NR2A subunit of the N-methyl D-aspartate receptors are required for potentiation at the mossy fiber to granule cell synapse and vestibulo-cerebellar motor learning.

Traditionally studies aimed at elucidating the molecular mechanisms underlying cerebellar motor learning have been focused on plasticity at the parallel fiber to Purkinje cell synapse. In recent years, however, the concept is emerging that formation and storage of memories are both distributed over multiple types of synapses at different sites. Here, we examined the potential role of potentiation at the mossy fiber to granule cell synapse, which occurs upstream to plasticity in Purkinje cells. We show that null-mutants of N-methyl d-aspartate-NR2A receptors (NMDA-NR2A(-/-) mice) have impaired induction of postsynaptic long-term potentiation (LTP) at the mossy fiber terminals and a reduced ability to raise the granule cell synaptic excitation, while the basic excitatory output of the mossy fibers is unaffected. In addition, we demonstrate that these NR2A(-/-) mutants as well as mutants in which the C terminal in the NR2A subunit is selectively truncated (NR2A(ΔC/ΔC) mice) have deficits in phase reversal adaptation of their vestibulo-ocular reflex (VOR), while their basic eye movement performance is similar to that of wild type littermates. These results indicate that NMDA-NR2A mediated potentiation at the mossy fiber to granule cell synapse is not required for basic motor performance, and they raise the possibility that it may contribute to some forms of vestibulo-cerebellar memory formation.

In vivo mouse inferior olive neurons exhibit heterogeneous subthreshold oscillations and spiking patterns.

In vitro whole-cell recordings of the inferior olive have demonstrated that its neurons are electrotonically coupled and have a tendency to oscillate. However, it remains to be shown to what extent subthreshold oscillations do indeed occur in the inferior olive in vivo and whether its spatiotemporal firing pattern may be dynamically generated by including or excluding different types of oscillatory neurons. Here, we did whole-cell recordings of olivary neurons in vivo to investigate the relation between their subthreshold activities and their spiking behavior in an intact brain. The vast majority of neurons (85%) showed subthreshold oscillatory activities. The frequencies of these subthreshold oscillations were used to distinguish four main olivary subtypes by statistical means. Type I showed both sinusoidal subthreshold oscillations (SSTOs) and low-threshold Ca(2+) oscillations (LTOs) (16%); type II showed only sinusoidal subthreshold oscillations (13%); type III showed only low-threshold Ca(2+) oscillations (56%); and type IV did not reveal any subthreshold oscillations (15%). These subthreshold oscillation frequencies were strongly correlated with the frequencies of preferred spiking. The frequency characteristics of the subthreshold oscillations and spiking behavior of virtually all olivary neurons were stable throughout the recordings. However, the occurrence of spontaneous or evoked action potentials modified the subthreshold oscillation by resetting the phase of its peak toward 90 degrees . Together, these findings indicate that the inferior olive in intact mammals offers a rich repertoire of different neurons with relatively stable frequency settings, which can be used to generate and reset temporal firing patterns in a dynamically coupled ensemble.

Analysis of Cx36 knockout does not support tenet that olivary gap junctions are required for complex spike synchronization and normal motor performance.

Electrotonic coupling by gap junctions between neurons in the inferior olive has been claimed to underly complex spike (CS) synchrony of Purkinje cells in the cerebellar cortex and thereby to play a role in the coordination of movements. Here, we investigated the motor performance of mice that lack connexin36 (Cx36), which appears necessary for functional olivary gap junctions. Cx36 null-mutants are not ataxic, they show a normal performance on the accelerating rotorod, and they have a regular walking pattern. In addition, they show normal compensatory eye movements during sinusoidal visual and/or vestibular stimulation. To find out whether the normal motor performance in mutants reflects normal CS activity or some compensatory mechanism downstream of the cerebellar cortex, we determined the CS firing rate, climbing-fiber pause, and degree of CS synchrony. None of these parameters in the mutants differed from those in wildtype littermates. Finally, we investigated whether the role of coupling becomes apparent under challenging conditions, such as during application of the tremorgenic drug harmaline, which specifically turns olivary neurons into an oscillatory state at a high frequency. In both the mutants and wildtypes this application induced tremors of a similar duration with similar peak frequencies and amplitudes. Thus surprisingly, the present data does not support the notion that electrotonic coupling by gap junctions underlies synchronization of olivary spike activity and that these gap junctions are essential for normal motor performance.

A reexamination of the role of GABA in the mammalian suprachiasmatic nucleus.

Three independent electrophysiological approaches in hypothalamic slices were used to test the hypothesis that gamma-amino butyric acid (GABA)A receptor activation excites suprachiasmatic nucleus (SCN) neurons during the subjective day, consistent with a recent report. First, multiple-unit recordings during either the subjective day or night showed that GABA or muscimol inhibited firing activity of the SCN population in a dose-dependent manner. Second, cell-attached recordings during the subjective day demonstrated an inhibitory effect of bath- or microapplied GABA on action currents of single SCN neurons. Third, gramicidin perforated-patch recordings showed that bicuculline increased the spontaneous firing rate during the subjective day. Therefore, electrophysiological data obtained by three different experimental methods provide evidence that GABA is inhibitory rather than excitatory during the subjective day.

