banner
Home
Research
Techniques
Outcomes
Publications
People
Funding
Alumni
Location
Contact
Scrapbook
Techniques

Optogenetics. Genetic or Arch Optoviral approaches are used to express light-activated molecules in specific cells (e.g. channelrhodopsin or archaerhodopsin) to define the role of these neurons in circuits that mediate behavior and endocrine responses. Such experiments are performed in vivo or (as done here) in vitro, where action potential firing is precisely  suppressed by a pulse of laser light.

 
patchedcellHistology and morphometry.  We use many approaches to study the cellular anatomy of the molecules, cells and circuits we study; immunohistochemistry, phalloidin staining for actin filaments, antibody staining of live or fixed cells, intracellular dye injections and morphometric analysis of the surface area of single cells or neurons imaged in live tissue slices. We also use Diolistic labeling of single cells and various methods for tacing axonal pathways.

shrinkDigital imaging.  We perform many forms of imaging in live cells and slices, including; dynamic analysis of changes in cell volume using morphometric approaches; ion concentration imaging (e.g. Calcium) in live cells or slices, fluorescence detection combined with electrophysiology.    
Tissue culture and molecular biology.  Our experiments also involve the use of organotypic slice cultures, cultured cell lines, in-vitro transfection of these preparations, as well as standard molecular biology techniques such as western blotting and RT-PCR for detection of specific mRNA species in single cells and tissue extracts.


Electrophysiology.  The Bourque Lab is first and foremost an electrophysiology lab. Most trainees will gain experience with many different electrophysiological techniques as required by their particular project, including:


Extracellular single-unit recording. Here, a glass pipettes filledextracellular with saline solution is used to record action potentials fired by a neuron at the tip of the pipette. The electrode is positioned using a micromanipulator. What one sees on the oscilloscope or computer is shown at right. The advantage of this technique is that the neuron is not  damaged by the recording process and one records the natural spontaneous electrical activity of the cell in a relatively non-invase way. Recordings such as these can last many hours.    


VIcurveIntracellular (sharp electrode) recording. This approach uses a fine-tipped (~100 nm) glass micropipette inserted into the neuron, allowing direct recording of electrical events generated by the neuron (membrane potential, resistance, time constant, synaptic potentials and action potentials). Current can be injected to change the membrane potential of the cell (left). When the neuron is depolarized past "threshold", action potentials are generated. Voltage-current analysis can be performed by plotting voltage (at arrow) as a function of current. The slope of the linear part of the curve provides a measure of the input resistance of the cell.  Although this technique is relatively difficult to perform (compared with patch clamp), sharp electrode recordings allow recordings to be obtained for long periods without the common problems of dialysis and run-down that affect neurons recorded by patch clamp.


            Unlike many other techniques, electrophysiological experiments provide data and information that can be observed in real time. No need to wait an hour or a day to know if the experiment worked! The animation shown above illustrates a measurement of the neurons' V-I profile as done in real time. Evidently protocols such as this are usually  performed at a slightly slower rate, and must be repeated often during an experiment. But the information they provide can be interpreted on-line.  



Patch clamp
single channel recording. Here, a larger tipped (diameter 1-2 microns) glass pipette is pressed against the membrane of a neuron while it is observed using a microscope. Gentle suction is applied and the high resistance seal that forms (a so-called "gigaseal") allows one to record the activity of individual ion channel proteins as the pore opens and closes to regulate singlechanther flux of ions (and thus electrical current) across the membrane. In the example shown at right we see several spontaneous openings of a single channel. In one of the sweeps we see that two channels are opened simultaneously, leading to a brief period of time when the current recorded jumps to total amplitude that is twice as large as that carried when a single channel is opened (implying that there are at least two channels in the patch!).                



suctionPatch clamp -whole cell recording. Here, a gigaseal is formed as described for single channel recording. Except that a brief pulse of current is used to destroy the small patch of membrane that lies below the tip of the pipette. As a result, direct electrical contact is made between the inside of the pipette and the interior of the cell. This "whole cell" configuration allows high quality current-clamp and voltage-clamp recordings to be made from neurons. One advantage of this mechanotechnique is that drugs, dyes or fluorescent probes (e.g. Calcium indicators) can be easily delivered to the interior of the cell. Because this technique is most often performed while observing the cell with a microscope, it is easy to combine electrophysiological experiments with simultaneous imaging. In the example shown at right, the volume of a supraoptic nucleus neuron was reduced by applying suction to a recording pipette. As can be seen by the graphical representation of an equivalent experiment, the decrease in cell volume caused by suction is associated with an increase in the membrane conductance (G) of the cell as measured by voltage clamp. This indicates that cell shrinking is associated with the opening of ion channels.This finding provides insight into one of the mechanisms underlying osmosensory transduction in these neurons.


 
drugstepFast drug application. Whole cell recordings are used to record the simultaneous activity of many ion channels, or fast responses involving a subset of ion channels activated by fast drug delivery using a fast stepper system or responses evoked by neurotreansmitter release induced by the activation of synaptic afferents. In the movie shown at left, a tiny neuron (not visible) at the tip of the patch pipette is being repeatedly exposed to either control or drug-containing solution (purple). The solutions are delivered via an assembly of multiple square glass pipettes (3 in this case) which is rapidly moved using a computer-controlled piezoelectric device while the response of the neuron is being recorded.


epsp

Synaptic Responses.
Our research involves analysis of spontaneous and evoked synaptic responses. In the example shown at right, voltage responses are recorded in current-clamp from a supraoptic nucleus neuron while an afferent nucleus is activates by a brief (1 ms) electrical pulse (stim). In some sweeps no response occurs, while in others an excitatory postsynaptic potential (EPSP) is visible. In a few sweeps the evoked EPSP generates an action potential. We also frequently examine the effects of physiological stimulation of afferents. For example, we examine the effects of osmotic or thermal stimulation of central sensory neurons in one nucleus while recording changes in synaptic activity relayed to downstream neurons. An example of this approach is shown in the Research page.



sliceCells and tissues.  Our work is performed on  models that replicate functions present in the  human brain. We study acutely isolated or cultured somata or isolated nerve terminals to define the roles of specific types of ion channels and receptors in identified cells and cellular compartments. Recordings in acute slices,  superfused explants or organotypic slice cultures are used to study neuron-glia interactions, and the integration of cellular, synaptic and network properties in-situ.  

 


       Home | Research | Techniques | Outcomes | Publications | people | Funding | alumni | Location | Contact                              ©Bourque 2012