Electrophysiologic assessment of olfactory and gustatory function

Handbook of Clinical Neurology, Vol. 164 (3rd series) Smell and Taste R.L. Doty, Editor https://doi.org/10.1016/B978-0-444-63855-7.00016-2 Copyright © 2019 Elsevier B.V. All rights reserved

Chapter 16

Electrophysiologic assessment of olfactory and gustatory function HILMAR GUDZIOL* AND ORLANDO GUNTINAS-LICHIUS Department of Otorhinolaryngology, University Hospital, Jena, Germany

Abstract This chapter reviews approaches for assessing human and gustatory function using electrophysiologic methods. Its focus is on changes in electrical signals, including summated generator potentials that occur after nasal or oral stimulation. In the first part of the review, we describe tools available to the clinician for assessing olfactory and nasotrigeminal function, including modern electroencephalography (EEG) analysis of brain responses both in the time domain and in the time-frequency (TF) domain. Particular attention is paid to chemosensory event-related potentials (CSERPs) and their potential use in medical–legal cases. Additionally, we focus on the changes of summated generator potentials from the olfactory and respiratory nasal epithelium that could provide new diagnostic insights. In the second part, we describe gustatory event-related potentials (gCSERPs) obtained using a relatively new computer controlled gustometer. A device for presenting different pulses of electrical current to the tongue is also described, with weaker pulses likely reflecting gCSERPs and stronger ones trigeminal CSERPs. Finally, summated generator potentials from the surface of the tongue during gustatory stimulation are described that may prove useful for examining peripheral taste function.

INTRODUCTION This chapter describes state-of-the-art electrophysiologic methods for assessing chemosensation. The focus is on electroencephalographic (EEG) responses to chemical stimuli measured from the scalp and peripheral summated generator potentials measured from the surface of olfactory and gustatory epithelia. The first section of the chapter focuses on the nasal olfactory and trigeminal systems, whereas the second focuses on lingual chemosensory systems. Emphasis is placed on the assessment of olfactory or gustatory disorders. Strengths and weaknesses of the techniques are discussed, as are areas of uncertainty and the need for future studies.

ELECTROPHYSIOLOGIC ASSESSMENT OF NASAL OLFACTORY AND TRIGEMINAL FUNCTION EEG responses QUANTITATIVE CHANGES OF OLFACTORY ELECTROENCEPHALOGRAPHY

In relaxed wakefulness, eye-opening suppresses the a rhythm of the electroencephalography (EEG) signal. This phenomenon, known as Berger effect (Berger, 1929, 1932), can also be evoked by odorants (Gudziol and Gramowski, 1982). Quantitative changes in EEG responses during the application of odorants can be measured by frequency analysis, e.g., by fast Fourier

*Correspondence to: Hilmar Gudziol, Prof. Dr. Med., ENT Clinic, University Hospital, Am Klinikum 1, Jena, Thuringia, 07747, Germany. Tel: +49-3641-9-329301, Fax: +49-3641-9-329302, E-mail: [email protected]

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transform (FFT). It has been shown that the power of a (8–13 Hz) and b (13–64 Hz) frequency bands is reduced at various scalp locations during the presentation of different odorants (van Toller, 1987). Significant differences have also been shown in the power of a frequency bands at frontal and central recording sites, depending upon the involved odorant (Hummel et al., 1989). Bonanni et al. (2006) investigated quantitative changes of EEG signal during olfactory stimulation in 25 posttraumatic anosmics. By means of a flowolfactometer, odorous air containing 0.5% (v/v) benzaldehyde was presented during inspiration. In a subgroup of eight patients with visually unrecognizable blocked a waves induced by olfactory stimulation, no change in the EEG power in all frequency bands was observed using FFT. In a subgroup with a positive odor-induced Berger effect, the y band power at the central electrode localizations (Fz, Cz, Pz) was higher after stimulation, whereas the a band power was lower at electrodes O1, O2, T5, T6. Although all patients were anosmic and denied any olfactory perception during olfactory stimulation, EEG changes occurred. The basis of this phenomenon is not known but could reflect some residual olfactory function or recovered sense of smell or an unintended irritation. Increases in EEG amplitudes at the a frequency have also been shown during odor stimulation (Toller et al., 1993). Moreover, changes of power spectra have been investigated in relation to pleasant and unpleasant odors. In one study, the unpleasant-smelling odorant valeric acid evoked increased a power at frontal and parietal electrode locations in comparison to the pleasantsmelling odorant phenyl ethyl alcohol (PEA) (Brauchli et al., 1995). In another study, Owen and Patterson (2002) found that, of 36 subjects tested, 15 liked, 8 disliked, and 13 were indifferent to the odor of damascenone (fruity, berry smell). Those who disliked the odor tended to have higher a and y power responses at frontal EEG electrodes compared to those who liked or were indifferent to this odor. Interestingly, this phenomenon can be shown with imagery without the presentation of an odor, suggesting that the general level of activity likely reflects cognitive more than olfactory processes (Lorig, 1989). That being said, EEG power investigations of hedonic aspects of odor perception are rare and inconsistent, and the extant studies are confounded by odorant species. Clearly, more studies are needed in this area.

CONTINGENT NEGATIVE VARIATION After an initial stimulus, and in expectation and in preparation for responding to a second stimulus, a slowly rising negative shift of the EEG baseline appears. After the second stimulus is presented, this response immediately

drops away. Walter termed this negative expectancy potential the contingent negative variation (CNV) (Walter, 1964). A few studies have used CNV to assess olfactory function. When the first stimulus is an odorant and the second is a tone or a light flash, the direct CNV slowly builds up and stops at the second stimulus. If two different odorants (e.g., A or B) are intermittently used as the first stimulus, but the second stimulus follows only one of the odorants, an elective CNV develops following only that odorant. In other words, cognitive performance in discriminating the two odorants is thus captured (Gerull et al., 1981; Lorig and Roberts, 1990). Mrowinski et al. (2002) recorded CNVs from 25 normosmic adults and from 16 patients with anosmia. In normosmics, the direct CNV was found in 84% and the selective CNV in 92%. No CNVs were observed in patients with anosmia. The authors recommended recording the CNV as an additional robust and objective olfactometric tool—a tool particularly useful for medicolegal examinations.

CHEMOSENSORY EVENT-RELATED POTENTIALS The University of Erlangen-Nuremberg, Germany, was the cradle for the development of modern electrophysiologic chemosensory assessment procedures. In 1966, Finkenzeller was the first to report a successful recording of an olfactory chemosensory event-related potential (oCSERP) (Finkenzeller, 1966). Kobal and Plattig (1978), also from the same university, introduced new flow-olfactometer technology ideal for measuring oCSERPs. In 1981, Kobal published his pioneering electrophysiologic investigations on the human sense of smell with a self-made olfactometer (Kobal, 1981). Since then, the circuit principles and both the hardware and software have continuously improved. Olfactometers useful for such measurement have been commercially available since 1990, with most being manufactured by Burghart Medical Technology (Wedel, Germany). These olfactometers still use the switching principle described by Kobal (1981). This principle makes it possible to rapidly shift from an odorless airstream to an odorized airstream without noticeable pressure fluctuations. The embedded rectangular-shaped stimulus can be varied in odor quality, concentration, and duration, and trains of such stimuli can be presented at different interstimulus intervals. The humidified and temperature-controlled gas streams pass continuously through a Teflon tube into one nostril. A relative high airflow and a short distance from the switching point to the exit of the olfactometer provide a rapid and steep stimulus onset essential for the emergence of the CSERP. Although most odorants stimulate both olfactory and trigeminal afferents, a few primarily stimulate the

ELECTROPHYSIOLOGIC ASSESSMENT OF OLFACTORY AND GUSTATORY FUNCTION olfactory system (e.g., vanillin, PEA, low concentrations of H2S). Odorless gaseous CO2 is commonly employed to assess the nasal trigeminal system. In clinical practice, scalp recording sites usually include five electrodes (international 10/20 positions Fz, Cz, Pz, C3, and C4) referenced to linked earlobes (A1 + A2). In most cases, the measurement of reliable CSERPs requires the recording and averaging of 10–30 consecutive trials (Rombaux et al., 2009). Blinking artifacts are registered at Fp2, and EEG epochs contaminated by artifacts are discarded. The single-subject mean wave is considered as a present CSERP when two different observers independently agree. The peaks of the negative–positive complex of the CSERP are then manually measured (latencies and amplitudes), usually based on the following criteria: the largest negative peak between 200 and 700 ms is considered to be N1; the P2 peak is measured between 300 and 800 ms (Fig. 16.1) (Hummel et al., 2000; Lascano et al., 2017). The early components (P1 and N1) mostly represent stimulus properties (e.g. odorant concentration), whereas the late components (P2 and P3)

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represent higher-order endogenous responses, i.e., are related to novelty, familiarity, pleasantness, or significance (Kobal et al., 1992; Pause et al., 1996; Lorig, 2000; Rombaux et al., 2012; Pellegrino et al., 2017). Influence of stimulus concentration and duration on CSERPs Kobal concluded in 1981 that only the stimulus onset is decisive for latencies and amplitudes of the oCSERP (Kobal, 1981). A few years later, three studies found that a shortening of CSERP latencies occurs with higher odor concentrations, whereas CSERP amplitudes were not impacted by stimulus concentration (Thiele and Kobal, 1984; Pause et al., 1997; Covington et al., 1999). Only one study has reported that higher odor concentrations result in larger amplitudes and shorter latencies of oCSERPs (Tateyama et al., 1998). Wang et al. (2002) found in 12 healthy adults that larger N1-P3 oCSERP amplitudes correlated with increasing stimulus durations (stimulus strengths), whereas latencies

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D Fig. 16.1. Olfactory chemosensory event-related potentials (CSERPs). CSERPs are usually recorded from Pz, Fz, and Cz referenced to the linked mastoid electrodes (M1/M2) or earlobes (A1/A2). (A) Single trace evoked potential (EP) response recorded at a parietal electrode, showing an unstable initial component (P1) followed by N1 and P2–P3 complex observed between 300 and 1000 ms after stimulation with the unpleasant odor hydrogen sulfide. (B) High density EP topographical analysis of 12 healthy controls portrayed as a butterfly plot of 64 superimposed electrodes. (C) and (D) Scalp topographies of the three main components and their probable corresponding intracranial generators with an initial ipsilateral activation of the anterior temporal lobe, followed by bilateral activation of mesial posterior temporal structures. Adapted with permission from Lascano, A.M., Hummel, T., Lacroix, J.S., et al., 2010. Spatio-temporal dynamics of olfactory processing in the human brain: an event-related source imaging study. Neuroscience 167, 700–708.

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were not affected. More recently, Frasnelli et al. (2006) also found, using variable stimulus durations, that longer stimulus durations led to larger CSERP amplitudes. No effect on CSERP latencies was found. In that same year, a larger study with 95 healthy adults showed that higher concentrations of H2S and CO2 were associated with larger amplitudes at P2, while latencies and amplitude at N1 were unchanged (Stuck et al., 2006). Frasnelli et al. (2003) found that both N1 and P3 amplitudes increased when CO2 stimulus concentrations were increased. Interestingly, the late complex of the trigeminal CSERP (tCSERP) was also involved in the encoding of CO2 concentration. No significant relation was found between tCSERP latencies and stimulus concentration. Influence of flow rate on CSERPs Nasal flow rate is important because it influences the number of chemosensory molecules reaching and being absorbed by the olfactory or respiratory mucosa. Thus, the strength of a chemosensory stimulus is dependent on the concentration of molecules in the air, on stimulus duration, and on the flow rate. Kobal and Hummel (1991) investigated this association in more detail and found that the intensity estimates and amplitudes N1-P2 of the oCSERP correlate with increasing odor concentrations and with increasing flow rates at a constant concentration. The stimulus duration was set at 200 ms (Kobal and Hummel, 1991). If the flow rate did not exceed 85 mL/s, no CSERP could be displayed (Kobal, 1981). A higher flow rate (8 L/min compared to 4 L/min) improved the signal-to-noise ratio (S/N ratio) of the oCSERPs, reflecting an increase in the steepness of stimulus onset (Han et al., 2018). For clinical use, a technical and physiologic compromise has been made using a flow rate of 140 mL/s. Influence of interstimulus interval and stimulus repetitions on CSERPs For the recording of the oCSERPs, interstimulus intervals (ISIs) of 30–40 s are typically employed, along with 16–20 stimulus repetitions. To increase the S/N ratio, more repeated stimulus applications seem advantageous. However, this requires extended recording time. Shortening the ISI increases the probability of unintended, adaptive, and habitual influences. In the past few years, some studies have examined the effect of shortening the recording time by decreasing ISIs (Kassab et al., 2009; Schaub and Damm, 2012). Long ISIs, usually over 30 s, are intended to reduce the negative effects of habituation on the amplitude and, thus, on the detectability of the CSERPs (Schriever et al., 2014). A typical 20-fold repetition of stimuli has been used in an effort to improve the S/N ratio. Long recording

times are difficult for both the subject and the experimenter, so numerous approaches to minimize this time have been evaluated. Standard recording of CSERPs requires about 1 h. If the ISI is shortened to 20 or 10 s, reliable CSERPs were observed in young normosmic adults. However, the latencies were longer and the amplitudes smaller (Kassab et al., 2009). When a 30 s ISI sequence and a pseudorandomized 15 s ISI sequence was used, CSERPs were computed in similar numbers for both young normosmic and older hyposmic adults. The advantage of this paradigm is that the recording time is considerably reduced. In this study, the S/N ratio was not different from the standard protocol (30 s ISI) (Schaub and Damm, 2012). In a large study by Whitcroft et al. (2017), ISIs were shortened to 10 s for the recording of CSERPs in 101 healthy adults and patients with impaired smelling ability. By concomitantly increasing the PEA stimulus repetitions, the S/N ratio improved significantly in normosmics and functionally anosmic patients but not in hyposmics. In accord with an earlier CSERP study applying longer ISIs, the proportion of detectable CSERPs in the three groups was unchanged (L€otsch and Hummel, 2006). Interestingly, the hyposmics showed shorter latencies at P1 and N1 and larger amplitudes at N1 and P2 than normosmics. The authors hypothesize that hyposmics might pay more attention to olfactory stimuli, i.e., that olfactory vigilance might be increased in hyposmics compared to normosmics. This is an interesting finding that should be confirmed with larger studies. Two studies have tested the extremes of stimulus repetitions. Boesveldt et al. (2007) used up to 160 stimulus repetitions with 25–35 s ISIs using H2S, PEA, and CO2 as stimuli. The S/N ratio in EEG responses to PEA increased significantly up to 80 averages. The number of stimuli for an optimal S/N ratio for tCSERP was 60 averages. The recording time rose to at least 100 min, generally an unacceptably long time, especially for a patient (Boesveldt et al., 2007). Wetter et al. found that it is possible to obtain oCSERPs with three single stimuli and extreme ISIs of 10 min under the assumption that for restless patients this made it easier to objectively verify their function (Wetter et al., 2004). The interpeak amplitude N1P2 of the tCSERPs becomes smaller in relation to shortening of the ISIs. Data indicate that ISI of less than 20 s in combination with 16 stimulus repetitions (CO2 50%, v/v) is of questionable value for clinical investigations (Hummel and Kobal, 1999). Test–retest reliability of CSERPs The parameters of the CSERPs show reasonably good test–retest reliability. Most test–retest Pearson

ELECTROPHYSIOLOGIC ASSESSMENT OF OLFACTORY AND GUSTATORY FUNCTION correlation coefficients have ranged between 0.4 and 0.75. CSERP reliability is comparable to that of auditory and visual ERPs, assisting the application of CSERPs in both research and clinical assessment (Thesen and Murphy, 2002; Welge-L€ ussen et al., 2003).

Attention/vigilance A traditional CSERPs test session requires about an hour. In such a session, both sides of the nose are tested with each of two odorants (H2S and PEA) and a trigeminal stimulus (CO2) at an ISI of 30 s with 16 stimulus repetitions. This is a long time period, particularly in a clinical setting when a subject’s attention tends to wax and wane. The following measures have been recommended to keep the level of vigilance high during such sessions: 1.

2. 3.

4.

5.

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Minimize the checking of the olfactometer and the EEG recording system during recording sessions, i.e., do not disrupt the subject’s attention. Explain the scheduled protocol to the subject before testing in a psychologically sensitive manner. During the recording, the subject should not speak, and barely move, blink and swallow. This has to be made clear to the subject beforehand. Decide whether or not both sides of the nose and multiple odorants should be employed in the same session to shorten the recording time. Employ a tracking game, e.g., by having subjects move a joystick to hold a white dot inside a larger moving square (Kobal et al., 1992); alternatively, by having subjects perform mental calculations (e.g., counting stimuli presented on a computer screen). Decide whether a different stimulation protocol should be used (e.g., shorter ISI, single stimulus or interspersed stimulus paradigm).

Influence of gender on CSERPs Various studies have assessed whether sex differences are present in CSERP parameters. None were found in the oCSERP amplitudes measured by Geisler and Murphy (2000). Similarly, in a tCSERP study by Hummel et al. (1998a), the N1/P2 amplitudes did not differ between men and women. However, other studies have reported larger P2 amplitudes in women than in men (Evans et al., 1995; Hummel et al., 1998a; Olofsson and Nordin, 2004; Stuck et al., 2006; Haehner et al., 2011). Haehner et al. reported that not only were the P2 amplitudes larger in women but that the latencies of N1 and P2 of tCSERPs were shorter (Haehner et al., 2011).

