Modulation of Murine Olivary Connexin 36 Gap Junctions by PKA and CaMKII.

The inferior olive (IO) is a nucleus located in the brainstem and it is part of the olivo-cerebellar loop. This circuit plays a fundamental role in generation and acquisition of coherent motor patterns and it relies on synchronous activation of groups of Purkinje cells (PC) in the cerebellar cortex. IO neurons integrate their intrinsic oscillatory activity with excitatory inputs coming from the somatosensory system and inhibitory feedback coming from the cerebellar nuclei. Alongside these chemical synaptic inputs, IO neurons are coupled to one another via connexin 36 (Cx36) containing gap junctions (GJs) that create a functional syncytium between neurons. Communication between olivary neurons is regulated by these GJs and their correct functioning contributes to coherent oscillations in the IO and proper motor learning. Here, we explore the cellular pathways that can regulate the coupling between olivary neurons. We combined in vitro electrophysiology and immunohistochemistry (IHC) on mouse acute brain slices to unravel the pathways that regulate olivary coupling. We found that enhancing the activity of the protein kinase A (PKA) pathway and blocking the Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathway can both down-regulate the size of the coupled network. However, these two kinases follow different mechanisms of action. Our results suggest that activation of the PKA pathway reduces the opening probability of the Cx36 GJs, whereas inhibition of the CaMKII pathway reduces the number of Cx36 GJs. The low densities of Cx36 proteins and electrical synapses in ßCaMKII knock-out mice point towards an essential role for this protein kinase in regulating the density of GJs in the IO. Thus, the level of olivary coupling is a dynamic process and regulated by a variety of enzymes modulating GJs expression, docking and activity.

Heterogeneous Expression of T-type Ca(2+) Channels Defines Different Neuronal Populations in the Inferior Olive of the Mouse.

The neurons in the inferior olive express subthreshold oscillations in their membrane potential. This oscillatory activity is known to drive synchronous activity in the cerebellar cortex and plays a role in motor learning and motor timing. In the past years, it was commonly thought that olivary neurons belonged to a unique population of oscillating units and that oscillation properties were exclusively dependent on network settings and/or synaptic inputs. The origin of olivary oscillations is now known to be a local phenomenon and is generated by a combination of conductances. In the present work, we show the existence of at least two neuronal populations that can be distinguished on the basis of the presence or absence of low-voltage activated Ca(2+) channels. The expression of this channel determines the oscillatory behavior of olivary neurons. Furthermore, the number of cells that express this channel is different between sub nuclei of the inferior olive. These findings clearly indicate the functional variability within and between olivary sub nuclei.

Climbing fiber burst size and olivary sub-threshold oscillations in a network setting.

The inferior olivary nucleus provides one of the two main inputs to the cerebellum: the so-called climbing fibers. Activation of climbing fibers is generally believed to be related to timing of motor commands and/or motor learning. Climbing fiber spikes lead to large all-or-none action potentials in cerebellar Purkinje cells, overriding any other ongoing activity and silencing these cells for a brief period of time afterwards. Empirical evidence shows that the climbing fiber can transmit a short burst of spikes as a result of an olivary cell somatic spike, potentially increasing the information being transferred to the cerebellum per climbing fiber activation. Previously reported results from in vitro studies suggested that the information encoded in the climbing fiber burst is related to the occurrence of the spike relative to the ongoing sub-threshold membrane potential oscillation of the olivary cell, i.e. that the phase of the oscillation is reflected in the size of the climbing fiber burst. We used a detailed three-compartmental model of an inferior olivary cell to further investigate the possible factors determining the size of the climbing fiber burst. Our findings suggest that the phase-dependency of the burst size is present but limited and that charge flow between soma and dendrite is a major determinant of the climbing fiber burst. From our findings it follows that phenomena such as cell ensemble synchrony can have a big effect on the climbing fiber burst size through dendrodendritic gap-junctional coupling between olivary cells.

Olivary subthreshold oscillations and burst activity revisited.

The inferior olive (IO) forms one of the major gateways for information that travels to the cerebellar cortex. Olivary neurons process sensory and motor signals that are subsequently relayed to Purkinje cells. The intrinsic subthreshold membrane potential oscillations of the olivary neurons are thought to be important for gating this flow of information. In vitro studies have revealed that the phase of the subthreshold oscillation determines the size of the olivary burst and may gate the information flow or encode the temporal state of the olivary network. Here, we investigated whether the same phenomenon occurred in murine olivary cells in an intact olivocerebellar system using the in vivo whole-cell recording technique. Our in vivo findings revealed that the number of wavelets within the olivary burst did not encode the timing of the spike relative to the phase of the oscillation but was related to the amplitude of the oscillation. Manipulating the oscillation amplitude by applying Harmaline confirmed the inverse relationship between the amplitude of oscillation and the number of wavelets within the olivary burst. Furthermore, we demonstrated that electrotonic coupling between olivary neurons affect this modulation of the olivary burst size. Based on these results, we suggest that the olivary burst size might reflect the "expectancy" of a spike to occur rather than the spike timing, and that this process requires the presence of gap junction coupling.

