Cephalic sensory influence on forelimb movement in newborn opossums, Monodelphis domestica
Graphical abstract
Highlights
► We studied head sensory systems that influence forelimb movement in newborn opossums. ► Electrophysiology, neuronal tracing and immunolocalization of NF200 were used. ► Vestibular and trigeminal systems, but not vision or olfaction, affect limb movement. ► An utriculovestibulospinal pathway likely controls the antigravity reflex. ► Trigeminal afferents convey facial touch to the cord directly or via reticular cells.
Introduction
The gray short-tailed opossum Monodelphis domestica is a small marsupial from the Didelphidae family increasingly used in biomedical research, notably to investigate nervous system development and regeneration. Like other marsupials this opossum is born in a very immature state. The newborn’s head makes up about one-third of the total body length. It is glabrous, and has a large snout with opened nostrils, but the eyes are unopened and covered by skin, and the external ears are not yet demarcated. Locomotion consists solely of rhythmical and alternating extension/flexion movements of low amplitude performed by the forelimbs while the trunk sways from side to side. The hindlimbs look almost like embryonic buds and are immobile. This simple and basic locomotion enables the newborn to climb on the mother’s belly, unaided by her, from the urogenital opening to a nipple where it attaches (Fadem et al., 1982, Cassidy et al., 1994, Pflieger et al., 1996). Overall the newborn opossum compares to a rat embryo of 13–14 days (see Smith, 2001, Smith, 2006, Lavallée and Pflieger, 2009) or to a mouse embryo of 11.5 days (Nagarajan et al., 2010), albeit with a slightly more developed brachial girdle and forelimbs and less developed pelvic girdle and hindlimbs. A large part of the opossum’s somatic and nervous development unfolds postnatally, explaining its usefulness as a model of mammalian sensorimotor development (review in Cabana, 2000).
The spinal cord of the neonatal opossum contains few supraspinal inputs. Projections to the cervical cord originate from the brainstem raphe and adjacent reticular formation (RF), the vestibular complex and, to a lesser extent, the spinal trigeminal nucleus (Sp5) and the paraventricular hypothalamic nucleus (Wang et al., 1992). The forelimb movements of the newborn opossum must be generated by the presumptive central pattern generators in the cord, as demonstrated for perinatal rodents (reviews in Clarac et al., 2004, Falgairolle et al., 2006). Nonetheless, the opossum being unaided by the mother in its progression to the nipple, some sensory guidance must exist for the animal to reach its target.
A guiding role for the auditory system is unlikely. Not only is the auditory meatus of the newborn opossum closed, but the presumptive middle ear is comprised of mesenchyme with the ossicles only partially formed (Sánchez-Villagra et al., 2002), and the cochlear component of the inner ear appears even more immature (unpublished observations). Evoked auditory potentials are not recorded before P24 in the brainstem (Reimer, 1996) or in the inferior colliculus (Aitkin et al., 1997). The auditory meatus opens around P30 (Cassidy et al., 1994).
A guiding role for the visual system is also unlikely. As mentioned, the eyes of the newborn opossum are covered by skin. Moreover, anatomical studies have shown that the first retinal fibers cross the optic chiasm only three days postnatally (Taylor and Guillery, 1994) and tectal axons arrive in the cervical cord around P16 (Wang et al., 1992). The eyelids open around P35, when visual placing reflexes begin to be expressed (Cassidy et al., 1994).
Studies in some marsupials could suggest a role for the olfactory system in guiding the newborn. When removing newborn quokkas or brushtail possums from the nipples and putting them elsewhere on the mother’s belly, whether the mother is facing downward or upward, most pups climbed back to the pouch if they were not too remote from it, indicating negative geotropism and/or chemoattraction from cues emanating from the pouch (Cannon et al., 1976, Veicht et al., 2000). Schneider et al. (2009) demonstrated that newborn wallabies climbing on a pelt are attracted to swabs soaked with pouch fluid and proposed, based on histological and immunohistochemical observations, that the olfactory or the vomeronasal organs are sufficiently mature to explain this response. However, electrophysiological recordings of olfactory bulb (OB) neurons following olfactory stimulation revealed that these neurons become functional only around the third postnatal week in wallabies (Ellendorff et al., 1988), when the glomeruli start to be well differentiated (Ashwell et al., 2008). After a morphological study of the olfactory epithelium in the bandicoot, Kratzing (1986) concluded it was too immature for the olfactory system to play a role in guiding the newborn to the teat. However, in neonatal M. domestica some olfactory receptor neurons and some cells in the main OB express the olfactory marker protein and other markers that indicate a certain degree of differentiation and the presence of connections between the two regions (Tarozzo et al., 1995, Shapiro et al., 1997). Nonetheless, significant development of the olfactory epithelium and bulbs occurs during the second to the fourth postnatal weeks (Brunjes et al., 1992). Thus these various studies in the opossum and other marsupials lead to believe that a role for the olfactory system in guiding the newborn cannot be excluded without further investigation, especially in the light of the behavioral demonstration that olfactory stimulation can induce locomotor-like movements in newborn rats (Fady et al., 1998).
