Methods and Approaches

The methods we use include

  • Electrophysiology (extra- and intracellular recordings), in vivo, in situ and in vitro. Single and dual electrode current clamp, voltage clamp and dynamic clamp.
  • Optical imaging (voltage-sensitive dyes)
  • Photoablation of identified neurons
  • Behavioral studies
  • Real-time Computer Modeling using MadSim
  • Immunocitochemistry
  • Voltammetry

Electrophysiology.
This is a photo of one of our electrophysiology rigs. We typically use an isolated ganglion preparation of the stomatogastric nervous system to study motor pattern generation and control. Multiple extra- and intracellular recordings are used to monitor and manipulate central pattern generating neurons.

Electrophysiology setup in the Stein Lab with 4 simultaneous intracellular recordings of neurons.

Optical imaging.
In addition to electrophysiology, we use voltage-sensitive dyes and Calcium-sensitive dyes to monitor the activities of individual neurons or multiple neurons. These dyes allow recording the neuronal membrane potential or Calcium changes with optical methods, i.e. the light emitted or absorbed by these dyes changes with the membrane potential or Calcium concentration and we can measure these changes.

Software tools used

Hardware

  • An Olympus BW51 fluorescent microscope is used to mount the preparation. A 20x Olympus objective with a numerical aperture of 0.95 gives us the best signal to noise ratio in the recorded optical data. A 10x objective with a numerical aperture of 0.3, however, is sufficient to record membrane potential changes of most neurons.
  • Data are recorded using a Micam02 from Scimedia. Best results for slow waveform changes (1 Hz oscillations of the membrane potential in pyloric neurons) were obtained with a sampling rate of 1.5ms per frame.

Sample data

  • Event-triggered average of optical recording (BV_Analyse format): Download here

Screenshots of sampled data



This is a single-sweep optical recording of three pattern generating neurons in the pyloric circuit of the stomatogastric ganglion.

This image show the simultaneous intracellular (bottom trace) and optical (top 3 traces) recordings of a pacemaker neuron in the stomatogastric ganglion. This neuron was injected with a Calcium-sensitive dye.

Photoablation.
Laser-photablation allows us to selectively remove individual neurons from the motor circuits in the stomatogastric ganglion. This way their influence on the motor pattern can be tested directly. We can also disable individual axons of a given neuron while keeping other axons or axonal compartments intact.

This image shows the electrophysiology rig during laser-photoablation of individual identified neurons.

Computer modeling

To achieve e better understanding of the mechanisms that generate and control rhythmic activity patterns, we combine our physiological recordings with computer modeling. We use a variety of different programs for this, including MadSim, which we created together with our colleagues from Ulm University. MadSim is a graphical user interface for modeling small neuronal networks (up to 100) neurons with Hodgkin-Huxley equations. Neurons are typically modeled as soma plus three-part dendrite. Axonal / Synaptical connections are represented as lines. MadSIm is available for free and maintained at Ulm University. You can download it here.


This figure shows the result of a network model generating the gastric mill motor pattern.

Behavioral studies.
A good part of our research uses the intact animal. We implant chronic recording electrodes to study long-term behavioral and sensory influences on the in vivo motor patterns. We use crabs and crayfish for our in vivo studies.

Rusty crayfish (Orconectes rusticus) female with babies hiding under her Pleon.


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