A role for the cerebellum in motor-triggered alleviation of anxiety.

Physical exercise is known to reduce anxiety, but the underlying brain mechanisms remain unclear. Here, we explore a hypothalamo-cerebello-amygdalar circuit that may mediate motor-dependent alleviation of anxiety. This three-neuron loop, in which the cerebellar dentate nucleus takes center stage, bridges the motor system with the emotional system. Subjecting animals to a constant rotarod engages glutamatergic cerebellar dentate neurons that drive PKCδ+ amygdalar neurons to elicit an anxiolytic effect. Moreover, challenging animals on an accelerated rather than a constant rotarod engages hypothalamic neurons that provide a superimposed anxiolytic effect via an orexinergic projection to the dentate neurons that activate the amygdala. Our findings reveal a cerebello-limbic pathway that may contribute to motor-triggered alleviation of anxiety and that may be optimally exploited during challenging physical exercise.

cATR Tracing Approach to Identify Individual Intermediary Neurons Based on Their Input and Output: A Proof-of-Concept Study Connecting Cerebellum and Central Hubs Implicated in Developmental Disorders.

Over the past decades, it has become increasingly clear that many neurodevelopmental disorders can be characterized by aberrations in the neuro-anatomical connectome of intermediary hubs. Yet, despite the advent in unidirectional transsynaptic tracing technologies, we are still lacking an efficient approach to identify individual neurons based on both their precise input and output relations, hampering our ability to elucidate the precise connectome in both the healthy and diseased condition. Here, we bridge this gap by combining anterograde transsynaptic- and retrograde (cATR) tracing in Ai14 reporter mice, using adeno-associated virus serotype 1 expressing Cre and cholera toxin subunit B as the anterograde and retrograde tracer, respectively. We have applied this innovative approach to selectively identify individual neurons in the brainstem that do not only receive input from one or more of the cerebellar nuclei (CN), but also project to the primary motor cortex (M1), the amygdala or the ventral tegmental area (VTA). Cells directly connecting CN to M1 were found mainly in the thalamus, while a large diversity of midbrain and brainstem areas connected the CN to the amygdala or VTA. Our data highlight that cATR allows for specific, yet brain-wide, identification of individual neurons that mediate information from a cerebellar nucleus to the cerebral cortex, amygdala or VTA via a disynaptic pathway. Given that the identified neurons in healthy subjects can be readily quantified, our data also form a solid foundation to make numerical comparisons with mouse mutants suffering from aberrations in their connectome due to a neurodevelopmental disorder.

Single-pulse stimulation of cerebellar nuclei stops epileptic thalamic activity.

BACKGROUND: Epileptic (absence) seizures in the cerebral cortex can be stopped by pharmacological and optogenetic stimulation of the cerebellar nuclei (CN) neurons that innervate the thalamus. However, it is unclear how such stimulation can modify underlying thalamo-cortical oscillations. HYPOTHESIS: Here we tested whether rhythmic synchronized thalamo-cortical activity during absence seizures can be desynchronized by single-pulse optogenetic stimulation of CN neurons to stop seizure activity. METHODS: We performed simultaneous thalamic single-cell and electrocorticographical recordings in awake tottering mice, a genetic model of absence epilepsy, to investigate the rhythmicity and synchronicity. Furthermore, we tested interictally the impact of single-pulse optogenetic CN stimulation on thalamic and cortical recordings. RESULTS: We show that thalamic firing is highly rhythmic and synchronized with cortical spike-and-wave discharges during absence seizures and that this phase-locked activity can be desynchronized upon single-pulse optogenetic stimulation of CN neurons. Notably, this stimulation of CN neurons was more effective in stopping seizures than direct, focal stimulation of groups of afferents innervating the thalamus. During interictal periods, CN stimulation evoked reliable but heterogeneous responses in thalamic cells in that they could show an increase or decrease in firing rate at various latencies, bi-phasic responses with an initial excitatory and subsequent inhibitory response, or no response at all. CONCLUSION: Our data indicate that stimulation of CN neurons and their fibers in thalamus evokes differential effects in its downstream pathways and desynchronizes phase-locked thalamic neuronal firing during seizures, revealing a neurobiological mechanism that may explain how cerebellar stimulation can stop seizures.

Temporal dynamics of the cerebello-cortical convergence in ventro-lateral motor thalamus.

KEY POINTS: Ventrolateral thalamus (VL) integrates information from cerebellar nuclei and motor cortical layer VI. Inputs from the cerebellar nuclei evoke large-amplitude responses that depress upon repetitive stimulation while layer VI inputs from motor cortex induce small-amplitude facilitating responses. We report that the spiking of VL neurons can be determined by the thalamic membrane potential, the frequency of cerebellar inputs and the duration of pauses after cerebellar high frequency stimulation. Inputs from motor cortical layer VI shift the VL membrane potential and modulate the VL spike output in response to cerebellar stimulation.  These results help us to decipher how the cerebellar output is integrated in VL and modulated by motor cortical input. ABSTRACT: Orchestrating complex movements requires well-timed interaction of cerebellar, thalamic and cerebral structures, but the mechanisms underlying the integration of cerebro-cerebellar information in motor thalamus remain largely unknown. Here we investigated how excitatory inputs from cerebellar nuclei (CN) and primary motor cortex layer VI (M1-L6) neurons may regulate the activity of neurons in the mouse ventrolateral (VL) thalamus. Using dual-optical stimulation of the CN and M1-L6 axons and in vitro whole-cell recordings of the responses in VL neurons, we studied the individual responses as well as the effects of combined CN and M1-L6 stimulation. Whereas CN inputs evoked large-amplitude responses that were depressed upon repetitive stimulation, M1-L6 inputs elicited small-amplitude responses that were facilitated upon repetitive stimulation. Moreover, pauses in CN stimuli could directly affect VL spiking probability, an effect that was modulated by VL membrane potential. When CN and M1-L6 pathways were co-activated, motor cortical afferents increased the thalamic spike output in response to cerebellar stimulation, indicating that CN and M1 synergistically, yet differentially, control the membrane potential and spiking pattern of VL neurons.

