Climbing Fiber Input Research Articles

Coding of stimulus strength via analog calcium signals in Purkinje cell dendrites of awake mice

The climbing fiber input to Purkinje cells acts as a teaching signal by triggering a massive influx of dendritic calcium that marks the occurrence of instructive stimuli during cerebellar learning. Here, we challenge the view that these calcium spikes are all-or-none and only signal whether the instructive stimulus has occurred, without providing parametric information about its features. We imaged ensembles of Purkinje cell dendrites in awake mice and measured their calcium responses to periocular airpuffs that serve as instructive stimuli during cerebellar-dependent eyeblink conditioning. Information about airpuff duration and pressure was encoded probabilistically across repeated trials, and in two additional signals in single trials: the synchrony of calcium spikes in the Purkinje cell population, and the amplitude of the calcium spikes, which was modulated by a non-climbing fiber pathway. These results indicate that calcium-based teaching signals in Purkinje cells contain analog information that encodes the strength of instructive stimuli trial-by-trial.

Intracellular calcium dynamics permit a Purkinje neuron model to perform toggle and gain computations upon its inputs.

Without synaptic input, Purkinje neurons can spontaneously fire in a repeating trimodal pattern that consists of tonic spiking, bursting and quiescence. Climbing fiber input (CF) switches Purkinje neurons out of the trimodal firing pattern and toggles them between a tonic firing and a quiescent state, while setting the gain of their response to Parallel Fiber (PF) input. The basis to this transition is unclear. We investigate it using a biophysical Purkinje cell model under conditions of CF and PF input. The model can replicate these toggle and gain functions, dependent upon a novel account of intracellular calcium dynamics that we hypothesize to be applicable in real Purkinje cells.

A matter of trial and error for motor learning

Climbing fiber inputs to cerebellar Purkinje cells are thought to carry error signals that can trigger motor learning across multiple time scales. A new study by Kimpo et al. finds that the potency of climbing fibers as instructive signals for adaptation of the vestibulo-ocular reflex depends on task conditions.

Global Scaling Down of Excitatory Postsynaptic Responses in Cerebellar Purkinje Cells Impairs Developmental Synapse Elimination

Synapse elimination is crucial for precise neural circuit formation during postnatal development. We examined how relative differences in synaptic strengths among competing inputs and/or absolute synaptic strengths contribute to climbing fiber (CF) to Purkinje cell (PC) synapse elimination in the cerebellum. We generated mice with PC-selective deletion of stargazin (TARP γ-2), the major AMPA receptor auxiliary subunit in PCs (γ-2 PC-KO mice). Whereas relative differences between "strong" and "weak" CF-mediated postsynaptic response are preserved, absolute strengths of CF inputs are scaled down globally in PCs of γ-2 PC-KO mice. Although the early phase of CF elimination is normal, dendritic translocation of the strongest CF and the late phase of CF elimination that requires Ca(2+)-dependent activation of Arc/Arg3.1 in PCs are both impaired in γ-2 PC-KO mice. We conclude that, although relative differences in CF synaptic inputs are initially essential, proper synaptic scaling is crucial for accomplishing CF synapse elimination.

βCaMKII regulates bidirectional long-term plasticity in cerebellar Purkinje cells by a CaMKII/PP2B switch mechanism