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Influence of aging on CSERPs A number of studies suggest that older persons have significantly longer oCSERP latencies than younger ones and that amplitudes decline in size over decades of life (Hummel et al., 1998a; Murphy et al., 1998, 2000; Stuck et al., 2006). However, this does not always seem to be the case. For example, Covington et al. (1999) described shorter latencies in older adults when stronger, compared to weaker, olfactory stimuli were employed. The authors speculated that the common shortening of CSERP latencies was due to odorant concentrations that exceeded the age-related latency prolongation. The elderly brain seems to slow down olfactory processing and has difficulty shifting attention to new stimuli. Suprathreshold odor concentrations can accelerate the speed of that processing independent of age. These discrepant findings should be clarified. That being said, the age-dependent olfactory recruitment phenomenon may have the power to be used as a diagnostic tool for age-related impairment of olfaction.

Active or passive chemosensory stimulus application When a subject breathes through the nose, the respiratory pressure fluctuations can disturb the steady stimulus airflow. This can produce turbulences that blur the boundary between the odorized and nonodorized airstreams. That is why Kobal recommended a special breathing technique in which subjects breathe through the mouth while employing active velopharyngeal closure (VC). Thesen and Murphy compared CSERPs from subjects who breathed naturally (with mixed nasal and oral breathing and no VC) to those who breathed only orally while performing VC (Thesen and Murphy, 2001). CSERPs of the two types of breathing were not significantly different. However, use of VC generated larger amplitudes of N1P2. Natural breathing showed only a trend toward shorter latencies of the late complex (P2 + P3) of CSERPs. This finding could reflect the habitual expectation that the next stimulus could be triggered by the olfactometer in the following inspiration. In a study of 40 subjects, Haehner et al. (2011) presented odors from a flow-olfactometer that were independent of the respiratory cycle to subjects who were practicing VC. Offline, the individual EEG epochs were assigned to either inspiration or expiration categories and then averaged. The OERPs were larger when the stimuli, including CO2, had been presented during inspiration. The N1 amplitudes were increased for all applied stimulants. In addition, during inspiration-synchronous trigeminal stimulation, the amplitude P2 was larger than during expiration. The latencies were not affected by

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the respiratory cycle. The results suggest that inspiration boosts the nasal chemosensory system for stimulants independent of the nasal inspiratory airflow. Lorig et al. (1996) found that oCSERPs amplitudes in response to 2% and 4% n-butanol concentrations were higher during mouth breathing and passive respirationasynchronous olfactory stimulation than during nasal breathing and active inspiration-synchronous stimulation. The authors attribute this finding to higher attention in respect to the next expected stimulus. This suggests that stimuli that are presented independent of the stage of the respiratory cycle may be a better measure of the integrity of the olfactory pathways. The authors of this chapter favor passive stimulation. Detectability of CSERP components The S/N ratio is sometimes so small that, even in normosmics, no CSERP components can be discriminated from background noise after conventional stimulation. Two components of the CSERPs should at least be detected in the averaged curve. L€ otsch and Hummel (2006) did not detect oCSERPs in 13 out of 40 normosmic subjects when stimulating with PEA. But three of them exhibited responses to H2S. In 8 of the remaining 10 subjects, the failed detection of oCSERPs was explained by a large number of artifacts such as high muscular activity, constant presence of eye blinks with low amplitudes, and low-frequency and high-amplitude brain waves. In two subjects no obvious reason for the absence of oCSERPs was detected. It was speculated that a low level of attention was the cause in these cases. The finding of missing oCSERPs in some normosmics has been confirmed by others. Whitcroft et al. (2017) found no oCSERPs in six out of 41 normosmics when stimulating with PEA, using a standard 30 s ISI. This corresponds to 15% of all test subjects. Functional anosmia is defined as very low olfactory test performance in persons with impaired olfaction but who are still able to perceive some odors. Patients with functional anosmia can also exhibit oCSERPs

(Hummel et al., 2007). L€otsch and Hummel (2006) could detect oCSERPs in eight out of 40 anosmics by stimulation with the PEA standard protocol (30 s ISI, 20 repetitions). The presence of oCSERPs in anosmia was confirmed by Whitcroft et al. (2017). Recently, they found oCSERPs in 14 out of 40 anosmics stimulated with PEA using a 30 s ISI and 20 stimulus repetitions (Table 16.1). Rombaux et al. (2009) reported a prevalence of oCSERPs of 2.7% in a cohort of 115 anosmics defined as a TDI score < 16.5. TDI score is an individual summated threshold-, discrimination- and identification score, distinguishing normosmia, hyposmia, and anosmia by Sniffin’Sticks® (Hummel et al., 2007). Not all hyposmics (defined as having an individual olfactory TDI score of <30.5 and >15.5) generate oCSERPs. The detection of oCSERPs is dependent on the severity of the hyposmia. L€otsch and Hummel (2006) identified in their 123 subjects cohort (40 normosmics, 40 hyposmics, 43 anosmics) a TDI score of 22.6 (95% confidence interval: 16.1–27.8) as the turning point where the probability of recordable oCSERPs exceeded 50%. Rombaux et al. (2009) confirmed this result. The mean TDI value of 229 hyposmic and anosmic patients was 24.19 (95% confidence interval: 22.9–25.4) for the detection of oCSERPs. Whether the presence or absence of oCSERPs has any significance for the prognosis of the impaired olfaction in hyposmics requires further study (Rombaux et al., 2007). In a subsequent study, Rombaux et al. (2010) assessed the prognostic value of the detectability of oCSERPs. A marked improvement of smell was seen in patients after postinfectious olfactory impairment during a mean period of 8.6 months. Regarding oCSERPs, they could show that the detectability of oCSERPs at the first examination was linked to a better prognosis of recovery of olfaction.

