Eolus. The cytoplasm of large (projection) and small (local) neurons is always stained. In the latter, theNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptEur J Neurosci. Author manuscript; available in PMC 2015 June 01.Garc -Cabezas and BarbasPagecytoplasm appears as a thin stripe around the nucleus but the pyramidal or oval shape of the cell can always be identified (Fig. 3).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptWe distinguished two types of neurons using these classical cellular criteria to determine the composition of the clear Pepstatin site central zone that we identified as layer IV in area 4, which was aided by tissue stained for SMI-32. We identified small and large neurons in Nissl stained tissue. One series of sections was labeled for SMI-32 and also counterstained for Nissl to label and sort out neurons in the inner `granular’ layer. We also identified types of glia according to the classical descriptions of Cajal (1896; 1899/2002/1913) and del R -Hortega (1919; 1921; 1928; 1932) as well as more recent workers (Ling et al., 1973; O’Kusky Colonnier, 1982a; Gabbott Stewart, 1987). Studies in the electron microscope in the monkey primary visual cortex yield estimates of neurons and glial cell types comparable to those obtained with the optical microscope without immunohistochemical markers (O’Kusky Colonnier, 1982a; SKF-96365 (hydrochloride) site Peters et al., 1991). Astrocytes have rounded to oval nuclei, homogeneous nucleoplasm and several rounded clumps of heterochromatin, some of which are located under the slightly irregular nuclear membrane. Frequently, the cytoplasm has pigmented granular inclusions close to the nucleus. Oligodendrocytes have a smooth nuclear membrane and a few clumps of heterochromatin and round and darkly stained nuclei, which usually are smaller than in astrocytes. Microglia have darkly stained elongated or polylobular nuclei without visible heterochromatin and the cytoplasm does not stain; in some cases there are granular inclusions close to the lobulations of the nucleus. Finally, we distinguished endothelial cells, which are not glia; they have rectangular to ovoid nuclei, mold to the shape of blood vessels, have smooth nuclear membrane and two to four nucleoli, one usually larger than the rest (Fig. 3). We estimated the number and density of neurons and glial cells in layer IV of area 4 and area 46 in Nissl-stained sections (n=4 cases) using the unbiased stereological method of the optical fractionator (Gundersen, 1986; Howard Reed, 1998) with the aid of a commercial system (StereoInvestigator; MicroBrightField, Inc.), as described in previous studies (Barbas et al., 2005; Medalla Barbas, 2006; Garc -Cabezas Barbas, 2013). Briefly, we analyzed a minimum of four evenly spaced brain sections for each area per case using systematic random sampling of layer IV. The stereological data included volume calculation for layer IV, which takes into consideration the sampled area and thickness of each section. We used as guard zones the top and bottom of each section (minimum 2 m for 15 m sections after shrinkage) and measured the actual mounted section thickness using the program software at each counting site. The counting frame/disector size (area= 50?0 m; height=5 m) and grid spacing (150?50 m) were set to employ a fraction to yield a coefficient of error of < 10 , as recommended (Gundersen, 1986; Howard Reed, 1998). For the sparse microglial cells, the coefficient of error w.Eolus. The cytoplasm of large (projection) and small (local) neurons is always stained. In the latter, theNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptEur J Neurosci. Author manuscript; available in PMC 2015 June 01.Garc -Cabezas and BarbasPagecytoplasm appears as a thin stripe around the nucleus but the pyramidal or oval shape of the cell can always be identified (Fig. 3).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptWe distinguished two types of neurons using these classical cellular criteria to determine the composition of the clear central zone that we identified as layer IV in area 4, which was aided by tissue stained for SMI-32. We identified small and large neurons in Nissl stained tissue. One series of sections was labeled for SMI-32 and also counterstained for Nissl to label and sort out neurons in the inner `granular' layer. We also identified types of glia according to the classical descriptions of Cajal (1896; 1899/2002/1913) and del R -Hortega (1919; 1921; 1928; 1932) as well as more recent workers (Ling et al., 1973; O'Kusky Colonnier, 1982a; Gabbott Stewart, 1987). Studies in the electron microscope in the monkey primary visual cortex yield estimates of neurons and glial cell types comparable to those obtained with the optical microscope without immunohistochemical markers (O'Kusky Colonnier, 1982a; Peters et al., 1991). Astrocytes have rounded to oval nuclei, homogeneous nucleoplasm and several rounded clumps of heterochromatin, some of which are located under the slightly irregular nuclear membrane. Frequently, the cytoplasm has pigmented granular inclusions close to the nucleus. Oligodendrocytes have a smooth nuclear membrane and a few clumps of heterochromatin and round and darkly stained nuclei, which usually are smaller than in astrocytes. Microglia have darkly stained elongated or polylobular nuclei without visible heterochromatin and the cytoplasm does not stain; in some cases there are granular inclusions close to the lobulations of the nucleus. Finally, we distinguished endothelial cells, which are not glia; they have rectangular to ovoid nuclei, mold to the shape of blood vessels, have smooth nuclear membrane and two to four nucleoli, one usually larger than the rest (Fig. 3). We estimated the number and density of neurons and glial cells in layer IV of area 4 and area 46 in Nissl-stained sections (n=4 cases) using the unbiased stereological method of the optical fractionator (Gundersen, 1986; Howard Reed, 1998) with the aid of a commercial system (StereoInvestigator; MicroBrightField, Inc.), as described in previous studies (Barbas et al., 2005; Medalla Barbas, 2006; Garc -Cabezas Barbas, 2013). Briefly, we analyzed a minimum of four evenly spaced brain sections for each area per case using systematic random sampling of layer IV. The stereological data included volume calculation for layer IV, which takes into consideration the sampled area and thickness of each section. We used as guard zones the top and bottom of each section (minimum 2 m for 15 m sections after shrinkage) and measured the actual mounted section thickness using the program software at each counting site. The counting frame/disector size (area= 50?0 m; height=5 m) and grid spacing (150?50 m) were set to employ a fraction to yield a coefficient of error of < 10 , as recommended (Gundersen, 1986; Howard Reed, 1998). For the sparse microglial cells, the coefficient of error w.
