Effects of intraocular tetrodotoxin on the postnatal development of the dorsal lateral geniculate nucleus of the rat: a Golgi analysis.

The postnatal development of the rat dorsal lateral geniculate nucleus (dLGn) during tetrodotoxin (TTX)-induced monocular impulse blockade was investigated by quantitative Golgi techniques. Beyond 14 days postnatal (dpn), the effectiveness of TTX was monitored by loss of the pupillary light reflex. By 21 dpn, Golgi analysis indicated that TTX had no effect on the pattern of dendritic branching of class A or class B neurons, although the number of dendritic spinous protrusions was reduced. No evidence of any TTX-induced loss of optic axons or neuronal degeneration in the dLGn was found, despite a 16% decrease in the size of the nucleus, suggesting a reduction in the growth of neuropil. These data indicate that optic impulses are important in mediating the proper growth of postsynaptic specializations in the dLGn during ontogenesis, but that the postnatal development of the dendritic arbor of neurons in the dLGn appears to be independent of retinal impulse activity.

Protein modification by amino acid addition is increased in crushed sciatic but not optic nerves.

Rat optic and sciatic nerves were crushed, and 10 minutes to 3 days later nerve segments between the crushed site and the cell body were removed and assayed for posttranslational protein modification by amino acid addition. Protein modification was comparable in intact optic and sciatic nerves, but in sciatic nerves increased to 1.6 times control levels 10 minutes after crushing and reached a maximum of ten times control levels by 2 hours. In optic nerves activity was decreased throughout the time course studied. The results indicate that, in a nerve which is capable of regeneration (sciatic), protein modification by the addition of amino acids increases immediately after injury, but a nerve incapable of regeneration (optic) is incapable of activating the modification reaction. These findings may be important in understanding the reasons for the lack of a regenerative response after injury to central mammalian nerves.

The effect of intraocular injection of tetrodotoxin on fast axonal transport of [3H]proline- and [3H]fucose-labeled materials in the developing rat optic nerve.

The fast axonal transport of [3H]proline-labeled proteins and [3H]fucose-labeled glycoproteins delivered to the dorsal lateral geniculate nucleus in the developing rat optic nerve was investigated during tetrodotoxin-induced monocular impulse blockade. Repeated intraocular injections of various dosages of tetrodotoxin or citrate buffer vehicle were made every two days in rats aged 5-21 days postnatal, and the accumulation of rapidly transported radioactivity in the lateral geniculate nucleus measured between three and twelve hours post-injection at each age. The effectiveness of prolonged tetrodotoxin treatment was monitored by loss of the pupillary light reflex and the level of cytochrome oxidase activity in the contralateral superior colliculus and dorsal lateral geniculate nucleus. Numbers of optic axons proximal to the chiasm and the frequency of retinal ganglion cells per unit distance from the optic disc were examined for signs of tetrodotoxin-induced degeneration of the retinofugal pathway. Tetrodotoxin-treatment reduced the amount of fucosyl glycoproteins, but not proline-labeled proteins, axonally transported to the lateral geniculate nucleus during the first three weeks of postnatal development. Other studies indicated that tetrodotoxin significantly reduced the incorporation of [3H]fucose into retinal proteins indicating that the reduction in transport was probably due to a decrease in precursor incorporation into retinal ganglion cells. Electron microscopy of ganglion cells at 21 days revealed dilated and vacuolated Golgi cisternae associated with tetrodotoxin treatment, suggesting that tetrodotoxin may alter fucose metabolism by secondarily disrupting Golgi organization. Other protein synthetic machinery in these cells, including ribosomes and rough endoplasmic reticulum, appeared normal throughout tetrodotoxin treatment. These data indicate that Na+-dependent optic impulse activity may be indirectly related to the axonal transport of glycoproteins during early postnatal development by mediating the incorporation of precursor into glycoproteins at the Golgi apparatus and their subsequent entrance into the fast transport system.

