Alpha-9 nicotinic acetylcholine receptors mediate

Physiology & Behavior 174 (2017) 114–119

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Alpha-9 nicotinic acetylcholine receptors mediate hypothermic responses elicited by provocative motion in mice Longlong Tu a, Lauren Poppi b, John Rudd a, Ethan T. Cresswell b, Doug W. Smith b, Alan Brichta b, Eugene Nalivaiko b,⁎ a b

School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China School of Biomedical Sciences and Pharmacy, University of Newcastle, Newcastle, NSW, Australia

H I G H L I G H T S • • • •

α9-AChR KO and WT mice were subjected to provocative motion and balance test. Motion stimuli caused hypothermic responses that were attenuated in KO mice. Motion stimuli caused cutaneous vasodilation that was attenuated in KO mice. KO mice required 25% more time to complete motor coordination/balance test.

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Article history: Received 7 December 2016 Received in revised form 6 March 2017 Accepted 10 March 2017 Available online 14 March 2017 Keywords: Motion sickness Hypothermia Efferent vestibular system Nicotinic acetylcholine receptor Cholinergic

a b s t r a c t Hypothermic responses accompany motion sickness in humans and can be elicited by provocative motion in rats. We aimed to determine the potential role in these responses of the efferent cholinergic vestibular innervation. To this end, we used knockout (KO) mice lacking α9 cholinoreceptor subunit predominantly expressed in the vestibular hair cells and CBA strain as a wild-type (WT) control. In WT mice, circular horizontal motion (1 Hz, 4 cm radius, 20 min) caused rapid and dramatic falls in core body temperature and surface head temperature associated with a transient rise in the tail temperature; these responses were substantially attenuated in KO mice; changes were (WT vs. KO): for the core body temperature − 5.2 ± 0.3 vs. − 2.9 ± 0.3 °C; for the head skin temperature − 3.3 ± 0.2 vs. −1.7 ± 0.2 °C; for the tail skin temperature + 3.9 ± 1.1 vs + 1.1 ± 1.2 °C. There was a close correlation in the time course of cooling the body and the surface of the head. KO mice also required 25% more time to complete a balance test. We conclude: i) that the integrity of cholinergic efferent vestibular system is essential for the full expression of motion-induced hypothermia in mice, and that the role of this system is likely facilitatory; ii) that the system is involvement in control of balance, but the involvement is not major; iii) that in mice, motion-induced body cooling is mediated via increased heat flow through vasodilated tail vasculature and (likely) via reduced thermogenesis. Our results support the idea that hypothermia is a biological correlate of a nausea-like state in animals. © 2017 Published by Elsevier Inc.

1. Introduction The phenomenon of motion sickness has been extensively studied and the currently accepted theory proposes that motion sickness develops when “sensory conflict” occurs between converging patterns of vestibular, visual, and proprioceptive inputs [10,11,18,32]. Precisely how these sensory modalities combine to produce motion sickness is not understood, but functional inner ear vestibular organs appear to be a prerequisite. For example, dogs are immune to motion sickness ⁎ Corresponding author at: School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW 2308, Australia. E-mail address: [email protected] (E. Nalivaiko).

http://dx.doi.org/10.1016/j.physbeh.2017.03.012 0031-9384/© 2017 Published by Elsevier Inc.

after bilateral labyrinthectomy [40], as are humans with bilateral vestibular deficiency [2,17]. It must be acknowledged that there is still some controversy in the field as sensitivity to both visual and vestibular stimulation has been reported in some individuals with bilateral vestibular deficiency [9,15]. A major component of vestibular organs is the sensory receptors or hair cells. These receptors convert (transduce) head movement into neural signals that are transmitted to the brain via the eighth cranial nerve. These signals play a crucial role in the pathogenesis of motion sickness and therefore modulation of hair cell output is likely to affect or even alleviate the deleterious effects of provocative motion. Vestibular hair cells not only transmit signals to afferent nerves but also receive efferent input from the brainstem cholinergic ‘group e’, part of the efferent vestibular system or EVS [8,31,34]. Although the

