Aged mice are less susceptible to motion sickness and show decreased efferent vestibular activity compared to young adults

Abstract Introduction The efferent vestibular system (EVS) is a feedback circuit thought to modulate vestibular afferent activity by inhibiting type II hair cells and exciting calyx‐bearing afferents in the peripheral vestibular organs. In a previous study, we suggested EVS activity may contribute to the effects of motion sickness. To determine an association between motion sickness and EVS activity, we examined the effects of provocative motion (PM) on c‐Fos expression in brainstem efferent vestibular nucleus (EVN) neurons that are the source of efferent innervation in the peripheral vestibular organs. Methods c‐Fos is an immediate early gene product expressed in stimulated neurons and is a well‐established marker of neuronal activation. To study the effects of PM, young adult C57/BL6 wild‐type (WT), aged WT, and young adult transgenic Chat‐gCaMP6f mice were exposed to PM, and tail temperature (T tail) was monitored using infrared imaging. After PM, we used immunohistochemistry to label EVN neurons to determine any changes in c‐Fos expression. All tissue was imaged using laser scanning confocal microscopy. Results Infrared recording of T tail during PM indicated that young adult WT and transgenic mice displayed a typical motion sickness response (tail warming), but not in aged WT mice. Similarly, brainstem EVN neurons showed increased expression of c‐Fos protein after PM in young adult WT and transgenic mice but not in aged cohorts. Conclusion We present evidence that motion sickness symptoms and increased activation of EVN neurons occur in young adult WT and transgenic mice in response to PM. In contrast, aged WT mice showed no signs of motion sickness and no change in c‐Fos expression when exposed to the same provocative stimulus.


INTRODUCTION
Motion sickness often occurs when there is a difference between actual and expected motion and is thought to result from discrepancies between the senses of balance (vestibular), proprioception, and vision (Golding & Gresty, 2005, 2015Oman, 1990;Reason & Brand, 1975). This "sensory conflict" theory was first proposed by Reason and Brand (1975); however, the exact neurobiological mechanism that allows sensory conflict to trigger motion sickness symptoms such as dizziness, sweating, excessive saliva production, nausea, and emesis is still unclear.
The vestibular system appears to be a key component in the development of motion sickness. Symptoms of motion sickness are abolished in rats (Morita et al., 1988), monkeys (Wilpizeski et al., 1987), and dogs (Money & Friedberg, 1964) that have had their vestibular organs removed (Money, 1970).
Susceptibility to motion sickness also appears to be age dependent.
Older humans (>50 years) are significantly less vulnerable to motion sickness than younger age groups, although infants and young children below 2 years old are thought to be invulnerable (Takeda et al., 2001). Therefore, age-related changes in motion sickness susceptibility are likely related to the well-documented deterioration in vestibular function in humans and reported in other animals including laboratory rats (McCaffrey & Graham, 1980;Morita et al., 1988) and mice (Tung et al., 2014). Documented vestibular deterioration includes loss of hair cells and associated nerve fibers, thus attenuating vestibular afferent input to brain centers involved in the coordination of movement (Bergström, 1973a(Bergström, , 1973bJi & Zhai, 2018;Rosenhall & Rubin, 1975;Zalewski, 2015). While reduction of motion sickness susceptibility could be regarded as a positive outcome of vestibular senescence, it should be remembered other outcomes such as imbalance and falls are major causes of injury in the elderly (Iwasaki & Yamasoba, 2015;Sloane et al., 1989).
In this study, we focused on the enigmatic feedback component of the vestibular system, the efferent vestibular system (EVS), which is a much less studied area in relation to motion sickness and aging.
The EVS originates bilaterally from the brainstem efferent vestibular nucleus (EVN) and provides rich, mainly cholinergic innervation to vestibular hair cells and afferent nerve terminals and parent fibers (Holt et al., 2011;Mathews et al., 2017;Poppi et al., 2020). The precise function of the EVS is still unclear, but evidence suggests the mammalian EVS has mixed effects, exciting primary vestibular afferents, and afferent calyx terminals in the periphery (Schneider et al., 2021) while simultaneously inhibiting type II hair cells (one of two hair cell types in the vestibular neuroepithelium) through the activation of alpha-9 nicotinic acetylcholine receptors and SK (small conductance) potassium channels (Poppi et al., 2018). Thus, the EVS has a surprisingly complex effect on the afferent sensory output from the vestibular organs of the inner ear. Previous results from our laboratory also suggest the involvement of EVS in motion sickness. Mice with an attenuated EVS, that is, alpha-9 nicotinic receptor knockouts, displayed reduced motion sickness symptoms (Tu et al., 2017).
A characteristic and obvious sign of motion sickness is emesis, which occurs in humans and other mammalian species such as monkeys (Ordy & Brizzee, 1980), dogs (Benchaoui et al., 2007), and shrews (Horn et al., 2014). One notable exception, however, is rodents such as rats and mice, which are unable to vomit. Therefore, to determine if these common laboratory animals experience motion sickness, other indicators are used. These include pica, the consumption of nonnutritious substances (Mitchell et al., 1977;Morita et al., 1988), increased defecation (Ossenkopp & Frisken, 1982), suppression of drinking (Haroutunian et al., 1976), and thermal responses such as transient increasing tail temperature (T tail ) and decreasing core body temperature Guimaraes et al., 2015;Ngampramuan et al., 2014). It has been previously reported by our group (Tu et al., 2017) and others (Rahman & Luebke, 2022) that these thermal responses are a robust and accurate indicator of motion sickness in laboratory mice and can be recorded simply using infrared imaging. We used this method to monitor the response to provocative motion (PM) in different mouse strains and an aged cohort.
The detection of c-Fos protein is regarded as a reliable marker for the study of neuronal activation following a behavioral stimulus.
c-Fos is an early-gene product produced by neurons after increased action potential discharge. The protein is generally concentrated in the cell nucleus and its production peaks approximately 90 min after an adequate stimulus (Hoffman et al., 1993;Kovács, 2008;Miller & Ruggiero, 1994;Zhu et al., 1995). Using fluorescent immunolabeling and confocal imaging, cfos mRNA and c-Fos protein expression can be precisely localized in activated cells. Despite its extensive use, c-Fos labeling, however, has a significant temporal resolution limitation. Due to its extended time-to-peak, using c-Fos expression to identify the exact time course of neuronal activation is challenging. Therefore, c-Fos detection is often better suited to identifying the involvement of brain regions and cell groups in the neurobiological processing of an applied stimulus (Kovács, 1998). Similar to a study of c-Fos expression in rat medial vestibular nucleus (MVN) after sinusoidal galvanic stimulation of the vestibular periphery (Holstein et al., 2012), we used c-Fos expression to study possible activity changes of the murine EVN after provocative horizontal orbital motion in young adult and aged mice. T tail recordings and c-Fos immunolabeling, but only the experimental groups were exposed to PM.

