Development of axonal transport of RNA and its precursors in the optic pathway of the chick embryo

The axonal migration of RNA has been investigated at various developmental stages in the optic pathway of chick embryo after intraocular administration of labeled uridine. The somatofugal movement of soluble RNA precursors has also been studied. An excess of radioactivity in RNA within the optic lobe contralateral to the injected eye relative to that within the ipsilateral lobe was found 3 days after ³H-uridine injection in 13- and 18-day-old embryos. In 9-day-old embryos no asymmetry in radioactive RNA from paired optic lobes developed even 6 days after isotope injection. At all stages no asymmetry was found 6 and 24 hr after isotope administration. These results suggest a slow retinotectal flow of RNA beginning at about Day 13 of incubation. In the newly hatched chick, 3 days after monocular ³H-uridine injection, the asymmetry in label within RNA from paired optic lobes was much greater than was that of embryos. At all stages studied a considerable asymmetry of radioactivity within nucleotides and nucleosides from paired optic lobes also developed 3 days after monocular ³H-uridine injection. However, this was at least as high in 9-day-old embryos as in newly hatched chicks. Intraocular injection of colchicine prior to ³H-uridine blocked the migration of labeled RNA in the newly hatched chick, suggesting such transport to be dependent on the integrity of neuronal microtubules. It is concluded that a significant proximodistal migration of RNA occurs in the optic pathway of chick embryos prior to the onset of electrical activity.     

In ovo chronic neurosteroid treatment affects the function and allosteric interactions of GABAA receptor modulatory sites

We investigated the effects of in ovo chronic administration of the endogenous neurosteroid epipregnanolone (5β-pregnan-3β-ol-20-one) on the GABA_A receptor complex present in chick optic lobe synaptic membranes. Chronic epipregnanolone treatment failed to exert any effect on the chick optic lobe total protein content and wet weight at the different doses tested. [³H]Flunitrazepam control binding remained unaltered after neurosteroid exposure, however, the positive allosteric modulation of this ligand by 4 μM allopregnanolone was reduced in a dose-dependent manner by neurosteroid treatment. Embryo exposure to 30 μM epipregnanolone decreased allopregnanolone EC₅₀ and E_max values. Analyses of saturation binding isotherms disclosed that such administration had no effect on K_d and B_max values for [³H]flunitrazepam and [³H]GABA binding. [³H]GABA binding modulation disclosed an increase in allopregnanolone EC₅₀ value with a decrease in its E_max value. Diazepam EC₅₀ and E_max values were enhanced, while low affinity sodium pentobarbital EC₅₀ value was reduced by epipregnanolone treatment. The investigation of the GABA_A receptor function revealed that administration of this neurosteroid reduces the efficacy of GABA to induce ³⁶Cl⁻ influx into microsacs prepared from chick optic lobe. These results indicate that endogenous neurosteroid epipregnanolone chronically administered in ovo produces homologous uncoupling between steroid modulatory sites, and those corresponding to benzodiazepine and GABA receptors. Thus epipregnanolone is able to induce heterologous changes in the allosteric linkage between benzodiazepine and barbiturate modulatory sites, and the GABA receptor site. Taken jointly with results on epipregnanolone enhancing effects on [³H]flunitrazepam and [³H]GABA binding, in the context of its endogenous synthesis, our present findings support this neurosteroid as the endogenous modulator of GABA_A receptor sites and function during chick optic lobe development.     

Putative neurotransmitters of the avian visual pathway.

