3.3.6. Discontinuous EAG recording

Discontinuous EAG does not require a gas chromatograph, but rather a stimulus delivery system for delivery of odours over the antenna at intervals as puff (sometimes referred to as puff stimulation). Stimulus delivery systems are commercially available as stand-alone devices or as accessories which can be installed on to a GC (Fig. 5a). It also requires a preparation of the stimulus in a manner different from natural volatile extracts (Syntech, 2004) as follows:

  1. Prepare a stock solution of 1μg/μl (equivalent to 1 mg/ml) by dissolving 5 mg of a pure compound of interest to the experimenter in 5 ml of solvent (e.g. hexane, dichloromethane, acetone ) and shake gently to dissolve the sample.
  2. Serially dilute the stock solution to prepare a range of doses (e.g. 100 ng/μl, 10 ng/μl, 1 ng/μl, etc.) to be tested.
  3. Place a cut piece of paper, preferably filter paper (3 cm long x1 cm wide), in the wide end of a standard Pasteur pipette.
  4. Apply a specified amount of the solution of the compound to be tested. For example apply 1 μl of a 1 μg/μl solution to test 1 μg of the compound, etc.
  5. Allow the solvent to evaporate for 30 s to several min and gently push the filter paper completely into the Pasteur pipette. Seal both ends of the pipette using Teflon tape or parafilm.
  6. Label the pipette by the side to indicate the stimulus type (code names for test compound and concentration).
  7. Prepare three control stimuli consisting of:
    7a. a clean Pasteur pipette,
    7b. a pipette containing filter paper only,
    7c. a pipette with filter paper with only solvent applied.
    These three controls check for pipette, filter paper and solvent contamination respectively.
  8. Place the smaller end of the pipette inside the hole on the side of the delivery tube (Fig. 5a) and connect its wider end to an air supply after removing the Teflon tape or parafilm.
  9. Apply the stimulus over the antenna by puffing using the EAG puff pedal for about 0.5-3 s, in the order; control stimulus followed by the test stimuli and finally control stimuli again.  You may randomize the application of the test and control stimuli on the antennae, and puff each test stimulus several times since only a fraction of the test compound is delivered in each puff. 
    Stimulus application should be done at intervals of 30-120 s to allow the antenna to recover from the previous stimulus.
  10. Record antenna signals as described in section 3.3.5. and open files later for analysis (see Fig. 6b for an example of a stimulus-puff recording).

EAG recordings are limited in their sensitivity and specificity to certain chemicals because responses to these chemicals are a summation of the total depolarisations elicited by the chemicals across the mounted antenna at the level of the olfactory receptor neurones (ORNs) contained within each receptor cells (sensillum) (Christensen, 2004). As such, chemicals which elicit action potentials on very small number of ORNs are often not detected in EAG recordings. A newer technique, single-sensillum recording (SSR) partially overcomes EAG limitations by recording antennal electrical activity at the level of ORNs (Millar and Haynes, 1998; Christensen, 2004). Details on the equipment type, experimental protocols and analysis of results in SSR studies have been well described by Christensen (2004).

Fig. 6b. A discontinuous (puff stimulation) electro-antennal recording output showing antennal responses to three different doses (10 ng, 10 pg and 10 μg respectively) of a single chemical applied over the antenna at approximately 1 min intervals.