Experimental animals and ethical statement
Genotyping was done in conjunction with Australian BioResources (Moss Vale, New South Wales) and the Garvan Institute (Sydney) using standard forward and reverse primer sequences recommended by Jackson Laboratory for these commercially available transgenic strains.
A recording chamber (40 × 25 × 30 cm [l × w × h]) with elevated red walls was placed on a laboratory orbital shaker (Model E0M6, Ratek, Australia), and a suspended infrared camera (FLIR-E50, Flir Systems, Wilsonville, OR, USA) was used for monitoring T tail by taking infrared images of the mouse from above.
All mice were habituated to the setup for 7 days prior to recordings to minimize stress induced by handling and exposure to a new environment, which can potentially mask elevated T tail response to PM. The chamber was thoroughly cleaned with 70% ethanol after each mouse.
Individual experimental mice were placed in the chamber for 5 min before recordings to acclimate them to their surroundings as indicated by baseline T tail . PM was generated for 15 min at 60 horizontal orbital revolutions per minute. Infrared images were taken every minute beginning just prior to PM onset (0 min) and then every minute during PM (1-15 min) and for 1 min post-PM (16 min) ( Figure 1a). After T tail recordings, mice were placed back into their home cage for a further 90 min, a time when c-Fos expression is expected to peak (Kovács, 1998(Kovács, , 2008. After this waiting period, the mice were transcardially perfused for immunohistochemical labeling (see below).
Control mice were treated similarly. They were habituated to the setup and placed in the recording chamber for the same amount of time as the PM group, and infrared images were also collected every minute.
However, control mice were not subject to PM. After 16 min in the recording chamber, control mice were also placed back into their home cage for 90 min before transcardial perfusion for immunohistochemical labeling.

