Analysis and Modeling of Frequency-Specific Habituation of the Goldfish Vestibulo-Ocular Reflex

Modification of the vestibulo-ocular reflex (VOR) by vestibular habituation is an important paradigm in the study of neural plasticity. The VOR is responsible for rotating the eyes to maintain the direction of gaze during head rotation. The response of the VOR to sinusoidal rotation is quantified by its gain (eye rotational velocity/head rotational velocity) and phase difference (eye velocity phase—inverted head velocity phase). The frequency response of the VOR in naïve animals has been previously modeled as a high-pass filter (HPF). A HPF passes signals above its corner frequency with gain 1 and phase 0 but decreases gain and increases phase lead (positive phase difference) as signal frequency decreases below its corner frequency. Modification of the VOR by habituation occurs after prolonged low-frequency rotation in the dark. Habituation causes a reduction in low-frequency VOR gain and has been simulated by increasing the corner frequency of the HPF model. This decreases gain not only at the habituating frequency but further decreases gain at all frequencies below the new corner frequency. It also causes phase lead to increase at all frequencies below the new corner frequency (up to some asymptotic value). We show that habituation of the goldfish VOR is not a broad frequency phenomena but is frequency specific. A decrease in VOR gain is produced primarily at the habituating frequency, and there is an increase in phase lead at nearby higher frequencies and a decrease in phase lead at nearby lower frequencies (phase crossover). Both the phase crossover and the frequency specific gain decrease make it impossible to simulate habituation of the VOR simply by increasing the corner frequency of the HPF model. The simplest way to simulate our data is to subtract the output of a band-pass filter (BPF) from the output of the HPF model of the naïve VOR. A BPF passes signals over a limited frequency range only. A BPF decreases gain and imparts a phase lag and lead, respectively, as frequency increases and decreases outside this range. Our model produces both the specific decrease in gain at the habituating frequency, and the phase crossover centered on the frequency of habituation. Our results suggest that VOR habituation may be similar to VOR adaptation (in which VOR modification is produced by visual-vestibular mismatch) in that both are frequency-specific phenomena.     

Simulating vestibular compensation using recurrent back-propagation

Vestibular compensation is simulated as learning in a dynamic neural network model of the horizontal vestibulo-ocular reflex (VOR). The bilateral, three-layered VOR model consists of nonlinear units representing horizontal canal afferents, vestibular nuclei (VN) neurons and eye muscle motoneurons. Dynamic processing takes place via commissural connections that link the VN bilaterally. The intact network is trained, using recurrent back-propagation, to produce the VOR with velocity storage integration. Compensation is simulated by removing vestibular afferent input from one side and retraining the network. The time course of simulated compensation matches that observed experimentally. The behavior of model VN neurons in the compensated network also matches real data, but only if connections at the motoneurons, as well as at the VN, are allowed to be plastic. The dynamic properties of real VN neurons in compensated and normal animals are found to differ when tested with sinusoidal but not with step stimuli. The model reproduces these conflicting data, and suggests that the disagreement may be due to VN neuron nonlinearity.     

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.     

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.     

Neural network models of velocity storage in the horizontal vestibulo-ocular reflex

The vestibulo-ocular reflex (VOR) produces compensatory eye movements by utilizing head rotational velocity signals from the semicircular canals to control contractions of the extraocular muscles. In mammals, the time course of horizontal VOR is longer than that of the canal signals driving it, revealing the presence of a central integrator known as velocity storage. Although the neurons mediating VOR have been described neurophysiologically, their properties, and the mechanism of velocity storage itself, remain unexplained. Recent models of integration in VOR are based on systems of linear elements, interconnected in arbitrary ways. The present study extends this work by modeling horizontal VOR as a learning network composed of nonlinear model neurons. Network architectures are based on the VOR arc (canal afferents, vestibular nucleus (VN) neurons and extraocular motoneurons) and have both forward and lateral connections. The networks learn to produce velocity storage integration by forming lateral (commissural) inhibitory feedback loops between VN neurons. These loops overlap and interact in a complex way, forming both fast and slow VN pathways. The networks exhibit some of the nonlinear properties of the actual VOR, such as dependency of decay rate and phase lag upon input magnitude, and skewing of the response to higher magnitude sinusoidal inputs. Model VN neurons resemble their real counterparts. Both have increased time constant and gain, and decreased spontaneous rate as compared to canal afferents. Also, both model and real VN neurons exhibit rectification and skew. The results suggest that lateral inhibitory interactions produce velocity storage and also determine the properties of neurons mediating VOR. The neural network models demonstrate how commissural inhibition may be organized along the VOR pathway.     

