Characteristics of olfactory ensheathing cells and microarray analysis in Tupaia belangeri (Wagner, 1841).

Tree shrews are most closely related to the primates and so possess a number of advantages in experimental studies; they have been used as an animal model in bacterial and virus infection, cancer, endocrine system disease, and certain nervous system diseases. Their olfactory ensheathing cells (OECs) are able to release several cytokines to promote neuronal survival, regeneration and remyelination. The present study used western blot analysis to identify antibody specificity in protein extracts from whole tree shrew brains to identify the specificity of p75 nerve growth factor receptor (NGFR) derived from rabbits (75 kDa). OECs were cultured and isolated, then stained and identified using the antibodies for p75NGFR. To investigate the capacity of OECs to express cytokines and growth factors, microarray technology was used, and the analysis revealed that OECs were able to express 9,821 genes. Of these genes, 44 genes were from the neurotrophic factor family, which may indicate their potential in transplantation in vivo. The present study considered the function of OECs as revealed by other studies, and may contribute to future research.

Basal physiological parameters in domesticated tree shrews (Tupaia belangeri chinensis).

Establishing non-human primate models of human diseases is an efficient way to narrow the large gap between basic studies and translational medicine. Multifold advantages such as simplicity of breeding, low cost of feeding and facility of operating make the tree shrew an ideal non-human primate model proxy. Additional features like vulnerability to stress and spontaneous diabetic characteristics also indicate that the tree shrew could be a potential new animal model of human diseases. However, basal physiological indexes of tree shrew, especially those related to human disease, have not been systematically reported. Accordingly, we established important basal physiological indexes of domesticated tree shrews including several factors: (1) body weight, (2) core body temperature and rhythm, (3) diet metabolism, (4) locomotor rhythm, (5) electroencephalogram, (6) glycometabolism and (7) serum and urinary hormone level and urinary cortisol rhythm. We compared the physiological parameters of domesticated tree shrew with that of rats and macaques. Results showed that (a) the core body temperature of the tree shrew was 39.59±0.05 ℃, which was higher than that of rats and macaques; (b) Compared with wild tree shrews, with two activity peaks, domesticated tree shrews had only one activity peak from 17:30 to 19:30; (c) Compared with rats, tree shrews had poor carbohydrate metabolism ability; and (d) Urinary cortisol rhythm indicated there were two peaks at 8:00 and 17:00 in domesticated tree shrews, which matched activity peaks in wild tree shrews. These results provided basal physiological indexes for domesticated tree shrews and laid an important foundation for diabetes and stress-related disease models established on tree shrews.

[Morphological characteristics and cryodamage of Chinese tree shrew (Tupaia belangeri chinensis) sperm].

The tree shrew (Tupaia belangeri chinensis) is a small non-rodent mammal, which is a relatively new experimental animal in medicine due to its close evolutionary relationship to primates and its rapid propagation. Sperm characteristics and cryopreservation in the tree shrew were the main contents of our spermatological research. Epididymal sperm were surgically harvested from male tree shrews captured from the Kunming area. The rate of testis weight to body weight was (1.05±0.07)%, volume of both testis was (1.12 ± 0.10) mL, total sperm from epididymis and vas deferens were 2.2-8.8×10(7), and sperm motility and acrosome integrity were (68.8 ± 3.9)% and (90.0 ± 2.1)%, respectively. Sperm ultrastructure of the tree shrew was examined by scanning electron microscopy and transmission electron microscopy. Tree shrew sperm had a round or oval shaped head of approximately 6.65×5.82 µm, and midpiece, principal piece, tail, and total sperm lengths were 13.39, 52.35, 65.74, and 73.05 µm, respectively. The mitochondria in the midpiece consisted of approximately 48 gyres and had a 9+9+2 axonemal pattern. After freezing and thawing, sperm showed partly intact acrosomes and plasma membrane defects, and sperm breakages, twists, and swellings were found. The tree shrew had similar ultrastructure with other mammalians except for the mitochondria number and the sperm size. Ultrastructural alteration is still the main cause resulting in poor sperm after cryopreservation.