Brain tissue collection and immunohistochemical labeling
Mice were deeply anaesthetized with an intraperitoneal mixture of ketamine (100 mg/kg) and xylazine (0.01 mL/g) and transcardially perfused using heparinized saline, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBA). Whole brains were dissected from the skull and postfixed for 3 h in fresh PFA, then rinsed three times for 5 min in PBS, and stored in PBS at 4 • C, for a week or less. Prior to sectioning, the brains were cryoprotected by immersion in 30% sucrose/PBS solution overnight, and coronal sections were cut at 50 µm using a Leica CM1950 freezing cryostat.
To label the EVN neurons, brainstem slices of adult and aged-adult C57BL/6 WT mice were incubated in primary antibodies of anti-ChAT

2.4
Imaging and cell counting EVN neurons in brainstem sections were imaged using a Nikon C1 confocal microscope with 20×, 40×, and 60× objectives. To count the number of ChAT-or GFP-and c-Fos-positive EVN neurons, the brainstem slices were imaged with a z-stack step size of 0.7 µm. Images were processed and reconstructed as maximum-intensity projections or three-dimensional reconstructions using ImageJ (NIH Image) software and EVN neurons were counted. For more details about EVN cell counting, see Lorincz et al. (2022).

Statistics
Statistical analysis was performed in GraphPad Prism software (Graph-Pad, CA, USA) version 9.4.0. Results of statistical analysis are presented as mean ± SEM. To determine the difference of c-Fos expression between groups, ordinary one-way ANOVA was used followed by Tukey's multiple comparisons test. Mann-Whitney U test was used for nonparametric behavioral T tail data. Statistical significance was set at p < .05.

F I G U R E 1
Experimental design and tail temperature (T tail ) response to provocative motion (PM). (a) Experimental design and timeline of experiments; schematic was created by BioRender.com. (b-d; h-j; and n-p) Infrared images of T tail responses to PM of C57BL/6; ChAT-gCaMP6 f ; and aged C57BL/6 mice, respectively. Images were taken during first 5 min of PM; between 5 and 10 min of PM (at peak tail temp), and after 15 min of PM. (e-g; k-m; and q-s) Infrared images of control responses (no PM) of C57BL/6; ChAT-gCaMP6 f ; and aged C57BL/6 mice, respectively, at the same time intervals as experimental groups. Plot (i) Mean ± SEM T tail responses of C57BL/6 experimental group during PM (black trace; n = 14) and control (no PM) group (gray trace; n = 3). Vertical dashed lines for all plots indicate the start and end point of PM for experimental groups only. Plot (ii) Mean ± SEM T tail plots show response of the ChAT-gCaMP6 f experimental group during PM (black trace; n = 5) and the T tail of the control group PM (gray trace; n = 2). An outlier (red dashed line) differed from the mean response to PM (n = 1). The outlier trace suggested a summed

T tail response to PM
The average T tail of young adult C57BL/6 mice (n = 14; Figure 1b-d) was initially 26.11 ± 0.73 • C and, after 3 min of PM, increased to 27.06 ± 0.76 • C. T tail reached a peak within 7 min (30.02 ± 0.74 • C) and thereafter began to drop gradually, as previously reported (Tu et al., 2017), to an average temperature of 27.83 ± 1.06 • C after 15 min PM (plot [i] in Figure 1). Chat-gCaMP6 f mice (n = 5) also exhibited similar pattern of T tail responses in response to PM (Figure 1h-j), from a slightly lower average temperature of 24.7 ± 0.3 • C at 0 min to 28.6 ± 1.1 • C after 3 min of PM, reaching a higher peak temperature of 32.38 ± 0.28 • C within 7 min of PM, which then decreased gradually to 29.40 ± 1.48 • C at 15 min of PM. In contrast, aged-C57BL/6 mice (n = 5; All three PM groups showed significantly different T tail response compared to controls over the PM period (U C57BL/6 WT = 0, p C57BL/6 WT < .0001; U Chat-gCaMP6f = 7.5, p Chat-gCaMP6f < .0001; U C57BL/6 aged = 12.5, p C57BL/6 aged < .0001).

Immunolabeling of EVN neurons and c-Fos following PM
The identification, labeling, and counting of EVN neurons were based on our recent study detailing the anatomy of the mouse EVS (Lorincz et al., 2022). Coronal sections (50 µm thick) of PFA-fixed brains were used for the immunohistochemical detection of c-Fos and labeling of EVN neurons in the brainstem (Figures 2 and 3). Choline acetyltransferase (ChAT) primary antibody was used to label EVN neurons in C57BL/6 (Figure 2a,b) and aged-C57BL6 mouse brain tissue response of a quicker onset, nonspecific, stress response added to a typical PM stress response. Plot (iii) Mean ± SEM T tail plots show response of aged C57BL/6 experimental group during PM (black trace; n = 5) and the T tail of the control group (gray trace; n = 3). An outlier (red dashed line) differed in response to mean PM (n = 1) and resembled more closely the young adult C57BL/6 experimental response in Plot i.