L. Tu et al. / Physiology & Behavior 174 (2017) 114–119

functional role of EVS in mammals is still poorly understood, preliminary evidence in mice [29] and more extensive non-mammalian studies suggest the EVS has a modulatory effect on hair cells [14]. This modulation of hair cells is achieved mainly through the principal EVS neurotransmitter, acetylcholine (ACh) [16] acting on nicotinic receptors containing the α9 subunit (α9-AChR). These particular receptors are found predominantly in the inner ear and specifically on the surface of type II hair cells [6,19]. In the current study we used wild type controls and the α9-AChR-knockout (α9−/−) mouse strain to explore the potential role of the EVS in physiological changes induced by provocative motion. The major challenge in preclinical studies of motion sickness is objective assessment of its principal symptom, nausea. Most laboratory animal species do not possess vomiting reflex, and conclusions about a nausea-like state in such animals rely on behavioral indices with relatively low temporal resolution (e.g. pica [41]), or on those which require training paradigms (e.g. conditioned taste aversion [7], conditioned gaping or retching [28,39]), or utilize complex analysis of behavioral clusters [13]. Assessing emetic episodes in the relatively few common laboratory species with a vomiting reflex (dog, cat, ferret, house musk shrew) also has limitation as the pharmacology and neural substrates of nausea and emesis are different [36]. Our recent studies have demonstrated that provocative motion causes robust and prominent hypothermic responses in rats [4,12,24]. We contend that these falls in body temperature represent a biomarker of a nausea-like state in laboratory animals for four reasons: 1) these responses are provoked by motion and by chemical emetic stimuli [1,3,4,12,24]; 2) differential pharmacological sensitivity of these responses in rats mirrors sensitivity in humans [12]; 3) in house musk shrews, motion-induced hypothermia precedes emetic episodes [24]; and 4) there is a clear parallel in hypothermic responses between animals and humans in underlying physiological mechanism - cutaneous vasodilatation that favors heat loss [22, 24]. It is currently unknown whether emetic stimuli trigger hypothermic responses in mice, and the first aim of our study was to test whether provocative motion reduces body temperature in this species, and to determine physiological mechanism that mediate this response. Second, comparing responses in α9-nAChR-knockout mice with wildtype controls, we addressed the question of whether these receptors influence afferent vestibular signals, and hence are potentially involved in the pathogenesis of motion sickness.

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procedures were done under isoflurane anesthesia (1.5%) and body temperature was maintained using a heating pad. After a midabdominal incision, a telemetric transmitter (TA-F10, DSI, St Paul, MN, USA) was placed into the peritoneal cavity. Marbofloxacin and buprenorphine (0.05 mg/kg) were administered post-surgically. Animals were allowed to recover for one week. 2.3. Experimental procedures, data collection and analysis On the day of experiment, the telemetric transmitter was magnetically activated, the mouse together with its home cage and a telemetric receiver, inserted under the cage, was placed on a laboratory orbital shaker (Model E0M6, Ratek, Australia). An infrared camera (FLIR-E50, Flir Systems, Wilsonville, OR, USA) was fixed above the cage. The cage top was removed, and cage height was extended to 50 cm by adding cardboard sides. This allowed an unobstructed view of the mouse from above, while preventing escape. A 15-min baseline activity recording (shaker OFF) was followed by 20 min of provocative motion (shaker ON, 1 Hz, 4-cm orbital displacement), and by 15-min recovery (shaker OFF). After the experiment, α9−/− and CBA mice were either euthanized by an overdose of Xylazine or by cervical dislocation, to compare cooling rate observed during provocative motion with cooling rate following death. The radiofrequency-modulated signal from the temperature transmitter was converted into an analog signal by means of the Temperature Analog Adaptor (DSI, St Paul, MN, USA) and acquired at 1 Hz using PowerLab-8s A/D converter and a Dell computer running Chart 7.0 software (ADInstruments, Sydney, Australia). Cranial skin and tail skin temperatures were measured off-line using ResearchIR software (Flir Systems, Wilsonvialle, OR, USA). Tail temperature was measured approximately 2 cm from the base of the tail; head skin temperature was measured from a circular region (8 mm diameter) at the middle of the head image. Infrared data were collected every 2 min; for compatibility, core body temperature data we averaged for 2-min periods. 2.4. Balance beam walk assay Two separate groups of α9−/− (n = 9) and CBA (n = 9) mice were trained to walk across a narrow beam (1800 mm in length, 12 mm wide, and mounted 600 mm from the floor as in [20]). On a separate day, mice of each genotype completed 4 trials of the balance beam walk task, with each trial being timed and the 4 trials averaged for each mouse.

