Strategic planning in an academic radiation medicine program.

BACKGROUND: In this paper, we report on the process of strategic planning in the Radiation Medicine Program (rmp) at the Princess Margaret Cancer Centre. The rmp conducted a strategic planning exercise to ensure that program priorities reflect the current health care environment, enable nimble responses to the increasing burden of cancer, and guide program operations until 2020. METHODS: Data collection was guided by a project charter that outlined the project goal and the roles and responsibilities of all participants. The process was managed by a multidisciplinary steering committee under the guidance of an external consultant and consisted of reviewing strategic planning documents from close collaborators and institutional partners, conducting interviews with key stakeholders, deploying a program-wide survey, facilitating an anonymous and confidential e-mail feedback box, and collecting information from group deliberations. RESULTS: The process of strategic planning took place from December 2014 to December 2015. Mission and vision statements were developed, and core values were defined. A final document, Strategic Roadmap to 2020, was established to guide programmatic pursuits during the ensuing 5 years, and an implementation plan was developed to guide the first year of operations. CONCLUSIONS: The strategic planning process provided an opportunity to mobilize staff talents and identify environmental opportunities, and helped to enable more effective use of resources in a rapidly changing health care environment. The process was valuable in allowing staff to consider and discuss the future, and in identifying strategic issues of the greatest importance to the program. Academic programs with similar mandates might find our report useful in guiding similar processes in their own organizations.

Chytrid infections exhibit historical spread and contemporary seasonality in a declining stream-breeding frog.

Species with extensive geographical ranges pose special challenges to assessing drivers of wildlife disease, necessitating collaborative and large-scale analyses. The imperilled foothill yellow-legged frog (Rana boylii) inhabits a wide geographical range and variable conditions in rivers of California and Oregon (USA), and is considered threatened by the pathogen Batrachochytrium dendrobatidis (Bd). To assess drivers of Bd infections over time and space, we compiled over 2000 datapoints from R. boylii museum specimens (collected 1897-2005) and field samples (2005-2021) spanning 9° of latitude. We observed a south-to-north spread of Bd detections beginning in the 1940s and increase in prevalence from the 1940s to 1970s, coinciding with extirpation from southern latitudes. We detected eight high-prevalence geographical clusters through time that span the species' geographical range. Field-sampled male R. boylii exhibited the highest prevalence, and juveniles sampled in autumn exhibited the highest loads. Bd infection risk was highest in lower elevation rain-dominated watersheds, and with cool temperatures and low stream-flow conditions at the end of the dry season. Through a holistic assessment of relationships between infection risk, geographical context and time, we identify the locations and time periods where Bd mitigation and monitoring will be critical for conservation of this imperilled species.

A fully automatic system to assess foot collapse on lateral weight-bearing foot radiographs: A pilot study.

BACKGROUND: Foot collapse is primarily diagnosed and monitored using lateral weight-bearing foot x-ray images. There are several well-validated measurements which aid assessment. However, these are subject to inter- and intra-user variability. OBJECTIVE: To develop and validate a software system for the fully automatic assessment of radiographic changes associated with foot collapse; automatically generating measurements for calcaneal tilt, cuboid height and Meary's angle. METHODS: This retrospective study was approved by the Health Research Authority (IRAS 244852). The system was developed using lateral weight-bearing foot x-ray images, and evaluated against manual measurements from five clinical experts. The system has two main components: (i) a Random Forest-based point-finder to outline the bones of interest; and (ii) a geometry-calculator to generate the measurements based on the point positions from the point-finder. The performance of the point-finder was assessed using the point-to-point error (i.e. the mean absolute distance between each found point and the equivalent ground truth point, averaged over all points per image). For assessing the performance of the geometry-calculator, linear mixed models were fitted to estimate clinical inter-observer agreement and to compare the performance of the software system to that of the clinical experts. RESULTS: A total of 200 images were collected from 79 subjects (mean age: 56.4 years ±12.9 SD, 30/49 females/males). There was good agreement among all clinical experts with intraclass correlation estimates between 0.78 and 0.86. The point-finder achieved a median point-to-point error of 2.2 mm. There was no significant difference between the clinical and automatically generated measurements using the point-finder points, suggesting that the fully automatically obtained measurements are in agreement with the manually obtained measurements. CONCLUSIONS: The proposed system can be used to support and automate radiographic image assessment for diagnosing and managing foot collapse, saving clinician time, and improving patient outcomes.

