MONITORING ACTIVITY IN NEURONAL POPULATIONS WITH SINGLE-CELL RESOLUTION IN A BEHAVING VERTEBRATE

Vertebrate behaviours are produced by activity in populations of neuro ns, but the techniques typically used to study activity allow only one or very few nerve cells to be monitored at a time. This limitation ha s prompted the development of methods of imaging activity in the nervo us system. The overall goal of these methods is to image neural activi ty noninvasively in populations of neurons, ideally with high spatial and temporal resolution. We have moved closer to this goal by using co nfocal calcium imaging to monitor neural activity in the transparent l arvae of zebrafish. Neurons were labelled either by backfilling from i njections of the calcium indicator (Calcium Green dextran) into muscle or spinal cord of larvae or by injections into blastomeres early in d evelopment. 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 ne urons from the live animal could also be reconstructed in three dimens ions for morphometric study. Neurons increased their fluorescence duri ng activity produced by direct electrical stimulation and during escap e behaviours elicited by an abrupt touch to the head or tail of the fi sh. The rise in calcium associated with a single action potential coul d be detected as an increase in fluorescence of at least 7-10%, but ne urons typically showed much larger increases during behaviour. Calcium signals in the dendrites, soma and nucleus could be resolved, especia lly when using the line-scanning mode, which provides 2-ms temporal re solution. The imaging was used to study activity in populations of mot oneurons 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 dur ing 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 act ivity of populations of neurons throughout the nervous system in both normal and mutant lines of fish. (C) 1998 Chapman & Hall.