Endocrine correlates of reproductive development in the male tree-shrew (Tupaia belangeri) and the effects of infantile exposure to exogenous androgens.

The developmental life-history of tree-shrews conforms with the general primate pattern. Consequently, elucidation of the tree-shrew's neuroendocrine reproductive axis could shed light on the mechanisms that underlie human pubertal development. In the present study, we examined plasma gonadotropin concentrations in male tree-shrews from birth to sexual maturity, and related them to changes in the androgenic and gametogenic status of the testis. A hypogonadotropic infantile phase, during which a stable population of primordial cells is established, extended from birth to approximately Day 30. Following a short juvenile phase (Days 30-40), a pubertal phase of accelerated reproductive development was initiated between Days 40-55. At this time, FSH and LH levels rose and testosterone concentrations reached peak levels coincident with the descent of the testes, accelerated growth in the reproductive tract and the onset of spermatogenesis. To test whether this developmental peak in testosterone secretion is an important determinant in the normal onset of puberty, we exposed male tree-shrews prematurely to high circulating androgen levels for various periods and then examined the impact on key components of the developing reproductive axis. The testosterone implants failed to initiate spermatogenesis and the testes remained in an infantile state for the duration of the treatment, whereas implant removal led to the development of full spermatogenic activity. In both normal and experimental situations, low levels of FSH were associated with a lack of spermatogenic activity while the progression of germ cell development was precisely correlated with rising FSH levels. Taken together, these data establish a comprehensive picture of reproductive development in the male tree-shrew, and also provide support for the hypothesis that FSH plays a primary role in the initiation of spermatogenesis.

Development of starburst cholinergic amacrine cells in the retina of Tupaia belangeri.

"Starburst" cholinergic amacrines specify the response of direction-selective ganglion cells to image motion. Here, development of cholinergic amacrines was studied in the tree shrew Tupaia belangeri (Scandentia) by immunohistochemistry with antibodies against choline acetyltransferase (ChAT) and neurofilament proteins. Starburst amacrines expressed ChAT much earlier than previously thought. From embryonic day 34 (E34) onward, orthotopic and displaced subpopulations segregated from a single cluster of immunoreactive precursor cells. Orthotopic starburst amacrines rapidly took up positions in the inner nuclear layer. Displaced starburst amacrines were first arranged in a monocellular row in the inner plexiform layer, and, with a delay of 1 week, they descended to the ganglion cell layer. Conversely, dendritic stratification of displaced amacrines slightly preceded that of orthotopic ones. Starburst amacrines expressed the medium-molecular-weight neurofilament protein (NF-M) from E34 to postnatal day 11 (P11) and coexpressed alpha-internexin from E36.5 to P11. Consequently, neurofilaments composed of alpha-internexin and NF-M may stabilize developing dendrites of starburst amacrines. During the first 2 postnatal weeks, subpopulations of anti-NF-M-labeled ganglion cells costratified with the preexisting dendritic strata of starburst amacrines in the ON sublamina, OFF sublamina, or both. Hence, anti-NF-M-labeled ganglion cells may include direction-selective ones. Thereafter, NF-M and alpha-internexin proteins disappeared from starburst amacrines, and NF-M immunoreactivity was lost in the dendrites of ganglion cells. Our findings suggest that NF-M and alpha-internexin are important for starburst amacrines and ganglion cells to recognize each other and, thus, contribute to the formation of early developing retinal circuits in the inner plexiform layer.

On the postnatal development of the striate cortex (V1) in the tree shrew (Tupaia belangeri).