Increased c-Fos expression after PM in young adult mice but not in aged mice
In C57BL/6 WT mice, cholinergic EVN neurons were labeled using goat anti-ChAT primary antibody and green Alexa 488 secondary antibody (Figure 3a,d). c-Fos labeling was visualized using rabbit anti c-Fos and red Alexa 594 antibodies (Figure 3b,e). Compared to C57BL/6 WT control group (Figure 3a-c), we found strong c-Fos expression in the C57BL/6 WT PM group (Figure 3d-f). The percentage of ChAT + c-Fos double-labeled EVN neurons was significantly higher in the C57BL/6 WT PM group compared to C57BL/6 WT control (p = .0071) (Figure 3c,f and Graph A in Figure 3).
In Chat-gCaMP6 f transgenic mice, EVN neurons were labeled using primary antibody against GFP in green (Alexa 488) (Figure 3g,j). Similar to WT mice, c-Fos was labeled using rabbit anti-c-Fos and red Alexa 594 antibodies (Figure 3h,k). As above, significantly higher ChAT + c-Fos EVN labeling (Graph B in Figure 3; p < .0001) was observed in the ChAT-gCaMP6 f-PM group compared to ChAT-gCaMP6 f-control group (Figure 3i,l).
In aged C57BL/6 WT mice, EVN neurons were visualized using the blue channel to avoid nonspecific fluorescence from lipofuscin (ChAT and lipofuscin are both localized in the cytoplasm) using goat anti-ChAT and blue Alexa 405 antibodies (Figure 3m,p). Since c-Fos labeling was easily distinguishable from lipofuscin, it was labeled green using rabbit anti-c-Fos and Alexa 488 antibodies (Figure 3n,q). In the aged cohort, C57BL/6 WT PM did not show significantly higher c-Fos expression in the EVN compared to their controls C57BL/6 WT control (Figure 3o,r and Graph C in Figure 3; p = .7424). Figure 4 shows the summary of c-Fos labeling in all groups.

DISCUSSION
In this study, we present evidence for three findings: (1)

Motion sickness in rodents and PM-induced thermal response
Our previous study (Tu et al., 2017) reported a typical T tail increase during horizontal orbital motion as a sign of motion sickness in CBA mice that peaked within 4-10 min after PM onset and then returned close to baseline of 24-25 • C despite continued rotation (Tu et al., 2017).
Our findings in a further two mouse strains are consistent with that study and another recent murine motion sickness study (Rahman & Luebke, 2022). In young adult C57BL/6 and transgenic ChAT-gCaMP6 f mice, we found similar T tail responses, although ChAT-gCaMP6 f mice showed an elevated peak T tail compared to C57BL/6 mice within 6-7 min of PM onset (Figure 4a).
It is unclear why there was a higher T tail response in the transgenic strain. Increased blood flow to the extremities, including the tail, can be a result of sympathetic nervous system activation during general stress (Fuller et al., 2010;Jansen et al., 1995). However, the relatively slow time-to-peak T tail in this strain (6−7 min) suggests it is PM induced for several reasons. First, the contribution of general stress was minimized by prior handling and habituation. Second, during general stress, time-to-peak T tail occurs more rapidly (1−2 min) compared to PM T tail (Rahman & Luebke, 2022). The slower PM time-to-peak T tail response is thought to be the result of the time taken for sensory conflict to trigger a coordinated program of body cooling. This program includes decreasing body core temperature and thermogenesis in brown adipose tissue while increasing temperature in the extremities, including the tail (Tu et al., 2017) as described in the shrew and rat (Marks et al., 2009;Ngampramuan et al., 2014). In addition to changes in T tail , other signs of motion sickness were observed in young adult mice. This included cessation of movement and exploratory behavior, tremor, often staying in one corner of the box and lowering their body to the floor of the chamber, and urinary and fecal incontinences (Rahman & Luebke, 2022;Tu et al., 2017;Yu et al., 2007). We made note of these additional behaviors but did not quantify them. By measuring T tail and observing behavioral signs, we were able to distinguish motion sickness symptoms from a general stress response. Thus, we interpret the total tail temperature response observed in the "outlier" of plot (ii) in Figure 1 (red plot) as a combination of a fast, initial temperature rise due to an unrelated stress response, as this individual did not show any initial signs of motion sickness during this early period (1-2 min).
Our results also support the idea that aging is a significant factor affecting susceptibility to motion sickness induced by PM in mice.
Aged C57BL/6 mice did not show the typical increase in T tail peaking (n = 4) and PM group (n = 5). ****Significant difference between the control and PM group (p = .0001). (m) EVN neurons labeled with ChAT antibody (blue) and (n) c-Fos (green) labeling in the control group of aged C57BL/6 mice. Arrowheads denote c-Fos-positive EVN neurons enlarged in the inset. Arrows denote lipofuscin autofluorescence. (o) Merged image of panels (m) and (n). (p) ChAT (blue) and (q) c-Fos (green) labeling in the experimental group of aged C57BL/6 mice after PM. Arrow denotes lipofuscin expressing EVN cell shown in the inset. (r) Merged image of panels (p) and (q). Graph C shows the percentage of c-Fos-positive EVN neurons in the control (n = 3) and PM group (n = 5). Scale bar: 50µm. groups. c-Fos was significantly different between the young adult C57BL/6 and aged C57BL/6 experimental groups (p < .0001) and the ChAT-gCaMP6 f and aged C57BL/6 experimental groups (p < .0001). There was no significant difference between young adult C57BL/6 and ChAT-gCaMP6 f experimental groups (p = .1730) or aged C57BL/6 control group and aged C57BL/6 experimental group.
at 6-7 min of PM, rather a much slower time course that only began after 6 min (Figure 4a and plot [iii] in Figure 1). Moreover, aged mice did not show the other typical symptoms of motion sickness; they did not stop moving and continued exhibiting exploratory behavior. These results are strikingly similar to observations of an age-related decrease in motion sickness susceptibility in humans (Lawther & Griffin, 1988;Paillard et al., 2013;Zhang et al., 2016) and rats (McCaffrey & Graham, 1980). In short, the lack of a typical change in T tail together with the absence of other motion sickness symptoms in aged mice likely reflects a decreased sensitivity to PM in this cohort.

Increased c-Fos expression in EVN neurons in response to PM
Previous studies in rats have shown increased c-Fos protein expression in central vestibular nuclei after vestibular stimulation (Liu et al., 1999(Liu et al., , 2000. Specifically, increased c-Fos expression was observed in the MVN, an important target of afferent vestibular input, after rotational motion (Cai et al., 2010). Moreover, increased c-Fos expression in the MVN was demonstrated after galvanic vestibular stimulation (Holstein et al., 2012). To date, there have been no studies of c-Fos expression in vestibular nuclei of mouse or any other animal's EVN following PM.
In this study, we found strong expression of c-Fos after PM in the mouse MVN, consistent with studies in rats, and we also observed However, the T tail response was more typical of young adult mice, reaching peak T tail 6-7 min after the onset of PM. This discrepancy in the aged outlier, between T tail and the lack of increased c-Fos expression or signs of motion sickness, suggests c-Fos expression in the EVN may be an even more robust indicator of motion sickness than T tail .

Effects of aging on the susceptibility of motion sickness
It has been previously reported in humans that gender and age are two major determinants of motion sickness susceptibility; women tend to be more susceptible (Kennedy et al., 1995), while aged individuals seem to be less prone (Paillard et al., 2013;Reason, 1978;Turner, 1999).
While sexual dimorphism is of interest, it was not an aim of this study to compare male and female mice since we only had access to males in the aged group. By chance, we had a predominantly female representation in young adult cohorts, but we suggest it should not influence our agedependent results significantly since Rahman and Luebke (2022) show there were no differences in PM response between young adult males and females.
The age-dependent susceptibility in humans appears to peak between 9 and 10 years and begins to decline into middle age and beyond (Cooper et al., 1997;Gahlinger, 2000;Golding, 2006). A similar age dependence occurs in rats (McCaffrey & Graham, 1980;Zhou et al., 2017). Our study of young adult mice (C57BL/6 and transgenic Chat-gCaMP6 f ) also showed significant sensitivity to PM, while C57BL/6 aged mice did not show the typical elevated T tail response.
All these age-related changes likely contribute to altered or reduced sensation of balance. In humans, this leads to serious clinical sequelae including falls and injuries (Agrawal et al., 2020). Paradoxically perhaps, it is these same changes that may also result in reduced or absent motion sickness symptoms in response to PM in aged individuals.
Our focus in this study has been the EVS, since we know aging impacts cholinergic systems and can result in significant changes such as cognitive decline in humans (Gallagher & Colombo, 1995).
The mechanisms involved include decreasing levels of ChAT, acetylcholinesterase (AChE) (Perry, 1980;Perry et al., 1992), and cholinergic muscarinic receptor binding (Perry, 1980). Moreover, age-related loss of cholinergic neurons has been reported in the forebrain in humans (Sarter & Bruno, 2004) and rats (Fischer et al., 1992). Similar cholinergic decline has been observed in the inner ear. For example, the number of efferent cholinergic auditory neurons in the gerbil was reduced with age, but perhaps surprisingly, not seen in the EVN (Radtke-Schuller et al., 2015). Although it was not our main aim to count EVN neurons in this study, we did not see any obvious decrease in EVN neuron number in aged mice, which is consistent with findings in the gerbil. Despite the apparent lack of impact on EVN neuronal numbers in aged rodents, it is still possible that ChAT and AChE and receptor binding levels in the EVS decrease with age, similar to cholinergic changes in other systems.