GLOBAL FIELD POWER AND GLOBAL MAP DISSIMILARITY In the search for specific biomarkers for Parkinson’s disease (PD), Iannilli et al. (2017) sought to obtain more

Table 16.1 Detectability of oCSERPs in two stimulus protocols (Whitcroft et al., 2017) Olfactory function Stimulus protocol

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Present Absent Present Absent

14 (35%) 26 9 (22.5%) 31

14 (70%) 6 11 (55%) 9

35 (85%) 6 37 (90%) 4

PEA: phenylethyl alcohol, ISI: interstimulus interval.

ELECTROPHYSIOLOGIC ASSESSMENT OF OLFACTORY AND GUSTATORY FUNCTION specific information from the unspecific early symptom of an olfactory loss using global analyses of olfactory EEG responses. With reference-independent EEG analysis, information was generated after averaging olfactory stimulation, related to the response strength, timing, and topography detected by global field power (GFP) and global map dissimilarity (GMD). An example of a normosmic subject is demonstrated in Fig. 16.2. The significantly shorter latency of the first peristimulus peak (PST1) at GFP maximum in patients with PD compared to hyposmics could be an interesting discriminator. The authors also point out the importance of the hyporesponse of the late ERP signal, visible in the lowest display of area 3 (A3), in patients with PD. These findings could be interpreted as suggesting that PD damages central olfaction-related brain regions more than peripheral structures.

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CHEMOSENSORY RESPONSE EEG ANALYSIS IN CONTINUOUS WAVELET TRANSFORM

TIME-FREQUENCY DOMAIN BY

In a study of 11 subjects, Huart et al. (2012) compared the detectability of oCSERPs in the time domain to those in the TF domain using the “Continuous Wavelet Transform” (CWT). Only a few subjects demonstrated oCSERPs. In the time domain, the discrimination between EEG segments with and without olfactory stimulation was in a range expected by chance. In the TF domain, the use of single trials and subsequent averaging enhanced phase-locked responses and identified two nonphase-locked olfactory EEG responses: a longlasting increase in the amplitude of low EEG frequencies centered around 5 Hz (OLF-TF1) and a desynchronization in the a-band (OLF-TF2). The S/N ratio and the

Fig. 16.2. (A) Example of an average of 64 EEG channels during olfactory stimulation of a normosmic subject. (a) Average 64-channel plot referred to the earlobe. (b) 64-channel butterfly plot referred to the electrodes average. (c) Relative global field power (GFP) function and global map dissimilarity (GMD). The interesting time frames were delimited by the maximum of GDM function; the correspondent area under the GFP was measured and called A1, A2, and A3. The peristimulus peak (PST) was measured in correspondence of the GFP, maximum within the chosen time frame and called PST1, PST2, and PST3, respectively. The average among all the electrodes was used as a reference for the butterfly plot. (B) Distribution of the electrodes on the scalp and relative electrodes scalp subregion. From Iannilli, E., Stephan, L., Hummel, T., et al., 2017. Olfactory impairment in Parkinson’s disease is a consequence of central nervous system decline. J Neurol 264, 1236–1246. Adapted with permission from Springer Nature.

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H. GUDZIOL AND O. GUNTINAS-LICHIUS CWT SINGLE was significantly stronger than the discrimination performance of oCSERPs. Normosmics and hyposmics could be discriminated with a sensitivity of 91% and a specificity of 82%, and normosmics and anosmics with a sensitivity of 91% and a specificity of 91%. The authors conclude that the TF analysis of the EEG responses to olfactory stimulation is an efficient tool for the objective assessment of olfactory function in patients. In a recent study, 20 young healthy and 18 elderly patients with impaired smelling ability were examined after olfactory and trigeminal stimulation for a change in the power of EEG responses (Schriever et al., 2017). The AUC (area under the curve) calculated by ROC (receiver operating characteristic) analysis of EEG power change determined by CWT SINGLE was found to reliably discriminate between normosmics and anosmics (Fig. 16.3). Hyposmics could not be distinguished. In another study from the same working group, the influence of stimulus concentration and flow rate on olfactory processing was investigated. EEG data were analyzed by averaging in the time domain and in single-trial TF domain (CWT SINGLE). Higher PEA concentrations resulted in increased brain oscillations in the slow frequency d-band (1–3 Hz) at 0–600 ms after stimulus onset. Higher airflow rates attenuated d-band oscillations at 900–1500 ms after stimulus onset and enhanced y band oscillations (5–9 Hz) at 300–600 ms after stimulus onset (Han et al., 2018).

response detectability increased significantly. The OLFTF1 clearly discriminated between unstimulated EEG segments and olfactory stimulated segments (with a sensitivity of 81.8% and a specificity of 90.9%) (Huart et al., 2012). Huart et al. (2013) examined the clinical usefulness of CWT of CSERPs. Thirty-three patients complaining of disturbed olfaction were classified by the TDI score as normosmic, hyposmic, or anosmic. For each patient, across-trial averaging of the EEG segments in the time domain (CSERP) and the TF domain (CWT AVERAGE) for olfactory and trigeminal stimulation was performed. To obtain a TF representation of both phase-locked and nonphase-locked EEG responses to trigeminal and olfactory stimulation, the TF transform was also applied to each single EEG segment (CWT SINGLE). These single-trial TF maps were then averaged across trials. In the across-trial averaging in time domain, due to olfactory stimulation, only 6 out of 11 normosmics, 4 out of 11 hyposmics, and 1 out of 11 anosmics had visually detectable CSERPs. The discrimination between the three groups of patients using the components of the olfactory and trigeminal CSERPs was poor. TF values of the phase-locked EEG responses to olfactory and trigeminal stimulation (CWT AVERAGE) showed, in normosmics, a small increase of low frequency activity. In hyposmics, this phase-locked activity was less pronounced. In anosmics this low frequency activity was hardly detectable. In contrast, the trigeminal phase-locked activity was well identifiable in all three. Its amplitude appeared reduced in the anosmic group. In normosmics, the CWT SINGLE represents a marked magnitude of low frequency EEG oscillations. In this Huart et al. study, that value was reduced in hyposmics and was no longer detectable in anosmics. The discrimination performance of these three study groups using

