Nervous System and Sensory Organs

Neurulation is the first step in the formation of the nervous system. It starts in the median part of the antero-posterior axis of the neurula, spreads simultaneously along this axis towards the rostral and caudal parts of the embryo, and finally forms a closed tube.

Neurulation was not affected by microgravity in Xenopus laevis embryos (Duprat et al. 1998). In contrast, experiments on board Mir in 1996, 1998, and 1999 showed retardation in the closure of the neural tube in Pleurodeles waltl (Figure 5-04). Also, microcephaly developed more frequently in 0-g embryos than in 1-g control embryos. Despite of these modifications, epidermal ciliated cells functioned normally, and each 0-g embryo rotated randomly clockwise or counter-clockwise around its anteroposterior axis as in 1-g controls. The five brain subdivisions were morphologically normal, and sense organs such as eye and ear developed normally (Gualandris-Parisot et al. 2001).

The cytological differentiation of neuronal and glial structures was investigated in neural precursor cells from Pleurodeles, isolated in culture immediately after neuronal induction at the early neurula stage. During microgravity exposure on a 16-day Foton biosatellite flight, they differentiated without significant abnormalities. They developed long neurites and normal networks. Some slight modifications were related to a faster differentiation of cells and to the formation of varicosities along neurites (Duprat et al. 1998).

3.1.1 Axonal Growth and Dendritic Morphology

Further development includes the outgrowth of neurites, the formation of neuronal networks, and the establishment of the neuromuscular synapses.

As for the early period of development, the effects of microgravity on nervous system development were considered in only a few animal species and specific tracts. While these effects on the early formation of the nervous system were mainly based on studies in the aquatic animals, axonal growth and dendritic morphology was also studied in rats. Studies in developing rats considered model tracts such as the projections from the vestibular system, or the retino-hypothalamic tract which connects the retina with the suprachiasmatic nucleus (SCN). These pathways are related to functions such as equilibrium control and control of circadian activity, respectively.

Rat embryos exposed to microgravity from the gestation day G9 to G19, which is the period when the vestibular system starts to become functional, showed that afferents from the posterior canal projecting to the medial vestibular nucleus developed similarly in microgravity, in hypergravity at 1.5 g, and in normal gravity. However, afferents from the saccule showed delayed development in microgravity compared to development in hypergravity and in controls (Bruce 2003). In particular, three hours after Shuttle landing, peripheral vestibular branches had developed similarly in the flight and control rat embryos. Central projections of semicircular canal receptors to the vertical vestibular nuclei reached similar states of development in the flight and control animals. However, central projections from the gravisensing organs receptors to the medial vestibular nucleus were more immature than in the controls (Bruce and Fritzsch 1997). This result suggests that gravity is required for appropriate synaptic development and fine-tuning of the projections from the gravity sensing receptors to the central nervous system.

These observations were supplemented by studies in the vestibular nuclei of neonate rats launched at postnatal day P8. Several tests during the 16-day Neurolab STS-90 mission revealed an absence of connections into the vestibular nuclei from the cerebellum, the main control center for balance and coordination of movement (Raymond et al. 2003).

3.1.2 Synapse Formation In Vivo and in Cell Cultures

The transfer of information between nerves and muscles and among nerve cells occurs mainly at the synaptic level. The establishment of synaptic contacts is one primary goal of development. For example, in vertebrates, motoneurons (also called motor neurons) are efferent neurons that originate in the spinal cord and synapse with muscle fibers to facilitate muscle contraction and with muscle spindles to modify proprioceptive sensitivity. During development of the neuromuscular system, outgrowing motoneurons find their muscle fiber to form the motor endplates.

