Imaging Neural Activity With Single Cell Resolution in an Intact ...

Reference:

Viol. &I//.

192: I SO- 153. (February,

1997)

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

R. FETCHO,

Depurtment

ofNeurobiology

KINGSLEY

J. A. COX,

AND

DONALD

in

M. O’MALLEY

and Behavior, State University ofNew York at Stony Brook, Stony Brook, New York I1 794-5230

Introduction

brightness due to the calcium influx associated with their activity. Thus, neural activity during behavior can be determined by watching for the increase in brightness in particular cells (O’Donovan et al., 1993; Fetch0 and O’Malley, 1995; Fetch0 et al., 1995; Cox and Fetcho, 1996; O’Malley et al., 1996). The transparency of the fish has allowed us to image activity in neuronal populations with single-cell resolution in an intact vertebrate. Our work so far has focused on the neuronal populations involved in the escape behavior. This behavior has been the subject of intensive study with single-cell recording techniques in the past (Faber and Korn, 1978; Fetch0 and Faber, 1988; Fetcho, 1992) but the ability to monitor groups of neurons is providing a new view of the relationships between neural activity and the escape behavior.

Most behaviors are produced by activity in populations of neurons, but the physiological approaches commonly 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 groupwhile 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 determination 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 behavior. The ideal situation would be one in which a population of neurons could be labeled and their activities observed with single-cell resolution in an intact, behaving animal. This ideal is difficult to achieve with vertebrates 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 indicator is used to monitor neural activity in intact fish. Neurons that are labeled with the indicator increase in

Labeling Central neurons were labeled with a calcium indicator, calcium green dextran. Neuronal activity causes an influx of calcium through voltage-dependent calcium channels, so the calcium levels sensed by the indicator can be used as a measure of activity. The neurons in larval, post-hatching zebrafish (Dunio rerio) were labeled in two ways. In the first, the indicator was injected into muscle or into the central nervous system, backfilling the neurons of interest. Calcium green dextran is taken up by the damaged processes of neurons, resulting in both retrograde and anterograde labeling (O’Donovan et al., 1993; Fetch0 and O’Malley, 1995). This approach led to a very robust labeling of populations of neurons. The neurons could be observed and individually identified in the spinal cord and in the brain of the living fish either by confocal microscopy or enhanced video imaging. The labeling was very similar to that seen in material that had

This paper was originally presented at a workshop titled Thr If’zl/zrrc of’ Aq~lic Rewun~i~ in SI)UW: Neurohiolo~~~. C~IIIIUY und Molcwlur Biokgy. The workshop, which was held at the Marine Biological Laboratory, Woods Hole, Massachusetts, from I3 to I5 May 1996, was sponsored by the Center for Advanced Studies in the Space Life Sciences at MBL and funded by the National Aeronautics and Space Administration under Cooperative Agreement NCC 2-896. 150

NEUROBIOLOGY/SENSORY

been cleared and stained with horseradish peroxidase (HRP). We could see many of the individually identifiable neurons of zebrafish (Bernhardt et al., 1990) including the three primary motoneurons (rostral, middle, and caudal-RoP, MiP, and CaP, respectively) located in spinal segments, and the Mauthner cell and its two serially homologous neurons (MiD2cm, MiD3cm) located in the hindbrain. Other identifiable spinal neuronal types (e.g., secondary motoneurons, Rohon Beard cells, dorsal root ganglion cells, circumferential descending (CID) cells, commissural posterior ascending (CoPa) cells) and brain neurons (e.g., vestibulospinal neurons, nucleus of the medial longitudinal fasciculus) were also evident. The relatively high fluorescence of calcium green at basal calcium levels produced well-labeled dendrites and axons. The quality of the labeling, and the high resolution and optical sectioning ability of the confocal microscope, allowed the collection of serial optical sections through the neurons of interest. Three-dimensional reconstructions of the cells from live fish were produced from optical sections using the VolVis program (Sobierajski et al., 1995). The reconstructions allowed a detailed examination of the neurons whose physiology was being studied optically. This allowed the unambiguous identification of the cells and also provided information about the relationships of the axons and dendrites of the imaged neurons. Although labeling by injection into the larval fish produced well-labeled neurons, the injection may in some instances disrupt portions of the neural circuits of interest. To circumvent this problem, we also labeled neurons by injecting calcium green dextran into blastomeres of one-, two-, four-, or eight-celled embryos. In this procedure, the indicator is labeling the progeny of the blastomere, just as lineage tracers are used in developmental studies. The fish were then raised to post-hatching larval stages. These injections produced larval fish in which a large fraction of cells contained the calcium indicator; from one-eighth to all of the cells should be labeled, depending on the number of blastomeres that had been formed at the time of injection. The proportion of labeled cells, in our experience, was roughly consistent with this expectation, although the intensity of the labeling of individual cells varied. The confocal microscope effectively removed fluorescence that was out of focus, and allowed visualization of the labeled cells in the intact animal. This approach has the advantage of not disrupting the neural circuitry, at least so far as we can tell. The lack of damage is evidenced by our ability to raise such blastomere-labeled fish to adulthood after imaging their neural activity. The labeling produced by blastomere injections is not, however, as intense as that by injection into larvae, so the type of each cell is more difficult to identify. Nevertheless, we have been able to observe

