Effect of delay between nuclear medicine scanning and sentinel node biopsy on outcome in patients with cutaneous melanoma.

BACKGROUND: Sentinel lymph node biopsy (SLNB) is an important staging tool for the management of melanoma. A multicentre study was done to validate previous findings that the timing of lymphoscintigraphy influences the accuracy of SLNB and patient outcomes, particularly survival. METHODS: Data were reviewed on patients undergoing SLNB for melanoma at three centres in the UK and Sweden, examining the effect of timing of SLNB after nuclear medicine scanning. Kaplan-Meier survival analysis was used to assess overall (OS), disease-specific (DSS) and progression-free (PFS) survival, stratified by timing of lymphoscintigraphy. Independent risk factors for survival were identified by Cox multivariable regression analysis. RESULTS: A total of 2270 patients were identified. Median follow-up was 56 months. Univariable analysis showed a 4·2 per cent absolute and 35·5 per cent relative benefit in DSS (hazard ratio 1·36, 95 per cent c.i. 1·05 to 1·74; P = 0·018) for 863 patients whose SLNB was performed up to 12 h after lymphoscintigraphy compared with 1407 patients who had surgery after more than 12 h. There were similar OS and PFS benefits (P = 0·036 and P = 0·022 respectively). Multivariable analysis identified timing of lymphoscintigraphy as an independent predictor of OS (P = 0·017) and DSS (P = 0·030). There was an excess of nodal recurrences as first site of recurrence in the group with delayed surgery (4·5 versus 2·5 per cent; P = 0·008). CONCLUSION: Delaying SLNB beyond 12 h after lymphoscintigraphy with 99 Tc-labelled nanocolloid has a significant negative survival impact in patients with melanoma.

ANTECEDENTES: La biopsia de ganglio centinela (sentinel lymph node biopsy, SLNB) es una técnica importante para la estadificación y tratamiento del melanoma. Se realizó un estudio multicéntrico para validar hallazgos previos según los cuales el momento de la linfogammagrafía (lymphoscintigraphy, LS) influye en la precisión de la SLNB y en los resultados de los pacientes, especialmente en la supervivencia. MÉTODOS: Se revisaron los datos de los pacientes a los que se realizó una SLNB por melanoma en 3 centros en el Reino Unido y Suecia, con especial atención al efecto del período entre la inyección el material radioactivo y la SLNB. Se realizó un análisis de supervivencia mediante el método de Kaplan-Meier para la supervivencia específica de la enfermedad (disease-specific survival, DSS), supervivencia global (overall survival, OS) y supervivencia libre de progresión (progression-free survival, PFS), todas ellas estratificadas por el momento de la LS. Los factores de riesgo independientes para la supervivencia se determinaron mediante un análisis de regresión multivariable de Cox. RESULTADOS: Se incluyeron 2.270 pacientes. La mediana de seguimiento fue de 49 meses. El análisis univariado mostró un beneficio absoluto del 4,2% y relativo del 35,5% (cociente de riesgos instantáneos, hazard ratio, HR: 1,36 (i.c. del 95% 1,05-1,74, P = 0.02)) en la DDS para los pacientes a los que la SLNB se realizó < 12 horas después de la LS (n = 863) en comparación con aquellos realizados > 12 horas (n = 1407). Se detectaron beneficios similares para la OS y la PFS (P = 0,04 y P = 0,02, respectivamente). El análisis multivariable identificó el tiempo entre la LS y la SLNB como un factor independiente de OS (P = 0,017) y DSS (P = 0,03). Hubo un aumento en las recidivas ganglionares como primer sitio de recidiva en el grupo de > 12 horas (2,5% versus 4,5%; P = 0,008). CONCLUSIÓN: Estos datos validan nuestra investigación previa y tienen implicaciones significativas para las unidades de melanoma, en el sentido de que retrasar la SLNB más allá de las 12 horas después de realizar la LS con nanocoloides marcados con Tc99 tiene un impacto negativo significativo en la supervivencia de los pacientes y debe evitarse. Se presenta la hipótesis de que la causa subyacente es la migración temporal del trazador que determina una SLNB incorrecta. .

