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 TMS according to international expert guidelines23.</li>
		<li>Exclude participants with implanted medical devices (e.g., cochlear implant, cardiac pacemaker, etc.), a personal or family history of seizure, headache, brain trauma, and neuroactive medication. Exclude participants considered &#34;anosmic&#34; according to the European Test of Olfactory Capabilities24.</li>
	</ol>
	</li>
	<li>Handedness: Check for right-handedness as assessed by the Edinburgh Handedness Inventory questionnaire25. 
	&#8203;NOTE. It is highly recommended to recruit only right-handed participants in studies assessing corticospinal excitability and effective connectivity in the motor system26,27.</li>
	<li>Information and informed consent: Give all participants basic information about the study objectives, procedures, and risks approved by the Ethics Committee and ask them to sign written informed consent.</li>
</ol>
2. Experimental procedure
<ol>
	<li>Patient installation: Ask the participant to sit on a comfortable chair (dental chair type) with both hands relaxed and pronated. Position the paricipant&#39;s head on a chin rest to minimize head movement during stimulation.</li>
	<li>Electromyography recordings
	<ol>
		<li>Prepare the participant&#39;s skin before electrode application using an exfoliant scrub to lightly abrade the areas and clean the areas using alcohol pads where electrodes will be applied.</li>
		<li>Apply two silver/silver chloride disposable recording electrodes with a belly-tendon montage of the first dorsal interosseous (FDI) muscle. Add the ground electrode to the styloid process of the ulna (Figure 1).</li>
		<li>Connect the electrodes to the amplifier with cables and the data acquisition system.</li>
		<li>Record the EMG signal using an analog-to-digital (AD) conversion system. Amplify and filter EMG signals (gain = 1000) using a bandwidth frequency between 10 Hz and 1 kHz. Digitize at a sampling rate of 2,000 Hz and store each EMG file for offline analysis.</li>
		<li>Check the quality of the signal displayed on the computer screen connected to the data acquisition system.</li>
	</ol>
	</li>
	<li>TMS coilM1 position.
	<ol>
		<li>Connect this coil to the A stimulator (Figure 1).</li>
		<li>Place a tight-fitting cap over the participant&#39;s head. Use a tape measure to perform nasion-inion, tragus-tragus, and head-circumference measurements based on standard cranial landmarks. Identify and mark with a pen the scalp vertex at the intersection of the mid-sagittal (nasion-inion) and interaural (tragus-tragus) lines28.</li>
		<li>Place tangentially to the scalp the first small figure-of-eight coil (inner diameter: 40 mm) over the presumed hand area of the left M1 (coilM1), which is 5 cm lateral from the vertex, with the handle pointing backward and laterally at a 45&#176; angle to the midsagittal line, resulting in a posterior-anterior current flow (monophasic current waveform). This orientation corresponds to a maximum induced current flowing within M1 within M129.</li>
		<li>Ensure that the placement of the coilM1 is optimal, in accordance with the most recent international recommendations30. Start by delivering a few single pulses at 30% of the maximum stimulator output (%MSO) and check that the stimulation produces an MEP as recorded by the EMG system and displayed on the computer screen connected to the data acquisition system.
		<ol>
			<li>If there are no visible responses, gradually increase the stimulation intensity (5 %MSO increments) until MEPs are observed. Then, test four spots around the first site by delivering multiple pulses. Determine the mean peak-to-peak MEP amplitude for each site.</li>
			<li>Select the location where the average peak-to-peak MEP amplitude is the highest. This is the so-called hotspot location for the participant30. Mark the coilM1 location on the cap to ensure proper coil placement throughout the experiment.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Resting motor threshold (rMT) and TMS intensities
	<ol>
		<li>Determine the resting motor threshold (rMT) defined as the TMS intensity that produces a 50% probability of eliciting an MEP23,30.
		<ol>
			<li>Use the available online freeware (TMS Motor Threshold Assessment Tool, MTAT 2.1), which is based on a maximum-likelihood parameter estimation using a sequential testing strategy29. The stimulation sequence always starts with the intensity set at 37 %MSO.</li>
			<li>Let one experimenter hold the coilM1 while another indicates whether the MEP amplitude is &#62; 0.05 mV. The predictive algorithm then determines the next stimulation intensity to be delivered and is stopped after 20 stimulations, which provides sufficient accuracy for the rMT estimation according to previous studies31-34.</li>
		</ol>
		</li>
		<li>Set the %MSO for the conditioning and the test pulse stimulation. Use the previously determined rMT value of the participant. 
