BRAIN RESEARCH ELSEVIER Brain Research 663 (1994) 61-68 Research report Combined retrograde labeling and calcium imaging in spinal cord and brainstem neurons of the lamprey Andrew D. McClellan a,*, Duane McPherson <1, Michael J. O'Donovan c a Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA b Department of Biology, Marquette University, Milwaukee, W153233, USA c Neural Control Lab, Rm. 3A68 Bldg. 49, NINDS Nil-l, Bethesda, MD 20892, USA Accepted 12 July 1994 Abstract Neurons in the brainstem and spinal cord of the lamprey were retrogradely labeled with Calcium Green-dextran, an indicator dye that increases its fluorescence when intracellular calcium levels increase. Optical signals could be recorded from these labeled neurons during spinal cord stimulation, nerve stimulation, or spontaneous activity, up to 4 days after dye application and for distances of 5-14 mm away from the application site. Optical signals were enhanced by 4-AP, a potassium channel blocker, and blocked by cadmium, a calcium channel blocker. Taken together, the results suggest that the optical signals recorded from labeled neurons were due to calcium influx during electrical activity. Thus, retrograde labeling with calcium indicator dyes may provide a general purpose method for simultaneously monitoring the activity-related changes of intracellular calcium in anatomically identified groups of neurons in the lamprey nervous system. Keywords: Optical imaging; Calcium channel; Calcium Green-dextran; Fluorescence; Lamprey I. Introduction Electrical signalling in nerve cells is accompanied by changes in membrane potential and generation of cur- rents in the extracellular space which can be recorded with various types of electrodes. For example, intracel- lular recordings with micropipettes can monitor the membrane potentials of single neurons, while extracel- lular electrodes are usually used to record activity in populations of neurons. Both types of recording tech- niques have their advantages and disadvantages. Intra- cellular electrodes can record action potentials as well as subthreshold types of activity (e.g. synaptic poten- tials), but the method is not well suited to monitor the activity patterns in groups of cells since only one or a few neurons can be recorded at a time. Extracellular electrodes can monitor the activity patterns in many * Corresponding author. Division of Biological Sciences, 105 Lefevre Hall, University of Missouri, Columbia, MO 65211, USA. Fax: (1) (314) 884-5020. 1 Present address: Dept. of Biology, SUNY Geneseo, Geneseo, NY 14454, USA. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00889-2 neurons at the same time, but it is often difficult to separate the patterns of activity in individual neurons and to know which neurons are contributing to the activity. In addition, extracellular electrodes are usually used to record action potentials, while subthreshold activity may go undetected. Because of the limitations in the above electrophysi- ological recording techniques, methods involving opti- cal recording have been developed which potentially allow one to monitor intracellular activity in as many neurons as can be visualized simultaneously in the field of view of a microscope [5,10]. For example, certain fluorescent dyes, such as the styryl dye RH-461, be- come incorporated into the membranes of nerve ceils after bath application or intraceIlular injection [5,11]. These dyes, when illuminated at an appropriate excita- tion wavelength, change their fluorescence in response to changes in electric field across the membrane that occur during electrical activity. These voltage-sensitive dyes generate optical signals that can be used to detect the occurrence of action potentials and sometimes subthreshold activity. However, the signal-to-noise ra- tio obtained from these dyes during electrical activity is