It is thought that modifications in the strength of synaptic connections onto cerebellar Purkinje cells (PCs) such as long-term depression (LTD) and long-term potentiation (LTP) contribute to learning. Repeated paired stimulations of parallel fibres (PFs) and climbing fibre (CF) evoke high-amplitude calcium signals that lead to LTD in PCs, while LTP is induced by low-amplitude calcium pulses as a result of PF input alone. Although the events following these stimulation protocols are becoming clearer, the precise interactions of the molecular machinery underlying LTD and LTP induction remain something of an enigma. Calcium-calmodulin dependent protein kinase II (CaMKII) is an important component of the signalling network that is responsible for cerebellar plasticity in PCs. The CaMKII holoenzyme contains isoforms such as αCaMKII and βCaMKII. Experiments with Camk2b knockout mice that lack the β isoform of CaMKII have demonstrated that βCaMKII regulates the direction of plasticity at PF-PC synapses [1]. Stimulation protocols that induce LTD in wild-type mice, which contain both α and βCaMKII, lead to LTP in knockout mice without βCaMKII, and vice versa. In previous work, we have developed a kinetic simulation of the phosphorylation and dephosphorylation of AMPA receptors by CaMKII and protein phosphatase 2B (PP2B) to investigate how βCaMKII controls bidirectional plasticity in cerebellar PCs [2]. This bidirectional plasticity model was based on our earlier model of CaMKII activation [3,4]. Because the βCaMKII, but not αCaMKII, isoforms can bind to filamentous actin (F-actin), we have included the binding of F-actin to CaMKII to simulate plasticity induction in wild-type mice, whereas the F-actin binding was omitted in the knockout mice that lack the β isoform. Our previous simulation results have demonstrated that the binding of F-actin to βCaMKII can contribute to the regulation of bidirectional plasticity at PF-PC synapses, as suggested by Van Woerden and collaborators [1]. However, we have investigated the induction of bidirectional plasticity only in the presence of calcium signals, i.e. during the early phase of LTD and LTP. But the experimental observations in [1] have demonstrated that the bidirectional plasticity is also observed after the offset of paired PF and CF inputs and PF input alone. To analyse the persistent AMPA receptor phosphorylation after calcium stimulation protocols that last 5 min, we modified our previous model [2] because it did not simulate the CaMKII dephosphorylation, and PP2B was rapidly inactivated with the interruption of calcium signals. Here, we allow the CaMKII subunits in the autonomous form, which are not bound to calcium-calmodulin but still retain some activity levels, to be dephosphorylated and switch to the inactive state of CaMKII. The kinase is therefore gradually inactivated after calcium stimulation stops. Moreover, calcium-calmodulin slowly dissociates from PP2B, leading to a gradual deactivation of PP2B. Results of computer simulations of our mathematical model suggest that the balance of CaMKII-mediated phosphorylation and PP2B-mediated dephosphorylation of AMPA receptors determines whether LTD or LTP occurs at the PF-PC synapse. PF activity alone evokes PP2B concentration levels higher than CaMKII, leading to LTP induction. Instead, LTD occurs when the CaMKII concentration levels surpass the PP2B concentration in response to the paired activation of PF and CF. Moreover, our recent simulation results replicate the experimental observations by Van Woerden et al. [1] not only in the early phase of LTD and LTP as shown in [2], but also in their intermediate and late phases after the termination of PF and CF stimulation and PF input alone. The mechanisms observed in our previous simulations with 5 min duration are also observed in simulations that last 100 min. The loss of F-actin binding in the knockout mice enhances the availability of active CaMKII when compared to the wild-type mice at low calcium concentrations. This favours the induction of LTD rather than LTP. In contrast, the reduction in the CaMKII concentration in the knockout mice leads to AMPA receptor dephosphorylation by PP2B for high calcium concentrations, leading to the induction of LTP instead of LTD. We again demonstrate that the binding of F-actin to βCaMKII can indeed contribute to the control of bidirectional plasticity at PF-PC synapses, as suggested in [1,2].

Supervised learning with complex spikes and spike-timing-dependent plasticity.

One distinctive feature of Purkinje cells is that they have two types of discharge: in addition to simple spikes they fire complex spikes in response to input from the climbing fibers. These complex spikes have an initial rapid burst of spikes and spikelets followed by a sustained depolarization; in some models of cerebellar function this climbing fiber input supervises learning in Purkinje cells. On the other hand, synaptic plasticity is often thought to rely on the timing of pre-synaptic and post-synaptic spikes. It is suggested here that the period of depolarization following a complex spike, combined with a simple spike-timing-dependent plasticity rule, gives a mechanism for the climbing fiber to supervise learning in the Purkinje cell. This proposal is illustrated using a simple simulation in which it is seen that the climbing fiber succeeds in supervising the learning.