2. Methods 2.5. Statistical analysis 2.1. Experimental animals and ethics statement As control animals, we used six background strain mice CBA/ CaJ,129SvEvTac (CBA), the CBA strain was regularly refreshed using an SvEvTac mouse line to prevent genetic drift. We also used six homozygous α9-subunit knockout (α9−/−) mice (JAX005696; CBACaJ;129SChrna9tm1Bedv). The original a9−/− mice from Jackson Labs were back-crossed onto the CBA background control strain. Heterozygous offspring were used to produce a homozygous knockout line. The α9−/− genotype was confirmed using standard PCR and the mutant primer sequence ‘CAC GAG ACT AGT GAG ACG TG’. Body weights of the two mouse strains were 23 ± 1 (CBA) and 24 ± 1 g, (α9−/−). All procedures described below were approved by the University of Newcastle Animal Care and Ethics Committee, and complied with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. 2.2. Surgery Prior to all experiments, mice were housed in temperaturecontrolled room at 22 °C, under 12/12 h artificial light cycle. Water and food were available ad libitum. Animals were housed individually from the day of surgery to the end of the experiment. Surgical

We used the last time-point of baseline recording as reference point for statistical analysis. Statistical differences within groups were determined using repeated one-way ANOVA; statistical differences between groups - using repeated measures two-way ANOVA, with “time” and “group” factors, followed by post-hoc Bonferroni test. Pearson's correlation was used to assess temporal relations between the core body and head temperature values. For the balance beam walk assay, groups were compared by Students' t-test. All data are expressed as mean ± S.E.M. Differences are considered statistically significantly at p b 0.05. We used Prism v.6 (GraphPad, CA, USA) for all statistical analyses. 3. Results Prior to the onset of motion stimuli, the temperature values for tail, core body, and head (Ttail, Tcore, and Thead) did not differ between CBA and α9−/− mice. Corresponding values were (CBA vs. α9−/−): 24.7 ± 0.2 vs. 24.5 ± 0.1 °C; 38.1 ± 0.2 vs. 37.8 ± 0.2 °C; and 32.5 ± 0.2 vs. 32.8 ± 0.2 °C for Ttail, Tcore, and Thead, respectively; p N 0.5 for each pair. Within two minutes of provocative motion onset, infrared images of CBA mice showed that Ttail started to rise and reached a peak of 28.5 ± 1.0 °C within 6 min, and then gradually returned to the baseline

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(Fig. 1A). Ttail values in CBA mice were significantly different (p b 0.01) from the baseline from 4 to 8 min after the onset of motion. While a slight increase in Ttail values was seen in α9−/− mice, these changes were not significant (p N 0.05). Therefore, significant differences between CBA and α9−/− mice were observed between 4 and 8 min of motion (Fig. 1A). Fig. 2 shows representative thermal images just before and 4 min after the onset of provocative motion of CBA (left images) and α9−/− mice (right images). Note the raised temperature of the CBA mouse tail. Provocative motion also caused rapid and substantial fall in the Tcore in both groups, with a significantly larger response in CBA mice than α9−/− (− 5.2 ± 0.3 vs. − 2.9 ± 0.3 °C; p b 0.01). In both groups, the temperature change was significantly lower compared to baseline (p b 0.01) at 4 min after onset of provocative motion until the end of experiment (Fig. 1B). Significant between-group differences were found from 8 min after onset of motion to the end of experiment. Analysis of thermal images revealed that changes in Thead mirrored falls of the Tcore (Fig. 1C). Head cooling was significantly larger in CBA vs. α9−/− groups (−3.3 ± 0.2 vs. −1.7 ± 0.2 °C, respectively; p b 0.01). Compared to baseline, Thead became significantly different (p b 0.01) starting from the 6th min of the provocation onset. Between-group difference was significant from 10 min after onset to the end of experiment (p b 0.01). Changes in Tcore and Thead temperature significantly correlated (r2 ranged from 0.92 to 0.95 and from 0.81 to 0.90 in CBA and α9−/− mice, respectively); example of such correlation for one CBA and one α9−/− mouse is shown in Fig. 3.

