Calcium channels in evoked neurotransmitter release at individual synapses and neurological disease

Synapses between neurons are critical sites of modulation and plasticity, both in health and in disease. We have devised a new way of investigating the events leading up to the release of chemical neurotransmitters at individual synapses. This is based on measuring, with fluorescence microscopy, rapid changes in the concentration of calcium ions, as well as the rate at which small vesicles containing chemical neurotransmitters are discharged. This approach will allow us to investigate how different channels that transmit calcium ions into the terminal control the release of vesicles, how they influence plasticity, and how synapses are influenced by other modulatory neurotransmitters acting upon presynaptic terminals. Moreover, it has recently been established that some inherited cases of migraine, ataxia (incoordination caused by abnormal cerebellar function) and epilepsy are due to mutations of one subtype of calcium channel. We will apply these novel methods to investigate how the desiase-linked mutations affect calcium signals in nerve terminals, and how they affect neurotransmitter release. By making advances at the cutting edge of basic neuroscience relevant to neurological disease this project may point the way to new approaches of treating several disabling neurological diseases caused by intermittent disturbances of synaptic function.

Synaptic release of neurotransmitters is triggered by Ca2+ entry via P/Q-, N-, and R-type Ca2+ channels. Although these channels differ in their biophysical and pharmacological properties, their contributions to the extensive heterogeneity in use-dependent control of neurotransmitter release exhibited by distinct synapses are poorly understood. This question is important to understand both the normal function of neuronal circuits and the mechanisms of several neurological disorders that are caused by mutations in Ca2+ channel genes (notably some forms of migraine, ataxia, and absence epilepsy). Hitherto, investigating the roles of distinct Ca2+ channel subtypes at small central synapses has been hampered by the difficulty of recording electrophysiologically and/or optically from individual nerve terminals. Most studies have therefore been based on recordings obtained from populations of synapses.

The main aim of this project is to understand the roles of different Ca2+ channels subtypes in mediating presynaptic control at small excitatory and inhibitory synapses through the application of a new imaging technique, which allows presynaptic Ca2+ dynamics and vesicular release to be studied simultaneously at a single-synapse level. I have developed and tested this methodology in the sponsor‘s laboratory.

Briefly, I patch-load neurons in dissociated cultures with the morphological tracer Alexa-594 and a fluorescent Ca2+ indicator (e.g. Fluo-4). This allows identification and recordings of Ca2+ dynamics in individual axonal boutons using fast confocal microscopy. In parallel, I load the same set of boutons with a styryl dye (SynaptoRed C1) to image action potential-dependent vesicular release. This approach allows me for the first time to directly probe modulation of neurotransmitter release by different Ca2+ channels at individual synapses.

In particular, I will aim to: (1) test the hypothesis that P/Q-type channels are more efficiently coupled to the release machinery than other channel sub-types; (2) establish how short-term plasticity (use-dependent facilitation/depression and modulation by G protein coupled receptors) correlates with the expression of different Ca2+ channel sub-types at individual nerve terminals; and (3) apply the methods and insights gained during this study to investigate the effects of familial hemiplegic migraine (FHM) - associated mutations of the CACNA1A gene, which encodes the {alpha}1 pore-forming subunit of P/Q-type channels.

The anticipated outcome will provide novel and important insights not only into the roles of different Ca2+ channels in neurotransmitter release, but also into the molecular mechanisms of FHM.