Biomedical subjects

D W Walker

Publications and source records attributed to D W Walker.

At least 73 records · Page 4Linked to original sources

PubMed1996

Cultured postnatal rat medial septal neurons respond to acute ethanol treatment and nerve growth factor by changing intracellular calcium levels.

Ethanol neurotoxicity results in the loss of neurons during the development of the nervous system. Nerve growth factor (NGF) can ameliorate the neurotoxic effects of ethanol (EtOH) in rat medial septal (MS) neurons. These experiments study the effects of EtOH and NGF on neuronal calcium (Ca2+) homeostasis in cultured postnatal day of birth (PO) rat MS neurons. Previously, we observed that EtOH and NGF modulate intracellular Ca2+ levels [Ca2+]i) in unstimulated and high potassium stimulated (30 mM KCl) cultured rat embryonic day 21 (E21) MS neurons (Webb et al., Brain Res 701:61-74, 1995). The purpose of the present study was to explore whether the effects of EtOH and NGF on Ca2+ homeostasis were altered by developmental stage. The hypotheses tested were the following: treatment with EtOH affects Ca2+ homeostasis in postnatal day of birth (PO) rat MS neurons by causing transient and persistent changes in [Ca2+]i; NGF modulates Ca2+ homeostasis in MS neurons by regulating [Ca2+]i; the action of NGF changes the response of MS neurons to EtOH, thus altering Ca2+ homeostasis; and that EtOH and/or NGF effects on Ca2+ homeostasis are developmentally regulated. Our results indicated that behaviorally relevant levels of EtOH caused a rapid transient increase in basal [Ca2+]i, whereas there was no effect of NGF on basal [Ca2+]i. Ethanol and NGF interacted, resulting in the lowering of [Ca2+]i. During stimulation with high K+, EtOH inhibited the change in [Ca2+]i. NGF partially ameliorated this effect of higher levels of EtOH, allowing [Ca2+]i to increase. NGF and the lowest level of EtOH potentiated the high K+ stimulated increase in [Ca2+]i. Ethanol and NGF effects on [Ca2+]i were different in the PO neurons compared with our previously published observations in E21 neurons. Therefore, these data suggest that EtOH neurotoxicity and NGF protection involve mechanisms that regulate neuronal Ca2+ homeostasis, and the magnitude of these effects depend on developmental stage.

Animals↗

PubMed1996

Long-term effects of chronic ethanol on muscarinic receptor binding in rat brain.

Effects of chronic ethanol treatment (CET) on muscarinic acetylcholine receptor (mAChR) binding properties were investigated via quantitative autoradiography in rats maintained on an ethanol-containing liquid diet for 28 weeks and withdrawn from ethanol for 8 weeks before harvesting of tissues. Controls received an identical diet in which sucrose was substituted isocalorically for ethanol. Maximal binding of the radiolabeled mAChR antagonist quinuclidinyl benzilate ([3H]QNB) was not reduced in hippocampal area CA1, dentate gyrus, neocortex, striatum, or thalamus, suggesting that CET results in no significant mAChR loss in these regions. Binding affinities of the cholinergic agonist carbachol to mAChRs were unaffected by CET in each of these regions, as determined by competitive displacement of [3H]QNB labeling. These results suggest that CET-induced functional deficits in brain cholinergic responses are not due to direct effects of CET on mAChR binding properties.

Animals↗

PubMed1995

Ethanol and nerve growth factor effects on calcium homeostasis in cultured embryonic rat medial septal neurons before and during depolarization.

Ethanol and nerve growth factor (NGF) affect the survival of cholinergic neurons in the rat medial septum. To investigate whether calcium (Ca2+) homeostasis in these neurons is affected by ethanol or NGF treatment, changes in intracellular free Ca2+ concentration ([Ca2+]i) were studied in embryonic (E21) cultured medial septal neurons before stimulation (basal) and during stimulation with high potassium (K+). Changes in [Ca2+]i across time were measured in cultures of neurons treated without ethanol or with 100, 200, 400, or 800 mg% ethanol with NGF (+NGF) or without NGF (-NGF). Changes in [Ca2+]i were analyzed from fluorescence images, using indo-1. The effect of ethanol or NGF treatment was to reduce the rise in basal [Ca2+]i. The combination of ethanol and NGF treatment in +NGF neurons led to increases in basal [Ca2+]i with the greatest increase in basal [Ca2+]i occurring with 200 mg% ethanol. The effect of ethanol or NGF was to increase [Ca2+]i during stimulation with high K+. The greatest increases in [Ca2+]i occurred with 100 and 800 mg% ethanol. Together, ethanol and NGF treatment in +NGF-treated neurons led to significantly greater increases or decreases in K+ stimulated changes in [Ca2+]i compared to similarly treated -NGF neurons. We conclude that in medial septal neurons (before and during depolarization) changes in Ca2+ homeostasis occur in the presence of ethanol or NGF. The changes in [Ca2+]i following ethanol treatment are greater when NGF is present.

