Basal somatodendritic dopamine release requires snare proteins Gabriel D. Fortin,* Catherine C. Desrosiers,* Nobuharu Yamaguchi and Louis-E ´ ric Trudeau* *Department of Pharmacology, Faculty of Medicine and  Faculty of Pharmacy, Universite ´ de Montre ´al, Montreal, Quebec, Canada Abstract Dopaminergic neurons have the capacity to release dopamine not only from their axon terminals, but also from their somato- dendritic compartment. The actual mechanism of somato- dendritic dopamine release has remained controversial. Here we established for the first time a rat primary neuron culture model to investigate this phenomenon and use it to study the mechanism under conditions of non-stimulated spontaneous firing (1–2 Hz). We found that we can selectively measure somatodendritic dopamine release by lowering extracellular calcium to 0.5 mM, thus confirming the previously established differential calcium sensitivity of somatodendritic and terminal release. Dopamine release measured under these conditions was dependent on firing activity and independent of reverse transport through the plasma membrane. We found that treat- ment with botulinum neurotoxins A and B strongly reduced so- matodendritic dopamine release, thus demonstrating the requirement for SNARE proteins SNAP-25 and synaptobrevin. Our work is the first to provide such direct and unambiguous evidence for the involvement of an exocytotic mechanism in basal spontaneous somatodendritic dopamine release. Keywords: botulinum toxins, cell culture, dopamine, exocy- tosis, somatodendritic, rat, SNARE proteins. J. Neurochem. (2006) 96, 1740–1749. Somatodendritic dopamine release has been established as a biological fact in the late 1970s (Bjorklund and Lindvall 1975; Geffen et al. 1976; Cheramy et al. 1981). However, the mechanism by which dopamine is released from the soma and dendrites of neurons has remained controversial. Com- patible with an exocytotic-like mechanism, numerous authors have confirmed that somatodendritic dopamine release is activity-dependent and sensitive to tetrodotoxin (Robertson et al. 1991; Kalivas and Duffy 1991; Santiago et al. 1992; Heeringa and Abercrombie 1995; Beckstead et al. 2004). Moreover, it is calcium-dependent (Beart and McDonald 1980; Elverfors et al. 1997; Chen and Rice 2001) and can be inhibited by blocking the vesicular monoamine transporter with reserpine (Elverfors and Nissbrandt 1991; Heeringa and Abercrombie 1995). Although these properties are charac- teristic of vesicular exocytotic release, observations of actual synaptic-like vesicles within dendrites have proven difficult (Hajdu et al. 1973; Wilson et al. 1977; Hattori et al. 1979; Wassef et al. 1981; Groves and Linder 1983). The endo- plasmic reticulum (ER) and its associated tubulovesicles have been proposed as possible storage sites for somato- dendritic dopamine. These structures indeed appear to contain dopamine (Groves and Linder 1983; Hattori et al. 1979), and similar structures have been shown to express proteins of the SNAP-receptor (SNARE) complex, otherwise known to be essential for exocytosis (Prekeris et al. 1999). Falkenburger et al. (2001) have recently shown in a midbrain slice preparation that the stimulation of glutamat- ergic afferent fibres from the subthalamic nucleus, either with carbachol or with high frequency electrical stimulation, induced somatodendritic dopamine release through a mech- anism requiring the plasma membrane dopamine transporter (DAT) (Falkenburger et al. 2001). On the contrary however, previous observers have reported that direct electrical stimulation of the substantia nigra in the slice or perfusion with high potassium induced extracellular dopamine accu- mulation that increased with DAT blockade (Elverfors et al. 1997; Chen and Rice 2001; Beckstead et al. 2004), impli- cating a mechanism that is completely independent from reverse transport. This apparent discrepancy is probably due to the fact that different stimulation conditions induce somatodendritic dopamine release through different mecha- nisms, including facilitation of reverse transport by glutamate Received November 21, 2005; accepted November 25, 2005. Address correspondence and reprint requests to Dr Louis-Eric Trud- eau, Department of Pharmacology, Faculty of Medicine, Universite ´ de Montre ´al, C.P. 6128, Succursale Centre-Ville, Montre ´al, Que ´bec, Canada, H3C 3J7. E-mail: louis-eric.trudeau@umontreal.ca Abbreviations used: BoNT, botulinum neurotoxin; DAT, dopamine transporter; EPSC and IPSC, excitatory and inhibitory postsynaptic currents; KRB, Krebs ringer buffer; SNARE, SNAP-receptor; TTX, tetrodotoxin; VAMP, synaptobrevin. Journal of Neurochemistry , 2006, 96, 1740–1749 doi:10.1111/j.1471-4159.2006.03699.x 1740 Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 1740–1749 Ó 2006 The Authors