Summated generator potential of nasal, olfactory, and respiratory epithelium ELECTRO-OLFACTOGRAM The sum of evoked generator potentials of olfactory receptor neurons can be recorded intranasally as an

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Fig. 16.3. EEG-power change (CWT SINGLE) after olfactory, trigeminal, and control stimulation. Displayed are the Cz EEGpower changes (group average including the 10 participants) after control (A), olfactory (B), and trigeminal (C) stimulation. The dashed black line marks the stimulus onset. An increase in low frequency within the ROI (red square) can be observed in b and c but not in a. The scale displays EEG-power change in percent compared to the prestimulus interval. Adapted with permission from Schriever, V.A., Han, P., Weise, S., et al., 2017. Time frequency analysis of olfactory induced EEG-power change. PLoS One 12, e0185596.

ELECTROPHYSIOLOGIC ASSESSMENT OF OLFACTORY AND GUSTATORY FUNCTION

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6.

The success rate for obtaining an EOG in a given subject is
75% (Lapid and Hummel, 2013). Increasing EOG amplitudes are correlated with increasing stimulus concentrations and perceived intensities (Lapid et al., 2009). The perception of olfactory stimuli decreases more quickly than the EOG signals; i.e., EOGs remain in response to stimuli that subjects can no longer perceive because of central habituation (Hummel et al., 1996, 2006). EOG amplitudes are higher when triggered by orthonasal than by retronasal stimulation (Hummel et al., 2006, 2017). Different recording sites are differentially tuned, implying that the distribution of human receptor subtypes is neither uniform nor random (Lapid and Hummel, 2013). Odorant hedonic tone appears to be mapped onto the olfactory epithelium (Lapid et al., 2011).

EOG recordings are not yet employed in routine clinical practice or in the assessment of neuropsychiatric disorders. In one study, patients with schizophrenia produced significantly higher EOG amplitudes following H2S stimulation than healthy subjects. The cause remains unclear. Future studies are needed to clarify whether such EOG abnormalities are found in new-onset untreated schizophrenia patients, patients with prodromal symptoms, or in patients with other related abnormalities (Turetsky et al., 2009).

NEGATIVE MUCOSA POTENTIAL Negative mucosal potentials (NMPs) are suitable to investigate the responsiveness of the peripheral trigeminal system (Kobal, 1985b; Th€ urauf et al., 1991). The recording technique is similar to EOG recording. A practical difference is that recordings take place at the more accessible respiratory epithelium of the main nasal cavity. NMPs can be recorded as a result of stimulation with CO2 but cannot be recorded in response to most olfactory stimuli (Thu et al., 1993). NMP amplitudes have been shown to be related to stimulus concentration, stimulus duration, and perceived stimulus intensity (Thu et al., 1993; Hummel et al., 1998b; Th€ urauf et al., 2002). The highest sensitivity exists in the anterior septum nasi. These findings are compatible with the idea that the trigeminal system acts as a guardian

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electro-olfactogram (EOG), a negative voltage transient. Although the olfactory epithelium is theoretically directly in contact with the environment, it is hidden behind the upper and middle nasal turbinates and is, therefore, difficult to access for recording. Some conclusions from EOG studies are as follows:

– 50 µV + NMP P1 Stimulus N1 EEG P2 0

4000

8000

12,000

16,000

Time (ms)

Fig. 16.4. Negative mucosa potential (NMP, top) and chemosensory event related potentials (tCSERP) (EEG, bottom) after painful stimulation of the nasal mucosa with CO2. The onset of the NMP is observed immediately after application of the painful stimulus, preceding the onset of the tCSERP. From L€ otsch, J., Hummel, T., Kraetsch, H., et al., 1997. The negative mucosal potential: separating central and peripheral effects of NSAIDs in man, Eur J Clin Pharmacol 52, 359–364. Adapted with permission from Springer Nature.

to alter or stop breathing to avert the inhalation of noxious irritants (Scheibe et al., 2006). The area under the NMP curve correlates highly with the amplitude N1P2 of the pain-related chemosensory evoked potentials (Fig. 16.4) (L€otsch et al., 1997; Hummel et al., 2003).

ELECTROPHYSIOLOGIC ASSESSMENT OF ORAL GUSTATORY FUNCTION EEG responses (gustatory and electrogustatory CSERPs) The development of a taste stimulation method applicable for artifact-free measurement of cortical gustatory responses took much longer than the development of the air-dilution flow-olfactometer. Switching the liquid tastant pulse to a tasteless liquid stream produced disturbing pressure fluctuations that confounded tactile stimulation with gustatory stimulation. Plattig sought to avoid this by using electric taste stimulation (Plattig, 1968a, 1968b/1969). Twenty years later, he published a paper in which a liquid-dilution flow-gustometer was shown