In order to determine how motion-induced hypothermia is related to a fall in body temperature after death, when neutrally-induced thermogenesis ceases, in a subset of animals (n = 5) we compared hypothermia induced by provocative motion with temperature fall following rapid euthanasia by cervical dislocation. As shown in Fig. 4, the time course of temperature fall was nearly identical for the first 16 min of these two conditions; after which cooling continued in euthanized mice but slowed in live mice. During baseline period all animals in both groups were active, and moved constantly around the cage with only a few pauses. This locomotor activity ceased soon after the onset of provocative motion, and the rest of the time mice of both strains remained largely motionless. Notably, their posture did not resemble tense freezing; rather they were laying down on the cage floor. The time when this immobility stage began corresponded to falls in the core body temperature to 37.7 ± 0.2 and 37.3 ± 0.4 °C for CBA and α9−/− mice, respectively (p = 0.27). There was no obvious postural/motor animalies when α9−/− were observed in their home cages. However, in the balance beam walk test α9−/− mice took significantly longer to traverse the beam compared their wild type CBA counterparts (10.3 ± 0.5 vs 7.2 ± 0.4 s, respectively; p = 0.013). 4. Discussion We have made the following novel findings: 1) in mice, provocative motion causes rapid and significant (N5 °C) hypothermic responses, with the initial phase of the temperature decrease comparable to that occurring immediately after death; 2) in CBA mice these responses are mediated, at least in part, by tail vasodilatation; 3) in α9−/− mice there is a suppression of motion-induced tail vasodilatation and substantial attenuation of associated hypothermia; 4) there is a close correlation in temporal course between the Tcore and Thead during provocative motion; and 5) α9−/− mice underperformed in the balance test compared to CBA mice. 4.1. Hypothermia as a biological correlate of motion sickness in animals Hypothermia during motion sickness is a well documented phenomenon; it has been reported as a consequence of provocative motion in humans, rats, musk shrews and now, mice (see [23] for review). We contend this represents a general biological correlate of motion sickness in animals (see Section 1 for supporting evidence). The initial trigger for the hypothermia is vestibular stimulation; in rats it could be prevented by bilateral labyrhynthectomy [27]. Our study is the first description of a similar physiological response in mice. Therefore, the existence of motion-induced hypothermia in four mammalian species suggests that this reaction may be conserved in many if not most mammals. In our recent review [23] we have proposed that motion-induced fall in the body temperature is possibly due to “unintended” activation of the evolutionary beneficial “defensive hypothermia” that normally accompanies intoxication [33]. This hypothermic reaction is possibly triggered by the output of the hypothetical “sensory conflict” circuitry [32], and is interpreted by the brain as a sign of toxin ingestion; this idea is consistent with the “toxic” theory of nausea during motion sickness [38]. The idea that hypothermic response to motion is a highly coordinated reaction is supported by the fact that it includes such diverse components as cognitive (altered perception of ambient temperature), behavioral (cold-seeking) and physiological (increased sweating and cutaneous vasodilation leading to heat loss and reduced thermogenesis) [23]. 4.2. Physiological hypothermia

Fig. 1. Time course of temperature changes in α9−/− (squares) and CBA (circles) mice induced by provocative motion. A – tail skin temperature; B – core body temperature; C – head skin temperature. Vertical lines indicate moments when the motion was turned on and off. ** - statistical differences between groups; p b 0.01.

mechanisms

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motion-induced

We noted differences in the magnitude of motion sickness-related hypothermia between rats and mice. In several previous rat studies conducted in four different laboratories but at similar ambient temperature,


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Fig. 2. Infrared images of one CBA and one α9−/− mouse just before the onset of provocative (top panels) motion and at 4 min after the onset (bottom panels). Noticeable tail warming occurred in CBA but not α9−/− mouse (arrows). Pseudo-color temperature scale is shown on the right in °C.

there is good agreement that core body temperature falls do not exceed 2 °C during 40-min provocation [4,12,22]. Compared to rats, hypothermic responses in mice were much more pronounced, especially in the CBA strain where they averaged twice the temperature fall (N 5 °C) in only half the time (20 min). One explanation for the large temperature differences between the two species could be due to the different provocative stimuli used (rotation around vertical axis in rats vs. orbital motion in mice). Nevertheless, it is also reasonable to suggest the differences in total body mass and/or body mass/surface area contributes to the faster temperature fall in smaller animals. In rodents, control of tail (cutaneous) vasculature is the principal mechanism of heat loss [25,26]. It is obvious that in our previous rat experiments [24] and in the current mouse study provocative motion caused falls in body temperature through this mechanism as evidenced by tail warming. It is however quite likely that cutaneous vasodilatation was not the only mechanism involved in this response. First, in rat studies, the onset of body cooling preceded the onset of tail vasodilation [24]. Second, in both species tail vasodilation was transient, while body temperature continued to fall even after the return of the tail temperature to the baseline. Third, in our study of α9−/− mice, tail warming was minimal and yet this strain still developed substantial hypothermia. Thus it is plausible to suggest the “sensory mismatch” signal activated a coordinated program of body cooling, including not only heatdissipating mechanism (tail vascular bed) but also reduced

