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

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 (Ttail ) 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 Ttail 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.

Molecular and Functional Changes to Postsynaptic Cholinergic Signaling in the Vestibular Sensory Organs of Aging C57BL/6 Mice.

Cholinergic circuits in the central nervous system are vulnerable to age-related functional decline, but it is not known if aging impacts cholinergic signaling in the vestibular sensory organs, which are critically important to balance maintenance and visual gaze stability. We have previously shown cholinergic neurotransmission between vestibular efferent terminals and type II mechanosensory hair cells requires the alpha9 (Chrna9) nicotinic receptor subunit. Homozygous knockout of the alpha9 subunit causes vestibulo-ocular reflex adaptation deficits that mirror those observed in aged mice. This prompted examination of cholinergic signaling in the vestibular sensory organs of aged mice. We confirmed older (>24 months) mice had impaired performance in a balance beam task compared to young (3-4 months) adult mice. While there was no qualitative loss of cholinergic axon varicosities in the crista ampullaris of old mice, qPCR analysis revealed reduced expression of nicotinic receptor subunit genes Chrna1, Chrna9, and Chrna10 in the cristae of old relative to young mice. Functionally, single-cell patch clamp recordings taken from type II vestibular hair cells exposed to acetylcholine show reduced conductance through alpha9/10 subunit-containing nicotinic receptors in older mice, despite preserved passive membrane properties and voltage-activated conductances. These findings suggest that cholinergic signaling in the peripheral vestibular sensory organs is vulnerable to aging processes, manifesting in dynamic molecular and functional age-related changes. Given the importance of these organs to our everyday activities, and the dramatic increase in fall incidence in the older, further investigation into the mechanisms of altered peripheral vestibular function in older humans is warranted.

Evolutionary Modifications Are Moderate in the Astroglial System of Actinopterygii as Revealed by GFAP Immunohistochemistry.

The present paper is the first comparative study on the astroglia of several actinopterygian species at different phylogenetical positions, teleosts (16 species), and non-teleosts (3 species), based on the immunohistochemical staining of GFAP (glial fibrillary acidic protein), the characteristic cytoskeletal intermediary filament protein, and immunohistochemical marker of astroglia. The question was, how the astroglial architecture reflexes the high diversity of this largest vertebrate group. The actinopterygian telencephalon has a so-called 'eversive' development in contrast to the 'evagination' found in sarcopterygii (including tetrapods). Several brain parts either have no equivalents in tetrapod vertebrates (e.g., torus longitudinalis, lobus inferior, lobus nervi vagi), or have rather different shapes (e.g., the cerebellum). GFAP was visualized applying DAKO polyclonal anti-GFAP serum. The study was focused mainly on the telencephalon (eversion), tectum (visual orientation), and cerebellum (motor coordination) where the evolutionary changes were most expected, but the other areas were also investigated. The predominant astroglial elements were tanycytes (long, thin, fiber-like cells). In the teleost telencephala a 'fan-shape' re-arrangement of radial glia reflects the eversion. In bichir, starlet, and gar, in which the eversion is less pronounced, the 'fan-shape' re-arrangement did not form. In the tectum the radial glial processes were immunostained, but in Ostariophysi and Euteleostei it did not extend into their deep segments. In the cerebellum Bergmann-like glia was found in each group, including non-teleosts, except for Cyprinidae. The vagal lobe was uniquely enlarged and layered in Cyprininae, and had a corresponding layered astroglial system, which left almost free of GFAP the zones of sensory and motor neurons. In conclusion, despite the diversity and evolutionary alterations of Actinopterygii brains, the diversity of the astroglial architecture is moderate. In contrast to Chondrichthyes and Amniotes; in Actinopterygii true astrocytes (stellate-shaped extraependymal cells) did not appear during evolution, and the expansion of GFAP-free areas was limited.

The Long and Winding Road-Vestibular Efferent Anatomy in Mice.

The precise functional role of the Efferent Vestibular System (EVS) is still unclear, but the auditory olivocochlear efferent system has served as a reasonable model on the effects of a cholinergic and peptidergic input on inner ear organs. However, it is important to appreciate the similarities and differences in the structure of the two efferent systems, especially within the same animal model. Here, we examine the anatomy of the mouse EVS, from its central origin in the Efferent Vestibular Nucleus (EVN) of the brainstem, to its peripheral terminations in the vestibular organs, and we compare these findings to known mouse olivocochlear anatomy. Using transgenic mouse lines and two different tracing strategies, we examine central and peripheral anatomical patterning, as well as the anatomical pathway of EVS axons as they leave the mouse brainstem. We separately tag the left and right efferent vestibular nuclei (EVN) using Cre-dependent, adeno-associated virus (AAV)-mediated expression of fluorescent reporters to map their central trajectory and their peripheral terminal fields. We couple this with Fluro-Gold retrograde labeling to quantify the proportion of ipsi- and contralaterally projecting cholinergic efferent neurons. As in some other mammals, the mouse EVN comprises one group of neurons located dorsal to the facial genu, close to the vestibular nuclei complex (VNC). There is an average of just 53 EVN neurons with rich dendritic arborizations towards the VNC. The majority of EVN neurons, 55%, project to the contralateral eighth nerve, crossing the midline rostral to the EVN, and 32% project to the ipsilateral eighth nerve. The vestibular organs, therefore, receive bilateral EVN innervation, but without the distinctive zonal innervation patterns suggested in gerbil. Similar to gerbil, however, our data also suggest that individual EVN neurons do not project bilaterally in mice. Taken together, these data provide a detailed map of EVN neurons from the brainstem to the periphery and strong anatomical support for a dominant contralateral efferent innervation in mammals.