The ability of homogenates of the chick optic lobe to accumulate a series of possible neurotransmitters has been studied. High affinity uptake of several possible neurotransmitters was examined in optic lobes of 21-day-old embryos that had a single eye removed on the third day of incubation and in 23-day-old chicks that had an eye removed at hatch. Embryonic enucleation resulted in severe reduction of development of the ability of the contralateral optic lobe to take up tritiated GABA, dopamine, choline, serotonin and glutamate from solutions around 10(-8)M. Unilateral eye removal of new-hatched chicks caused failure of the denervated optic lobe to grow, but only the uptake capacity for glutamate was significantly recuced. This deficit was apparent as early as 4 days after enucleation. The transport of other compounds was unimpaired. The uptake of glutamate by homogenates of the optic tract was 43% of that or the optic lobe. This was a much greater fraction than the corresponding value for other postulated neurotransmitters. These data suggest that glutamate may be the primary neurotransmitter of the fibers of the optic tract originating in the retinal ganglion cells.     

Biosynthesis of progesterone derived neurosteroids by developing avian CNS : in vitro effects on the gabaa receptor complex

It has been demonstrated in different vertebrate species that the GABA_A receptor complex is modulated by certain steroids. Theses results prompted work on the synthesis of these neurosteroids in the Central Nervous System. However, there are scarcely any studies analyzing their production or their modulatory effects on this receptor during development. In this work, the biosynthesis of [[14]C]progesterone metabolites as well as the characterization of their in vitro effects on the GABA_A receptor complex in developing chick optic lobe were investigated. Studies on progesterone metabolism indicated that this steroid was converted to 5β-pregnanedione, 5β-pregan-3β-ol-20-one, and a 20-hydroxy derivative. Radioactive progesterone was completely metabolized at early embryonic stages, and a great proportion of 5β-pregnanedione was converted to 5β-pregnan-3β-ol-20-one. Thus, it seems that some of the steroidogenic activities present in chick optic lobe are age-dependent, though greater at embryonic stages. Results from in vitro modulation of [[3]H]flunitrazepam binding by 5β-pregnan-3β-ol-20-one indicated that this steroid produces a one-component-concentration dependent enhancement above control binding. 5β-pregnan-3β-ol-20-one EC₅₀ values were 0.195±0.049, 0.101±0.017, 0.147±0.009, and 0.569±0.114 μM, and E_max were 22.37±1.57, 23.67±4.02, 29.01±1.08, and 15.11±2.67% at embryonic days 11, 14, hatching, and postnatal day 21, respectively.In conclusion, the biosynthesis of 5β-pregnan-3β-ol-20-one from progesterone in developing chick optic lobe, together with its ability to modulate the GABA_A receptor present in such tissues, suggests a physiological role of this neurosteroid in developing avian Central Nervous System.     

The involvement of nitric oxide in the secretion of β-endorphin from the pituitary intermediate lobe of the rat

Nitric oxide (NO) generated by the enzyme nitric oxide synthase (NOS) has been implicated in the regulation of a variety of endocrine functions. A number of biochemical and anatomical studies have demonstrated the presence of neuronal NOS (nNOS) in the neuroendocrine axis and have shown significant effects of NO on the release of hypothalamic and pituitary hormones. Using a C-terminal directed peptide antibody that is specific for nNOS we have found a predominance of nNOS in the neural lobe of the pituitary and in a single layer of epithelial cells, possibly a remnant of Rathke's pouch that form a border between the intermediate lobe and the anterior lobe. Furthermore, we have examined the effect of sodium nitroprusside (SNP), a donor of NO on the secretion of β-endorphin (β-EP) from the isolated neuro-intermediate lobe (NIL) of the rat and cultured rat melanotrophs. It was shown that in explant cultures of intact neuro-intermediate lobes, SNP (100 μM) was able to cause an inhibition of β-EP secretion. In the presence of sulpiride (10 μM), a dopamine D₂-receptor antagonist, there was a partial reversal of the SNP effect. On the other hand SNP did not affect β-EP secretion in primary cultures of melanotrophs that no longer possessed any innervation. Taken together these data suggest that NO has an indirect inhibitory effect on the secretion of β-EP by the intermediate lobe via the release of dopamine.     