Role of plasticity at different sites across the time course of cerebellar motor learning.

Learning comprises multiple components that probably involve cellular and synaptic plasticity at multiple sites. Different neural sites may play their largest roles at different times during behavioral learning. We have used motor learning in smooth pursuit eye movements of monkeys to determine how and when different components of learning occur in a known cerebellar circuit. The earliest learning occurs when one climbing-fiber response to a learning instruction causes simple-spike firing rate of Purkinje cells in the floccular complex of the cerebellum to be depressed transiently at the time of the instruction on the next trial. Trial-over-trial depression and the associated learning in eye movement are forgotten in <6 s, but facilitate long-term behavioral learning over a time scale of ∼5 min. During 100 repetitions of a learning instruction, simple-spike firing rate becomes progressively depressed in Purkinje cells that receive climbing-fiber inputs from the instruction. In Purkinje cells that prefer the opposite direction of pursuit and therefore do not receive climbing-fiber inputs related to the instruction, simple-spike responses undergo potentiation, but more weakly and more slowly. Analysis of the relationship between the learned changes in simple-spike firing and learning in eye velocity suggests an orderly progression of plasticity: first on Purkinje cells with complex-spike (CS) responses to the instruction, later on Purkinje cells with CS responses to the opposite direction of instruction, and last in sites outside the cerebellar cortex. Climbing-fiber inputs appear to play a fast and primary, but nonexclusive, role in pursuit learning.

Climbing fiber projections in relation to zebrin stripes in the ventral uvula in pigeons.

The cerebellum consists of sagittally oriented zones that are delineated by afferent input, Purkinje cell response properties, and the expression of molecular markers such as zebrin II (ZII). ZII is heterogeneously expressed in Purkinje cells such that there are parasagittal stripes of high expression (ZII+) interdigitated with stripes of little or no expression (ZII-). In pigeons, folium IXcd consists of seven pairs of ZII+/- stripes denoted P1+/- (medial) to P7+/- (lateral). In the present study we examined the climbing fiber input to the medial half of folium IXcd, the ventral uvula, which spans the medial two stripe pairs (P1+/- to P2+/-). Purkinje cells in the ventral uvula respond to patterns of optic flow resulting from self-motion through the environment along translational axes and their climbing fibers originate in the lateral half of the medial column in the inferior olive (mcIO). Using anterograde injections into this region of the mcIO, we found the following topographic relationship: climbing fibers from the caudal lateral mcIO were located in P1+ and medial P1- ZII stripes; climbing fibers from the rostral lateral mcIO were located in lateral P2+ and P2- ZII stripes, and climbing fibers from the middle lateral mcIO were located in lateral P1- and medial P2+ ZII stripes. These data complement our previous findings showing a topographic relationship between Purkinje cell responses to optic flow visual stimuli and ZII stripes. Taken together, we suggest that a ZII+/- stripe pair may represent a functional unit in the pigeon vestibulocerebellum.

A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice.

Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions.

Purkinje-cell plasticity and cerebellar motor learning are graded by complex-spike duration.