A putative 'pre-nervous' endocannabinoid system in early echinoderm development.

Embryos and larvae of sea urchins (Lytechinus variegatus, Strongylocentrotus droebachiensis, Strongylocentrotus purpuratus, Dendraster excentricus), and starfish (Pisaster ochraceus) were investigated for the presence of a functional endocannabinoid system. Anandamide (arachidonoyl ethanolamide, AEA), was measured in early L. variegatus embryos by liquid chromatography/mass spectrometry. AEA showed a strong developmental dynamic, increasing more than 5-fold between the 8-16 cell and mid-blastula 2 stage. 'Perturb-and-rescue' experiments in different sea urchin species and starfish showed that AEA blocked transition of embryos from the blastula to the gastrula stage, but had no effect on cleavage divisions, even at high doses. The non-selective cannabinoid receptor agonist, CP55940, had similar effects, but unlike AEA, also blocked cleavage divisions. CB1 antagonists, AEA transport inhibitors, and the cation channel transient membrane potential receptor V1 (TrpV1) agonist, arachidonoyl vanillic acid (arvanil), as well as arachidonoyl serotonin and dopamine (AA-5-HT, AA-DA) acted as rescue substances, partially or totally preventing abnormal embryonic phenotypes elicited by AEA or CP55940. Radioligand binding of [(3)H]CP55940 to membrane preparations from embryos/larvae failed to show significant binding, consistent with the lack of CB receptor orthologs in the sea urchin genome. However, when binding was conducted on whole cell lysates, a small amount of [(3)H]CP55940 binding was observed at the pluteus stage that was displaced by the CB2 antagonist, SR144528. Since AEA is known to bind with high affinity to TrpV1 and to certain G-protein-coupled receptors (GPCRs), the ability of arvanil, AA-5-HT and AA-DA to rescue embryos from AEA teratogenesis suggests that in sea urchins AEA and other endocannabinoids may utilize both Trp and GPCR orthologs. This possibility was explored using bioinformatic and phylogenetic tools to identify candidate orthologs in the S. purpuratus sea urchin genome. Candidate TrpA1 and TrpV1 orthologs were identified. The TrpA1 ortholog fell within a monophyletic clade, including both vertebrate and invertebrate orthologs, whereas the TrpV1 orthologs fell within two distinct TrpV-like invertebrate clades. One of the sea urchin TrpV orthologs was more closely related to the vertebrate epithelial calcium channels (TrpV5-6 family) than to the vertebrate TrpV1-4 family, as determined using profile-hidden Markov model (HMM) searches. Candidate dopamine and adrenergic GPCR orthologs were identified in the sea urchin genome, but no cannabinoid GPCRs were found, consistent with earlier studies. Candidate dopamine D(1), D(2) or alpha(1)-adrenergic receptor orthologs were identified as potential progenitors to the vertebrate cannabinoid receptors using HMM searches, depending on whether the multiple sequence alignment of CB receptor sequences consisted only of urochordate and cephalochordate sequences or also included vertebrate sequences.

pp60c-src in the developing cerebellum.

pp60c-src was localized in the cerebellum of developing chicken embryos by immunoperoxidase staining with antisera raised against bacterially expressed pp60v-src. Immunoreactivity (IR) appeared in the cerebellum of the chicken embryos at the time of neuronal differentiation. pp60c-src IR was detected in regions of the developing cerebellum where processes of developing neurons and glia are located. In the early embryo (stage 17), pp60c-src IR was localized in the marginal zone of the cerebellar plate. By stage 40, pp60c-src IR was localized in the process-rich molecular layer of the cerebellum and between the cells of the developing internal granular layer. Cell bodies of cerebellar neurons did not show pp60c-src IR at any stage of development. Mitotically active neuroepithelial cells of the metencephalon did not express pp60c-src before the onset of differentiation in the early embryo, nor did proliferating cells of the external granular layer express pp60c-src at later stages. Although it is not possible to ascertain whether pp60c-src is localized in developing neurons or glia at the light microscope level, the time of its appearance and pattern of distribution in the molecular layer is suggestive of a localization within the developing neuronal processes which compose the bulk of this layer. Biochemical analyses of pp60c-src in the developing cerebellum by the immune complex protein kinase activity and sensitivity of the kinase to inhibition by P1,P4-di(adenosine-5')tetraphosphate confirmed that the expression of pp60c-src coincided with the time of neuronal differentiation. We conclude from these results that in the central nervous systems, pp60c-src may be more important in an aspect of cell differentiation or a mature neuronal function than in the proliferation of neuronal or glial precursors.