A Rare Fourth Branch of the Marginal Mandibular Nerve.

This report describes a good example of the rare fourth branch of the marginal mandibular nerve. This case emphasizes the need for respecting the variation in the marginal mandibular nerve when carrying out surgery.

Microsurgical Reconstructions for Head and Neck Cancers in Elderly Aged >80 Years: An Analysis of Surgical Outcomes and Quality of Life.

BACKGROUND: The rising incidence of primary head and neck (H&N) cancers in the elderly presents a dilemma regarding the appropriateness of complex surgery in this assumed frail age group. With limited data on surgical morbidity, survival, and patient quality of life (QOL), this analysis aimed to broaden the understanding of safety and effectiveness of microsurgical treatment in very elderly H&N cancer patients. METHODS: A prospective database analysis was used to evaluate surgical outcomes (morbidity, survival, and QOL) in all patients aged 80 years and older undergoing microsurgical reconstruction for cutaneous and intra-oral H&N cancers between 2004 and 2014. Outcomes were assessed for their association with surgical, tumour, and patient variables. Comorbidities were categorized by the ACE27 index and postoperative morbidity by the Clavien-Dindo scoring system. QOL was analyzed using the UW-QOLv4. RESULTS: Of 720 microsurgical reconstructions, 96 patients were identified. Median survival was 25 months. The ACE27 index was the only variable significantly associated with survival with a 5-year survival of 59.2 % in the least comorbid group versus 19.7 % in the most comorbid group (p 0.015). ACE-27 showed influence on socioemotional QoL scores. Physical QOL scores were influenced by tumour and operative factors. Patients were found to value physical QOL over socioemotional. CONCLUSIONS: Microsurgical reconstructions are well tolerated in the very elderly patients and should be considered predominantly based on comorbidity. Tumour stage, flap type, and cancer site should still form part of the preoperative counseling due to their implication on postoperative physical function.

The Integration of Proteome-Wide PTM Data with Protein Structural and Sequence Features Identifies Phosphorylations that Mediate 14-3-3 Interactions.

14-3-3s are abundant proteins that regulate essentially all aspects of cell biology, including cell cycle, motility, metabolism, and cell death. 14-3-3s work by docking to phosphorylated Ser/Thr residues on a large network of client proteins and modulating client protein function in a variety of ways. In recent years, aided by improvements in proteomics, the discovery of 14-3-3 client proteins has far outpaced our ability to understand the biological impact of individual 14-3-3 interactions. The rate-limiting step in this process is often the identification of the individual phospho-serines/threonines that mediate 14-3-3 binding, which are difficult to distinguish from other phospho-sites by sequence alone. Furthermore, trial-and-error molecular approaches to identify these phosphorylations are costly and can take months or years to identify even a single 14-3-3 docking site phosphorylation. To help overcome this challenge, we used machine learning to analyze predictive features of 14-3-3 binding sites. We found that accounting for intrinsic protein disorder and the unbiased mass spectrometry identification rate of a given phosphorylation significantly improves the identification of 14-3-3 docking site phosphorylations across the proteome. We incorporated these features, coupled with consensus sequence prediction, into a publicly available web app, called "14-3-3 site-finder". We demonstrate the strength of this approach through its ability to identify 14-3-3 binding sites that do not conform to the loose consensus sequence of 14-3-3 docking phosphorylations, which we validate with 14-3-3 client proteins, including TNK1, CHEK1, MAPK7, and others. In addition, by using this approach, we identify a phosphorylation on A-kinase anchor protein-13 (AKAP13) at Ser2467 that dominantly controls its interaction with 14-3-3.

Discovery, validation and characterization of 1039 cattle single nucleotide polymorphisms.

