Disynaptic Inhibitory Cerebellar Control Over Caudal Medial Accessory Olive.

The olivocerebellar system, which is critical for sensorimotor performance and learning, functions through modules with feedback loops. The main feedback to the inferior olive comes from the cerebellar nuclei (CN), which are predominantly GABAergic and contralateral. However, for the subnucleus d of the caudomedial accessory olive (cdMAO), a crucial region for oculomotor and upper body movements, the source of GABAergic input has yet to be identified. Here, we demonstrate the existence of a disynaptic inhibitory projection from the medial CN (MCN) to the cdMAO via the superior colliculus (SC) by exploiting retrograde, anterograde, and transsynaptic viral tracing at the light microscopic level as well as anterograde classical and viral tracing combined with immunocytochemistry at the electron microscopic level. Retrograde tracing in Gad2-Cre mice reveals that the cdMAO receives GABAergic input from the contralateral SC. Anterograde transsynaptic tracing uncovered that the SC neurons receiving input from the contralateral MCN provide predominantly inhibitory projections to contralateral cdMAO, ipsilateral to the MCN. Following ultrastructural analysis of the monosynaptic projection about half of the SC terminals within the contralateral cdMAO are GABAergic. The disynaptic GABAergic projection from the MCN to the ipsilateral cdMAO mirrors that of the monosynaptic excitatory projection from the MCN to the contralateral cdMAO. Thus, while completing the map of inhibitory inputs to the olivary subnuclei, we established that the MCN inhibits the cdMAO via the contralateral SC, highlighting a potential push-pull mechanism in directional gaze control that appears unique in terms of laterality and polarity among olivocerebellar modules.

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.

Controlling absence seizures from the cerebellar nuclei via activation of the Gq signaling pathway.

Absence seizures (ASs) are characterized by pathological electrographic oscillations in the cerebral cortex and thalamus, which are called spike-and-wave discharges (SWDs). Subcortical structures, such as the cerebellum, may well contribute to the emergence of ASs, but the cellular and molecular underpinnings remain poorly understood. Here we show that the genetic ablation of P/Q-type calcium channels in cerebellar granule cells (quirky) or Purkinje cells (purky) leads to recurrent SWDs with the purky model showing the more severe phenotype. The quirky mouse model showed irregular action potential firing of their cerebellar nuclei (CN) neurons as well as rhythmic firing during the wave of their SWDs. The purky model also showed irregular CN firing, in addition to a reduced firing rate and rhythmicity during the spike of the SWDs. In both models, the incidence of SWDs could be decreased by increasing CN activity via activation of the Gq-coupled designer receptor exclusively activated by designer drugs (DREADDs) or via that of the Gq-coupled metabotropic glutamate receptor 1. In contrast, the incidence of SWDs was increased by decreasing CN activity via activation of the inhibitory Gi/o-coupled DREADD. Finally, disrupting CN rhythmic firing with a closed-loop channelrhodopsin-2 stimulation protocol confirmed that ongoing SWDs can be ceased by activating CN neurons. Together, our data highlight that P/Q-type calcium channels in cerebellar granule cells and Purkinje cells can be relevant for epileptogenesis, that Gq-coupled activation of CN neurons can exert anti-epileptic effects and that precisely timed activation of the CN can be used to stop ongoing SWDs.

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.

Cerebellar Purkinje cells can differentially modulate coherence between sensory and motor cortex depending on region and behavior.

Activity of sensory and motor cortices is essential for sensorimotor integration. In particular, coherence between these areas may indicate binding of critical functions like perception, motor planning, action, or sleep. Evidence is accumulating that cerebellar output modulates cortical activity and coherence, but how, when, and where it does so is unclear. We studied activity in and coherence between S1 and M1 cortices during whisker stimulation in the absence and presence of optogenetic Purkinje cell stimulation in crus 1 and 2 of awake mice, eliciting strong simple spike rate modulation. Without Purkinje cell stimulation, whisker stimulation triggers fast responses in S1 and M1 involving transient coherence in a broad spectrum. Simultaneous stimulation of Purkinje cells and whiskers affects amplitude and kinetics of sensory responses in S1 and M1 and alters the estimated S1-M1 coherence in theta and gamma bands, allowing bidirectional control dependent on behavioral context. These effects are absent when Purkinje cell activation is delayed by 20 ms. Focal stimulation of Purkinje cells revealed site specificity, with cells in medial crus 2 showing the most prominent and selective impact on estimated coherence, i.e., a strong suppression in the gamma but not the theta band. Granger causality analyses and computational modeling of the involved networks suggest that Purkinje cells control S1-M1 phase consistency predominantly via ventrolateral thalamus and M1. Our results indicate that activity of sensorimotor cortices can be dynamically and functionally modulated by specific cerebellar inputs, highlighting a widespread role of the cerebellum in coordinating sensorimotor behavior.