We have already provided evidence for a possible role of the vestibular system in steering the newborn opossum to a nipple. Indeed, a sensory epithelium is seen only in the utricle, but not yet in the more immature saccule or the semicircular canals, and vestibular ganglion cells project peripheral processes to the utricular macula (but not to the other components of the vestibular labyrinth) and central processes to parts of the brainstem vestibular complex that contain spinal-projecting cells (Pflieger and Cabana, 1996). This utriculo-vestibulospinal pathway has not been tested physiologically in newborn opossums. However, it has been known for decades that this pathway in mammals influences the activity of neck and limb muscles in relation to gravity clues (Wilson and Melvill-Jones, 1979). The finding of vestibulospinal axons in the cervical cord in newborn opossums contrasts with the later development of vestibulospinal projections reported in wallabies by McCluskey et al. (2008), but their tracer was applied to more caudal spinal segments, which may explain the later arrival of vestibular axons therein.
A study in newborn wallabies has revealed the presence in the snout skin of a dense network of fibers arising from the trigeminal ganglion (5G) and of central processes in the spinal trigeminal tract (sp5) down to the upper cervical cord (Waite et al., 1994). An earlier study using electron microscopy in neonatal opossums revealed fibers innervating mechanosensory Merkel cells in the snout skin (Jones and Munger, 1985), but the origin of this innervation in the 5G and the central projections were not investigated. These reports, combined to the existence of a projection from the Sp5 to the cervical cord in newborn opossums already mentioned (Wang et al., 1992), provide partial evidence for a role of touch and possibly other sensory modalities conveyed by the trigeminal system in locomotor guidance.
The first aim of the present study was to test physiologically the above sensory systems for a possible implication in locomotor control in newborn opossums. Using the same kind of in vitro preparation developed by Lavallée and Pflieger (2009), the minimal intensities of stimulation needed for these sensory brain regions to elicit forelimb movement have been determined. Since stimulation of the 5G revealed to be very efficient, we then aimed to verify the trigeminal innervation of the snout and the possible pathways by which trigeminal inputs are relayed to the spinal cord, using immunohistochemistry and anatomical tracing. These experiments also provided information on the other sensory systems.
Preliminary results have been presented in abstract form (Pflieger, 2008, Adadja and Pflieger, 2010).
Section snippets
Experimental procedures
Gray, short-tailed opossums M. domestica were obtained from a colony maintained at the Department according to Fadem et al. (1982) and Kraus and Fadem (1987) (for further details, see Cassidy et al., 1994). All procedures followed the guidelines of the NIH and the Canadian Council on Animal Care as approved by the University Animals Ethics Committee.
Results
The average minimal intensity of stimulation of the dorsal spinal cord at C4 necessary to induce a forelimb movement comparable to that performed by the intact newborn opossum was 0.4 ± 0.04 μA (0.2–0.6 μA; Table 1). The average intensity was similar whether the left of the right forelimb responded, therefore the data for both limbs were pooled (I + C). The value of 0.4 ± 0.04 μA is thus used as threshold (T) with which the brain stimulation intensities below are compared. It can be noted that a single
Discussion
In the present study we compared the influence of three head sensory systems, vestibular, trigeminal and olfactory, on forelimb movement in order to determine which system is most likely to guide the newborn opossum, M. domestica, from the birth canal to the nipple where it attaches. In in vitro preparations of the neuraxis with the limbs attached to the carcass, we measured the minimal intensities of stimulation of different brain regions needed to induce forelimb movement similar to what is
Conclusion
As summarized in Fig. 7, the results obtained from the present physiological, immunohistochemical and tract-tracing experiments in the opossum M. domestica do not support the olfactory system as a major guide of newborn locomotor behavior, but support our previous suggestion of the implication of the vestibular system. More significantly, these results show a much better developed trigeminal system than suspected.
Acknowledgments
This work was undertaken by T.A. in partial fulfillment of the requirements for the M.Sc. degree. It is funded by Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC #312015) and the Canadian Foundation for Innovation (#10442) to JFP, and NSERC (#3595) to TC.
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