Histological serial sections, three-dimensional reconstructions and morphometry served to study the postnatal development of V1 in tree shrews. The main objectives were to evaluate the expansion of V1, the implications of its growth on the occipital cortex and, vice versa, the effects of the expanding neocortex on the topography of V1. The future V1 was identified on postnatal day 1 by its granular layer IV, covering the superior surface of the occipital cortices including the poles. A subdivision of layer IV, distinctive for the binocular part, was evident in the central region. V1 expanded continuously with age into all directions succeeded by the maturation of layering. The monocular part was recognized from day 15 onward, after the binocular part had reached its medial border. In reference to the retinotopic map of V1, regions emerged in a coherent temporo-spatial sequence delineating the retinal topography in a central to peripheral gradient beginning with the visual streak representation. The growth of V1 was greatest until tree shrews open their eyes, culminated during adolescence, and completed after a subsequent decrease in the young adult. Simultaneous expansion of the neocortex induced a shifting of V1. Translation and elongation of V1 entailed that the occipital cortex covered the superior colliculi along with a downward rotation of the poles. The enlargement of the occipital part of the hemispheres was in addition associated with the formation of a small occipital horn in the lateral ventricles, indicating an incipient 'true' occipital lobe harbouring mainly cortices involved in visual functions.

Extracellular matrix and development of lamination in the dorsal lateral geniculate nucleus in the tree shrew (Tupaia belangeri).

In the tree shrew (Tupaia belangeri), the cytoarchitectonic lamination of the lateral geniculate nucleus cannot be detected at birth; it only appears during the early postnatal period. However, a laminated pattern was revealed with rapid Golgi staining and retinal afferents were segregated into the appropriate laminae well before cytoarchitectonic lamination could be seen. Both observations indicate that the extracellular matrix may play a role in the separation of lateral geniculate nucleus cells into laminae. In the present study, the organization of the extracellular matrix was investigated during development using immunohistochemical and in situ hybridization techniques. For immunohistochemistry, peanut agglutinin (PNA) lectin and antibodies against tenascin (TN) were chosen, while for in situ hybridization, mTN riboprobes were used, simultaneously, with antibodies against Vimentin (Vim) and microtubule associated protein (MAP-2). The results showed that the pattern of PNA-binding glycoproteins and that of tenascin were relatively similar, although tenascin appeared later and disappeared earlier. The first interlaminar spaces to be detected were those between layers innervated by opposite eyes. The TN specific mRNA was detected in the lateral geniculate nucleus at P0, but was no longer visible at P7. By comparing TN mRNA and Vim or MAP-2 stainings a correspondence could be observed. The extracellular matrix lamination therefore seems to precede cytoarchitectonic lamination, suggesting that the extracellular matrix may play a role in the development of laminated structures. The TN-producing cells seem to be developing astrocytes and neurons.

Active emmetropization--evidence for its existence and ramifications for clinical practice.

There is increasing evidence from animal studies in support of the concept of an active emmetropization mechanism which has potentially important clinical ramifications for the management of refractive errors. Recent research into refractive development and emmetropization is reviewed, with emphasis given to work involving the chick, tree shrew and monkey, which represent the three most widely used animal models in this field. The findings of this research are reviewed in a clinical context. Compensatory eye growth responses to focusing errors imposed by lenses represent the most compelling evidence for active emmetropization. These observations are complemented by other evidence showing recovery from induced refractive errors such as form-deprivation myopia. Of the animals listed above, chicks show the most impressive emmetropization, being able to compensate fully (using choroidal and scleral mechanisms) to lens powers ranging from +15 D to -10 D. The range of lens powers eliciting appropriate compensatory responses is narrower in the tree shrew and monkey, and the response patterns generally are also more complex to interpret. These data relate to young animals and together indicate refractive plasticity during development. Extrapolation of these findings to humans predicts that natural emmetropization will be inhibited in neonates by early intervention with prescription lenses, and that refractive correction of myopia will lead to accelerated progression. This convincing evidence for active emmetropization warrants due consideration in developing clinical management strategies for refractive errors.

Behavioral measures of auditory thresholds in developing tree shrews (Tupaia belangeri).

