Active reversal of motor memories reveals rules governing memory encoding

Learning systems must be able to store memories reliably, yet be able to modify them when new learning is required. At the mechanistic level, new learning may either reverse the cellular events mediating the storage of old memories or mask the old memories with additional cellular changes that preserve the old cellular events in a latent form. Behavioral evidence about whether reversal or masking occurs in a particular circuit can constrain the cellular mechanisms used to store memories. Here we examine these constraints for a simple cerebellum-dependent learning task, motor learning in the vestibulo-ocular reflex (VOR). Learning can change the amplitude of the VOR in two opposite directions. Contrary to previous models about memory encoding by the cerebellum, our results indicate that these behavioral changes are implemented by different plasticity mechanisms, which reverse each other with unequal efficacy.     

Impairment of LTD and cerebellar learning by Purkinje cell-specific ablation of cGMP-dependent protein kinase I

The molecular basis for cerebellar plasticity and motor learning remains controversial. Cerebellar Purkinje cells (PCs) contain a high concentration of cGMP-dependent protein kinase type I (cGKI). To investigate the function of cGKI in long-term depression (LTD) and cerebellar learning, we have generated conditional knockout mice lacking cGKI selectively in PCs. These cGKI mutants had a normal cerebellar morphology and intact synaptic calcium signaling, but strongly reduced LTD. Interestingly, no defects in general behavior and motor performance could be detected in the LTD-deficient mice, but the mutants exhibited an impaired adaptation of the vestibulo-ocular reflex (VOR). These results indicate that cGKI in PCs is dispensable for general motor coordination, but that it is required for cerebellar LTD and specific forms of motor learning, namely the adaptation of the VOR.     

Synaptic Plasticity in Medial Vestibular Nucleus Neurons: Comparison with Computational Requirements of VOR Adaptation

Background

Vestibulo-ocular reflex (VOR) gain adaptation, a longstanding experimental model of cerebellar learning, utilizes sites of plasticity in both cerebellar cortex and brainstem. However, the mechanisms by which the activity of cortical Purkinje cells may guide synaptic plasticity in brainstem vestibular neurons are unclear. Theoretical analyses indicate that vestibular plasticity should depend upon the correlation between Purkinje cell and vestibular afferent inputs, so that, in gain-down learning for example, increased cortical activity should induce long-term depression (LTD) at vestibular synapses.

Methodology/Principal Findings

Here we expressed this correlational learning rule in its simplest form, as an anti-Hebbian, heterosynaptic spike-timing dependent plasticity interaction between excitatory (vestibular) and inhibitory (floccular) inputs converging on medial vestibular nucleus (MVN) neurons (input-spike-timing dependent plasticity, iSTDP). To test this rule, we stimulated vestibular afferents to evoke EPSCs in rat MVN neurons in vitro. Control EPSC recordings were followed by an induction protocol where membrane hyperpolarizing pulses, mimicking IPSPs evoked by flocculus inputs, were paired with single vestibular nerve stimuli. A robust LTD developed at vestibular synapses when the afferent EPSPs coincided with membrane hyperpolarisation, while EPSPs occurring before or after the simulated IPSPs induced no lasting change. Furthermore, the iSTDP rule also successfully predicted the effects of a complex protocol using EPSP trains designed to mimic classical conditioning.

Conclusions

These results, in strong support of theoretical predictions, suggest that the cerebellum alters the strength of vestibular synapses on MVN neurons through hetero-synaptic, anti-Hebbian iSTDP. Since the iSTDP rule does not depend on post-synaptic firing, it suggests a possible mechanism for VOR adaptation without compromising gaze-holding and VOR performance in vivo.     

