Indistinguishable odour enantiomers: Differences between peripheral and central-nervous electrophysiological responses

The ability of humans to discriminate enantiomeric odour pairs is substance –specific. Current literature suggests that psychophysical discrimination of odour enantiomers mainly depends on the peripheral processing at the level of the olfactory sensory neurons (OSN). To study the influence of central processing in discrimination, we investigated differences in the electrophysiological responses to psychophysically indistinguishable (+)- and (−)- rose oxide enantiomers at peripheral and central-nervous levels in humans. We recorded the electro-olfactogram (EOG) from the olfactory epithelium and the EEG-derived olfactory event-related potentials (OERP). Results from a psychophysical three alternative forced choice test indicated indistinguishability of the two odour enantiomers. In a total of 19 young participants EOG could be recorded in 74 and OERP in 95% of subjects. Significantly different EOG amplitudes and latencies were recorded in response to the 2 stimuli. However, no such differences in amplitude or latency emerged for the OERP. In conclusion, although the pair of enantiomer could be discriminated at a peripheral level this did not lead to a central-nervous/cognitive differentiation of the two stimuli.


Participants
A prospective study was conducted at the Smell and Taste Clinic at the Department of Otolaryngology of the "Technische Universität" (TU) Dresden. The study was performed according to the Declaration of Helsinki and approved by the Ethics Committee of the Medical Faculty at the TU Dresden (application number: EK361082016). All experiments were undertaken after subjects had provided written informed consent. Only participants between the age of 18 and 40 years with a normal sense of smell ascertained by means of Sniffin' Sticks 1 were included in the study.
The following exclusion criteria applied: pregnancy, previous head trauma leading to unconsciousness, chronic/acute rhinosinusitis, neurological diseases, any systemic disease associated with smell disorders like chronic renal failure or thyroid disorders, and impaired sense of smell. In addition, only participants who were clearly able to discriminate the two enantiomers were included in the study (i.e., with a discrimination score > 4 out of 9). Participants were instructed to only drink water one hour prior to the experiment and not to wear any scented products on the day of testing. A total number of 12 participants (6 women, 6 men) with an average age of 27.2 years (range: 21 to 40 years, SD: 5.1) was tested.

Odor stimuli and delivery
A pair of distinguishable odour enantiomers was used as stimulus 2 : (+)-limonene (50% v/v) (Sigma-Aldrich, CAS 5989-27-5) and (−)-limonene (50% v/v) (Sigma-Aldrich, CAS, 5989-54-8). Odours were always presented to one nostril using a computer-controlled air-dilution olfactometer (OM6b; Burghart, Wedel, Germany) capable of delivering odorants without altering mechanical or thermal conditions inside the nasal cavity. Stimuli were embedded in a constantly flowing air stream of 3 controlled temperature (36.5° C) and humidity (80% relative humidity) which was directed into the nasal cavity by means of a Teflon® tubing (8 cm length, 4/2 mm outer/inner diameter). Total flow rate was set at 6 L/min. With a sharp stimulus onset required, two thirds of the maximum stimulus concentration were reached at the olfactometer's outlet within 20 ms. During experiments subjects were seated comfortably in an air-conditioned room. White noise of approximately 50 dB SPL was used to mask clicking sounds associated with odour presentation. Further, participants were instructed to perform a tracking task on a video monitor during stimulation to keep them in an awake and vigilant state during the recordings. Before exposing participants to the odorants, a specific breathing technique (velopharyngeal closure) was trained in order to avoid respiratory flow inside the nasal cavity. Thirty presentations of each odor enantiomer with a duration of 500ms were randomly presented to each participant. The inter stimulus interval was set at 20 (±4) s during which odourless air was delivered. Participants were instructed to control their eye blinking as much as possible.

EEG setting
EEG was recorded during stimuli presentation from Cz, according to the international 10-20 system using an 8-channel amplifier (Nihon Kohden, Tokyo, Japan) referenced to linked earlobes (A1, A2). Blink artefacts were monitored from an additional recording site (Fp2). Stimulus-linked EEG-segments of 2048 s were digitally recorded at a frequency of 250 Hz (low-pass filter 15 Hz).
For recordings, pre-analysis was applied using Letswave 5 software (http://www.nocions.org/letswave5/). To establish baseline, recordings started 200 ms prior to stimulus onset. The epochs were inspected by eye for any kind of artefacts (blinks, muscle contractions etc.). Epochs containing amplitudes of 100 µV or higher were discarded. Clean epochs were baseline corrected, also filtered with a low-pass filter (30 Hz) and averaged in the time 4 domain. Then, the peak amplitudes (N1, P2, n1p2) and peak latencies (ln1, lp2) were measured using Letswave 5 software.

Behavioural testing
The ability to discriminate between the two enantiomers was assessed using 9 trials of a 3-AFC discrimination test. The participants had to discriminate the target odour [(+)-limonene from two identical non-target odours [(-)-limonene)]. The stimulus duration was set at 500 ms with an interstimulus interval (ISI) of 6s and approximately 20s (±4) between presentations of each triplet.
Subsequently, participants were also asked to provide ratings of odour intensity using a computerized visual analogue scale (VAS) ranging from 0 to 100 units (0 -"stimulus not perceived" to 100 -"very intense").

Behavioural data
All subjects showed a normal sense of smell with an average TDI score of 37.3 points (SD: 2.3). The average discrimination performance for the target odour was 6.2 (SD: 1.3). Applying a one sample t-test, this result differed significantly from chance level (t [11]) = 8.66, p < 0.001). Regarding the intensity ratings, no significant differences emerged between the two enantiomers [∅ intensity ---Please insert Figure S1 about here -