Anti-malaria drug mefloquine induces motor learning deficits in humans.

Mefloquine (a marketed anti-malaria drug) prophylaxis has a high risk of causing adverse events. Interestingly, animal studies have shown that mefloquine imposes a major deficit in motor learning skills by affecting the connexin 36 gap junctions of the inferior olive. We were therefore interested in assessing whether mefloquine might induce similar effects in humans. The main aim of this study was to investigate the effect of mefloquine on olivary-related motor performance and motor learning tasks in humans. We subjected nine participants to voluntary motor timing (dart throwing task), perceptual timing (rhythm perceptual task) and reflex timing tasks (eye-blink task) before and 24 h after the intake of mefloquine. The influence of mefloquine on motor learning was assessed by subjecting participants with and without mefloquine intake (controls: n = 11 vs mefloquine: n = 8) to an eye-blink conditioning task. Voluntary motor performance, perceptual timing, and reflex blinking were not affected by mefloquine use. However, the influence of mefloquine on motor learning was substantial; both learning speed as well as learning capacity was impaired by mefloquine use. Our data suggest that mefloquine disturbs motor learning skills. This adverse effect can have clinical as well as social clinical implications for mefloquine users. Therefore, this side-effect of mefloquine should be further investigated and recognized by clinicians.

Intrinsic plasticity complements long-term potentiation in parallel fiber input gain control in cerebellar Purkinje cells.

Synaptic gain control and information storage in neural networks are mediated by alterations in synaptic transmission, such as in long-term potentiation (LTP). Here, we show using both in vitro and in vivo recordings from the rat cerebellum that tetanization protocols for the induction of LTP at parallel fiber (PF)-to-Purkinje cell synapses can also evoke increases in intrinsic excitability. This form of intrinsic plasticity shares with LTP a requirement for the activation of protein phosphatases 1, 2A, and 2B for induction. Purkinje cell intrinsic plasticity resembles CA1 hippocampal pyramidal cell intrinsic plasticity in that it requires activity of protein kinase A (PKA) and casein kinase 2 (CK2) and is mediated by a downregulation of SK-type calcium-sensitive K conductances. In addition, Purkinje cell intrinsic plasticity similarly results in enhanced spine calcium signaling. However, there are fundamental differences: first, while in the hippocampus increases in excitability result in a higher probability for LTP induction, intrinsic plasticity in Purkinje cells lowers the probability for subsequent LTP induction. Second, intrinsic plasticity raises the spontaneous spike frequency of Purkinje cells. The latter effect does not impair tonic spike firing in the target neurons of inhibitory Purkinje cell projections in the deep cerebellar nuclei, but lowers the Purkinje cell signal-to-noise ratio, thus reducing the PF readout. These observations suggest that intrinsic plasticity accompanies LTP of active PF synapses, while it reduces at weaker, nonpotentiated synapses the probability for subsequent potentiation and lowers the impact on the Purkinje cell output.

Role of olivary electrical coupling in cerebellar motor learning.

The level of electrotonic coupling in the inferior olive is extremely high, but its functional role in cerebellar motor control remains elusive. Here, we subjected mice that lack olivary coupling to paradigms that require learning-dependent timing. Cx36-deficient mice showed impaired timing of both locomotion and eye-blink responses that were conditioned to a tone. The latencies of their olivary spike activities in response to the unconditioned stimulus were significantly more variable than those in wild-types. Whole-cell recordings of olivary neurons in vivo showed that these differences in spike timing result at least in part from altered interactions with their subthreshold oscillations. These results, combined with analyses of olivary activities in computer simulations at both the cellular and systems level, suggest that electrotonic coupling among olivary neurons by gap junctions is essential for proper timing of their action potentials and thereby for learning-dependent timing in cerebellar motor control.

Estradiol improves cerebellar memory formation by activating estrogen receptor beta.