Distributed parallel processing in the vertical vestibulo-ocular reflex: Learning networks compared to tensor theory

The vestibulo-ocular reflex (VOR) is capable of producing compensatory eye movements in three dimensions. It utilizes the head rotational velocity signals from the semicircular canals to control the contractions of the extraocular muscles. Since canal and muscle coordinate frames are not orthogonal and differ from one another, a sensorimotor transformation must be produced by the VOR neural network. Tensor theory has been used to construct a linear transformation that can model the three-dimensional behavior of the VOR. But tensor theory does not take the distributed, redundant nature of the VOR neural network into account. It suggests that the neurons subserving the VOR, such as vestibular nucleus neurons, should have specific sensitivity-vectors. Actual data, however, are not in accord. Data from the cat show that the sensitivity-vectors of vestibular nucleus neurons, rather than aligning with any specific vectors, are dispersed widely. As an alternative to tensor theory, we modeled the vertical VOR as a three-layered neural network programmed using the back-propagation learning algorithm. Units in mature networks had divergent sensitivity-vectors which resembled those of actual vestibular nucleus neurons in the cat. This similarity suggests that the VOR sensorimotor transformation may be represented redundantly rather than uniquely. The results demonstrate how vestibular nucleus neurons can encode the VOR sensorimotor transformation in a distributed manner.     

Time constants of vestibular nuclei neurons in the goldfish: A model with ocular proprioception

A simple model of the vestibular-ocular reflex with a proprioceptive eye velocity feedback loop is used to simulate recent data on the vestibular responses of neurons in the vestibular nuclei of spinal goldfish. The data support the hypothesis that a proprioceptive feedback loop elongates the vestibular nucleus time constant to equal that of the slow phase eye movements of vestibular nystagmus.     

Intrinsic and synaptic plasticity in the vestibular system

The vestibular system provides an attractive model for understanding how changes in cellular and synaptic activity influence learning and memory in a quantifiable behavior, the vestibulo-ocular reflex. The vestibulo-ocular reflex produces eye movements that compensate for head motion; simple yet powerful forms of motor learning calibrate the circuit throughout life. Learning in the vestibulo-ocular reflex depends initially on the activity of Purkinje cells in the cerebellar flocculus, but consolidated memories appear to be stored downstream of Purkinje cells, probably in the vestibular nuclei. Recent studies have demonstrated that the neurons of the vestibular nucleus possess the capacity for both synaptic and intrinsic plasticity. Mechanistic analyses of a novel form of firing rate potentiation in neurons of the vestibular nucleus have revealed new rules of plasticity that could apply to spontaneously firing neurons in other parts of the brain.     

The vestibulo-ocular reflex and velocity storage in spinocerebellar ataxia 8

The autosomal dominant spinocerebellar ataxias (SCAs) are a group of neurodegenerative diseases characterized by progressive instability of posture and gait, incoordination, ocular motor dysfunction, and dysarthria due to degeneration of cerebellar and brainstem neurons. Among the more than 20 genetically distinct subtypes, SCA8 is one of several wherein clinical observations indicate that cerebellar dysfunction is primary, and there is little evidence for other CNS involvement. The aim of the present work was to study the decay of the horizontal vestibulo-ocular reflex (VOR) after a short period of constant acceleration to understand the pathophysiology of the VOR due to cerebellar Purkinje cell degeneration in SCA8. The VOR was recorded in patients with genetically defined SCA8 during rotation in the dark. Moderate to severely affected patients had a qualitatively intact VOR, but there were quantitative differences in the gain and dynamics compared to normal controls. During angular velocity ramp rotations, there was a reversal in the direction of the VOR that was more pronounced in SCA8 compared to controls. Modeling studies indicate that there are significant changes in the velocity storage network, including abnormal feedback of an eye position signal into the network that contributes to this reversal. These and other results will help to identify features that are diagnostic for SCA subtypes and provide new information about selective vulnerability of neurons controlling vestibular reflexes.     

