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1.  Cortical inputs to the middle temporal visual area in New World owl monkeys 
Eye and brain  2014;2015(7):1-15.
We made eight retrograde tracer injections into the middle temporal visual area (MT) of three New World owl monkeys (Aotus nancymaae). These injections were placed across the representation of the retina in MT to allow us to compare the locations of labeled cells in other areas in order to provide evidence for any retinotopic organization in those areas. Four regions projected to MT: 1) early visual areas, including V1, V2, V3, the dorsolateral visual area, and the dorsomedial visual area, provided topographically organized inputs to MT; 2) all areas in the MT complex (the middle temporal crescent, the middle superior temporal area, and the fundal areas of the superior temporal sulcus) projected to MT. Somewhat variably across injections, neurons were labeled in other parts of the temporal lobe; 3) regions in the location of the medial visual area, the posterior parietal cortex, and the lateral sulcus provided other inputs to MT; 4) finally, projections from the frontal eye field, frontal visual field, and prefrontal cortex were also labeled by our injections. These results further establish the sources of input to MT, and provide direct evidence within and across cases for retinotopic patterns of projections from early visual areas to MT.
PMCID: PMC4302954  PMID: 25620872
middle temporal area; visual cortex; parietal cortex
2.  A Rapid and Reliable Method of Counting Neurons and Other Cells in Brain Tissue: A Comparison of Flow Cytometry and Manual Counting Methods 
It is of critical importance to understand the numbers and distributions of neurons and non-neurons in the cerebral cortex because cell numbers are reduced with normal aging and by diseases of the CNS. The isotropic fractionator method provides a faster way of estimating numbers of total cells and neurons in whole brains and dissected brain parts. Several comparative studies have illustrated the accuracy and utility of the isotropic fractionator method, yet it is a relatively new methodology, and there is opportunity to adjust procedures to optimize its efficiency and minimize error. In the present study, we use 142 samples from a dissected baboon cortical hemisphere to evaluate if isotropic fractionator counts using a Neubauer counting chamber and fluorescence microscopy could be accurately reproduced using flow cytometry methods. We find greater repeatability in flow cytometry counts, and no evidence of constant or proportional bias when comparing microscopy to flow cytometry counts. We conclude that cell number estimation using a flow cytometer is more efficient and more precise than comparable counts using a Neubauer chamber on a fluorescence microscope. This method for higher throughput, precise estimation of cell numbers has the potential to rapidly advance research in post-mortem human brains and vastly improve our understanding of cortical and subcortical structures in normal, injured, aged, and diseased brains.
PMCID: PMC2841487  PMID: 20300202
isotropic fractionator; flow cytometry; Neubauer chamber; neuron number
3.  Use of flow cytometry for high-throughput cell population estimates in brain tissue 
The large size of primate brains is an impediment to obtaining high-resolution cell number maps of the cortex in humans and non-human primates. We present a rapid, flow cytometry-based cell counting method that can be used to estimate cell numbers from homogenized brain tissue samples comprising the entire cortical sheet. The new method, called the flow fractionator, is based on the isotropic fractionator (IF) method (Herculano-Houzel and Lent, 2005), but substitutes flow cytometry analysis for manual, microscope analysis using a Neubauer counting chamber. We show that our flow cytometry-based method for total cell estimation in homogenized brain tissue provides comparable data to that obtained using a counting chamber on a microscope. The advantages of the flow fractionator over existing methods are improved precision of cell number estimates and improved speed of analysis.
