The concomitant activity of neurons in the brain can either potentiate or depress functional connections (synapses) between neurons in a persistent manner. These changes have properties that have made them the premiere cellular model to study regulation of memory mechanisms and learning. The overall aim of this research is to characterize, in dendrites and, for the first time, in dendritic spines of individual neurons, the non-linear and spatially inhomogeneous interactions of membrane potential signals and calcium signals that represent the first fundamental step in the induction of synaptic plasticity. Experiments will be conducted with brain slices prepared from rat hippocampus, a region with an essential role in learning and the establishment of new memories. The dendrites and spines of individual neurons will be explored with a novel imaging technique using a voltage sensitive dye, so that electrical activity can be monitored simultaneously from the entire dendritic arbor. Answers to some of the questions articulated in this project would further our understanding of experience related changes in the strength of functional connections in the brain, and are likely to lead to novel ideas about mechanisms that underlie learning and memory formation. Thus, the results of this analysis will bear on the basic neuroscience of higher nervous activity. The broader impacts of this research will be realized primarily by contributing to better understanding of the brain, thereby promoting the progress of fundamental science. Better understanding of brain mechanisms has clear and widely documented positive effects on developing the most advanced concepts used in information processing and computational technologies which, in turn, have enormous impact on almost all aspects of modern human activities. The impacts of this research will also be realized through teaching and mentoring of graduate and MD/PhD students and by providing opportunities for the participation of underrepresented minorities in research.
The major outcome of our NSF funded research is the development of a new tool in cellular neurophysiology: laser-based optical recording of electrical signaling in individual neurons using organic voltage-sensitive dyes (Vm-imaging). The sensitivity of this measurement technique has reached a level that permits single trial optical recordings of electrical signals from all parts of an individual neuron, including axons, terminal dendrites, and individual dendritic spines (sites of functional contacts between neurons). This development overcomes the prior absolute methodological barrier that prevented the analysis of electrical signaling in thin axonal and dendritic processes of individual nerve cells and in dendritic spines for many decades; these structures have never been probed for electrical signals before. At the same time, understanding electrical signaling and functional organization of individual neurons and how they process and encode information is fundamental to understanding how brain works. This methodological developments, including the apparatus and the procedures necessary to introduce and utilize Vm-imaging in other laboratories has been described in detail (Popovic et al., 2012. JoVE, in press). We first utilized laser-based Vm-imaging to provide new understanding on how axonal spikes propagates into the dendritic tree. Our results indicated that the rapid time course of the action potential in dendrites may be a critical determinant for the precise regulation of spike timing-dependent synaptic plasticity, a process closely related to learning and memory formation (Holthoff et al., 2010). Another series of experiments was carried out on the analysis of electrical events that encode information in individual axons. Axons are long process of nerve cells that mediate communication and control within the organism. Mammalian neurons have developed a complex ion channel clustering mechanism in axons to optimize rapid signaling. It has been discovered recently that intricate details of the spatial pattern of channel clustering play a critical role in signal processing in the axon. The electrical properties of axons, however, have been difficult to study using electrodes because axons are very small in diameter. We took advantage of a critical methodological improvement described above to study electrical correlates of channel clustering in the axon of cortical neurons. Voltage imaging revealed the location and length of the axonal site for nerve impulse initiation as well as the pattern of saltatory conduction in myelinated axons in the form of dynamic spatial maps of transmembrane potential. The results demonstrated that it was possible to measure and characterize, for the first time, the site of nerve impulse initiation in the axon as well as to reveal ionic control of spike propagation into axon collaterals (Foust et al., 2010; Popovic et al., 2011; Foust et al., 2011). In another study, we provided evidence for an important hypothesis that the morphology of cortical dendritic spines (anatomically miniscule structures that mediate functional contacts between neurons) might participate in modifying synaptic efficacy that underlies plasticity and possibly learning and memory mechanisms. To examine a specific question of the transfer of dendritic signals to synapses of spines, we took advantage of a high-sensitivity Vm imaging technique and carried out optical measurements of electrical signals from dendritic spines . The results showed that spine neck does not filter membrane potential signals as they spread from the dendrites into the spine heads. The functional significance of these results is related to the prominent role of bAPs in dendritic signal integration in general and in synaptic plasticity underlying memory formation in particular (Popovic et al., 2012). The project provided a stimulating environment for PhD students and postdoctoral fellows who participated in this research. Two graduate students (Amanda Foust and Amanda Casale) constructed two separate Vm-imaging setups under the guidance of the PI of this project. This is a unique practical experience which has enabled them to carry out experiments for their PhD project which they both successfully completed in 2012. Thus, the opportunity provided by NSF funding directly broadened the participation of women in science. Two postdoctoral fellows (Marko Popovic and Xin Gao) participated in experimental design, data analysis and interpretation as well as in writing scientific papers under the mentorship of the PI. This is a critical experience in the educational process for young scientists. Further, our results have been reported on scientific meetings; the results are are published and well received. The methodology developed under this NSF funding is now a key component (high-speed imaging from individual neurons) in a wider avant-garde strategy to implement all-optical investigation of living neural circuits using light-activated ionchannels and imaging technologies.