The postnatal development of the rat primary visual cortex during optic nerve impulse blockade by intraocular tetrodotoxin: a quantitative electron microscopic analysis.

The effect of tetrodotoxin (TTX)-induced monocular impulse blockade on various parameters of synaptogenesis during the first 3 postnatal weeks of the developing rat visual cortex was investigated by quantitative electron microscopy. During the injection period, beyond 14 days postnatal (dpn), the effectiveness of TTX in blocking optic nerve impulses was monitored by loss of the pupillary light reflex. Between 5 and 21 dpn, TTX treatment reduced the number of type I axodendritic synapses by approximately 23%, when compared to sham-injected controls. These reductions were found in layers III, IV, and the superficial region of layer V. Layer IV exhibited the greatest decrease (24%) while layers III and V showed reductions of 20% and 18%, respectively. At 21 dpn, the number of type II axodendritic synapses decreased by 19% in the same layers, but no reductions were found at earlier ages. TTX also reduced the mean number of synaptic vesicles within type I and type II terminals by 27% and 15%, respectively. At 9 dpn, reductions were first found in layers IV and V, but by 21 dpn significant decreases were found in layers II/III, IV and V. TTX had no effect on the length of the postsynaptic density of both synaptic types or on cortical thickness at any age. These data indicate that optic impluses are important mediators of synaptogenesis in the developing visual cortex, the loss of which induces localized and specific synaptic alterations, possibly due to a change in cortical circuitry.

Effects of intraocular tetrodotoxin on dendritic spines in the developing rat visual cortex: a Golgi analysis.

The effect of tetrodotoxin (TTX)-induced monocular impulse blockade on the growth of dendritic spines in the developing rat primary visual cortex was analysed by quantitative Golgi techniques. Between 5 and 21 days postnatal (dpn), rats were injected with TTX every 2 days into the right eye to chronically eliminate optic impulses. Effectiveness of TTX was monitored by loss of the pupillary light reflex. At 21 dpn, the number of spines located on the portion of the apical dendrite within layers III, IV and the superficial region of layer V was reduced by approximately 26%. These decreases were found on the apical dendrites of both large and medium sized pyramidal cells. TTX also reduced the number of spines on the proximal portion of oblique dendrites in layer IV by 16%, yet did not change the number of spines on basilar dendrites. No evidence of transneuronal degeneration was seen following long-term TTX treatment. These data indicate that dendritic spine development in the visual cortex is sensitive to the loss of optic impulses and that the decrease in spine population is principally due to a reduction in spine growth.

Intraocular kainic acid injection suppresses fast axonal transport in the developing rat optic nerve.

A single intraocular injection of 1 or 3 nmol kainic acid (KA) into the right eye of rats aged 5 days postnatal (5 dpn) significantly reduced the incorporation of 3H-proline into retinal proteins and suppressed the amount of 3H-proline-labeled materials fast axonally transported in the optic nerve for at least 2 weeks thereafter. Intraocular KA injection within this dose range had no adverse effect on the optic axon population compared to normal nerves determined at 21 dpn; however, doses above 3 nmol (i.e., 6 nmol) caused significant axonal degeneration. Although partially recovered by 21 dpn this effect of KA on protein synthesis and axonal transport suggests that, as in the adult, retinal ganglion cells are also KA-sensitive during postnatal development.

Inhibition of axoplasmic transport in the developing visual system of the rat: IV. Quantitative Golgi, electron microscopic, and histochemical analyses of the maturation of the visual cortex.

