Climbing Fiber Signaling and Cerebellar Gain Control

The physiology of climbing fiber signals in cerebellar Purkinje cells has been studied since the early days of electrophysiology. Both the climbing fiber-evoked complex spike and the role of climbing fiber activity in the induction of long-term depression (LTD) at parallel fiber-Purkinje cell synapses have become hallmark features of cerebellar physiology. However, the key role of climbing fiber signaling in cerebellar motor learning has been challenged by recent reports of forms of synaptic and non-synaptic plasticity in the cerebellar cortex that do not involve climbing fiber activity, but might well play a role in cerebellar learning. Moreover, cerebellar LTD does not seem to strictly require climbing fiber activity. These observations make it necessary to re-evaluate the role of climbing fiber signaling in cerebellar function. Here, we argue that climbing fiber signaling is about adjusting relative probabilities for the induction of LTD and long-term potentiation (LTP) at parallel fiber synapses. Complex spike-associated, dendritic calcium transients control postsynaptic LTD and LTP induction. High calcium transients, provided by complex spike activity, do not only favor postsynaptic LTD induction, but simultaneously trigger retrograde cannabinoid signaling, which blocks the induction of presynaptic LTP. Plasticity of the climbing fiber input itself provides additional means to fine-tune complex spike associated calcium signaling and thus to adjust the gain of heterosynaptic climbing fiber control. In addition to dendritic calcium transients, climbing fiber activity leads to the release of the neuropeptide corticotropin-releasing factor (CRF), which facilitates LTD induction at both parallel fiber and climbing fiber synapses.

, which are required for the induction of cerebellar LTD .
More recent observations suggest that the role of climbing fi ber signaling is more complex than that of an invariant 'teacher' signal contributing to LTD induction and, therefore, to cerebellar motor learning. Here, we will discuss recent evidence showing that climbing fi ber synapses onto cerebellar Purkinje cells show forms of plasticity as well. Moreover, we will review recent observations on types of synaptic and non-synaptic cerebellar plasticity that do not depend on climbing fi ber signaling, suggesting that motor learning is not exclusively linked to climbing fi ber activity. Rather, a picture emerges, in which the presence or absence of climbing fi ber activity infl uences induction probabilities for various types of plasticity, thus orchestrating Purkinje cell output patterns and cerebellar gain control.

