Donald M. O'Malley

Monitoring activity in neuronal populations with single-cell resolution in a behaving vertebrate.

Vertebrate behaviours are produced by activity in populations of neurons, but the techniques typically used to study activity allow only one or very few nerve cells to be monitored at a time. This limitation has prompted the development of methods of imaging activity in the nervous system. The overall goal of these methods is to image neural activity non-invasively in populations of neurons, ideally with high spatial and temporal resolution. We have moved closer to this goal by using confocal calcium imaging to monitor neural activity in the transparent larvae of zebrafish. Neurons were labelled either by backfilling from injections of the calcium indicator (Calcium Green dextran) into muscle or spinal cord of larvae or by injections into blastomeres early in development. The labelled neurons were bright enough at resting calcium levels to allow the identification of individual neurons in the live, intact fish, based upon their dendritic and axonal morphology. The neurons from the live animal could also be reconstructed in three dimensions for morphometric study. Neurons increased their fluorescence during activity produced by direct electrical stimulation and during escape behaviours elicited by an abrupt touch to the head or tail of the fish. The rise in calcium associated with a single action potential could be detected as an increase in fluorescence of at least 7--10%, but neurons typically showed much larger increases during behaviour. Calcium signals in the dendrites, soma and nucleus could be resolved, especially when using the line-scanning mode, which provides 2-ms temporal resolution. The imaging was used to study activity in populations of motoneurons and hindbrain neurons during the escape behaviour fish use to avoid predators. We found a massive activation of the motoneuron pool and a differential activation of populations of hindbrain neurons during escapes. The latter finding confirms predictions that the activity pattern of hindbrain neurons may help to determine the directionality of the escape. This approach should prove useful for studying the activity of populations of neurons throughout the nervous system in both normal and mutant lines of fish. 1998

Imaging neuronal networks in behaving animals.

Neuronal activity has recently been imaged with single-cell resolution in behaving vertebrates. This was accomplished by using fluorescent calcium indicators in conjunction with confocal or two-photon microscopy. These optical techniques, along with other new approaches for imaging synaptic activity, second messengers, and neurotransmitters and their receptors offer great promise for the study of neuronal networks at high resolution in vivo.

Regulation of M current by intracellular calcium in bullfrog sympathetic ganglion neurons

Regulation of M current (lM) by intracellular free calcium was studied in dissociated bullfrog sympathetic ganglion B cells using whole-cell recording, intracellular perfusion, and confocal calcium imaging. BAPTA (20 mM) and appropriate amounts of calcium were added to pipette solutions to clamp calcium at different levels. A high concentration of BAPTA itself mildly inhibited lM. Intracellular perfusion effectively controlled cellular free calcium; this was confirmed by confocal imaging with the calcium indicator fluo-3. In a calcium-free environment (no calcium added to either side of the cell membrane), average lM was 166 pA. Raising intracellular free calcium to 60 nM or higher reversibly enhanced lM by more than 100%. The maximum M conductance doubled upon raising calcium from 0 to 120 nM, and was accompanied by a -11 mV shift of the half-activation voltage. The kinetics of the closing and reopening relaxations of lM were also altered by raising calcium. Enhancement of lM by calcium required ATP in the pipette. TEA (5 mM) and d-tubocurarine (d-TC; 100 microM) did not alter the calcium effect, indicating that it was the M current being modulated and not other K+ currents. High calcium (450 nM) reduced lM. The up- and downregulation of lM paralleled the increases and decreases of fluorescence intensity observed via calcium imaging. Changing extracellular calcium had no significant effect on lM or cellular fluorescence. The role of calcium in muscarinic and peptidergic modulation of lM was also explored. Muscarine (1 or 10 microM) inhibited lM less at zero calcium than at higher calcium. Nearly complete suppression occurred with 120 nM calcium in the presence of 20 mM BAPTA. lM overrecovered upon washout of muscarine at 120 nM calcium, while little overrecovery of lM developed at zero calcium. Similar effects were observed at zero and 120 nM calcium when using the peptide LHRH to inhibit lM. We conclude that the absolute level of free calcium determines the size of lM, and that a minimum sustained level of calcium is required both for optimal suppression of lM by muscarine and for overrecovery. While our data suggest that resting calcium levels play a permissive role in muscarinic suppression, an additional role for agonist-induced calcium increases cannot be ruled out.

Photostimulation with caged neurotransmitters using fiber optic lightguides

Caged neurotransmitters are molecules that are transformed to a neuroactive state by exposure to light of an appropriate wavelength and intensity. Use of these substances has centered on in vitro bath application and subsequent activation using light from lasers or flashlamps that is delivered into the preparation through microscope optics. We have tested a new and simpler method, using finely tapered fiberoptic lightguides, that promises to expand the use of caged compounds for in vitro and in vivo experimentation. We demonstrated the feasibility and flexibility of this method for caged neurotransmitter delivery using a range of ex vitro, in vitro and in vivo approaches. The degree and timing of uncaging could be controlled by manipulating the wavelength, intensity and timing of the light projected into the optical fiber. Because of the small size of the light guide and the ability to control light exposure at the source, this new method promises greater control over the spatial and temporal delivery of neuroactive substances than simple bath or iontophoretic application, and enables delivery of conventional neurotransmitters with a spatial and temporal resolution closer to that of the natural neuronal circuitry. In addition, this new method allows the application of normally labile substances, such as the free radical gas nitric oxide, by the photoconversion of photosensitive precursors.

Visualization of calcium activity in nerve cells

Calcium plays an important role in many cellular functions. This investigation of its role in nervous system behavior uses imaging techniques, confocal microscopy, and the VolVis volume visualization system. The use of VolVis provided substantial assistance in the study of cellular calcium dynamics and in monitoring neural network activity. It yielded geometric details that facilitated our understanding of the behavior of calcium indicators inside nerve cells and led to a new view of the calcium permeability of the nuclear envelope. It has also produced anatomical details that significantly facilitated the development of a technique to directly visualize the activity of neuron populations while simultaneously observing vertebrate behavior. >

Imaging Neural Activity With Single Cell Resolution in an Intact, Behaving Vertebrate

JOSEPH R. FETCHO, KINGSLEY J. A. COX, AND DONALD M. O’MALLEY Depurtment ofNeurobiology and Behavior, State University ofNew York at Stony Brook, Stony Brook, New York I1 794-5230 Introduction Most behaviors are produced by activity in popula- tions of neurons, but the physiological approaches com- monly used to study neural circuits allow the activity of only one or very few neurons to be monitored at a time. What is needed are approaches that allow the monitoring of activity in a group of cells-preferably a large group- while simultaneously permitting the identification and the recording of activity from each cell. Progress along these lines has been made with the use of electrode arrays (Wilson and McNaughton, 1994). An alternative, very promising approach-i.e., imaging-offers an easy de- termination of both the activity and the identity of cells (Wu et ul., 1994; O’Donovan et al., 1993). In this method, the neurons are labeled with an indicator dye that signals their activity, and the dye is then used to monitor the cells that are active during a particular be- havior. The ideal situation would be one in which a pop- ulation of neurons could be labeled and their activities observed with single-cell resolution in an intact, behav- ing animal. This ideal is difficult to achieve with verte- brates because most of them are opaque, so the neurons cannot be seen in the intact animal. Notable exceptions are the larvae of many fishes, which are transparent and thus especially suitable for imaging neurons. We have developed approaches in which a fluorescent calcium in- dicator is used to monitor neural activity in intact fish. Neurons that are labeled with the indicator increase in