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The nuclei are the main output structures of the cerebellum. Each and every cerebellar cortical computation reaches several areas of the brain by means of cerebellar nuclei processing and integration. Nevertheless, our knowledge of these structures is still limited compared to the cerebellar cortex. Here, we present a mouse genetic inducible fate-mapping study characterizing rhombic lip-derived glutamatergic neurons of the nuclei, the most conspicuous family of long-range cerebellar efferent neurons. Glutamatergic neurons mainly occupy dorsal and lateral territories of the lateral and interposed nuclei, as well as the entire medial nucleus. In mice, they are born starting from about embryonic day 9.5, with a peak between 10.5 and 12.5, and invade the nuclei with a lateral-to-medial progression. While some markers label a heterogeneous population of neurons sharing a common location (BRN2), others appear to be lineage specific (TBR1, LMX1a, and MEIS2). A comparative analysis of TBR1 and LMX1a distributions reveals an incomplete overlap in their expression domains, in keeping with the existence of separate efferent subpopulations. Finally, some tagged glutamatergic progenitors are not labeled by any of the markers used in this study, disclosing further complexity. Taken together, our results obtained in late embryonic nuclei shed light on the heterogeneity of the excitatory neuron pool, underlying the diversity in connectivity and functions of this largely unexplored cerebellar territory. Our findings contribute to laying the groundwork for a comprehensive functional analysis of nuclear neuron subpopulations., (© 2024 The Author(s). Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

The neurons of the three cerebellar nuclei (CN) are the primary output neurons of the cerebellum. The excitatory neurons (e) of the medial (m) CN (eCNm) were recently divided into molecularly defined subdomains in the adult; however, how they are established during development is not known. We define molecular subdomains of the mouse embryonic eCNm using single-cell RNA-sequencing and spatial expression analysis, showing that they evolve during embryogenesis to prefigure the adult. Furthermore, eCNm are transcriptionally divergent from cells in the other nuclei by embryonic day 14.5. We previously showed that loss of the homeobox genes En1 and En2 leads to loss of approximately half of the embryonic eCNm. We demonstrate that mutation of En1/2 in the embryonic eCNm results in death of specific posterior eCNm molecular subdomains and downregulation of TBR2 (EOMES) in an anterior embryonic subdomain, as well as reduced synaptic gene expression. We further reveal a similar function for EN1/2 in mediating TBR2 expression, neuron differentiation and survival in the other excitatory neurons (granule and unipolar brush cells). Thus, our work defines embryonic eCNm molecular diversity and reveals conserved roles for EN1/2 in the cerebellar excitatory neuron lineage., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Whisker stimulation in awake mice evokes transient suppression of simple spike probability in crus I/II Purkinje cells. Here, we investigated how simple spike suppression arises synaptically, what it encodes, and how it affects cerebellar output. In vitro, monosynaptic parallel fiber (PF)-excitatory postsynaptic currents (EPSCs) facilitated strongly, whereas disynaptic inhibitory postsynaptic currents (IPSCs) remained stable, maximizing relative inhibitory strength at the onset of PF activity. Short-term plasticity thus favors the inhibition of Purkinje spikes before PFs facilitate. In vivo, whisker stimulation evoked a 2-6 ms synchronous spike suppression, just 6-8 ms (∼4 synaptic delays) after sensory onset, whereas active whisker movements elicited broadly timed spike rate increases that did not modulate sensory-evoked suppression. Firing in the cerebellar nuclei (CbN) inversely correlated with disinhibition from sensory-evoked simple spike suppressions but was decoupled from slow, non-synchronous movement-associated elevations of Purkinje firing rates. Synchrony thus allows the CbN to high-pass filter Purkinje inputs, facilitating sensory-evoked cerebellar outputs that can drive movements., Competing Interests: Declaration of interests I.M.R. is a member of the Neuron Cell Press advisory board., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The brain is the seat of body weight homeostasis. However, our inability to control the increasing prevalence of obesity highlights a need to look beyond canonical feeding pathways to broaden our understanding of body weight control 1-3 . Here we used a reverse-translational approach to identify and anatomically, molecularly and functionally characterize a neural ensemble that promotes satiation. Unbiased, task-based functional magnetic resonance imaging revealed marked differences in cerebellar responses to food in people with a genetic disorder characterized by insatiable appetite. Transcriptomic analyses in mice revealed molecularly and topographically -distinct neurons in the anterior deep cerebellar nuclei (aDCN) that are activated by feeding or nutrient infusion in the gut. Selective activation of aDCN neurons substantially decreased food intake by reducing meal size without compensatory changes to metabolic rate. We found that aDCN activity terminates food intake by increasing striatal dopamine levels and attenuating the phasic dopamine response to subsequent food consumption. Our study defines a conserved satiation centre that may represent a novel therapeutic target for the management of excessive eating, and underscores the utility of a 'bedside-to-bench' approach for the identification of neural circuits that influence behaviour., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The role of the cerebellum in predictive motor control and coordination has been thoroughly studied during movements of a single body part. In the real world, however, actions are often more complex. Here, we show that a small area in the rostral anterior interpositus nucleus (rAIN) of the mouse cerebellum is responsible for generating a predictive motor synergy that serves to protect the eye by precisely coordinating muscles of the eyelid, neck, and forelimb. Within the rAIN region, we discovered a new functional category of neurons with unique properties specialized for control of motor synergies. These neurons integrated inhibitory cutaneous inputs from multiple parts of the body, and their activity was correlated with the vigor of the defensive motor synergy on a trial-by-trial basis. We propose that some regions of the cerebellum are organized in poly-somatotopic "action maps" to reduce dimensionality and simplify motor control during ethologically relevant behaviors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Understanding the relationship between the local field potential (LFP) and single neurons is essential if we are to understand network dynamics and the entrainment of neuronal activity. Here, we investigated the interaction between the LFP and single neurons recorded in the rat cerebellar nuclei (CN), which are part of the sensorimotor network, in freely moving rats. During movement, the LFP displayed persistent oscillations in the theta band frequency, whereas CN neurons displayed intermittent oscillations in the same frequency band contingent on the instantaneous LFP power; the neurons oscillated primarily when the concurrent LFP power was either high or low. Quantification of the relative instantaneous frequency and phase locking showed that CN neurons exhibited phase locked rhythmic activity at a frequency similar to that of the LFP or at a shifted frequency during high and low LFP power, respectively. We suggest that this nonlinear interaction between cerebellar neurons and the LFP power, which occurs solely during movement, contributes to the shaping of cerebellar output patterns. NEW & NOTEWORTHY We studied the interaction between single neurons and the LFP in the cerebellar nuclei of freely moving rats. We show that during movement, the neurons oscillated in the theta frequency band contingent on the concurrent LFP oscillation power in the same band; the neurons oscillated primarily when the LFP power was either high or low. We are the first to demonstrate a nonlinear, state-dependent entrainment of single neurons to the LFP.