		NOTE: Here, the intensity for the first conditioning stimulation (coilDLPFC) was set to 110% of the rMT19,20. The intensity of the test stimulation (coilM1) was set at 120% of the rMT, an intensity that differs slightly from previous studies that used a TS intensity that evoked a MEP of ~1 mV in all participants19,20 . This fixed peak-to-peak intensity occurs at very different points on the input-output recruitment curves due to the high inter-subject variability in motor output35. Therefore, the stimulation intensity could be optimized using 120% RMT intensity across individuals.</li>
	</ol>
	</li>
	<li>TMS coilDLPFC positioning
	<ol>
		<li>Connect this coil to the B stimulator (Figure 1).</li>
		<li>Use the recently updated scalp heuristic to locate the region of the scalp corresponding to the left DLPFC36,37 to estimate the position of the second small figure-of-eight coil (internal diameter: 40 mm) over the DLPFC (coilDLPFC). Download the online Excel Spreadsheet Calculation Tool36 and enter the nasion-inion and tragus-tragus distances and the head circumference in centimeters as inputs. Report the XLA and the YLA distances directly on the participant&#39;s head.</li>
		<li>Place tangentially to the scalp the coilDLPFC over the presumed left DLPFC location, with the handle pointing downward and laterally at a -45&#176; angle to the mid-sagittal line. Mark the coilDLPFC placement on the cap to ensure proper coil placement throughout the experiment. 
		NOTE: This scalp-based targeting method for both coilM1 and coilDLPFC locations is not optimal. In fact, it is known to be less accurate than the neuronavigation method used to target the brain areas of interest based on individual T1 anatomical magnetic resonance imaging (MRI)38.</li>
	</ol>
	</li>
	<li>Delay between the conditioning and test pulses: Set this delay to 10 ms on the pulse generator device. 
	NOTE: Here, the delay is fixed at 10 ms based on previous studies showing an inhibitory influence from the left DLPFC to the left M1 at this interval19,20. This inhibitory effect observed at 10 ms is likely due to the activation of the basal ganglia via the DLPFC projections to the pre-SMA, thereby exerting an indirect influence on M139. The delay can be adjusted in the code according to the user&#39;s needs. For example, a longer interstimulation interval (i.e., 25 ms) could be used to investigate polysynaptic indirect cortico-subcortical-cortical circuits connecting DLPFC to M119. Furthermore, differential facilitatory/inhibitory influences have been demonstrated using dual-site ppTMS between multiple cortical areas, with intervals ranging from 1 ms to 150 ms40,41. Thus, the fact that the interval can be adjusted opens the way to a wide range of possibilities for future research studies.</li>
	<li>Olfactometer settings
	<ol>
		<li>Select odorants with pleasant and unpleasant hedonic values. Dilute in advance the odorants individually in mineral oil to create iso-intense perception. 
		NOTE: Here, the selection and concentration of odorants (i.e., isoamyl acetate and butyric acid diluted to 0.6% and 0.11% vol/vol concentrations, respectively) were based on previous studies by our group using the same olfactometer setup and odorants42,43. A pilot study confirms that the positive and the negative odors did not differ in terms of intensity but were opposite in hedonic value. In the control condition (i.e., no odorant), only airflow is delivered to the participant.</li>
		<li>Write the code to deliver the odorants. For each trial, indicate the total duration of the trial, the odorant to be delivered, the flow rate of the odorant controller (in milliliters per minute), the flow rate of the carrier air regulator (in milliliters per minute ), and the flow rate of the suction regulator. 
		NOTE: The order of the odorant delivered can be randomized between positive, negative, and no-odor. Here, each trial has a duration of 12 s. The order of odor delivered was pseudo-randomized. In addition, based on a pilot experiment, the flow rate of the odor controller was set to 200 mL/min, the flow rate of the carrier air regulator at 500 mL/min, and the flow rate of the suction regulator at 100 mL/min.</li>
		<li>Position the nasal cannula near the participant&#39;s nostrils to measure nasal breathing. Instruct the participant to breathe normally through the nose.</li>
		<li>Turn on the portable air compressor, the olfactometer case, and the PC containing the software. Check all the cable connections (Figure 1). 
		NOTE: The olfactometer used in the current study has been described in detail in a previous publication44 but has been modified here to allow TMS triggering with variable delays after inspiration onset detection.Briefly, the device is composed of several modules, including 1) an air source and air treatment system coming from a portable air compressor, 2) a stimulation system including electronic and pneumatic devices, 3) a homemade mixing head coupled to a delivery system that allows the diffusion of odorants in the participant&#39;s nose, 4) a respiratory sensory system that triggers the olfactometer according to the nasal respiration measurement with a nasal cannula and 5) a software control system44.</li>