Enhancement of both long-term depression induction and optokinetic response adaptation in mice lacking delphilin

In the cerebellum, Delphilin is expressed selectively in Purkinje cells (PCs) and is localized exclusively at parallel fiber (PF) synapses, where it interacts with glutamate receptor (GluR) delta2 that is essential for long-term depression (LTD), motor learning and cerebellar wiring. Delphilin ablation exerted little effect on the synaptic localization of GluRdelta2. There were no detectable abnormalities in cerebellar histology, PC cytology and PC synapse formation in contrast to GluRdelta2 mutant mice. However, LTD induction was facilitated at PF-PC synapses in Delphilin mutant mice. Intracellular Ca(2+) required for the induction of LTD appeared to be reduced in the mutant mice, while Ca(2+) influx through voltage-gated Ca(2+) channels and metabotropic GluR1-mediated slow synaptic response were similar between wild-type and mutant mice. We further showed that the gain-increase adaptation of the optokinetic response (OKR) was enhanced in the mutant mice. These findings are compatible with the idea that LTD induction at PF-PC synapses is a crucial rate-limiting step in OKR gain-increase adaptation, a simple form of motor learning. As exemplified in this study, enhancing synaptic plasticity at a specific synaptic site of a neural network is a useful approach to understanding the roles of multiple plasticity mechanisms at various cerebellar synapses in motor control and learning.     

Multiple memory mechanisms in the cerebellum?

Long-term potentiation (LTP) and long-term depression (LTD) are arguably two of the most widely discussed cellular plasticity mechanisms for learning and memory. However, the extent to which they are required for behavioral plasticity and learning is not clear. In this issue of Neuron, Boyden et al. use mice lacking CaMKIV and Hansel et al. use mice lacking alphaCaMKII to assess the contribution of LTD to cerebellar learning.     

Insights into the encoding of memories through the circuitry of fear

Associative learning induces physical changes to a network of cells, known as the memory engram. Fear is widely used as a model to understand the circuit motifs that underpin associative memories. Recent advances suggest that the distinct circuitry engaged by different conditioned stimuli (e.g. tone vs. context) can provide insights into what information is being encoded in the fear engram. Moreover, as the fear memory matures, the circuitry engaged indicates how information is remodelled after learning and hints at potential mechanisms for consolidation. Finally, we propose that the consolidation of fear memories involves plasticity of engram cells through coordinated activity between brain regions, and the inherent characteristics of the circuitry may mediate this process.     

Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory

Plasticity of the nervous system is dependent on mechanisms that regulate the strength of synaptic transmission. Excitatory synapses in the brain undergo long-term potentiation (LTP) and long-term depression (LTD), cellular models of learning and memory. Protein phosphorylation is required for the induction of many forms of synaptic plasticity, including LTP and LTD. However, the critical kinase substrates that mediate plasticity have not been identified. We previously reported that phosphorylation of the GluR1 subunit of AMPA receptors, which mediate rapid excitatory transmission in the brain, is modulated during LTP and LTD. To test if GluR1 phosphorylation is necessary for plasticity and learning and memory, we generated mice with knockin mutations in the GluR1 phosphorylation sites. The phosphomutant mice show deficits in LTD and LTP and have memory defects in spatial learning tasks. These results demonstrate that phosphorylation of GluR1 is critical for LTD and LTP expression and the retention of memories.     

Cerebellar Motor Learning: When Is Cortical Plasticity Not Enough?

Classical Marr-Albus theories of cerebellar learning employ only cortical sites of plasticity. However, tests of these theories using adaptive calibration of the vestibulo–ocular reflex (VOR) have indicated plasticity in both cerebellar cortex and the brainstem. To resolve this long-standing conflict, we attempted to identify the computational role of the brainstem site, by using an adaptive filter version of the cerebellar microcircuit to model VOR calibration for changes in the oculomotor plant. With only cortical plasticity, introducing a realistic delay in the retinal-slip error signal of 100 ms prevented learning at frequencies higher than 2.5 Hz, although the VOR itself is accurate up to at least 25 Hz. However, the introduction of an additional brainstem site of plasticity, driven by the correlation between cerebellar and vestibular inputs, overcame the 2.5 Hz limitation and allowed learning of accurate high-frequency gains. This “cortex-first” learning mechanism is consistent with a wide variety of evidence concerning the role of the flocculus in VOR calibration, and complements rather than replaces the previously proposed “brainstem-first” mechanism that operates when ocular tracking mechanisms are effective. These results (i) describe a process whereby information originally learnt in one area of the brain (cerebellar cortex) can be transferred and expressed in another (brainstem), and (ii) indicate for the first time why a brainstem site of plasticity is actually required by Marr-Albus type models when high-frequency gains must be learned in the presence of error delay.     