151

BIOLOGY

identifiable classes of spinal neurons (e.g., Rohon Beard cells) and some neurons, such as olfactory epithelial cells, are very brightly labeled (Cox and Fetcho, 1996). Responses

of Neurons

We initially used electrical stimuli to determine whether the indicator was responsive in the neurons and to demonstrate that we could resolve differential responses in adjacent cells (Fetch0 and O’Malley, 1995). Motoneurons in larval fish, typically 3-5-days old, were backfilled by injections of calcium green dextran into muscle and then, a day or more later, were embedded in agar and observed with a Biorad MRC 600 confocal imaging system using a Zeiss IM 35 inverted microscope. The fish remain healthy in the agar, probably because they depend on cutaneous respiration at these early stages. An electrical stimulus applied to the skin overlying the muscle near the site of calcium green injection could increase the fluorescence of the labeled motoneurons. A single stimulus typically produced increases of 5%-10%; additional stimuli, applied in rapid succession, further increased the fluorescence (over 100% change for a train of 40 stimuli in some cells) consistent with the accumulation of calcium expected upon repeated activation of the motoneurons. By varying the strength of the electrical stimulus, we could detect differential responses in adjacent motoneurons, with one cell showing a large increase in fluorescence (e.g., 70%) and an immediately adjacent one showing none. Thus, we could resolve differences in adjacent cells which were roughly 10 pm diameter and only about 1 pm apart. Having confirmed that the cells behaved as expected to electrical stimulation, we examined their responses during escapes (Fetch0 and O’Malley, 1995). Escape behaviors were elicited by an abrupt touch applied to the head or tail with a piezoelectric tapping device. In the escape behavior, a touch on one side of the head leads to a massive, rapid C-bend to the opposite side of the body, which turns the fish away from a threatening stimulus. When we imaged spinal motoneurons on the side of a Cbend during the escape, we found a massive activation, with every cell that we imaged responding during the escape bend. In individual cells, the increases in fluorescence ranged from 16% to 15 1%. The activated motoneurons included the larger primary motoneurons, as well as the smaller secondary ones. Most of our observations were obtained with low temporal resolution, usually 400 ms for each image. However, we could achieve resolutions on the millisecond time scale of synaptic events by taking advantage of the line-scanning ability of the confocal microscope. This allowed us to look at one line through a group of cells every 2 ms, and revealed a synchronous activation of the motoneurons. The pri-

152

FUTURE

OF

AQUATIC

mary motoneurons innervate extensive regions ofthe axial muscle and are known to be important in escapes, based upon evidence that they receive a monosynaptic input from the reticulospinal Mauthner cell that initiates the escape (Myers et al., 1986; Westerfield el ul., 1986; Fetch0 and Faber, 1988; Liu and Westerfield, 1988). The secondary motoneurons are more numerous and their role in escapes has been less clear. Our data indicate that the motoneurons activated in escapes include both primary and secondary pools (Fetch0 and O’Malley, 1995). We observed a similar massive activation of cells in ventral cord that had been labeled by blastomere injections and imaged in the larval fish (Cox and Fetcho, 1996). The identity of these cells in the blastomere-labeled fish was not certain because of the less complete filling of cells from blastomere injections. But, based upon their locations, they were most likely motoneurons. The observations from backfills and blastomere injections are consistent with the massive activation of muscle that occurs during the escape bend. The most powerful C-bends occur in response to stimulation of the head (Eaton et al., 1984; Foreman and Eaton, 1993). These bends are the most forceful motor behavior produced by the fish, so such a large recruitment of motoneurons is not surprising. We began our imaging studies with motoneurons, because enough was known about their activity patterns that we could use previous data to evaluate the reliability of the approach. More recently we have begun to study other, less well understood systems, including the olfactory system, reticulospinal neurons, and spinal interneurons. Our studies of reticulospinal neurons, for example, have demonstrated that the brain as well as the spinal cord, can be examined by in viva imaging. We have used the approach to evaluate predictions about the pattern of activation of the Mauthner cell and its serially homologous neurons in hindbrain (Fetch0 et al., 1995; O’Malley et al., 1996). The hindbrain consists of a series of segments that contain repeated, morphologically similar neurons in successivesegments.One of these setsof serially repeated cells includes the reticulospinal Mauthner cell (in hindbrain segment 4), which is known to play a role in escapes,and two reticulospinal cells (MiD2cm, MiD3cm in segments5 and 6 respectively) that are very like the Mauthner cell in their dendritic structure and axonal projection. The functional role of the Mauthnerlike cells was unknown, but Foreman and Eaton (1993) predicted that the observed variability in the strength of the C-bend, in response to sensory stimuli at different locations relative to the fish, might be determined in part by variability in the activation neurons in the set (including the Mauthner cell, MiD2cm, and MiD3cm). We have examined and confirmed their predictions by calcium imaging, which has allowed us to study the func-

