Retinal functional development is sensitive to environmental enrichment: a role for BDNF.

Retina has long been considered less plastic than cortex or hippocampus, the very sites of experience-dependent plasticity. Now, we show that retinal development is responsive to the experience provided by an enriched environment (EE): the maturation of retinal acuity, which is a sensitive index of retinal circuitry development, is strongly accelerated in EE rats. This effect is present also in rats exposed to EE up to P10, that is before eye opening, suggesting that factors sufficient to trigger retinal acuity development are affected by EE during the first days of life. Brain derived neurotrophic factor (BDNF) is precociously expressed in the ganglion cell layer of EE with respect to non-EE rats and reduction of BDNF expression in EE animals counteracts EE effects on retinal acuity. Thus, EE controls the development of retinal circuitry, and this action depends on retinal BDNF expression.

Bcl-2 overexpression per se does not promote regeneration of neonatal crushed optic fibers.

We have explored whether overexpression of the bcl-2 gene 'per se' can promote regeneration of retinal ganglion cells (RGCs) after optic nerve axotomy in developing transgenic mice. We have used newborn mice (postnatal day 5) because at this age the central nervous system environment is more permissive for regeneration than in adults, thus, maximizing the probability to detect a regeneration-promoting role of bcl-2. Thirty days postsurgery we found that in mice overexpressing bcl-2, a high proportion of retinal ganglion cells survived and also that some fibers in the proximal stump of the optic nerve were preserved. However, the optic nerve of transgenic mice does not show signs of regeneration. On the contrary, in the presence of Schwann cell transplants, there are signs of fiber regrowth. Indeed, many axonal terminals cross the crush site and reach the chiasm in both wild type and transgenic mice nerves. These results suggest that bcl-2 overexpression is not sufficient 'per se' to increase the regenerative potentiality of axotomized RGCs.

Brain-derived neurotrophic factor is an anterograde survival factor in the rat visual system.

BACKGROUND: The neurotrophins, which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5 and NT-6, are a family of proteins that play fundamental roles in the differentiation, survival and maintenance of peripheral and central neurons. Much research has focused on the role of neurotrophins as target-derived, retrogradely transported trophic molecules. Although there is recent evidence that BDNF and NT-3 can be transported in an anterograde direction along peripheral and central axons, there is as yet no conclusive evidence that these anterograde factors have direct post-synaptic actions. RESULTS: We report that BDNF travels in an anterograde direction along the optic nerve. The anterogradely transported BDNF had rapid effects on retinal target neurons in the superior colliculus and lateral geniculate nucleus of the brain. When endogenous BDNF within the developing superior colliculus was neutralised, the rate of programmed neuronal death increased. Conversely, provision of an afferent supply of BDNF prevented the degeneration of geniculate neurons after removal of their cortical target. CONCLUSIONS: BDNF released from retinal ganglion cells acts as a survival factor for post-synaptic neurons in retinal target fields.

Optic nerve crush: axonal responses in wild-type and bcl-2 transgenic mice.

Retinal ganglion cells of transgenic mice overexpressing the anti-apoptotic protein Bcl-2 in neurons show a dramatic increase of survival rate after axotomy. We used this experimental system to test the regenerative potentials of central neurons after reduction of nonpermissive environmental factors. Survival of retinal ganglion cells 1 month after intracranial crush of the optic nerve was found to be 100% in adult bcl-2 mice and 44% in matched wild-type (wt) mice. In the optic nerve, and particularly at the crush site, fibers regrowing spontaneously or simply sprouting were absent in both wt and bcl-2 mice. We attempted to stimulate regeneration implanting in the crushed nerves hybridoma cells secreting antibodies that neutralize central myelin proteins, shown to inhibit regeneration (IN-1 antibodies) (Caroni and Schwab, 1988). Again, we found that regeneration of fibers beyond the site of crush was virtually absent in the optic nerves of both wt and bcl-2 mice. However, in bcl-2 animals treated with IN-1 antibodies, fibers showed sprouting in the proximity of the hybridoma implant. These results suggest that neurons overexpressing bcl-2 are capable of surviving axotomy and sprout when faced with an environment in which inhibition of regeneration has been reduced. Nevertheless, extensive regeneration does not occur, possibly because other factors act by preventing it.

Protection of retinal ganglion cells and preservation of function after optic nerve lesion in bcl-2 transgenic mice.