Neurotransmitters as growth regulatory signals: role of receptors and second messengers.

In the adult nervous system, neurotransmitters act as chemical mediators of intercellular communication by the activation of specific receptors and second messengers in postsynaptic cells. This specialized role may have evolved from more primitive functions in lower organisms where these substances were used as both intra- and intercellular signalling devices. This view derives from the finding that a number of 'classical' neurotransmitters are present in primitive organisms and early embryos in the absence of a nervous system, and pharmacological evidence that these substances regulate morphogenetic activities such as proliferation, differentiation, cell motility and metamorphosis. These phylogenetically old functions may be reiterated in the developing nervous system and in the humoral functions of neurotransmitters outside the nervous system. This review will provide evidence for this hypothesis based on the commonality of signal transduction mechanisms used in primitive organisms, early embryos and non-neuronal cells, and relate these relationships to the functions of neurotransmitters in the developing nervous system. This discussion has generally been limited to neurotransmitters where non-neuronal functions have been studied and information regarding the involvement of receptors and second messenger pathways is available.

Prenatal stress in the rat alters 5-HT1A receptor binding in the ventral hippocampus.

Exposure of a pregnant woman to physical and/or psychological stress might affect her offspring by promoting the development of various learning, behavioral and/or mood disorders in later life. The 5-HT1A and 5-HT2A receptors are prominently implicated in the modulation of anxiety and mood-related behaviors. Using a semi-quantitative radiolabel immunocytochemical analysis (immunobinding), we studied the effect of prenatal stress on binding of these two receptor subtypes in the hippocampus of 4-week-old male and female Fischer 344 rats. Levels of 5-HT1A immunobinding in the ventral hippocampus, which is primarily implicated in emotional processing, were significantly decreased in male offspring after prenatal stress. A trend towards a decrease was observed in the ventral hippocampus of females. In contrast, 5-HT1A immunobinding within the dorsal hippocampus, which is mainly related to learning and memory, was not affected by prenatal stress in offspring of either gender. Likewise, no significant differences between control and prenatally stressed rats were observed for levels of 5-HT2A immunobinding in either part of the hippocampus or gender. The observed reduction in hippocampal 5-HT1A receptor binding in male offspring after prenatal stress may have important consequences for adult anxiety- and depressive-like behavior.

p59fyn in rat brain is localized in developing axonal tracts and subpopulations of adult neurons and glia.

Expression of the c-fyn proto-oncogene was analysed in the developing and adult rat brain by subcellular fractionation and immunocytochemistry using polyclonal antibodies specific for its protein product (p59fyn). Immunoperoxidase staining revealed widespread localization of p59fyn in developing axonal tracts throughout the fetal (E18) rat brain. fyn immunoreactivity was not observed in most axon-rich regions of the adult brain, but continued to be expressed at elevated levels in the adult olfactory and vomeronasal systems. At other sites in the adult rat brain, fyn immunoreactivity was restricted to cell bodies of neuronal subpopulations, especially those in brain stem and hypothalamic nuclei, and to subpopulations of glial cells along axonal tracts in the medulla, optic nerve and white matter of the spinal cord. In the peripheral nervous system, fyn staining was also prominent in Schwann cells. Subcellular fractionation of fetal and adult rat brain confirmed the immunocytochemical localization, demonstrating an enrichment of p59fyn in membranes from a fetal brain fraction containing nerve growth cones, and lower levels in adult brain where there was only a small enrichment in synaptosomal membranes. The developmental regulation of p59fyn suggests that the fyn tyrosine kinase may serve separate cellular roles in axonal growth and specialized functions of mature neurons and glia.

