Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus.

We have engineered a set of useful tools that facilitate targeted single copy knock-in (KI) at the hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) locus. We employed fine scale mapping to delineate the precise breakpoint location at the Hprt1(b-m3) locus allowing allele specific PCR assays to be established. Our suite of tools contains four targeting expression vectors and a complementing series of embryonic stem cell lines. Two of these vectors encode enhanced green fluorescent protein (EGFP) driven by the human cytomegalovirus immediate-early enhancer/modified chicken beta-actin (CAG) promoter, whereas the other two permit flexible combinations of a chosen promoter combined with a reporter and/or gene of choice. We have validated our tools as part of the Pleiades Promoter Project (http://www.pleiades.org), with the generation of brain-specific EGFP positive germline mouse strains.

The homeodomain protein Arix promotes protein kinase A-dependent activation of the dopamine beta-hydroxylase promoter through multiple elements and interaction with the coactivator cAMP-response element-binding protein-binding protein.

The differentiation and maintenance of a neurotransmitter phenotype is guided by the interaction of exogenous cues with intrinsic genetic machinery. For the noradrenergic phenotype, these influences combine to activate the expression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH). In this study, we evaluate the molecular mechanisms by which the transcription factor Arix/Phox2a contributes to DBH gene transcription. We have evaluated the contribution of individual homeodomain binding sites in the rat DBH promoter region and find that all are essential for both basal and cAMP-dependent protein kinase A (PKA)-stimulated transcription. Using mammalian one-hybrid and two-hybrid systems, we demonstrate that recruitment of Arix to the positions of homeodomain core recognition sites 1 and 2 at -153 to -166 of the DBH gene restores complete responsiveness of the promoter to PKA in SHSY-5Y neuroblastoma and HepG2 hepatoma cells. Intracellular Arix-Arix interactions are evident and may contribute to the interdependence of homeodomain binding sites. Analysis of functional domains of Arix reveals an N-terminal activation domain and a C-terminal repression domain. The N terminus of Arix contains an amino acid motif similar to a region in Brachyury and Pax9 transcription factors. The N-terminal activation domain of Arix interacts with the transcriptional co-activator, cAMP-response element-binding protein-binding protein, which potentiates transcription from the DBH promoter in a PKA-dependent manner. The present study supports the hypothesis that the paired-like homeodomain protein, Arix, acts as a critical phenotype-specific regulator of the DBH promoter by serving as an integrator of signal-dependent transcription activators within the network of the general transcription machinery.

AP1 proteins mediate the cAMP response of the dopamine beta-hydroxylase gene.

Neurotransmitter biosynthesis is regulated by environmental stimuli, which transmit intracellular signals via second messengers and protein kinase pathways. For the catecholamine biosynthetic enzymes, dopamine beta-hydroxylase and tyrosine hydroxylase, regulation of gene expression by cyclic AMP, diacyl glycerol, and Ca2+ leads to increased neurotransmitter biosynthesis. In this report, we demonstrate that the cAMP-mediated regulation of transcription from the dopamine beta-hydroxylase promoter is mediated by the AP1 proteins c-Fos, c-Jun, and JunD. Following treatment of cultured cells with cAMP, protein complexes bound to the dopamine beta-hydroxylase AP1/cAMP response element element change from consisting of c-Jun and JunD to include c-Fos, c-Jun, and JunD. The homeodomain protein Arix is also a component of this DNA-protein complex, binding to the adjacent homeodomain recognition sites. Transfection of a dominant negative JunD expression plasmid inhibits cAMP-mediated expression of the dopamine beta-hydroxylase promoter construct in PC12 and CATH.a cells. In addition to the role of c-Fos in regulating dopamine beta-hydroxylase gene expression in response to cAMP, a second pathway, involving Rap1/B-Raf is involved. These experiments illustrate an unusual divergence of cAMP-dependent protein kinase signaling through multiple pathways that then reconverge on a single element in the dopamine beta-hydroxylase promoter to elicit activation of gene expression.