PMCID: PMC3394395  PMID: 22798947
flow cytometry; Neubauer chamber; nuclear suspension; cell counting
4.  Cortical Connections of the Visual Pulvinar Complex in Prosimian Galagos (Otolemur garnetti) 
The pulvinar complex of prosimian primates is not as architectonically differentiated as that of anthropoid primates. Thus, the functional subdivisions of the complex have been more difficult to determine. In the present study, we related patterns of connections of cortical visual areas (primary visual area, V1; secondary visual area, V2; and middle temporal visual area, MT) as well as the superior colliculus of the visual midbrain, with subdivisions of the pulvinar complex of prosimian galagos (Otolemur garnetti) that were revealed in brain sections processed for cell bodies (Nissl), cytochrome oxidase, or myelin. As in other primates, the architectonic methods allowed us to distinguish the lateral pulvinar (PL) and inferior pulvinar (PI) as major divisions of the visual pulvinar. The connection patterns further allowed us to divide PI into a large central nucleus (PIc), a medial nucleus (PIm), and a posterior nucleus (PIp). Both PL and PIc have separate topographic patterns of connections with V1 and V2. A third, posterior division of PI, PIp, does not appear to project to V1 and V2 and is further distinguished by receiving inputs from the superior colliculus. All these subdivisions of PI project to MT. The evidence suggests that PL of galagos contains a single, large nucleus, as in monkeys, and that PI may have only three subdivisions, rather than the four subdivisions of monkeys. In addition, the cortical projections of PI nuclei are more widespread than those in monkeys. Thus, the pulvinar nuclei in prosimian primates and anthropoid primates have evolved along somewhat different paths.
PMCID: PMC3710459  PMID: 19795374
superior colliculus; visual cortex; middle temporal area; area 17; area 18; primate evolution; thalamus
5.  Cell and neuron densities in the primary motor cortex of primates 
Cell and neuron densities vary across the cortical sheet in a predictable manner across different primate species (Collins et al., 2010b). Primary motor cortex, M1, is characterized by lower neuron densities relative to other cortical areas. M1 contains a motor representation map of contralateral body parts from tail to tongue in a mediolateral sequence. Different functional movement representations within M1 likely require specialized microcircuitry for control of different body parts, and these differences in circuitry may be reflected by variation in cell and neuron densities. Here we determined cell and neuron densities for multiple sub-regions of M1 in six primate species, using the semi-automated flow fractionator method. The results verify previous reports of lower overall neuron densities in M1 compared to other parts of cortex in the six primate species examined. The most lateral regions of M1 that correspond to face and hand movement representations, are more neuron dense relative to medial locations in M1, which suggests differences in cortical circuitry within movement zones.
PMCID: PMC3583034  PMID: 23450743
M1; flow fractionator; isotropic fractionator; movement
6.  Comparison of Area 17 Cellular Composition in Laboratory and Wild-Caught Rats Including Diurnal and Nocturnal Species 
Brain, Behavior and Evolution  2011;77(2):116-130.
In this study we examine the size of primary sensory areas in the neocortex and the cellular composition of area 17/V1 in three rodent groups: laboratory nocturnal Norway rats (Long-Evans; Rattus norvegicus), wild-caught nocturnal Norway rats (R. norvegicus), and laboratory diurnal Nile grass rats (Arvicanthis niloticus). Specifically, we used areal measures of myeloarchitecture of the primary sensory areas to compare area size and the isotropic fractionator method to estimate the number of neurons and nonneurons in area 17 in each species. Our results demonstrate that the percentage of cortex devoted to area 17 is significantly greater and the percentage of cortex devoted to S1 is significantly smaller in the diurnal Nile grass rat compared with the nocturnal Norway rat groups. Further, the laboratory rodent groups have a greater percentage of cortex devoted to auditory cortex compared with the wild-caught group. We also demonstrate that wild-caught rats have a greater density of neurons in area 17 compared to laboratory-reared animals. However, there were no other clear cellular composition differences in area 17 or differences in the percentage of brain weight devoted to area 17 between nocturnal and diurnal rats. Thus, there are differences in primary sensory area size between diurnal versus nocturnal and laboratory versus wild-caught rat groups and cellular density between wild-caught and laboratory rat groups. Our results demonstrate that the differences in the size and cellular composition of cortical areas do not fit with what would be expected based on brain scaling differences alone, and have a consistent relationship with lifestyle and sensory morphology.
PMCID: PMC3094678  PMID: 21525748
Architecture; Area 17; Cellular composition; Evolution; Isotropic fractionator; Vision
7.  Cellular Scaling Rules for the Brains of an Extended Number of Primate Species 
Brain, Behavior and Evolution  2010;76(1):32-44.
