Nonvisual photic responsiveness in newly hatched pigeons (Columba livia).

Overt behavioral arousal was elicited by light stimulation in pigeon hatchlings. The sensitivity is not mediated via the retina or by direct stimulation of the brain, but rather it is most likely a dermal sensitivity.

The development of the oculomotor nuclear complex in the Japanese quail embryo.

The development of the oculomotor nuclear complex was studied in the Japanese quail. In hatchlings, this complex was found to consist of four subnuclei: The accessory, the dorsolateral, the dorsomedial, and the ventromedial. An arciform subnucleus or central subnucleus was not found in this species. All subnuclei were made up of homogeneous cell populations. The oculomotor primordia can first be recognized at day 3 of incubation, and a clear subdivision within this primordia is apparent on day 5 (total incubation period = 17 days). At this time the accessory subnucleus can be discerned. By day 6, a horizontally oriented dorsal cell mass, the anlagen of the dorsolateral and dorsomedial subnuclei, is seen, as is a vertically oriented ventral cell mass, the anlagen of the ventromedial subnucleus. The boundaries between the subnuclei are not yet distinct at this age, however. By day 7 of incubation, all four subnuclei can be detected the ventromedial subnuclei, are first seen on day 5, at which time the oculomotor commissure begins to appear. Migratory traffic is extensive during days 6 and 7 of incubation, with wide bands of migrating cells typically seen, spanning the entire dorsal-ventral extent of the oculomotor complex. These cells do not appear to be associated exclusively with the ventromedial subnuclei. In many instances, migratory cells appear to be affiliated with the dorsomedial cell group. By day 10 of incubation, migration has ceased and the oculomotor complex has attained its definitive configuration. These observations are discussed with comparative reference to earlier studies of chick and duck embryos and hatchings.

Influence of nerve growth factor on chick trigeminal motor nucleus explants.

Explants of the metencephalic basal plate from stage 11 (40-hour) chick embryos containing the trigeminal (V) motor nucleus were cultured in standard control medium, in medium supplemented with nerve growth factor (NGF), in medium supplemented with NGF and specific antibodies to NGF (anti-NGF), and in medium supplemented with anti-NGF alone. The explants grown in the presence of NGF displayed an enhanced density and complexity of neuritic outgrowth, with this growth significantly surpassing that seen in the control group (p less than .001). The explants grown in NGF plus anti-NGF and those grown in anti-NGF alone did not differ from controls. The results indicate that this early cholinergic population is specifically responsive to NGF. This finding is consistent with recent studies in which NGF receptor binding has been found in this and other early brainstem and spinal cord motor neuron populations. The possible relevance of these observations to the normal sequence involved in the development of the V motor nucleus is discussed, particularly as they may relate to the relationship between the V ganglion and the developing V motor population.

Early development and migration of the trigeminal motor nucleus in the chick embryo.

The development of the trigeminal motor nucleus in the chick embryo was studied using autoradiographic, cell staining, fiber staining, and axonal transport techniques. It was found that this nucleus arises very early in neurogenesis, with the first cells produced at 48 hours of incubation (stage 12), peak cell production at 50--56 hours (stage 15), and neuroblast proliferation completed by 72 hours (stage 18). As has been described in mammalian embryos, the primordial trigeminal cells move from the ventricular layer to accumulate as part of the common medial column, and later migrate in a ventrolateral direction to form the definitive lateral motor nucleus. The first identifiable component of the trigeminal system is the semilunar ganglion, which flanks the neural tube at stage 12, and sends afferents into the metencephalon by stage 13. By stage 12-13, the medial column cells are first apparent, and at stage 14, a few of these medial column cells have moved to begin formation of a lateral nucleus. At this time, a thin motor root can be seen exiting the brainstem. During subsequent stages, migratory traffic from medial to lateral regions increases, with cells frequently moving in association with fiber processes in the marginal zone. These fibers are presumed to emanate from secondary sensory, reticular, and medial column neuroblasts. By day 5, the medial column is greatly depleted and by day 6--7, the definitive lateral motor nucleus is formed. Beginning at 5 days, the dorsal motor nucleus can be detected, with cells from the lateral nucleus appearing to stream in a dorsomedial direction for its formation. Injections of horseradish peroxidase (HRP) into the mandibular process of the first visceral arch resulted in retrograde labeling of lateral nucleus cells as early as 3.5 days of incubation. In addition, migrating cells, intermediate between medial column and lateral nucleus, were similarly labeled. These observations indicate that processes of the lateral nucleus cells and those of migrating cells are well into their peripheral field at this age, but we cannot conclude that neuromuscular affiliations have been established, due to the possibility of HRP diffusion and growth cone uptake.