Cytokine effects on cortical neuron MAP-2 immunoreactivity: implications for schizophrenia.

BACKGROUND: Cytokines demonstrate diverse actions in the brain and modulate systemic and central nervous system (CNS) responses to injury, infection, and inflammation. Cytokines in the CNS are elevated during infection and ischemia, two neurodevelopmental insults associated with increased schizophrenia risk. We hypothesize that cytokine-mediated neuronal injury during development may contribute to schizophrenia pathophysiology, causing subtle alterations in neuronal number and density. METHODS: We examined cytokine regulation of neuronal number in embryonic day 18 rat cortical cultures using MAP-2 immunohistochemistry. Mixed cultures derived from frontal cortex were fixed and stained after 48-hour exposure to the proinflammatory interleukin-1beta (IL-1beta), interleukin-6 (IL-6), or tumor necrosis factor-alpha (TNF-alpha; 0, 10, 100, or 1000 units/mL). RESULTS: IL-1beta (maximum effect 35%) and IL-6 (maximum effect 29%) produced dose-dependent decreases in the number of cells (neurons) immunoreactive for MAP-2 antibody, suggesting decreased neuronal survival. TNF-alpha also tended to decrease MAP-2 immunostaining at the highest dose tested. CONCLUSIONS: Our data suggest a role for cytokines in the modulation of neuronal survival during neurodevelopment, a finding potentially relevant to schizophrenia pathophysiology. If cytokine-mediated neuronal injury proves to be a common response to gestational insults associated with increased schizophrenia risk, the pharmacologic modulation of these molecules may have clinical utility.

Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. II. Synaptogenesis.

Synaptogenesis was studied in the monoamine (MA) cell groups locus coeruleus (LC), dorsal and medial raphe nuclei (RN) and substantia nigra, zona compacta (SN) between day 18 of gestation and postnatal day 60 using ethanolic phosphotungstic acid (E-PTA) to visualize synaptic profiles. Nuclear area, and cellular packing density (inversely proportional to area of neuropil) were also determined. As determined using the E-PTA method, synaptogenesis begins in the neuropil of the SN first, on or before 18 days of gestation, and in the LC and RN at 19 days. Synaptogenesis on MA cell perikarya is first observed in the SN, on or before 18 days, and in the LC and RN at 20 days. The onset of somatic synaptogenesis coincides with the beginning of nuclear growth and development of the neuropil (decrease in cellular packing density) in all MA cell areas, raising the possibility of common factors in the initiation of these processes. Nonsynaptic contacts precede the appearance of synaptic profiles both in the neuropil and on the somata of the MA cells of the LC, RN and SN, and may represent precursors of mature synapses or desmosome-like contacts. Somatosomatic nonsynaptic contacts occur only prenatally between adjacent MA neurons in the LC, RN and SN. Although some synaptogenesis occurs prenatally in these MA cell groups (indiciating that these parts of the MA circuitry may be functional before birth), most of this synaptogenesis occurs postnatally and continues into adulthood. Since such synaptogenesis does not begin until 2-4 days prior to birth, whereas these neurons and their processes exhibit MA fluorescence as early as 12-14 days of gestation, they apparently are capable of synthesizing transmitter and proliferating terminals before they themselves are innervated.

3H-thymidine long survival autoradiography as a method for dating the time of neuronal origin in the chick embryo: the locus coeruleus and cerebellar Purkinje cells.

Contrary to previous assumptions, we have found that a single dose of 3H-thymidine (25 muCi), injected into the yolk sac of White Leghorn chick eggs on 2 days of incubation (d.i.) only remains available for DNA-synthesizing (proliferating) cells for 48 hours following the time of injection. This finding now makes it possible to date the time of neuronal origin in the avian embryo using a single injection of isotope and a long survival time (30 days posthatch) as in mammalian studies where 3H-thymidine is only available as a short "pulse." Using this method, we have determined that neurons in the chick locus coeruleus (LC) cease proliferation on 2-6 d.i. with a peak of neuronal genesis on 3-5 d.i. In addition, neuronal genesis is not homogeneous throughout the LC cell population, but occurs in a predominantly caudorostral gradient. Conversely, the cerebellar Purkinje cells cease division on 3-8 d.i. with a peak of heavy labeling on 4-6 d.i., 1 day later than that observed in the LC.