Animals↗

PubMed1995

Perturbation of target-directed neurite outgrowth in embryonic CNS co-cultures grown in the presence of ethanol.

Studies were conducted to determine the influence of ethanol on target-directed fiber outgrowth in culture, using embryonic chick spinal cord-muscle, and fetal rat septal-hippocampal co-cultured explants. Process extension from the spinal cord and septal explants in control cultures was selectively oriented toward the appropriate target tissue. Ethanol in the culture medium (500 mg/dl) eliminated this target-oriented outgrowth in both systems, although the overall extent of neurite outgrowth was not affected. In an effort to further characterize the source of this disruption, target explants were grown alone, with and without ethanol, and the target-conditioned culture media was subsequently harvested and placed on newly plated spinal cord or septal explants, to determine whether ethanol decreased the target production of soluble substances. To determine whether deposition of substrate-bound materials by the target tissue was affected by ethanol, spinal cord or septal explants were plated in wells which had previously been occupied by the appropriate target tissue. These studies revealed that ethanol significantly inhibited production of soluble and substrate-bound materials by muscle explants, but not by hippocampal explants. It was concluded that the ethanol-induced loss of target-directed neurite outgrowth in the spinal cord explants could be accounted for primarily by the attenuated production of neurotropic/neurotropic substances by the muscle tissue. The loss of target-directionality in the septal explants appeared to be due to other factors, possibly related to ethanol-induced compromise of the capacity of the septal neurons to respond appropriately to target-derived neurotrophic/neurotropic substances. The implications of these results for the fetal alcohol syndrome are considered.

Animals↗

PubMed1995

Chronic ethanol administration decreases brain-derived neurotrophic factor gene expression in the rat hippocampus.

We have previously demonstrated that chronic ethanol consumption decreases neurotrophic activity in hippocampal extracts, as assessed by a chick dorsal root ganglia bioassay, but has no effect on hippocampal NGF mRNA or NGF protein levels. We presently report that hippocampal mRNAs encoding neurotrophin-3 and basic fibroblast growth factor are also unaffected. However, in contrast, brain-derived neurotrophic factor mRNA is reliably decreased, thereby suggesting that ethanol-induced damage of the septohippocampal system may at least partially result from an ethanol-induced decrease in hippocampal brain-derived neurotrophic factor expression.

Animals↗

PubMed1995

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.

Aging↗

PubMed1995

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.

Animals↗

PubMed1995

A method for labeling embryonic rat medial septal region projection neurons, in vitro, using fluorescent tracers.

A retrograde labeling method is described in which rat embryonic (E18, E21) and postnatal (P1) medial septal neurons were labeled with succinyl wheat germ agglutin-fluorescein, fluorescent green microspheres, or 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil) following in vitro hippocampal injections. The brains were removed and immediately immersed in oxygenated Tyrode solution. Dye was pressure injected into the hippocampus bilaterally. After incubating the brain in oxygenated Tyrode, the medial septal region was removed. The neurons were dissociated and cultured at medium density in 35 mm dishes with a hole in the bottom covered by a coverslip with a grid. The neurons were observed with a low light system, and cell counts were made at 5, 24, and 48 h. Labeled and unlabeled neurons showed considerable neurite outgrowth and acetylcholinesterase activity in culture. Highly reproducible labeling was obtained, with Dil giving the best results. Dil labeled the neurons in vitro, was retained during culture for 1 week, and was compatible with cell survival.

Acetylcholinesterase↗

PubMed1995

Functional development of fetal limb muscles: a review of the roles of activity, nerves and hormones.