Multicellular organisms face the necessity of removing superfluous or injured cells during normal development, tissue turn-over and in response to damaging conditions. These finalised killings occur throughout a process, commonly called programmed cell death (PCD), which is placed under strict cellular control. PCD is regulated by the products of the expression of a number of genes. This fact raises the intriguing possibility of inhibiting such degenerative processes by operating on some of the controlling genes. Central neurons of transgenic mice overexpressing bcl-2, a powerful inhibitor of PCD, are remarkably resistant to degeneration induced by noxious stimuli. We have explored the ate of retinal ganglion cells and of their axons, when such transgenic animals have been challenged by a lesion of the optic nerve. These results have direct bearing on the possibility of attaining functional restoration of the injured pathway.

The visual response of retinal ganglion cells is not altered by optic nerve transection in transgenic mice overexpressing Bcl-2.

Attempts to rescue retinal ganglion cells from retrograde degeneration have had limited success, and the residual function of surviving neurons is not known. Recently, it has been found that axotomized retinal ganglion cells die by apoptotic mechanisms. We have used adult transgenic mice overexpressing the Bcl-2 protein, a powerful inhibitor of apoptosis, as a model for preventing injury-induced cell death in vivo. Several months after axotomy, the majority of retinal ganglion cells survived and exhibited normal visual responses. In control wild-type mice, the vast majority of axotomized retinal ganglion cells degenerated, and the physiological responses were abolished. These results suggest that strategies aimed at increasing Bcl-2 expression, or mimicking its function, might effectively counteract trauma-induced cell death in the central nervous system. Neuronal survival is a necessary condition in the challenge for promoting regeneration and eventually restoring neuronal function.

Long-term survival of retinal ganglion cells following optic nerve section in adult bcl-2 transgenic mice.

The bcl-2 gene codes for a protein that acts as a powerful inhibitor of active cell death. Since the transection of the optic nerve in adult mammalians starts a massive process of degeneration in retinal ganglion cells, we investigated whether the overexpression of bcl-2 in adult transgenic mice can protect the axotomized ganglion cells. We performed intracranial optic nerve transection on both wild type and transgenic adult mice, and we tested cell survival 2 or 3.5 months after axotomy. The percentage of surviving ganglion cells after optic nerve section was computed by combining the counts of the optic nerve fibres in intact nerves with the cell density measures of the ganglion cell layer of axotomized retinae. From these data we found that in transgenic mice approximately 65% of ganglion cells survived 3.5 months after axotomy. In contrast, 2 months after surgery, < 10% of ganglion cells were left in wild type retinae. We have also examined the morphology and fine structure of the proximal stump of the sectioned optic nerves by light and electron microscopy. In the transgenic mice a very large number of axons survived after surgery and they still exhibited fairly normal morphology and ultrastructure. On the other hand the wild type transected nerves had only a few visible axons that displayed clear signs of degeneration. We conclude that the overexpression of Bcl-2 protein in central neurons is a very effective strategy to ensure long-term survival in axotomized cells.

Protection of retinal ganglion cells from natural and axotomy-induced cell death in neonatal transgenic mice overexpressing bcl-2.

Approximately half of the retinal ganglion cells (RGCs) present in the rodent retina at birth normally die during early development. Overexpression of the photo-oncogene bcl-2 recently has been shown to rescue some neuronal populations from natural cell death and from degeneration induced by axotomy of nerves within the peripheral nervous system. Here we study in vivo the role of the overexpression of bcl-2 in the natural cell death of RGCs and in the degenerative process induced in these cells by transection of the optic nerve. We find that in newborn bcl-2 transgenic mice, the number of RGCs undergoing natural cell death is considerably lower than in wild-type pups. Consistently, a vast majority (90%) of the ganglion cells found in the retina of neonatal transgenics are maintained in adulthood, whereas only 40% survive in wild-type mice. After transection of the optic nerve, the number of degenerating ganglion cells, determined by counting pyknotic nuclei or nuclei with fragmented DNA, is substantially reduced in transgenic mice. In wild-type animals, almost 50% of ganglion cells degenerate in the 24 hr after the lesion, whereas almost the entire ganglion cell population survives axotomy in transgenic mice. Therefore, overexpression of bcl-2 is effective in preventing degeneration of this neuronal population, raising the possibility that ganglion cells are dependent on the endogenous expression of bcl-2 for survival. The remarkable rescue capacity of bcl-2 overexpression in these neurons makes it an interesting model for studying natural cell death and responses to injury in the CNS.

A developmentally regulated nerve growth factor-induced gene, VGF, is expressed in geniculocortical afferents during synaptogenesis.