Ontogeny of cholinergic neurons in the mouse forebrain.

The development of cholinergic neurons in the mouse forebrain was studied by immunocytochemistry with a monoclonal antibody to choline acetyltransferase (ChAT), the rate-limiting enzyme for acetylcholine synthesis. Since this antibody stained dividing cells in ventricular germinal zones as well as differentiating neurons, likely routes of migration could be inferred on the basis of the location of immunoreactive (IR) cells at different gestational ages. Germinal zones for cholinergic cells were observed in all ventricular zones of the forebrain with the ventral zones generating the earliest cells by gestational day 13.5 (GD13.5). On GD14, ChAT IR cells were visible in the germinal zones of the eye, olfactory ventricle, anterior horn, and dorsolateral aspect of the lateral ventricle, lateral ganglionic eminence, ventro- and dorsolateral third ventricle, and in the pineal anlage (epiphysis). ChAT IR neurons continued to develop in these and additional germinal zones on GD15, including the medial, dorsal, and dorsomedial walls of the lateral ventricle, and the medial and dorsal ganglionic eminence. On GD16, ChAT IR neurons were located in the prelimbic, pyriform, and parietal cortices and the lamina terminalis, and a cluster of IR cells was observed in the ventricular zone of the caudatopallial angle. On GD17-18, neurons in the anterior olfactory nucleus, olfactory tubercle, horizontal and vertical nucleus of the diagonal band, and medial septal nucleus stained more darkly and were multipolar, whereas immature bipolar neurons appeared to continue their migration into the hippocampus and along major fiber tracts, such as the corpus callosum, external capsule, fornix and anterior commissure. This study provides a comprehensive view of the zones of origin, probable routes of migration, and final destination of cholinergic neurons in the mouse forebrain.

Distribution of glucagonlike peptide I (GLP-I), glucagon, and glicentin in the rat brain: an immunocytochemical study.

Although glucagonlike immunoreactants (GLIs) are present in the central nervous system of several mammalian species, their structural relationship with pancreatic proglucagon is not defined, and their precise anatomical distribution has not been studied extensively. To obtain further information about the structure and biological significance of brain GLIs, the anatomical distribution of three different antigenic determinants of pancreatic proglucagon--glucagonlike peptide I (GLP-I), glucagon, and glicentin--was mapped in the brain of colchicine-treated rats by immunocytochemistry using the avidin-biotin-peroxidase method. Neuronal cell bodies immunoreactive with antisera specific for GLP-I, glucagon, and glicentin were found only in the caudal medulla oblongata. Within the caudal medulla immunostained cell bodies were found at levels from approximately 0.55 mm rostral to the obex to 0.45 mm caudal to the obex, and were located within the nucleus of the solitary tract (NTS) and the dorsal (MdD) and ventral (MdV) parts of the medullary reticular nucleus. The NTS contained three times more immunoreactive cell bodies than the MdD and MdV, and these cell bodies were located in the midline, medial, and lateral subnuclei of the caudal third of the NTS. Immunostaining of the same cell bodies in paired adjacent sections incubated with GLP-I and glucagon antisera or glucagon and glicentin antisera provided evidence for coexistence of the three antigens within the same neurons of the NTS. Nerve fibers and terminals immunoreactive with GLP-I, glucagon, and glicentin antisera were widely distributed throughout the rat brain and there was no discernible difference in the distribution of fibers and terminals immunoreactive with each of the three antisera. The highest densities of immunostained fibers and terminals were observed in the hypothalamus, thalamus, and septal regions, and the lowest in the cortex and hindbrain. The localization of neuronal cell bodies containing GLP-I, glucagon, and glicentin within the NTS and the MdD and MdV, and the extensive distribution of immunoreactive fibers and terminals throughout the rat brain suggest a role for these peptides in the integration of autonomic as well as central nervous system functions.

Disregulation of urokinase plasminogen activator gene in breast cancer.

