Evidence for vestibular regulation of autonomic functions in a mouse ...

Evidence for vestibular regulation of autonomic functions in a mouse genetic model Dean M. Murakami*†, Linda Erkman‡, Ola Hermanson§, Michael G. Rosenfeld†‡, and Charles A. Fuller* *Section of Neurobiology, Physiology, and Behavior, University of California, Davis, CA 95619-8519; ‡Department of Medicine, University of California at San Diego, La Jolla, CA 92093-0648; and §Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, SE-171 77 Stockholm, Sweden Contributed by Michael G. Rosenfeld, October 28, 2002

Physiological responses to changes in the gravitational field and body position, as well as symptoms of patients with anxietyrelated disorders, have indicated an interrelationship between vestibular function and stress responses. However, the relative significance of cochlear and vestibular information in autonomic regulation remains unresolved because of the difficulties in distinguishing the relative contributions of other proprioceptive and interoceptive inputs, including vagal and somatic information. To investigate the role of cochlear and vestibular function in central and physiological responses, we have examined the effects of increased gravity in wild-type mice and mice lacking the POU homeodomain transcription factor Brn-3.1 (Brn-3b兾Pou4f3). The only known phenotype of the Brn-3.1ⴚ/ⴚ mouse is related to hearing and balance functions, owing to the failure of cochlear and vestibular hair cells to differentiate properly. Here, we show that normal physiological responses to increased gravity (2G exposure), such as a dramatic drop in body temperature and concomitant circadian adjustment, were completely absent in Brn-3.1ⴚ/ⴚ mice. In line with the lack of autonomic responses, the massive increase in neuronal activity after 2G exposure normally detected in wildtype mice was virtually abolished in Brn-3.1ⴚ/ⴚ mice. Our results suggest that cochlear and vestibular hair cells are the primary regulators of autonomic responses to altered gravity and provide genetic evidence that these cells are sufficient to alter neural activity in regions involved in autonomic and neuroendocrine control. circadian rhythm 兩 gravity 兩 temperature regulation 兩 Brn-3.1 兩 parabrachial

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ne experimental system in which to study the contribution of the vestibular system to autonomic responses is brought by alterations of gravitational conditions. Previous studies have shown that exposure to hypergravic environments affects the body temperature, heart rate, activity, and their respective circadian rhythms in rats and mice, and activates the hypothalamo-pituitary-adrenal axis (1–3). It is further well established that hypergravity activates vestibular nuclei, as well as nuclei involved in autonomic regulation, such as the nucleus of the solitary tract (NST), parabrachial nucleus (PB), paraventricular nucleus of the hypothalamus (PVH), and the central nucleus of amygdala (CNA) (4, 5). The peripheral vestibular system and hair cells in the cochlea have traditionally been referred to as the ‘‘gravity-sensing organs’’ (6), but psychophysiological studies on body posture and acceleration have suggested the existence of gravity receptors in the trunk of the body (7). Thus, it has been generally assumed that the central and autonomic effects of gravity are the result of an integration of peripheral sensory and internal visceral afferent information. Stimulation of the cochlear and vestibular systems activates brain structures involved in autonomic regulation similar to those activated by altered gravity, including PB (8, 9). Consistent with these data, it has been shown that the vestibular system alone or in conjunction with gravity receptors in the body exert certain effects on autonomic regulation. For example, results from physiological studies have suggested a vestibulosympathetic ref lex regulating cardiovascular activity under 17078 –17082 兩 PNAS 兩 December 24, 2002 兩 vol. 99 兩 no. 26