Neural recordings and behavioral observations in the monkey vestibulo-ocular reflex constrain the cellular mechanisms for cerebellum-dependent behavioral learning

Recordings from the cerebellum under behavioral conditions that cause learning in the vestibulo-ocular reflex (VOR) constrain the cellular mechanisms that could mediate learning. Analysis of the complex-spike responses of Purkinje cells demonstrates a mismatch between the properties of cerebellar long-term depression (LTD) in vitro and the signals available to guide learning in vivo. To resolve this mismatch, it may be necessary to assume that there are multiple cellular mechanisms of VOR learning, including both depression and potentiation.     

Depression of the vestibulo-ocular and optokinetic responses by intrafloccular microinjection of GABA-A and GABA-B agonists in the rabbit

The functional implication of the cerebellar flocculus in regulation of the VOR and OKR gain has mostly been studied by lesion experiments, and the hypotheses derived from these experiments are not always in line with one another. In the present study, a reversible method was used to inhibit floccular Purkinje cells. The GABA-A agonist muscimol or the GABA-B agonist baclofen were bilaterally injected into the flocculus of rabbits, and the effects of these injections on the gain of the VOR and OKR were studied. Both drugs induced a reduction by at least 50% of the gain of the VOR in light and darkness, and of the OKR. Although GABA-A and GABA-B receptors are known to have different cerebellar localizations, muscimol and baclofen injections resulted in quantitatively similar effects. It is suggested that these GABA-agonists cause either direct or indirect inhibition of floccular Purkinje cells, thus reducing modulation of the firing rate of these neurons by afferent mossy and climbing fibers. Because the flocular Purkinje cells act out of phase with the vestibular neurons which drive the oculomotor neurons, a reduced output of floccular Purkinje cells would result in a reduction of the VOR and OKR gain. These experiments provide strong evidence that the cerebellar flocculus has a positive influence on the basic VOR and OKR gain.     

Targeted in vivo mutations of the AMPA receptor subunit GluR2 and its interacting protein PICK1 eliminate cerebellar long-term depression

Cerebellar long-term depression (LTD) is a major form of synaptic plasticity that is thought to be critical for certain types of motor learning. Phosphorylation of the AMPA receptor subunit GluR2 on serine-880 as well as interaction of GluR2 with PICK1 have been suggested to contribute to the endocytic removal of postsynaptic AMPA receptors during LTD. Here, we show that targeted mutation of PICK1, the GluR2 C-terminal PDZ ligand, or the GluR2 PKC phosphorylation site eliminates cerebellar LTD in mice. LTD can be rescued in cerebellar cultures from mice lacking PICK1 by transfection of wild-type PICK1 but not by a PDZ mutant or a BAR domain mutant deficient in lipid binding, indicating the importance of these domains in PICK1 function. These results demonstrate that PICK1-GluR2 PDZ-based interactions and GluR2 phosphorylation are required for LTD expression in the cerebellum.     