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to be able to generate gustatory CSERPs (gCSERP) with various liquid tastants (Plattig, 1989). The constant flow of liquid across the tongue was unpleasant because the subjects were not allowed to swallow. The liquid had to drop out of the mouth into a collection vessel. Therefore, approaches to more comfortable means of presenting stimuli were sought, most notably the presentation of tastants in the form of gases. The principle of a gaseous gustometer was first introduced in 1905 by Sternberg (1905). However, for an electrophysiologic examination, subsequent workers had to develop a means of applying the stimulus in a more systematic manner. Ideally, artifact-free rectangularshaped taste pulses are needed, in principle, analogous to the pulses employed in oCSERPs. One approach to achieve this end was to add sour-tasting acetic acid vapor into the constant airstream of an olfactometer. Then pulses of sour-tasting stimuli could be applied artifactfree by air dilution flow-olfactometer, and gCSERPs could be derived (Kobal, 1985a). The first systematic investigation of a gCSERP was carried out with a commercial gustometer in 2010 (Hummel et al., 2010). The latencies of P1 and N1 became shorter with increasing acetic acid vapor concentration. At the same time, the amplitude of P2 increased. In general, women elicited larger amplitudes and shorter latencies of gCSERPs than did men. Amplitude P1 exhibited a frontocentral maximum while amplitude P2 was largest at parietal sites. Test–retest reliability for amplitudes and latencies at position Cz, expressed as the coefficient of correlation, ranged between 0.77 and 0.97. The stimulator made it possible to test each side of the tongue separately. Detectable gCSERPs exhibited shorter response latencies after right- than after left-sided stimulation. More recently, to stimulate regions of the tongue with liquids of small volumes, a computer-controlled gustometer was constructed and made commercially available (Gu002/GM05, Burghart Medical Technology, Wedel, Germany). This device sprays intermixed tasting stimuli into a continuous tasteless spray pulse series on the tongue. The sequence of tasting and tasteless pulses, the pulse duration, and the interstimulus interval are programmable. The temperature control and the regular spray sequence are presented in a constant airstream, avoiding additional thermal or mechanical sensations. The subjects adapt to the slight mechanical puffs. The gustometer can be used in a reliable manner with a sufficient rise time to elicit gCSERP. Using this device, gCSERPs and gustatory event-related magnetic fields (gERMFs) have been recorded simultaneously (Fig. 16.5) (Iannilli et al., 2014). In this study, the spatio-temporal correlates of taste processing in the human primary gustatory cortex were assessed. Iannilli et al. (2014) used sucrose and sodium chloride solutions

as stimuli (stimulus duration: 250 ms, average ISI 30 s, repetition 80 times). The maximum amplitude of the first negative peak of the gCSERP arose around a latency of 250 ms on the left side at the temporal, central, and parietal electrodes. A larger positive deflection revealed its maximum amplitude at a latency of 650 ms at the centroparietal lead position. A source analysis revealed that the left and right anterior/middle part of the insula was related to primary taste perception. The posterior part of the insula was more responsible for secondary gustatory processing. In another study, gCSERPs were generated by NaCl and umani solutions using the same computer-controlled gustometer (Singh et al., 2011). The amplitudes of both gCSERPs were larger in the right than in the left hemisphere. It has been suggested that the right hemisphere dominates gustatory processing. The setting of subjectively proven loss of taste after surgical chorda tympani sectioning would be one means by which to objectify this one-sided local ageusia. Using an electro-gustometer, Ohla et al. (2010) generated both presumed gustatory and somatosensory ERPs using electrical anodal pulses (Fig. 16.6). These findings support the earlier observations by Plattig (1968a,b) and others that electrical stimuli can produce clear electrogustatory ERPs. Electrical stimuli are more practical than liquid stimuli since their characteristics are more easily manipulated (e.g., stimulus intensity, duration, location).

Summated generator potential from lingual fungiform papillae (electro-tastegram or ETG) Feldman et al. (2003) first demonstrated in 2003 that a biologic potential could be reproducibly measured from the surface of the human tongue. They used NaCl solutions flowing over the tongue surface in a special stimulation chamber (Feldman et al., 2009). Recently, Sollai et al. (2017) performed an electrophysiologic recording from the dorsal surface of the tongue. They were able to measure gustatory responses to a bitter compound, 6-npropylthiouracil (PROP). The subjects were classified as supertaster, medium-taster, and nontaster corresponding to the perceived bitter sensation of PROP. A low-cost, disposable, and noninvasive device evoked negative monophasic potentials, characterized by a rapid initial depolarization followed by a slow decline (Fig. 16.7). The authors called this potential the electro-tastegram (ETG) based on the term EOG for the olfactory summated generator potential. Unlike the EOG, the recording site of the ETG is easily accessible. The amplitude of the signal was found to be linearly correlated both with the density of the fungiform papillae at the recording area on the tip of the tongue and also with intensity of perceived bitterness. If it is confirmed that the ETG corresponds to the

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N1 LPC – 4mV CP1 60

ms

+ 50 fT A179 60 ERMF

ms

ERP

ERMF

ERP

CDR [F-distributed] 114

100 90 80

Fig. 16.5. Topographical distributions of gERMFs and gCSERPs. Center top: Electrical and magnetic fields distributions on the scalp in the time range highlighted as gray shadow on the signal registered at the position CP1 for EEG and A179 for MEG. For each time range, the relative source position is depicted. The F-statistical probability to detect the source is referred to the color-coded bar. The N1 peak corresponds to the first negativity in the ERP signal and the LPC (late positive component) corresponds to the late positive peak. They were labeled following the conventional nomenclature of event-related potentials. Adapted from Iannilli, E., Noennig, N., Hummel, T., et al., 2014. Spatio-temporal correlates of taste processing in the human primary gustatory cortex. Neuroscience 273, 92–99.

summated response of stimulated taste cells, it could evolve into an objective quantitative tool for studying the peripheral taste function in humans. For a clinical application of the ETG, further studies with other taste solutions in various concentrations in healthy subjects and patients with taste disorders are needed.

BRIEF EVALUATION OF THE PRESENTED METHODS FROM A CLINICAL POINT OF VIEW ad Quantitative changes of olfactory electroencephalography Quantitative EEG power investigations are a relatively robust diagnostic tool for assessment of the chemosensory functions. It is important to use pure olfactory stimulation (e.g., H2S, vanillin, or PEA) and also pure trigeminal stimulation (CO2) to gain best results. The examiner needs some experience with EEG recording and analyzing especially by FFT.

ad Contingent negative variation CNV is an additional objective olfactometric tool, also useful for medical expert reviews in case of legal and insurance issues. The method is clinically verified. Important for the interpretation of the results are, first, an artifactfree stimulus application by a flow-olfactometer and, second, an artifact-free EEG recording and analysis. ad Chemosensory event related potentials (CSERPs) in the time domain Recording and averaging of olfactory and trigeminal CSERPs are the most common methods for the objective evaluation of chemical senses. Many clinical as well as experimental studies using CSERPs are published. Prerequisites for valid EEG potential derivation are artifact-free stimulation without mechanical and thermal stimulus-synchronous irritation using a flow-olfactometer by Kobal and also an artifact-free EEG recording usually with five electrodes (Fz, Cz, Pz, C3, and C4). To improve the S/N ratio in the EEG, a number of measures are necessary: reasonable

