Systemic delivery of (1R-1-benzo thiophen-5-yl-2[2-diethylamino)-ethoxy] ethanol hydrochloride (T-588) prevented long-term depression (LTD) of the parallel fiber (PF)-Purkinje cell (PC) synapse induced by conjunctive climbing fiber and PF stimulation in vivo.However, similar concentrations of T-588 in the brains of behaving mice and rats affected neither motor learning in the rotorod test nor the learning of motor timing during classical conditioning of the eyeblink reflex. Rats given doses of T-588 that prevented PF-PC LTD were as proficient as controls in learning to adapt the timing of their conditioned eyeblink response to a 150-or 350-ms change in the timing of the paradigm. The experiment indicates that PF-PC LTD under control of the climbing fibers is not required for general motor adaptation or the learning of response timing in two common models of motor learning for which the cerebellum has been implicated. Alternative mechanisms for motor timing and possible functions for LTD in protection from excitotoxicity are discussed.cerebellum ͉ eyeblink ͉ field potentials ͉ rotorod A longstanding hypothesis concerning cerebellar function has been the proposal that it serves as the repository for new motor skills through plasticity of the parallel fiber (PF)-Purkinje cell (PC) synapse (1, 2). In the prevailing hypothesis of ''cerebellar learning,'' the acquisition of new skills is controlled by climbing fibers (CFs), which function to depress the strength of the PF-PC synapse when the two afferents are conjointly active. The cerebellar learning hypothesis has inspired much research since the demonstration, in vivo, that near-synchronous activation of CFs and PFs by direct electrical stimulation depressed the strength of the excitatory potentials triggered by PFs in PCs (3). Because the depression required repeated pairings of CF and PF synaptic input and was retained for many hours, the phenomenon has been termed cerebellar long-term depression (LTD) and has been invoked to explain the acquisition and lifetime retention of many motor skills, including learned motor timing, reflex adaptation, and procedural and implicit learning (4).A substantial body of evidence has established the necessary intracellular events that produce PC LTD in vitro, and these events have been assumed to relate to motor learning in vivo. Moreover, a recent experiment showed that mouse PCs genetically modified to lack protein kinase C (PKC) function could not undergo LTD (5, 6); when such transgenic mice were tested in classical eyeblink conditioning, they were unable to learn the appropriate motor timing of conditioned eyeblinks [conditioned response (CR)] (7). That finding, complemented by the finding that cerebellar decortication also impaired the learned motor timing of CRs (8), has sustained the hypothesis that PF-PC LTD is required for motor learning. However, it is also evident that, up to this time, it has not been determined whether LTD occurs in awake animals as they acquire and retain new motor skills. Thus, it has been diffic...

SUMMARY Electrical synapses are formed by gap junctions and permit electrical coupling that shapes the synchrony of neuronal ensembles. Here, we provide the first direct demonstration of receptormediated strengthening of electrical coupling in mammalian brain. Electrical coupling in the inferior olive of rats was strengthened by activation of NMDA-type glutamate-receptors (NMDARs), which were found at synaptic loci and at extrasynaptic loci 20–100 nm proximal to gap junctions. Electrical coupling was strengthened by pharmacological and synaptic activation of NMDARs, while co-stimulation of ionotropic non-NMDAR glutamate-receptors transiently antagonized the effect of NMDAR activation. NMDAR-dependent strengthening (i) occurred despite increased input conductance, (ii) induced Ca2+-influx microdomains near dendritic spines, (iii) required activation of the Ca2+/calmodulin-dependent protein-kinase II, (iv) was restricted to neurons that were weakly coupled, and thus, (v) strengthened coupling mainly between non-adjacent neurons. This provided a mechanism to expand the synchronization of rhythmic membrane potential oscillations by chemical neurotransmitter input.

Inferior olive (IO) neurons are electrically coupled by cytosolic pores formed by the neuron-specific connexin 36 (Cx36). Electrical coupling in the IO figures prominently in current views about brain control of movement. However, a role for Cx36 in movement has been questioned and not definitively demonstrated. Previous reports have shown that embryonic deletion of the Cx36 gene resulted in almost complete loss of cytosolic and electrical coupling in the IO without an obvious deficit in movement, possibly due to developmental compensations in ionic conductances that can confound the approach of embryonic gene deletion. We used a replication-incompetent lentiviral vector to stably express a dominant-negative Cx36 mutant in the IO of adult rats. We show that interneuronal cytosolic coupling is severely reduced by the mutant Cx36, without effect on neuron morphology or electrical properties. Multisite electromyography revealed that blocking Cx36 in the IO impaired the coherence of muscle firing during harmaline tremor without affecting its rhythm. The data demonstrate that gap junction coupling within the IO mediated by Cx36 adds 10 -20 ms of precision to the fine temporal coordination of muscle firing during movement. E lectrical synapses permit neuronal ensembles to fire synchronously with millisecond precision. Connexin 36 (Cx36) is the neuron-specific protein (1) that forms electrical synapses mediated by gap junction pores that link the cytosol of neighboring neurons (2, 3). Although outnumbered by chemical synapses, electrical synapses are distributed widely throughout the brain. Nevertheless, it has not been possible to establish a behavioral function for Cx36 primarily due to the lack of agents that specifically block connexins (4).In adult brain, Cx36 and electrical synapses are most prevalent in the inferior olive (IO), where dendritic gap junctions underlie electrotonic coupling (5-7). The IO is a precerebellar nucleus that emits rhythmic activity patterns punctuated by a high degree of synchrony, as measured by multiple intracranial microelectrodes (8-10). The spatial pattern of IO synchrony is modulated during skilled movement (9), suggesting that electrical synapses are important for motor coordination. However, embryonic deletion of Cx36 did not detectably impair gross motricity (11) or the tremor produced by harmaline (11, 12), a movement that is generated by rhythmic output from the IO (13,14). A recent analysis showed that compensatory alterations in ionic conductances might account for the single-cell rhythmicity of electrically uncoupled IO neurons (15) and potentially explain why harmaline tremor persisted in the absence of electrical synapses (11,12). Thus, the role of Cx36 in the IO for movement remains a compelling question.To circumvent the morphological and electrophysiological compensations that can confound embryonic gene deletion, we used gene transfer into the adult brain with a replicationincompetent and self-inactivating lentiviral vector (LV; refs. 16-18) to deliver a dominant nega...