Contactin supports synaptic plasticity associated with hippocampal long-term depression but not potentiation

BACKGROUND: Changes in synaptic efficacy are believed to mediate the processes of learning and memory formation. Accumulating evidence implicates cell adhesion molecules in activity-dependent synaptic modifications associated with long-term potentiation (LTP); however, there is no precedence for the selective role of this molecule class in long-term depression (LTD). The mechanisms that modulate these processes still remain unclear. RESULTS: We report a novel role for glycosylphosphatidyl inositol (GPI)-anchored contactin in hippocampal CA1 synaptic plasticity. Contactin selectively supports paired-pulse facilitation (PPF) and NMDA (N-methyl-D-aspartate) receptor-dependent LTD but is not required for synaptic morphology, basal transmission, or LTP. Molecular analyses indicate that contactin is essential for the membrane and synaptic targeting of the contactin-associated protein (Caspr/paranodin) and for the proper distribution of a presumptive ligand, receptor protein tyrosine phosphatase beta (RPTPbeta)/phosphacan. CONCLUSIONS: These results indicate that contactin plays a selective role in synaptic plasticity and identify PPF and LTD, but not LTP, as contactin-dependent processes. Engagement of the contactin-Caspr complex with RPTPbeta may thus regulate cell-cell interactions contributing to specific synaptic plasticity forms.     

A New Rapid Protocol for Eyeblink Conditioning to Assess Cerebellar Motor Learning

Mice with spontaneous and induced mutations causing cerebellar phenotypes have provided key insights into how motor-related memories are stored in cerebellar circuits. Delayed eyeblink conditioning is a form of associative motor learning that depends on the cerebellum. However, neurochemical investigation of the underlying mechanisms has been hampered by the long training period (usually several days) required to establish conditioning. Here, we report a new rapid-training protocol that reliably induced delayed eyeblink conditioning within a single day. The associative memory formation depended on the expression of the δ2 glutamate receptor (GluD2) in cerebellar Purkinje cells. It lasted for several weeks, but could be erased by extinction sessions in a single day. In addition, using the rapid protocol, we found that eyeblink conditioning could be induced in juvenile mice at postnatal day 21, and that the Sindbis-virus-mediated expression of GluD2 could rescue the impaired eyeblink conditioning in GluD2-null mice in vivo.     

Altered cortical synaptic morphology and impaired memory consolidation in forebrain- specific dominant-negative PAK transgenic mice

Molecular and cellular mechanisms for memory consolidation in the cortex are poorly known. To study the relationships between synaptic structure and function in the cortex and consolidation of long-term memory, we have generated transgenic mice in which catalytic activity of PAK, a critical regulator of actin remodeling, is inhibited in the postnatal forebrain. Cortical neurons in these mice displayed fewer dendritic spines and an increased proportion of larger synapses compared to wild-type controls. These alterations in basal synaptic morphology correlated with enhanced mean synaptic strength and impaired bidirectional synaptic modifiability (enhanced LTP and reduced LTD) in the cortex. By contrast, spine morphology and synaptic plasticity were normal in the hippocampus of these mice. Importantly, these mice exhibited specific deficits in the consolidation phase of hippocampus-dependent memory. Thus, our results provide evidence for critical relationships between synaptic morphology and bidirectional modifiability of synaptic strength in the cortex and consolidation of long-term memory.     

Sensory stimulation-dependent plasticity in the cerebellar cortex of alert mice

In vitro studies have supported the occurrence of cerebellar long-term depression (LTD), an interaction between the parallel fibers and Purkinje cells (PCs) that requires the combined activation of the parallel and climbing fibers. To demonstrate the existence of LTD in alert animals, we investigated the plasticity of local field potentials (LFPs) evoked by electrical stimulation of the whisker pad. The recorded LFP showed two major negative waves corresponding to trigeminal (broken into the N2 and N3 components) and cortical responses. PC unitary extracellular recording showed that N2 and N3 occurred concurrently with PC evoked simple spikes, followed by an evoked complex spike. Polarity inversion of the N3 component at the PC level and N3 amplitude reduction after electrical stimulation of the parallel fiber volley applied on the surface of the cerebellum 2 ms earlier strongly suggest that N3 was related to the parallel fiber-PC synapse activity. LFP measurements elicited by single whisker pad stimulus were performed before and after trains of electrical stimuli given at a frequency of 8 Hz for 10 min. We demonstrated that during this later situation, the stimulation of the PC by parallel and climbing fibers was reinforced. After 8-Hz stimulation, we observed long-term modifications (lasting at least 30 min) characterized by a specific decrease of the N3 amplitude accompanied by an increase of the N2 and N3 latency peaks. These plastic modifications indicated the existence of cerebellar LTD in alert animals involving both timing and synaptic modulations. These results corroborate the idea that LTD may underlie basic physiological functions related to calcium-dependent synaptic plasticity in the cerebellum.     