Patterns of extraocular innervation by the oculomotor complex in the chick.

The horseradish peroxidase retrograde tracer technique was used to map the projection pattern of the oculomotor nuclear complex to the extraocular muscles in the chick embryo. The following projection pattern was found: The dorsolateral oculomotor subnucleus innervates the ipsilateral inferior rectus muscle, the dorsomedial subnucleus innervates the ipsilateral medial rectus muscle, a lateral division of the ventromedial subnucleus innervates the ipsilateral inferior oblique muscle, and a medial division of the ventromedial subnucleus innervates the contralateral superior rectus muscle. The so-called central nucleus also innervates the contralateral superior rectus muscle. This pattern was extremely discrete, with virtually no overlapping representations. These results provide the first evidence for a functional medial-lateral subdivision of the ventromedial subnucleus. This pattern relates to the unusual development of this subnucleus and suggests that only part of the primordium for this cell group migrates across the midline during its ontogeny, rather than all of it, as was previously believed. The subnuclear organization of the avian oculomotor complex is also considered in comparison to such functional organization in other species.

Developmental relationships between trigeminal ganglia and trigeminal motoneurons in chick embryos. I. Ganglion development is necessary for motoneuron migration.

The migration and early development of trigeminal (V) motoneurons were studied in chick embryos in which two different populations of primary trigeminal sensory neurons had been removed prior to the birthdate of the V motoneurons. Ablation of mesencephalic neural crest cells, which eliminates monosynaptic sensory input, did not affect the migration, early development, or later differentiation of the V motoneurons. However, when the anlagen of the V ganglion were removed, the V motor root did not exit from the brainstem and the V motor nucleus did not develop. Although the neurons of the V ganglion do not innervate adult V motoneurons, these populations are related developmentally. In those embryos in which the V ganglion did not develop, medial column cells, which are midline, postmitotic, premigratory V motoneurons, and a few medial, elongated cells (possibly migratory) were present until days 5-6, but these cells did not complete their lateral migration to form the lateral nucleus of V. In cases where the ganglion anlagen were not completely removed, the number of postmigratory V motoneurons was positively correlated to the size of the ganglion remnant. There also was a correlation between the axial position of the postmigratory V motoneurons and the ganglion remnants. If a caudal remnant developed, only caudal V motoneurons, whose axons reached the ganglion, migrated; if a rostral remnant developed, only rostral V motoneurons, with axons reaching this remnant, migrated. Additionally, if the central axons of the ganglion remnant entered the metencephalon in either dorsal or ventral ectopic positions, the V motor nucleus was located in a corresponding aberrant position. Thus, some characteristic of the V ganglion cells appears to guide the motor axons and somas to their final brainstem position.

Developmental relationships between trigeminal ganglia and trigeminal motoneurons in chick embryos. II. Ganglion axon ingrowth guides motoneuron migration.