The neuronal organization of the supracapsular part of the stria terminalis in the rat: the dorsal component of the extended amygdala.

In the present normal anatomical light and electron microscopic study in the rat, histochemical (Nissl, Timm, Golgi) or immunocytochemical (microtubule-associated protein type 2, glutamate decarboxylase, glutamate receptor subunit 1, synaptophysin) stains were used to analyse neurons embedded within the stria terminalis and their associated neuropil. These cells are closely related to the bed nucleus of the stria terminalis and the centromedial amygdala, and have been termed the "supracapsular part of the bed nucleus of the stria terminalis". The largest part of this neuronal complex is located in the ventrolateral part of the stria, where it appears as a round or oval "lateral pocket" in virtually any type of light microscopic preparation because of its collection of neuronal cell bodies and dense neuropil, in addition to a lacework of unmyelinated axons. A much smaller but still distinct "medial pocket" is located in the medial corner of the stria. The large lateral subdivision of the supracapsular stria terminalis is directly continuous with the lateral bed nucleus of the stria terminalis and extends to the central amygdaloid nucleus, containing a column of neurons that is only broken up into cell clusters at the most caudal levels of the stria as it drops vertically toward the amygdala. The considerably smaller medial subdivision appears, in turn, to be directly continuous with the medial part of the bed nucleus of the stria terminalis. The medial column tapers off more rapidly than the lateral part, so that as the middle levels are approached, only small interrupted clusters of cells are seen. Solitary neurons can also be found in practically every part of the stria terminalis except among the ventrally located axons of the commissural component. Most of the neurons are small to medium in size, as viewed in transverse sections of the stria, but larger neurons are also encountered. In sections parallel to the stria, many neurons are fusiform in appearance. The dendrites are often aligned in a longitudinal fashion; many of the dendrites related to the cells in the lateral pocket are moderately to densely spined, whereas those in the medial pocket are more sparsely spined. The neuropil in both the lateral and medial pockets is characterized by boutons, bundles of unmyelinated axons, and dendrites. Based on their vesicle content, the boutons are divided into three major types: (A) round or slightly oval, agranular vesicles of uniform size; (B) pleomorphic, agranular vesicles, many of which are flattened; and (C) pleomorphic agranular vesicles, some of which are considerably larger than the ones in type B boutons. Type A boutons establish contacts with both dendritic spines and shafts, whereas types B and C usually contact dendritic shafts and sometimes somata. These synaptic components are similar to those described earlier for the central and medial amygdaloid nuclei. Overall, our results support the contention advanced in 1923 by Johnston [J. comp. Neurol. 35, 337481] that the cells accompanying the stria terminalis are interconnecting columns of a macrostructure encompassing the bed nucleus of the stria terminalis and centromedial amygdala. More recently, it has been appreciated that columns of neurons below the globus pallidus also belong to this macrostructure [Alheid G. F. et al. (1995) In The Rat Nervous System, 2nd edn, pp. 495 578, Academic, San Diego; de Olmos J. S. et al. (1985) In The Rat Nervous System, pp. 223-334, Academic, Sydney], which has been named the "extended amygdala".

The homeodomain protein Arix interacts synergistically with cyclic AMP to regulate expression of neurotransmitter biosynthetic genes.

Transcription of the neurotransmitter biosynthetic genes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH) is regulated by cell type-specific transcription factors, including the homeoprotein Arix, and second messengers, including cyclic AMP. The cis-acting regulatory sites of the DBH gene which respond to Arix and cAMP lie adjacent to each other, between bases -180 and -150, in a regulatory element named DB1. Neither Arix nor cyclic AMP analogs alone effectively stimulate transcription from the DBH promoter in non-neuronal cell cultures. However, when Arix is present together with cAMP, transcription is substantially activated. Synergistic transcription from the DBH promoter can also be elicited by cotransfection of Arix with an expression vector encoding the catalytic subunit of protein kinase A. Nuclear extracts from PC12 cells display a cAMP-induced complex binding to the DB1 element, and antisera to transcription factors CREB, CREM, Fos, and Jun indicate that these proteins, or closely related family members, interact with DB1. A dominant negative construct of CREB inhibits the response of the DBH promoter to protein kinase A. These results demonstrate a synergistic interaction between a homeodomain protein and the cAMP signal transduction system and suggest that similar interactions may regulate the tissue-specific expression of neuroendocrine genes.