Neurons of the rat suprachiasmatic nucleus show a circadian rhythm in membrane properties that is lost during prolonged whole-cell recording.

The suprachiasmatic nucleus is commonly considered to contain the main pacemaker of behavioral and hormonal circadian rhythms. Using whole-cell patch-clamp recordings, the membrane properties of suprachiasmatic nucleus neurons were investigated in order to get more insight in membrane physiological mechanisms underlying the circadian rhythm in firing activity. Circadian rhythmicity could not be detected either in spontaneous firing rate or in other membrane properties when whole-cell measurements were made following an initial phase shortly after membrane rupture. However, this apparent lack of rhythmicity was not due to an unhealthy slice preparation or to seal formation, as a clear day/night difference in firing rate was found in cell-attached recordings. Furthermore, in a subsequent series of whole-cell recordings, membrane properties were assessed directly after membrane rupture, and in this series we did find a significant day/night difference in spontaneous firing rate, input resistance and frequency adaptation. As concerns the participation of different subpopulations of suprachiasmatic nucleus neurons expressing circadian rhythmicity, cluster I neurons exhibited strong rhythmicity, whereas no day/night differences were found in cluster II neurons. Vasopressin-containing cells form a subpopulation of cluster I neurons and showed a more pronounced circadian rhythmicity than the total population of cluster I neurons. In addition to their strong rhythm in spontaneous firing rate they also displayed a day/night difference in membrane potential.

Electrophysiological and morphological heterogeneity of neurons in slices of rat suprachiasmatic nucleus.

1. Whole cell patch clamp recordings of neurons in slices of the suprachiasmatic nucleus (SCN) were made in order to assess their electrophysiological and morphological heterogeneity. This assessment was accomplished by (i) quantification of intrinsic membrane properties recorded in current clamp mode, (ii) studying frequency distributions of these properties, (iii) grouping of cells based on visual inspection of data records, and (iv) use of cluster analysis methods. 2. Marked heterogeneity was found in the resting membrane potential, input resistance, time constant, rate of frequency adaptation, size of rebound depolarization (low-threshold Ca2+ potential) and regularity of firing. The frequency distribution of these membrane properties deviated significantly from a normal distribution. Other parameters, including spike amplitude and width, amplitude and rising slope of the spike after-hyperpolarization (AHP) and amplitude of the spike train AHP, showed considerable variability as well but generally obeyed a normal distribution. 3. Visual inspection of the data led to partitioning of cells into three clusters, viz. cluster I characterized by monophasic spike AHPs and irregular firing in the frequency range from 1.5 to 5.0 Hz; cluster II with biphasic spike AHPs and regular firing in the same range; and cluster III with large rebound depolarizations and biphasic spike AHPs. In a post hoc analysis, these clusters also appeared to differ in other membrane properties. This grouping was confirmed by hierarchical tree clustering and multidimensional scaling. 4. The light microscopic properties of recorded neurons were studied by biocytin labelling. Neurons had monopolar, bipolar or multipolar branching patterns and were often varicose. Axons sometimes originated from distal dendritic segments and usually branched into multiple collaterals. Many cells with extra-SCN projections also possessed intranuclear axon collaterals. We found no morphological differences between clusters except that cluster III neurons possessed more axon collaterals than cluster I or II cells. 5. These results suggest that SCN neurons are heterogeneous in some basic as well as active membrane properties and can be partitioned into at least three clusters. Cluster I and II cells fire spontaneously in a regular and irregular mode, respectively, and sustain prolonged spike trains. In contrast, cluster III cells have low firing rates but may adopt a burst-like firing mode when receiving appropriate input. While all clusters transmit output to target cells within and outside SCN, cluster III cells in particular are suggested to affect excitability of large numbers of SCN neurons by their extensive local network of axon collaterals.

Functional characterization of the H-current in SCN neurons in subjective day and night: a whole-cell patch-clamp study in acutely prepared brain slices.

Neurons of the rat suprachiasmatic nucleus (SCN) exhibit a circadian rhythm in spontaneous firing rate. In this whole-cell patch-clamp study in slices, we examined the possibility that H-current (IH) contributes to the spontaneous firing rate of SCN neurons. Most of our experiments were performed during the subjective day, because this is the time epoch during which one would expect the largest excitatory effect of IH if it were to fluctuate in a circadian rhythm. Current-clamp experiments showed that blockade of IH by Cs+ (1 mM) did not influence the spontaneous firing rate and resting membrane potential. Voltage-clamp experiments revealed that IH, when activated at the resting membrane potential, is probably too small in magnitude and too slow in activation to make a significant contribution to the spontaneous firing rate. Both results suggest that IH does not significantly contribute to the spontaneous firing of SCN neurons. In addition, we investigated whether the kinetics and voltage dependence of IH were modulated in a circadian manner. However, no substantial day-night differences in IH were found. We conclude that IH, as recorded in whole-cell mode, does not contribute significantly to spontaneous firing in most SCN neurons and that this current, is more likely to be involved in 'rescuing' SCN neurons from large and long-lasting hyperpolarizations by depolarizing the membrane.