Animals that are immature at birth with respect to postural and locomotor control (e.g. cats, rats) possess incompletely differentiated 'fast-twitch' and 'slow-twitch' muscles at birth; full development proceeds slowly in the postnatal period and involves myogenic, hormonal, neural and behavioural factors. The gradual emergence of specific motor patterns and the exercise of individual muscle groups is thought to play a major role in the final development of each muscle and the fibre types which comprise them. In contrast, precocial species such as the sheep are born with skeletal muscles, especially those of the limbs, which are fully differentiated at birth. The relative importance of neural and hormonal factors in allowing this functional specialization to occur in the presumed absence of significant load-bearing exercise in the intrauterine environment is unclear. In this brief review, the changes which occur in contractile function and fibre type differentiation during the last one-third of gestation in fetal sheep are described, and some of the factors which influence this development are considered.

Animals↗

PubMed1995

Hypoxic inhibition of breathing and motor activity in the foetus and newborn.

In fetal animals hypoxia of rapid onset causes cessation of breathing movements, electro-ocular activity and decrease of muscle tone. These effects last several hours and are in contrast to the hypernoea and behavioural activation which occurs during hypoxia soon after birth and in the adult. Transection and lesion studies in fetal sheep suggest that hypoxia activates a descending inhibition of respiratory and other motor activities which either originates in the pons or is conveyed to medullary and spinal levels of the neuraxis by fibres through the pons in the region of the Kolliker-Fuse nucleus. Recently, using FOS immunohistochemistry we have identified cells in the medial parabrachial complex which are activated by hypoxia in fetal sheep, but not newborn lambs. It is proposed that these cells have descending inhibitory connections with respiratory and spinal motor pathways, but the precise anatomy and neurochemistry of such pathways is unknown. It is not known if the parabrachial cells are directly sensitive to low Po2 or receive input from other centres or peripheral receptors which monitor arterial Po2 in the foetus. Nor is it known why these cells are not activated by low Po2 after birth.

Adult↗

PubMed1995

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)

Animals↗

PubMed1995

Cardiovascular responses to heat stress in late gestation fetal sheep.

Heat stress during pregnancy in sheep is associated with respiratory alkalosis in both the mother and fetus, and, if prolonged, fetal growth is retarded. In seven pregnant sheep at 130-137 days gestation we used 15 microns diameter radioactive microspheres to determine the effect of raising the environmental temperature from 20 to 43 degrees C for 8 h on uteroplacental blood flows and the distribution of cardiac output in the ewe and fetus. Fetal cardiac output increased slightly from 47.0 +/- 3.2 (mean +/- S.E.M.) to 54.0 +/- 3.6 ml min-1 (100 g tissue)-1, fetal arterial pressure and heart rate were unchanged, and total vascular conductance in the fetus increased significantly from 12397 +/- 1111 to 14732 +/- 1569 ml min-1 kg-1 mmHg-1 (P < 0.01). Tissue blood flows (in ml min-1 (100 g)-1) increased significantly (P < 0.05) in the fetal body (e.g. nasal mucosa, torso and foreleg skin, adrenal, thyroid and thymus glands, brown and omental fats, heart, urinary bladder and carcass) and the fetal brain (e.g. cerebellum, cerebral grey matter, cervical spinal cord and pituitary gland). These regional vasodilatations occurred despite a significant fall (P < 0.01) in fetal arterial O2 saturation (55.2 +/- 1.8 vs. 38.6 +/- 2.4%), PO2 (18.1 +/- 0.7 vs. 13.5 +/- 0.8 mmHg) and PCO2 (51.0 +/- 1.8 vs. 36.1 +/- 2.3 mmHg); under normothermic conditions hypoxia is associated with peripheral vasoconstriction. Because hypocapnia would also be expected to cause cerebral vasoconstriction it is suggested that during hyperthermia, hypoxia- and hypocapnia-induced vasoconstrictions are reduced by the release of vasodilator substances, or a decrease of sympathoadrenal effector responses. Blood flow to the fetal and maternal sides of the placenta did not change during the heat stress, suggesting that perfusion-dependent transfer of heat from fetus to mother across the placenta does not increase under hyperthermic conditions.

Animals↗

PubMed1995

Oxygen, glucose, and lactate uptake by fetus and placenta during prolonged hypoxemia.