Cortical input to the basal forebrain.

The arborization pattern and postsynaptic targets of corticofugal axons in basal forebrain areas have been studied by the combination of anatomical tract-tracing and pre- and postembedding immunocytochemistry. The anterograde neuronal tracer Phaseolus vulgaris leucoagglutinin was iontophoretically delivered into different neocortical (frontal, parietal, occipital), allocortical (piriform) and mesocortical (insular, prefrontal) areas in rats. To identify the transmitter phenotype in pre- or postsynaptic elements, the tracer staining was combined with immunolabeling for either glutamate or GABA, or with immunolabeling for choline acetyltransferase or parvalbumin. Tracer injections into medial and ventral prefrontal areas gave rise to dense terminal arborizations in extended basal forebrain areas, particularly in the horizontal limb of the diagonal band and the region ventral to it. Terminals were also found to a lesser extent in the ventral part of the substantia innominata and in ventral pallidal areas adjoining ventral striatal territories. Similarly, labeled fibers from the piriform and insular cortices were found to reach lateral and ventral parts of the substantia innominata, where terminal varicosities were evident. In contrast, descending fibers from neocortical areas were smooth, devoid of terminal varicosities, and restricted to the myelinated fascicles of the internal capsule en route to more caudal targets. Ultrastructural studies obtained indicated that corticofugal axon terminals in the basal forebrain areas form synaptic contact primarily with dendritic spines or small dendritic branches (89%); the remaining axon terminals established synapses with dendritic shafts. All tracer labeled axon terminals were immunonegative for GABA, and in the cases investigated, were found to contain glutamate immunoreactivity. In material stained for the anterograde tracer and choline acetyltransferase, a total of 63 Phaseolus vulgaris leucoagglutinin varicosities closely associated with cholinergic profiles were selected for electron microscopic analysis. From this material, 37 varicosities were identified as establishing asymmetric synaptic contacts with neurons that were immunonegative for choline acetyltransferase, including spines and small dendrites (87%) or dendritic shafts (13%). Unequivocal evidence for synaptic interactions between tracer labeled terminals and cholinergic profiles could not be obtained in the remaining cases. From material stained for the anterograde tracer and parvalbumin, 40% of the labeled terminals investigated were found to establish synapses with parvalbumin-positive elements; these contacts were on dendritic shafts and were of the asymmetrical type. The present data suggest that corticofugal axons innervate forebrain neurons that are primarily inhibitory and non-cholinergic; local forebrain axonal arborizations of these cells may represent a mechanism by which prefrontal cortical areas control basal forebrain cholinergic neurons outside the traditional boundaries of pallidal areas.

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.

Amygdaloid input to transiently tyrosine hydroxylase immunoreactive neurons in the bed nucleus of the stria terminalis of the rat.

Several studies have reported transient expression of tyrosine hydroxylase in a subpopulation of neurons in the bed nucleus of stria terminalis of preadolescent rats. The tyrosine hydroxylase immunoreactive (TH) neurons, which are of small to medium size and often display a typical bipolar configuration, are confined to the intermediate part of the lateral bed nucleus. By the use of a combination of experimental tracer techniques and immunocytochemical methods, we have demonstrated that these neurons receive a significant number of amygdaloid afferents, which establish mostly symmetric synaptic contacts on the cell bodies and sparsely spined dendritic shafts of the TH neurons. TH neurons also receive a small number of tyrosine hydroxylase-positive terminals of unspecified origin.

Chronic prenatal ethanol exposure alters the normal ontogeny of choline acetyltransferase activity in the rat septohippocampal system.