What are the rules relating the size of the brain and its structures to the number of cells that compose them and their average sizes? We have shown previously that the cerebral cortex, cerebellum and the remaining brain structures increase in size as a linear function of their numbers of neurons and non-neuronal cells across 6 species of primates. Here we describe that the cellular composition of the same brain structures of 5 other primate species, as well as humans, conform to the scaling rules identified previously, and that the updated power functions for the extended sample are similar to those determined earlier. Accounting for phylogenetic relatedness in the combined dataset does not affect the scaling slopes that apply to the cerebral cortex and cerebellum, but alters the slope for the remaining brain structures to a value that is similar to that observed in rodents, which raises the possibility that the neuronal scaling rules for these structures are shared among rodents and primates. The conformity of the new set of primate species to the previous rules strongly suggests that the cellular scaling rules we have identified apply to primates in general, including humans, and not only to particular subgroups of primate species. In contrast, the allometric rules relating body and brain size are highly sensitive to the particular species sampled, suggesting that brain size is neither determined by body size nor together with it, but is rather only loosely correlated with body size.
PMCID: PMC2980814  PMID: 20926854
Allometry; Brain size; Evolution; Glia, number; Neurons, number; Primates
8.  Conversion Analysis for Mutation Detection in MLH1 and MSH2 in Patients With Colorectal Cancer 
The accurate identification and interpretation of germline mutations in mismatch repair genes in colorectal cancer cases is critical for clinical management. Current data suggest that mismatch repair mutations are highly heterogeneous and that many mutations are not detected when conventional DNA sequencing alone is used.
To evaluate the potential of conversion analysis compared with DNA sequencing alone to detect heterogeneous germline mutations in MLH1, MSH2, and MSH6 in colorectal cancer patients.
Design, Setting, and Participants
Multicenter study with patients who participate in the Colon Cancer Family Registry. Mutation analyses were performed in participant samples determined to have a high probability of carrying mismatch repair germline mutations. Samples from a total of 64 hereditary nonpolyposis colorectal cancer cases, 8 hereditary nonpolyposis colorectal cancer–like cases, and 17 cases diagnosed prior to age 50 years were analyzed from June 2002 to June 2003.
Main Outcome Measures
Classification of family members as carriers or noncarriers of germline mutations in MLH1, MSH2, or MSH6; mutation data from conversion analysis compared with genomic DNA sequencing.
Genomic DNA sequencing identified 28 likely deleterious exon mutations, 4 in-frame deletion mutations, 16 missense changes, and 22 putative splice site mutations. Conversion analysis identified all mutations detected by genomic DNA sequencing—plus an additional exon mutation, 12 large genomic deletions, and 1 exon duplication mutation—yielding an increase of 33% (14/42) in diagnostic yield of deleterious mutations. Conversion analysis also showed that 4 of 16 missense changes resulted in exon skipping in transcripts and that 17 of 22 putative splice site mutations affected splicing or mRNA transcript stability. Conversion analysis provided an increase of 56% (35/63) in the diagnostic yield of genetic testing compared with genomic DNA sequencing alone.
The data confirm the heterogeneity of mismatch repair mutations and reveal that many mutations in colorectal cancer cases would be missed using conventional genomic DNA sequencing alone. Conversion analysis substantially increases the diagnostic yield of genetic testing for mismatch repair mutations in patients diagnosed as having colorectal cancer.