During normal development, the number of synapses undergoes a period of overproduction followed by a significant reduction to a standard level. In mature neurons, synaptic proteins are highly concentrated in axon endings where they help to regulate neurotransmitter release. Their distribution experiences significant modifications during development. At early stages, they are distributed throughout the neuron, but with increasing maturity they concentrate in the axon endings. Two of the most understood synaptic proteins are synaptophysin found at the synaptic vesicles and SNAP-25, a protein that probably functions in synaptic vesicle exocytose. Adaptive processes to altered gravitational conditions have to consider modifications at the synaptic level, including the formation of contacts between neurons and muscles, as well as the formation of proteins such as synaptophysin or SNAP-25 involved in the information transmission.

This assumption of a g-sensitivity of synapse formation was revealed to be true, but the effects were related to some sites within the brain and showed a time window of sensitivity. In P8 rat neonates that developed for 16 days in microgravity during Neurolab, a reduced growth of motor neuron terminals was observed (Figure 5-05). At launch, more than 75% of motor endplates were innervated by multiple motor nerve terminals. During spaceflight, reduction of terminal numbers proceeded as on ground, so that after landing all but one terminal per endplate was eliminated. However, the frequency of complex branching patterns, which is a marker for advanced developmental progress, was significantly higher in ground (44 ± 3%) than in flight (16 ± 1%) neonate rats (Riley and Wong-Riley 2003).

In the P8 neonate group of the same flight, expression of proteins linked in the synaptic transmission was determined for the hippocampus as well as for the vestibular and cochlear nuclei. During and after the STS-90 flight, the cellular distribution of synaptophysin and SNAP-25 in the

Figure 5-05. Morphological development of neuromuscular connections during gravity deprivation. Spaceflight animals (0G) revealed a depressed development of axonal terminals compared to ground controls (1G). At onset of the mission, more than 75% of motor endplates had a multiple innervation of the immature muscle fiber. Further development of the ground control (1G) was normal; motoneurons and muscle fibers increased their size, the multiple innervations disappeared. Animals from the microgravity group (OG) revealed disappearance of multiple innervations but a depressed growth of neurons and muscle fibers. Adapted from Rilev and Wong-Riley (2003).

vestibular and cochlear nuclei differed significantly from those of the 1-g control neonate rats. The ground animals revealed a more developed type of distribution, whereas synaptic proteins were more distributed throughout the neurons in the flight neonate rats, characterizing a more immature status (Raymond et al. 2003). In contrast, the hippocampus of these neonate rats orbiting in space between postnatal stage P8 and P24 revealed no significant difference in the staining of synaptophysin and SNAP-25 (Temple et al. 2003).

In co-cultures of spinal neurons and myocytes (muscle fibers) isolated from Xenopus laevis embryos that were exposed to simulated microgravity, the formation of ACh receptor patches" was strongly affected depending on the level of maturity of this system at onset of microgravity. Inhibition of incidence and area of these patches was obvious if nerve contact took place during or shortly before onset of simulated microgravity (Gruener and Hoeger 1990) (Figure 5-06).

Figure 5-06. Physiological development of neuromuscular connections during gravity deprivation. Effects of clinostat rotation on the area (top diagram) and incidence (bottom diagram) of nerve-induced acetylcholine receptor patches, or ACh NARP, in myocytes in mature (A: maturity before clinostat rotation onset), immature (B: synaptic contacts developed just before onset of rotation) and de-novo formed synapses (C: synapses formed during clinostat rotation). Clinostat rotation was performed at 1 or 10 revolutions per minute (rpm); 0 rpm indicates no rotation. Note the significant effects of simulated microgravity in sets B and C and their absence if maturation occurred before onset of clinostat rotation. Adapted from Gruener and Hoeger (1990).

11 Acetylcholine (ACh) is one of the neurotransmitters. After being released into the nerve terminal, ACh binds to the post-synaptic ACh receptor, resulting in a transient increase in membrane permeability to Na, K, Ca, and Mg, leading to an endplate potential (EPP).

These observations were confirmed in space-flown cell cultures (Gruener et al. 1994). Surprisingly, the changes in the receptor's cellular organization by clinostat rotation did not alter the ACh receptor single channel properties. Indeed, the mean open-time and conductance of the ACh receptor channel were statistically not different from control values (Reitstetter and Gruener 1994).

3.1.3 Vestibular Apparatus

At the beginning of the era of space biology, many experiments studied the effects of microgravity on the vestibular apparatus, and in particular the otoliths or otoconia. Otoliths, or "ear stones", are calcium carbonate crystals found in the inner ear of most fish and vertebrates (see Figure 1-13). Pressure or shear motion of the otoliths on the hair cells of the macula (the most sensitive area of the inner ear) provides sensory inputs about the orientation of the head relative to gravity. Bony fish were the first choice of species for developmental studies of the vestibular apparatus because they possess species-specific solid otoliths of constant shape that grow in layers. This specific feature allows for a clear-cut quantification of microgravity effects on the developing otolith.

After experiments on board Salyut-5, Russian scientists claimed that the development of the vestibular apparatus of Brachyodanio rerio was not affected by spaceflight. The fine structure of the receptor epithelium and the otolith apparatus, as well as the ional composition of the intravestibular fluid, remained unchanged. Studies in Fundulus heteroclitus developed on board Skylab and the Cosmos-782 biosatellite confirmed these observations. Also, no changes were observed in young fish launched before the earliest stage of development of the vestibular apparatus had appeared (Vinnikov et al. 1983). In the swordtail fish Xiphophorus helleri, however, otolith growth in slowly growing embryos was retarded, but growth was augmented in fast growing embryos. The otoliths of juveniles developed in microgravity in the same way as on the ground (Wiederhold et al. 2003). Retarded otolith development was also observed in Danio rerio during exposure to simulated weightlessness in a rotating bioreactor (Moorman et al. 1999).

Studies in aquatic amphibians are more difficult to perform because they possess many otoconia. In Xenopus laevis, tadpoles launched at the embryonic stage before hatching or shortly thereafter, the expression of CalBindin, a marker for maturity, was similar in vestibular cells compared to ground controls. Furthermore, morphometric investigations of cell size and number in the otolith maculae revealed no difference between flight and ground tadpoles (Horn et al. 2006). These recent results confirmed earlier observations on tadpoles (Ross 1993).

Otoconia from 0-g exposed Xenopus tadpoles were reported as 30% larger than those from 1-g controls (Lychalcov 1991), while their basic shapes remained unaffected after a 10-day microgravity exposure on board the ISS (Horn et al. 2003). During the IML-2 mission, embryos of newt Cynops pyrrhogaster were sent in orbit before any stones were formed. After the flight, otoliths and otoconia from the utricle and saccule were found to be larger compared to those from ground animals (Wiederhold et al. 1997). This increased size of otoconia might be the basis of a sensitization of the developing vestibular system by spaceflight, which was observed in Xenopus tadpoles (see below) and young fish Oreochromis (Sebastian et al. 2001).

3.1.4 Other Sensory Organs

During the STS-72 flight, microgravity affected the retina of neonatal rats, probably by degeneration of cells or parts of individual cell types. In the age- and weight-matched test animals, the most obvious defects observed in all the three test populations launched when they had reached postnatal days P5, P8, and P15 were the absence of the outer segments of rods, a decreased thickness of the inner plexiform layer, and a reduced number of retinal ganglion cells (Tombran-Tinlc and Barnstable 2005). As the affected sites of the retina are involved in visual transduction and first steps of visual information processing, it is likely that vision would have been strongly disturbed in these animals.

This rather discouraging report is completely opposite to studies on the embryonic eye of the Japanese quail Coturnix japónica. Fertilized eggs were launched on STS-76, incubated at 39-40°C on board Mir, and embryos were fixed in microgravity on specific days, ranging from embryonic days E0 to El6. Their eyes were less affected by microgravity than those of the animals during the STS-72 mission described above. Indeed, eye weight, eye, corneal, and scleral ring diameters, numbers of bones in scleral rings, transparency of corneas, and corneal innervation were indistinguishable from the ground controls except for the corneal diameter of E16 eyes (Barrett et al. 2000).

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