The development of hearing in tree shrews (Tupaia belangeri) was tested through behavioral measures in the range of 0.3-60 kHz from the first postnatal thresholds detectable through to those of weanlings. The onset of hearing was defined using unconditioned startle responses, while, later on, pinna reflexes and head movements were used as behavioral indicators of sound perception. Startle responses could be evoked from day 16 after birth (DAB) onwards, indicating that the hearing system is capable of transferring airborne sound, although no reactions to tones were recorded at this age. The first unconditioned reactions to tones were found at DAB 18 in the range of 1-5 kHz (range of mother-infant call). Responses were restricted to the range of greatest sensitivity in adults (1-10 kHz). An increase in sensitivity was detected from DAB 18-38. The lowest tested frequency (0.3 kHz) reached adult levels earlier than higher frequencies. No further improvement of auditory thresholds could be observed from DAB 38 (end of nutritional weaning) to DAB 42. Results were related to recent findings on the development of acoustical behavior and of the peripheral and central auditory system of tree shrews and other mammals.

Postnatal development of central nervous alpha 2-adrenergic binding sites: an in vitro autoradiography study in the tree shrew.

The postnatal development of the alpha 2-adrenoceptor pattern was investigated by in vitro receptor autoradiography with the antagonist [3H]rauwolscine in the brains of tree shrews (Tupaia belangeri). At birth, high numbers of [3H]rauwolscine binding sites are diffusely distributed in the whole brain with exception of the neocortex which is very weakly labeled at this time. While the number of [3H]rauwolscine binding sites in the cerebellum decreases to low levels during the first three postnatal weeks, several brain regions show a significant increase in binding sites which are progressively concentrated in distinct nuclei. In the medulla oblongata, the diffuse labeling pattern changes so that binding sites become centralized in the dorsomedial nuclei. In the pons, similar changes can be observed with a moderate labeling of the locus coeruleus on postnatal day 10 and a strong labeling in the adult. In the thalamus, a transient appearance of high numbers of [3H]rauwolscine binding sites can be observed during the second and third postnatal week in specific nuclei. In the preoptic area and hypothalamus, there are only minor postnatal changes but the numbers of [3H]rauwolscine binding sites decrease between postnatal day 5 and adulthood. The high number of binding sites in the limbic system does not significantly change after birth. In the neocortex and the superior colliculus, the [3H]rauwolscine labeling pattern which is characteristic for the adult is achieved not before the third postnatal week. Competition experiments demonstrate that [3H]RAUW binds with high affinity to alpha 2-adrenoceptors in the postnatal as well as in the adult brain. Therefore, this study demonstrates region specific developmental profiles of the pattern of alpha 2-adrenoceptors in the postnatal tree shrew brain.

Postnatal development of 3H-rauwolscine binding sites in the dorsal lateral geniculate nucleus and the striate cortex of the tree shrew (Tupaia belangeri).

Noradrenaline has been shown to play an important role within the visual system of the brain. To analyze the postnatal development of alpha2-noradrenergic receptors in the visual system of tree shrews, we localized and quantified binding sites for the antagonist [3H]-rauwolscine by in vitro-autoradiography in the dorsal lateral geniculate nucleus and the striate cortex at different postnatal ages. At birth, the dorsal lateral geniculate nucleus is only slightly labeled by [3H]-rauwolscine. During the postnatal period, the number of binding sites increases to reach a maximum around postnatal day 20. Since the young tree shrews open their eyes at approximately day 19, it appears that this high concentration of alpha2-adrenoceptors is related to eye opening. In the adult animal, [3H]-rauwolscine labeling shows a laminated pattern in the dorsal lateral geniculate nucleus. Laminae 1, 2, and 3 are more strongly labeled than laminae 4, 5, and 6. In the striate cortex, the pattern of [3H]-rauwolscine-binding sites changes dramatically during the early postnatal period. Immediately after birth, there is only one layer, located within the subplate zone, which is labeled. From postnatal day 5 onwards, all cortical layers which can be distinguished on histologically stained sections reveal [3H]-rauwolscine-binding sites, but in layer IV, which is known to receive major inputs from the dorsal lateral geniculate nucleus, there is very little labeling during the first two postnatal weeks. In this layer, a large number of [3H]-rauwolscine-binding sites occurs between postnatal day 15 and 20, that is slightly before and around the time of eye opening.(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal development of area 17 callosal connections in Tupaia.

