Identification of neural networks that contribute to motion sickness through principal components analysis of fos labeling induced by galvanic vestibular stimulation

Motion sickness is a complex condition that includes both overt signs (e.g., vomiting) and more covert symptoms (e.g., anxiety and foreboding). The neural pathways that mediate these signs and symptoms are yet to identified. This study mapped the distribution of c-fos protein (Fos)-like immunoreactivity elicited during a galvanic vestibular stimulation paradigm that is known to induce motion sickness in felines. A principal components analysis was used to identify networks of neurons activated during this stimulus paradigm from functional correlations between Fos labeling in different nuclei. This analysis identified five principal components (neural networks) that accounted for greater than 95% of the variance in Fos labeling. Two of the components were correlated with the severity of motion sickness symptoms, and likely participated in generating the overt signs of the condition. One of these networks included neurons in locus coeruleus, medial, inferior and lateral vestibular nuclei, lateral nucleus tractus solitarius, medial parabrachial nucleus and periaqueductal gray. The second included neurons in the superior vestibular nucleus, precerebellar nuclei, periaqueductal gray, and parabrachial nuclei, with weaker associations of raphe nuclei. Three additional components (networks) were also identified that were not correlated with the severity of motion sickness symptoms. These networks likely mediated the covert aspects of motion sickness, such as affective components. The identification of five statistically independent component networks associated with the development of motion sickness provides an opportunity to consider, in network activation dimensions, the complex progression of signs and symptoms that are precipitated in provocative environments. Similar methodology can be used to parse the neural networks that mediate other complex responses to environmental stimuli. © 2014 Balaban et al

Photomicrographs of Fos-labeled neurons in a response type 1 animal (C52).

<p>In each row, a rectangular box on the left diagram (generated from photomontages of sections taken using a 4X objective) shows the region depicted at higher magnification to the right. Scale bars on the right photomicrographs designate 500 µm. <b>A,</b> Fos labeling in nucleus tractus solitarius, approximately 13.5 mm posterior to stereotaxic zero. <b>B,</b> Fos labeling in the rostral portion of the medial vestibular nucleus, approximately 6 mm posterior to stereotaxic zero. <b>C,</b> Fos labeling in the periaqueductal gray, approximately 3 mm rostral to stereotaxic zero. Abbreviations are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086730#pone-0086730-g001" target="_blank">Figs. 1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086730#pone-0086730-g002" target="_blank">2</a>, with the following additions: III, oculomotor nucleus; MR, magnocellular portion of the red nucleus; PAG, periaqueductal gray; SC, superior colliculus.</p

Behaviors exhibited by animals during galvanic vestibular stimulation: <b><i>A,</i></b> sinusoidal head roll at the frequency of the stimulus; <b><i>B,</i></b> nystagmus; <b><i>C,</i></b> frequent licking; <b><i>D,</i></b> retching; <b><i>E,</i></b> excessive salivation; <b><i>F,</i></b> sinusoidal pinna movement at the frequency of the stimulus; <b><i>G,</i></b> vocalization; <b><i>H,</i></b> panting; <b><i>I,</i></b> thrashing in the restraint bag, presumably as an attempt to escape the stimulus; <b><i>J,</i></b> defecation during the stimulation session; <b><i>K,</i></b> urination during the stimulation session; <b><i>L,</i></b> sinusoidal limb movements at the frequency of the stimulus; <b><i>M,</i></b> sedation (sleeping during the majority of the stimulation session).

<p>The behaviors were graded as either being overt <b>(++)</b> or only weakly perceptible <b>(+)</b>. Blank cells indicate that the behavior was not present. Based on these behaviors, we classified the stimulus as being highly effective in generating responses (1), moderately effective in generating responses (2), or ineffective (3). The later category includes two animals (C83 and C84) that served as unstimulated controls. A score sum was generated by assigning a score of 2 to overt symptoms (++), and a score of 1 to weak symptoms (+).</p

Correlation of labeling with symptom score and component loadings from principal component analysis with equamax rotation.

<p><b>Bold type</b> is used to highlight regions with strong negative or positive correlations between the number of Fos-immunopositive neurons and symptom severity scores. <b>Bold type</b> also designates large positive component loadings; <b><i>bold and italicized type</i></b> designates negative component loadings. <i>TPH2: Tryptophan hydroxylase 2,</i> a marker of brain serotoninergic neurons.</p

Locations of Fos-labeled neurons in two animals exhibiting strong symptoms of motion sickness (response type 1) during galvanic vestibular stimulation.

<p>Neuronal locations were plotted on photomontages of sections taken using a 4X objective. Sections (A, E) are from animal C52, whereas (B–D) are from animal C39. The sections were located at the following approximate distances posterior to stereotaxic zero, in accordance with Berman’s atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086730#pone.0086730-Berman1" target="_blank">[85]</a>: A, 13.5 mm; B, 10 mm; C, 9 mm; D, 7 mm; E, 3 mm. <i>Abbreviations</i>: BC, brachium conjunctivum; CN, cochlear nuclei; DMV, dorsal motor nucleus of the vagus; DRNL, lateral division of dorsal raphe nucleus; DRNM, medial division of dorsal raphe nucleus; EC, external cuneate nucleus; G, genu of facial nerve; IO, inferior olivary nucleus; LRN, lateral reticular nucleus; PBN, parabrachial nucleus; PH, prepositus hypoglossi; RB, restiform body; RM, raphe magnus; RO, raphe obscurus; RP, raphe pallidus; SNV, spinal trigeminal nucleus; STN, subtrigeminal nucleus; STV, spinal trigeminal tract; VI, abducens nucleus; VII, facial nucleus; VIN, inferior vestibular nucleus; VLD, dorsal division of lateral vestibular nucleus; VLV, ventral division of lateral vestibular nucleus; VMN, medial vestibular nucleus; XII, hypoglossal nucleus.</p

Characteristics about the animals used in these experiments, as well as the maximal voltage delivered to the labyrinths to induce motion sickness and the period of acclimation to experimental conditions prior to the stimulation session.

<p>Characteristics about the animals used in these experiments, as well as the maximal voltage delivered to the labyrinths to induce motion sickness and the period of acclimation to experimental conditions prior to the stimulation session.</p

Photomicrograph illustrating examples of neurons that were immunopositive for Fos (solid black arrows), TPH-2 (solid gray arrows) and both TPH-2 and Fos (open arrow).

<p>A rectangular box on the inset diagram indicates the region of the dorsal raphe nucleus depicted in the photomicrograph. The scale bar represents 250 µm.</p

Correlation of labeling with symptom score and component loadings from principal component analysis with equamax rotation, with nuclear subdivisions included.

<p><b>Bold type</b> is used to highlight regions with strong negative or positive correlations between the number of Fos-immunopositive neurons and symptom severity scores. <b>Bold type</b> also designates large positive component loadings; <b><i>bold and italicized type</i></b> designates negative component loadings. <i>TPH2: Tryptophan hydroxylase 2,</i> a marker of brain serotoninergic neurons.</p