		<li>Calibration: Proceed to the calibration phase (about 20 s.), which allows for calibrating the participant&#39;s respiratory signal and adjusting the detection thresholds of the expiratory and inspiratory phases. In this software, the expiratory phase is positive, and the inspiratory phase is negative.</li>
		<li>Odor hedonic and intensity ratings: Deliver the two odorants in a randomized order and ask participants to rate the hedonic value and intensity of each odorant on visual analog scales ranging from 1 &#34;not at all pleasant&#34; to 9 &#34;extremely pleasant&#34; and from 1 &#34;not at all intense&#34; to &#34;extremely intense.&#34;</li>
	</ol>
	</li>
	<li>Combining olfactometer and TMS: Set the delay between the detection of the inspiration phase and the trigger for sending the TMS at 600 ms. 
	NOTE: The setting of the delay is important and has to be determined according to the literature and the needs of the user. In this protocol, the delay was set at 600 ms, which has been shown to be the maximum conscious perceptual representation of odors45. For single-pulse TMS condition, this trigger immediately activates the A stimulator, and a pulse is delivered by the coil positioned on the left M1 to evoke an unconditioned MEP.For dual-coil TMS condition, this trigger is sent to two different devices (via two coaxial cables connected by a T-connection): the first one immediately activates the B stimulator and a conditioning pulse is delivered by the coil positioned on the left DLPFC; the second one is received by a pulse generator which makes it possible to induce a fixed delay before activating the A stimulator, thus delivering a test-stimulation through the coil positioned on the left M1 to evoke a conditioned MEP (Figure 1).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65714/65714fig01.jpg" /> 
Figure 1: Experimental setup. The bold lines represent pneumatic connections. An air compressor is connected to the olfactometer to generate different air flows. A regulator controls the pressure, and the input air flow is directed to 3 channels (through 3 mass regulators): one for the air conveyor (blue line), one for the aspiration system (brown line) to clean and help control the stimulation time and the last one for the odorants44. Two U-shaped tubes contain the odorants (green: pleasant; red: unpleasant) in which they are conditioned under pressure in the saturated steam state, ensuring an odorized air flow with stable intensity over time. The mixing head is used to mix the clean and odorized air streams. The airflow (odorized or pure) is delivered to the nostrils through two tubes (gray lines) attached to a nasal cannula, which is also used to record nasal breathing (purple line). Based on the respiratory signal, as soon as the inhalation phase is detected, for the spTMS condition a trigger is sent to a pulse generator device used to set a delay (here: 10 ms), then to a TMS stimulator A connected to CoilM1 applied over the left M1 hand muscle representation, while the TMS stimulator B is turned off. For the dsTMS condition, a trigger is immediately sent to the TMS stimulator B connected to the CoilDLPFC applied over the left DLPFC, and the pulse generator device is used to set a delay (here: 10 ms) before triggering the TMS stimulator A connected to the CoilM1. The respiratory signal and MEP amplitude acquired by the EMG system are recorded by software installed on a PC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65714/65714fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Measurements
<ol>
	<li>Run the custom-made coding script in the olfactometer software (see step 2.7.2) to deliver all combinations of spTMS and dsTMS with pleasant and unpleasant odors and no-odors occurring in a random order. 
	&#8203;NOTE: Here, 20 trials were recorded for each condition (120 trials in total). The experiment was divided into 6 blocks of 20 trials each. The number of trials for each condition can be changed according to the user&#39;s needs.</li>
</ol>
4. Data analyses
<ol>
	<li>For each participant, condition, and trial, extract the peak-to-peak MEP amplitude. This can be done using one of the open-source Toolboxes available online46,47.</li>
	<li>Normalize the data by calculating an MEP ratio expressing MEPs elicited by the test stimulation in dsTMS trials relative to MEPs elicited by the test stimulation in spTMS trials12. Do this separately for each participant and for each odor condition (i.e., no-odor, positive odor, and negative odor). After this procedure, interpret the results as follows: MEP ratios above 1 indicate a facilitatory influence of the DLPFC on M1, whereas MEP ratios below 1 indicate an inhibitory influence of the DLPFC on M1.</li>
</ol>

