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Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan
Received for publication May 22, 2006.
Accepted for publication May 25, 2006.
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2-
(type 1 or 2) (Gong et al., 2001
; Piechan et al., 2004), raising the possibility that these agents act through modulation of voltage sensitive Ca2+ channels. Indeed, in some studies, but not in all, pregabalin and gabapentin cause partial inhibition of Ca2+ currents.
The contribution by (Micheva et al., 2006) in the current issue takes a fresh look at the action of pregabalin. The authors examine the effects of pregabalin on presynaptic function of cultured hippocampal neurons using a powerful technique to follow the trafficking of synaptic vesicles in individual boutons. The method uses amphipathic fluorescent dyes (FM dyes) that label endocytic vesicles in living neurons (Betz and Bewick, 1992; Rizzoli and Betz, 2005). When added to the extracellular medium, these membrane-impermeant probes equilibrate between the aqueous phase and the outer leaflet of the plasma membrane in which they become highly fluorescent (Fig. 1, C1). Upon endocytosis that follows exocytosis, the labeled membrane is internalized. When removed from the extracellular medium, the dye is retained by the endocytic vesicles in the bouton but is lost from the plasma membrane (Fig. 1, C2). Endocytic vesicles are transformed within approximately 1 min into synaptic vesicles containing the dye. Most importantly, these recycled synaptic vesicles lose the probe upon subsequent exocytosis when the vesicle membrane is exposed to the extracellular medium (Fig. 1, C3).
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This technique has permitted the dynamics of the exocytic/endocytic cycle of synaptic vesicles to be investigated at individual boutons. In the cultured hippocampal neurons used in the study by Micheva et al. (2006), each bouton contains approximately 200 synaptic vesicles (Fig. 1A). Half of these vesicles recycle upon stimulation at 37°C and are labeled upon incubation with FM dye (Fig. 1D) (Micheva and Smith, 2005). By labeling boutons by prior electrical stimulation in the presence of FM dye and then washing the cultures of extracellular dye, the subsequent exocytosis of synaptic vesicles can be followed by measuring the decrease in bouton fluorescence.
The present study by Micheva et al. (2006) made use of the unique strengths of this system to provide new insights into the action of pregabalin. By measuring fluorescence intensity changes in hundreds of individual boutons per experimental group with a time resolution of seconds, mechanistic insights were achieved despite the intrinsic variability of synaptic release. In fact, the remarkable statistical precision of the experiments in the hands of these investigators permitted the discovery of heretofore-unappreciated aspects of pregabalin effects.
Micheva et al. (2006) revealed a small inhibition of synaptic vesicle exocytosis at high stimulus frequency in both glutamatergic and GABAergic neurons at clinically relevant concentrations of pregabalin. The investigators were surprised to find that pregabalin targets a subpopulation of recycling vesicles, the readily releasable pool. This pool of vesicles is reduced by approximately 25%. Vesicles in the readily releasable pool are the first to be released upon electrical stimulation in the presence of Ca2+ and are specifically released in a Ca2+-independent manner in response to hypertonic sucrose (Rosenmund and Stevens, 1996). Consistent with these results is the reduction in spontaneous synaptic vesicle exocytosis, which is known to be independent of Ca2+ and probably comes from the same readily releasable pool. These effects are unlikely to be caused by inhibition of voltage sensitive Ca2+ channels. They probably reflect a new action of pregabalin on the vesicular trafficking pathway in neurons, perhaps mediated by an unknown function of the auxiliary Ca2+ channel subunit 2-. The effect of pregabalin to reduce the readily releasable pool is likely to have important functional implications. The reduction is predicted to decrease the probability of synaptic vesicle fusion even during a single action potential (Rosenmund and Stevens, 1996).
Another intriguing aspect of the study by Micheva et al. (2006) was the finding that an NMDA antagonist prevented the pregabalin-induced inhibition of transmission. Although NMDA receptors are primarily postsynaptic, there is also compelling evidence for presynaptic NMDA receptors (MacDermott et al., 1999). Thus, the NMDA receptor-mediated effect could originate either postsynaptically through a retrograde signaling pathway (Micheva et al., 2003) or presynaptically. It will be important in future studies to determine the mechanism by which NMDA receptor activation participates in pregabalin action.
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Footnotes |
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Please see the related article on page 467.
ABBREVIATIONS: FM 4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide; NMDA, N-methyl-D-aspartate.
Address correspondence to: Ronald W. Holz, Department of Pharmacology, University of Michigan Medical School, 2301 MSRB III, Ann Arbor, MI 48109-0632. E-mail holz{at}umich.edu
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References |
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TopAbstractReferences |
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Gong HC, Hang J, Kohler W, Li L, and Su TZ (2001) Tissue-specific expression and gabapentin-binding properties of calcium channel A2 subunit subtypes. J Membrane Biol 184: 35-43.[CrossRef][Medline]
Holz RW and Fisher SK (2006) Synaptic transmission and cellular signaling: an overview, in Basic Neurochemistry, 7th ed (Siegel GJ, Albers RW, Brady ST, and Price DL eds) pp 167-183, Elsevier Academic Press, San Diego.
MacDermott AB, Role LW, and Siegelbaum SA (1999) Presynaptic ionotropic receptors and the control of transmitter release. Annu Rev Neurosci 22: 443-485.[CrossRef][Medline]
Micheva KD, Buchanan J, Holz RW, and Smith SJ (2003) Retrograde regulation of synaptic vesicle endocytosis and recycling. Nat Neurosci 6: 925-932.[CrossRef][Medline]
Micheva KD and Smith SJ (2005) Strong effects of subphysiological temperature on the function and plasticity of mammalian presynaptic terminals. J Neurosci 25: 7481-7488.
Micheva KD, Taylor CP, and Smith SJ (2006) Pregabalin reduces the release of synaptic vesicles from cultured hippocampal neurons. Mol Pharmacol 70: 467-476.
Murthy VN, Schikorski T, Stevens CF, and Zhu Y (2001) Inactivity produces increases in neurotransmitter release and synapse size. Neuron 32: 673-682.[CrossRef][Medline]
Piechan JL, Donevan SD, Taylor CP, Dickerson MR, and Li Z (2004) Pregabalin, a novel anticonvulsant, analgesic and anxiolytic drug, exhibits class-specifric A2-1 and A2-2 calcium channel subunit binding. Soc Neurosci Abstr 30: 115.11.
Rizzoli SO and Betz WJ (2005) Synaptic vesicle pools. Nat Rev Neurosci 6: 57-69.[CrossRef][Medline]
Rosenmund C and Stevens CF (1996) Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16: 1197-1207.[CrossRef][Medline]
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50% of the 200 SV in boutons recycle at 37°C. [Reproduced from Micheva KD and Smith SJ (2005) Strong effects of subphysiological temperature on the function and plasticity of mammalian presynaptic terminals.