Influence of Magnitude and Duration of Altered Gravity and Readaptation to 1 g on the Structure and Function of the Utricle in Toadfish, Opsanus tau.

Gravity has remained constant during animal evolution and the neural sensory systems detecting acceleration forces have remained remarkably conserved among vertebrates. The utricular organ senses the sum of inertial force due to head translation and head tilt relative to gravitational vertical. Change in gravitational force would be expected to have profound effects on how an organism maintains equilibrium. We characterize the physiology of utricular afferents to applied accelerations in the oyster toadfish, Opsanus tau, in normal 1 g to establish benchmarks, after 1-32-day exposures to 2.24 g (resultant) via centrifugation (hypergravity, HG), after 4- and 16-day exposures to 1.12 g (resultant), and following 1-8 days recovery to HG exposures to study re-adaptation to 1 g. Afferents were also examined during activation of efferent vestibular pathway. Centrifugation at 2.24 g included 228°/s constant angular velocity component, and thus horizontal canal afferent responses to yaw rotation were recorded as an internal control in each fish. Afferents studied after 228°/s rotation for 4 and 16 days without centripetal acceleration, called On-Center-Control, were indistinguishable from their control counterparts. Principal response to HG was an adjustment of afferent sensitivity as a function of magnitude and duration of exposure: an initial robust increase at 3-4 days followed by a significant decrease from 16 to 32 days. Initial increase observed after 4 days of HG took >4 days in 1 g to recover, and the decrease observed after 16 days of HG took >2 days to readapt to 1 g. Hair cells in striola and medial extrastriola macula regions were serially reconstructed in 3D from thin sections using transmission electron microscopy in control fish and fish exposed to 4 and 16 days of HG. Despite the highly significant differences in afferent physiology, synaptic body counts quantified in the same fish were equivalent in their inter-animal variability and averages. No clear role of the efferent pathway as a feedback mechanism regulating afferent behavior to HG was found. Transfer from 1 g to HG imparts profound effects on gravitational sensitivity of utricular afferents and the accompanying transfer from the HG back to the 1 g resembles in part (as an analog) the transfer from 1 g to the micrograms.

Morphology of the utricular otolith organ in the toadfish, Opsanus tau.

The utricle provides the vestibular reflex pathways with the sensory codes of inertial acceleration of self-motion and head orientation with respect to gravity to control balance and equilibrium. Here we present an anatomical description of this structure in the adult oyster toadfish and establish a morphological basis for interpretation of subsequent functional studies. Light, scanning, and transmission electron microscopy techniques were applied to visualize the sensory epithelium at varying levels of detail, its neural innervation and its synaptic organization. Scanning electron microscopy was used to visualize otolith mass and morphological polarization patterns of hair cells. Afferent nerve fibers were visualized following labeling with biocytin, and light microscope images were used to make three-dimensional (3-D) reconstructions of individual labeled afferents to identify dendritic morphology with respect to epithelial location. Transmission electron micrographs were compiled to create a serial 3-D reconstruction of a labeled afferent over a segment of its dendritic field and to examine the cell-afferent synaptic contacts. Major observations are: a well-defined striola, medial and lateral extra-striolar regions with a zonal organization of hair bundles; prominent lacinia projecting laterally; dependence of hair cell density on macular location; narrow afferent dendritic fields that follow the hair bundle polarization; synaptic specializations issued by afferents are typically directed towards a limited number of 7-13 hair cells, but larger dendritic fields in the medial extra-striola can be associated with > 20 hair cells also; and hair cell synaptic bodies can be confined to only an individual afferent or can synapse upon several afferents.

Adaptive Changes in the Vestibular System of Land Snail to a 30-Day Spaceflight and Readaptation on Return to Earth.

The vestibular system receives a permanent influence from gravity and reflexively controls equilibrium. If we assume gravity has remained constant during the species' evolution, will its sensory system adapt to abrupt loss of that force? We address this question in the land snail Helix lucorum exposed to 30 days of near weightlessness aboard the Bion-M1 satellite, and studied geotactic behavior of postflight snails, differential gene expressions in statocyst transcriptome, and electrophysiological responses of mechanoreceptors to applied tilts. Each approach revealed plastic changes in the snail's vestibular system assumed in response to spaceflight. Absence of light during the mission also affected statocyst physiology, as revealed by comparison to dark-conditioned control groups. Readaptation to normal tilt responses occurred at ~20 h following return to Earth. Despite the permanence of gravity, the snail responded in a compensatory manner to its loss and readapted once gravity was restored.

Neural response in vestibular organ of Helix aspersa to centrifugation and re-adaptation to normal gravity.

Gravity plays a key role in shaping the vestibular sensitivity (VS) of terrestrial organisms. We studied VS changes in the statocyst of the gastropod Helix aspersa immediately after 4-, 16-, and 32-day exposures to a 1.4G hypergravic field or following a 7-day recovery period. In the same animals we measured latencies of behavioral "negative gravitaxis" responses to a head-down pitch before and after centrifugation and found significant delays after 16- and 32-day runs. In an isolated neural preparation we recorded the electrophysiological responses of the statocyst nerve to static tilt (±19°) and sinusoids (±12°; 0.1 Hz). Spike sorting software was used to separate individual sensory cells' patterns out of a common trace. In correspondence with behavior we observed a VS decrease in animals after 16- (p < 0.05) and 32-day (p < 0.01) centrifugations. These findings reveal the capability of statoreceptors to adjust their sensitivity in response to a prolonged change in the force of gravity. Interestingly, background discharge rate increased after 16 and 32 days in hypergravity and continued to rise through the recovery period. This result indicates that adaptive mechanisms to novel gravity levels were long lasting, and re-adaptation from hypergravity is a more complex process than just "return to normal".

Functional changes in the snail statocyst system elicited by microgravity.

BACKGROUND: The mollusk statocyst is a mechanosensing organ detecting the animal's orientation with respect to gravity. This system has clear similarities to its vertebrate counterparts: a weight-lending mass, an epithelial layer containing small supporting cells and the large sensory hair cells, and an output eliciting compensatory body reflexes to perturbations. METHODOLOGY/PRINCIPAL FINDINGS: In terrestrial gastropod snail we studied the impact of 16- (Foton M-2) and 12-day (Foton M-3) exposure to microgravity in unmanned orbital missions on: (i) the whole animal behavior (Helix lucorum L.), (ii) the statoreceptor responses to tilt in an isolated neural preparation (Helix lucorum L.), and (iii) the differential expression of the Helix pedal peptide (HPep) and the tetrapeptide FMRFamide genes in neural structures (Helix aspersa L.). Experiments were performed 13-42 hours after return to Earth. Latency of body re-orientation to sudden 90° head-down pitch was significantly reduced in postflight snails indicating an enhanced negative gravitaxis response. Statoreceptor responses to tilt in postflight snails were independent of motion direction, in contrast to a directional preference observed in control animals. Positive relation between tilt velocity and firing rate was observed in both control and postflight snails, but the response magnitude was significantly larger in postflight snails indicating an enhanced sensitivity to acceleration. A significant increase in mRNA expression of the gene encoding HPep, a peptide linked to ciliary beating, in statoreceptors was observed in postflight snails; no differential expression of the gene encoding FMRFamide, a possible neurotransmission modulator, was observed. CONCLUSIONS/SIGNIFICANCE: Upregulation of statocyst function in snails following microgravity exposure parallels that observed in vertebrates suggesting fundamental principles underlie gravi-sensing and the organism's ability to adapt to gravity changes. This simple animal model offers the possibility to describe general subcellular mechanisms of nervous system's response to conditions on Earth and in space.