Behavioral learning is mediated by cellular plasticity such as changes in the strength of synapses at specific sites in neural circuits. The theory of cerebellar motor learning1,2,3 relies on movement errors signaled by climbing-fiber inputs to cause long-term depression of synapses from parallel fibers to Purkinje cells4,5. Yet, a recent review6 has called into question the widely-held view that the climbing fiber input is an “all-or-none” event. In anesthetized animals, there is wide variation in the duration of the complex-spike (CS) caused in Purkinje cells by a climbing fiber input7. Further, the duration of electrically-controlled bursts in climbing fibers grades the amount of plasticity in Purkinje cells8,9. The duration of bursts depends on the “state” of the inferior olive and therefore could be correlated across climbing fibers8,10. Here, we provide a potential functional context for these mechanisms during motor learning in behaving monkeys. The magnitudes of both plasticity and motor learning depend on the duration of the CS responses. Further, the duration of CS responses appears to be a meaningful signal that is correlated across the Purkinje cell population during motor learning. We suggest that during learning, longer bursts in climbing fibers lead to longer duration CS responses in Purkinje cells, more calcium entry into Purkinje cells, larger synaptic depression, and stronger learning. The same graded impact of instructive signals for plasticity and learning could occur throughout the nervous system.

Plastic corollary discharge predicts sensory consequences of movements in a cerebellum-like circuit.

The capacity to predict the sensory consequences of movements is critical for sensory, motor, and cognitive function. Though it is hypothesized that internal signals related to motor commands, known as corollary discharge, serve to generate such predictions, this process remains poorly understood at the neural circuit level. Here we demonstrate that neurons in the electrosensory lobe (ELL) of weakly electric mormyrid fish generate negative images of the sensory consequences of the fish's own movements based on ascending spinal corollary discharge signals. These results generalize previous findings describing mechanisms for generating negative images of the effects of the fish's specialized electric organ discharge (EOD) and suggest that a cerebellum-like circuit endowed with associative synaptic plasticity acting on corollary discharge can solve the complex and ubiquitous problem of predicting sensory consequences of movements.

Third Trimester‐Equivalent Ethanol Exposure Does Not Alter Complex Spikes and Climbing Fiber Long‐Term Depression in Cerebellar Purkinje Neurons from Juvenile Rats

Studies indicate that exposure to ethanol (EtOH) during fetal development damages cerebellar Purkinje cells (PCs). PC proximal dendrites receive glutamatergic input from climbing fibers (CFs) originating at the inferior olive. CF input produces a characteristic response in PCs known as the complex spike (CS). During the first 2 weeks of life in rodents (equivalent to the human third trimester of pregnancy), CF-PC synapses undergo profound refinement. Here, we characterized the impact of EtOH exposure during this period on CF-evoked responses in PCs. Using vapor chambers, neonatal rat pups and their mothers were exposed to air or EtOH for 4 h/d between postnatal day 2 (P2) and P12 (pup serum EtOH concentration, 0.16 g/dl). The function of CF-PC synapses was characterized using patch-clamp electrophysiological techniques in acute slices from the cerebellar vermis. Experiments were performed soon after EtOH withdrawal, when perisomatic CFs are still being eliminated (P15 to P17), and after weaning when CF dendritic translocation is almost complete (P21 to P34). Neither the baseline characteristics of the CS (Na(+) spike amplitude, area, coastline index, and afterhyperpolarization [AHP] amplitude) nor the type-1 metabotropic glutamate receptor (mGluR1)-mediated component of both the CS and AHP were significantly affected by EtOH exposure at P15 to P17 or P21 to P34. The mGluR1-dependent long-term depression (LTD) of CF-evoked excitatory postsynaptic currents was not significantly affected by EtOH exposure at P21 to P34. EtOH exposure during the third trimester equivalent neither affected basal characteristics of the CS nor CF-LTD at rat cerebellar PCs from juvenile rats.