Recovery of tectal nicotinic-cholinergic receptor sites during optic nerve regeneration in goldfish

The concentration of cholinergic nicotinic-like sites as measured by alphabungarotoxin (αBuTX) binding, decreased in the goldfish (Carassius auratus) optic tectum after optic nerve disconnection. Initially, the rate of loss of sites is greater than the rate of tissue or protein degradation in experiments where disconnection was achieved either by unilateral optic nerve crush or by enucleation of one eye. When the crushed optic nerve is allowed to regenerate and form behaviorally potent connections, the number and concentration of these sites appears restored. Pharmacological studies indicate that the αBuTX binding site in the goldfish optic tectum has a drug binding profile similar to that seen at central or peripheral αBuTX sites in other species.     

On the formation of eye dominance stripes and patches in the doubly-innervated optic tectum of the chick

By surgically dividing the region of the presumptive optic chiasm in chick embryos on the third day of incubation (around stage 15), we have been able to induce substantial numbers of optic nerve fibers to grow aberrantly into the ipsilateral optic tract. As a result, many of the visual centers that are normally innervated only by fibers from the contralateral retina received fibers from both eyes. The proportion of fibers going to each tectal lobe varied from case to case, but in about one-third of the animals the tectal lobes received approximately equal numbers of fibers from each eye. In animals that survived until embryonic days 17-19 (which is beyond the period of retinal ganglion cell death) labeling of the two eyes with WGA-HRP and [3H]proline respectively, revealed a pattern of sharply defined eye dominance stripes or patches in the stratum griseum et fibrosum superficiale (SGFS) of the optic tectum, and in the ventral lateral geniculate nucleus. Less clearly segregated eye dominance zones were seen in the ectomammillary nucleus and the nucleus externus. The size and distribution of the stripes varied depending on the number of fibers projecting from each eye to a given tectal lobe; the minimum size was about 75 micron, while the maximum was large enough to occupy almost the entire tectal lobe. In animals in which the tectal input from the two eyes was roughly equal, the stripes varied in width between 75 micron and about one-third of the surface of the tectal lobe. The orientation of the stripes was consistently orthogonal to the direction of fiber ingrowth from the optic tract. From the earliest stages of optic fiber ingrowth, the fibers from the two eyes are completely intermixed in the stratum opticum (SO). However, on embryonic day 12, shortly after they have begun to penetrate into the SGFS, they are already segregated into stripes, although the stripe borders are very fuzzy. This suggests that the fibers from the two eyes may overlap at this stage. The phase of stripe formation coincides with that of naturally occurring retinal ganglion cell death, and we suggest that the two processes are interlinked.     

Gaba-stimulated chloride uptake during avian CNS development: modulation by neurosteroids

In the present report we studied the GABA-stimulated ³⁶Cl⁻ uptake during chick optic lobe development in order to establish the ontogenetic profile of the functional GABA_A receptor complex. A concentration-dependent stimulation of ³⁶Cl⁻ influx by GABA was demonstrated, starting at developmental stages as early as 10 days of incubation. The maximal GABA-induced ³⁶Cl⁻ uptake changed significantly during ontogeny with highest values near hatching. However, GABA potency to stimulate ion influx remained unchanged. We also examined the effect of two neurosteroids, allopregnanolone and epipregnanolone, on GABA-stimulated ³⁶Cl⁻ influx at three developmental stages (embryonic day 14, post-hatching day 1 and adult stage). Both steroids enhanced ion uptake in a concentration-dependent manner, exerting greater stimulatory effects at early developmental stages. Allopregnanolone displayed EC₅₀ values lower than epipregnanolone at all three time points and was also more potent at post-hatching stages. Analysis of the GABA concentration-effect curve disclosed that both steroid decreased EC₅₀ values for GABA stimulation while E_max levels were unaffected. In conclusion, our results showed an early appearance of the GABA-associated chloride channel together with the ability of neurosteroids to modulate GABA-gating of such channel.     

