RESUMO
Virus-based methods for labelling populations of cortical neurons, when combined with cell-type specific recombinant promoters and techniques allowing temporal control of gene expression, provide neuroscience with new opportunities to examine the connectivity between brain regions and how this connectivity is modified by experience or disease. However, to take full advantage of these technical advances, it is necessary to develop new methods for quantification of the axonal projections revealed. Here we describe a method for quantitative analysis of axonal projection patterns emanating from populations of labelled cells, using transmitted light bright field microscopy. A single high resolution image of an area to be analysed is first acquired using mosaic extended focus image microscopy. This image is then analysed by specifically developed image processing algorithms that identify and track axon segments present. For quantitative analysis, measurement grids consisting of a user-defined number of individual elements are placed over an area of interest, with the computer-based method then returning the summed length of the axon segments in each element. Axon density plots can thus be generated. We present an example from rat brain showing, over a whole coronal section, axon densities emanating from a population of layer 2/3 somatosensory neurons.
Assuntos
Axônios/ultraestrutura , Mapeamento Encefálico/métodos , Contagem de Células/métodos , Citometria por Imagem/métodos , Microscopia/métodos , Coloração e Rotulagem/métodos , Algoritmos , Animais , Animais Recém-Nascidos , Axônios/fisiologia , Mapeamento Encefálico/instrumentação , Contagem de Células/instrumentação , Forma Celular/fisiologia , Tamanho Celular , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Citometria por Imagem/instrumentação , Processamento de Imagem Assistida por Computador , Lentivirus/genética , Microscopia/instrumentação , Células Piramidais/fisiologia , Ratos , Ratos Wistar , Córtex Somatossensorial/citologia , Córtex Somatossensorial/fisiologia , Coloração e Rotulagem/instrumentaçãoRESUMO
Group B streptococci (GBS) express various surface antigens designated c, R, and X antigens. A new R-like surface protein from Streptococcus agalactiae strain Compton R has been identified by using a polyclonal antiserum raised against the R protein fraction of this strain to screen a lambda Zap library. DNA sequence analysis of positive clones allowed the prediction of the primary structure of a 105-kDa protein designated BPS protein (group B protective surface protein) that exhibited typical features of streptococcal surface proteins such as a signal sequence and a membrane anchor region but did not show significant similarity with other known sequences. Immunogold electron microscopy using a BPS-specific antiserum confirmed the surface location of BPS protein on S. agalactiae strain Compton R. Anti-BPS antibodies did not cross-react with R1 and R4 proteins expressed by two variant type III GBS strains but reacted with the parental streptococcal strain in Western blot and immunoprecipitation analyses. Separate R3 and BPS immunoprecipitation bands were observed when a cell extract of strain Compton R was tested with an antiserum against Compton R previously cross-absorbed to remove R4 antibodies. Immunization of mice with recombinant BPS protein by the subcutaneous route produced an efficient antigen-specific response, and immunized animals survived challenge with a lethal dose of a virulent strain. Therefore, BPS protein represents a new R-like protective antigen of GBS.