Development and organization of polarity-specific segregation of primary vestibular afferent fibers in mice

A striking feature of vestibular hair cells is the polarized arrangement of their stereocilia as the basis for their directional sensitivity. In mammals, each of the vestibular end organs is characterized by a distinct distribution of these polarized cells. We utilized the technique of post-fixation transganglionic neuronal tracing with fluorescent lipid soluble dyes in embryonic and postnatal mice to investigate whether these polarity characteristics correlate with the pattern of connections between the endorgans and their central targets; the vestibular nuclei and cerebellum. We found that the cerebellar and brainstem projections develop independently from each other and have a non-overlapping distribution of neurons and afferents from E11.5 on. In addition, we show that the vestibular fibers projecting to the cerebellum originate preferentially from the lateral half of the utricular macula and the medial half of the saccular macula. In contrast, the brainstem vestibular afferents originate primarily from the medial half of the utricular macula and the lateral half of the saccular macula. This indicates that the line of hair cell polarity reversal within the striola region segregates almost mutually exclusive central projections. A possible interpretation of this feature is that this macular organization provides an inhibitory side-loop through the cerebellum to produce synergistic tuning effects in the vestibular nuclei. The canal cristae project to the brainstem vestibular nuclei and cerebellum, but the projection to the vestibulocerebellum originates preferentially from the superior half of each of the cristae. The reason for this pattern is not clear, but it may compensate for unequal activation of crista hair cells or may be an evolutionary atavism reflecting a different polarity organization in ancestral vertebrate ears.     

L9 modulation of gill withdrawal reflex habituation in Aplysia.

Repeated tactile stimulation of the siphon in Aphysia normally results in habituation of the gill withdrawal reflex and a concomitant decrease in the amplitude of the excitatory synaptic input ot gill motor neurons in the abdominal ganglion. It was found, however, that induced low-level tonic activity in motor neuron L9, which does not itself elicit a gill withdrawal movement, prevented habituation of the reflex from occurring. Further, in preparations already habituated, this tonic low-level activity brought about a reversal of habituation. Although tonic L9 activity prevented the occurrence of habituation or brought about its reversal, it did not interfere with the synaptic decremental process which normally accompanies gill reflex habituation. Motor neurons L7 and LDG1 were found not to possess this ability of L9 to modulate gill reflex habituation. Evidence suggests that L9's modulatory effect is mediated in the periphery, in the gill and not centrally in the abdominal ganglion.     

The phenolic profile of maize primary cell wall changes in cellulose-deficient cell cultures

Cultured maize cells habituated to grow in the presence of the cellulose synthesis inhibitor dichlobenil (DCB) have a modified cell wall in which the amounts of cellulose are reduced and the amounts of arabinoxylan increased. This paper examines the contribution of cell wall-esterified hydroxycinnamates to the mechanism of DCB habituation. For this purpose, differences in the phenolic composition of DCB-habituated and non-habituated cell walls, throughout the cell culture cycle and the habituation process were characterized by HPLC. DCB habituation was accompanied by a net enrichment in cell wall phenolics irrespective of the cell culture phase. The amount of monomeric phenolics was 2-fold higher in habituated cell walls. Moreover, habituated cell walls were notably enriched in p-coumaric acid. Dehydrodimers were 5–6-fold enhanced as a result of DCB habituation and the steep increase in 8,5′-diferulic acid in habituated cell walls would suggest that this dehydrodimer plays a role in DCB habituation. In summary, the results obtained indicate that cell wall phenolics increased as a consequence of DCB habituation, and suggest that they would play a role in maintaining the functionality of a cellulose impoverished cell wall.     

A matrix analysis for a conjugate vestibulo-ocular reflex

The technique of matrix analysis is used to compare the connectivity between vestibular neurons and oculomotor neurons of the two eyes that would generate a conjugate vestibulo-ocular reflex (VOR). The technique shows that the connectivity is normally anatomically symmetric. The technique is also used to determine the types and loci of adaptation within the VOR that will maintain conjugacy. Adaptation is divided into1) that evoked by changes in visual feedback, which requires VOR or system-specific changes and2) that produced by changes in the canals or muscles, which requires deficit-specific adaptation. In the former case, the adaptation could best be achieved by an additive alteration of the vestibularmotoneuron projections. In the latter case, the appropriate adaptations would be serial, multiplicative changes, applied at the level of the vestibular neurons when the canals are at fault or at the level of the motoneurons of the eye whose muscles are impaired. The analysis thus suggests multiple loci of plasticity within the VOR, specialized for adapting to different deficits.     

Contribution of neurons to habituation to mechanical stimulation in Caenorhabditis elegans

In Caenorhabditis elegans, a light touch induces a locomotor response. Repeated touches, however, result in an attenuation of response, that is, habituation. Withdrawal responses elicited by anterior touch are controlled by anterior mechanosensory neurons (AVM and ALMs), and by four pairs of interneurons (AVA, AVB, AVD, and PVC) (Chalfie et al., 1985; White et al., 1986). To identify the neurons that participate in habituation, we ablated these neurons with a laser microbeam and investigated the resulting habituation of the operated animals. The animals lacking both left and right homologues AVDLR were habituated more rapidly than intact animals. We propose that chemical synapses at AVD play a critical role in the habituation of intact animals.     