The cerebellum processes neural signals related to rewarding and aversive stimuli, suggesting that the cerebellum supports nonmotor functions in cognitive and emotional domains. Catecholamines are a class of neuromodulatory neurotransmitters well known for encoding such salient stimuli. Catecholaminergic modulation of classical cerebellar functions have been demonstrated. However, a role for cerebellar catecholamines in modulating cerebellar nonmotor functions is unknown. Using biochemical methods in male mice, we comprehensively mapped TH + fibers throughout the entire cerebellum and known precerebellar nuclei. Using electrochemical (fast scan cyclic voltammetry), and viral/genetic methods to selectively delete Th in fibers innervating the lateral cerebellar nucleus (LCN), we interrogated sources and functional roles of catecholamines innervating the LCN, which is known for its role in supporting cognition. The LCN has the most TH + fibers in cerebellum, as well as the most change in rostrocaudal expression among the cerebellar nuclei. Norepinephrine is the major catecholamine measured in LCN. Distinct catecholaminergic projections to LCN arise only from locus coeruleus, and a subset of Purkinje cells that are positive for staining of TH. LC stimulation was sufficient to produce catecholamine release in LCN. Deletion of Th in fibers innervating LCN (LCN- Th-cKO ) resulted in impaired sensorimotor integration, associative fear learning, response inhibition, and working memory in LCN- Th-cKO mice. Strikingly, selective inhibition of excitatory LCN output neurons with inhibitory designer receptor exclusively activated by designer drugs led to facilitation of learning on the same working memory task impaired in LCN- Th-cKO mice. Collectively, these data demonstrate a role for LCN catecholamines in cognitive behaviors. SIGNIFICANCE STATEMENT Here, we report on interrogating sources and functional roles of catecholamines innervating the lateral nucleus of the cerebellum (LCN). We map and quantify expression of TH, the rate-limiting enzyme in catecholamine synthesis, in the entire cerebellar system, including several precerebellar nuclei. We used cyclic voltammetry and pharmacology to demonstrate sufficiency of LC stimulation to produce catecholamine release in LCN. We used advanced viral techniques to map and selectively KO catecholaminergic neurotransmission to the LCN, and characterized significant cognitive deficits related to this manipulation. Finally, we show that inhibition of excitatory LCN neurons with designer receptor exclusively activated by designer drugs, designed to mimic Gi-coupled catecholamine GPCR signaling, results in facilitation of a working memory task impaired in LCN-specific TH KO mice., (Copyright © 2021 the authors.)