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

JB is a board member of the Brain Stimulation Section (STEP) of the French Association of Biological Psychiatry and Neuropsychopharmacology (AFPBN), of the European Society of Brain Stimulation (ESBS), and reports academic research grants in the field of brain stimulation from CIHR (Canada), ANR and PHRC (France). Other authors have nothing to disclose.

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 parameters (placement, intensities) and olfactometer parameters (odorant selection, timing relative to respiratory phases).
This combination of spTMS and dsTMS with an olfactometer can be adapted in many ways, depending on the user&#39;s needs, and has clear methodological advantages. As mentioned in the introduction, two methodological aspects seemed crucial for a more in-depth investigation of the mechanistic basis of the interactions between the olfactory and motor systems. The first was the possibility of presenting different odor conditions (pleasant/unpleasant/no odor) within the same experimental phase. This is now feasible because&#160;it is possible to specify on a trial-by-trial basis which odorant will be delivered to the subject at a constant intensity. This is a crucial point, as it allows us to eliminate the systematic intra-individual changes in MEPs amplitude within and between stimulus blocks observed in previous studies, even at relatively long interstimulus intervals48,49.
Indeed, the application of a TMS pulse to M1 allows the quantification of the observed changes in corticospinal excitability with undeniable temporal accuracy. However, a very large number of factors can modulate corticospinal excitability, and these need to be controlled as much as possible. For example, the simple fact of voluntary inspiration or exhalation (a motor act) modifies the corticospinal excitability of non-respiratory finger muscles50.
The second was the possibility to control and synchronize several factors with the respiratory phases. These include the precise duration and timing of odor diffusion to the participants and the timing of the TMS pulse. More importantly, these different parameters can be modified according to the user&#39;s needs, opening the way for future studies.
The method presented here opens the way for a wide range of future research and broader questions in the field of olfaction. First, no study has yet examined the temporal precision of the modulation of corticospinal excitability in response to an olfactory stimulus. Is this modulation very early (i.e., before the emergence of perceptual odor representations, estimated to be between 300 ms and 500 ms after odor onset45) or later (i.e., when odor representations are extended to larger areas associated with emotional, semantic, and memory processing45)? Is the timing of changes in corticospinal excitability the same depending on the hedonic value of the odor? Unpleasant odors, such as pain, often signal potential danger, elicit a faster response to quickly avoid or escape negative situations51,52, and thus modulate corticospinal excitability earlier than positive odors. However, this remains speculative. By delivering the TMS pulse at different times after the onset of both positive and negative odors and comparing the changes in corticospinal excitability, the current protocol can address this question. Furthermore, although&#160;the focus of the present protocol was on the modulation of corticospinal excitability by targeting M1, the TMS technique, due to its high temporal resolution, can also be used to investigate the causal brain-behavior relationships and the time course of other areas during olfactory processes, due to its high temporal resolution53. Similarly, in the current protocol, we evaluated the effective connectivity between the DLPFC and M1 because there is evidence in the literature that modulations of this connectivity may occur during odor perception. However, other cortico-cortical or cortico-subcortical-cortical networks may be modulated during olfaction or motor control processes, and the connectivity within these networks can be easily assessed with this new method. The only change would then be the location of the coils toward the targeted cortical areas. For example, the orbitofrontal cortex has been shown to be involved in coding for odor hedonic value and odor perception54, and a recent dual-site TMS study showed that this area has an inhibitory influence on M1 at rest12. Probing changes in the effective connectivity between the orbitofrontal cortex and M1 during perception of positive and negative odors is an interesting avenue of study for a better understanding of the mechanisms behind the interactions between olfactory and motor systems.
