Proceedings of The Physiological Society

University College London 2006 (2006) Proc Physiol Soc 3, WA1


Combining two-photon imaging with electrophysiology in vivo: from the synapse to the network

Jason N. D. Kerr1

1. Cell Physiology / Biomedical Optics, Max Planck Institute for Medical Research, Heidelberg, Germany.

Understanding how information is represented and processed in the mammalian neocortex requires measurement of spatiotemporal activity patterns in identified networks of neurons in vivo. What will be required is the ability to be simultaneously record both input and output of cortical microcircuits with single-cell and single-spike resolution. Recently, two-photon laser scanning microscopy (2PLSM) has provided a viewing window into the in vivo brain. The advantages of multi-photon laser excitation combined with in vivo bulk labeling techniques have been exploited to image both cellular and subcellular structures within the mammalian brain on time scales ranging from milliseconds to weeks. Bulk loading of brain tissue with Acetoxymethyl (AM) ester derivatives of calcium indicators has become a potentially powerful tool in the quest to understand encoding of information in neuronal populations. Several issues arise with the use of this technique: 1) all tissue and structures are labeled with these dyes requiring specific counterstaining with either genetically encoded labels or additional dyes such as astrocyte specific sulforhodamine 101. 2) because of sparse neuronal action-potential (AP) firing in many cortical areas, it is therefore necessary to ensure the detection of single AP evoked calcium signals. In addition, there is a compromise between the spatial/temporal resolution and signal to noise ratio of signal detection. Combining these imaging techniques with simultaneous targeted electrophysiological recordings allows for the probing of neuronal circuits as well as the calibration of neuronal electrical signals with imaging data. Here I will present work that combines both 2PLSM imaging of on-going neuronal population activity and various electrophysiological techniques to simultaneously record neuronal output activity from local neuronal populations with single cell and single AP resolution. In addition, because the neuropil is also loaded using this loading technique, I will also present data showing that the ongoing neuropil signal represents axonal activity and reflects a volume averaged input signal to the local circuit. I will describe how both targeted cell attached and whole-cell recording techniques were used to establish that AP activity is reliably resolved with single-cell and single-AP resolution in bulk-loaded neurons. This made it possible to optically extract AP patterns, representing ‘‘output’’ activity, in local neuronal circuits. These results revealed that spatial organization of active neurons was not stable but displayed considerable heterogeneity over the time course of minutes. This heterogeneity indicated that spontaneous activity does not emerge exclusively in a particular subset of neurons but rather is generated by a continually changing subpopulation of neurons. Spontaneous calcium signals in the neuropil were tightly correlated to electrocorticogram and intracellular membrane potentials of neurons embedded within the local network. This optical encephalogram (OEG) represents bulk calcium signals in axonal structures, and provides a measure of local input activity. Because input–output relationships are of key importance for the understanding of signal processing in neuronal circuits, this optical approach should enable the study of input–output transformations during sensory input and how they are affected by varying levels of background activity such as they occur during different behavioral states.

Where applicable, experiments conform with Society ethical requirements