In animal models of fetal alcohol syndrome (FAS), the hippocampus has been shown to be especially sensitive to the effects of prenatal ethanol exposure, exhibiting neuronal loss and alterations in neuritic process elaboration. We have characterized the influence of chronic prenatal ethanol treatment (CPET) on the postnatal expression of choline acetyltransferase (ChAT) in the hippocampus and the septal area that contains neurons that provide the primary cholinergic innervation to the hippocampus. On gestation days 1-22, pregnant rats were either fed an ethanol-containing liquid diet, pair-fed a calorically equivalent sucrose-containing diet, or given rat chow ad libitum. In Chow control animals, the ontogenetic progression of ChAT activity in the septal area and hippocampus was characterized by a significant period of upregulation during the 2nd and 3rd postnatal weeks, exhibiting and an approximate 5-fold increase (septal area) and 7-fold increase (hippocampus) by postnatal day 21 (P21). At P14, ethanol exposure reduced septal and hippocampal ChAT activity levels, compared with those of pair-fed offspring. ChAT activity reached control levels by P21 in ethanol-exposed pups, suggesting that the earlier decline in activity may reflect a delay in the ontogenetic upregulation. In addition, there was a trend toward increased septal and hippocampal ChAT activities at P1 and P7 in both liquid diet groups. This liquid diet-stimulated increase may mask the effects of ethanol on early postnatal ChAT expression in the septohippocampal system. The results suggest that prenatal ethanol exposure may influence factors that regulate the developmental expression of ChAT in the septohippocampal system.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal ethanol exposure alters neurotrophic activity in the developing rat hippocampus.

Extract made from hippocampus of rat pups exposed prenatally to an ethanol-supplemented diet was found to contain more neurotrophic activity at postnatal day 21 than that from animals exposed to control diets, when quantified in a dorsal root ganglion bioassay. This apparent upregulation was specific to hippocampal extract (cerebellar and forebrain/midbrain extracts were also assessed), and to this age (P1, P7, P14 and P60 extracts were also tested). It was suggested that this upregulation may be indicative of, or secondary to, trauma resulting from fetal ethanol exposure. It is speculated that such departures from the normal developmental timetable could contribute to anomalies seen in the fetal alcohol syndrome.

Alterations in responsiveness to ethanol and neurotrophic substances in fetal septohippocampal neurons following chronic prenatal ethanol exposure.

Pregnant Long-Evans rats were maintained on three diets: 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 gestation day 18, the fetuses were taken and cultures of septal and hippocampal neurons prepared. Neuronal survival and neurite outgrowth were compared in cultures from the three diet groups, using the following media supplements: ethanol (1.2, 1.8 or 2.4 g/dl), neurotrophic factors (nerve growth factor [NGF] with the septal cultures, basic fibroblast growth factor [bFGF] with the hippocampal cultures), or ethanol plus neurotrophic factors. Both the septal and hippocampal neurons responded to ethanol in a dose-dependent manner. The neurons from both populations from fetuses which had been exposed prenatally to ethanol, however, tolerated considerably higher ethanol concentrations before decreases in survival or outgrowth were seen. These ethanol-exposed neuronal populations were also less responsive to neurotrophic factors: in hippocampal cultures, process outgrowth was significantly enhanced by bFGF in control but not ethanol-derived cultures, and in septal and hippocampal cultures, the neurotrophic factors significantly ameliorated ethanol neurotoxicity in control cultures, but not in those from the ethanol-exposed fetuses. The possible relevance of these observations to the fetal alcohol syndrome is discussed.

Responsiveness of cultured septal and hippocampal neurons to ethanol and neurotrophic substances.