Disregulation of urokinase plasminogen activator (uPA) was assessed in 134 breast cancer specimens. Overexpression of uPA was determined by immunohistochemistry using the specific monoclonal antibody, #394. Gene amplification was assessed by differential polymerase chain reaction, using primers designed to amplify a 111 bp segment of the uPA gene. Overexpression of uPA was detected in 33% of breast cancers, including 4 of 21 in situ carcinomas, 7 of 14 lobular and 2 of 10 tubular carcinomas. Overexpression of uPA did not correlate with the presence or absence of oestrogen receptors. uPA gene amplification was not detected in any cancer. We conclude that uPA gene amplification is not a major mechanism instigating uPA overexpression in breast cancer, and that overexpression is likely to be controlled by other mechanisms.

Prenatal ontogeny of the GABAergic system in the rat brain: an immunocytochemical study.

Prenatal development of the GABAergic system in the rat brain has been studied using an antiserum to GABA-glutaraldehyde-hemocyanin conjugates, specific for GABAergic neurons. The gamma-aminobutyric acid (GABA) system has been found to differentiate very early relative to other transmitter-identified neurons, such that by embryonic day 13 a well developed fiber network exists in the brainstem, mesencephalon and diencephalon, including a large projection in the posterior commissure and adjacent areas on the surface of the mesencephalon and tectum. Although no cell bodies are visible at this time, it appears that these fibers originate from the caudal brainstem and spinal cord. GABAergic cell bodies begin to appear on embryonic day 14 in the lateral cortical anlage. By embryonic day 16, they are also visible in the basal forebrain and in all regions of cortex where they are located in three zones: in layer I, below the cortical plate, and in the intermediate zone. Also contained in the outer part of layer I is a dense fiber plexus which stains intensely for GABA. These fibers may be part of the first contingent of cortical afferents to invade the telencephalic vesicle, an event which is thought to be a stimulus for the beginning of neuronal differentiation in this region. By E18, two bands of immunoreactivity are visible in layer I, which probably contain both cell bodies and fibers. The trajectories taken by growing GABAergic fibers in the brainstem, mesencephalon and diencephalon at embryonic day 13 and at subsequent stages of development are coincident with regions of both monoaminergic and peptidergic differentiation and appear to correspond to recently reported patterns of benzodiazepine receptors which appear slightly later. The early differentiation of the GABAergic system could indicate a trophic role for GABA in early brain development, possibly involving receptors for this neurotransmitter or related substances.

Optimization of tyrosine hydroxylase immunocytochemistry in paraffin sections using pretreatment with proteolytic enzymes.

Immunocytochemical localization of tyrosine hydroxylase (TH) was performed on paraffin sections pretreated with various proteolytic enzymes. It was found that pretreatment with trypsin (1.2 mg/ml) for 5 min resulted in a dramatic increase in the number of TH-positive terminals throughout the brain, especially in the cerebellum, which contains fine preterminal and terminal axons that are difficult to stain. This pretreatment also led to a significant reduction in background staining and allowed for the use of the TH antiserum at high working dilutions. Several other proteolytic enzymes were tested and only chymotrypsin was nearly as effective as trypsin with respect to TH staining.

Immunocytochemical localization of glucocorticoid receptors in midgestation murine embryos and human embryonic cultured cells.

Glucocorticoid receptors have been localized immunocytochemically in the developing mouse secondary palatal shelves and in cultured human embryonic palatal mesenchyme cells. In the midgestation embryo, receptors are found in the highest concentration in the palatal mesenchymal cells, suggesting that they play a major role in normal development as well as in glucocorticoid-induced cleft palate. The presence of these receptors in cultured human embryonic palatal cells also suggests that development of the human secondary palate may be dependent on glucocorticoids.

Serotonin uptake in the ectoplacental cone and placenta of the mouse.