INTRODUCTION
The fi rst detailed characterization of excitatory synaptic responses to climbing fi ber stimulation resulted from intracellular Purkinje cell recordings performed in anaesthetized cats by Eccles, Llinas and Sasaki, working at the time at the Australian National University in Canberra (Eccles et al., 1964(Eccles et al., , 1966. Stimulation of the climbing fi ber input, or the contralateral inferior olive (from which climbing fi bers originate) led to an all-or-none 'giant' or 'complex' spike, composed of an initial fast action potential, followed by smaller spikelets superimposed on a sustained depolarization (Eccles et al., 1966). In the mature cerebellum, this massive, excitatory response pattern results from the activity of only one climbing fi ber input (Ramón y Cajal, 1911) that remains after a period of developmental elimination of surplus climbing fi bers, which in rats occurs during the fi rst 3 weeks of postnatal life (Crépel et al., 1976).
The characteristic complex spike provides a hallmark feature of cerebellar physiology, particularly because of its seemingly invariant nature (for review, see Schmolesky et al., 2002). Another hallmark feature is the role assigned to climbing fi ber activity in cerebellar motor learning. It is widely assumed that long-term depression (LTD) at parallel fi ber-Purkinje cell synapses, a type of synaptic plasticity that requires co-activation of the parallel fi ber and the climbing fi ber input, provides the cellular correlate of forms of motor learning. Climbing fi ber activity results in large, widespread calcium transients in Purkinje cell dendrites (Miyakawa et al., 1992; whereas parallel fi ber synapses contact spines on secondary and tertiary dendritic branches (Strata and Rossi, 1998). Whereas parallel fi ber stimulation causes a graded excitatory postsynaptic potential (EPSP) in Purkinje cells, climbing fi ber activity is monitored in somatic recordings as an all-or-none complex spike (Eccles et al., 1964(Eccles et al., , 1966, characterized by a fast sodium spike that is followed by typically two to three spikelets riding on top of a depolarization plateau (Figure 1). Purkinje cell complex spikes occur at low frequencies around 1 Hz, but can reach frequencies up to 11 Hz when nociceptive stimulation is applied (Ekerot et al., 1987). Each complex spike can be associated with high-frequency fi ring of spikes in the climbing fi ber itself, which can reach frequencies up to 500 Hz (Maruta et al., 2007). In Purkinje cell dendrites, climbing fi ber activation evokes calcium spikes, which have been characterized in early intradendritic recordings (Llinas and Sugimori, 1980). For a long time, however, the origin of the different somatically recorded complex spike components remained unclear (as discussed in Schmolesky et al., 2002). Recent somato-dendritic double-patch recordings demonstrate that dendritically recorded calcium spikes show no obvious relation to the number and occurrence of spikelets within the simultaneously recorded complex spike (Davie et al., 2008). These observations suggest that the typical, somatically recorded complex spike waveform is locally generated, whereas in the dendrite, climbing fi ber activity evokes isolated calcium spikes. Simultaneous somatic and dendritic patch-clamp recordings from our lab are shown in Figure 1. In the soma, climbing fi ber stimulation evokes complex spikes (black traces). In contrast, the dendritic recordings (blue traces) obtained at distances of about 75 µm ( Figure 1A) and 100 µm ( Figure 1B) from the soma, respectively, reveal local climbing fi ber responses, which do not show FIGURE 1 | Climbing fi ber responses monitored by simultaneous double patch-clamp recordings from the soma and dendrites of cerebellar Purkinje cells. (A) Paired recordings from the soma (black trace) and the dendrite (blue trace). The dendritic patch electrode was located at about 75 µm distance from the soma. (B) In this recording, which was obtained from a different Purkinje cell, a more distal location (ca. 100 µm) was selected for the dendritic patch electrode. Small arrows in (A) and (B) point towards small, delayed spikelets seen in the dendritic recordings, that likely refl ect sodium action potentials generated in the axon (see corresponding, but larger spikes in the somatic recordings), which passively spread into the dendrite. (C) At a far more proximal location (ca. 10 µm), the dendritic recording resembles the complex spike recorded in the soma, but already at this distance an attenuation of the initial, fast sodium spike can be seen. (D) Recording confi guration of the experiments. This picture was assembled for illustration purposes only. The recordings were obtained from cerebellar slices prepared from P28-35 Sprague-Dawley rats. Slices were perfused with standard ACSF (as described in Coesmans et al., 2004) bubbled with 95% O 2 and 5% CO 2 . The recordings were performed at near-physiological temperature (31-34°C) using an EPC-10 amplifi er (HEKA Electronics, Germany). Currents were fi ltered at 3 kHz, digitized at 10 kHz, and acquired using Fitmaster software. The recording electrodes were fi lled with a solution containing (in mM): 9 KCl, 10 KOH, 120 K gluconate, 3.48 MgCl 2 , 10 HEPES, 4 NaCl, 4 Na 2 ATP, 0.4 Na 3 GTP, and 17.5 sucrose (pH 7.25). Patch electrodes used for somatic recordings had electrode resistances of 3-4 MΩ, and patch electrodes used for dendritic recordings had electrode resistances of 7-10 MΩ. For climbing fi ber stimulation, glass pipettes were used that were fi lled with ACSF. the spikelets that are characteristic for the somatically recorded complex spike. An exception to this are small spikelets that occasionally occur during the late stages of the dendritic climbing fi ber response ( Figures 1A,B: arrows). These spikelets coincide with late, higher amplitude spikes seen in the somatic recordings. In Purkinje cells, sodium action potentials are initiated in the axon, and passively spread into the dendrite, where their amplitude decreases with increasing distance from the soma (Stuart and Häusser, 1994). The small spikelets seen in our dendritic recordings likely represent those attenuated sodium spikes.

CLIMBING FIBER ACTIVITY EVOKES DENDRITIC CALCIUM TRANSIENTS
Climbing fi bers form so-called 'en passant' synapses with their target Purkinje cells, whose number and distribution along the axis of the Purkinje cell primary dendrite allow for excitatory action throughout the dendritic tree. The most obvious consequence of this unusually tight synaptic contact formed by a single climbing fi ber input is a large, widespread calcium transient that accompanies complex spikes (Miyakawa et al., 1992;Ross and Werman, 1987). Climbing fi ber-evoked calcium transients not only can be recorded in the primary dendrites (an example of a calcium transient recorded in a primary dendrite spine is shown in Figure 2), but also in secondary and tertiary branches (Miyakawa et al., 1992;Ross and Werman, 1987). Such 'out-of-territory' calcium signaling has also been described in cerebellar Purkinje cells of mormyrid fi sh (Han et al., 2007), in which the separation of climbing fi ber and parallel fi ber input territories is even more pronounced. The dendritic tree of these cells is palisade-shaped, with a horizontal dendrite (contacted by the climbing fi ber input), and vertical dendrites (contacted by parallel fi bers) that show a much lower degree of branching as compared to their mammalian counterparts. Climbing fi ber stimulation results in calcium transients in both the horizontal dendrite and the vertical dendrites (Han  et al., 2007). In both mammalian and mormyrid Purkinje cells, calcium infl ux associated with dendritic calcium spikes is partially mediated by P/Q-type voltage-dependent calcium channels (Han et al., 2007;Usowicz et al., 1992;Watanabe et al., 1998), suggesting that these channels provide the regenerative component needed for the spatial spread of the calcium signal. In spines contacted by parallel fi bers, coincident parallel fi ber and climbing fi ber activity results in supralinear calcium transients (Wang et al., 2000). These spine calcium signals are largest when the parallel fi ber activation precedes climbing fi ber activation by 50-200 ms, and depend on calcium release from IP 3 -sensitive calcium stores when the parallel fi ber input is weakly activated (Wang et al., 2000). Stronger parallel fi ber stimulation results in the activation of more parallel fi bers. In this scenario, supralinear calcium signaling is not restricted to individual spines and is mediated by the activation of voltage-dependent calcium channels (Wang et al., 2000). A similar enhancement in dendritic calcium signaling at parallel fi ber input sites has been found in mormyrid Purkinje cells upon parallel fi ber and climbing fi ber stimulation (Han et al., 2007). These observations show that calcium transients evoked by climbing fi ber activity do not only invade the parallel fi ber input territory, but that they contribute to a local amplifi cation of calcium transients at parallel fi ber input sites, even in spines located on distal branchlets that are contacted by parallel fi bers (Wang et al., 2000). This is a remarkable fi nding, as the climbing fi ber input itself only contacts spines on the primary dendrite (Strata and Rossi, 1998). The amplitude of the climbing fi ber-evoked calcium transient itself also depends on the state of the membrane potential. Synaptic activity of the climbing fi ber or the granule cell input can shift the membrane potential to an 'UP' state, while inhibitory inputs can cause a transition towards a 'DOWN' state (Loewenstein et al., 2005; but see Schonewille et al., 2006). Dendritic climbing fi ber-evoked calcium transients are smaller following a 'DOWN' state (Rokni and Yarom, 2009). If these bidirectional state transitions occur in vivo, 'UP' states could provide an optimal time window for enhanced dendritic calcium signaling.