changes in posture (10), but potential participation of putative somatic gravity receptors has remained ambiguous. Interestingly, a large proportion of patients suffering from anxietyrelated disorders show vestibular dysfunction compared with controls, and observations of correlations between vestibular dysfunction and stress and anxiety in humans have been noted for more than two millenniums (11, 12). Although vestibular stimulation seems to have a major inf luence on the activity of autonomic nuclei, it has been difficult so far to rule out putative primary or secondary contributions of other proprioceptive and interoceptive stimuli because of the lack of a model system where the phenomenon could be studied without invasive surgery. Brn-3.1⫺/⫺ mice seem to provide an appropriate genetic model to determine the significance of the peripheral vestibular system for physiological responses to altered gravitational fields. Brn-3.1 is a POU homeodomain-containing gene that is required for the development of hair cells in the cochlea and vestibular organs. Targeted deletion of the Brn-3.1 gene in mice results in deafness and balance problems because of the failure of sensorineural hair cells to differentiate (13, 14). No morphological or molecular abnormalities in the central nervous system and peripheral sensory ganglia have so far been associated with Brn-3.1 gene deletion, except for spiral and vestibular ganglia, which degenerate probably because of the lack of trophic support from hair cells (13). The present study used short- and long-term centrifugation to create a hyperdynamic environment (2G) in which to study physiological responses and identify activated neurons as assessed by c-Fos induction in wild-type and Brn-3.1⫺/⫺ mice. Materials and Methods Animals and Housing. We have previously described the gener-

ation and phenotypic analysis of the mouse line used in this study (13). Their genetic background is a mixture of C57Bl6 ⫻ 129, resulting from the cross of F1 parents with a 50% C57Bl6, 50% 129 genetic makeup. To study physiological responses to hypergravity, wild-type mice (⫹兾⫹, n ⫽ 5; ⫹兾⫺, n ⫽ 3) and Brn-3.1⫺/⫺ littermates (n ⫽ 6) were implanted with biotelemetry units (Minimitter) into the peritoneum to monitor the circadian rhythms of body temperature (Tb). Mice were individually housed in standard plastic mouse cages. The cages were placed in isolation modules housed on a 4.5-m-diameter animal centrifuge. These modules provided a normal light兾 dark cycle (LD 12:12), ventilation, and visual isolation. The animals were provided with water and food ad libitum. A receiver beneath the animal cage was interfaced to a computer to collect measurements every 5 min. The centrifuge modules were mounted with one degree of freedom so that the gravAbbreviations: NST, nucleus of the solitary tract; PB, parabrachial nucleus; PVH, paraventricular nucleus of the hypothalamus; CNA, central nucleus of amygdala; Tb, body temperature. †To

whom correspondence may be addressed. E-mail: [email protected] or [email protected].

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itational vector was always directed perpendicular to the cage f loor. Centrifugation Protocol. After recovery from surgery, data were

recorded at 1G in LD 12:12 for 2 weeks to entrain circadian rhythms, synchronize all mice to the same lighting condition, and establish baseline measurements (mean daily Tb, and the amplitude, phase, and period of their circadian rhythms). The mice were then exposed to 21 days of continuous 2G via centrifugation. The centrifuge was stopped for 10–15 min each day for animal health checks and husbandry.

Data Analysis. The mean daily body temperature was the compiled average of all of the measurements taken over each 24-h period (day). A least-squares harmonic regression was used to measure the amplitude and phase of the rhythms for each mouse. In addition, periodigram analysis was used to measure the period of the rhythms to determine whether the measured physiological variables were entrained to the light兾dark cycle. Specific comparisons were made between wild-type and Brn-3.1⫺/⫺ mice by using averages of the daily mean and circadian amplitude of Tb during 4 consecutive days during 1G (control), early 2G, late 2G, and 1G recovery. The early 2G period was measured for 4 days beginning the day after 2G exposure. The late 2G period was measured for 4 days during the third week of 2G exposure. The 1G recovery period was measured following the first week of recovery at 1G after 2G exposure. The measures between wild-type and Brn-3.1⫺/⫺ mice were statistically compared using SYSTAT ANOVA (Systat, Evanston, IL). Neuronal Responses. To study neural responses via c-Fos induc-

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tion, two additional experimental groups of mice (⫹兾⫹, n ⫽ 3; ⫺兾⫺, n ⫽ 4) were exposed to 2 h of 2G via centrifugation at CT 6 (i.e., 6 h after light onset). Two control groups of mice (⫹兾⫹, n ⫽ 3; ⫺兾⫺, n ⫽ 4) remained in vivarium 1G conditions. All mice were killed at the same circadian time immediately after the centrifugation period by barbiturate overdose, then transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde. The brains were removed, postfixed in 4% paraformaldehyde, and sectioned at 40 ␮m through the brainstem and forebrain. The sections were immunostained using anti-c-Fos antibody from Oncogene Science, followed by Streptavidinperoxidase (Abbott) and True Blue (Kirkegaard & Perry Laboratories) as the chromogen. The sections were mounted, counterstained with thionine, and examined with an Olympus microscope equipped with a CCD camera (Optronics International, Chelmsford, MA) interfaced to Avid VIDEOSHOP software (Avid Technology, Tewksbury, MA). The nomenclature used follows a stereotaxic atlas of the mouse brain (15). Results Temperature Regulation and Circadian Rhythm. Increased gravity is

