This work was supported by the Fondation de France, Grant N&#176;: 00123049/WB-2021-35902 (a grant received by J.B. and N.M.). The authors would like to thank the Fondation Pierre Deniker for its support (grant received by C.N.) and the staff of the Neuro-Immersion platform for their valuable help in designing the setup.

All experimental procedures described in the following sections have been approved by an Ethics Committee (CPP Ile de France VII, Paris, France, protocol number 2022-A01967-36) in accordance with the Declaration of Helsinki. All participants provided written informed consent before study enrollment.
1. Participant recruitment
<ol>
	<li>Inclusion/exclusion criteria.
	<ol>
		<li>Include adult (&#62; 18 years) participants. Screen all participants for any contraindications to T...

Effect of Odor on Brain Excitability Assessed by a Breath-Synchronized Olfactometer

The protocol above describes a novel method combining the use of a breath-synchronized&#160;olfactometer with single- and dual-coil TMS to investigate changes in corticospinal excitability and effective connectivity depending on the hedonic value of the odorants. This setup will allow for objectively discriminating the pleasantness value of an odorant in a given participant, indicating the biological impact of the odorant on brain effective connectivity and reactivity. The critical steps in this protocol involve both TMS...

It is widely accepted that olfactory stimulation elicits automatic reactions and motor behaviors. For example, in humans, the existence of an avoidance motor response (leaning away from the odor source) occurring 500 ms after negative odor onset has been recently demonstrated1. By recording freely moving human participants exploring odors emanating from flasks, Chalen&#231;on et al. (2022) showed that motor behaviors (i.e., speed of approach to the nose and withdrawal of the flask containing the odorant) are closely linked to odor hedonics2. Moreover, a close link between processing in the olfactory system and activity in the motor cortex has been recently demonstrated in humans by using electroencephalography1. Specifically, approximately 350 ms after the onset of negative odors, a specific mu rhythm desynchronization, known to reflect action preparation processes, was observed over and within the primary motor cortex (M1), shortly followed by a behavioral backward movement1. Strengthening the idea of a relationship between the olfactory and motor systems, another recent study showed that exposure to a pleasant odorant increased corticospinal excitability compared to a no-odor condition3. In this study, single-pulse transcranial magnetic stimulation (spTMS) was applied to M1 to evoke a motor-evoked potential (MEP) in a target hand muscle, recorded peripherally with electromyography (EMG) during odor perception. Exposure to the pleasant odorant was provided passively&#160;by paper strips sodden with pure bergamot essential oil and placed on a metal holder under the nose3. In this context, it remains unclear whether the facilitation of the corticospinal excitability is due to the pleasant odorant stimulation or to unspecific behavioral effects such as sniffing and teeth clenching4,5. Furthermore, it is still unknown how an unpleasant odorant modulates M1 excitability probed by TMS.
In summary, this highlights the need to develop a method that offers the following advantages over existing techniques used in previous studies3,6: (1) randomizing the presentation of different odor conditions (pleasant/unpleasant/no-odor) within the same experimental phase, (2) precisely synchronizing odorant presentation and TMS timing according to the human nasal breathing phases (inspiration and expiration) when studying the motor system.
TMS can also be used as a tool to investigate cortico-cortical interactions, also called effective connectivity, between multiple cortical areas and M1 with a high temporal resolution7,8,9,10,11,12. Here, we use a dual-site TMS (dsTMS) paradigm, in which a first-conditioning stimulation (CS) activates a target cortical area, and a second-test stimulation (TS) is applied over M1 using another coil to evoke an MEP. The effect of the CS is evaluated by normalizing the amplitude of the conditioned MEP (dsTMS condition) to the amplitude of the unconditioned MEP (spTMS condition)13. Then, negative ratio values indicate&#160;suppressive cortico-cortical interactions, while positive ratio values indicate facilitatory cortico-cortical interactions between the two stimulated areas. The dsTMS paradigm thus provides a unique opportunity to identify the nature (i.e., facilitatory or suppressive), the strength, and the modulations of the effective connectivity between the preactivated area and M1. Importantly, cortico-cortical interactions reflect a complex balance of facilitation and suppression that may be modulated in different timing and mental states or tasks7,14.
To our knowledge, the relatively new dsTMS paradigm has never been used to investigate cortico-cortical interactions during odor perception with different hedonic values. However, neuroimaging studies have shown that exposure to pleasant and unpleasant odorants induces connectivity changes in areas involved in emotion, decision-making, and action control, including the supplementary motor area, the anterior cingulate cortex, and the dorsolateral prefrontal cortex (DLPFC)15,16. Indeed, the DLPFC is a key node mediating emotional control, sensory processing, and higher-level aspects of motor control, such as preparatory processes17,18,19. In addition, both human and animal studies have provided evidence that the DLPFC has diverse neuronal projections to M117,18,20,21,22. Depending on the context, these DLPFC projections can either facilitate or inhibit M1 activity7,19,20. Thus, it seems possible that the effective connectivity between DLPFC and M1 is modulated during odor presentation and that pleasant and unpleasant odorants recruit separated cortical networks, leading to a differential effect on DLPFC-M1 connectivity.
Here, we propose a new method suitable for the methodologically rigorous study of the modulations of corticospinal excitability and effective connectivity that might occur during the perception of pleasant and unpleasant odors, all delivered in synchrony with human nasal breathing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acquisition board (8 channels)&#160;</td>
			<td>National Instrument</td>
			<td>NI USB-6009&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Air compressor</td>
			<td>Jun-Air&#160;</td>
			<td>Model6-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol prep pads</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Butyric acid</td>
			<td>Sigma-Aldrich</td>
			<td>B103500</td>
			<td>Negative odorant</td>
		</tr>
		<tr>
			<td>Desktop computer</td>
			<td>Dell</td>
			<td>Latitude 3520</td>
			<td></td>
		</tr>
		<tr>
			<td>EMG system</td>
			<td>Biopac System</td>
			<td>MP150</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoamyl acetate</td>
			<td>Sigma-Aldrich</td>
			<td>W205508</td>
			<td>Positive odorant</td>
		</tr>
		<tr>
			<td>Nasal cannula</td>
			<td>SEBAC France</td>
			<td>O1320</td>
			<td></td>
		</tr>
		<tr>
			<td>Programmable pulse generator</td>
			<td>A.M.P.I</td>
			<td>&#160;Master-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Surface electrodes</td>
			<td>Kendall Medi-trace</td>
			<td>FS327</td>
			<td></td>
		</tr>
		<tr>
			<td>TMS coil (X2)</td>
			<td>MagStim</td>
			<td>D40 Alpha B.I. coil&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>TMS machine</td>
			<td>MagStim</td>
			<td>Bistim2</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube 6 mm x 20 m</td>
			<td>Radiospare</td>
			<td>686-2671</td>
			<td>Pneumatic connection</td>
		</tr>
		<tr>
			<td>USB-RS232</td>
			<td>Radiospare</td>
			<td>687-7806</td>
			<td></td>
		</tr>
		<tr>
			<td>U-shaped tubes</td>
			<td>VS technologies</td>
			<td>VS110115</td>
			<td></td>
		</tr>
	</tbody>
</table>

