Abstract
The cerebellum has often been referred to as the "neuronal machine", so called for the elegant architecture of neuronal circuitry and necessarily clockwork precision of signal transmission therein (Eccles 1967; Ito 2006). To maintain such precision, many synapses within the cerebellar micro-circuitry are highly plastic and frequency dependant. In particular, short-term synaptic plasticity (STP) at an important excitatory synaptic pathway between cerebellar granule cells (CGCs) and Purkinje neurons (PN), called the "parallel fibre to Purkinje neuron synapse" (PF-PN), is an integral feature of the synapse and for cerebellar function as a whole (Dittman, Kreitzer et al. 2000; Boyden, Katoh et al. 2004).
During transmission at PF-PN synapses, the dynamics of elevated pre-synaptic calcium (or residual calcium) dramatically impact the STP exhibited by the synapse (Zucker and Regehr 2002). Multiple cellular mechanisms function cooperatively to carefully control residual calcium dynamics and two principle pre-synaptic mechanisms expressed in CGCs include the sodium calcium exchanger (NCX) and plasma membrane calcium ATPase (PMCA) (Blaustein, Juhaszova et al. 2002; Ivannikov, Sugimori et al. 2010). Both PMCA and NCX proteins function to remove elevated intracellular calcium and their cooperative activity is thought to be critical for maintaining PF-PN synaptic behaviour (Regehr 1997; Empson, Garside et al. 2007). However, characterisation of NCX activity in pre-synaptic calcium control, its influence on synaptic transmission, and how it might interplay with PMCA activity at PF-PN synapses remains to be established.
This research aimed to understand the cooperative activity of NCX and PMCA2 and its functional impact on PF-PN synaptic behaviour. To do this, calcium fluorescent imaging and electrophysiological recordings were made from the mouse cerebellum in vitro, whilst PMCA2 and NCX were sequentially removed by either pharmacological or genetic manipulation to assess their individual and combined activities.
Two fluorescent calcium imaging techniques using a genetically encoded calcium indicator, GCaMP2 and a conventional calcium indicator, Calcium Green-1 Dextran, were utilised to determine the influence that cooperative NCX and PMCA2 activity has on PF pre-synaptic calcium dynamics. The impact that cooperative NCX and PMCA2 activity had on PF-PN synaptic behaviour was addressed using patch clamp electrophysiology to record PF-PN post-synaptic currents and assess STP of the synapse. To supplement experimental studies, a computational modelling approach was used throughout to aid interpretation of calcium fluorescent imaging experiments. The model simulated pre-synaptic calcium dynamics to provide a theoretical basis for how calcium efflux characteristics exhibited by NCX and PMCA2 dictate their cooperative interplay for pre-synaptic calcium control.
This investigation has provided strong evidence for an important cooperative interplay of pre-synaptic NCX and PMCA2 activity, capable of influencing PF-PN synapse behaviour. The extent of cooperation between NCX and PMCA2 activity depends upon on pre-synaptic calcium load (hence pre-synaptic activity) and this influences the behaviour of the PF-PN synapse. It is proposed that the kinetic balance for this cooperative activity within the PF pre-synaptic terminal is predominantly governed by the kinetic properties that each mechanism exhibits, including their calcium affinity for activation and maximal efflux capacities.