How have complex brains evolved from simple circuits? Here we investigated brain region evolution at cell-type resolution in the cerebellar nuclei, the output structures of the cerebellum. Using single-nucleus RNA sequencing in mice, chickens, and humans, as well as STARmap spatial transcriptomic analysis and whole-central nervous system projection tracing, we identified a conserved cell-type set containing two region-specific excitatory neuron classes and three region-invariant inhibitory neuron classes. This set constitutes an archetypal cerebellar nucleus that was repeatedly duplicated to form new regions. The excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in the massively expanded human lateral nucleus. Our data suggest a model of brain region evolution by duplication and divergence of entire cell-type sets., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Both spike rate and timing can transmit information in the brain. Phase response curves (PRCs) quantify how a neuron transforms input to output by spike timing. PRCs exhibit strong firing-rate adaptation, but its mechanism and relevance for network output are poorly understood. Using our Purkinje cell (PC) model, we demonstrate that the rate adaptation is caused by rate-dependent subthreshold membrane potentials efficiently regulating the activation of Na + channels. Then, we use a realistic PC network model to examine how rate-dependent responses synchronize spikes in the scenario of reciprocal inhibition-caused high-frequency oscillations. The changes in PRC cause oscillations and spike correlations only at high firing rates. The causal role of the PRC is confirmed using a simpler coupled oscillator network model. This mechanism enables transient oscillations between fast-spiking neurons that thereby form PC assemblies. Our work demonstrates that rate adaptation of PRCs can spatio-temporally organize the PC input to cerebellar nuclei., Competing Interests: YZ, SH, ED No competing interests declared, (© 2020, Zang et al.)

Modifications in the sensitivity of neural elements allow the brain to adapt its functions to varying demands. Frequency-dependent short-term synaptic depression (STD) provides a dynamic gain-control mechanism enabling adaptation to different background conditions alongside enhanced sensitivity to input-driven changes in activity. In contrast, synapses displaying frequency-invariant transmission can faithfully transfer ongoing presynaptic rates enabling linear processing, deemed critical for many functions. However, rigid frequency-invariant transmission may lead to runaway dynamics and low sensitivity to changes in rate. Here, I investigated the Purkinje cell to deep cerebellar nuclei neuron synapses (PC_DCNs), which display frequency invariance, and yet, PCs maintain background activity at disparate rates, even at rest. Using protracted PC_DCN activation (120 s) to mimic background activity in cerebellar slices from mature mice of both sexes, I identified a previously unrecognized, frequency-dependent, slow STD (S-STD), adapting IPSC amplitudes in tens of seconds to minutes. However, after changes in activation rates, over a behavior-relevant second-long time window, S-STD enabled scaled linear encoding of PC rates in synaptic charge transfer and DCN spiking activity. Combined electrophysiology, optogenetics, and statistical analysis suggested that S-STD mechanism is input-specific, involving decreased ready-to-release quanta, and distinct from faster short-term plasticity (f-STP). Accordingly, an S-STD component with a scaling effect (i.e., activity-dependent release sites inactivation), extending a model explaining PC_DCN release on shorter timescales using balanced f-STP, reproduced the experimental results. Thus, these results elucidates a novel slow gain-control mechanism able to support linear transfer of behavior-driven/learned PC rates concurrently with background activity adaptation, and furthermore, provides an alternative pathway to refine PC output. SIGNIFICANCE STATEMENT The brain can adapt to varying demands by dynamically changing the gain of its synapses; however, some tasks require ongoing linear transfer of presynaptic rates, seemingly incompatible with nonlinear gain adaptation. Here, I report a novel slow gain-control mechanism enabling scaled linear encoding of presynaptic rates over behavior-relevant time windows, and adaptation to background activity at the Purkinje to deep cerebellar nuclear neurons synapses (PC_DCNs). A previously unrecognized PC_DCNs slow and frequency-dependent short-term synaptic depression (S-STD) mediates this process. Experimental evidence and simulations suggested that scaled linear encoding emerges from the combination of S-STD slow dynamics and frequency-invariant transmission at faster timescales. These results demonstrate a mechanism reconciling rate code with background activity adaptation and suitable for flexibly tuning PCs output via background activity modulation., (Copyright © 2020 the authors.)

The cerebellum and the basal ganglia play an important role in the control of voluntary eye movement associated with complex behavior, but little is known about how cerebellar projections project to cortical eye movement areas. Here we used retrograde transneuronal transport of rabies virus to identify neurons in the cerebellar nuclei that project via the thalamus to supplementary eye field (SEF) of the frontal cortex of macaques. After rabies injections into the SEF, many neurons in the restricted region, the ventral aspects of the dentate nucleus (DN), the caudal pole of the DN, and the posterior interpositus nucleus (PIN) were labeled disynaptically via the thalamus, whereas no neuron labeling was found in the anterior interpositus nucleus (AIN). The distribution of the labeled neurons was dorsoventrally different from that of DN and PIN neurons labeled from the motor cortex. In the basal ganglia, a large number of labeled neurons were confined to the dorsomedial portion of the internal segment of the globus pallidus (GPi) as more neurons were labeled in the inner portion of the GPi (GPii) than in the outer portion of the GPi (GPio). This is the first evidence of a projection between cerebellum/basal ganglia and the SEF that could enable the cerebellum to modulate the cognitive control of voluntary eye movement.