well known to elicit a rapid and dramatic fall in Tb and also affect the circadian temperature rhythm (3). Transgenic mice harboring overexpression of uncoupling proteins 2 and 3 have previously been shown to respond to hypergravity with a normal drop in Tb and reduction in circadian amplitude (2). These autonomic responses to hypergravity thus seem very robust, and the anatomical and molecular substrates underlying this physiological response have remained obscure. We therefore initially studied the effects on Tb and circadian amplitude as a response on increased gravity (2G) in wild-type and Brn-3.1⫺/⫺ mice. No significant difference in Tb during the 1G control period was seen in wild-type and Brn-3.1⫺/⫺ mice, as illustrated by the similar mean daily Tb ⬇37°C and circadian rhythm amplitude in a wild-type versus a Brn-3.1⫺/⫺ mouse (Fig. 1A). At 2G, wildtype mice exhibited a dramatic fall in mean Tb relative to baseline, followed by a gradual recovery and an additional Murakami et al.

Fig. 1. Changes in body temperature in response to altered gravitational fields. (A) Line plots during 5 days of the control 1G period demonstrating a similar mean daily body temperature and circadian rhythm between wild-type and Brn-3.1⫺/⫺ mice. (B) Line plots illustrating the changes after exposure to 2G in a wild-type and a Brn-3.1⫺/⫺ mouse. At 2G, the wild-type mouse exhibits an immediate and dramatic fall in mean daily body temperature from 36.5°C down to 30°C, and a gradual recovery. In addition, there is a loss of circadian rhythm amplitude that lasts 5– 6 days. The Brn-3.1⫺/⫺ mouse shows no immediate drop in mean body temperature after 2G exposure and only a slight attenuation of circadian rhythm amplitude. The arrowheads in B show the time point where the gravity conditions were altered.

reduction in circadian rhythm amplitude that lasted ⬇7 days (Fig. 1B). In contrast, the Brn-3.1⫺/⫺ mice failed to show the decrease in the mean Tb immediately after exposure to 2G, and only a minor effect on circadian amplitude was observed (Fig. PNAS 兩 December 24, 2002 兩 vol. 99 兩 no. 26 兩 17079

Table 1. Relative c-Fos immunoreactivity after 2G exposure Region Spinal vestibular nucleus Medial vestibular nucleus PB Locus coeruleus Nucleus of the solitary tract Dorsal raphe nucleus PVH CNA

Wild-type

Brn-3.1⫺/⫺

⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹

⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹

Illustration of the relative differences in c-Fos staining of wild-type and Brn-3.1⫺/⫺ mice after 2 h of 2G exposure. The numbers of c-Fos immunoreactive cells in the most reactive section of each nucleus were categorized as follows: low (⫺), 1–10 cells; light (⫹), 11–50 cells; medium (⫹⫹), 51–100 cells; high (⫹⫹⫹), ⬎100 cells.

immediate response in body temperature to 2G stimulation was severely impaired in the Brn-3.1⫺/⫺ mice.

Fig. 2. Line graphs of mean daily and circadian amplitude summarizing the results of all wild-type and Brn-3.1⫺/⫺ mice in each gravitational condition (1G control period, early 2G, late 2G, and 1G recovery). The bars represent the standard error. Wild-type and Brn-3.1⫺/⫺ mice exhibit equivalent mean and circadian amplitude during the 1G baseline. During the early exposure to 2G, the wild-type mice show a significant reduction in mean daily amplitude and a loss of circadian amplitude. However, Brn-3.1⫺/⫺ mice did not exhibit a significant change in mean daily or circadian amplitude immediately after 2G exposure. No differences were found between wild-type and Brn-3.1⫺/⫺ mice after 2 weeks of chronic 2G or during the 1G recovery.

