Mapping of presynaptic nicotinic acetylcholine receptors using fluorescence imaging of neuritic calcium Romain Girod a , Mark Jareb b , Jason Moss a , Lorna Role a, * a Department of Anatomy and Cell Biology, The Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, 1051 Riverside Drive PI, Annex 807, New York, NY 10032, USA b Department of Biology, Sacred Heart University, Fairfield, CT 06432, USA Received 24 July 2001; received in revised form 6 August 2002; accepted 6 August 2002 Abstract Neuronal nicotinic receptors (nAChRs) appear to function at both pre- and postsynaptic sites, to modulate the release of neurotransmitter, and to mediate synaptic transmission, respectively. Localization of functional nAChRs at presynaptic structures has only been possible under the best of circumstances where the presynaptic structure is very large allowing direct nAChR channel recording. We report here a novel and simple method that allows the visualization of stimulus-evoked changes in Fura-2 fluorescence in the presynaptic structures of essentially any neuron type in vitro. Following ‘loading’ of all neurons by incubation with the calcium-sensitive dye, Fura-2-AM, we selectively reduced the fluorescent signal in the postsynaptic neuron by injecting the Fura-2 quenching agent, Mn 2 , into the postsynaptic neuron. After quenching, nicotine treatment elicits calcium transients that can be observed in spatially distinct regions of neurite bundles contacting the Mn 2 -infused neuron. Thus, the approach described allows one to readily map the distribution of activated nAChRs on presynaptic inputs in vitro. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Calcium; Presynaptic receptors; Nicotine 1. Introduction Neuronal nicotinic acetycholine receptors (nAChRs) are involved in numerous behaviors and functions as varied as attention, homeostasis, addiction, learning and memory. A diversity of nAChR subtypes appears to underlie this diversity of function; eight different a subunits (a2  /a9) and three different b subunits (b2  / b4) can be used to form functional nAChRs. Similar to the muscle nAChRs, neuronal nAChRs are thought to be pentameric (Anand et al., 1991; Cooper et al., 1991). Hence, the theoretical number of nAChRs with a distinct subunit composition is greater than 100 000. Work from heterologous expression systems suggest that only a small subset of the many possible nAChR subunit combinations can form functional nAChRs, yet the diversity can still be quite extensive(Boulter et al., 1987; Couturier et al., 1990; rev. Role and Berg, 1996). For example, neurons cultured from chick lumbar sympathetic ganglia (LSG), which as a population encode relatively high levels of a3, a5, a7, b2, and b4 subunit mRNA, express at least seven functionally distinguishable nAChRs on their soma (Moss et al., 1989; Moss and Role, 1993; Yu and Role, 1998a,b). In addition to this diversity of nAChR subtypes, the subcellular localization of the nAChR to pre- versus postsynaptic sites affects the functional consequences of nAChR activation. A number of studies have demon- strated direct nAChR-mediated synaptic transmission in regions such as the cerebral cortex, hippocampus, and ventral tegmental area (rev. Jones et al., 1999). In addition, activation of nAChRs has been shown to modulate the release of a wide variety of neurotrans- mitters from presynaptic structures (rev. Role and Berg, * Corresponding author. Tel.:  /1-212-305-8117; fax:  /1-212-795- 0700. E-mail address: lwr1@columbia.edu (L. Role). Journal of Neuroscience Methods 122 (2003) 109 /122 www.elsevier.com/locate/jneumeth 0165-0270/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII:S0165-0270(02)00232-7