In addition, this method proposes a new way to reliably assess odor hedonic perception in a non-verbal or conscious manner. This could pave the way for clinical investigations aimed at understanding abnormal interactions between processing in the olfactory and motor systems. For example, the current method could be used in patients with neuropsychiatric disorders such as major depressive disorder (MDD), which has been associated with alterations in olfactory function, including hedonic perception of odors and maladaptive approach and avoidance behaviors55. Furthermore, as&#160;the left DLPFC has been shown to be hypoactive in MDD patients56 and the DLPFC-M1 connectivity is modulated during approach-avoidance behaviors19, the combination of TMS and an olfactometer may be a promising potential tool to elucidate neurophysiological indicators of dysfunctional connectivity between DLPFC and M1 in MDD patients. Neurophysiological findings can then be correlated with clinical symptomatology, such as the severity of depression or the olfactory anhedonia score, defined as the reduced ability to experience pleasure, found in patients with MDD57. Finally, if abnormalities in effective connectivity are revealed in these patients using the method presented here and correlate with clinical symptoms, dual-site TMS could be used repeatedly to neuromodulate DLPFC-M1 connectivity and improve clinical symptoms, a protocol called paired-associative cortico-cortical stimulation58,59.
Although the present method and results provide a proof of concept for future investigations into the neural mechanisms underlying the interactions between the olfactory and motor systems, some limitations and considerations must be mentioned. First, to increase the reliability and reproducibility of the measurements, the targeted brain areas should be precisely based on anatomical and functional areas (this is especially true for the DLPFC target). Second, as mentioned above and as demonstrated by E-field computational modeling, the scalp-based targeting method used to position the coils is suboptimal compared to MRI guidance60. To maximize the accuracy and precision of TMS positioning, a neuronavigation system that co-registers the patient&#39;s head and structural magnetic resonance imaging (MRI) scan and provides real-time feedback on the coil position should be used38. In addition, computational E-field dosimetry has been shown to provide more efficient and focused stimulation by determining the individual coil placement that maximizes E-field delivery to a specific brain target61. A third point to consider when interpreting the results related to MEP amplitude. Indeed, MEP amplitude is known to reflect&#160;intrinsic different neural inputs to the corticospinal cells, including transcortical elements, and the activity of the spinal motoneuron pool62,63,64. Therefore, the modulation of corticospinal excitability and effective connectivity observed during the exposure to a pleasant odor provides a partial picture of the more complex supraspinal and spinal networks that are likely to be involved in the modulation of the MEP amplitude. Results should be interpreted with caution.

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><tbody><tr><td>Acquisition board (8 channels)&amp;nbsp;</td><td>National Instrument</td><td>NI USB-6009&amp;nbsp;</td><td></td></tr><tr><td>Air compressor</td><td>Jun-Air&amp;nbsp;</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>&amp;nbsp;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&amp;nbsp;</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.