RESEARCH

IN SPACE

tion of a set of cells that has been difficult to study with more conventional techniques (Fetch0 et al., 1995; O’Malley et al., 1996). Our observations suggestthat the hindbrain consists of serial sets of functionally similar neurons that may act together in various combinations to produce behavioral variability. Conclusions Although still in its infancy, the use of calcium imaging in the larval zebrafish offers the possibility of studying the activity of populations of neurons with singlecell resolution anywhere in the brain or spinal cord of an intact, behaving vertebrate. This will provide important information about the relationships between the activity of neuronal populations and behavior in the normal animal, but there are other advantages of the zebrafish as well. Thousands of mutant lines of zebrafish have now been generated by saturation mutagenesis, including many with sensory and motor deficits (Mullins et al., 1994; Driever et al., 1994). The calcium imaging approach should prove useful in analyzing the functional deficits of these mutant lines. The transparency of the fish will also facilitate the optical killing of particular, fluorescently labeled neurons. The behavior and neuronal activity of the fish can be studied both before and after particular neurons or sets of neurons have been killed, allowing a more causal link between neurons and behavior. It may also be possible to use local optical uncaging of neuroactive substances to activate or inhibit neurons. This would allow noninvasive perturbations of activity. The combination of imaging activity, genetics, and cell ablation will permit a powerful analysis of the neural basisof behavior in a vertebrate. Acknowledgments Supported by NIH NS26539, NS-09 113, and the Howard Hughes Medical Institute. Literature Cited Bernhardt, R. R., A. B. Chitnis, L. Lindamer, and J. Y. Kuwada. 1990. Identification of spinal neurons in the embryonic and larval zebrafish. J. Comnp. Ncurol. 302: 603-6 16. Cox, K. J. A., and J. R. Fetcho. 1996. Labeling blastomeres with a calcium indicator: a non-invasive method of visualizing neuronal activity in zebrafish. J. Nauwci. Methods 68: 185% I9 1. Driever, W., D. Stemple, A. Schier, and S. Solinica-Krezel. 1994. Zebrafish: genetic tools for studying vertebrate development. Trends Gcwet. 10: 152-159. Differential acEaton, R. C., J. Nissanov, and C. M. Wieland. 1984. tivation of Mauthner and non-Mauthner startle circuits in zebrafish: implications for functional substitution. J. Camp. Phyxiol. 155: 813-820. Faber, D. S., and H. Korn, eds. 1978. Nrurohiok~gy of‘the Muuthnw Cell. Raven Press, New York.

NEUROBIOLOGY/SENSORY Fetcho, J. R. 1992. Excitation of motoneurons by the Mauthner axon in goldfish: complexities in a “simple” reticulospinal pathway. J. Nwophyiol. 67: I574- 1586. Fetcho, J. R., and D. S. Faber. 1988. Identification of motoneurons and interneurons in the spinal network for escapes initiated by the Mauthner cell in goldfish. J. Nmrmci. 8: 4 192-42 13. Fetcho, J. R.,and D. M. O’Malley. 1995. Visualization ofactive ncural circuitry in the spinal cord of intact zebrafish. J. Nclrro~~~k~~.riol. 73: 399-406. Fetcho, J. R., Y.-H. Kao, and D. M. O’Malley. 1995. Functional roles of serially homologous reticulospinal neurons studied by in viva confocal calcium imaging in zebrafish. Sot. Nu/,o.w~. A&u. 21: 687. Foreman, M. B., and R. C. Eaton. 1993. The direction change concept for reticulospinal control of goldfish escape. J. Ncwosc~i. 13: 4101-4113. Liu, D. W., and M. Westerfield. 1988. Function of identified motoneurons and coordination of primary and secondary motor systems during zebrafish swimming. J. t’h.vsiol. (Lord) 403: 73-89. Mullins, M. C., M. Hammerschmidt, P. Haffter, C. Nusslein-Volhard. 1994. Large-scale mutagenesis in the zebrafish: in search ofgenes controlling development in a vertebrate. Cwr. Bid. 4: 189-202.

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