1B). Both groups of mice showed the same mean temperature and circadian amplitude after 2 weeks of chronic 2G (Fig. 2). During the 1G recovery period, both groups responded with an increase in mean Tb and circadian amplitude (Fig. 2). Thus, the

Hypergravity-Induced c-Fos Immunoreactivity. The levels of the protein product of the immediate early gene c-fos, c-Fos, are normally low in neurons but can be rapidly increased by various stimuli (16). Immunohistochemical detection of c-Fos thus provides a very valuable tool for histological detection and anatomical analysis of increased neuronal activity (17–19). As described (4, 5), wild-type mice displayed high c-Fos immunoreactivity after exposure to hypergravity in many brain regions, such as vestibular nuclei, PB, locus coeruleus, NST, dorsal raphe nucleus, PVH, and CNA (Fig. 3 B, E, and H; data not shown). The relative number of c-Fos immunoreactive neurons within a specific region was compared between wildtype and Brn-3.1⫺/⫺ mice in each condition (1G control, 2 h of 2G) and are summarized in Table 1. A detailed anatomical investigation revealed that c-Fos labeling was highly restricted to specific subregions. Thus, in the vestibular complex, virtually all neurons in the spinal and medial vestibular nuclei exhibited c-Fos-positive neurons, whereas no c-Fos-immunoreactive neurons were found in the lateral and superior vestibular nuclei (Fig.

Fig. 3. Lack of c-Fos induction in the vestibular and autonomic nuclei of Brn-3.1⫺/⫺ mice after exposure to hypergravity. Shown are low-power micrographs of transverse sections from wild-type and Brn-3.1⫺/⫺ mice demonstrating the differences in c-Fos staining between the experimental groups. At 1G, no significant c-Fos labeling is seen in any brain region (A, D, and G), whereas after 2 h of 2G exposure, massive c-Fos labeling is shown in the spinal vestibular nucleus (B), specific subnuclei of the parabrachial nucleus (E), and the central nucleus of the amygdala (H). Very little c-Fos staining was seen in these regions in Brn-3.1⫺/⫺ mice after 2G exposure (C, F, and I). MVN, medial vestibular nucleus; DL, dorsal lateral parabrachial nucleus; LCA, lateral crescent area; CNA, central nucleus of amygdala. (Scale bar, 200 ␮m.) 17080 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.252652299

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3B). In PB, the lateral crescent area, and the dorsal and outer external subnuclei showed massive c-Fos labeling (Fig. 3E). In the PVH, the labeling was localized mainly to the parvocellular subdivisions (Fig. 4 A–C). In stark contrast to the wild-type mice, Brn-3.1⫺/⫺ mice showed virtually no increase in c-Fos labeling in the vestibular complex and PB after 2 h of 2G stimulation (Fig. 3 C, F, and I; data not shown). Very small numbers of lightly labeled cells could be detected in NST, PVH, and CNA, suggesting a minor vagal afferent pathway of activation. No significant numbers of c-Fos-labeled neurons were found in the 1G control mice (wild-type and Brn-3.1⫺/⫺ mice; Figs. 3 A, D, and G and 4 A and D). Thus, Brn-3.1 and functional hair cells are required for proper immediate central response to increased gravity. Discussion Previous studies have suggested that cochlear and vestibular systems contribute to the central and autonomic responses to altered gravitation and body position (20–22). However, clinical studies on posture and balance have implicated receptors in the trunk in the cognitive response to altered gravity (7, 23), and it has been a general assumption that visceral and somatic proprioceptors contribute significantly to the responses to increased gravity. Our investigation is an experimental study on responses to hypergravity in a genetic model of cochlear and vestibular dysfunction, and our results, in combination with previous studies, strongly suggest that cochlear and vestibular hair cells are the primary mediators of gravity influences on autonomic function and brain regions involved in autonomic regulation and stress mechanisms. Somatic gravity receptors located in the trunk have been suggested to respond to indirect changes in blood flow and kidney function secondary to changes in posture and acceleration. The evidence for such somatic receptors for gravity is mainly based on psychophysiological studies on cognitive experience of balance and body motion in humans (cf. ref. 24). Further, experimental studies based on pharmacologically or surgically induced vestibular dysfunction have suggested that somatic gravity-sensing systems can compensate to some extent for loss of vestibular information. However, the experimental settings and conditions in these studies are completely different Murakami et al.