combining a breath-synchronized olfactometer with brain simulation to study the impact of odors on corticospinal excitability and effective connectivity

It is widely accepted that olfactory stimulation elicits motor behaviors, such as approaching pleasant odorants and avoiding unpleasant ones, in animals and humans. Recently, studies using electroencephalography and transcranial magnetic stimulation (TMS) have demonstrated a strong link between processing in the olfactory system and activity in the motor cortex in humans. To better understand the interactions between the olfactory and the motor systems and to overcome some of the previous methodological limitations, we developed a new method combining an olfactometer that synchronizes the random order presentation of odorants with different hedonic values and the TMS (single- and dual-coil) triggering with nasal breathing phases. This method allows probing the modulations of corticospinal excitability and effective ipsilateral connectivity between the dorsolateral prefrontal cortex and the primary motor cortex that could occur during pleasant and unpleasant odor perception. The application of this method will allow for objectively discriminating the pleasantness value of an odorant in a given participant, indicating the biological impact of the odorant on brain effective connectivity and excitability. In addition, this could pave the way for clinical investigations in patients with neurological or neuropsychiatric disorders who may exhibit&#160;odor hedonic alterations and maladaptive approach-avoidance behaviors.

Author Spotlight: Exploring Olfactory Influences on Corticospinal Excitability - Insights and Innovations in Neurological Research 

Combining a Breath-Synchronized Olfactometer with Brain Simulation to Study the Impact of Odors on Corticospinal Excitability and Effective Connectivity

Here we aim to investigate the relationship between olfactory and motor system in the brain. We are particularly interested in exploring how pleasant and unpleasant odors will impact corticospinal accessibility and effective connectivity in both normal and pathological condition. Exploring the mechanistic basis of olfactory motor interactions requires careful consideration of two methodological aspects.First, it is imperative to deliver different olfactory condition within the same experimental phase. Second, precise control and synchronization of olfactory and TMS stimulations with respiratory phases is required. We present a new method designed for the rigorous investigations of the modulations of corticospinal excitability and effective connectivity that might occur during the perception of pleasant and unpleasant odors.All these stimulations are delivered in synchrony with human nasal breathing. This method paved the way for clinical investigation exploring aberrant interactions between olfactory and motor system. We are currently applying these methods to patients with psychiatric disorders that are associated with alterations in the autonomic perception of odors and maladaptive approach or avoidance behaviors.

The representative data presented here reflect recordings from participants after completing the step-by-step protocol above to provide a preliminary insight into what we might expect.
Figure 2&#160;shows an example of a representative participant&#39;s respiratory signals recorded with the olfactometer software. The expiratory and inspiratory phases are well detected when the thresholds are crossed. The odorant is triggered immediately after the expiration phase ...

Watch this Scientific Journal Video about Exploring Olfactory Influences on Corticospinal Excitability — Insights and Innovations in Neurological Research at JoVE.com

This paper describes using a breath-synchronized olfactometer to trigger single- and dual-coil transcranial magnetic stimulation (TMS) during odorant presentation synchronized to human nasal breathing. This combination allows us to objectively investigate how pleasant and unpleasant odors impact corticospinal excitability and brain-effective connectivity in a given individual.

It is widely accepted that olfactory stimulation elicits motor behaviors, such as approaching pleasant odorants and avoiding unpleasant ones, in animals ...

combining-breath-synchronized-olfactometer-with-brain-simulation-to