PMCID: PMC2933041  PMID: 15713769
9.  Large-Scale Reorganization in the Somatosensory Cortex and Thalamus after Sensory Loss in Macaque Monkeys 
Adult brains undergo large-scale plastic changes following peripheral and central injuries. Although it has been shown that both the cortical and thalamic representations can reorganize, uncertainties exist regarding the extent, nature, and time course of changes at each level. We have determined how cortical representations in the somatosensory area 3b and the ventroposterior (VP) nucleus of thalamus are affected by long standing unilateral dorsal column lesions at cervical levels in macaque monkeys. In monkeys with recovery periods of 22-23 months, the intact face inputs expanded into the deafferented hand region of area 3b following complete or partial lesions of the dorsal columns. The expansion of the face region could extend all the way medially into the leg and foot representations. In the same monkeys, similar expansions of the face representation take place in the VP nucleus of the thalamus, indicating that both these processing levels undergo similar reorganizations. The receptive fields of the expanded representations were similar in somatosensory cortex and thalamus. In two monkeys, we determined the extent of the brain reorganization immediately after dorsal column lesions. In these monkeys, the deafferented regions of area 3b and the VP nucleus became unresponsive to the peripheral touch immediately after the lesion. No reorganization was seen in the cortex or the VP nucleus. A comparison of the extents of deafferentation across the monkeys shows that even if the dorsal column lesion is partial, preserving most of the hand representation, it is sufficient to induce an expansion of the face representation.
PMCID: PMC2613515  PMID: 18945912
Plasticity; Spinal cord injury; Dorsal Column; Primate; Microelectrode; Mapping; Receptive Field
10.  Geometric morphometrics defines shape differences in the cortical area map of C57BL/6J and DBA/2J inbred mice 
BMC Neuroscience  2006;7:63.
We previously described planar areal differences in adult mouse visual, somatosensory, and neocortex that collectively discriminated C57BL/6J and DBA/2J inbred strain identity. Here we use a novel application of established methods of two-dimensional geometric morphometrics to examine shape differences in the cortical area maps of these inbred strains.
We used Procrustes superimposition to align a reliable set of landmarks in the plane of the cortical sheet from tangential sections stained for the cytochrome oxidase enzyme. Procrustes superimposition translates landmark configurations to a common origin, scales them to a common size, and rotates them to minimize an estimate of error. Remaining variation represents shape differences. We compared the variation in shape between C57BL/6J and DBA/2J relative to that within each strain using a permutation test of Goodall's F statistic. Significant differences in shape in the posterior medial barrel subfield (PMBSF), as well as differences in shape across primary sensory areas, characterize the cortical area maps of these common inbred, isogenic strains.
C57BL/6J and DBA/2J have markedly different cortical area maps, in both size and shape. These differences suggest polymorphism in genetic factors underlying cortical specification, even between common isogenic strains. Comparing cortical phenotypes between normally varying inbred mice or between genetically modified mice can identify genetic contributions to cortical specification. Geometric morphometric analysis of shape represents an additional quantitative tool for the study of cortical development, regardless of whether it is studied from phenotype to gene or gene to phenotype.
PMCID: PMC1578583  PMID: 16978410
11.  Variation in the cortical area map of C57BL/6J and DBA/2J inbred mice predicts strain identity 
BMC Neuroscience  2005;6:18.
Recent discoveries suggest that arealization of the mammalian cortical sheet develops in a manner consonant with principles established for embryonic patterning of the body. Signaling centers release morphogens that determine regional growth and tissue identity by regulating regional expression of transcription factors. Research on mouse cortex has identified several candidate morphogens that affect anteroposterior or mediolateral cortical regionalization as well as mitogenesis. Inbred strains of laboratory mice can be exploited to study cortical area map formation if there are significant phenotypic differences with which to correlate gene polymorphism or expression data. Here we describe differences in the cortical area map of two commonly used inbred strains of laboratory mice, C57BL/6J and DBA/2J. Complete cortical hemispheres from adult mice were dissected and stained for the cytochrome oxidase enzyme in order to measure histochemically defined cortical areas.
C57BL/6J has the larger neocortex, relatively larger primary visual cortex (V1), but relatively smaller posterior medial barrel subfield of the primary somatosensory cortex (PMBSF). The sample of C57BL/6J and DBA/2J mice can be discriminated with 90% accuracy on the basis of these three size dimensions.
C57BL/6J and DBA/2J have markedly different cortical area maps, suggesting that inbred strains harbor enough phenotypic variation to encourage a forward genetic approach to understanding cortical development, complementing other approaches.
PMCID: PMC1079866  PMID: 15774010

Results 1-11 (11)