In the chick embryo the trigeminal (V) sensory ganglion cells send axons into the metencephalon a few hours before the V motoneurons migrate from the midline to form a lateral nucleus adjacent to the ingrowing sensory axons. This relationship suggests that the ganglion axons may influence the initiation and direction of V motoneuron migration. In the present experiment the development of the ganglion axons was retarded by removing the neural crest anlage of the V ganglion. Subsequently, V ganglion cells which were derived from the ectodermal placode anlage sent axons into the metencephalon up to 2 days later than normal. The lateral migration of the V motoneurons was similarly delayed, commencing only after the central axons from the placodal ganglia penetrated the metencephalon. This study demonstrates that the presence of V ganglion perikarya alone is not sufficient to guide the appropriate migration of V motoneurons. This migration occurs only after the axons from the V sensory ganglion cells have penetrated the brainstem.

Developmental relationships between trigeminal ganglia and trigeminal motoneurons in chick embryos. III. Ganglion perikarya direct motor axon growth in the periphery.

The previous study in this series demonstrated that the ingrowth of the central axons of the trigeminal (V) ganglion is prerequisite to V motor axon outgrowth and somatic translocation. In the present experiment we determined whether further interactions with V ganglion cell bodies were required by V motoneurons after the V ganglion innervates the brainstem. Soon after the ganglion axons had penetrated the brainstem they were severed, and a barrier, either permeable or impermeable, was placed between the ganglion cell bodies and the metencephalon. V motor axons grew along aberrant pathways to circumvent the impermeable barriers, many rerouting to reach the V ganglion. Only those V motor nerves which contacted the V ganglion distal to the barrier reached their target musculature in the mandible. The pattern of migration of V motoneurons was normal regardless of the V motor nerve trajectory, but the cell bodies of those axons which did not reach a muscle were not fully differentiated. When permeable barriers (Millipore filters) were implanted, the nerves followed two types of trajectories. If the pore size of the filter was small (0.45 and 0.025 microns), the V motor nerves grew identically to those observed in embryos in which impermeable barriers had been implanted. If the pore size of the filter was large (8.0 and 0.08 microns), the V motor nerve grew along its normal path directly to the barrier. Small axonal bundles from these nerves frequently grew into the filter toward the distal V ganglion. These results indicate that V motor axons preferentially grow to the V ganglion perikarya after exiting from the brainstem. Contact with the V ganglion always results in V motor nerve growth to the mandible while growth of the V motor axons to aberrant target sites only occurs when the axons fail to contact the V ganglion cells distal to the barrier.

Specific responsiveness of chick trigeminal motor nucleus explants to target-conditioned media.

Explants of the neural tube from stage 11 chick embryos containing the metencephalic trigeminal (V) motor nucleus were cultured in standard control medium, in medium conditioned by appropriate target musculature (the mandibular process of the first visceral arch that gives rise to jaw musculature innervated by motor V) or in medium conditioned by inappropriate target musculature (rostral limb bud tissue). The appropriate and inappropriate muscle tissues were of the same developmental stage (stage 22) and were in similar states of differentiation. At this point in vivo, both are just beginning to be innervated. The neuritic outgrowth from the explants was quantified after 6 days in vitro. While explants from all three groups appeared healthy and exhibited some neuritic outgrowth, the density and complexity of this growth was significantly greater in the group cultured with the appropriate (jaw) muscle-conditioned medium. Growth in this group significantly surpassed that of both the control and the inappropriate muscle-conditioned medium group did not differ from the control group. These results demonstrate a specific responsiveness of the trigeminal motor nucleus population to its appropriate target tissue. Since relatively small amounts of the muscle-conditioned medium were used with each explant, it is suggested that there is a high degree of sensitivity of this population to factors present in their target at the time innervation would normally be occurring. It is hypothesized that such selective responsiveness may play a role in guiding or sustaining growth during normal neurogenesis.

Influence of prenatal ethanol exposure on cholinergic development in the rat striatum.