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 threshold and diffuses for 5 s. The TMS pulse is triggered with a delay (600 ms) after the inspiration phase threshold.
This result demonstrates that the method developed here can precisely synchronize odorant diffusion and TMS timing according to human nasal breathing phases.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65714/65714fig02.jpg" /> 
Figure 2: Example of raw data from respiratory recordings for a representative participant. The expiration phase is detected when a threshold (represented by the red line) is crossed. Inspiration phase is detected when a threshold (represented by the blue line) is crossed. The odorant is triggered immediately after the expiration phase threshold and diffuses for 5 s, as shown by the green line. The TMS pulse is triggered with a delay (600 ms) after the inspiration phase threshold. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65714/65714fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3 illustrates the results obtained from EMG data recordings from the right FDI muscle (MEP recordings), according to the conditions (spTMS and dsTMS) and the odor hedonic values (no-odorant, positive odorant and negative odorants) for a representative participant. The peak-to-peak amplitude of the MEPs evoked by spTMS (Figure 3A) and by dsTMS (Figure 3B) varied according to the hedonic value of the odorant. When the results are normalized (Figure 3C), all MEP ratios are below 1, indicating a suppressive effect of the left DLPFC on the left M1.This result demonstrates that the method developed here allows the investigations of modulations of corticospinal excitability and effective connectivity that occur during pleasant and unpleasant odor perception, all delivered in a synchronized manner with human nasal breathing. These results are preliminary and deserve further investigation to conclude on the specific effects of odors hedonic values on corticospinal excitability and effective connectivity.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65714/65714fig03.jpg" /> 
Figure 3: Example of typical raw recordings from the right FDI muscle of a participant. (A) spTMS condition with positive odorant (green), negative odorant (orange), and the no-odorant conditions (gray).&#160;(B)&#160;dsTMS condition, with positive odorant (green), negative odorant (orange) and the no-odorant conditions (gray). (C)&#160;MEP ratios obtained after the normalization procedure for a representative participant. The three MEP ratios are below 1, indicating an inhibitory influence of the DLPFC on M1. Raw MEP traces represent a single trial recording. Bar graphs show the mean, the standard deviation, and the individual MEP value of the 20 trials obtained in each condition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65714/65714fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

962 Views.  Université Claude Bernard Lyon 1. Hier willen we de relatie tussen reukzin en motoriek in de hersenen onderzoeken. We zijn vooral geïnteresseerd in het onderzoeken van hoe aangename en onaangename geuren de corticospinale toegankelijkheid en effectieve connectiviteit in zowel normale als pathologische toestand zullen beïnvloeden. Het onderzoeken van de mechanistische basis van olfactorische motorische interacties vereist een zorgvuldige afweging van twee methodologische aspecten.Ten eerste is het absoluut noodzakelij...

Video: Auteur in de schijnwerpers: onderzoek naar olfactorische invloeden op corticospinale prikkelbaarheid - inzichten en innovaties in neurologisch onderzoek 

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they yield data relevant to the cognitive processing that occurs under ecologically valid conditions. These two goals often are at odds, since well controlled stimuli often are too repetitive to sustain subjects' motivation. Studies employing electroencephalography (EEG) are often especially sensitive to this dilemma between ecological validity and experimental control: attaining sufficient signal-to-noise in physiological averages demands large numbers of repeated trials within lengthy recording sessions, limiting the subject pool to individuals with the ability and patience to perform a set task over and over again. This constraint severely limits researchers' ability to investigate younger populations as well as clinical populations associated with heightened anxiety or attentional abnormalities. Even adult, non-clinical subjects may not be able to achieve their typical levels of performance or cognitive engagement: an unmotivated subject for whom an experimental task is little more than a chore is not the same, behaviourally, cognitively, or neurally, as a subject who is intrinsically motivated and engaged with the task. A growing body of literature demonstrates that embedding experiments within video games may provide a way between the horns of this dilemma between experimental control and ecological validity. The narrative of a game provides a more realistic context in which tasks occur, enhancing their ecological validity (Chaytor &amp; Schmitter-Edgecombe, 2003). Moreover, this context provides motivation to complete tasks. In our game, subjects perform various missions to collect resources, fend off pirates, intercept communications or facilitate diplomatic relations. In so doing, they also perform an array of cognitive tasks, including a Posner attention-shifting paradigm (Posner, 1980), a go/no-go test of motor inhibition, a psychophysical motion coherence threshold task, the Embedded Figures Test (Witkin, 1950, 1954) and a theory-of-mind (Wimmer &amp; Perner, 1983) task. The game software automatically registers game stimuli and subjects' actions and responses in a log file, and sends event codes to synchronise with physiological data recorders. Thus the game can be combined with physiological measures such as EEG or fMRI, and with moment-to-moment tracking of gaze. Gaze tracking can verify subjects' compliance with behavioural tasks (e.g. fixation) and overt attention to experimental stimuli, and also physiological arousal as reflected in pupil dilation (Bradley et al., 2008). At great enough sampling frequencies, gaze tracking may also help assess covert attention as reflected in microsaccades - eye movements that are too small to foveate a new object, but are as rapid in onset and have the same relationship between angular distance and peak velocity as do saccades that traverse greater distances. The distribution of directions of microsaccades correlates with the (otherwise) covert direction of attention (Hafed &amp; Clark, 2002).

Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Controlled Odor Mimic Permeation Systems for Olfactory Training and Field Testing

The Controlled Odor Mimic Permeation System (COMPS) was developed to provide a convenient field testing method of odor delivery at controlled and reproducible rates. COMPS are composed of an odorant of interest on an absorbent material sealed inside of a permeable polymer bag. The permeable layer allows for a constant release of the odorant over a given amount of time. The permeable bag is further stored in a secondary, impermeable bag. The double-containment procedure allows for equilibration of the odorant from the permeable bag but within the impermeable outer layer, resulting in an instant and reproducible source of odorant vapor upon removal from the outer packaging. COMPS are used in both olfactory testing for experimental scenarios and for olfactory detection training, such as with detection canines. COMPS can be used to contain a wide range of odorants (e.g., narcotics powders) and provide a controlled release of the associated odorants. Odor availability from COMPS is expressed in terms of permeation rate (i.e., the rate of the odorant vapor released from a COMPS per unit time) and is typically measured by gravimetric means. The permeation rate for a given mass or volume of odorant can be adjusted as needed by varying the bag thickness, surface area, and/or polymer type. The available odor concentration from a COMPS can also be measured by headspace analysis techniques such as solid phase microextraction with gas chromatography/mass spectrometry (SPME-GC/MS).

The Controlled Odor Mimic Permeation System is a simple, field-portable, low-cost method of odor delivery for olfactory testing and training. It is constructed of an odorant retained on an adsorbent material and contained inside of a permeable polymer bag allowing controlled release of the odorant vapor over time.

The Controlled Odor Mimic Permeation System (COMPS) was developed to provide a convenient field testing method of odor delivery at controlled and ...

Chemistry

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Designing a Flexible, Cost-Effective Olfactometer for Rodent Olfactory Experiments

Constructing an Olfactometer for Rodent Olfactory Behavior Studies

The use of olfactometers to study rodent behavior and brain activity during olfactory tasks is crucial for understanding brain circuits. These sophisticated devices allow researchers to precisely control and deliver odor stimuli, enabling the investigation of complex olfactory processes in rodents. Although commercially available olfactometers are convenient, they present challenges when technical issues arise, often requiring costly assistance and potentially disrupting research timelines. This article details the construction of a custom olfactometer specifically designed for mouse olfactory behavior experiments, providing a comprehensive list of parts and step-by-step instructions. The olfactometer is controlled through MATLAB, offering a user-friendly interface for researchers. Importantly, the open-source code allows users to modify and adapt the system, tailoring behavioral tasks to meet specific experimental needs. Building a customized olfactometer empowers users with the knowledge and capability to perform custom experimental design and troubleshooting independently, saving both time and resources. This approach not only enhances research flexibility but also fosters a deeper understanding of the equipment's functionality, ultimately leading to more robust and reliable olfactory studies in rodents.

Constructing an Olfactometer for Rodent Olfactory Behavior Studies Near-Infrared Spectroscopy Hyperscanning Study in Psychological Counseling

This protocol describes the construction of an olfactometer for go/no-go olfactory behavior experiments. Step-by-step instructions, along with images, are provided to ensure the successful construction of the olfactometer. Information for troubleshooting issues encountered during the process is also included.

The use of olfactometers to study rodent behavior and brain activity during olfactory tasks is crucial for understanding brain circuits. These ...

Behavior

A Free-breathing fMRI Method to Study Human Olfactory Function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

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

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