We identified approximately 13 000 putative single nucleotide polymorphisms (SNPs) by comparison of repeat-masked BAC-end sequences from the cattle RPCI-42 BAC library with whole-genome shotgun contigs of cattle genome assembly Btau 1.0. Genotyping of a subset of these SNPs was performed on a panel containing 186 DNA samples from 18 cattle breeds including 43 trios. Of 1039 SNPs confirmed as polymorphic in the panel, 998 had minor allele frequency > or =0.25 among unrelated individuals of at least one breed. When Btau 4.0 became available, 974 of these validated SNPs were assigned in silico to known cattle chromosomes, while 41 SNPs were mapped to unassigned sequence scaffolds, yielding one SNP every approximately 3 Mbp on average. Twenty-four SNPs identified in Btau 1.0 were not mapped to Btau 4.0. Of the 1015 SNPs mapped to Btau 4.0, 959 SNPs had nucleotide bases identical in Btau 4.0 and Btau 1.0 contigs, whereas 56 bases were changed, resulting in the loss of the in silico SNP in Btau 4.0. Because these 1039 SNPs were all directly confirmed by genotyping on the multi-breed panel, it is likely that the original polymorphisms were correctly identified. The 1039 validated SNPs identified in this study represent a new and useful resource for genome-wide association studies and applications in animal breeding.

Scrapie resistance in ARQ sheep.

Variation in the ovine prion protein amino acid sequence influences scrapie progression, with sheep homozygous for A(136)R(154)Q(171) considered susceptible. This study examined the association of survival time of scrapie-exposed ARQ sheep with variation elsewhere in the ovine prion gene. Four single nucleotide polymorphism alleles were associated with prolonged survival. One nonsynonymous allele (T112) was associated with an additional 687 days of survival for scrapie-exposed sheep compared to M112 sheep (odds ratio, 42.5; P = 0.00014). The only two sheep homozygous for T112 (TARQ) did not develop scrapie, suggesting that the allelic effect may be additive. These results provide evidence that TARQ sheep are genetically resistant to development of classical scrapie.

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.

Karyotypic analysis of adult pluripotent stem cells.

Three categories of precursor cells have been identified in postnatal mammals: tissue-committed progenitor cells, germ layer lineage-committed stem cells and lineage-uncommitted pluripotent stem cells. Progenitor cells are the immediate precursors of differentiated tissues. Germ layer lineage stem cells can be induced to form multiple cell types belonging to their respective ectodermal, mesodermal, and endodermal embryological lineages. Pluripotent stem cells will form somatic cell types from all three primary germ layer lineages. Progenitor cells demonstrate a finite life span before replicative senescence and cell death occur. Both germ layer lineage stem cells and pluripotent stem cells are telomerase positive and display extensive capabilities for self-renewal. Stem cells which undergo such extensive replication have the potential for undergoing mutations that may subsequently alter cellular functions. Gross mutations in the genome may be visualized as chromosomal aneuploidy and/or chromosomes that appear aberrant. This study was designed to determine whether any gross genomic mutations occurred within the adult pluripotent stem cells. Karyotypic analysis was performed using pluripotent stem cells purified from adult male rats using established procedures. Giemsa Banding was used in conjunction with light microscopy to visualize metaphase chromosome spreads. To date over 800 metaphase spreads have been analyzed. We found that the metaphase spreads averaged 42 chromosomes and concluded that these pluripotent stem cells isolated from adult rats have a normal karyotype.

Incidence of infection in 39-month-old ewes with TMEM154 diplotypes "1 1," "1 3," and "3 3" after natural exposure to ovine progressive pneumonia virus.