This study investigated the influence of ethanol exposure throughout gestation on cholinergic development within the rat striatal region. Pregnant Long-Evans rats were maintained on three diets throughout gestation: A liquid diet in which ethanol accounted for 35-39% of the total calories, a similar diet with the isocaloric substitution of sucrose for ethanol, and a lab chow control diet. At postnatal days 14 and 60 (P14 and P60), the striatal regions of the offspring were analyzed for the number of cholinergic neurons, via choline acetyltransferase (ChAT) immunostaining. The area of the striatum was also measured in these animals. At P14, P21, and P60, ChAT activity was assessed in the same region. These analyses revealed a significant increase in the number of cholinergic striatal neurons at P14 in the animals which had been exposed prenatally to ethanol. This increase was transient, however, with equal numbers of ChAT-positive cells found in all three groups by adulthood (P60). The brain weights of the ethanol-exposed animals were significantly reduced at P14 and P21, but were comparable to controls by P60. There were no significant differences in the striatal area or the overall volume of the region assessed, however, at either P14 or P60. Although there were some increases in ChAT activity across the ages viewed (most notably between P14 and P21), there were no effects of diet on ChAT activity at any age assessed. It is proposed that the increased numbers of cholinergic neurons could be a function of errors in migration, enhanced neurogenesis, diminished cell death, alterations in gene expression, or increased cell survival as a result of alterations in neurotrophic factor production or availability.

Influence of chronic prenatal ethanol on cholinergic neurons of the septohippocampal system.

This study characterized the influence of full-term gestational ethanol exposure on choline acetyltransferase (ChAT)-immunoreactive neurons that project to the hippocampus, within the medial septal (MS) nucleus and the vertical limb of the diagonal band of Broca (DBv). On gestation days 1-22, pregnant dams were fed either a vitamin fortified ethanol-containing liquid diet, pair fed a calorically equivalent sucrose-containing diet, or given rat chow ad libitum. In a previous study, we found that chronic prenatal exposure to ethanol, in this manner, resulted in a significant decline in the ontogenetic upregulation of ChAT activity in the septal area during the second postnatal week, but was followed by recovery to control levels by adulthood. On postnatal days 14 and 60 (P14 and P60) the brains were prepared for ChAT immunocytochemistry. Ethanol exposure had little influence on the number of ChAT-positive neurons in the MS nucleus of animals at either age. Ethanol exposure had no effect on neuronal size or ChAT staining intensity of MS or DBv neurons when compared to chow-fed offspring. Although age-related increases in cholinergic neuronal numbers and decreases in neuronal size were observed between juvenile and adult animals, prenatal ethanol exposure did not appear to influence these postnatal changes in the population as a whole. Overall, these findings suggest that the anatomical maturation of septal cholinergic neurons may be relatively insensitive to prenatal ethanol exposure under conditions of a vitamin-rich dietary supplementation, while biochemical development within this region may be more susceptible to early ethanol influences.

Tectal projection of displaced ganglion cells in avian retina.

Labeled displaced ganglion cells (DGC's) were observed in chick embryos (day 12 to 18) and hatchings following discrete tectal injections of horseradish peroxidase (HRP). Average DGC diameter was 10 to 15 mu, approximately twice the size of orthotopic ganglion cells. Six to 12 DGC's were found per 15 mu section. These findings conclusively demonstrate a tectal projection for the DGC.

Up-regulation of high-affinity neurotrophin receptor, trk B-like protein on western blots of rat cortex after chronic ethanol treatment.