Low threshold voltage activated Kv1 potassium channels play key roles in regulating action potential (AP) threshold, neural excitability, and synaptic transmission. Kv1 channels are highly expressed in the cerebellum and mutations of human Kv1 genes are associated to episodic forms of ataxia (EAT-1). Besides the well-established role of Kv1 channels in controlling the cerebellar basket-Purkinje cells synapses, Kv1 channels are expressed by the deep cerebellar nuclear neurons (DCNs) where they regulate the activity of principal DCNs carrying the cerebellar output. DCNs include as well GABAergic neurons serving important functions, such as those forming the inhibitory nucleo-olivary pathway, the nucleo-cortical DCNs providing feed-back inhibition to the cerebellar cortex, and those targeting principal DCNs, but whether their function is regulated by Kv1 channels remains unclear. Here, using cerebellar slices from mature GAD67-GFP mice to identify putative GABAergic-DCNs (GAD + DCN) we show that specific Kv1 channel blockers (dendrotoxin-alpha/I/K, DTXs) hyperpolarized the threshold of somatic action potentials, increased the spontaneous firing rate and hampered evoked high frequency repetitive responses of GAD + DCNs. Moreover, DTXs induced somatic depolarization and tonic firing in previously silent, putative nucleo-cortical DCNs. These results reveal a novel role of Kv1 channels in regulating GABAergic-DCNs activity and thereby, cerebellar function at multiple levels.

The cerebellum drives motor coordination and sequencing of actions at the millisecond timescale through adaptive control of cerebellar nuclear output. Cerebellar nuclei integrate high-frequency information from both the cerebellar cortex and the two main excitatory inputs of the cerebellum: the mossy fibers and the climbing fiber collaterals. However, how nuclear cells process rate and timing of inputs carried by these inputs is still debated. Here, we investigate the influence of the cerebellar cortical output, the Purkinje cells, on identified cerebellar nuclei neurons in vivo in male mice. Using transgenic mice expressing Channelrhodopsin2 specifically in Purkinje cells and tetrode recordings in the medial nucleus, we identified two main groups of neurons based on the waveform of their action potentials. These two groups of neurons coincide with glutamatergic and GABAergic neurons identified by optotagging after Chrimson expression in VGLUT2-cre and GAD-cre mice, respectively. The glutamatergic-like neurons fire at high rate and respond to both rate and timing of Purkinje cell population inputs, whereas GABAergic-like neurons only respond to the mean population firing rate of Purkinje cells at high frequencies. Moreover, synchronous activation of Purkinje cells can entrain the glutamatergic-like, but not the GABAergic-like, cells over a wide range of frequencies. Our results suggest that the downstream effect of synchronous and rhythmic Purkinje cell discharges depends on the type of cerebellar nuclei neurons targeted. SIGNIFICANCE STATEMENT Motor coordination and skilled movements are driven by the permanent discharge of neurons from the cerebellar nuclei that communicate cerebellar computation to other brain areas. Here, we set out to study how specific subtypes of cerebellar nuclear neurons of the medial nucleus are controlled by Purkinje cells, the sole output of the cerebellar cortex. We could isolate different subtypes of nuclear cell that differentially encode Purkinje cell inhibition. Purkinje cell stimulation entrains glutamatergic projection cells at their firing frequency, whereas GABAergic neurons are only inhibited. These differential coding strategies may favor temporal precision of cerebellar excitatory outputs associated with specific features of movement control while setting the global level of cerebellar activity through inhibition via rate coding mechanisms., (Copyright © 2020 the authors.)

For neural systems to function effectively, the numbers of each cell type must be proportioned properly during development. We found that conditional knockout of the mouse homeobox genes En1 and En2 in the excitatory cerebellar nuclei neurons (eCN) leads to reduced postnatal growth of the cerebellar cortex. A subset of medial and intermediate eCN are lost in the mutants, with an associated cell non-autonomous loss of their presynaptic partner Purkinje cells by birth leading to proportional scaling down of neuron production in the postnatal cerebellar cortex. Genetic killing of embryonic eCN throughout the cerebellum also leads to loss of Purkinje cells and reduced postnatal growth but throughout the cerebellar cortex. Thus, the eCN play a key role in scaling the size of the cerebellum by influencing the survival of their Purkinje cell partners, which in turn regulate production of granule cells and interneurons via the amount of sonic hedgehog secreted., Competing Interests: RW, NB, AL, AK, AW, ZL, DS, AR, KD, GO, ZW, MT, AJ No competing interests declared, (© 2019, Willett et al.)

In week-old rats, somatosensory input arises predominantly from external stimuli or from sensory feedback (reafference) associated with myoclonic twitches during active sleep. A previous study suggested that the brainstem motor structures that produce twitches also send motor copies (or corollary discharge, CD) to the cerebellum. We tested this possibility by recording from two precerebellar nuclei-the inferior olive (IO) and lateral reticular nucleus (LRN). In most IO and LRN neurons, twitch-related activity peaked sharply around twitch onset, consistent with CD. Next, we identified twitch-production areas in the midbrain that project independently to the IO and LRN. Finally, we blocked calcium-activated slow potassium (SK) channels in the IO to explain how broadly tuned brainstem motor signals can be transformed into precise CD signals. We conclude that the precerebellar nuclei convey a diversity of sleep-related neural activity to the developing cerebellum to enable processing of convergent input from CD and reafferent signals., Competing Interests: DM, GS, MB No competing interests declared, (© 2018, Mukherjee et al.)

The basal interstitial nucleus (BIN) in the white matter of the vestibulocerebellum has been defined more than three decades ago, but has since been largely ignored. It is still unclear which neurotransmitters are being used by BIN neurons, how these neurons are connected to the rest of the brain and what their activity patterns look like. Here, we studied BIN neurons in a range of mammals, including macaque, human, rat, mouse, rabbit, and ferret, using tracing, immunohistological and electrophysiological approaches. We show that BIN neurons are GABAergic and glycinergic, that in primates they also express the marker for cholinergic neurons choline acetyl transferase (ChAT), that they project with beaded fibers to the glomeruli in the granular layer of the ipsilateral floccular complex, and that they are driven by excitation from the ipsilateral and contralateral medio-dorsal medullary gigantocellular reticular formation. Systematic analysis of codistribution of the inhibitory synapse marker VIAAT, BIN axons, and Golgi cell marker mGluR2 indicate that BIN axon terminals complement Golgi cell axon terminals in glomeruli, accounting for a considerable proportion ( > 20%) of the inhibitory terminals in the granule cell layer of the floccular complex. Together, these data show that BIN neurons represent a novel and relevant inhibitory input to the part of the vestibulocerebellum that controls compensatory and smooth pursuit eye movements., (© 2018 Wiley Periodicals, Inc.)

Classical eyeblink conditioning is an experimental model widely used for the study of the neuronal mechanisms underlying the acquisition of new motor and cognitive skills. There are two principal interpretations of the role of the cerebellum in the learning of eyelid conditioned responses (CRs). One considers that the cerebellum is the place where this learning is acquired and stored, while the second suggests that the cerebellum is mostly involved in the proper performance of acquired CRs, implying that there must be other brain areas involved in the learning process. We checked the timing of cerebellar interpositus nucleus (IPN) neurons' firing rate with eyelid CRs in both wild-type (WT) and Lurcher (a model of cerebellar cortex degeneration) mice. We used delay and trace conditioning paradigms. WT mice presented a better execution for delay vs. trace conditioning and also for these two paradigms than did Lurcher mice. IPN neurons were activated during CRs following the activation of the orbicularis oculi muscle. Firing patterns of IPN neurons were altered in Lurcher mice. In conclusion, the cerebellum seems to be mostly related with the performance of conditioned responses, rather than with their acquisition.

The ability to flexibly adjust movement timing is important for everyday life. Although the basal ganglia and cerebellum have been implicated in monitoring of supra- and sub-second intervals, respectively, the underlying neuronal mechanism remains unclear. Here, we show that in monkeys trained to generate a self-initiated saccade at instructed timing following a visual cue, neurons in the caudate nucleus kept track of passage of time throughout the delay period, while those in the cerebellar dentate nucleus were recruited only during the last part of the delay period. Conversely, neuronal correlates of trial-by-trial variation of self-timing emerged earlier in the cerebellum than the striatum. Local inactivation of respective recording sites confirmed the difference in their relative contributions to supra- and sub-second intervals. These results suggest that the basal ganglia may measure elapsed time relative to the intended interval, while the cerebellum might be responsible for the fine adjustment of self-timing., Competing Interests: JK, TS, SO, MT No competing interests declared

Outputs from the cerebellar nuclei (CN) are important for generating and controlling movement. The activity of CN neurons is controlled not only by excitatory inputs from mossy and climbing fibers and by γ-aminobutyric acid (GABA)-based inhibitory transmission from Purkinje cells in the cerebellar cortex but is also modulated by inputs from other brain regions, including serotonergic fibers that originate in the dorsal raphe nuclei. We examined the modulatory effects of serotonin (5-HT) on GABAergic synapses during development, using rat cerebellar slices. As previously reported, 5-HT presynaptically decreased the amplitudes of stimulation-evoked inhibitory postsynaptic currents (IPSCs) in CN neurons, with this effect being stronger in slices from younger animals (postnatal days [P] 11-13) than in slices from older animals (P19-21). GABA release probabilities accordingly exhibited significant decreases from P11-13 to P19-21. Although there was a strong correlation between the GABA release probability and the magnitude of 5-HT-induced inhibition, manipulating the release probability by changing extracellular Ca 2+ concentrations failed to control the extent of 5-HT-induced inhibition. We also found that the IPSCs exhibited slower kinetics at P11-13 than at P19-21. Pharmacological and molecular biological tests revealed that IPSC kinetics were largely determined by the prevalence of α 1 subunits within GABA A receptors. In summary, pre- and postsynaptic developmental changes in serotonergic modulation and GABAergic synaptic transmission occur during the second to third postnatal weeks and may significantly contribute to the formation of normal adult cerebellar function.