Emergence of a 600-Hz buzz UP state Purkinje cell firing in alert mice

Purkinje cell (PC) firing represents the sole output from the cerebellar cortex onto the deep cerebellar and vestibular nuclei. Here, we explored the different modes of PC firing in alert mice by extracellular recording. We confirm the existence of a tonic and/or bursting and quiescent modes corresponding to UP and DOWN state, respectively. We demonstrate the existence of a novel 600-Hz buzz UP state of firing characterized by simple spikes (SS) of very small amplitude. Climbing fiber (CF) input is able to switch the 600-Hz buzz to the DOWN state, as for the classical UP-to-DOWN state transition. Conversely, the CF input can initiate a typical SS pattern terminating into 600-Hz buzz. The 600-Hz buzz was transiently suppressed by whisker pad stimulation demonstrating that it remained responsive to peripheral input. It must not be mistaken for a DOWN state or the sign of PC inhibition. Complex spike (CS) frequency was increased during the 600-Hz buzz, indicating that this PC output actively contributes to the cerebello-olivary loop by triggering a disinhibition of the inferior olive. During the 600-Hz buzz, the first depolarizing component of the CS was reduced and the second depolarizing component was suppressed. Consistent with our experimental observations, using a 559-compartment single-PC model – in which PC UP state (of about −43mV) was obtained by the combined action of large tonic AMPA conductances and counterbalancing GABAergic inhibition – removal of this inhibition produced the 600-Hz buzz; the simulated buzz frequency decreased following an artificial CS.

A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo

Calcium imaging of individual neurons is widely used for monitoring their activity in vitro and in vivo. Synthetic fluorescent calcium indicator dyes are commonly used, but the resulting calcium signals sometimes suffer from a low signal-to-noise ratio (SNR). Therefore, it is difficult to detect signals caused by single action potentials (APs) particularly from neurons in vivo. Here we showed that a recently developed calcium indicator dye, Cal-520, is sufficiently sensitive to reliably detect single APs both in vitro and in vivo. In neocortical neurons, calcium signals were linearly correlated with the number of APs, and the SNR was > 6 for in vitro slice preparations and > 1.6 for in vivo anesthetised mice. In cerebellar Purkinje cells, dendritic calcium transients evoked by climbing fiber inputs were clearly observed in anesthetised mice with a high SNR and fast decay time. These characteristics of Cal-520 are a great advantage over those of Oregon Green BAPTA-1, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo.

Chapter 7 - Decorrelation Learning in the Cerebellum: Computational Analysis and Experimental Questions

Many cerebellar models use a form of synaptic plasticity that implements decorrelation learning. Parallel fibers carrying signals positively correlated with climbing-fiber input have their synapses weakened (long-term depression), whereas those carrying signals negatively correlated with climbing input have their synapses strengthened (long-term potentiation). Learning therefore ceases when all parallel-fiber signals have been decorrelated from climbing-fiber input. This is a computationally powerful rule for supervised learning and can be cast in a spike-timing dependent plasticity form for comparison with experimental evidence. Decorrelation learning is particularly well suited to sensory prediction, for example, in the reafference problem where external sensory signals are interfered with by reafferent signals from the organism's own movements, and the required circuit appears similar to the one found to mediate classical eye blink conditioning. However, for certain stimuli, avoidance is a much better option than simple prediction, and decorrelation learning can also be used to acquire appropriate avoidance movements. One example of a stimulus to be avoided is retinal slip that degrades visual processing, and decorrelation learning appears to play a role in the vestibulo-ocular reflex that stabilizes gaze in the face of unpredicted head movements. Decorrelation learning is thus suitable for both sensory prediction and motor control. It may also be well suited for generic spatial and temporal coordination, because of its ability to remove the unwanted side effects of movement. Finally, because it can be used with any kind of time-varying signal, the cerebellum could play a role in cognitive processing.

Spike timing-dependent selective strengthening of single climbing fibre inputs to Purkinje cells during cerebellar development

Shaping functional neural circuits in developing brain involves activity-dependent refinement of early-formed redundant synapses. In the developing cerebellum, a one-to-one connection between a climbing fibre (CF) and a Purkinje cell (PC) is established by selective strengthening of a single CF followed by elimination of surplus CFs. Here we investigate developmental changes in CF-mediated responses in PCs by using in vivo whole-cell recordings and two-photon Ca2+ imaging. We show that each neonatal PC receives temporally clustered inputs from multiple CFs and temporal integration of these inputs is required to induce burst spiking and Ca2+ rise in PCs. Importantly, a single CF input closest to PC’s spike output is selectively strengthened during postnatal development. This spike timing-dependent selective strengthening is much less prominent in PC-selective P/Q-type voltage-dependent Ca2+ channel knockout mice. Thus, spike timing- and Ca2+-dependent plasticity appears to underlie the selection of a single ‘winner’ CF and the establishment of mature CF–PC connections.