Our aim was to compare the effects of short (4 h) and prolonged (24 h) periods of reduced uterine blood flow (RUBF) on fetal and placental uptake of O2, glucose, and lactate. In pregnant sheep, uterine and umbilical blood flows were measured under normal conditions and after 4 and 24 h of RUBF. A 50% reduction in uterine blood flow caused a 56% reduction in fetal arterial O2 saturation (SaO2). Umbilical blood flow increased from 325 +/- 33 to 378 +/- 32 ml.min-1.kg-1 (P < 0.05) after 4 h but was not different from pre-RUBF values after 24 h. O2 uptake by the gravid uterus was not altered by RUBF, due to an increase (84%) in uterine O2 extraction. Similarly, uteroplacental and fetal O2 consumptions and fetal glucose uptake were not affected by RUBF, whereas uteroplacental glucose uptake was significantly reduced after 4 h (by 42%) and 24 h (by 58%) of RUBF. Fetal lactate uptake was greatly reduced from 78.7 +/- 15.5 to -167 +/- 57 mumol.min-1.kg-1 after 4 h and to -198 +/- 80 mumol.min-1.kg-1 after 24 h of RUBF; negative values indicate placental lactate uptake from the fetal circulation. Thus, although RUBF significantly reduced fetal SaO2, fetal and uteroplacental O2 consumptions did not change. In addition, although fetal glucose uptake was not altered by RUBF, during RUBF the placenta became a major site of lactate clearance from the fetal circulation.

Animals↗

PubMed1994

D2 dopamine receptor mRNA distribution in cholinergic and somatostatinergic cells of the rat caudate-putamen and nucleus accumbens.

An in situ hybridization procedure that identifies cells expressing D2 dopamine receptor mRNA was combined in double-labelling studies with immunohistochemical procedures that identify cells expressing either choline acetyltransferase (ChAT) or somatostatin. D2 receptor mRNA was detected in almost all of the ChAT positive caudate-putamen cells, approximately half of the ChAT positive nucleus accumbens cells and none of the somatostatin-positive cells in either brain region.

Acetylcholine↗

PubMed1994

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.

Acetylcholinesterase↗

PubMed1994

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.

Animals↗

PubMed1994

Forehead and forearm skin blood flows in newborn infants measured by laser Doppler flowmetry: short-term variability and relationship to sleep states.

Laser doppler flowmeters were used to measure blood flow in the skin of the forehead and volar surface of the forearm of infants at 2-9 days of age, and at 8-12 weeks of age. At both ages mean skin blood flow was higher during active sleep compared to quiet sleep. In infants up to 9 days of age, mean skin blood flow was higher in forehead skin compared to forearm skin. Skin blood flow was highly variable at both recording sites, and was significantly higher and more variable during active compared to quiet sleep in the forehead, but not in the forearm. The results indicate that recordings of skin blood flow over at least two cycles of active and quiet sleep are needed to describe the normal variation with sleep state. The factors which regulate forehead and forearm skin blood flows may differ in importance between the two sites, perhaps reflecting the different thermoregulatory importance of cutaneous blood flow in the forehead and forearm.

Forearm↗

PubMed1994

Chronic alcohol ingestion: nerve growth factor gene expression and neurotrophic activity in rat hippocampus.

Chronic ethanol treatment induces memory deficits accompanied by anatomical and biochemical changes in basal forebrain and hippocampus. Cholinergic neurons in the septohippocampal pathway are especially vulnerable to alcohol neurotoxicity. Several studies showed that an adequate supply of neurotrophins, such as Nerve Growth Factor and Brain-Derived Neurotrophic Factor, is required for the normal function and survival of cholinergic neurons in basal forebrain and medial septal nuclei. We tested the hypothesis that chronic alcohol ingestion may alter the gene expression level of Nerve Growth Factor in hippocampus, the major source of neurotrophins to the cholinergic neurons in the septohippocampal pathway. We measured Nerve Growth Factor protein and Nerve Growth Factor mRNA contents using sensitive two-site ELISA and Northern analysis. We also tested the endogenous neurotrophic activity, including and excluding Nerve Growth Factor, contained in 5%, 2%, 1%, 0.5% and 0.1% (w/v) hippocampal tissue extracts on sympathetic ganglia neurons. Twenty-eight weeks of chronic ethanol treatment did not reduce Nerve Growth Factor protein, Nerve Growth Factor mRNA, or total neurotrophic activity contained in the rat hippocampus when measured on sympathetic ganglia neurons.

Alcoholism↗