Detecting changes in calcium influx which contribute to synaptic modulation in mammalian brain slice.
The control of neurotransmitter release by modulation of presynaptic calcium influx was investigated at the granule cell to Purkinje cell synapse in rat cerebellar slices. Excitatory post-synaptic currents were measured using whole cell voltage clamp, and changes in presynaptic Ca influx were determined with the Ca-sensitive dye mag-fura-5. Single stimuli of the parallel fibers evoked rapid changes in mag-fura-5 fluorescence which increased from 10 to 90% in 1.4 msec, and then decayed within hundreds of milliseconds to prestimulus levels. These fluorescence changes were unaffected by disruption of internal stores with ryanodine or thapsigargin, and were reduced by 79% by the calcium channel toxin omega-conotoxin-MVIIC. We conclude that these signals result from calcium entry into presynaptic terminals through voltage gated calcium channels opened by action potentials. These fluorescence signals allow us to quantitate changes in calcium influx. We used this approach to study the enhancement of stimulus-evoked synaptic currents by 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor and antagonist of adenosine receptors. Both enhancement of calcium influx into presynaptic terminals, and reduction in the firing threshold of the parallel fibers, were found to contribute to IBMX-mediated synaptic enhancement. Changes in presynaptic calcium influx were also quantified with a novel method, which is unaffected by changes in fiber threshold. These studies illustrate some of the difficulties encountered when determining the factors responsible for synaptic enhancement and demonstrate how measurements of presynaptic calcium influx can contribute to our understanding of synaptic modulation. The approach described here promises to be widely useful in elucidating the role of calcium influx in the modulation of synapses in brain slice.
Sabatini
B L
BL
Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
Regehr
W G
WG
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D002531:Cerebellum / Q000502:physiology*; D004594:Electrophysiology; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000378:metabolism*; D058915:Purinergic P1 Receptor Antagonists; D051381:Rats; D013050:Spectrometry, Fluorescence; D009435:Synaptic Transmission / Q000502:physiology*
D015220:Calcium Channels; D005456:Fluorescent Dyes; D010726:Phosphodiesterase Inhibitors; D058915:Purinergic P1 Receptor Antagonists; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine