HEALTH-RELATEDNESS OF RESEARCH Despite the U.S. Surgeon General's advisory in 2005, warning women that no amount of alcohol can be considered safe for the fetus, 11-13% of women report drinking during pregnancy and 3% binge drink or drink frequently. It is estimated that no fewer than 1% of babies are born with some measurable alcohol-induced damage. Prenatal alcohol exposure can have variable effects on the developing nervous system, from a severe constellation of birth defects called Fetal Alcohol Syndrome (FAS) to more subtle but still disabling cognitive and behavioral impairments that are collectively referred to as Fetal Alcohol Spectrum Disorders (FASD). The developing brain is particularly sensitive to alcohol throughout gestation, even when exposure is limited to only one trimester. Neurologic defects associated with prenatal alcohol include craniofacial abnormalities, microencephaly, cerebellar hypoplasia, and agenesis of the corpus callosum. Key histopathologies suggest ethanol disrupts neural cell proliferation, neuronal migration, the outgrowth of axons and dendrites and the guidance of axons toward their synaptic targets. Efforts to prevent FAS and FASD have been unsuccessful, in part, because women may drink socially before they know they are pregnant and because alcoholics are often unable to stop drinking during pregnancy. Clinical management of both populations of patients would be improved by the availability of pharmacologic therapeutics that could block or reverse the damaging effects of alcohol on the fetus. A number of potential approaches are under investigation, but progress has been limited by our poor understanding of the basic mechanisms underlying ethanol effects on cells in the developing brain. Dr. Lindsley is a Developmental Neurobiologist whose research is focused on identifying the cellular mechanisms underlying disruption of neuronal development by ethanol with the goal of identifying targets for development of therapeutics to prevent or ameliorate FASD. TECHNIQUES EMPLOYED The use of low-density embryonic rat hippocampal pyramidal neuron cultures is integral to the objectives of Dr. Lindsley's research. These cultures are the most extensively characterized primary culture of mammalian CNS neurons. Neurons in these cultures develop axons, dendrites and synapses according to a stereotyped sequence of events that closely mimics their development in vivo. In addition, the culture conditions permit high-resolution imaging of living neurons extending processes and turning in response to diffusible guidance molecules. Dr. Lindsley's lab employs real-time monitoring of calcium flux in growth cones, confocal microscopy, immunocytochemistry, electrophysiology and transient transfection with molecular reagents to manipulate expression of key signaling intermediates regulating cytoskeletal organization. Research in Dr. Lindsley's laboratory has revealed remarkable similarities between effects of ethanol on neurons in theses cultures and its effects in situ. KEY DISCOVERIES AND CURRENT RESEARCH AIMS Results of studies in Dr. Lindsley's laboratory have demonstrated that ethanol has differential effects on growing axons and dendrites and that the timing, dose and duration of ethanol exposure all influence its effects on process outgrowth. These factors also affect cell survival in the presence of ethanol, and whether the neuron can recover upon ethanol withdrawal. More recent studies using confocal microscopy and high temporal resolution digital time-lapse images of axonal growth have shown that ethanol delays initial formation of axons, disrupts their periodic retractions and changes the organization of F-actin in their growing tips (called growth cones). These findings have provided novel insights that bear on several key questions of importance to public health, including "when are developing neurons most vulnerable to ethanol's effects?" and "can neurons recover from the morphologic effects of ethanol exposure during their development?" Dr. Lindsley's laboratory was the first to demonstrate that when immature neurons are exposed to ethanol their newly developed axons respond abnormally to certain guidance factors. Considered together, these finding are consistent with disruption of cellular mechanisms that regulate motile activities (e.g. dynamics of actin and microtubules). Currently, Dr. Lindsley's laboratory is testing two specific hypotheses regarding the mechanisms underlying effects of ethanol on axon growth and guidance, 1) that ethanol disruption of growth cone actin and microtubule organization involves signaling via small RhoGTPases, especially RhoA, Rac1 and Cdc42, and 2) that modulation of calcium signaling contributes to effects of ethanol on process growth and guidance. In collaboration with investigators outside of AMC, additional studies are planned to test experimental agents (currently in preclinical trials to protect against birth defects caused by prenatal ethanol), to determine their ability to prevent ethanol disruption of axon growth and guidance.
Click on this link to view an example of live-cell time-lapse imaging that shows typical neuron development over a 7 hour period beginning about 7 hours after being removed from the brain.
The neuron in the movie was removed from the hippocampus of an embryonic rat and placed in culture. Five-hundred images were captured with a digital camera attached to the microscope, at a rate of 1 image every 2.5 mins. First, the neuron makes several short processes (these are the precursors of dendrites), then one rapidly elongates to become the axon. This sequence of development is characteristic of neurons in the intact brain, as is the highly dynamic and motile behavior of the growth cones (at the tips of each process). The cell body diameter is approx. 20 micrometers.
Detailed methods: Cell culture: Low-density dissociated-cell cultures were prepared from embryonic rat hippocampi as described by Kaech and Banker (Nature Protocols 1(5): 2406-2415, 2006). Neurons plated on poly-D-lysine coated glass coverslips were affixed with sterile silicone grease to the substrate of a Smart Slide dish (WaferGen), with cells facing up. The culture medium was serum-free MEM with N2 supplements (Invitrogen) and 0.6% wt/vol glucose, conditioned for 2 days by confluent rat cortical astrocytes. Smart Slide-50 (WaferGen) maintained cultures at optimal temperature, pH and optical clarity.
Microscopy: IX-70 Olympus inverted microscope, Ludl Stage with insert modified to accommodate the Smart Slide dish, UPlanFl 10x/0.30 Phase objective, 1/2" Dage MTI CCD-300-RC B+W camera and Uniblitz shutter. Automated capture was programmed using ImageProPlus software with ScopePro and StagePro modules.