GLUTAMATERGIC TRANSMISSION AT CLIMBING FIBER SYNAPSES: NEW PLAYERS IN SIGHT
Excitatory postsynaptic currents (EPSCs) at climbing fi ber synapses are largely mediated by the activation of AMPA receptors (Konnerth et al., 1990;Llano et al., 1991;Perkel et al., 1990) that contain GluR2 subunits and are therefore not permeable to calcium (Hollmann et al., 1991). In the following, we will focus on two additional types of glutamate receptors, whose contribution to climbing fi ber signaling (and potential role in calcium signaling) has only recently been fully appreciated: N-methyl-D-aspartate (NMDA) receptors and metabotropic glutamate receptors.

NMDA RECEPTORS
At many types of excitatory synapses, NMDA receptors provide a major source of calcium infl ux and are therefore considered as key players in synaptic plasticity (Bliss and Collingridge, 1993). Purkinje cells, in contrast, were until recently assumed to lack functional NMDA receptors. Both NR1 and NR2 subunits are required to form functional NMDA receptors. In Purkinje cells, NR1 subunits are expressed at all ages, from birth throughout adulthood, as consist-ently shown by numerous morphological studies (Monyer et al., 1992(Monyer et al., , 1994Moriyoshi et al., 1991;Petralia et al., 1994). During the fi rst postnatal week in rodents, a juvenile form of the NR2 subunit (the NR2D subtype) has clearly been demonstrated in Purkinje cells (Momiyama et al., 1996). However, during this period the resulting NMDA receptors do not contribute to synaptic transmission at parallel fi ber or climbing fi ber inputs (Lachamp et al., 2005;Llano et al., 1991). In mature Purkinje cells, in situ hybridization and immunohistochemical studies reached contradicting conclusions regarding the expression of NR2 subunits (Monyer et al., 1994;Watanabe et al., 1994;Yamada et al., 2001; but see Akazawa et al., 1994;Thompson et al., 2000). As no NMDA currents were detected in whole-cell patch-clamp recordings (Farrant and Cull-Candy, 1991;Konnerth et al., 1990;Llano et al., 1991), the notion became widely accepted that Purkinje cells lack functional NMDA receptors.
Recently, this question has been re-examined in older animals (>8-week-old mice) using more selective pharmacological tools (NBQX instead of CNQX as an AMPA receptor antagonist) and a new generation of antibodies. It now appears in the light of these more recent results (Piochon et al., 2007;Renzi et al., 2007) that mature Purkinje cells express functional NMDA receptors from the end of the third postnatal week on, which contain NR2A and/or NR2B subunits. These NMDA receptors are activated by climbing fi ber stimulation and contribute to the complex spike waveform, by infl uencing the number and timing of spikelets, as well as the afterdepolarization plateau (Piochon et al., 2007). Thus, in mature Purkinje cells, functional NMDA receptors are expressed at climbing fi ber synapses, and might well contribute to climbing fi berevoked calcium signaling.