Increased susceptibility to cortical spreading depression and epileptiform activity in a mouse model for FHM2.

Migraine is a highly prevalent, debilitating, episodic headache disorder affecting roughly 15% of the population. Familial hemiplegic migraine type 2 (FHM2) is a rare subtype of migraine caused by mutations in the ATP1A2 gene, encoding the α2 isoform of the Na+/K+-ATPase, predominantly expressed in astrocytes. Differential comorbidities such as epilepsy and psychiatric disorders manifest in patients. Using a mouse model harboring the G301R disease-mutation in the α2 isoform, we set to unravel whether α2+/G301R mice show an increased susceptibility for epilepsy and cortical spreading depression (CSD). We performed in vivo experiments involving cortical application of KCl in awake head-restrained male and female mice of different age groups (adult and aged). Interestingly, α2+/G301R mice indeed showed an increased susceptibility to both CSD and epileptiform activity, closely replicating symptoms in FHM2 patients harboring the G301R and other FHM2-causing mutations. Additionally, this epileptiform activity was superimposed on CSDs. The age-related alteration towards CSD indicates the influence of female sex hormones on migraine pathophysiology. Therefore, the FHM2, α2+/G301R mouse model can be utilized to broaden our understanding of generalized epilepsy and comorbidity hereof in migraine, and may be utilized toward future selection of possible treatment options for migraine.

Cerebellar involvement in migraine.

Background Although there is a great wealth of knowledge about the neurobiological processes underlying migraine and its accompanying symptoms, the mechanisms by which an attack starts remain elusive, and the disease remains undertreated. Although the vast majority of literature focuses on the involvement of the trigeminovascular systems and higher systems it innervates, such as thalamic and hypothalamic nuclei, several lines of evidence implicate the cerebellum in the pathophysiology of migraine. Aim In this review, we aim to summarize potential cerebellar involvement seen from different perspectives including the results from imaging studies, cerebellar connectivity to migraine-related brain structures, comorbidity with disorders implying cerebellar dysfunction, similarities in triggers precipitating both such disorders, and migraine and cerebellar expression of migraine-related genes and neuropeptides. We aim to inspire an increase in interest for future research on the subject. Conclusion It is hoped that future studies can provide an answer as to how the cerebellum may be involved and whether treatment options specifically targeting the cerebellum could provide alleviation of this disorder.

PRRT2-dependent dyskinesia: cerebellar, paroxysmal and persistent.

In an elegant publication in Cell Research, Tan and colleagues showed that ablation of PRRT2 in cerebellar granule cells is sufficient to induce paroxysmal kinesigenic dyskinesia. PRRT2 turns out to downregulate the presynaptic SNARE complex in granule cell axons, which in turn controls the activity patterns of Purkinje cells, the sole output of the cerebellar cortex.

Corrigendum: Synchronicity and Rhythmicity of Purkinje Cell Firing during Generalized Spike-and-Wave Discharges in a Natural Mouse Model of Absence Epilepsy.

[This corrects the article DOI: 10.3389/fncel.2017.00346.].

Synchronicity and Rhythmicity of Purkinje Cell Firing during Generalized Spike-and-Wave Discharges in a Natural Mouse Model of Absence Epilepsy.

Absence epilepsy is characterized by the occurrence of generalized spike and wave discharges (GSWDs) in electrocorticographical (ECoG) recordings representing oscillatory activity in thalamocortical networks. The oscillatory nature of GSWDs has been shown to be reflected in the simple spike activity of cerebellar Purkinje cells and in the activity of their target neurons in the cerebellar nuclei, but it is unclear to what extent complex spike activity is implicated in generalized epilepsy. Purkinje cell complex spike firing is elicited by climbing fiber activation and reflects action potential firing in the inferior olive. Here, we investigated to what extent modulation of complex spike firing is reflected in the temporal patterns of seizures. Extracellular single-unit recordings in awake, head-restrained homozygous tottering mice, which suffer from a mutation in the voltage-gated CaV2.1 calcium channel, revealed that a substantial proportion of Purkinje cells (26%) showed increased complex spike activity and rhythmicity during GSWDs. Moreover, Purkinje cells, recorded either electrophysiologically or by using Ca2+-imaging, showed a significant increase in complex spike synchronicity for both adjacent and remote Purkinje cells during ictal events. These seizure-related changes in firing frequency, rhythmicity and synchronicity were most prominent in the lateral cerebellum, a region known to receive cerebral input via the inferior olive. These data indicate profound and widespread changes in olivary firing that are most likely induced by seizure-related activity changes in the thalamocortical network, thereby highlighting the possibility that olivary neurons can compensate for pathological brain-state changes by dampening oscillations.