Learning motor skills is critical for motor abilities such as driving a car or playing piano. The speed at which we learn those skills is subject to many factors. Yet, it is not known to what extent gonadal hormones can affect the achievement of accurate movements in time and space. Here we demonstrate via different lines of evidence that estradiol promotes plasticity in the cerebellar cortex underlying motor learning. First, we show that estradiol enhances induction of long-term potentiation at the parallel fiber to Purkinje cell synapse, whereas it does not affect long-term depression; second, we show that estradiol activation of estrogen receptor beta receptors in Purkinje cells significantly improves gain-decrease adaptation of the vestibulo-ocular reflex, whereas it does not affect general eye movement performance; and third, we show that estradiol increases the density of parallel fiber to Purkinje cell synapses, whereas it does not affect the density of climbing fiber synapses. We conclude that estradiol can improve motor skills by potentiating cerebellar plasticity and synapse formation. These processes may be advantageous during periods of high estradiol levels of the estrous cycle or pregnancy.

Zonal organization of the mouse flocculus: physiology, input, and output.

The zones of the flocculus have been mapped in many species with a noticeable exception, the mouse. Here, the functional map of the mouse was constructed via extracellular recordings followed by tracer injections of biotinylated-dextran-amine and immunohistochemistry for heat-shock protein-25. Zones were identified based on the Purkinje cell complex spike modulation occurring in response to optokinetic stimulation. In zones 1 and 3 Purkinje cells responded best to rotation about a horizontal axis oriented at 135 degrees ipsilateral azimuth, whereas in zones 2 and 4 they responded best to rotation about the vertical axis. The tracing experiments showed that Purkinje cells of zone 1 projected to the parvicellular part of lateral cerebellar nucleus and superior vestibular nucleus, while Purkinje cells of zone 3 projected to group Y and the superior vestibular nucleus. Purkinje cells of zones 2 and 4 projected to the magnocellular and parvicellular parts of the medial vestibular nucleus, while some also innervated the lateral vestibular nucleus or nucleus prepositus hypoglossi. The climbing fiber inputs to Purkinje cells in zones 1 and 3 were derived from neurons in the ventrolateral outgrowth of the contralateral inferior olive, whereas those in zones 2 and 4 were derived from the contralateral caudal dorsal cap. Purkinje cells in zones 1 and 2, but not in zones 3 and 4, were positively labeled for heat-shock protein-25. The present study illustrates that Purkinje cells in the murine flocculus are organized in discrete zones with specific functions, specific input - output relations, and a specific histochemical signature.

Otolith deprivation induces optokinetic compensation.

According to the multisensory integration theory vestibular, optokinetic and proprioceptive inputs act in concert to maintain a stable retinal image of the visual world. Yet, it remains elusive to what extent the otolith organs contribute to this process and whether a specific loss of otolith input is compensated for. Here we investigated the compensatory eye movements in tilted mice, which lack otoconia because of a mutation in otopetrin 1. Tilted mice showed very small displacements of the eyes in the orbit during static roll paradigms, suggesting the absence of functional otolith organs. Independent of head position with respect to gravity, the gain and phase lead of angular vestibuloocular reflex of tilted mice were decreased and increased, respectively (frequencies 0.2 to 1 Hz and peak accelerations 8 to 197 degrees /s2, respectively). Furthermore, lack of otolith input increases the dependency of the vestibular system on stimulus frequency. In contrast, the gain of optokinetic reflex in tilted mice was significantly higher in the low-frequency range than in control mice, regardless of the position of the mice in space or the plane of the eye movements. To explain these results, a simple model was used in which a multisensory integration unit was embedded. With this model, we were able to simulate all the behaviors observed. Thus our data and the model support the presence of the multisensory integration system and revealed a compensatory enhanced optokinetic reflex in tilted mice, indicating an adaptive synergism in the processing of otolith and visually driven signals.

Diurnal modulation of pacemaker potentials and calcium current in the mammalian circadian clock.

The central biological clock of the mammalian brain is located in the suprachiasmatic nucleus. This hypothalamic region contains neurons that generate a circadian rhythm on a single-cell basis. Clock cells transmit their circadian timing signals to other brain areas by diurnal modulation of their spontaneous firing rate. The intracellular mechanism underlying rhythm generation is thought to consist of one or more self-regulating molecular loops, but it is unknown how these loops interact with the plasma membrane to modulate the ionic conductances that regulate firing behaviour. Here we demonstrate a diurnal modulation of Ca2+ current in suprachiasmatic neurons. This current strongly contributes to the generation of spontaneous oscillations in membrane potential, which occur selectively during daytime and are tightly coupled to spike generation. Thus, day-night modulation of Ca2+ current is a central step in transducing the intracellular cycling of molecular clocks to the rhythm in spontaneous firing rate.