METABOTROPIC GLUTAMATE RECEPTORS
Similar to parallel fi ber burst stimulation, climbing fi ber stimulation can evoke a slow excitatory current that is triggered by the activation of type 1 metabotropic glutamate receptors (mGluR1) and can be signifi cantly enhanced when glutamate uptake is blocked (Dzubay and Otis, 2002). Such mGluR1-mediated potentials are also evoked by climbing fi ber stimulation in the presence of an mGluR agonist in the bath, suggesting agonist binding and a widespread, dendritic calcium transient as key triggers for this type of slow excitatory signaling (Yuan et al., 2007). In this scenario, climbing fi ber activity could facilitate mGluR1 potentials well beyond the climbing fi ber input territory, providing that the climbing fi ber-evoked calcium signal coincides with suffi ciently high local glutamate transients. A related phenomenon has been described earlier, in which parallel fi ber activation elicits mGluR1 potentials when the climbing fi ber input was stimulated up to 90 s prior to the parallel fi ber input (Batchelor and Garthwaite, 1997). Adding the calcium chelator EGTA to the recording electrode abolished the slow potentials, while they could be triggered when substituting photolytic calcium uncaging for climbing fi ber stimulation. The examples provided in these studies (Batchelor and Garthwaite, 1997;Yuan et al., 2007) show that calcium signaling is required to trigger mGluR1 potentials. Recent evidence suggests that this calcium sensitivity results from the involvement of TRPC cation channels that mediate the slow, excitatory conductances. Both TRPC1 (Kim et al., 2003) and TRPC3 channels (Hartmann et al., 2008) have been suggested to mediate the slow current. TRPC channels open in response to G-protein-coupled receptor activation and/or the occurrence of calcium surges. This activation pattern principally enables TRPC channels to mediate capacitive calcium entry after release of calcium from intracellular stores (Montell et al., 2002). However, the calcium transient associated with slow, mGluR1 potentials is not blocked in Purkinje cells obtained from TRPC1 −/− or TRPC3 −/− mice (Hartmann et al., 2008). These observations suggest that mGluR1 potentials might be triggered by calcium surges, but that signifi cant components of the associated calcium infl ux are not contributed by TRPC channels.
These novel fi ndings are crucial for the present discussion of the consequences of climbing fi ber signaling for cerebellar plasticity and function, because mGluR1 potentials signifi cantly enhance complex spike associated calcium transients (Yuan et al., 2007), and might therefore facilitate the induction of parallel fi ber LTD. It has been suggested that such an increase in the LTD induction probability occurs when mGluR1 potentials are enhanced after blockade of glutamate transporters (Brasnjo and Otis, 2001). However, the cause for the enhanced probability of LTD induction remains unclear. Both TRPC channels (but see Hartmann et al., 2008) and IP 3 -mediated calcium release from internal stores could contribute to more pronounced calcium signaling. Moreover, a stronger mGluR1 activation would also result in enhanced PKC activation. All of these factors could enhance the LTD induction probability. Despite of these remaining uncertainties with regard to the type of TRPC channels involved and the origin of the calcium signal associated with slow excitatory potentials, new aspects of mGluR1 signaling at climbing fi ber synapses emerge. First, mGluR1-triggered potentials can be evoked by climbing fi ber signaling, and second, these slow potentials enhance calcium signaling and are likely to facilitate LTD induction.

PLASTICITY OF CLIMBING FIBER-PURKINJE CELL SYNAPSES
Marr-Albus-Ito models of cerebellar motor learning describe the parallel fi ber input to Purkinje cells as the site in the cerebellar network at which the learning events take place (Albus, 1971;Ito, 1984;Marr, 1969). In these classic models, the climbing fi ber plays a crucial role as well, but is seen as a 'teacher' that signals errors and disturbances in sensomotoric function, rather than an additional site of information storage. The long prevailing dogma of the 'invariant' climbing fi ber response resulted from the high probability of release at climbing fi ber terminals (Dittman and Regehr, 1998;Hashimoto and Kano, 1998;Silver et al., 1998), as well as the all-or-none character of climbing fi ber signaling (Eccles et al., 1966). These features make climbing fi ber transmission both extremely reliable and forceful, and distinguish it from transmission at most other types of central nervous system synapses.
Nevertheless, synaptic plasticity exists at climbing fi ber synapses as well: LTD of climbing fi ber EPSCs (recorded in voltage-clamp mode) can be induced using low-frequency (5 Hz, 30 s) climbing fi ber stimulation (Carta et al., 2006;Hansel and Linden, 2000). In current-clamp mode, LTD is associated with an alteration in the complex spike waveform (Hansel and Linden, 2000), a reduction in the complex spike afterhyperpolarization (Schmolesky et al., 2005), and a long-term depression of climbing fi ber evoked calcium transients (Weber et al., 2003). Figure 3 illustrates two crucial aspects of climbing fi ber LTD: climbing fi ber tetanization leads to a reduction in the amplitude of the slow spikelets that make up the late component of a complex spike (Figure 3A), and the associated calcium transients (Figure 3B). Climbing fi ber LTD is postsynaptically induced and expressed (Shen et al., 2002). The biochemical cascade for the induction of climbing fi ber LTD shares elements with the LTD induction cascade at parallel fi ber synapses: at both types of synapses, a postsynaptic calcium surge, activation of mGluR1 receptors, and activation of protein kinase C (PKC) are required for LTD induction (Hansel and Linden, 2000). Climbing fi ber LTD induction is also PKA-dependent (Schmolesky et al., 2007), which has not been tested for parallel fi ber LTD yet.
It remains to be determined whether climbing fi ber plasticity can play a similar role in motor learning as assumed for parallel fi ber LTD. However, the reduction of calcium transients accompanying climbing fi ber LTD (Weber et al., 2003) has a signifi cant effect on the LTD induction probability at parallel fi ber synapses (Coesmans et al., 2004) and might therefore provide a critical component of cerebellar gain control (see below).

