Ly (Ghashghaei Barbas, 2001; Medalla Barbas, 2006).Eur J Neurosci. Author manuscript; available in PMC 2015 June 01.Garc -Cabezas and BarbasPageWe first identified the areal and laminar borders of area 4 in Nissl-stained sections (n=4 cases) using the architectonic descriptions in maps from previous studies (Gatter Powell, 1978; get Leupeptin (hemisulfate) Barbas Pandya, 1987; Morecraft et al., 2012), as well as for area 46 (Barbas Pandya, 1989) (Figs. 1 and 2). Area 4 is easily identified in Nissl stained sections by the characteristic clusters of giant H 4065 side effects pyramidal neurons, or Betz cells, in the upper part of layer V. The anterior border of area 4 abuts caudal area 6, which has scattered large pyramidal neurons that are not as large as Betz cells. At the posterior border of area 4 lies somatosensory area 3 which does not have large pyramidal neurons as in the motor cortex (Fig. 1A). How to identify a layer that many believe does not exist The challenge is to identify layer IV in area 4 for analysis. In Nissl stained sections through area 4, in between the largest pyramidal neurons in layer III and the clusters of Betz cells in layer V there is a thin and pale band of tissue that corresponds to layer IV (Fig. 1B) (Gatter Powell, 1978). To clearly delineate layer IV we also used tissue stained for SMI-32 (n=3 cases), an antibody for a non-phosphorylated intermediate neurofilament protein (Sternberger Monoclonals; Lutherville, MD, USA). In the primate cortex SMI-32 labels a subset of pyramidal projection neurons in layers III and V, and to a lesser extent pyramidal neurons in layers II and VI (Campbell Morrison, 1989). Labeled neurons are especially prominent at the bottom part of layer III and the upper part of layer V. The selective labeling of pyramidal projection neurons allows easy delineation of cortical layers, and in particular layer IV, where neurons do not stain for SMI-32 (Fig. 1C). This band of unstained tissue in area 4 is continuous with the similarly unstained band in `granular’ area 3 (Fig. 1D). The central band of cortex with unstained neurons is invaded focally by stained pyramidal neurons from the adjacent layers (Fig. 1E and F). We also identified the borders of prefrontal area 46 for comparison in the same cases. Area 46 lies within the banks of the principal sulcus and adjacent cortex (Fig. 2A) and has six well-delineated layers, including a dense granular layer IV (Fig. 2B) (Barbas Pandya, 1989). SMI-32 labels pyramidal neurons in layers III, V and VI and clearly delineates layer IV (Fig. 2C). The clear zone in area 46 is also invaded focally by stained pyramidal neurons from the adjacent layers, as in area 4, though to a lesser extent (Fig. 2D). We used the above criteria to identify layer IV in areas 4 and 46. But there is yet another way to identify clearly neurons that reside in layer IV in the cortex. The basis for this identification dates back to classical studies, which apparently have been forgotten by most. In his classic studies, Cajal distinguished the nuclear features of large projection neurons from small pyramids and other interneurons (Ram y Cajal, 1896; 1899/2002). Briefly, large pyramidal neurons have a single, large, rounded and well-defined nucleolus. In contrast, small neurons, including small pyramids and small inhibitory neurons, have several nucleoli, or have two or more rod-like clumps of chromatin that may coalesce in the center of the nucleus. But small neurons do not show a clearly differentiated nucl.Ly (Ghashghaei Barbas, 2001; Medalla Barbas, 2006).Eur J Neurosci. Author manuscript; available in PMC 2015 June 01.Garc -Cabezas and BarbasPageWe first identified the areal and laminar borders of area 4 in Nissl-stained sections (n=4 cases) using the architectonic descriptions in maps from previous studies (Gatter Powell, 1978; Barbas Pandya, 1987; Morecraft et al., 2012), as well as for area 46 (Barbas Pandya, 1989) (Figs. 1 and 2). Area 4 is easily identified in Nissl stained sections by the characteristic clusters of giant pyramidal neurons, or Betz cells, in the upper part of layer V. The anterior border of area 4 abuts caudal area 6, which has scattered large pyramidal neurons that are not as large as Betz cells. At the posterior border of area 4 lies somatosensory area 3 which does not have large pyramidal neurons as in the motor cortex (Fig. 1A). How to identify a layer that many believe does not exist The challenge is to identify layer IV in area 4 for analysis. In Nissl stained sections through area 4, in between the largest pyramidal neurons in layer III and the clusters of Betz cells in layer V there is a thin and pale band of tissue that corresponds to layer IV (Fig. 1B) (Gatter Powell, 1978). To clearly delineate layer IV we also used tissue stained for SMI-32 (n=3 cases), an antibody for a non-phosphorylated intermediate neurofilament protein (Sternberger Monoclonals; Lutherville, MD, USA). In the primate cortex SMI-32 labels a subset of pyramidal projection neurons in layers III and V, and to a lesser extent pyramidal neurons in layers II and VI (Campbell Morrison, 1989). Labeled neurons are especially prominent at the bottom part of layer III and the upper part of layer V. The selective labeling of pyramidal projection neurons allows easy delineation of cortical layers, and in particular layer IV, where neurons do not stain for SMI-32 (Fig. 1C). This band of unstained tissue in area 4 is continuous with the similarly unstained band in `granular’ area 3 (Fig. 1D). The central band of cortex with unstained neurons is invaded focally by stained pyramidal neurons from the adjacent layers (Fig. 1E and F). We also identified the borders of prefrontal area 46 for comparison in the same cases. Area 46 lies within the banks of the principal sulcus and adjacent cortex (Fig. 2A) and has six well-delineated layers, including a dense granular layer IV (Fig. 2B) (Barbas Pandya, 1989). SMI-32 labels pyramidal neurons in layers III, V and VI and clearly delineates layer IV (Fig. 2C). The clear zone in area 46 is also invaded focally by stained pyramidal neurons from the adjacent layers, as in area 4, though to a lesser extent (Fig. 2D). We used the above criteria to identify layer IV in areas 4 and 46. But there is yet another way to identify clearly neurons that reside in layer IV in the cortex. The basis for this identification dates back to classical studies, which apparently have been forgotten by most. In his classic studies, Cajal distinguished the nuclear features of large projection neurons from small pyramids and other interneurons (Ram y Cajal, 1896; 1899/2002). Briefly, large pyramidal neurons have a single, large, rounded and well-defined nucleolus. In contrast, small neurons, including small pyramids and small inhibitory neurons, have several nucleoli, or have two or more rod-like clumps of chromatin that may coalesce in the center of the nucleus. But small neurons do not show a clearly differentiated nucl.