A cortico-cerebellar loop for motor planning.

Persistent and ramping neural activity in the frontal cortex anticipates specific movements1-6. Preparatory activity is distributed across several brain regions7,8, but it is unclear which brain areas are involved and how this activity is mediated by multi-regional interactions. The cerebellum is thought to be primarily involved in the short-timescale control of movement9-12; however, roles for this structure in cognitive processes have also been proposed13-16. In humans, cerebellar damage can cause defects in planning and working memory13. Here we show that persistent representation of information in the frontal cortex during motor planning is dependent on the cerebellum. Mice performed a sensory discrimination task in which they used short-term memory to plan a future directional movement. A transient perturbation in the medial deep cerebellar nucleus (fastigial nucleus) disrupted subsequent correct responses without hampering movement execution. Preparatory activity was observed in both the frontal cortex and the cerebellar nuclei, seconds before the onset of movement. The silencing of frontal cortex activity abolished preparatory activity in the cerebellar nuclei, and fastigial activity was necessary to maintain cortical preparatory activity. Fastigial output selectively targeted the behaviourally relevant part of the frontal cortex through the thalamus, thus closing a cortico-cerebellar loop. Our results support the view that persistent neural dynamics during motor planning is maintained by neural circuits that span multiple brain regions17, and that cerebellar computations extend beyond online motor control13-15,18.

Cerebellar output controls generalized spike-and-wave discharge occurrence.

OBJECTIVE: Disrupting thalamocortical activity patterns has proven to be a promising approach to stop generalized spike-and-wave discharges (GSWDs) characteristic of absence seizures. Here, we investigated to what extent modulation of neuronal firing in cerebellar nuclei (CN), which are anatomically in an advantageous position to disrupt cortical oscillations through their innervation of a wide variety of thalamic nuclei, is effective in controlling absence seizures. METHODS: Two unrelated mouse models of generalized absence seizures were used: the natural mutant tottering, which is characterized by a missense mutation in Cacna1a, and inbred C3H/HeOuJ. While simultaneously recording single CN neuron activity and electrocorticogram in awake animals, we investigated to what extent pharmacologically increased or decreased CN neuron activity could modulate GSWD occurrence as well as short-lasting, on-demand CN stimulation could disrupt epileptic seizures. RESULTS: We found that a subset of CN neurons show phase-locked oscillatory firing during GSWDs and that manipulating this activity modulates GSWD occurrence. Inhibiting CN neuron action potential firing by local application of the γ-aminobutyric acid type A (GABA-A) agonist muscimol increased GSWD occurrence up to 37-fold, whereas increasing the frequency and regularity of CN neuron firing with the use of GABA-A antagonist gabazine decimated its occurrence. A single short-lasting (30-300 milliseconds) optogenetic stimulation of CN neuron activity abruptly stopped GSWDs, even when applied unilaterally. Using a closed-loop system, GSWDs were detected and stopped within 500 milliseconds. INTERPRETATION: CN neurons are potent modulators of pathological oscillations in thalamocortical network activity during absence seizures, and their potential therapeutic benefit for controlling other types of generalized epilepsies should be evaluated.

Impact of conventional anesthesia on auditory brainstem responses in mice.

Anesthesia is known to affect the auditory brainstem response (ABR) in animals often used in hearing research. This study describes the differences in ABRs between awake and anesthetized FVB/N mice. Intracranial electrodes connected to a head fixation pedestal were used for click-evoked ABR recordings. This pedestal served to immobilize mice, either awake or under anesthesia, in a 'free' sound field. The presence of myogenic noise in the awake condition obviously increases recording time. However it is demonstrated that recording times can be significantly reduced by increasing the stimulus repetition rate from 23 up to 80 impulses per second. This causes only a small but significant increase in absolute peak latencies in the awake condition, but has no significant effect on the overall ABR-waveform, nor on the ABR-threshold, nor on the ABR interpeak latencies, nor on the absolute peak latencies in the anesthetized condition. Anesthesia with ketamine/xylazine caused a significant prolongation of ABR-peak latencies and interpeak latencies as well as a significant upward shift (8.0+/-1.8 dB) of ABR-thresholds as compared to the awake condition. Under anesthesia the measurement accuracy of peak latencies, interpeak latencies and thresholds decreases. In conclusion, the awake condition is preferable for more accurate measurements of ABR characteristics, in spite of the myogenic noise concomitant with this condition.