We previously reported that the total neurotrophic activity of hippocampal extracts was significantly (25-50%) reduced after 21-28 weeks of chronic ethanol treatment (CET) [23]. To test whether the level of a neurotrophic factor (i.e., ligand itself) is compromised, we measured nerve growth factor (NGF) protein and NGF mRNA contents using ELISA and Northern analysis. We reported that CET did not appear to reduce NGF protein, NGF mRNA or total neurotrophic activity when measured on sympathetic ganglia neurons [4]. We also observed that both NT-3 mRNA and bFGF mRNA levels were unaffected, but the BDNF mRNA levels was significantly reduced in CET rat hippocampus [18]. Neuronal degeneration and reduction of total neurotrophic activity after CET appear to be induced, at least partially, by compromised transcription of BDNF gene. CET may also induce functional changes in receptors for the neurotrophic factors. To investigate possible changes in neurotrophic factor-receptors, we examined Western blots (immunoblots) of rat cortex after 28 weeks of CET. After sonication and ultra-centrifugation, the supernatant of crude lysates of the cortex from individual animals was subjected to SDS-PAGE, electrotransfered to nitrocellulose membrane, incubated with anti-trk B antibody and secondary antibody conjugated to alkaline phosphatase, and reacted with chemiluminescent substrate. The membranes were then exposed to Kodak XAR film. Compared to controls (n = 6), CET rats (n = 6) appeared to have significantly higher band intensity (P < 0.01) of trk B-like protein at about 145 kDa, which suggests an up-regulation of trk B-like proteins to compensate the compromised level of certain subset (i.e., BDNF or NT-4/5, but not NGF) of neurotrophins in cortex.

A technique for improved resolution of [3H]thymidine autoradiography in the avian embryo: a preliminary report.

A technique is described for producing pulse-like effects in [3H]thymidine autoradiography in chick embryos. This procedure involves combining thymidine with reserpine, which temporarily inhibits thymidine incorporation. The concept is to introduce thymidine, allow time for its incorporation, then introduce reserpine, so that subsequent uptake is inhibited, and a relatively discrete label will appear in cells generated at the time of thymidine administration. The best results occurred when thymidine was administered 12 h prior to reserpine, or when the two were introduced simultaneously. A dose of 0.004 mg of reserpine produced the suppression, but had no deleterious effects, in terms of embryonic survival or in long-lasting changes in cell numbers, as reflected by cell counts in the trochlear nucleus, a population which was undergoing proliferation at the time of reserpine injection. Thus, this technique appears to hold considerable promise for improving the precision of the autoradiography procedure in avian embryos.

Species specificity in the responsiveness of chick embryo neural tube explants to target-conditioned medium.

Explants from the metencephalic region of 40-h chick embryo neural tubes containing the trigeminal (V) motor nucleus were cultured in appropriate target muscle-conditioned media (MCM) derived from chick, quail and rat embryos. Enhanced neuritic outgrowth was found only in the presence of chick MCM, indicating that this early, initial responsiveness to target-released materials within this system is species-specific.

The retrograde transport of horseradish peroxidase from the developing limb of the chick embryo.

Chick embryos ranging in age from 4.0 to 18 days of incubation and 1-2-day-old hatchlings received injections of HRP solutions directly into the leg musculature. After survival periods of from 5 to 25 h motoneurons and cells in the spinal sensory ganglia were found to be stained with the HRP reaction product. It was found that the first appearance of a positive HRP reaction coincided with the time when nerve processes are first detected in the limb-bud by silver techniques (i.e. at 4.5 days of incubation). Only neurons with processes in the region of injection showed a positive reaction. The demonstration of a retrograde transport mechanism in neurons and axons which are still undergoing growth and differentiation provides a possible mechanism for the peripheral regulation of certain features of CNS neurogenesis. The application of this technique during development may also allow one to map neuroanatomical pathways during their formation.

Modulation of ethanol neurotoxicity by nerve growth factor.

Dorsal root ganglion (DRG) neurons were cultured with varying concentrations of ethanol and NGF. At low concentrations of NGF (0.1 ng/ml) moderate initial ethanol levels (250 mg/dl) significantly suppressed neurite outgrowth. Higher NGF concentrations (5 ng/ml) protected against this neurotoxicity. At this higher NGF concentration, neuronal survival was not significantly affected by exposure to 0.25-4 g/dl ethanol, although survival was significantly diminished at 5 and 6 g/dl. Neurite outgrowth was a more sensitive indicator of ethanol neurotoxicity in this population, with significant decreases in process extension seen with 1 g/dl ethanol. When cultures were supplemented with 10 ng/ml NGF, however, process elaboration was significantly greater at 1 g/dl ethanol than that measured with 5 ng/ml NGF, and in fact did not differ from NGF controls. These studies indicate that NGF can provide neuroprotective effects against ethanol toxicity under these conditions. The results are discussed in relation to other recent reports of trophic factor neuroprotection.