Cerebellar Purkinje cell activity drives motor learning

The climbing fiber input to the cerebellar cortex is thought to provide instructive signals that drive the induction of motor skill learning. We found that optogenetic activation of Purkinje cells, the sole output neurons of the cerebellar cortex, can also drive motor learning in mice. This dual control over the induction of learning by climbing fibers and Purkinje cells can expand the learning capacity of motor circuits.

Structural basis of cerebellar microcircuits in the rat.

The topography of the cerebellar cortex is described by at least three different maps, with the basic units of each map termed "microzones," "patches," and "bands." These are defined, respectively, by different patterns of climbing fiber input, mossy fiber input, and Purkinje cell (PC) phenotype. Based on embryological development, the "one-map" hypothesis proposes that the basic units of each map align in the adult animal and the aim of the present study was to test this possibility. In barbiturate anesthetized adult rats, nanoinjections of bidirectional tracer (Retrobeads and biotinylated dextran amine) were made into somatotopically identified regions within the hindlimb C1 zone in copula pyramidis. Injection sites were mapped relative to PC bands defined by the molecular marker zebrin II and were correlated with the pattern of retrograde cell labeling within the inferior olive and in the basilar pontine nuclei to determine connectivity of microzones and patches, respectively, and also with the distributions of biotinylated dextran amine-labeled PC terminals in the cerebellar nuclei. Zebrin bands were found to be related to both climbing fiber and mossy fiber inputs and also to cortical representation of different parts of the ipsilateral hindpaw, indicating a precise spatial organization within cerebellar microcircuitry. This precise connectivity extends to PC terminal fields in the cerebellar nuclei and olivonuclear projections. These findings strongly support the one-map hypothesis and suggest that, at the microcircuit level of resolution, the cerebellar cortex has a common plan of spatial organization for major inputs, outputs, and PC phenotype.

Short‐term synaptic plasticity and the ‘active calcium’ hypothesis at a central synapse

Short‐term synaptic plasticity and the ‘active calcium’ hypothesis at a central synapse

An adaptive filter model of cerebellar zone C3 as a basis for safe limb control?

The review asks how the adaptive filter model of the cerebellum might be relevant to experimental work on zone C3, one of the most extensively studied regions of cerebellar cortex. As far as features of the cerebellar microcircuit are concerned, the model appears to fit very well with electrophysiological discoveries concerning the importance of molecular layer interneurons and their plasticity, the significance of long-term potentiation and the striking number of silent parallel fibre synapses. Regarding external connectivity and functionality, a key feature of the adaptive filter model is its use of the decorrelation algorithm, which renders it uniquely suited to problems of sensory noise cancellation. However, this capacity can be extended to the avoidance of sensory interference, by appropriate movements of, for example, the eyes in the vestibulo-ocular reflex. Avoidance becomes particularly important when painful signals are involved, and as the climbing fibre input to zone C3 is extremely responsive to nociceptive stimuli, it is proposed that one function of this zone is the avoidance of pain by, for example, adjusting movements of the body to avoid self-harm. This hypothesis appears consistent with evidence from humans and animals concerning the role of the intermediate cerebellum in classically conditioned withdrawal reflexes, but further experiments focusing on conditioned avoidance are required to test the hypothesis more stringently. The proposed architecture may also be useful for automatic self-adjusting damage avoidance in robots, an important consideration for next generation 'soft' robots designed to interact with people.