Dissociated septal and hippocampal neurons from E18 fetal rats were cultured with varying concentrations of ethanol (0.6-2.4 g/dl) and in cultures containing ethanol plus nerve growth factor (NGF) or basic fibroblast growth factor (bFGF). These substances have been shown to provide neurotrophic support for these populations and to afford neuroprotection against certain toxic substances or conditions applied to some neuronal populations. Both the septal and hippocampal neurons responded to ethanol in a dose-dependent manner. Survival of septal neurons was generally unaffected by initial ethanol concentrations of 0.6 and 1.2 g/dl but was considerably impaired by higher concentrations (1.8 and 2.4 g/dl), while neurite outgrowth was compromised by all ethanol concentrations except the lowest one applied. The hippocampal neurons survived ethanol concentrations up to 2.4 g/dl, although process extension was decreased in concentrations of 1.2 g/dl and higher. NGF or bFGF in the culture medium (in cultures without ethanol) did not affect neuronal survival or process outgrowth in either population, probably owing to the relatively high plating densities of the cultures. NGF did tend to have a moderate ameliorative effect on the ethanol neurotoxicity in the septal cultures, however, and was slightly effective in this regard in hippocampal cultures at intermediate ethanol concentrations (1.8 g/dl). High concentrations of ethanol (2.4 g/dl) reduced the proportion of cholinergic cells in the septal preparations by approximately 50%. This neuronal loss could be reversed by inclusion of high concentrations of NGF in the culture medium (100 ng/ml) but not by a lower concentration (20 ng/ml). bFGF provided some protection against ethanol cytotoxicity with respect to both populations. The implications of these results for studies of fetal alcohol effects are discussed, as well as their relation to prior reports of trophic factor neuroprotection.

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.

Chronic ethanol alters CNS cholinergic and cerebellar development in chick embryos.

Chick embryos were given daily injections of ethanol (approximately 30 mg/day) either chronically from embryonic days 4 to 15 (E4-E15) or E18, or acutely from E15 to E18. Untreated and saline-injected embryos served as controls. Although morphological indicators of developmental age were not different among groups, chronic ethanol reduced weights of several brain regions. Similar to rodent models of prenatal ethanol exposure, chronic ethanol treatment reduced cerebellar Purkinje cell numbers compared to controls. Chronic but not acute ethanol exposure resulted in a significant reduction in choline acetyltransferase activity in the optic tectum (OT) and forebrain (FB) compared with controls. This study demonstrates that the chick embryo is a viable model to investigate the effects of ethanol exposure on CNS development. Unlike the mammalian fetus, the avian embryo is isolated from maternal interactions and may prove more useful in investigating the mechanisms by which ethanol directly influences brain development.

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.

Neurotrophic activity in embryonic chick brain: early appearance and differential regional distribution.

Homogenate extracts were prepared from various regions of the embryonic chick brain from E6 to E19. The extracts were applied to cultured dorsal root ganglia (DRGs), sympathetic ganglion cells, PC12 cells and E5 spinal cord neurons. Potent neurotrophic activity with respect to DRGs was found in an extract from the forebrain (FBX) from the earliest stage assayed (E6). This activity was comparable to that of nerve growth factor (NGF), and it was not appreciably altered during later stages. By E8, extracts prepared from the optic lobe also induced responsiveness, although to a lesser extent than the FBX and NGF. At E12-19, extracts from the hippocampus (HCX) and cerebellum (CBX) were also tested. Activity found in the HCX resembled that in the FBX, but responsiveness to the CBX was similar to that in control (unsupplemented) cultures. The responsiveness of sympathetic neurons in the FBX was comparable to that with NGF. The FBX induced only moderate differentiation of PC12 cells, and spinal cord neurons were not responsive. Antibodies to NGF and basic fibroblast growth factor did not diminish the influence of the FBX, and the FBX and NGF when combined had synergistic effects. The results demonstrate potent neurotrophin-like activity in the embryonic brain at much earlier stages than had previously been seen, with differential regional and temporal distributions.

Quantitative mapping of glutamate presynaptic terminals in the supraoptic nucleus and surrounding hypothalamus.