Studies on the mechanism of sprouting of noradrenergic terminals in rat and mouse cerebellum after neonatal 6-hydroxydopa

KOSTRZEWA, R. M., J. W. KLARA, J. ROBERTSON AND L. C. WALKER. Studies on the mechanism of sprouting ofnoradrenergic terminals in rat and mouse cerebellum after neonatal 6-hydroxydopa. BRAIN RES. BULL. 3(5) 525–531, 1978.—The effect of various pharmacologic agents on the noradrenergic innervation of rat cerebellum was observed. It was found that the neurotoxin 6-hydroxydopa (6-OHDOPA), when given to rats at birth, caused a 46% reduction at 5 weeks of age in tyrosine hydroxylase activity in the locus coeruleus, the nucleus of origin for noradrenergic fibers innervating the cerebellum. At the same time, however, both tyrosine hydroxylase activity and NE content were elevated by 50% in the cerebellum. By treating gravid mice with the 6-OHDOPA, which crosses the placental barrier to affect the brains of developing pups, a dissociation has been shown between the elevated cerebellar NE levels and reduced telencephalic NE content. None of the other assorted pharmacological agents—namely amphetamine, metaraminol, apomorphine, α-methyl-ρ-tyrosine, L-dihydroxyphenylalanine and tyramine—when given at birth, caused a permanent elevation in cerebellar NE content. This series of studies suggests that a reduced number of noradrenergic perikarya are providing a greater innervation of the cerebellum than in control rats. Also, alteration of the telencephalic noradrenergic fibers, which are also derived from the locus coeruleus, does not appear to be a necessary event for the initiation of sprouting of noradrenergic fibers in the cerebellum. Because none of the acute-acting pharmacological agents caused a permanent elevation of NE in the cerebellum, it appears that damage, and not mere stimulation or blockade, is a necessary event for initiation of sprouting.     

Connexin α1 and Cell Proliferation in the Developing Chick Retina

During the formation of the eye, high levels of connexin α1 (connexin 43) are expressed within the tissues of the cornea, lens, and neural retina. In order to determine whether connexin α1 plays a role in the regulation of cell proliferation we have used a novel antisense technique to reduce its expression early in development (embryonic days 2–4). Application of Pluronic gel, containing antisense oligodeoxynucleotides (ODNs) to connexin α1, to one eye of early chick embryos results in a rapid and significant reduction of α1 protein which lasts for 24–48 h. Embryos grown for 48 h, after ODN application to one eye, showed a marked reduction in the diameter of the treated, compared to that of the contralateral untreated, eye. Sections cut from the treated eyes showed that the retina was also reduced in size. TUNEL labeling and staining with propidium iodide showed that apoptosis within the retinae of both treated and untreated eyes was rare and thus that the reduction in the area of the retina brought about by antisense ODNs directed at connexin α1 was unlikely to be the result of increased cell death. However, the number of mitotic figures in the ventricular zone of the antisense-treated retinae revealed by propidium iodide staining was significantly reduced (P < 0.0001) to 53 ± 3.5% (n = 5) of that in the contralateral untreated control eyes. Embryos in which one eye was sham operated, treated with pluronic gel, or treated with sense ODN showed no significant changes in eye size or in the number of mitotic figures within the neural retina. These results point to a role for connexin α1-mediated gap-junctional communication in controlling the early wave of neurogenesis in the chick retina.     

Development of gaba binding sites in chick embryo optic lobe: Effect of triton X-100