Activity of cat cerebellar neurons in penicillin epilepsy and amphetamine treatment.

The discharge pattern of cerebellar Purkinje cells and fastigial neurons was studied after acute amphetamine treatment in immobilized cats, as well as during generalized penicillin-induced epilepsy. There was a marked reversible decrease in spontaneous firing rate of Purkinje cells and an increase in spontaneous firing rate of fastigial neurons after acute d-1 amphetamine administration (5 mg/kg, s.c.). The discharge pattern of Purkinje cells showed tendency towards inhibition, while the fastigial neurons showed less clear tendency towards disinhibition in the course of epilepsy induced by parenteral administration of penicillin (400.000-500.000 I.U./kg, i.m.). Moreover, acute amphetamine treatment (5 mg/kg, s.c.) performed after the development of penicillin-induced epileptic episodes elicited a prominent suppression of Purkinje cell discharges associated with a parallel increase in discharges of fastigial neurons. These results suggest that the changes in discharge rate of cerebellar corticonuclear neurons induced by amphetamine contribute to suppression of seizural activity in the feline model of generalized epilepsy.     

Habituation of bean (Phaseolus vulgaris) cell cultures to Quinclorac and analysis of the subsequent cell wall modifications

Background and Aims

The herbicide quinclorac has been reported to inhibit incorporation of glucose both into cellulose and other cell wall polysaccharides. However, further work has failed to detect any apparent effect of this herbicide on the synthesis of the wall. In order to elucidate whether quinclorac elicits the inhibition of cellulose biosynthesis directly, in this study bean cell calli were habituated to grow on lethal concentrations of the herbicide and the modifications in cell wall composition due to the habituation process were analysed.

Methods

Fourier transform infrared spectroscopy associated with multivariate analysis, cell wall fractionation techniques, biochemical analyses and the immunolocation of different cell wall components with specific monoclonal antibodies were used to characterize the cell walls of quinclorac-habituated cells.

Key Results

Quinclorac-habituated cells were more irregularly shaped than non-habituated cells and they accumulated an extracellular material, which was more abundant as the level of habituation rose. Habituated cells did not show any decrease in cellulose content, but cell wall fractionation revealed that changes occurred in the distribution and post-depositional modifications of homogalacturonan and rhamnogalacturonan I during the habituation process. Therefore, since the action of quinclorac on the cell wall does not seem to be due to a direct inhibition of any cell wall component, it is suggested that the effect of quinclorac on the cell wall could be due to a side-effect of the herbicide.

Conclusions

Long-term modifications of the cell wall caused by the habituation of bean cell cultures to quinclorac did not resemble those of bean cells habituated to the well-known cellulose biosynthesis inhibitors dichlobenil or isoxaben. Quinclorac does not seem to act primarily as an inhibitor of cellulose biosynthesis.Key words: Quinclorac, herbicide, Phaseolus vulgaris, cell culture habituation, primary cell wall, cellulose, FTIR spectroscopy     

The fractional-order dynamics of brainstem vestibulo-oculomotor neurons

The vestibulo-ocular reflex (VOR) and other oculomotor subsystems such as pursuit and saccades are ultimately mediated in the brainstem by premotor neurons in the vestibular and prepositus nuclei that relay eye movement commands to extraocular motoneurons. The premotor neurons receive vestibular signals from canal afferents. Canal afferent frequency responses have a component that can be characterized as a fractional-order differentiation (d ^k x/dt _k where k is a nonnegative real number). This article extends the use of fractional calculus to describe the dynamics of motor and premotor neurons. It suggests that the oculomotor integrator, which converts eye velocity into eye position commands, may be of fractional order. This order is less than one, and the velocity commands have order one or greater, so the resulting net output of motor and premotor neurons can be described as fractional differentiation relative to eye position. The fractional derivative dynamics of motor and premotor neurons may serve to compensate fractional integral dynamics of the eye. Fractional differentiation can be used to account for the constant phase shift across frequencies, and the apparent decrease in time constant as VOR and pursuit frequency increases, that are observed for motor and premotor neurons. Fractional integration can reproduce the time course of motor and premotor neuron saccade-related activity, and the complex dynamics of the eye. Insight into the nature of fractional dynamics can be gained through simulations in which fractional-order differentiators and integrators are approximated by sums of integer-order high-pass and low-pass filters, respectively. Fractional dynamics may be applicable not only to the oculomotor system, but to motor control systems in general.     