The goal of the present study was to investigate the pattern of maturation of callosal projecting neurons in a well-studied mammalian visual system with unique structural and functional properties. Studies of the distribution pattern of interhemispheric connections in the adult tree shrew primary visual cortex reveal not only a high concentration of labeled neurons along the area 17/18 border, as in standard experimental animals such as the cat and monkey, but also numerous callosal projecting neurons in the adjacent dorsal part of area 17, which largely corresponds to the binocular visual field (Kretz and Rager, Exp. Brain Res. 82:271, '90). Callosal projections were anatomically traced in 11 tree shrews (Tupaia belangeri) at various ages between postnatal day 7 (7, 9, 10, 13, 15, 17, 19, and 26 days old) and adulthood (107 days old). In each animal, four injections of wheat germ agglutinin conjugated to horseradish peroxidase were made in a standard configuration into the striate cortex of one hemisphere. In young tree shrews only 7 and 9 days old, heavily labeled terminal axon structures could be seen in the white matter and in layer VI of the opposite hemisphere. Only a few labeled neurons, however, were detected in layer III. The small number of labeled neurons indicated that early in postnatal development, only a few callosal axons had invaded the upper cortical layers. By 10 days of age, the number of supragranular neurons was increasing and the maximal value was counted in a 13-day-old tree shrew. A sharp decline in the number of labeled supragranular neurons was noticed--about 94% in our case--between days 13 and 15. In animals more than 15 days old, the distribution pattern and the density of the neurons looked like the pattern seen in the adult Tupaia brain. The labeled cells were mostly concentrated in layers II and III. The majority of neurons resembled typical pyramidal cells. However, some of the neurons in sublayer IIIc had elongated cell bodies oriented parallel to the laminar boundaries. In contrast to the supragranular cells found in all stages investigated, small populations of labeled cells in layer VI were observed in 9- to 17-day-old tree shrews only. In young postnatal animals 7 to 13 days old, a peculiar cell type was labeled on the ipsilateral side. In coronal sections these cell bodies formed a continuous band that extended from the ventricular wall to the subcortical white matter. These cells might belong to a population of cells still in migration.

Development of acetylcholinesterase activity in the lateral geniculate nucleus.

The pattern of acetylcholinesterase activity in the tree shrew (Tupaia belangeri) lateral geniculate nucleus (LGN) undergoes a number of striking changes during postnatal development. The adult tree shrew LGN is made up of six cellular layers divided by relatively cell-free interlaminar zones. At birth, however, the nucleus appears unlaminated when processed with conventional Nissl-staining techniques. The cellular lamination appears during the first postnatal week. The eyes open much later, typically at the end of the third week after birth. In the adult tree shrew, acetylcholinesterase (AChE) activity is found throughout the nucleus (both within and between the six cellular layers). In most sections examined, reaction product is slightly more intense in the lateral cell layers (4, 5, and 6). This is in sharp contrast to the pattern at birth (postnatal day zero, or P0). The detectable AChE activity at this age is apparently found in inchoate layers 1-2 and 4-5. Within these pairs, areas innervated by the ipsilateral eye (i.e., incipient layers 1 and 5) appear to contain more reaction product. From P0 to P4, the density of AChE activity increases in layers 1-2 and 4-5 and becomes detectable in the barely evident layers 3 and (usually) 6 at this age. By the middle of the second postnatal week, after laminae are clearly apparent with a Nissl stain, AChE activity has increased and is mainly associated with each cellular layer in the nucleus. During the third week after birth this pattern undergoes a radical shift. The most intense AChE activity is now in the interlaminar zones. Finally, as the adult pattern emerges, AChE activity increases in the cellular layers and all areas of the nucleus exhibit relatively high levels of AChE activity. Superimposed on this changing laminar pattern of AChE activity are changes related to the retinotopic map within the nucleus. Portions of the LGN representing central vision develop their characteristic pattern of activity several days ahead of the regions representing more peripheral visual field locations. AChE activity is also found transiently in the optic tract near the LGN during the first 3 postnatal weeks. Two (possibly three) groups of AChE-carrying fibers can be traced from the optic chiasm to their apparent sites of termination (or origin) in the parabigeminal nucleus, ventral lateral geniculate nucleus, and dorsal LGN. The activity present in the optic tract disappears shortly after eye opening.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure and postnatal development of photoreceptors and their synapses in the retina of the tree shrew (Tupaia belangeri).