The effects of chronic ethanol consumption on neurotrophins and their receptors in the rat hippocampus and basal forebrain.

Damage to the basal forebrain frequently results in deficits in learning and memory. Mnenonic dysfunction also occurs following prolonged ethanol consumption in humans and in animal models of chronic ethanol intake, accompanied by specific abnormalities in synaptic transmission between the basal forebrain and hippocampus. The integrity of at least some of the reciprocal neuronal connections between these brain regions is influenced by target-derived neurotrophic factors. We used a semiquantitative reverse transcription polymerase chain reaction technique to measure the messenger RNA for neurotrophins BDNF and NGF, and for their receptors trkB, trkA, and the low affinity receptor, p75(NTR) in the hippocampus and basal forebrain of rats after 28 weeks of alcohol consumption without malnutrition. This chronic ethanol treatment (CET) resulted in a marked and selective reduction in basal forebrain trkA mRNA. Western blotting revealed a similar reduction of basal forebrain trkA protein. CET effects on basal forebrain trkA may reflect impaired NGF signaling that could compromise septohippocampal synaptic connections, cholinergic differentiation, and emergent functional abilities dependent on these properties.

Ethanol neurotoxicity in vitro: effects of GM1 ganglioside and protein synthesis inhibition.

Cultures of septal and hippocampal neurons from fetal rat and dorsal root ganglion neurons from embryonic chick were pretreated with GM1 ganglioside or cycloheximide and then supplemented with toxic concentrations of ethanol. GM1 provided significant protection against ethanol neurotoxicity in each population. The inhibition of protein synthesis by cycloheximide, however, which protects against cell death resulting from withdrawal of neurotrophic factor support, did not ameliorate ethanol-induced neuronal loss.

Cultured postnatal rat septohippocampal neurons change intracellular calcium in response to ethanol and nerve growth factor.

Ethanol exposure affects cellular mechanisms involved in the regulation of calcium (Ca2+) homeostasis. Neurotrophins, such as nerve growth factor (NGF), stabilize intracellular Ca2+([Ca2+]i) during a variety of neurotoxic insults. In this study, changes in [Ca2+]i during treatment with ethanol and NGF were measured at the cell body of neurons using the Ca2+ indicator indo-1. Cultured postnatal day-of-birth (P0) septohippocampal (SH) neurons that were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI), increased [Ca2+]i in response to ethanol. This response was dose-related. P0 SH neurons treated with NGF had lower [Ca2+]i than neurons withdrawn from NGF, implying that NGF may modulate Ca2+ homeostasis in these neurons. NGF also prevented the dose-related increase in [Ca2+]i in ethanol-treated SH neurons. The SH neurons increased [Ca2+]i when they were stimulated with 30 mM potassium chloride (KCl). Ethanol inhibited the potassium-stimulated change in [Ca2+]i but the combination of ethanol and NGF caused [Ca2+]i to increase with 100 mg% and 400 mg% ethanol and to decrease to a lower level with 200 mg% ethanol. These data were compared to data from previously published similar aged medial septal (MS) neurons (B. Webb, S.S. Suarez, M.B. Heaton, D.W. Walker, Clin. Exp. Res. 20 (1996) 1385-1394) and with embryonic gestational day 21 (E21) SH neurons (B. Webb, S.S. Suarez, M.B. Heaton, D.W. Walker, Brain Res. 729 (1996) 176-189). Differences in [Ca2+]i responses were observed in ethanol and NGF-treated postnatal SH neurons compared with P0 MS neurons and E21 SH neurons. Of these differences, most occurred during the combined treatment with ethanol and NGF compared with either treatment alone.