Hypothermia-induced dystonia and abnormal cerebellar activity in a mouse model with a single disease-mutation in the sodium-potassium pump.

Mutations in the neuron-specific α3 isoform of the Na+/K+-ATPase are found in patients suffering from Rapid onset Dystonia Parkinsonism and Alternating Hemiplegia of Childhood, two closely related movement disorders. We show that mice harboring a heterozygous hot spot disease mutation, D801Y (α3+/D801Y), suffer abrupt hypothermia-induced dystonia identified by electromyographic recordings. Single-neuron in vivo recordings in awake α3+/D801Y mice revealed irregular firing of Purkinje cells and their synaptic targets, the deep cerebellar nuclei neurons, which was further exacerbated during dystonia and evolved into abnormal high-frequency burst-like firing. Biophysically, we show that the D-to-Y mutation abolished pump-mediated Na+/K+ exchange, but allowed the pumps to bind Na+ and become phosphorylated. These findings implicate aberrant cerebellar activity in α3 isoform-related dystonia and add to the functional understanding of the scarce and severe mutations in the α3 isoform Na+/K+-ATPase.

Controlling Cerebellar Output to Treat Refractory Epilepsy.

Generalized epilepsy is characterized by recurrent seizures caused by oscillatory neuronal firing throughout thalamocortical networks. Current therapeutic approaches often intervene at the level of the thalamus or cerebral cortex to ameliorate seizures. We review here the therapeutic potential of cerebellar stimulation. The cerebellum forms a prominent ascending input to the thalamus and, whereas stimulation of the foliated cerebellar cortex exerts inconsistent results, stimulation of the centrally located cerebellar nuclei (CN) reliably stops generalized seizures in experimental models. Stimulation of this area indicates that the period of stimulation with respect to the phase of the oscillations in thalamocortical networks can optimize its effect, opening up the possibility of developing on-demand deep brain stimulation (DBS) treatments.

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.

Expert versus proxy rating of verbal communicative ability of people with aphasia after stroke.

In randomized clinical trials of aphasia treatment, a functional outcome measure like the Amsterdam-Nijmegen Everyday Language Test (ANELT), administered by speech-language therapists, is often used. However, the agreement between this expert rating and the judgment of the proxy about the quality of the daily life communication of the person with aphasia is largely unknown. We examined the association between ANELT scores by speech-language therapists and proxy judgments on the Partner Communication Questionnaire both at 3 and 6 months in 39 people with aphasia after stroke. We also determined which factors affected the level of agreement between expert and proxy judgment of the communicative ability at 6 months in 53 people with aphasia. We found moderate agreement (at 3 months r = .662; p = < .0001 and at 6 months r = .565; p = .0001), with proxies rating slightly higher than experts. Less severe aphasia, measured with the Aphasia Severity Rating Scale, was associated with better agreement. In conclusion, although proxies were slightly more positive than experts, we found moderate agreement between expert and proxy rating of verbal communicative ability of people with aphasia after stroke, especially in milder cases.

Purkinje cell-specific ablation of Cav2.1 channels is sufficient to cause cerebellar ataxia in mice.

The Cacna1a gene encodes the α(1A) subunit of voltage-gated Ca(V)2.1 Ca(2+) channels that are involved in neurotransmission at central synapses. Ca(V)2.1-α(1)-knockout (α1KO) mice, which lack Ca(V)2.1 channels in all neurons, have a very severe phenotype of cerebellar ataxia and dystonia, and usually die around postnatal day 20. This early lethality, combined with the wide expression of Ca(V)2.1 channels throughout the cerebellar cortex and nuclei, prohibited determination of the contribution of particular cerebellar cell types to the development of the severe neurobiological phenotype in Cacna1a mutant mice. Here, we crossed conditional Cacna1a mice with transgenic mice expressing Cre recombinase, driven by the Purkinje cell-specific Pcp2 promoter, to specifically ablate the Ca(V)2.1-α(1A) subunit and thereby Ca(V)2.1 channels in Purkinje cells. Purkinje cell Ca(V)2.1-α(1A)-knockout (PCα1KO) mice aged without difficulties, rescuing the lethal phenotype seen in α1KO mice. PCα1KO mice exhibited cerebellar ataxia starting around P12, much earlier than the first signs of progressive Purkinje cell loss, which appears in these mice between P30 and P45. Secondary cell loss was observed in the granular and molecular layers of the cerebellum and the volume of all individual cerebellar nuclei was reduced. In this mouse model with a cell type-specific ablation of Ca(V)2.1 channels, we show that ablation of Ca(V)2.1 channels restricted to Purkinje cells is sufficient to cause cerebellar ataxia. We demonstrate that spatial ablation of Ca(V)2.1 channels may help in unraveling mechanisms of human disease.