Some properties of the synapses studied in this thesis

In this thesis (chapters 6 and 7) I studied the mossy fibre to granule cell as well as the parallel fibre to Purkinje cell synapses and will therefore briefly describe some of the main properties of these synapses.

1.9,3,1 The mossy fibre to granule cell synapse

Most of the mossy fibre to granule cell synapse formation in the rat takes place in the first three weeks after birth, when granule cells migrate from the external

granule cell layer along Bergmann radial glia through the molecular layer (where their processes form the parallel fibres) towards their final destination, the granule cell layer (Altman, 1972a; Hamori and Somogyi, 1983). Here the mossy fibres form excitatory synapses with granule cells and the maturation of the cerebellar glomeruli takes place (Hamori and Somogyi, 1983). Mossy fibres release glutamate as their neurotransmitter, which acts on postsynaptic AMP A and NMDA receptors (Garthwaite and Brodbelt, 1989; Silver et al., 1992; D'Angelo et al., 1993). The AMP A component is very brief, with a 10-90% rise time of around 200 ps, and a decay time constant of 1-2 ms (Silver et ah, 1992). The NMDA component of the postsynaptic current changes during postnatal development (Rumbaugh and Vicini, 1999; Cathala et ah, 2000), as the NR2 subunit expression of the NMDA receptors in granule cells changes during the first two postnatal weeks (Akazawa et ah, 1994; Farrant et ah, 1994; Monyer et ah, 1994; Watanabe et ah, 1994; Wang et ah, 1995; Takahashi et ah, 1996b): granule cells in rat cerebellum undergo a switch in NR2 expression from NR2B (from birth until around P I4) to NR2A/NR2C (starting gradually from around P8) to mainly NR2C (in the adult). The NRl subunit is expressed throughout this period, but the first mossy fibre synapses form only at the end of the first postnatal week (Altman, 1972b).

The properties of the mossy fibre to granule cell synapse can be modulated in the short term by, for example, metabotropic glutamate and G ABA receptors. G ABA released from Golgi cell terminals can spill over onto GABAb receptors on the mossy fibre terminal, decreasing the amount of glutamate release and thus the amplitude of the postsynaptic current (Mitchell and Silver, 2000a). Conversely, glutamate released from mossy fibres has also been shown to spill over and activate mGluRs (of type I and II) on neighbouring Golgi cell axon terminals, decreasing G ABA release and thus reducing the inhibition onto granule cells (Mitchell and Silver, 2000b). Additionally, granule cells express metabotropic glutamate receptors of groups I and III (Vetter et al., 1999), which couple to the PLC-IP3/DAG and the

cAMP pathways.

The mossy fibre to granule cell synapse exhibits a longer lasting plasticity, i.e. an mGluR dependent form of long-term potentiation (LTP), which requires NMDA receptor activation (rossi et al., 1996; D'Angelo et al., 1999; Armano et al., 2000). It can be elicited either by (1) application of an mGluR agonist (t-ACPD; for around 2 minutes) while NMDA receptors are synaptically activated at low frequency (0.1 Hz, recorded in voltage clamp; Rossi et al., 1996), by (2) theta burst stimulation ( 8 bursts of 10 stimuli at 100 Hz repeated every 250 ms) or a 1 second train (100 Hz) while the granule cell is depolarized (-^0 mV; to allow NMDA receptor activation; recorded in voltage clamp; D ’Angelo et al., 1999) or by (3) theta burst stimulation (4 bursts of 10 stimuli at 100 Hz repeated every 250 ms) when recorded in current clamp (resting potential was -70 mV; Armano et al., 2000). After any of these protocols the amplitudes of the non-NMDA and the NMDA components of the EPSC/EPSP were potentiated (for >45 minutes) by around 40-100 %. Experiments using pharmacological blockers show that this form of LTP requires the activation of mGluRs, NMDA receptors and PKC, as well as a rise in the intracellular calcium concentration (D'Angelo et al., 1999). LTP protocols at the mossy fibre to granule cell synapse did not only augment the synaptic strength, but increased also the intrinsic excitability of the granule cell (i.e. the threshold to fire an action potential was decreased and the input resistance was increased; Armano et al., 2000). However, LTP was not induced if GABAergic inhibition was intact, i.e. when recordings were made in bicuculline-free solution.

1.9,3,2 The parallel fibre to Purkinje cell synapse

Parallel fibres release glutamate when granule cells fire, which activates ionotropic AMP A receptors (Konnerth et al., 1990; Perkel et al., 1990; Llano et al, 1991), metabotropic glutamate receptors (Batchelor et al, 1994; Tempia et al, 2001) and possibly also glutamate transporters (Canepari et al, 2001) on the Purkinje cell.

The parallel fibre to Purkinje cell EPSC is thus mediated by a fast rising and decaying AMP A component (as documented in chapter 6) and a slowly rising and decaying mGluR (mGluRl) component. Synaptic activation of the mGluRl receptors (which belong, as mentioned in section 1.1.2, to the group I of mGluRs) leads to an intracellular calcium concentration rise via IP3 signalling in the Purkinje cell spine (Finch and Augustine, 1998; Takechi et al., 1998), to opening of a cation channel (Canepari et al., 2001) and to release of endogenous cannabinoids, which can act on presynaptic CBl receptors to reduce the amount of neurotransmitter release from parallel fibres (Kreitzer and Regehr, 2001), climbing fibres (Maejima et al., 2001) and intemeurone axon terminals (Diana et al., 2002). Purkinje cell spines also express the 52 glutamate receptors (Landsend et al., 1997; Zhao et al., 1997), which may be important during synaptogenesis and plasticity. There are no functional postsynaptic NMDA receptors at this synapse after about PIO (Rosenmund et al., 1992; Momiyama et al., 1996; Cull-Candy et al., 1998), but it has recently been shown that the presynaptic parallel fibre terminals express NMDA receptors (Casado et al., 2000). The parallel fibre also expresses GABAb and A1 receptors which, when activated by G ABA and adenosine respectively, modulate presynaptic calcium channels and result in reduced glutamate release (Takahashi et al., 1995b; Dittman and Regehr, 1996, 1997).

The granule cell to Purkinje cell synapse shows short term plasticity (on a second time scale: paired pulse facilitation (PPF; Konnerth et al., 1990; Atluri and Regehr, 1996; Kreitzer and Regehr, 2000)) and long term (hour time scale) potentiation (LTP; Salin et al, 1996) and depression (LTD; Ito and Kano, 1982; Ito, 2001)). The short term facilitation can be elicited by a pair of stimuli (less than about

1 second apart; the facilitation decays with a time constant of around 2 0 0 ms) or a train of stimuli (for example 10 stimuli at 1-50 Hz as in Kreitzer and Regehr, 2000). It facilitates the EPSC up to 2.5 times over the millisecond to second time scale and is critically dependent on the residual calcium concentration in the presynaptic

terminal (Atluri and Regehr, 1996; Kreitzer and Regehr, 2000; Carter et al, 2002). LTP can be induced by low frequency stimulation of the parallel fibres (about 2-8 Hz) and is dependent on presynaptic activation of calcium sensitive adenylate cyclase and PKA activation (Salin et al, 1996). LTP is not only induced but also expressed presynaptically (reviewed by Hansel et a l, 2001). Purkinje cell to parallel fibre LTD can be induced by simultaneous low frequency stimulation of parallel fibres and climbing fibres (Ito and Kano, 1982). It depresses the synapse by up to 50% and is dependent on (1) calcium influx via voltage gated calcium channels (thought to be the result of climbing fibre activity), (2) activation of postsynaptic AMP A and mGluRl, and presynaptic NMDA receptors (Casado et al, 2002) by glutamate (as result of parallel fibre activity) and (3) transient PKC activation. Postsynaptic glutamate transporters appear to regulate mGluR (and possibly also presynaptic NMDA receptor) activation and induction of LTD (Brasnjo and Otis, 2001). Long term depression seems to result from a reduction of the number of AMP A receptors in the Purkinje cell membrane due to clathrin mediated endocytosis (reviewed by Hansel et al, 2001). Purkinje cell to parallel fibre LTD appears thus to be induced partly pre- and partly postsynaptically, and expressed postsynaptically. It has long been postulated to play an important role in motor learning (Marr, 1969).