thermogenesis in the brown adipose tissue (BAT). This notion is indirectly supported by studies in musk shrews where provocative motion reduced the temperature difference between interscapular and lumbar regions [24], which is indicative of reduced BAT thermogenesis [21]. It is of major interest that motion-induced hypothermia has a similar time course as body cooling after euthanasia. While the mechanisms of hypothermia were different here - increase in the transcutaneous heat loss in the former vs. cessation of neutrally-induced thermogenesis associated with circulatory stasis (i.e. minimal heat loss from the skin) in the latter case – we believe that our observation is worth presenting here as it underscores that motion-induced hypothermia was indeed very substantial. In conclusion, it now clear that tail vasodilation is not the only physiological mechanism that mediate hypothermia induced by provocative motion. It is plausible that nausea-like state may be also associated with reduction in BAT activity; to clarify this, further experiments with direct monitoring of BAT temperature are required. 4.3. Differences between CBA and α9−/− mice Our most intriguing and novel observation is the difference in hypothermic and cutaneous vasodilator responses to motion found between CBA and α9−/− mice. Reduced responses in the latter strain could be potentially caused by three factors: 1) differences in vestibular afferent mechanisms (detection and transmission of information from motion

Fig. 3. Correlation between changes in core body temperature and surface head temperature in one CBA mouse (A; r2 = 0.89) and one α9−/− mouse (B; r2 = 0.81) during provocative motion.

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Fig. 4. Time course of the body cooling in CBA mice was similar during provocative motion in live animals (squares) and post-euthanasia (triangles). Vertical line indices the start of motion stimuli or the moment of cervical dislocation.

sensors); 2) differences in central processing (detection of “sensory mismatch” and routing it to the central thermoregulatory network); or 3) differences in the efferent pathways to the thermo-effectors (skin vasculature and BAT). Lack of differences in baseline body or skin temperatures between the two strains suggests that the absence α9-AChR expression does not cause any gross disturbances in the thermoregulatory homeostatic mechanisms. Much more important and relevant here is the fact that α9-AChR subunits are predominantly expressed in cochlear and vestibular organs, particularly in type II hair cells [6,19]. Consequently, the most likely neural substrate responsible for physiological differences reported here are the synaptic connection between the efferent vestibular system (EVS) and type II hair cells. Data on the functional role of the EVS in mammals is limited. Classic studies using direct electrical stimulation of the efferent vestibular neurons in anesthetized squirrel monkey caused consistent increase in the background vestibular activity [8]. Therefore, a compromised EVS would reduce or eliminate this excitatory effect, and this is a potential explanation of why motion-induced responses were attenuated in our α9−/− mice. Recently, Hubner and colleagues [14] demonstrated that in α9−/− mice, whose EVS is impaired, there was a moderate reduction in the sensitivity of the vestibular-ocular reflex (VOR). In human studies it has been reported that lower VOR sensitivity is associated with reduced susceptibility to motion sickness [35,37]. Therefore, we propose that reduced hypothermic responses to provocative motion stimuli in α9−/− mice were the consequence of abolished synaptic transmission between the EVS and type II hair cells. This possibility is supported by our latest in vitro findings which demonstrate that type II hair cells are insensitive to cholinergic stimulation in α9−/− mice [30]. In addition to impaired VOR sensitivity, Hubher [14] also found a major deterioration of VOR adaptation in α9−/− mice. VOR adaptation is a form neural plasticity that allows for the long-term adjustment of VOR gain to compensate for changed viewing conditions. While significantly impaired VOR adaptation could be another potential explanation of our results, this runs counter to human studies that report VOR adaptation associated with amelioration of motion discomfort [5]. It should be noted, however, that this human study used altered viewing conditions and therefore does not readily equate to the standard viewing conditions used in our experiments. Evidence from our current and cited Hubner's [14] experiments suggests that α9−/− mice might have bilateral vestibular deficit. While we did not observe any obvious gross motor disturbances in knockout animals, subjecting them to specialized motor balance and coordination test [20] revealed that they require 25% more time to cross a narrow beam. The most likely explanation of this fact it that they have subtle impairment of balance – an abnormality that also made them less susceptible to provocative motion. 4.4. Conclusions and perspectives Our results favor the notion that hypothermia following emetic provocations reflects a nausea-like state in experimental animals and