Conspecifics vary consistently in their behavioural responses towards environment stimuli such as exposure to novel objects; ethologists often refer to this variability as animal personality. The neurological mechanisms underlying animal personality traits remain largely unknown, but linking the individual variation in emotional expression to brain structural and neurochemical factors is attracting renewed interest. While considerable research has focused on hormonal and neurotransmitter effects on behavioural responses, less is known about how individual variation in the number of specific neuron populations contributes to individual variation in behaviour. The basolateral amygdala (BLA) and the central nuclei of the amygdala (CeA) mediate emotional processing by regulating behavioural responses of animals in a potentially threatening situation. As such, these structures are good candidates for evaluating the relationship between neuronal populations and behavioural traits. We now show that individual American mink (Neovison vison) reacting more boldly towards novelty have more neurons in the BLA than do their more timid conspecifics, suggesting that a developmental pattern of the number of amygdala neurons can influence behavioural traits of an adult animal. Furthermore, post hoc correlations revealed that individuals performing with higher arousal, as reflected by their frequency of startle behaviour, have more CeA neurons. Our results support a direct link between the number of neurons in amygdala regions and aspects of animal personality.

The excitatory synapses of the rat deep cerebellar nuclei (DCN) were quantitatively analyzed by vesicular glutamate transporter 1 and 2 (vGluT1 and vGluT2) immunolabeling. We calculated the number and sizes of the labeled boutons and compared them between lateral/dentate nucleus (LN/DN), posterior interposed nucleus (PIN), anterior interposed nucleus (AIN), and medial nucleus (MN). The density of vGluT1+ boutons differs significantly within these nuclei. In contrast, the vGluT2+ bouton density is more similar between different nuclei. The phylogenetically newer DCN (LN/DN and PIN) have a 39% higher density of vGluT1+ boutons than the phylogenetically older DCN (AIN and MN). The volume of vGluT1+ boutons does not differ between the DCN, however the average volume of vGluT2+ boutons is larger in MN. In summary, our current results confirm and extend our previous findings showing that the increase in dendritic and axonal wiring in phylogenetically newer DCN is associated with an increase in vGluT1+ bouton density., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Many daily behaviors rely critically on estimates of our body motion. Such estimates must be computed by combining neck proprioceptive signals with vestibular signals that have been transformed from a head- to a body-centered reference frame. Recent studies showed that deep cerebellar neurons in the rostral fastigial nucleus (rFN) reflect these computations, but whether they explicitly encode estimates of body motion remains unclear. A key limitation in addressing this question is that, to date, cell tuning properties have only been characterized for a restricted set of motions across head-re-body orientations in the horizontal plane. Here we examined, for the first time, how 3D spatiotemporal tuning for translational motion varies with head-re-body orientation in both horizontal and vertical planes in the rFN of male macaques. While vestibular coding was profoundly influenced by head-re-body position in both planes, neurons typically reflected at most a partial transformation. However, their tuning shifts were not random but followed the specific spatial trajectories predicted for a 3D transformation. We show that these properties facilitate the linear decoding of fully body-centered motion representations in 3D with a broad range of temporal characteristics from small groups of 5-7 cells. These results demonstrate that the vestibular reference frame transformation required to compute body motion is indeed encoded by cerebellar neurons. We propose that maintaining partially transformed rFN responses with different spatiotemporal properties facilitates the creation of downstream body motion representations with a range of dynamic characteristics, consistent with the functional requirements for tasks such as postural control and reaching. SIGNIFICANCE STATEMENT Estimates of body motion are essential for many daily activities. Vestibular signals are important contributors to such estimates but must be transformed from a head- to a body-centered reference frame. Here, we provide the first direct demonstration that the cerebellum computes this transformation fully in 3D. We show that the output of these computations is reflected in the tuning properties of deep cerebellar rostral fastigial nucleus neurons in a specific distributed fashion that facilitates the efficient creation of body-centered translation estimates with a broad range of temporal properties (i.e., from acceleration to position). These findings support an important role for the rostral fastigial nucleus as a source of body translation estimates functionally relevant for behaviors ranging from postural control to perception., Competing Interests: The authors declare no competing financial interests., (Copyright © 2018 the authors 0270-6474/18/383584-19$15.00/0.)