Neurogenic potential of the vestibular nuclei and behavioural recovery time course in the adult cat are governed by the nature of the vestibular damage

Functional and reactive neurogenesis and astrogenesis are observed in deafferented vestibular nuclei after unilateral vestibular nerve section in adult cats. The newborn cells survive up to one month and contribute actively to the successful recovery of posturo-locomotor functions. This study investigates whether the nature of vestibular deafferentation has an incidence on the neurogenic potential of the vestibular nuclei, and on the time course of behavioural recovery. Three animal models that mimic different vestibular pathologies were used: unilateral and permanent suppression of vestibular input by unilateral vestibular neurectomy (UVN), or by unilateral labyrinthectomy (UL, the mechanical destruction of peripheral vestibular receptors), or unilateral and reversible blockade of vestibular nerve input using tetrodotoxin (TTX). Neurogenesis and astrogenesis were revealed in the vestibular nuclei using bromodeoxyuridine (BrdU) as a newborn cell marker, while glial fibrillary acidic protein (GFAP) and glutamate decarboxylase 67 (GAD67) were used to identify astrocytes and GABAergic neurons, respectively. Spontaneous nystagmus and posturo-locomotor tests (static and dynamic balance performance) were carried out to quantify the behavioural recovery process. Results showed that the nature of vestibular loss determined the cellular plastic events occurring in the vestibular nuclei and affected the time course of behavioural recovery. Interestingly, the deafferented vestibular nuclei express neurogenic potential after acute and total vestibular loss only (UVN), while non-structural plastic processes are involved when the vestibular deafferentation is less drastic (UL, TTX). This is the first experimental evidence that the vestibular complex in the brainstem can become neurogenic under specific injury. These new data are of interest for understanding the factors favouring the expression of functional neurogenesis in adult mammals in a brain repair perspective, and are of clinical relevance in vestibular pathology.     

Bilateral otolith contribution to spatial coding in the vestibular system

Recent work on the coding of spatial information in central otolith neurons has significantly advanced our knowledge of signal transformation from head-fixed otolith coordinates to space-centered coordinates during motion. In this review, emphasis is placed on the neural mechanisms by which signals generated at the bilateral labyrinths are recognized as gravity-dependent spatial information and in turn as substrate for otolithic reflexes. We first focus on the spatiotemporal neuronal response patterns (i.e. one- and two-dimensional neurons) to pure otolith stimulation, as assessed by single unit recording from the vestibular nucleus in labyrinth-intact animals. These spatiotemporal features are also analyzed in association with other electrophysiological properties to evaluate their role in the central construction of a spatial frame of reference in the otolith system. Data derived from animals with elimination of inputs from one labyrinth then provide evidence that during vestibular stimulation signals arising from a single utricle are operative at the level of both the ipsilateral and contralateral vestibular nuclei. Hemilabyrinthectomy also revealed neural asymmetries in spontaneous activity, response dynamics and spatial coding behavior between neuronal subpopulations on the two sides and as a result suggested a segregation of otolith signals reaching the ipsilateral and contralateral vestibular nuclei. Recent studies have confirmed and extended previous observations that the recovery of resting activity within the vestibular nuclear complex during vestibular compensation is related to changes in both intrinsic membrane properties and capacities to respond to extracellular factors. The bilateral imbalance provides the basis for deranged spatial coding and motor deficits accompanying hemilabyrinthectomy. Taken together, these experimental findings indicate that in the normal state converging inputs from bilateral vestibular labyrinths are essential to spatiotemporal signal transformation at the central otolith neurons during low-frequency head movements.     

Potentiation of mossy fiber EPSCs in the cerebellar nuclei by NMDA receptor activation followed by postinhibitory rebound current

Behavioral and computational studies predict that synaptic plasticity of excitatory mossy fiber inputs to cerebellar nuclear neurons is required for associative learning, but standard tetanization protocols fail to potentiate nuclear cell EPSCs in mouse cerebellar slices. Nuclear neurons fire action potentials spontaneously unless strongly inhibited by Purkinje neurons, raising the possibility that plasticity-triggering signals in these cells differ from those at classical Hebbian synapses. Based on predictions of neuronal activity during delay eyelid conditioning, we developed quasi-physiological induction protocols consisting of high-frequency mossy fiber stimulation and postsynaptic hyperpolarization. Robust, NMDA receptor-dependent potentiation of nuclear cell EPSCs occurred with protocols including a 150-250 ms hyperpolarization in which mossy fiber stimulation preceded a postinhibitory rebound depolarization. Mossy fiber stimulation potentiated EPSCs even when postsynaptic spiking was prevented by voltage-clamp, as long as rebound current was evoked. These data suggest that Purkinje cell inhibition guides the strengthening of excitatory synapses in the cerebellar nuclei.