The expression of the nerve growth factor-inducible gene VGF has been examined by in situ hybridization. Western blot and immunohistochemical studies in the developing and adult rat central nervous system, with particular emphasis on the visual system. Both the messenger RNA and the protein are particularly abundant in the developing dorsal lateral geniculate nucleus, appearing, respectively, at embryonal day 16 and 18. After its onset at E16, VGF messenger RNA expression increases progressively in the dorsal lateral geniculate nucleus and remains high during the first two post-natal weeks; afterwards, it gradually decreases and, at the offset of the plasticity period, it reaches very low levels maintained in adulthood. A similar time course has been observed for VGF protein in the dorsal lateral geniculate nucleus area, by semi-quantitative Western blots. In addition to the presence of the protein in the geniculate neurons, a strong, transient immunoreactivity has been found at the embryonic cortical subplate at E18, reflecting the presence of the antigen in axonal terminals originating from thalamic neurons. Interestingly, we found that the blockade of afferent electrical activity by intraocular injection of tetrodotoxin strongly reduces the level of VGF messenger RNA in the dorsal lateral geniculate nucleus. Although the function of the VGF protein is not known, it had been previously proposed that VGF could be a precursor for neuropeptide/s. The spatiotemporal expression of VGF, together with the observation of a regulation by electrical activity, suggest that this protein may be relevant in the process of synaptogenesis and/or synaptic stabilization in the developing geniculocortical connections.

Pyrolytic carbon coating enhances Teflon and Dacron fabric compatibility with endothelial cell growth.

Compatibility with endothelial cell attachment and growth appears to be an important requisite of vascular prosthetic materials, possibly influencing thrombosis, pseudointimal hyperplasia, and accelerated atherosclerosis at the site of blood-material interaction. Since deposition of pyrolytic carbon (PC) on prosthetic surfaces has been associated with enhanced hemocompatibility, in the present study we assessed whether a thin layer (0.5 microns) of PC deposited onto materials such as knitted Teflon and Dacron enhanced endothelial cell attachment and growth. Cultured human umbilical vein endothelial cells (HUVEC) were seeded at a density of 4.5 x 10(4) cells/cm2 on PC-coated and uncoated grafts. In order to quantify endothelial cell attachment on the fabrics, the area of Teflon and Dacron fabrics covered by endothelial cells was estimated on day 2 after seeding using the point counting method in scanning electron micrographs. Subsequently, on days 2 and 4 after seeding, endothelial cell proliferation was measured both as number of endothelial cells and as total proteins of the endothelial cells covering the fabrics. On day 2 endothelial cell growth on PC-coated fabrics was greater (mean +/- SE; area 42.3 +/- 9.9 mm2, n = 6; cell number 3.9 x 10(4) +/- 3.03 x 10(3) cells, n = 4; total proteins 14.9 +/- 1.2 micrograms, n = 4) than on uncoated fabrics (area 10.6 +/- 4.6 mm2, n = 6; cell number 2.9 x 10(4) +/- 4.3 x 10(3) cells, n = 4; total proteins 11.3 +/- 1.7 micrograms, n = 4; P less than 0.001, less than 0.05 and less than 0.05, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Monocular deprivation in split-chiasm kittens does not impair the development of visuo-motor behaviour.

Development of visual acuity is known to be impaired by early monocular deprivation. This may be due to lack of visual experience or to abnormal binocular competition. We report here the results of behavioural experiments on monocularly deprived kittens in which the optic chiasm had been split. In these kittens it has been found that visual acuity of the deprived eye is as good as the visual acuity of the non-deprived eye, namely 2-2.5 c/deg.

Impairment of stereoacuity in cats with oculomotor proprioceptive deafferentation.

Stereoacuity was measured with an operant conditioning technique in adult cats with unilateral deafferentation of oculomotor proprioception (section of the ophthalmic branch of the fifth cranial nerve). Binocular stereoacuity was found not to exceed monocular depth sensitivity, in contrast to what is obtained in normal cats. The results confirm previous findings and indicate a role of oculomotor proprioception in binocular depth perception of the cat.

Deafferentation of oculomotor proprioception affects depth discrimination in adult cats.

Depth discrimination was tested behaviourally in adult cats prior to and after chronic section of the ophthalmic branch of the Vth cranial nerve, that contains the majority of oculomotor proprioceptive fibers. Binocular depth discrimination was considerably impaired following either unilateral or bilateral oculomotor proprioceptive deafferentation.