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Fig. 16.6. Grand-averaged baseline corrected ERPs plotted from three electrode clusters (stimulus onset at 0 ms). Electric stimulation evoked an ERP with a first positive peak (P1) at around 130 ms over frontal electrodes (A), followed by a negative peak (N1) at around 200 ms over central electrodes (B), and a second broader positive component (LPC) with a maximum at around 390 ms over central electrodes (C). Left (dotted lines) and right (solid lines) side of stimulation exhibited similar responses. Bar graphs show the peak latencies and amplitudes of each ERP component from the respective electrode cluster (indicated in topographies) and time period (blue shades) with standard errors of the mean. All components exhibited significantly enhanced amplitudes and shorter latencies for high intensity (red) as compared to low intensity (blue) stimulation. Ellipses on the topographical voltage maps for each component indicate approximate electrode locations of the clusters as used for statistical analyses and display of ERP waveforms. Adapted with permission from Ohla, K., Toepel, U., le Coutre, J., et al., 2010. Electrical neuroimaging reveals intensity-dependent activation of human cortical gustatory and somatosensory areas by electric taste. Biol Psychol 85, 446–455.

stimulation repetition, suprathreshold stimulus concentration, adaptation-avoiding ISIs, high intranasal flow rate, maintaining high vigilance and passive stimulus application during VC. A traditional CSERPs test session requires about an hour. In such a session, both sides of the nose are tested with each of two odorants (H2S and PEA) and a trigeminal stimulus (CO2) at an ISI of 30 s with 16 stimulus repetitions. The parameters of the CSERPs show reasonably good test–retest reliability. Advantages: The method is clinically proven and also established for legal and insurance related questions.

Limitations: It demands time, material, and personnel. Only phase-locked EEG responses produce recognizable CSERP after averaging, meaning that all nonphaselocked EEG activations are lost. Evidence: Only when an oCSERP is present the intactness of the sense of smell can be assumed, not vice versa! ad. Global field power (GFP) and global map dissimilarity (GMD) To date, these tools are not used in a clinical routine setting.

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Fig. 16.7. Examples of bioelectrical signals recorded from the human tongue and of the subsequent waveform analysis. (A) The original signal of a representative supertaster, medium taster, and nontaster. (B) The fitting of each original signal applied from the beginning of depolarization up to the next 15 s. (C) The first derivative calculated for each fitting. The maximum value of the first derivative, indicated by an arrow in each panel, was used as an index of depolarization rate of the original signal. The waveform analysis was performed for all participants (n ¼ 43). (D) Drawing showing how the electrodes were positioned for the differential electrophysiologic recordings: one in contact with the ventral surface of the tongue and one adhering to the dorsal surface; a third disposable adhesive electrode (the ground terminal) was placed on the skin of the cheek. The electrode is positioned on the dorsal surface of tongue in a way leaving a circular area of the tip of the tongue uncovered. This is the same area where PROP stimulation was delivered during recordings and the density (No. fungiform papillae/cm2) of fungiform papillae was calculated. (E) Response to a sequence of three stimulations (15 s): blank filter paper (control 1), paper impregnated with 30 mL of spring water (control 2), and paper impregnated with 30 mL of PROP solution. Adapted with permission from Sollai, G., Melis, M., Pani, D., et al., 2017. First objective evaluation of taste sensitivity to 6-n-propylthiouracil (PROP), a paradigm gustatory stimulus in humans. Sci Rep 7, 40353.

ad Chemosensory response EEG analysis in TF domain by Continuous Wavelet Transform (CWT).

ad Summated generator potential of the olfactory and respiratory nasal epithelium

Up to now, this method is used only by a few working groups without clinical relevance. There is a great potency as an additional olfactometric tool to detect nonphase-locked EEG responses that may emerge as transient increase (synchronization) or as transient decrease (desynchronization) of the power of EEG or as the result of temporal jitter. Discrimination performance using CWT SINGLE was significantly stronger than the discrimination performance of oCSERPs. Normosmics and hyposmics can be discriminated with a sensitivity of 91% and a specificity of 82%. Normosmics and anosmics can be discriminated with a sensitivity of 91% and a specificity of 91%.

To date, EOGs and NMP are not used in clinical routine practice. Few research groups are exploiting the unique ability to examine the functioning of olfactory and trigeminal receptor cell clusters in the nasal cavity. ad Electrophysiologic assessment of oral gustatory function (gustatory and electro-gustatory CSERPs) Only recently, a modern computer-controlled gustometer has been commercially available to stimulate regions of the tongue with different taste solutions. Using this technique with repetitions of same stimuli, gustatory CSERPs can be recorded and averaged from

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the ongoing EEG analogous to oCSERPs. So far, mainly basic research is being conducted with this new technology. Limitations: The purchase price is high and the examination procedure is not comfortable for the test person. When using anodic weak current pulses that elicit an electro-taste percept, electro-gustatory CSERPs (egCSERPs) can be evoked. Presented stimulus duration is longer than the largest latency of the evoked potential. This is important to avoid a superposition of electric OFF response with components of the egCSERPs. Advantages: Stimulation equipment is not expensive. The test procedure is more comfortable for the subject than one with taste solutions. Limitations: No clinical application is known to date. There is still a discussion as to whether the electrical taste is a gustatory or a somatosensory phenomenon. ad Summated generator potential from lingual fungiform papillae (ETG) ETG has only been used in the laboratory so far. The amplitude of the negative monophasic potential was dependent on the density of the fungiform papillae and the intensity of the taste solution. Advantages: A lowcost, disposable, and noninvasive device. Limitations: No clinical application is realized until now.

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