The temporal course of the development of GABA receptor sites in chick optic lobe was studied as a parameter of neuronal differentiation in the central nervous system. At 10 days of incubation, specific [3H]GABA binding was of 0.08 pmol/optic lobe and increased 7-8 fold between 12 and 16 days of incubation, reaching at 16 days a value of 0.60 pmol/optic lobe. This coincides with the period of arrival of the retinal fibers to the optic lobe. After this stage, the number of GABA binding sites decreased to a value of 0.35 pmol/optic lobe at hatching. After hatching a new increase appeared which reached at 5 days post-hatching a value of 0.87 pmol/optic lobe. Scatchard analysis of the saturation binding data obtained at 16 days of incubation and at hatching revealed the presence of two binding sites: one with high affinity and the other with low affinity, while at 12 days of incubation, the earliest stage examined, only the low-affinity binding site appeared. The high-affinity binding site for [3H]GABA was inhibited by muscimol, GABA, and bicuculline (IC50: 0.006, 0.002 and 10 microM, respectively). These values correspond to the potencies shown by those compounds in the binding to the synaptic GABA receptor. Treatment of the synaptic membranes with Triton X-100 showed a marked increase in the amount of specific [3H]GABA binding after 16 days of incubation reaching a 3-fold increase at hatching. These results suggest that endogenous inhibitors of the higher affinity binding site, probably appear during this period.     

Histamine-immunoreactive neurons in the brain of the cockroach Leucophaea maderae

Histamine is the neurotransmitter of insect photoreceptor cells but has also been found in a small number of interneurons in the insect brain. In order to investigate whether the accessory medulla (AMe), the putative circadian pacemaker of the cockroach Leucophaea maderae receives direct visual input from histaminergic photoreceptors, we analyzed the distribution of histamine-like immunoreactivity in the optic lobe and midbrain of the cockroach. Intense immunostaining was detected in photoreceptor cells of the compound eye, which terminated in the first optic neuropil, the lamina, and in a distal layer of the medulla, the second optic neuropil. Histamine immunostaining in parts of the AMe, however, originated from a centrifugal neuron of the midbrain. Within the midbrain 21–23 bilaterally symmetric pairs of cell bodies were stained. Most areas of the brain were innervated by one or more of these neurons, but the protocerebral bridge and the mushroom bodies were devoid of histamine immunoreactivity. The branching patterns of most histamine-immunoreactive neurons could be reconstructed individually. While the majority of identified neurons arborized in both brain hemispheres, five cells were local neurons of the antennal lobe. A comparison with other insect species shows striking similarities in the position of certain histamine-immunoreactive neurons, but considerable variations in the presence and branching patterns of others. The data suggest a role for histamine in a non-photic input to the circadian system of the cockroach.     

T Lymphocyte infiltration in the brain following anterior chamber inoculation of HSV-1

Following inoculation of the KOS strain of herpes simplex virus type 1 (HSV-1) into one anterior chamber of euthymic BALB/c mice, virus spreads from the injected eye to the central nervous system and from the central nervous system to the optic nerve and retina of only the uninoculated eye. In contrast, in athymic BALB/c mice or mice depleted of both CD4⁺ and CD8⁺ T cells, virus spreads to the optic nerve and retina of both the injected eye and the uninjected eye. To determine the location in the central nervous system where spread of virus to the optic nerve and retina of the injected eye is prevented, euthymic BALB/c mice were injected with a mixture of KOS and RH116, a mutant of KOS that contains the Escherichia coli β-galactosidase (β-gal) gene. Several animals were sacrified each day; serial frozen sections of the brain were prepared and sequential sections were stained for β-gal or for T cells. At all sites except the suprachiasmatic nuclei, virus and T cells arrived at approximately the same time. However, at day 5 post inoculation (PI), T cells were present in both the ipsilateral and the contralateral suprachiasmatic nuclei, but only the ipsilateral suprachiasmatic nucleus was virus-positive. Since virus spreads from the ipsilateral suprachiasmatic nucleus to the contralateral optic nerve, these results suggest that T cells infiltrating the area of the contralateral suprachiasmatic nucleus prior to the arrival of virus at this site prevent virus spread into the optic nerve of the inoculated eye.     