4.4
EVN activation during PM, a possible contribution to nausea symptoms Vestibular afferents and one of their major central targets, MVN neurons, are reported to project to the EVN (Chi et al., 2007;Li et al., 2005;Liu et al., 2021;Wang et al., 2013). Indeed, the MVN has been shown to have direct excitatory projections (Liu et al., 2021) via ipsilateral glutamatergic (Mathews et al., 2015) closed-loop circuits to EVN neurons (Chi et al., 2007;Li et al., 2005;Liu et al., 2021;Wang et al., 2013;Zhou et al., 2018). There also appear to be reciprocal connections since EVN neurons provide extensive dendritic branching toward the MVN (Lorincz et al., 2022). Therefore, in young adult mice, PM would produce a significant volley of afferent vestibular input to the MVN that, in turn, would activate EVN cells and therefore increase their c-Fos expression via glutamatergic short-loop projections. In contrast, since aging impacts vestibular hair cells and afferent fibers (Bergström, 1973a(Bergström, , 1973bRichter, 1980;Rosenhall, 1973;Rosenhall & Rubin, 1975), we conjecture that overall vestibular input to the central nervous system is reduced. This reduction would directly impact the MVN/EVN circuitry. Therefore, the stimulus induced by PM would be similarly diminished by aging. The lack of c-Fos expression in aged mice would support this notion, as do the attenuated motion sickness symptoms, including the significantly altered T tail in the first 6-7 min of the PM exposure.
Taken together, our results highlight a potential link between EVS activation and the generation of motion sickness symptoms. For example, elevated activity of the EVN, in response to PM exposure, is strongly associated with symptoms of motion sickness. Similarly, compromised EVS activity corresponds with diminished or the absence of motion sickness symptoms (Tu et al., 2017). Nevertheless, the precise link between EVN neurons and motion sickness symptoms has yet to be determined. The chemoreceptor trigger zone (CTZ) or area postrema is known to control nausea and emesis and is located in the floor of the fourth ventricle of the dorsal medulla oblongata. Its main function is monitoring the cerebrospinal fluid (CSF) and blood for emetic agents and toxins. Since the CTZ and its receptors lie outside the blood-brain barrier, it is able to sample larger molecules within the CSF or those that diffuse out of nearby blood vessels (Borison, 1989). CTZ receptors are sensitive to dopamine, histamine, serotonin, encephalins, substance-P, acetylcholine (Miller & Leslie, 1994), and PACAP (pituitary adenylate cyclase-activating polypeptide). Indeed, PACAP-positive fibers are found within the CTZ (Hannibal, 2002).
Although the main fast EVN neurotransmitter is AChE, together with neuropeptides such as calcitonin gene-related peptide (CGRP) and enkephalins (Perachio & Kevetter, 1989), our preliminary unpublished data show the presence of substance-P and PACAP in EVN neurons.
As we have previously reported, a characteristic anatomical feature of the EVN is its close proximity to a blood vessel, which often bisects it (Lorincz et al., 2022). It is possible that during the EVN activation, some of the neurochemicals mentioned above (especially neuropeptides CGRP, enkephalins, and PACAP) could enter the bloodstream and trigger the CTZ, to generate nausea-like symptoms and even emesis.
A more direct association between the EVN and CTZ has been shown with viral tracing studies that suggest the CTZ provides projections to