Novel presynaptic mechanisms for coincidence detection in synaptic plasticity

Long-term plasticity typically relies on postsynaptic NMDA receptors to detect the coincidence of pre- and postsynaptic activity. Recent studies, however, have revealed forms of plasticity that depend on coincidence detection by presynaptic NMDA receptors. In the amygdala, cortical afferent associative presynaptic long-term potentiation (LTP) requires activation of presynaptic NMDA receptors by simultaneous thalamic and cortical afferents. Surprisingly, both types of afferent can also undergo postsynaptically induced NMDA-receptor-dependent LTP. In the neocortex, spike-timing-dependent long-term depression (LTD) requires simultaneous activation of presynaptic NMDA autoreceptors and retrograde signalling by endocannabinoids. In cerebellar LTD, presynaptic NMDA receptor activation suggests that similar presynaptic mechanisms may exist. Recent studies also indicate the existence of presynaptic coincidence detection that is independent of NMDA receptors, suggesting that such mechanisms have a widespread role in plasticity.     

Phospholipase Cbeta4 and protein kinase Calpha and/or protein kinase CbetaI are involved in the induction of long term depression in cerebellar Purkinje cells

Activation of the type-1 metabotropic glutamate receptor (mGluR1) signaling pathway in the cerebellum involves activation of phospholipase C (PLC) and protein kinase C (PKC) for the induction of cerebellar long term depression (LTD). The PLC and PKC isoforms that are involved in LTD remain unclear, however. One previous study found no change in LTD in PKCgamma-deficient mice, thus, in the present study, we examined cerebellar LTD in PLCbeta4-deficient mice. Immunohistochemical and Western blot analyses of cerebellum from wild-type mice revealed that PLCbeta1 was expressed weakly and uniformly, PLCbeta2 was not detected, PLCbeta3 was expressed predominantly in caudal cerebellum (lobes 7-10), and PLCbeta4 was expressed uniformly throughout. In PLCbeta4-deficient mice, expression of total PLCbeta, the mGluR1-mediated Ca(2+) response, and LTD induction were greatly reduced in rostral cerebellum (lobes 1-6). Furthermore, we used immunohistochemistry to localize PKCalpha, -betaI, -betaII, and -gamma in mouse cerebellar Purkinje cells during LTD induction. Both PKCalpha and PKCbetaI were found to be translocated to the plasmamembrane under these conditions. Taken together, these results suggest that mGluR1-mediated activation of PLCbeta4 in rostral cerebellar Purkinje cells induced LTD via PKCalpha and/or PKCbetaI.     

Activity dependent protein degradation is critical for the formation and stability of fear memory in the amygdala

Protein degradation through the ubiquitin-proteasome system [UPS] plays a critical role in some forms of synaptic plasticity. However, its role in memory formation in the amygdala, a site critical for the formation of fear memories, currently remains unknown. Here we provide the first evidence that protein degradation through the UPS is critically engaged at amygdala synapses during memory formation and retrieval. Fear conditioning results in NMDA-dependent increases in degradation-specific polyubiquitination in the amygdala, targeting proteins involved in translational control and synaptic structure and blocking the degradation of these proteins significantly impairs long-term memory. Furthermore, retrieval of fear memory results in a second wave of NMDA-dependent polyubiquitination that targets proteins involved in translational silencing and synaptic structure and is critical for memory updating following recall. These results indicate that UPS-mediated protein degradation is a major regulator of synaptic plasticity necessary for the formation and stability of long-term memories at amygdala synapses.     