DEVELOPMENTAL CLIMBING FIBER PLASTICITY
Climbing fi ber synaptic plasticity has also been observed in the developing cerebellum, where it might play a role in the activitydependent elimination of surplus climbing fi bers, and the stabilization of the remaining 'winner' climbing fi ber input. This pruning process is typically completed at the end of the third postnatal week (Crépel et al., 1976;Lohof et al., 1996). Recent studies suggest that long-term potentiation (LTP) and LTD can be observed at climbing fi ber synapses during postnatal development (Bosman et al., 2008;Ohtsuki and Hirano, 2008). In P4-11 Purkinje cells, pairing of climbing fi ber stimulation and Purkinje cell depolarization leads to LTP at 'large' climbing fi ber inputs, which are suffi ciently strong to evoke spike fi ring in Purkinje cells, but induces LTD at 'small' climbing fi ber inputs (Bosman et al., 2008). As multiple climbing fi ber inputs share innervation fi elds on Purkinje cell dendrites (Scelfo et al., 2003;Sugihara, 2005), it is conceivable that LTP and LTD at developing climbing fi ber synapses refl ect a direct synaptic competition of neighbouring climbing fi ber inputs, at the end of which the potentiated input is stabilized and becomes the 'winner' , whereas the depressed synaptic inputs are eventually eliminated (Bosman et al., 2008). The LTP described in this study is calciumdependent, but does not require the activation of NMDA receptors. The potentiation is mediated by an increase in the single channel conductance of AMPA receptors, suggesting a postsynaptic induction and expression mechanism (Bosman et al., 2008). Another study also described that in postnatal development (P5-9), climbing fi ber stimulation leads to LTP at strong climbing fi ber inputs, and LTD at weak climbing fi ber inputs (Ohtsuki and Hirano, 2008). In this study, however, LTP and LTD were accompanied by changes in the paired-pulse depression ratio and alterations in the frequency of asynchronous EPSCs, indicating that both types of plasticity are presynaptically expressed. LTP (but not LTD) induction requires a postsynaptic calcium transient, suggesting the involvement of a retrograde messenger (Ohtsuki and Hirano, 2008). Whether or not the different observations made in these two studies can be explained by slight differences in the stimulation protocols (for example, the second study applied unpaired  (2008) show that bidirectional climbing fi ber plasticity exists during postnatal development. These forms of climbing fi ber plasticity might be critically involved in the elimination of surplus climbing fi bers.

PARALLEL FIBER PLASTICITY UNDER HETEROSYNAPTIC CLIMBING FIBER CONTROL
The classic Marr-Albus-Ito theories of cerebellar motor learning suggest that synaptic plasticity at parallel fi ber synapses (the learning site) depends on activity at the heterosynaptic climbing fi ber synapses (the instructor site). In agreement with this theoretical framework, Masao Ito and colleagues described in the early 1980s a form of LTD at parallel fi ber synapses that is induced following paired parallel fi ber and climbing fi ber activity (Ito and Kano, 1982;. As fi rst suggested by Albus (Albus, 1971), parallel fi ber LTD provides an attractive candidate mechanism for cerebellar motor learning, as it is expected to result in a disinhibition of the target cells of inhibitory Purkinje cell projections in the deep cerebellar nuclei (DCN) or vestibular nuclei. Parallel fi ber LTD induction depends on activation of the mGluR1/ PKC signaling cascade (for review see Hansel and Bear, 2008) and activation of the α isoform of calcium/calmodulin-dependent kinase II (αCaMKII; Hansel et al., 2006). Climbing fi ber signaling triggers dendritic calcium transients and contributes to parallel fi ber LTD induction  by activating these induction cascades. Co-activation of parallel fi ber and climbing fi ber inputs causes

FIGURE 3 | Climbing fi ber LTD affects bidirectional parallel fi ber plasticity. (A)
A 5-Hz climbing fi ber tetanization for 30 s evokes climbing fi ber LTD, which is monitored here as a reduction in the amplitude of the fi rst slow spike component (arrow). (B) Climbing fi ber LTD is accompanied by a reduction in complex spike-associated calcium transients (red trace: before climbing fi ber tetanization). Calcium transients were recorded in the region of interest (red box). Fluorescence signals were monitored using a cooled CCD camera (Quantix, Roper Scientifi c), and the calcium indicator dye Oregon Green BAPTA-2 (200 µM). (C) After induction of climbing fi ber LTD (open dots; CF stimulation at 5 Hz, 30 s), subsequent application of the parallel fi ber LTD protocol (PF + CF stimulation at 1 Hz, 5 min) induces LTP instead (closed dots). (D) 'Inverse' calcium thresholds in the cerebellum: a higher calcium threshold has to be reached for LTD than for LTP induction. Climbing fi ber stimulation contributes calcium to reach this higher threshold, whereas climbing fi ber LTD lowers the amplitude of this calcium transient (red arrows). supralinear calcium signaling in parallel fi ber spines (Wang et al., 2000), thus providing a coincidence detection mechanism that might be required to reach a critical calcium threshold for LTD induction. A somewhat puzzling observation has been that strong parallel fi ber activation on its own can trigger parallel fi ber LTD in the absence of climbing fi ber activity (Eilers et al., 1997;Hartell, 1996). Similarly, climbing fi ber stimulation can be replaced by somatic depolarization (Linden et al., 1991). These fi ndings suggest that climbing fi ber activity indeed facilitates LTD induction by amplifying local calcium transients, but that there is no specifi c requirement for climbing fi ber-evoked calcium signals. This conclusion is supported by recent recordings from cerebellar Purkinje cells of mormyrid fi sh (introduced above). Parallel fi ber stimulation alone at enhanced stimulus strength (increase in the pulse duration) can induce parallel fi ber LTD. When the climbing fi ber is co-stimulated, however, a lower stimulus strength (as applied to monitor test responses before and after tetanization) is suffi cient for LTD induction (Han et al., 2007). These observations suggest that parallel fi ber LTD can be induced in the absence of climbing fi ber activity, which might nevertheless have an important role in facilitating the induction process. The impact of climbing fi ber signaling on parallel fi ber plasticity becomes more obvious when taking LTP into consideration. Both pre-and postsynaptically expressed types of LTP have been described at parallel fi ber synapses (Lev-Ram et al., 2002;Salin et al., 1996). LTP can be induced when applying the same lowfrequency/low-intensity parallel fi ber stimulation protocol used for LTD induction, when climbing fi ber stimulation is omitted (Lev-Ram et al., 2002). Just like LTD, this form of LTP is postsynaptically induced and expressed (Coesmans et al., 2004;Lev-Ram et al., 2002) and might therefore function as a reversal mechanism for LTD. LTP induction depends on lower calcium transients than LTD induction (Coesmans et al., 2004; Figure 3D), and requires the activation of protein phosphatases PP1, PP2A and PP2B (Belmeguenai and Hansel, 2005). Therefore, at the level of calcium signaling and kinase/phosphatase activation requirements, cerebellar bidirectional synaptic plasticity seems to be governed by induction rules that provide a mirror image of those described at glutamatergic synapses in hippocampal and neocortical pyramidal cells (Jörntell and Hansel, 2006). Moreover, a unique motif in cerebellar plasticity is the heterosynaptic control of parallel fi ber plasticity by the climbing fi ber input. The effi cacy of this control function becomes obvious when looking at the consequences of LTD at the climbing fi ber input itself (Hansel and Linden, 2000). Climbing fi ber LTD is accompanied by a reduction in complex spike-associated calcium transients (Weber et al., 2003). This reduction in calcium signaling is suffi ciently strong to reverse the polarity of parallel fi ber plasticity after previous climbing fi ber LTD induction (Coesmans et al., 2004). This metaplastic interaction is illustrated in Figure 3C: when climbing fi ber LTD is induced fi rst, subsequent application of the parallel fi ber LTD induction protocol results in LTP induction instead. The most likely explanation for this sign reversal is that climbing fi ber LTD reduced the activitydependent calcium signal below the threshold for LTD induction (Figure 3D).
In addition to its role in postsynaptic parallel fi ber plasticity, climbing fi ber signaling also affects a form of presynaptic parallel fi ber LTP that results from brief parallel fi ber tetanization (e.g. 8 Hz for 15 s; Salin et al., 1996). Presynaptic parallel fi ber LTP is induced by activation of adenylyl cyclase I (Storm et al., 1998), production of cAMP and the subsequent activation of cAMP-dependent protein kinase (PKA; Chen and Regehr, 1997;Salin et al., 1996). More recent observations show that climbing fi ber-evoked calcium signaling can trigger the release of endocannabinoids from Purkinje cell dendrites (Brenowitz and Regehr, 2003), which bind to CB1 receptors at parallel fi ber terminals and suppress LTP induction by interfering with the adenylyl cyclase/PKA cascade (Van Beugen et al., 2006). It has been suggested that endocannabinoid signaling facilitates the induction of postsynaptic LTD (Safo and Regehr, 2005). The inhibitory action of CB1 receptor activation on presynaptic LTP might well contribute to this facilitation of LTD, assuming that activity-dependent postsynaptic alterations are often accompanied by presynaptic changes. In this scenario, climbing fi ber-evoked calcium transients do not only promote postsynaptic LTD, but in addition provide a 'safety lock' mechanism that prevents that presynaptic LTP and postsynaptic LTD occur at the same time (Van Beugen et al., 2006). Under some conditions, coincident parallel fi ber activity and retrograde endocannabinoid signaling might even promote the induction of a presynaptic form of LTD (Qiu and Knöpfel, 2009), thus aligning pre-and postsynaptic changes. These recent reports show that the climbing fi ber input heterosynaptically affects four forms of parallel fi ber plasticity: postsynaptic LTD, postsynaptic LTP, presynaptic LTP, and presynaptic LTD.

THE OTHER CLIMBING FIBER SIGNAL: CORTICOTROPIN-RELEASING FACTOR
The complex spike-associated calcium transients in Purkinje cell dendrites are certainly the best characterized contribution of climbing fi ber signaling to cerebellar plasticity. However, it should not be overlooked that climbing fi ber activity can additionally result in the release of the neuropeptide corticotropin-releasing factor (CRF) from climbing fi ber terminals (Barmack and Young, 1990;Tian and Bishop, 2003). CRF can bind to type 1 and/or type 2 CRF receptors expressed in Purkinje cells. Whereas type 2 CRF receptors are not expressed in spines, type 1 receptors, which are G-protein coupled and lead to the activation of adenylyl cyclase/PKA and PKC pathways (Grammatopoulos et al., 2001), are located in the dendrite across from parallel fi ber terminals, and in non-synaptic regions (Swinny et al., 2003). CRF signaling has been shown to be critically involved in parallel fi ber LTD induction as the CRF receptor antagonists α-helical CRF-(9-41) (α-h CRF) and astressin prevent LTD (Miyata et al., 1999). Type-1 CRF receptors are not expressed in the dendrite across from climbing fi ber terminals (Swinny et al., 2003), and yet climbing fi ber LTD is blocked, too, in the presence of astressin (Schmolesky et al., 2007), suggesting that diffusion to adjacent receptors is suffi cient. It is possible that CRF signaling facilitates LTD induction at both climbing fi ber and parallel fi ber synapses by activating the PKC signaling cascade and, at least in the case of climbing fi ber LTD, the PKA signaling cascade (Schmolesky et al., 2007). These results show that the climbing fi ber input exerts a control function over parallel fi ber plasticity not only through the calcium transients associated with complex spike activity, but also by the activity-dependent release of the neuropeptide CRF (Figure 4). Moreover, it seems that the same factors involved in parallel fi ber LTD (here: high calcium, CRF receptor activation) promote LTD at the climbing fi ber input as well. cerebellar motor learning? To slightly clear the fog, it might be useful to take a step back and have a look at the evidence at hand. Granule cells provide massive excitatory input to Purkinje cells via ascending granule cell axons on the one hand, and parallel fi ber synapses on the other. These two sets of granule cell input, however, seem to play different roles in cerebellar processing and gain control. Synapses of the ascending axons do not show forms of long-term plasticity (Sims and Hartell, 2006). In contrast, there is a high degree of pre-and postsynaptic plasticity at parallel fi ber synapses, but about 85% of these synapses are functionally silent (Isope and Barbour, 2002). It has therefore been suggested that granule cells predominantly activate Purkinje cells through the hardwired ascending axon input, while the parallel fi ber input allows for acquired control using fi ne-tuned recruiting of parallel fi ber synapses (Rokni et al., 2008). These observations suggest a high degree of functional specialization of the two different sets of synaptic contacts provided by granule cells, and support the view that the parallel fi ber system plays a key role in cerebellar adaptations. But under what conditions does parallel fi ber plasticity occur, and what are its functional consequences? A very elegant study has been provided by Jörntell and Ekerot, who showed that parallel fi ber receptive fi elds in adult cats can be bidirectionally modifi ed after parallel fi ber stimulation in vivo (Jörntell and Ekerot, 2002). Paired parallel fi ber and climbing fi ber stimulation causes a long-term decrease in the receptive fi eld size of Purkinje cells, while unpaired parallel fi ber stimulation causes a lasting increase. While this study provides an example of plasticity of sensory inputs to cerebellar Purkinje cells, without immediately obvious consequences for motor control, there are three aspects that are highly relevant for the present discussion. First, the stimulus