Axonal transport of [S]Methionine labeled proteins in Xenopus optic nerve: Phases of transport and the effects of nerve crush on protein patterns

Axonal transport of proteins in the Xenopus optic nerve was examined by labeling proteins in the eye with [³⁵S]methionine injected intraocularly and then analyzing the labeled proteins in the eye, nerve, and tectum on linear gradient SDS polyacrylamide gels at different times after the injection. Because the optic nerve in Xenopus is short, in order to distinguish transported proteins from locally synthesized proteins, the optic nerve on one side of the animal was crushed at the orbit (to stop axonal transport) 5–30 min prior to injection and the crushed and normal nerve segments were compared. Proteins in the intact nerve which were absent in the crushed nerve were identified as axonally transported proteins. By such criteria several waves corresponding to transported material moving at 6mm/day, 1.6–2.8 mm/day, and approximately 0.2 mm/day were detected in the nerve. The most rapid phases of transport could be further resolved in the optic tectum into 3 additional components at 60–96 mm/day, 30–48 mm/day, and 6–11 mm/day.Analysis of labeled proteins in the crushed nerves distal to the crush, near the injury site, revealed several locally synthesized proteins (mol. wt. 54,000, 48,000, 43,000 daltons) which were not present in normal, uninjured nerves. Such proteins are probably synthesized by glia in response to injury.     

A critical period in the development of tectal neurons in the chick, as revealed by early enucleation

To further study the existence of a critical trophic period in the development of the chick optic tectum¹⁷, during which the presence of retinal synapses is essential to the continued growth of tectal neurons, we have unilaterally enucleated embryos between stages 14–20 and allowed survival until stages 35–43. If the critical trophic period is between stages 40–44, as previously reported¹⁷, then we reasoned that early removal of the eye might not have any effect on tectal development until the critical period. We assessed tectal neuron survival by staining for degeneration in the efferent projections of tectal neurons. In early enucleates, degeneration was present from stages 37–43, and the severity of the degeneration was much reduced in comparison to animals enucleated during the critical period.These findings substantiate the proposition that there is a critical period late in chick tectal development. However, because the degeneration in tectal projections is less intense than in animals enucleated during the critical period, we suggest that the early enucleation has permitted axons from the remaining eye to be routed to the deafferented tectum, where they may help to sustain a portion of the tectal neurons through the critical period. Moreover, the somewhat earlier appearance of degeneration in tectal efferent pathways of early enucleates suggests that a subtle trophic relationship between retina and tectum may exist prior to stage 40, even though this relationship is not revealed when enucleations are performed later, as between stages 35–40 (ref. 17).     

The specification of the retino-tectal projection in the chick

The specificity of the connections between the retina and the optic tectum has been studied in the chick by ablating between 15% and 75% of the optic cup (usually during the third day of incubation) and subsequently determining the distribution, within the tectum, of the synapses formed by the axons of the surviving ganglion cells. This was done towards the end of the incubation period, or shortly after hatching, either autoradiographically following the injection of a tritiated amino acid into the eye, or using a variant of the Nauta-Gygax method after sectioning the optic nerve. In every case in which the initial retinal lesion was placed after Stage 12/13 (i.e., late on the second day of incubation) the surviving ganglion cells could be shown to have formed synapses in only a limited region of the contralateral optic tectum; and as far as could be determined from an examination of the cell loss in the isthmo-optic nucleus (in which the centrifugal fibers to the retina have their origin) the remaining portion of the neural retina consistently projected only to the homotopic region of the tectum (i.e., the region to which it would normally have been expected to project). In several cases it was found that the axons had passed over a heterotopic region of the tectum in order to reach a more distant region in which they had formed synapses. After a lesion of the optic vesicle before Stage 11 the surviving ganglion cells appeared to innervate all parts of the tectum; since the earliest retinal ganglion cells are formed at Stage 11/12 it would seem that in the chick, as in Xenopus, there is a clear temporal co-incidence between the withdrawal of the first ganglion cells from the cell-cycle and the establishment of the topographic specification of the retino-tectal projection. Preliminary studies of the projection of the retina upon the optic tectum, and of the tectum upon the isthmo-optic nucleus, in four to five week old chicks, indicates that the organization of these connections (and hence, presumably, that of the centrifugal fibers from the isthmo-optic nucleus to the retina) is essentially the same as that in the pigeon. However, there is a significant difference in the overall morphology of the isthmo-optic nucleus in the two species: as can best be seen in parasagittal sections, the isthmo-optic nucleus of the chick lacks a well-developed “anterior limb,” so that the representation of the horizontal meridian lies close to the junction of the “body” of the nucleus and its “posterior limb”.     