Although the hypothalamus is generally regarded to have low levels of glutamate receptors, anatomical and physiological studies have provided consistent evidence implicating glutamate as a potential transmitter for the control of neuroendocrine cell activity. To clarify the extent of the contribution of synapses utilizing glutamate for control of vasopressin/oxytocin neuroendocrine cells, we mapped the density and location of glutamate immunoreactive terminals in the supraoptic nucleus and surrounding hypothalamus. Colloidal gold particle densities in presynaptic terminals were measured from electron micrographs of: (1) the magnocellular neuroendocrine cell perikarya (main body of the supraoptic nucleus), (2) the dendritic field of the magnocellular neuroendocrine cells (ventral dendritic neuropil) and (3) the hypothalamic perinuclear zone dorsal to the supraoptic nucleus. In addition, serial sections were stained, alternatively, for glutamate or GABA to determine glutamate staining in GABA cells. Terminals with high glutamate immunoreactivity were clearly distinguished from the glutamate precursor staining found in GABA terminals and were abundant at all rostral-caudal levels within each region. The number of glutamate terminals identified in each region was similar but represented a very high proportion of all terminals in the ventral dendritic neuropil (38%) vs. the main body of the supraoptic nucleus and the perinuclear zone (20-22%). The regional variation in the relative proportion of glutamate terminals was determined largely by differences in the number of non-glutamate terminals within each region. Glutamate and GABA terminals together accounted for over two-thirds of the innervation of vasopressin/oxytocin neuroendocrine cells. No systematic relationship was observed between excitatory and inhibitory inputs on the same cell. These results suggest that glutamate is the predominant excitatory transmitter used for control of vasopressin/oxytocin cells. The relative contribution of glutamate neurotransmission to a particular region will depend, in part, on the number and type of competing non-glutamate terminals.

Ethanol exposure affects trophic factor activity and responsiveness in chick embryo.

Chick embryos were chronically exposed to either ethanol (approximately 30 mg/d) or saline, from E4-E13. Homogenate extract was prepared from forebrain tissue from E16 experimental and control embryos and was applied to cultured dorsal root ganglia (DRG). Neurotrophic activity in the forebrain extract (FBX) was significantly reduced in the ethanol-treated embryos compared to saline controls, both in terms of influences on neuronal survival and process elaboration. In addition, E8-9 DRGs from embryos exposed to ethanol from E4 were less viable in the presence of NGF than were those from controls. DRG survival in the presence of E16 FBX (from untreated embryos) was not different following ethanol treatment, but neurite production was significantly reduced. These results suggest that neurotrophic factor content and responsiveness may be appreciably altered following chronic prenatal ethanol exposure. Such alteration could underlie certain CNS anomalies seen in the fetal alcohol syndrome.

Total blood cholesterol and contributory risk factors in an adolescent population.

Total blood cholesterol (TBC) levels and contributory risk factors in an adolescent population were investigated. Existing TBC screening records were reviewed on 452 10th grade students in two schools. The sample consisted of 52% males and 48% females whose mean age was 15.47 years. Blood samples were analyzed by the Reflotron. Risk factors investigated included age, gender, ethnicity, individual and family history of high cholesterol, history of high blood pressure, smoking tobacco products, and oral contraceptive use. The sample mean for TBC was 150.61. The only significant factors identified by ANOVA were gender and use of oral contraceptives. Females had higher TBC levels than males, and females who used oral contraceptives had higher TBC levels than nonusers.

PIP: Total blood cholesterol levels were screened by a fingerstick-desktop method in 452 10th grade students from 2 schools as part of a cholesterol task force that included health education on risk factors. The children averaged 15.47 years. The Reflotron analyzer (Boehringer Mannheim Diagnostics) used non-fasting blood. The overall mean TBC level was 150.61 mg/dl. By risk groups, 76% were low risk, with TBC levels ranging from 100-169; 11% were moderate risk, ranging 1270-185; and 12% were high risk, with levels ranging from 170-400. Girls' levels averaged 160.15, significantly higher than boys' 141.95, consistent with prior published reports. The 10 girls using oral contraceptives had a mean TBC of 188.20, compared to 158.85 for nonusers. There were no significant differences for other potential risk groups: ethnicity, history of high blood pressure, current smoking, individual or family history of high cholesterol. School A had a higher socioeconomic background than School B. The mean cholesterol level for School A was 149.02, compared to 152.08 for School B. Schools need more health education on risk factors and lifestyle, and preventive health intervention.