title: A novel unbiased counting method for the quantification of synapses in the mouse brain.
abstract: BACKGROUND
  The numerical density of synapses and their ultrastructural features are best assessed with electron microscopy. Counting is done within counting frames placed on a pair of sections (disector technique). But this requires that the thin sections are taken from comparable brain regions and the disectors are placed in a uniform random fashion. Small brain areas like the polymorph layer of the mouse dentate gyrus are difficult to encounter, and manually moving the microscope stage for placing the micrographs seems arbitrary.
  
  
  METHODS
  Here the polymorph layer was approximated with 20μm thin, Nissl-stained vibratome sections. The subsequent vibratome section was processed for electron microscopy and serially thin sectioned. The microscope stage was moved using a random number generator, placing at least 20 disectors onto a pair of sections. The numerical synapse density, the numerical density of dense-core vesicles, and other ultrastructural features were compared between mice that had been kept in an enriched environment and mice kept under standard housing conditions.
  
  
  RESULTS
  Environmental enrichment significantly decreased the numerical density of dense-core vesicles and synaptic cleft widths within the polymorph layer, associated with behavioral improvement in the Morris water maze, a hippocampus-dependent task of spatial learning and memory.
  
  
  METHODS
  This procedure was easy to handle and enabled us to produce thin sections in small, defined brain areas. Furthermore, placing the disectors with random numbers excluded observer bias.
  
  
  CONCLUSIONS
  Our procedure provides an uncomplicated way of assessing numerical densities in small brain areas in an unbiased manner.
authors.affiliation: Research Unit of Translational Neurogastroenterology, Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Universitätsplatz 4, 8010 Graz, Austria. Electronic address: florian.reichmann@medunigraz.at.
authors.forename: Florian
authors.initials: F
authors.lastname: Reichmann
authors.affiliation: Research Unit of Translational Neurogastroenterology, Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Universitätsplatz 4, 8010 Graz, Austria.
authors.forename: Evelin
authors.initials: E
authors.lastname: Painsipp
authors.affiliation: Research Unit of Translational Neurogastroenterology, Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Universitätsplatz 4, 8010 Graz, Austria. Electronic address: peter.holzer@medunigraz.at.
authors.forename: Peter
authors.initials: P
authors.lastname: Holzer
authors.affiliation: Research Unit Electron Microscopic Techniques, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kummer
authors.affiliation: Research Unit Electron Microscopic Techniques, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Bock
authors.affiliation: Research Unit Electron Microscopic Techniques, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. Electronic address: gerd.leitinger@medunigraz.at.
authors.forename: Gerd
authors.initials: G
authors.lastname: Leitinger
mesh_terms: D000818:Animals; D018891:Dentate Gyrus / Q000648:ultrastructure*; D004777:Environment; D005260:Female; D006799:Housing, Animal; D018782:Maze Learning; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron / Q000379:methods*; D010363:Pattern Recognition, Automated / Q000379:methods*; D022142:Secretory Vesicles / Q000648:ultrastructure; D012984:Software; D065852:Spatial Memory; D013569:Synapses / Q000648:ultrastructure*
keywords: Dense core vesicle; Dentate gyrus; Disector; Electron microscopy; Environmental enrichment; Mouse