Development of the avian accessory optic system: effects of embryonic optic lesions

Representative cross-sections of the nuclei ectomammillaris (EM) from both normal and optically lesioned chick embryos (45 h of incubation, stage 12), were analyzed and compared on days 6, 8, 10, 12, 14 and 16 of incubation. An identifiable EM is clearly present at 8 days, in both normal and lesioned embryos, and increases in cell number and area up to embryonic day 12. However, embryos with partial or complete unilateral optic ablations demonstrate an apparent acceleration in cell death rate when compared with normals, from days 12-16, when a relatively mature and stable form of EM is apparently reached. Thus, early optic lesions do not affect the morphology of EM until day 12. These data also indicate that embryonic ipsilateral pathways to EM may persist and even expand when one eye primordium is removed or partially lesioned.     

Biochemical and morphological studies of synaptogenesis in the avian sympathetic cell column

The development of the preganglionic sympathetic cell column (nucleus of Terni) was studied in the spinal cord of the chick, utilizing morphological and biochemical techniques. The first synapses observed in relation to the neurons of the nucleus of Terni were at 10 days in ovo and were of the axodendritic type. The first axosomatic synapses did not appear until the 12th day. Axonal varicosities of the noradrenergic type, as visualized by formaldehyde-induced fluorescence, first appeared in the nucleus of Terni at 12 days in ovo. Biochemical assays of serotonin (5-HT) and norepinephrine (NE) demonstrated large amounts of these amines at 7–9 days in ovo. Their concentration gradually fell to a nadir at 12 days and began rising at 15 days, reaching a peak at the time of hatching. Kinetic studies of the uptake of exogenous amines revealed specific uptake of 5-HT beginning at 8 days in ovo with aK_m of 2.5 × 10⁻⁷ M and of NE beginning at 10 days with aK_m of 1.4 × 10⁻⁷ M. Autoradiographic studies utilizing [³H]5-HT showed label to be confined to the lateral funiculus at 8 days, while at 10 days and thereafter labeled varicosities were seen in apposition to the cells of the nucleus of Terni as well as those of the ventral horn. [³H]NE labeled varicosities were not found in the lateral funiculus until 10 days in ovo and in the nucleus of Terni and ventral horn until 12 days.These findings of a relatively late innervation of the preganglionic neurons of the nucleus of Terni are compared with the observations that these neurons themselves begin to innervate the peripheral sympathetic ganglia much earlier, 7–8 days, and, at this time, possess significant amounts of choline acetyltransferase. It would thus appear that innervation of the peripheral ganglia is not preceded by innervation of the preganglionic neurons, although the maintenance of peripheral connections may be dependent upon such subsequent innervation.     

The effects of tgf- ; IGF, insulin-like growth factor ; DTT, dithiothreitol. EDTA, ethylenediaminetetraacetic acid ; PMSF, phenylmethylsulfonyl fluoride ; TBS, tris buffer saline. SDS-PAGE, sodium dodecyl sulfate-polyacrilamide gel electrophoresis.1 on chick embryo retina development in vitro

This paper studies the effect exerted by TGF-β₁ on the development of chick embryo retina cultured in vitro. The addition of TGF-β₁ to retinal explants inhibited DNA synthesis, measured as ³H-thymidine incorporation into trichloroacetic acid-insoluble fraction, while it increased both wet weight and protein content, in particular that of extracellular matrix proteins. Lastly, in explants treated with TGF-β₁ an increment in the level of fibronectin was demonstrated by means of Western blotting analysis.