Aquaporin-4 water channels and synaptic plasticity in the hippocampus

Aquaporin-4 (AQP4) is the major water channel expressed in the central nervous system (CNS) and is primarily expressed in glial cells. Many studies have shown that AQP4 regulates the response of the CNS to insults or injury, but far less is known about the potential for AQP4 to influence synaptic plasticity or behavior. Recent studies have examined long-term potentiation (LTP), long-term depression (LTD), and behavior in AQP4 knockout (KO) and wild-type mice to gain more insight into its potential role. The results showed a selective effect of AQP4 deletion on LTP of the Schaffer collateral pathway in hippocampus using an LTP induction protocol that simulates pyramidal cell firing during theta oscillations (theta-burst stimulation; TBS). However, LTP produced by a different induction protocol was unaffected. There was also a defect in LTD after low frequency stimulation (LFS) in AQP4 KO mice. Interestingly, some slices from AQP4 KO mice exhibited LTD after TBS instead of LTP, or LTP following LFS instead of LTD. These data suggest that AQP4 and astrocytes influence the polarity of long-term synaptic plasticity (potentiation or depression). These potentially powerful roles expand the influence of AQP4 and astrocytes beyond the original suggestions related to regulation of extracellular potassium and water balance. Remarkably, AQP4 KO mice did not show deficits in basal transmission, suggesting specificity for long-term synaptic plasticity. The mechanism appears to be related to neurotrophins and specifically brain-derived neurotrophic factor (BDNF) because pharmacological blockade of neurotrophin trk receptors or scavenging ligands such as BDNF restored plasticity. The in vitro studies predicted effects in vivo of AQP4 deletion because AQP4 KO mice performed worse using a task that requires memory for the location of objects (object placement). However, performance on other hippocampal-dependent tasks was spared. The results suggest an unanticipated and selective role of AQP4 in synaptic plasticity and spatial memory, and underscore the growing appreciation of the role of glial cells in functions typically attributed to neurons. Implications for epilepsy are discussed because of the previous evidence that AQP4 influences seizures, and the role of synaptic plasticity in epileptogenesis.     

Cross axis VOR induced by pursuit training in monkeys: further properties of adaptive responses

We have shown recently in alert monkeys that repeated interaction between the pursuit and vestibular systems in the orthogonal plane induces adaptive changes in the VOR. To examine further properties of adaptive cross axis VOR induced by pursuit training, sinusoidal whole body rotation was applied either in the pitch or yaw plane while presenting a target spot that moved orthogonally to the rotation plane with either 90 degrees phase-lead or 90 degrees phase-lag to the chair signal. After one hour of training at 0.5 Hz (+/- 10 degrees), considerable phase-shift was observed in orthogonal eye movement responses consistent with the training paradigms by identical chair rotation in complete darkness, with further lead at lower frequencies and lag at higher frequencies. However, gains (eye/chair) induced by phase- shift pursuit training was different during pitch and yaw rotation. Although frequency tuning was maintained during pitch in the phase-shift paradigms, it was not maintained during yaw, resulting in higher gains at lower stimulus frequencies compared to the gains during yaw. This difference may reflect otolith contribution during pitch rotation. To understand further the nature of signals that induce adaptive cross axis VOR, we examined interaction of pursuit, whole field-visual pattern and vestibular stimuli. Magnitudes of the cross axis VOR with a spot alone on one hand and with a spot and pattern moving together in the same plane on the other during chair rotation were similar, and when one of the two visual stimuli was stationary during chair rotation, our well trained monkeys did not induce the cross axis VOR. These results suggest that the cross axis VOR induced by pursuit training shares common mechanisms with the cross axis VOR induced by whole field-slip stimuli and that if conflicting information is given between the two visual stimuli, adaptive changes are inhibited. Horizontal GVPs were recorded in the cerebellar floccular lobe during pitch rotation coupled with horizontal pursuit stimuli. These GVPs did not respond to pitch in the dark before training, but responded after 60 min of pursuit training with eye velocity sensitivities similar to those before training. Adaptive change in the VOR was specific to smooth eye movements but not to saccades in our paradigms.