Laser-induced native fluorescence (LINF) imaging of serotonin depletion in depolarized neurons

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Abstract

Since certain neurotransmitters exhibit native fluorescence we can monitor this property to disclose intracellular changes that result from neurotransmitter release. Isolated Retzius neurons of the leech are known to release serotonin (5-HT) during depolarization. Using intensified CCD technology coupled with UV laser (305 nm) excitation we observed depolarization and calcium-dependent reductions in native fluorescence in the axon, as well as in the cortex of the cell body. When taken together with data obtained from single-cell capillary electrophoresis, we demonstrate that this laser-induced native fluorescence can be reliably used to study spatial and temporal changes in intracellular transmitter content that accompany calcium-dependent secretion.

Introduction

During the past 5 years there has been a significant growth in methodologies that permit visualization of the activity of synaptic terminals. Perhaps one of the biggest advances was the development of the fluorescent styryl dyes (FM 1-43) by Bill Betz and his colleagues (Betz and Bewick, 1992; Betz et al., 1992a, Betz et al., 1992b, Betz et al., 1996; Betz and Wu, 1995; Henkel et al., 1996; Wu and Betz, 1996). During synaptic activity, FM 1-43 is taken up into synaptic vesicles during endocytosis, where it remains until the FM 1-43 filled vesicle discharges its contents during a subsequent round of exocytosis. The use of FM 1-43 has significantly increased our knowledge of the regulation of exocytosis and endocytosis (Ryan et al., 1993, Ryan et al., 1996a, Ryan et al., 1996b; Wu and Betz, 1996; Betz et al., 1996).

Limitations of this FM 1-43 method however, are that it is necessary to stimulate nerve terminals to obtain initial labeling, that an exogenous molecule must be added to the preparation and that it has been reported to work poorly in acutely isolated slices of CNS tissue. As an alternative approach, Kay and colleagues have developed a vital nerve terminal staining method which makes use of the high concentration of zinc in mossy fiber nerve terminals (Budde et al., 1997). They demonstrate that addition of a zinc-sensitive fluorescent probe, N-(6-methoxy-8-quinoly)-p-carboxybenzoylsulphonamide (TFLZn) to unstimulated slices labels mossy fiber terminals (Budde et al., 1997). Further, they demonstrate that electrical stimulation causes an activity and calcium-dependent reduction in fluorescence as zinc and TFLZn are expelled during exocytosis.

Since many neurotransmitters exhibit native fluorescence, (Chang and Yeung, 1995; Tan et al., 1995; Lillard et al., 1996; Lillard and Yeung, 1997; Maiti et al., 1997; Shear et al., 1997; Tong and Yeung, 1997; Tan et al., 1997), the goal of this study was to ask whether it is possible to detect synaptic activity based on fluorescence changes associated with the loss of transmitter in nerve terminals without the need to apply exogenous markers. We have previously shown that it is possible to image exogenously applied 5-HT that was taken up into astrocytes. Using laser-induced native fluorescence (LINF) imaging, we detected puncta of 5-HT within living astrocytes (Tan et al., 1995). To test the feasibility of detecting 5-HT release from a cell we have previously imaged mast cells. During degranulation, there is a transient increase in fluorescence associated with the release of 5-HT (Lillard and Yeung, 1997). Apparently, the 5-HT is stored in mast cell granules at such high concentrations that there is significant self-quenching. Thus, during degranulation, the 5-HT concentration decreases and fluorescence transiently increases.

Since 5-HT absorbs and fluoresces at UV wavelengths, a microscope is needed to observe the fluorescence of this transmitter in which quartz optics are utilized to permit passage of the UV photons. An alternative approach is to use multiphoton microscopy in which three photons at a longer wavelength (700 nm) are simultaneously absorbed by the fluorochrome (Maiti et al., 1997; Shear et al., 1997). While this overcomes some of the problems associated with delivering UV energy, it still requires UV transmitting optics for the emitted light. Also, focusing becomes more critical and the imaging speed is reduced. It was suggested that cellular damage is reduced in multiphoton excitation compared to an equivalent UV photon (Maiti et al., 1997; Shear et al., 1997), but such has not been conclusively demonstrated. Instead we have used a low power CW UV laser source together with intensified CCD imaging technology to study the native fluorescence of 5-HT within identified neurons.

In the leech, each ganglion is known to contain a pair of visually identified 5-HT containing neurons, the Retzius cells. These neurons are known to take up 5-HT precursors, to store 5-HT and to release 5-HT following depolarization and calcium entry (Henderson, 1983; Henderson et al., 1983; Kuffler et al., 1987). Because of the extensive characterization of the serotonergic properties of these cells (Henderson, 1983; Henderson et al., 1983; Kuffler et al., 1987; Fernandez de Miguel and Drapeau, 1995; Haydon and Drapeau, 1995; Catarsi and Drapeau, 1996) we have used them to ask whether we can utilize laser-induced native fluorescence (LINF) to image neurotransmitter depletion associated with synaptic activity.

Section snippets

Cell isolation and culture

Retzius cells were isolated from red-belly leeches (Macrobdella) by a method similar to that previously described (Dietzel et al., 1986). Ganglia were dissected in normal leech Ringer (NLR, see Section 2.4) and then desheated in culture medium composed of Leibowitz's l-15 medium (no phenol red) supplemented with heat-inactivated fetal bovine serum (2% v/v), d-glucose (6 mg/ml), and gentamicin (100 μg/ml). Individual Retzius neurons were visually identified and then removed using fire-polished

Results and discussion

An inverted microscope equipped with a 10× quartz objective was used for all imaging experiments. Based on the fluorescent properties of 5-HT (Fig. 1) we used the 305 nm laser line from an argon ion laser for sample excitation. While 5-HT also has an excitation maxima at 250 nm, we choose the longer excitation wavelength to reduce the fluorescence contributions from proteins (Tan et al., 1995). This laser line (4 mW) was isolated with an external quartz prism and was directed with a pair of

Acknowledgements

Vladimir Parpura and Wei Tong contributed equally to this work. The authors would like to thank Dr Alan Kay for comments on this manuscript. This work was supported by a grant from the Biotechnology Council at Iowa State University, the NIH (NS24233) and the US department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, contract W7405-Eng-82.

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