that α9−/− mice experienced a lower intensity nausea-like state compared to CBA mice. The major reason for this difference between the strains may be the impaired cholinergic transmission between the EVS and vestibular type II hair cells that normally express the α9-nAChRs. A conservative explanation of this phenomenon would be as follows. EVS activation of α9-AChR associated with type II hair cells maintains their contribution to the mechanism of motion detection. Therefore, a lack of such a contribution in α9−/− mice leads to reduction of all reactions triggered by motion, including “defensive hypothermia” [23]. If so, pharmacological agents that selectively block α9-AChR in the vestibular type II hair cells may represent a novel potential class of anti-motion sickness drugs. It is in fact tempting to speculate that therapeutic effect of scopolamine (a non-specific muscarinic antagonist and a common anti-motion sickness medication) could be mediated, at least in part, by the peripheral vestibular action. Finally, we believe that the finding of highly correlated changes between the head temperature (Thead) and the core body temperature (Tcore) is of substantial interest to both thermoregulatory and emesis fields. There is no subcutaneous fat surrounding the skull, and thus skin temperature in this region is determined by: i) the temperature of the underlying tissue (brain); ii) the temperature of the incoming blood; and iii) the extent of cutaneous vasoconstriction. From our infrared tail data, it is obvious that cutaneous vasoconstriction could not account for the head cooling. That the incoming blood was cooler is obvious from our telemetric data; this same cooler blood also supplied brain that likely became cooler as well; in support, in our rat study we observed fall in hypothalamic temperature during provocative motion [4]. It is clear that motion-induced hypothermia could be reliably assessed in a non-invasive way, simply by recording the head temperature by infrared thermography. Since this phenomenon is the only known occasion (with the exception of cold exposure) when exogeneous provocation causes fall in body temperature, this could be a simple, inexpensive and non-invasive way to determine and monitor a motion-induced nausea-like state in experimental animals. Currently there is no real-time and consistent physiological biomarker of motion sickness for preclinical studies. References [1] M. Ary, W. Chesarek, S.M. Sorensen, P. Lomax, Naltrexone-induced hypothermia in the rat, Eur. J. Pharmacol. 39 (1976) 215–220. [2] B.S. Cheung, I.P. Howard, K.E. Money, Visually-induced sickness in normal and bilaterally labyrinthine-defective subjects, Aviat. Space Environ. Med. 62 (1991) 527–531. [3] N.R. Cutler, R.M. Post, W.E. Bunney Jr., Apomorphine hypothermia: an index of central dopamine receptor function in man, Commun. Psychopharmacol. 3 (1979) 375–382. [4] F. Del Vecchio, E. Nalivaiko, M. Cerri, M. Luppi, R. Amici, Provocative motion causes fall in brain temperature and affects sleep in rats, Exp. Brain Res. 232 (2014) 2591–2599. [5] J.L. Demer, F.I. Porter, J. Goldberg, H.A. Jenkins, K. Schmidt, Adaptation to telescopic spectacles: vestibulo-ocular reflex plasticity, Invest. Ophthalmol. Vis. Sci. 30 (1989) 159–170. [6] A.B. Elgoyhen, D.S. Johnson, J. Boulter, D.E. Vetter, S. Heinemann, Alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells, Cell 79 (1994) 705–715. [7] J. Garcia, W.G. Hankins, On the origin of food aversion paradigms, in: L.M. Barker, M.R. Best, M. Domjan (Eds.), Learning Mechanisms in Food Selection, Baylor University Press, Waco, Texas 1977, pp. 3–22. [8] J.M. Goldberg, C. Fernandez, Efferent vestibular system in the squirrel monkey: anatomical location and influence on afferent activity, J. Neurophysiol. 43 (1980) 986–1025. [9] J. F. Golding. Motion sickness. In: Handbook of Clinical Neurology. vol. 137. Neurootology. J Furman & T Lempert. Elsevier Publishers. p. 371–90. [10] J.F. Golding, M.A. Gresty, Motion sickness, Curr. Opin. Neurol. 18 (2005) 29–34. [11] J.F. Golding, M.A. Gresty, Pathophysiology and treatment of motion sickness, Curr. Opin. Neurol. 28 (2015) 83–88. [12] D.D. Guimaraes, P.L.R. Andrews, J.A. Rudd, V.A. Braga, E. Nalivaiko, Ondansetron and promethazine have differential effects on hypothermic responses to lithium chloride administration and to provocative motion in rats, Temperature 2 (2015) 543–553. [13] C.C. Horn, S. Henry, K. Meyers, M.S. Magnusson, Behavioral patterns associated with chemotherapy-induced emesis: a potential signature for nausea in musk shrews, Front. Neurosci. 5 (2011).

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