Intraocularly injected colchicine suppresses axonal transport within the developing rat's optic nerve throughout the critical period of visual system development. This results in a stunting of retinofugal terminals and relay neurons in the lateral geniculate nucleus. The present study focuses upon the effects of this unique form of developmental deprivation on the maturation of the visual cortex. Colchicine, in concentrations of from 10(-5) to 10(-2) M, was injected into the eyes of albino rats at birth or at 5, 10, or 15 days of age. Litters were killed at 5 to 50 days after this single injection, and the brains were processed for Nissl, rapid Golgi, histochemical, or electron microscopic analysis. The following results were obtained: Planimetry of coronal sections of the striate cortex revealed a reduction in the thickness of the cortex and in the ratio of neuropil area to neuronal soma area contralateral to the injected eye which was confined principally to layer IV, lower layer III, and upper layer V. This effect was inversely related to postnatal age at injection and directly proportional to colchicine concentration. A rapid Golgi analysis of 51 pairs of layer V pyramidal neurons in control and experimental cortex demonstrated a reduction in the number and size of spines along the portion of the apical dendrite passing through lower layer III and IV following colchicine administration at birth or 5 or 10 days of age but no significant change in the branching pattern of the entire dendritic arbor. Electron microscopy revealed a reduction in the number of small, asymmetric synaptic complexes with the result that the average size of remaining profiles was increased in layers III and IV. Histochemical analysis of cortical succinic dehydrogenase and cytochrome oxidase revealed a distinct band of intense enzyme activity in lower layers III and IV in normal cortex at 20-30 days of age. This band was significantly reduced in intensity after neonatal injection of colchicine as shown by densitometric measurements and comparison of experimental and control cortex. It is concluded that the geniculocortical projection, while not affected directly by colchicine administration, is altered by the secondary effects of axonal transport suppression, leading to an alteration in the establishment of cortical synaptic patterns and arborizations of their postsynaptic neurons whose dendrites are located in those layers recipient to this projection.

An ultrastructural analysis of the development of foetal rat retina transplanted to the occipital cortex, a site lacking appropriate target neurons for optic fibres.

Foetal retina was removed from donor rats at 15 days of gestation and transplanted to the occipital cortex of neonatal host rats. The purpose of this procedure was to examine the development of retinal neurons and photoreceptors, and document synaptic patterns during maturation of the transplanted retina in an environment lacking a normal target for optic axons. Host animals were sacrificed at 5, 10, 15, 20 and 30 days and samples of cortex containing the transplant were subjected to a light and electron microscopic analysis. During early stages of development, (5 days) the retina assumes a radial orientation with the scleral (outer) surface located centrally and the vitreal (inner) surface occupying the periphery. Numerous mitotic figures are found at the centre of the transplant and columns of primitive neuroblasts appear to radiate out from this zone. By 10 to 15 days after transplantation the retinal tissue contains numerous small rosettes each of which displays a histotypic organization with recognizable layers of sensory cells and their centrally-projecting processes, an outer limiting membrane, made up of a network of zonulae adherentes, and a rudimentary outer and inner plexiform layer which delineate the cells of the inner nuclear layer. Ultrastructural analysis of such rosettes confirmed the presence of typical bipolar, amacrine, horizontal and ganglion cells, but revealed that while the plexiform layers were occupied by numerous processes from these neurons, few if any, of these exhibited synaptic vesicles. By 20 to 30 days following transplantation sensory cells have completely differentiated, giving rise to prominent inner and outer segments which display typical cilia, centrioles and basal bodies, together with numerous stacked lamellae of photoreceptors which were contorted, presumably due to growth in an abnormal site. It should be further emphasized that these structures developed in the absence of pigment cells. Synaptic development ensues during this period to form characteristic dyads within the outer and inner plexiform layers. Additionally, clusters of amacrine to amacrine contacts occurred in the inner plexiform layer and were found to be increased relative to other types of junctions. In general, synaptogenesis takes place in the outer and inner plexiform layers and all categories of retinal synapses are established, but the process was found to be significantly delayed in comparison to normal retina at the same stage of development. Quantitative analysis revealed a reduced number of presumptive ganglion cells in proportion to the other categories of neurons. Optic fibres remained small and failed to myelinate. It is suggested that lack of an appropriate target for optic axons induced this alteration and may be indirectly related to the delay in the onset of synaptic development.