A&S Graduate Studies
[PAST EVENT] Wiktor Phillips, Applied Science - Ph.D. Dissertation Defense
Abstract: Breathing is an important rhythmic motor behavior whose underlying neural mechanisms can be studied in vitro. The study of breathing rhythms in vitro has depended upon reduced preparations of the brainstem that both retain respiratory-?active neuronal populations and spontaneously generate respiratory-?related motor output from cranial and spinal motor nerves. Brainstem-?spinal cord en bloc preparations and transverse medullary slices of the brainstem have greatly improved the ability of researchers to experimentally access and thus characterizations important in respiratory rhythmogenesis. These existing in vitro preparations are, however, not without their limitations. For example, the window of time within which experiments may be conducted is limited to several hours.Moreover, these preparations are poorly suited for studying sub-cellular ion channel distributions and synaptic integration in dendrites of rhythmically active respiratory interneurons because of tortuous tissue properties in slices and en bloc, which limits imaging approaches. Therefore, there is a need for an alternative experimental approach. Acute transverse slices of the medulla containing the preB?tzinger complex (preB?tC) have been exploited for the last 25 years as a model to study the neural basis of inspiratory rhythm generation. Here we transduce such preparations into a novel organotypic slice culture that retains bilaterally synchronized rhythmic activity for up to four weeks in vitro. Properties of this culture model of inspiratory rhythm are compared to analogous acute slice preparations and the rhythm is confirmed to be generated by neurons with similar electrophysiological and pharmacological properties. The improved optical environment of the cultured brain tissue permits detailed quantitative calcium imaging experiments, which are subsequently used to examine the sub cellular distribution of a transient potassium current, IA, in rhythmically active preB?tCinterneurons. IA is found on the dendrites of these rhythmically active neurons,where it influences the electrotonic properties of dendrites and has the ability to counteract depolarizing inputs, such as post-?synaptic excitatory potentials, that are temporally sparse in their occurrence (i.e., do not summate). These results suggest that excitatory input can be transiently inhibited by IA prior to its steady-?stateinactivation, which would occur as temporally and spatially summating synapticinputs cause persistent depolarization. Thus, rhythmically active interneurons are equipped to appropriately integrate the activity state of the inspiratory network,inhibiting spurious inputs and yet yielding to synaptic inputs that summate, which thus coordinates the orderly recruitment of network constituents for rhythmic inspiratory bursts. In sum, the work presented here demonstrates the viability and potential usefulness of a new experimental model of respiratory rhythm generation,and further leverages its advantages to answer questions about dendritic synaptic integration that could not previously be addressed in the acute slice models of respiration. We argue that this new organotypic slice culture will have widespread applicability in studies of respiratory rhythm generation.
Bio: Wiktor Phillips grew up in Williamsburg, VA and attended William & Mary from 2006 until 2010, earning a bachelor?s degree in Neuroscience. After a year hiatus living in Poland, Wiktor began graduate studies at the Department of Applied Science in the laboratory of Dr. Christopher Del Negro. He was initially trained in electrophysiology, but also received instruction in primary cell culturing techniques via collaboration with the laboratory of Dr. Nadine Kabbani at the Krasnow Institute of George Mason University. His work with calcium signaling in the growth cones of developing hippocampal neuron axons provided the foundational knowledge to pursue more advanced culturing techniques utilizing brainstem slices containing respiratory pattern generating networks. In 2013, he began a collaboration with Dr. Jens C. Rekling at the University of Copenhagen in order to learn brain slice culturing techniques and ultimately went on to develop a novel in vitro model of inspiratory rhythmogenesis. Using this culture model, Wiktor has investigated the integrative properties of dendrites in rhythmically active neurons of the preB?tzinger complex?the kernel for respiratory rhythm generation.
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