The neurotransmitter serotonin (5-HT) was localized in the ectoplacental cone (EPC) and placenta of the day 9-12 (E9-12) mouse embryo in vivo and in whole embryo cultures, using immunocytochemistry with a specific 5-HT antiserum. In uncultured conceptuses, 5-HT immunoreactivity (5-HT IR) was most intense in the EPC at E9 (2-7 somites), particularly in giant cells around the periphery. Nuclear staining was observed in lightly staining giant cells and in small cells in the core of the cone. By E10 (18-24 somites) 5-HT IR in the placenta was less intense and almost exclusively limited to giant cells, where it was localized to chromatin-like material in nuclei. The same pattern and level of 5-HT IR persisted through E12. In the placenta, 5-HT IR appeared to be most intense in giant cells located near aggregations of platelets in decidual blood vessels. 5-HT IR was enhanced in cultured conceptuses, and further increased when exogenous 5-HT was added to the culture medium. Immunoreactivity was greatly reduced by adding the 5-HT uptake inhibitor fluoxetine to the culture medium, or culturing conceptuses in medium containing 5-HT depleted rat serum. Thus, 5-HT was apparently taken up from the culture medium. In conceptuses exposed to exogenous 5-HT, immunoreactivity in the placenta appeared as a gradient from the giant cells to the inner layers, suggesting that these cells may transport 5-HT toward the embryo. No evidence of 5-HT synthesis by the EPC/placenta was found. These results suggest that 5-HT present in the EPC/placenta is due to uptake, not synthesis. Possible sources and functions of 5-HT in the developing placenta are discussed.

Morphogenetic roles of acetylcholine.

In the adult nervous system, neurotransmitters mediate cellular communication within neuronal circuits. In developing tissues and primitive organisms, neurotransmitters subserve growth regulatory and morphogenetic functions. Accumulated evidence suggests that acetylcholine, (ACh), released from growing axons, regulates growth, differentiation, and plasticity of developing central nervous system neurons. In addition to intrinsic cholinergic neurons, the cerebral cortex and hippocampus receive extensive innervation from cholinergic neurons in the basal forebrain, beginning prenatally and continuing throughout the period of active growth and synaptogenesis. Acute exposure to ethanol in early gestation (which prevents formation of basal forebrain cholinergic neurons) or neonatal lesioning of basal forebrain cholinergic neurons, significantly compromises cortical development and produces persistent impairment of cognitive functions. Neonatal visual deprivation alters developmental expression of muscarinic acetylcholine receptors (mAChR) in visual cortex, whereas local infusion of mAChR antagonists impairs plasticity of visual cortical neurons. These findings raise the possibility that exposure to environmental neurotoxins that affect cholinergic systems may seriously compromise brain development and have long-lasting morphologic, neurochemical, and functional consequences.

An invertebrate model of the developmental neurotoxicity of insecticides: effects of chlorpyrifos and dieldrin in sea urchin embryos and larvae.

Chlorpyrifos targets mammalian brain development through a combination of effects directed at cholinergic receptors and intracellular signaling cascades that are involved in cell differentiation. We used sea urchin embryos as an invertebrate model system to explore the cellular mechanisms underlying the actions of chlorpyrifos and to delineate the critical period of developmental vulnerability. Sea urchin embryos and larvae were exposed to chlorpyrifos at different stages of development ranging from early cell cleavages through the prism stage. Although early cleavages were unaffected even at high chlorpyrifos concentrations, micromolar concentrations added at the mid-blastula stage evoked a prominent change in cell phenotype and overall larval structure, with appearance of pigmented cells followed by their accumulation in an extralarval cap that was extruded from the animal pole. At higher concentrations (20-40 microM), these abnormal cells constituted over 90% of the total cell number. Studies with cholinergic receptor blocking agents and protein kinase C inhibitors indicated two distinct types of effects, one mediated through stimulation of nicotinic cholinergic receptors and the other targeting intracellular signaling. The effects of chlorpyrifos were not mimicked by chlorpyrifos oxon, the active metabolite that inhibits cholinesterase, nor by nonorganophosphate cholinesterase inhibitors. Dieldrin, an organochlorine that targets GABA(A )receptors, was similarly ineffective. The effects of chlorpyrifos and its underlying cholinergic and signaling-related mechanisms parallel prior findings in mammalian embryonic central nervous system. Invertebrate test systems may thus provide both a screening procedure for potential neuroteratogenesis by organophosphate-related compounds, as well as a system with which to uncover novel mechanisms underlying developmental vulnerability.