IS CLIMBING FIBER SIGNALING INVOLVED IN CEREBELLAR MOTOR LEARNING?
Numerous studies using genetically modifi ed mice suggest a correlation between parallel fi ber LTD and cerebellar motor learning (De Zeeuw and Yeo, 2005). However, LTD has never been demonstrated during motor learning in behaving animals. It has indeed been claimed that parallel fi ber LTD might not be involved in motor learning at all, based on the observation that motor learning (eyeblink conditioning) is intact when parallel fi ber LTD is pharmacologically inhibited (Welsh et al., 2005). Even if LTD is involved in motor learning, the contribution of the climbing fi ber input remains unclear, as LTD can be induced in the absence of climbing fi ber activity, as long as the parallel fi ber input is suffi ciently active (Eilers et al., 1997;Han et al., 2007;Hartell, 1996). Moreover, when reviewing recent developments in cerebellar plasticity research, it becomes obvious that several types of plasticity that have been characterized do not require climbing fi ber activity for induction. This holds true for presynaptic LTP (Salin et al., 1996) and postsynaptic LTP (Lev-Ram et al., 2002). Climbing fi ber activity is only required for the induction of LTD at both climbing fi ber (Hansel and Linden, 2000) and parallel fi ber inputs (Ito and Kano, 1982; as well as for rebound potentiation at interneuron -Purkinje cell synapses (Kano et al., 1992). While climbing fi ber activity-dependent parallel fi ber LTD has been the predominant model for cerebellar motor learning, these more recently discovered types of plasticity could well be involved in cerebellar learning as well, but are independent of climbing fi ber activity.
So what is the role of parallel fi ber plasticity, and specially climbing fi ber activity-dependent parallel fi ber plasticity, in FIGURE 4 | Climbing fi ber activity facilitates LTD induction at parallel fi ber synapses. For simplicity, climbing fi ber and parallel fi ber terminals are shown to contact the same postsynaptic compartment. The LTD induction cascade is shown in blue: large calcium transients promote the activation of αCaMKII and PKC. A PKC-mediated phosphorylation of the AMPA receptor subunit GluR2 triggers the internalization of GluR2 subunits. The LTP cascade is shown in yellow: lower calcium transients promote phosphatase activation (only PP2B is directly calcium-regulated). Eventually, GluR2 subunits are delivered to the membrane. Climbing fi ber activity facilitates LTD induction by elevating the overall calcium transient (calcium sources are not shown), and by releasing the neuropeptide CRF. CRF binding to type 1 CRF receptors (CRF-R1) facilitates the activation of PKC and PKA. The activation of PKA is a required step for the induction of climbing fi ber LTD. It has not been determined yet, whether the same holds true for parallel fi ber LTD.

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July 2009 | Volume 3 | Article 4 | 9 Ohtsuki et al. Cerebellar computation: climbing fi ber signaling protocols applied suggest that both LTD (paired stimulation) and LTP (unpaired stimulation) phenomena contribute to the decrease and increase, respectively, of the receptive fi eld sizes. If so, this study demonstrates that LTD and LTP are involved in a form of cerebellar learning that can be monitored in vivo. Second, this study elegantly shows that LTD and LTP can perfectly complement each other in cerebellar information storage, without the need to classify one as the 'learning' mechanism and the other as a tool used for 'extinction' or 'reversal'. In other words, in some types of cerebellar learning, depression and potentiation simply provide two sides of the same coin, allowing for bidirectional adaptations. Third, this study clearly demonstrates that climbing fi ber activity exerts a crucial function in the control of bidirectional cerebellar plasticity. This latter observation is in line with the widespread notion that enhanced climbing fi ber activity precedes cerebellar learning, acting as an 'error detector' , or as a 'teacher' (for review see Simpson et al., 1996). In this view, climbing fi ber activity signals the need for adjusting the gain values of cerebellar sensory inputs and/or motor output control.

CONCLUSION
In a recent paper published in Frontiers in Neuroscience, Rodolfo Llinas and Yosef Yarom review the histology and physiology of the cerebellar cortex, concluding that 'the cerebellum should be regarded as a control machine rather than a learning machine' (Rokni et al., 2008). We do not agree with this assessment. In our view, the cerebellum certainly acts as a control machine, but on top of that the cerebellum (particularly the cerebellar cortex) provides a giant switchboard for associative learning. Currently, the existing evidence does not seem to allow for a defi nite conclusion. Our more learning-biased view results from close inspection of the cerebellar circuitry and its capacity for information storage based on both in vitro and in vivo studies (see also Hansel et al., 2001;Jörntell and Hansel, 2006). Parallel fi ber to Purkinje cell synapses are perfect candidate locations for the storage of motor memories, because of their ability to bidirectionally adjust synaptic gain both pre-and postsynaptically. Although not strictly required, elevated climbing fi ber activity facilitates the induction of parallel fi ber LTD by enhancing dendritic calcium signals and by releasing the neuropeptide CRF from climbing fi ber terminals. Climbing fi ber activity also suppresses presynaptic LTP by triggering the release of endocannabinoids from Purkinje cell dendrites. The complexity of this 'orchestration' of parallel fi ber plasticity by the climbing fi ber input shows after induction of LTD at the climbing fi ber input itself: the accompanying reduction in complex spike-associated calcium transients shifts the relative probabilities for the induction of LTD and LTP, respectively, at the parallel fi ber input. Plasticity residing at the parallel fi ber synapses is likely complemented by additional types of cerebellar plasticity, such as plasticity at inhibitory synapses onto Purkinje cells, and intrinsic plasticity mechanisms found in several types of neurons within the cerebellum. It remains to be seen how the cerebellum puts these features to use, but its underlying circuitry seems very well suited for activity-dependent information storage and learning.