The "all cone" retina of the tree shrew (Tupaia belangeri) was examined in the adult and early postnatal stages by light and electron microscopy. Rods are not as rare as previously thought, but make up about 4% of the photoreceptors. They are relatively short and narrow cells, which stain (toluidine blue) more intensively and lie more proximal than cones. Among the cones three morphological varieties could be distinguished. Most cones stain lightly but have a light or a dark giant mitochondrion in their inner segment; a third type stains darker but occurs only rarely. All cones possess extensive radial processes ("lateral fins") around the basal part of their inner segments. Such fins are well known from reptiles and birds, but have only once been described in a mammal (gray squirrel). The maturation of the retina in Tupaia belangeri proceeds centrifugally, i.e., from the vitreal to the scleral side, as in most mammals. A few synapses are already present at birth in the outer and inner plexiform layers, but seem to be more advanced in the latter. Such early synapses are small and have only few synaptic vesicles; they appear almost mature by day 14. The light-sensitive outer segments develop last. The first disks are seen by day 10, but regular membrane stacks are only present by day 18. Thus, it seems that the retina is functional when the young first open their eyes, which occurs around day 18.

Ultrastructure of the developing tree shrew lateral geniculate nucleus.

We examined the ultrastructural development of the lateral geniculate nucleus (LGN) in postnatal tree shrews to distinguish which features, if any, show a correlation with the development of cell layers. Our data indicate that synaptogenesis has begun at birth (PO) which is prior to the development of cell layers. At postnatal day 8 (P8), when laminar borders can be distinguished, the majority of synaptic profiles are still immature. Although some mature synaptic profiles can be identified at P8 and especially at P15, complex synaptic arrangements characteristic of adults are absent at that time. Growth cones are present at all 3 ages but are less prevalent at P8 and P15 than at birth and appear to be present in slightly higher concentrations in the interlaminar spaces. These results suggest that LGN cell layer formation does not correlate with the beginning of synaptogenesis but that it may correlate with an increase in growth cones in the interlaminar spaces.

The role of retinogeniculate afferents in the development of connections between visual cortex and the dorsal lateral geniculate nucleus.

The role of retinogeniculate afferents in the development of patterns of connections between visual cortex and the lateral geniculate nucleus (LGN) was addressed by studying the effect of bilateral enucleation at birth on those patterns of connections in tree shrew. In normal adult tree shrews there are six LGN cell layers separated by cell-sparse interlaminar spaces. The reciprocal connections between the LGN and visual cortex are restricted to a column running across all six LGN layers; the geniculocortical projection arises from the cell layers while the corticogeniculate projection terminates primarily in the interlaminar spaces. At birth, when the experimental animals were bilaterally enucleated, the retinogeniculate fibers have begun to segregate by eye but neither the cytological characteristics of individual layers nor the interlaminar spaces have yet formed, and the corticogeniculate fibers have not entered the nucleus. Bilateral enucleation does not prevent the development of the cytological characteristics of individual layers but the interlaminar spaces do not develop. The results of [3H]proline/HRP injections into visual cortex in animals bilaterally enucleated at birth indicate that in the absence of retinogeniculate fibers, and thus interlaminar spaces, the corticogeniculate fibers do not concentrate at the laminar borders but instead spread across all six LGN cell layers. Despite the failure of this projection to concentrate at laminar borders, the corticogeniculate fibers do terminate within a restricted projection column.