OXYGEN DYNAMICS IN THE EPILEPTIC BRAIN: FROM THE LARGER SCALE HIPPOCAMPAL FORMATION TO INDIVIDUAL NEURONS
Open Access
- Author:
- Ingram, Justin
- Graduate Program:
- Neuroscience
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 29, 2011
- Committee Members:
- Steven Schiff, Dissertation Advisor/Co-Advisor
Steven Schiff, Committee Chair/Co-Chair
Bruce Gluckman, Committee Member
Jian Xu, Committee Member
Bernhard Luscher, Committee Member
James Robert Connor, Committee Member
Ping Li, Committee Member - Keywords:
- FRET
Epilepsy
seizure
hypoxia
hippocampus
liposomes - Abstract:
- Oxygen (O2) is an essential requirement for the mammalian brain. An adequate and continuous supply is necessary to produce the cell’s primary source of energy. With minimal O2 storage ability, a tightly regulated balance between supply and demand is required to maintain proper brain functioning and if this balance becomes altered, so does physiological activity. Although all mammalian cells perish during prolonged states of O2 deprivation, the initial cellular response will inevitably cause membrane depolarization. This hypoxic depolarization becomes increasingly important during pathological brain states such as epilepsy, where excessive metabolic demands temporally deplete local O2 availability. We hypothesize that these metabolic changes in [O¬2] are important for influencing network excitability and may be more than just a consequence of an epileptic attack, but also a potential cause. To date, understanding the relationship between neuronal activity and O¬2 dynamics has been restricted by technical limitations of current sensing techniques. The lack of resolution, both spatially and temporally, as well as specificity, has hindered our understanding of brain metabolism. These dynamics are critical for understanding the basic cellular responses to metabolic changes as well as decoding the complex signals generated by neuroimaging techniques (e.g. intrinsic optical signaling, functional magnetic resonance imaging) that localize brain activity based off such changes In order to improve the spatiotemporal limitations of O2 sensing, we have designed a ratiometric fluorescence resonance energy transfer (FRET) excited optical sensor that allows interstitial [O¬2] to be monitored from an entire hippocampal slice to microdomains around single cells with high spatial and temporal resolution. In comparison with other direct O¬2 sensing techniques (polarography, probes encapsulated by biologically localized embedding, etc.), our sensors are easily calibrated to allow quantitative measurements, do not require stirring, and most importantly do not consume O¬2. Each ratiometric sensing device is fabricated from an optode matrix consisting of the oxygen sensitive dye platinum (II) octaethylporphine ketone (PtOEPK), coupled with O2 insensitive nano-quantum dots (NQDs) entrapped within a highly permeable and biologically inert polymer matrix. Using such sensors we probed entire hippocampal slices to microdomains around individual cells, focusing on the interactions between excitatory and inhibitory neurons within the CA1 to understand how metabolic induced hypoxia is influenced by the initiation, propagation, and termination of spontaneously induced 4-AP seizure events. With simultaneous optical and electrical recordings we found the majority of O2 metabolism to be localized within the pyramidal cell body layer of the CA1, and not necessarily dependent on neuronal firing activity as hypoxic changes occur up to several seconds before seizure onset can be determined electrically. This preseizure hypoxic change was also artificially induced by changing the bath [O2] perfusing our slices. With our FRET sensors we have quantified a narrow range of [O2,], similar to those found prior to epileptiform activity induced by 4-AP, which can destabilize a seemingly unexcitable network into increased burst firing and even into a seizure-like state.