Electrophysiology

Most of the projects in our lab rely on extracellular recording techniques.  Our current recording setup and data acquisition system consists of a  233MHz Pentium II PC running  Experimenter's Workbench 32  (DataWave Technologies), 2 Lynx-8 Amplifiers (NeuraLynx), and 2 custom built 8 channel headstages(Frederick Haer & Co.) Our setup allows us to record up to 16 channels simultaneously (The EWB32 software can handle up to 32 channels).   All of our recording is extracellular, and we either fabricate our own carbon fiber microelectrodes or use commercially available arrays of 3-8  tungsten microelectrodes (Frederick Haer & Co.)  These arrays allow us to record neuronal activity over larger local populations of neurons than is possible with single electrodes.  We also use multiple arrays to record simultaneously from different neuronal regions, including Primary and Secondary Somatosensory Cortex, Thalamus, and NeoStriatum.  We then use cross-correlation analysis to examine the temporal structure of  firing patterns from these neurons.   Data analysis is performed on a super-cool 266 MHz PII PC for speedy number crunching.

We are also now using LabView™ Virtual Instrumentation software (National Instruments) running on a dedicated 600MHz Athlon (K-7) PC with a 21" monitor to fulfill various new instrumentation needs.   Our first virtual instrument was a 4-channel oscilloscope customized for viewing neuronal signals & interactions.

Histochemistry and Tissue Processing

Various techniques are employed to process tissue for visualization of injected tracer dyes.  Brains that have been injected with biotinylated dextran amine, or BDA (Molecular Probes), must be histochemically processed to visualize the injection site and varicosities distributed through the cortex, neostriatum, thalamus, and other brain regions.  The protocol involves two incubations in histochemical solutions:  the first is in an avidin-biotin complex (ABC) (Vector Labs), which binds to biotin in the BDA.  The second is a peroxide-chromogen solution which yields a colored precipitate where the ABC, and therefore the BDA, is present in the tissue.

Some tissue is processed for the presence of cytochrome oxidase (CO), a metabolic enzyme.   The cortical columns representing specific vibrissae, sometimes called whisker barrels, typically show an elevated level of CO staining.  We use CO processed tissue on serial tissue sections to verify that our BDA and/or Fluoro-ruby (FR) injections have been directed into a specific whisker barrel or barrels in the cortex.

Microscopy and Anatomical Reconstruction

The research microscope in the Lab is equipped with a Minnesota Datametrics optical encoder.  When an investigator examines a processed slide with this microscope, the optical encoder outputs the X-Y coordinates of the stage (relative to a pre-determined zero point) to a computer.  As the investigator moves the microscope stage, the encoder registers new X-Y coordinates.  When desired, a footpedal is pressed which sends the current microscope stage coordinates to a computer.  Software which accompanies this apparatus (MDPlot) can receive the coordinates and record them, enabling anatomical boundaries to be traced (via a connect-the-dots method), and more importantly terminal varicosities to be "plotted" as a pattern of dots on the screen.  These plots can then be printed.  The MDPlot software, and another separate software package, Gradient Density Analysis (GDA), developed in house by Stephane Roy, together enable us to obtain useful measurements from the arborizations and anatomical units in the brain including area, terminal count, and terminal density.  This cutting edge technique lets us determine, objectively, the degree of overlap the arborizations produce from two separate, isolated injections of BDA and Fluoro-ruby, a fluorescent anterograde neuronal tracer dye.  The use of double (or more) tracer injections lets us ask and answer unique questions about how different anatomical units are organized and how they interact.

The lab recently acquired a SenSys CCD (Digital) Camera for use with our microscope.   This camera is equipped with a CRI LCD color cube which allows color image capture.   The 450MHz Pentium III and IPLab software (Scanalytics) which accompanies the camera allows us to z-section tissue and capture features which might not otherwise be clearly visible.

With the addition of a color printer, we are now able to produce hard copies (i.e. paper printouts) of plots of cases injected with both BDA and FR.  The color printouts allow the clear visualization of neighboring and/or overlapping areas of different tracer-dye arborizations.

Other Departmental Resources

Electron Microscope

Confocal Microscope - The Department posesses a Zeiss confocal microscope which our lab uses from time to time.  We have found the unorthodox method of scanning the cytochrome oxidase stained tissue -- tissue normally viewed using light microscopy -- with the 488 wavelength laser to be an effective method of visualizing darkly stained areas.   The resulting black and white digital image increases contrast between the whisker barrels and the septae, or inbetween areas, which lack the elevated levels of cytochrome oxidase.

Photography Lab - The Department has ample facilities for B&W developing and printmaking and color slide making.

Histology Lab - A freezing microtome, vibratome, and cryostat are available.  In addition equipment for paraffin processing is in the histology lab.