Production and well-being of sheep and goats in many countries are harmfully impacted by small ruminant lentiviruses (SRLV) that cause incurable, progressive diseases. Susceptibility to ovine progressive pneumonia virus (OPPV), the North American form of SRLV, is influenced by variants of the ovine transmembrane protein 154 gene (TMEM154). The experimental objective was to estimate additive and dominance effects of TMEM154 haplotypes 1 and 3 on susceptibility of breeding ewes to infection after natural exposure to OPPV from birth to 39 mo of age. Sires and dams were heterozygous for TMEM154 haplotypes 1 and 3, producing ewe lambs with diplotypes "1 1," "1 3," and "3 3." These lambs were raised by mature, infected dams to ensure natural, maternal exposure to OPPV. Ewe lambs (n = 108) were kept for breeding and joined an infected flock of ewes to guarantee natural, nonmaternal exposure to OPPV. Ewes were bred to lamb at 1, 2, and 3 yr of age. Serum samples were collected at breeding, 1 mo before lambing and shortly after weaning each year to monitor infection status to 39 mo of age. During the experiment, 9 of the 108 ewes died while uninfected and data collected on these ewes were not analyzed. Infection status of the remaining 99 ewes at 39 mo of age was analyzed using logistic regression procedures. Effects of ewe type of birth, ewe type of rearing, and breed type of dam were not detected (P > 0.10), and the estimated sire variance component was nil. Ewe diplotype affected infection status (P < 0.0001), as did additive (P < 0.0001) and dominance (P < 0.0022) effects. Predicted probabilities of infection for ewes with diplotypes "1 1," "1 3," and "3 3" were 0.10, 0.88, and 0.89, respectively, and confidence intervals for diplotypes "1 3" and "3 3" were distinct from "1 1." Haplotype 3 was completely dominant to haplotype 1 at 39 mo of age. The probability of infection for ewes with either diplotype "1 3" or "3 3" averaged 8.5 times that of ewes with diplotype "1 1." Diplotype "1 3" and "3 3" ewes were highly susceptible to nonmaternal transmission of OPPV, in contrast to diplotype "1 1" ewes. Therefore, the distribution of ewes with diplotypes "1 1," "1 3," and "3 3" within a flock will influence the number of infections caused by each route of transmission. Selection and mating strategies can be implemented to produce sheep that are genetically less susceptible to OPPV infection.

Mobilization of vancomycin resistance by transposon-mediated fusion of a VanA plasmid with an Enterococcus faecium sex pheromone-response plasmid.

A striking feature of recent outbreaks of vancomycin-resistant (VmR) enterococci is the apparent horizontal dissemination of resistance determinants. The plasmids pHKK702 and pHKK703 from Enterococcus faecium clinical isolate R7 have been implicated in the conjugal transfer of VmR. pHKK702 is a 41-kb plasmid that contains an element indistinguishable from the glycopeptide-resistance transposon Tn1546. pHKK703 is an approx. 55-kb putative sex pheromone-response plasmid that is required for conjugative mobilization of pHKK702. During experiments in which strain R7 was used as a donor, a highly conjugative VmR transconjugant was isolated that formed constitutive cellular aggregates. Restriction analyses and DNA hybridizations revealed that the transconjugant harbored a single plasmid of approx. 92 kb and this plasmid (pHKK701) was composed of DNA from both pHKK702 and pHKK703. Results from DNA sequence analyses showed that a 39-kb composite transposon (Tn5506) from pHKK702 had inserted into pHKK703. The left end of Tn5506 contained a single insertion sequence (IS) element, IS1216V2, whereas the right end was composed of a tandem IS structure consisting of the novel 1065-bp IS1252 nested within an IS1216V1 element. Transposition of Tn5506 from pHKK702 to pHKK703 created an 8-bp target sequence duplication at the site of insertion and interrupted an ORF (ORFX) that was 91% identical to that of prgX, a gene proposed to negatively regulate sex pheromone response of the E.faecalis plasmid, pCF10. We propose that the interruption of ORFX by Tn5506 led to the constitutive cellular aggregation phenotype and thereby enhanced the efficiency with which VmR was transferred. Similar IS1216V-mediated transposition events may contribute to the horizontal spread of glycopeptide resistance among enterococci in nature.

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.

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.

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.

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.

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.

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.