c is similar to its dorsal bank counterpart, although the lamination is greatly attenuated.
Areas 23a/b are not uniform in the dorsoventral plane or “z” axis. The dorsal areas d23a/b are relatively thinner than their ventral counterparts and have a slightly more dense layer Va. As shown in (NeuN), area v23b has much thicker layers IIIc, IV and V and it has many more NFP-ir neurons (SMI32) in layers IIIc and Va and they are substantially larger than is the case for area d23b. The NeuN and SMI32 images were merged by reducing the opacity of the SMI32 image and magnifying both by X1.5 to show the exact position of the SMI32-ir neurons in both areas. Although all pairs of NeuN and SMI32 images were so co-registered to assure exact co-registration of images, the present ones are provided for these areas to show the accurate co-registration in the presence of an intervening layer IV. This method emphasizes that the density of NFP-ir neurons is very substantially higher in layer IIIc of v23b and layers IIIc-V are thicker than is the case for areas d23a or d23b.
Area 31 surrounds the spls and has the most extensive layers II, IIIab, and IV of any cingulate area. Comparison of areas 31 and 7m in shows much greater NFP-ir in layer IIIc of area 7m, a dense layer IV, and a thinner layer Vb. Higher magnification of layers IIIc-Va shows that neurons throughout mid-cortical layers of area 7m appear to be stacked. The example of one such formation at the arrow was 37 neurons in length from layer IV to its apex in layer IIIc and its total length was 375 μm. Of course, neurons form similar aggregates in all cingulate areas, however, those in area 7m are much longer and contribute in a meaningful way to the low-magnification cytoarchitecture.
Architecture of the retrosplenial areas 29 and 30 has been thoroughly documented in both human and monkey, however, a few comments in the present context including parvalbumin expression need consideration. The highest level of NFP expression in retrosplenial cortex is in layer VI and to a lesser extent in layer V of area 29l as shown in . The granular neurons of layer III/IV in both areas 29l and 29m do not express these proteins. Since the laminar position of SMI32-ir can be difficult to assess, the images were merged with NeuN as shown in to show the exact position of labeled neurons. The only NFP-ir in superficial layers of area 29 is associated with labeled dendrites from deep lying pyramidal neurons. Area 29l has extremely rich parvalbumin expression making this marker ideal for locating the border between 29l and 29m. Indeed, the labeling of somata and processes is so dense that laminar differentiation is not possible in the former. Finally, area 30 is dysgranular as shown in human (Vogt et al., 2001). The variability of layer IV, interdigitation of layer IIIc neurons with those in layer Va, and heavy expression of NFPs by large layer IIIc pyramids is the same as in human. Interestingly, parvalbumin expression is almost entirely located in layers II-III with a predominance in layer II. This pattern of labeling is not present in any anterior or midcingulate area.
Figure 12 Retrosplenial areas are well defined with NeuN, SMI32 and parvalbumin. Indeed, the massive intrinsic parvalbumin system is very robust in area 29l. Neurons in the granular layer (i.e., layer III/IV) do not express NFP and this is documented by merging (more ...)
Horizontal Plane of Section
Horizontal sections show similar architectures at high magnifications, however, borders between areas on the gyral surface can be identified in macrophotographs of single sections and confirm observations of different architectures in separate coronal sections. shows a macrophotograph through the dorsal part of the cingulate gyrus with progressive differentiation from rostral area 24b to caudal area 31. Layer Va in the NeuN sample (A.) is emphasized with arrows to show the progression from thin and relatively sparse in area 24b (1) to a thicker and more neuron dense composition particularly in areas p24b′, 23d, and d23b (3, 4). Progressive changes in the expression of neurofilament proteins are also shown (B.) where arrows are used to emphasize changes in layer IIIc. Before arrow #5 the SMI32 immunoreactive neurons are sparse in layer IIIc and tend to be solitary. The border of areas p24b′ and 23d is heralded by heavy expression of NFP by layer IIIc pyramidal neurons starting at #5. The sixth arrow emphasizes the intermingling of NFP-expressing neurons in layer IIIc with those in layer Va in dysgranular area 23d. Area d23b has a more dense expression of neurons in both layers IIIc and Va, while those in both layers of area 31 are among the highest in the cingulate gyrus. Each of these laminar observations can be verified above with high magnification and in the coronal plane of section.
Figure 13 The dorsal part of the cingulate gyrus shows progressive differentiation from area 24b to area 31. The asterisk on the medial surface orients to the same dimple in each section. Layer Va in NeuN (A.) is emphasized with arrows to show progression from (more ...)