Distribution of GFAP in Squamata: Extended Immunonegative Areas, Astrocytes, High Diversity, and Their Bearing on Evolution.

Squamata is one of the richest and most diverse extant groups. The present study investigates the glial fibrillary acidic protein (GFAP)-immunopositive elements of five lizard and three snake species; each represents a different family. The study continues our former studies on bird, turtle, and caiman brains. Although several studies have been published on lizards, they usually only investigated one species. Almost no data are available on snakes. The animals were transcardially perfused. Immunoperoxidase reactions were performed with a mouse monoclonal anti-GFAP (Novocastra). The original radial ependymoglia is enmeshed by secondary, non-radial processes almost beyond recognition in several brain areas like in other reptiles. Astrocytes occur but only as complementary elements like in caiman but unlike in turtles, where astrocytes are absent. In most species, extended areas are free of GFAP-a meaningful difference from other reptiles. The predominance of astrocytes and the presence of areas free of GFAP immunopositivity are characteristic of birds and mammals; therefore, they must be apomorphic features of Squamata, which appeared independently from the evolution of avian glia. However, these features show a high diversity; in some lizards, they are even absent. There was no principal difference between the glial structures of snakes and lizards. In conclusion, the glial structure of Squamata seems to be the most apomorphic one among reptiles. The high diversity suggests that its evolution is still intense. The comparison of identical brain areas with different GFAP contents in different species may promote understanding the role of GFAP in neuronal networks. Our findings are in accordance with the supposal based on our previous studies that the GFAP-free areas expand during evolution.

No rapid and demarcating astroglial reaction to stab wounds in Agama and Gecko lizards and the caiman Paleosuchus - it is confined to birds and mammals.

The present study proves that rapid and demarcating astroglial reactions are confined to birds and mammals. To understand the function of post-lesion astroglial reaction, the phylogenetical aspects are also to be investigated. Considering the regenerative capabilities, reptiles represent an intermediate position between the brain regeneration-permissive fishes and amphibians and the almost non-permissive birds and mammals. Damage is followed by a rapid astroglial reaction in the mammalian and avian brain, which is held as an impediment of regeneration. In other vertebrates the reactions were usually observed following long survival periods together with signs of regeneration, therefore they can be regarded as concomitant phenomena of regeneration. The present study applies short post-lesion periods comparable to those seen in mammals and birds for astroglial reactions. Two species of lizards were used: gecko (leopard gecko, Eublepharis macularius, Blyth, 1854) and agama (bearded dragon, Pogona vitticeps, Ahl, 1926). The gecko brain is rich in GFAP whereas the agama brain is quite poor in this. Crocodilia, the closest extant relatives of birds were represented in this study by Cuvier's dwarf caiman (Paleosuchus palpebrosus, Cuvier, 1807). The post-lesion astroglial reactions of crocodilians have never been investigated. The injuries were stab wounds in the telencephalon. The survival periods lasted 3, 7, 10 or 14 days. Immunoperoxidase reactions were performed applying anti-GFAP, anti-vimentin and anti-nestin reagents. No rapid and demarcating astroglial reaction resembling that of mammalian or avian brains was found. Alterations of the perivascular immunoreactivities of laminin and ß-dystroglycan as indicators of glio-vascular decoupling proved that the lesions were effective on astroglia. The capability of rapid and demarcating astroglial reaction seems to be confined to mammals and birds and to appear by separate, parallel evolution in them.

Cerebrovascular ß-dystroglycan immunoreactivity in vertebrates: not detected in anurans and in the teleosts Ostariophysi and Euteleostei.

The aim of the present paper was to check for the presence of cerebrovascular dystroglycan in vertebrates, because dystroglycan, which is localized in the vascular astroglial end-feet, has a pivotal function in glio-vascular connections. In mammalian brains, the immunoreactivity of ß-dystroglycan subunit delineates the vessels. The results of the present study demonstrate similar patterns in other vertebrates, except for anurans and the teleost groups Ostariophysi and Euteleostei. In this study, we investigated 1 or 2 representative species of the main groups of Chondrichthyes, teleost and non-teleost ray-finned fishes, urodeles, anurans, and reptiles. We also investigated 5 mammalian and 3 bird species. Animals were obtained from breeders or fishermen. The presence of ß-dystroglycan was investigated immunohistochemically in free-floating sections. Pre-embedding electron microscopical immunohistochemistry on Heterodontus japonicus shark brains demonstrated that in Elasmobranchii, ß-dystroglycan is also localized in the perivascular glial end-feet despite the different construction of their blood-brain barrier. The results indicated that the cerebrovascular ß-dystroglycan immunoreactivity disappeared separately in anurans, and in teleosts, in the latter group before its division to Ostariophysi and Euteleostei. Immunohistochemistry in muscles and western blots from brain homogenates, however, detected the presence of ß-dystroglycan, even in anurans and all teleosts. A possible explanation is that in the glial end-feet, ß-dystroglycan is masked in these animals, or disappeared during adaptation to the freshwater habitat.