Thisstudy aims to determine whether dentate neurons can be translaminarlyneuromorphotopologically classified as ventrolateral or dorsomedial type. Adulthuman dentate interneuron 2D binary images are analyzed. The analysis isperformed on both real and virtual neuron samples and 29 parameters are used.They are divided into the classes: neuron surface, shape, length, branching andcomplexity. Clustering is performed by an algorithm that employs predictor extraction (matrix attractor analysis/non-negative matrix factorization and cluster analysis of predictor factors - separate unifactor analysis/Student’s t-test and MANOVA) and multivariate cluster analysis (cluster analysis, principal component analysis, factor analysis with pro/varimax rotation, Fisher’s linear discriminant analysis and feed-forward backpropagation artificial neural networks). The separate unifactor analysis extracted as significant the following predictors from the natural cell sample: the Npd (p< 0:05), and from the virtual cell sample: the Adt (p< 0.05),Do (p< 0.001), Ms (p< 0.01), Dwdth (p< 0:001), Npd (p< 0:05), Nsd (p< 0.001), Nt/hod (p< 0.001), Nmax (p< 0.01), Ds (p< 0.001), Cdf (Nt/hod)st (p< 0.05). For the multidimensional analysis, with the exception of the Fisher’s linear discriminant analysis which gave a false positive result, all other analyses rejected the translaminar dentate neuron classification. Thus, dentate neurons cannot be classified into ventrolateral/dorsomedial neuromorphotopological subtypes. Although some differences were found to exist, they are not sufficient to carry this classification. The methods of multidimensional statistical analysis are again shown to be the best for such kinds of analysis.

Key Points: The inferior olive sends instructive motor signals to the cerebellum via the climbing fibre projection, which sends collaterals directly to large premotor neurons of the mouse cerebellar nuclei (CbN cells). Optogenetic activation of inferior olivary axons in vitro evokes EPSCs in CbN cells of several hundred pA to more than 1 nA. The inputs are three-fold larger at younger ages, 12 to 14 days old, than at 2 months old, suggesting a strong functional role for this pathway earlier in development. The EPSCs are multipeaked, owing to burst firing in several olivary afferents that fire asynchronously. The convergence of climbing fibre collaterals onto CbN cells decreases from ∼40 to ∼8, which is consistent with the formation of closed-loop circuits in which each CbN neuron receives input from 4-7 collaterals from inferior olivary neurons as well as from all 30-50 Purkinje cells that are innervated by those olivary neurons., Abstract: The inferior olive conveys instructive signals to the cerebellum that drive sensorimotor learning. Inferior olivary neurons transmit their signals via climbing fibres, which powerfully excite Purkinje cells, evoking complex spikes and depressing parallel fibre synapses. Additionally, however, these climbing fibres send collaterals to the cerebellar nuclei (CbN). In vivo and in vitro data suggest that climbing fibre collateral excitation is weak in adult mice, raising the question of whether the primary role of this pathway may be developmental. We therefore examined climbing fibre collateral input to large premotor CbN cells over development by virally expressing channelrhodopsin in the inferior olive. In acute cerebellar slices from postnatal day (P)12-14 mice, light-evoked EPSCs were large (> 1 nA at -70 mV). The amplitude of these EPSCs decreased over development, reaching a plateau of ∼350 pA at P20-60. Trains of EPSCs (5 Hz) depressed strongly throughout development, whereas convergence estimates indicated that the total number of functional afferents decreased with age. EPSC waveforms consisted of multiple peaks, probably resulting from action potential bursts in single collaterals and variable times to spike threshold in converging afferents. Activating climbing fibre collaterals evoked well-timed increases in firing probability in CbN neurons, especially in younger mice. The initially strong input, followed by the decrement in synaptic strength coinciding with the pruning of climbing fibres in the cerebellar cortex, implicates the climbing fibre collateral pathway in early postnatal development. Additionally, the persistence of substantial synaptic input at least to P60 suggests that this pathway may function in cerebellar processing into adulthood., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Regular scaling of brain networks during evolution has been proposed to be the major process leading to enlarged brains. Alternative views, however, suggest that deviations from regular scaling were crucial to the evolution of the primate brain and the emergence of different cerebrotypes. Here, we examined the scaling within the major link between the cerebellum and the cerebral cortex by studying the deep cerebellar nuclei (DCN). We compared the major axonal and dendritic wiring in the DCN of rodents and monkeys in search of regular scaling. We were able to confirm regular scaling within the density of neurons, the general dendritic length per neuron and the Purkinje cell axon length. However, we also observed specific modification of the scaling rules within the primates' largest and phylogenetically newest DCN, the dentate nucleus (LN/dentate). Our analysis shows a deviation from regular scaling in the predicted dendritic length per neuron in the LN/dentate. This reduction in the dendritic length is also associated with a smaller dendritic region-of-influence of these neurons. We also detected specific changes in the dendritic diameter distribution, supporting the theory that there is a shift in the neuronal population of the LN/dentate towards neurons that exhibit spatially restricted, clustered branching trees. The smaller dendritic fields would enable a larger number of network modules to be accommodated in the primate LN/dentate and would provide an explanation for the unique folded structure of the primate LN/dentate. Our results show that, in some brain regions, connectivity maximization (i.e., an increase of dendritic fields) is not the sole optimum and that increases in the number of network modules may be important for the emergence of a divergent primate cerebrotype.

