Rationale: Use of integrated technologies allows the physiologist to monitor multiple cellular parameters in synchrony and real-time. These include mitochondrial health, Ca²⁺ homeostasis and membrane potential. The former is vital to cells such as cardiac myocytes and neurons which are vulnerable to mitochondrial deficiency, which can lead to possible heart failure and neural degeneration respectively. Integrated technology platforms developed and available in the BioCurrents Research Center (BRC) provide the ability to correlate mitochondrial function with cellular activity. The collective aim of these studies is to assess metabolic changes in single cells during periods of excitation and oxidative stress. This will have important implications towards developing interventions that target mitochondrial cytopathies.

1A	1B

Results:
Metabolic basis of Neuronal Disease

Previous work on cellular bioenergetics, particularly oxygen consumption, has only been able to assess metabolic parameters at the level of cell populations. This leads to poor temporal relationships in regards to drug responsiveness as well as inclusion of glial metabolism.  By using non-invasive oxygen microsensors (Fig. 1A)  in the self-referencing mode, the BRC routinely assesses oxygen consumption from single neurons (Fig. 1A).  Pharmacological agents are now applied via computer controlled syringe pumps to synchronize the time of application and to avoid thermal drift of the apparatus (Fig. 1B). In this example, higher current values signify higher oxygen consumption, while oligomycin blocks state III respiration and antimycin inhibits the respiratory chain.

We have now combined self-referencing with our spinning-disk confocal microscope in order to analyze additional cellular parameters in real-time (Fig. 2). By using our preferred ex vivo model of embryonic neurons in primary culture, we have simultaneously measured TMRE fluorescence to assess mitochondrial membrane potential (Dy_mito; Fig. 2B),  fluo-4 fluorescence to assess cytosolic Ca²⁺ (Fig. 2A), and oxygen flux from single neurons (Fig. 3). These measurements are all performed at 37^oC under varying states of excitation and under pathophysiological conditions. Fig. 3 shows the progressive increase in the concentration of the neurotransmitter glutamate (arrows), which stimulates neurons and ultimately leads to cell death. Labeling of the oxygen probe with the fluorochrome, TMRE (Fig. 2B), has proved advantageous for the control of probe position under low light conditions.

2A & B	3

In conjunction with the Mattson Laboratory and the National Institute on Aging (NIH), this new platform is currently being utilized for the study of glutamate excitotoxicity. Excitotoxicity plays a possible role in the delayed neuronal death that occurs in vivo during ischemic events within the brain and may also contribute to the pathology associated with stroke, epilepsy, traumatic brain injury and multiple sclerosis. However, the exact mechanism of cell death during glutamate exposure remains unresolved. Primarily, sustained stimulation of NMDA receptors by glutamate leads to a profound increase in intracellular Ca²⁺ (Fig. 2, 3). Energy-utilizing pumps on the plasma membrane help to maintain this homeostatic balance. Mitochondria also sequester Ca²⁺ but excess Ca²⁺ leads to a cessation of oxidative phosphorylation, depolarization of mitochondrial membrane potential (Fig. 2, 3) and generation of significant reactive oxygen species (ROS). The decrease in ATP production, associated with glutamate stimulation, causes the pumps to fail, further Ca²⁺ increase, and ultimately cell death. So far, our data suggests that glutamate does indeed elicit maximal respiratory demand in cells and that this is soon followed by loss of mitochondrial membrane potential and consequent cell death. Speculation remains as to the causative agent of glutamate-induced cell death, particularly whether the cell is unable to meet the metabolic demand associated with glutamate stimulation.

The BRC is not only attempting to identify the causative agents behind disease processes but also the physiological basis behind neuroprotective interventions. Integrated technologies developed in the BRC provide the ability to measure mitochondrial coupling (ratio of ATP synthesis to oxygen consumption) in single cells. The degree of coupling is indicative of the mitochondria's ability to fulfill the energetic needs of the cell.  In collaboration with the Jonas Laboratory at the Yale School of Medicine  we are assessing the metabolic changes associated with upregulation of the protein BCL-xL (Fig. 4A, B). BCL-xL localizes to mitochondrial membranes (Fig. 4B; mitochondria shown in red; BCL-xL in green) and promotes cell survival in neurons during periods of stress but its mechanism of action remains unidentified. 

4A & B

5

6A: O2 microsensor &myocyte	6B:Ca2+ dynamics myocyte	6C: mitochondrial ATP

It is anticipated that combining the above technologies will help to uncover, not just the bioenergetic basis to disease-related and cytoprotective interventions, but generate novel targets for drug discovery and unique strategies for disease control.

Integrated approach to signal analysis

Our Data Acquisition and Signal Analysis projects are also utliizing the integrated imaging/electrochemical platform. Here, fast (millisecond-second) ion flux events are recorded from single neurons that are spontaneously depolarizing in culture. These flux events are correlated temporally with cellular activity by simultaneously measuring intracellular Ca²⁺ using imaging of Ca²⁺ reporters.