We thank Connie King and Emilia McFarland for technical assistance and Mary Frazer for help with animal care. This study was supported in part by National Aeronautics and Space Administration Grants NAG2944 and NAGW-4552 (to D.M.M. and C.A.F.) and National Institutes of Health Grant NS34934 (to M.G.R.). M.G.R. is an investigator with the Howard Hughes Medical Institute. PNAS 兩 December 24, 2002 兩 vol. 99 兩 no. 26 兩 17081

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Fig. 4. High-power micrographs demonstrating the nuclear localization and density of the c-Fos labeling in the paraventricular hypothalamic nucleus (A–C) and the locus coeruleus (D–F) of wild-type (A, B, D, and E) and Brn-3.1⫺/⫺ (C and F) mice after 1G (A and D) and 2G (B, C, E, and F) exposure. PVHpc, PVH– parvocellular; LC, locus coeruleus. (Scale bar, 100 ␮m.)

from the approach in the present study, and the cognitive experience of gravity is difficult to assess in mice. Our results do not exclude the existence of somatic gravity receptors influencing certain cognitive aspects of posture and balance but provide strong genetic evidence for a primary role for the peripheral vestibular system in the regulation of specific autonomic responses, such as body temperature and circadian rhythm, to altered gravity. Indeed, the minor numbers of hypergravityinduced c-Fos immunoreactive neurons, primarily in NTS and CNA in the Brn-3.1⫺/⫺ mice, actually suggest that there is an intact vagal afferent component but that the activity is not sufficient to induce the hypergravity-mediated drop in temperature and massive neuronal activation. Previous studies have demonstrated that direct stimulation of the vestibular nerve results in increased sympathetic activity for the maintenance of homeostasis, an example of which is provided by the regulation of blood pressure by head tilt in cats (21). The major autonomic target for neurons in the vestibular complex seems to be PB, which in turn is connected to all brain regions that show c-Fos immunoreactivity in response to hypergravity (4, 5, 25, 26). Activation of parabrachial neurons has profound effects on autonomic regulation, including cardiovascular regulation and blood f low, due to both ascending and descending efferent projections (27, 28). Many activated neurons were detected in the dorsal lateral subnucleus of PB (DL, Fig. 3E). Neurons in DL produce the inhibitory opioid dynorphin and project heavily to the median preoptic nucleus in the anterior hypothalamus (29). Neurons in median preoptic nucleus have profound effects on blood f low and the distribution of blood f low to viscera versus muscles and to deep versus superficial capillary beds (30, 31), and may thus be involved in the changes in blood f low and body temperature in response to altered gravity. Furthermore, neurons in the lateral crescent area and external lateral subnucleus of PB, which showed massive activation after hypergravity (Fig. 3E), project heavily to CNA (32). The external lateral subnucleus of PB–CNA projection has been heavily implicated in aversive behavior and learning (33), and neurons in CNA exert profound influence on heart rate (34), providing further putative neuronal substrates for the effects of altered gravitation. Lesions of CNA also abolish taste-aversive behavior associated with hypergravity-induced motion sickness (35). In addition, neurons in PB are reciprocally connected to NST, locus coeruleus, dorsal raphe nucleus, and PVH, all brain areas well known to be involved in autonomic regulation, hypothalamopituitary-adrenal activity, and various aspects of stress mechanisms and anxiety (25, 28, 36). Because Brn-3.1⫺/⫺ mice show no neuronal aberrations in PB or any of the activated central regions as assessed by morphological analysis after thionin staining and calbindin immunohistochemistry (unpublished observations), our results suggest that information about the gravitational field is transmitted primarily by means of an activation of a cochleovestibulo-parabrachial pathway, which in turn leads to activation of multiple central structures governing the proper autonomic, endocrine, and emotional responses. Further, patients suffering from anxiety-related disorders and impaired stress response show increased incidence of vestibular dysfunction (11, 37, 38). This study contributes to the definition of mechanisms through which the vestibular system influences autonomic functions, an issue with important implications in space biology and psychiatric medicine.

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