Premotor circuits driving extraocular motoneurons and downstream motor outputs of cerebellar nuclei are well known. However, there is, as yet, no unequivocal account of cerebellar output pathways controlling eye movements in primates. Using retrograde transneuronal transfer of rabies virus from the lateral rectus (LR) eye muscle, we studied polysynaptic pathways to LR motoneurons in primates. Injections were placed either into the central or distal muscle portion, to identify innervation differences of LR motoneurons supplying singly innervated (SIFs) or multiply innervated muscle fibers (MIFs). We found that SIF motoneurons receive major cerebellar 'output channels' bilaterally, while oligosynaptic cerebellar innervation of MIF motoneurons is negligible and/or more indirect. Inputs originate from the fastigial nuclei di- and trisynaptically, and from a circumscribed rostral portion of the ventrolateral interpositus posterior and from the caudal pole of the dentate nuclei trisynaptically. While disynaptic cerebellar inputs to LR motoneurons stem exclusively from the caudal fastigial region involved in saccades, pursuit and convergence (via its projections to brainstem oculomotor populations), minor trisynaptic inputs from the rostral fastigial nucleus, which contributes to gaze shifts, may reflect access to vestibular and reticular eye-head control pathways. Trisynaptic inputs to LR motoneurons from the rostral ventrolateral interpositus posterior, involved in divergence (far-response), is likely mediated by projections to the supraoculomotor area, contributing to LR motoneuron activation during divergence. Trisynaptic inputs to LR motoneurons from the caudal dentate, which also innervates disynaptically the frontal and parietal eye fields, can be explained by its superior colliculus projections, and likely target saccade-related burst neurons., (© 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Background: Diolistic labeling is increasingly utilized in neuroscience as an efficient, reproducible method for visualization of neuronal morphology. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery allows for non-toxic fluorescent labeling of multiple neurons in both living and fixed tissue. Since first described, this labeling method has been modified to fit a variety of research goals and laboratory settings., New Method: Diolistic labeling has traditionally relied on commercially available devices for the propulsion of coated micro-particles into tissue sections. Recently, laboratory built biolistic devices have been developed which allow for increased availability and customization. Here, we discuss a custom biolistic device and provide a detailed protocol for its use., Results: Using custom diolistic labeling we have characterized alterations in neuronal morphology of the lateral/dentate nucleus of the rat cerebellum. Comparisons were made in developing rat pups exposed to abnormally high levels of 5-methyloxytryptamine (5-MT) pre-and postnatally. Using quantitative software; dendritic morphology, architecture, and synaptic connections, were analyzed., Comparison With Existing Method(s): The rapid nature of custom diolistics coupled with passive diffusion of dyes and compatibility with confocal microscopy, provides an unparalleled opportunity to examine features of neuronal cells at high spatial resolution in a three-dimensional tissue environment., Conclusions: While decreasing the associated costs, the laboratory-built device also overcomes many of the obstacles associated with traditional morphological labeling, to allow for reliable and reproducible neuronal labeling. The versatility of this method allows for its adaptation to a variety of laboratory settings and neuroscience related research goals., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The cerebellum, a hindbrain motor center, also participates in regulating nonsomatic visceral activities such as feeding control. However, the underlying neural mechanism is largely unknown. Here, we investigate whether the cerebellar medial nucleus (MN), one of the final outputs of the cerebellum, could directly project to and modulate the feeding-related neurons in the ventromedial hypothalamic nucleus (VMN), which has been traditionally implicated in feeding behavior, energy balance, and body weight regulation. The retrograde tracing results show that both GABAergic and glutamatergic projection neurons in the cerebellar MN send direct projections to the VMN. Electrical stimulation of cerebellar MN elicits an inhibitory, excitatory or biphasic response of VMN neurons. Interestingly, the VMN neurons modulated by cerebellar MN afferents not only receive phasic and tonic inputs from the gastric vagal nerves, but also are sensitive to peripheral glycemia and ghrelin signals. Moreover, a summation of inputs from the cerebellar MN and gastric vagal afferents occurs on single glycemia/ghrelin-sensitive neurons in the VMN, and the immunostaining result show that the axons from the cerebellar MN and the projections from the nucleus tractus solitarius, which conveys the gastric vagal inputs to hypothalamus, converge on single VMN glycemia/ghrelin-sensitive neurons. These results demonstrate that the somatic information forwarded by the cerebellar MN, together with the feeding signals from periphery, converge onto single VMN neurons, suggesting that a somatic-visceral integration related to feeding may occur in the VMN and the cerebellum may actively participate in the feeding regulation through the direct cerebellar MN-VMN projections.

Aims: Primary aim in this study is to investigate whether external and internal border neurons of adult human dentate nucleus express the same neuromorphological features or belong to a different morphological types i.e. whether can be classified not only by way of their topology as external and internal, but also based on their morphological features or in addition to their topology also by way of their morphology. Secondary aim is to determine and compare various methodologies in order to perform the first aim in a more accurate and efficient manner., Material and Methods: Blocks of tissue were cut out from the adult human cerebellum and stained according to the Kopsch-Bubenaite method. Border neurons of the dentate nucleus were investigated and digitized under the light microscope and processed thereafter. Seventeen parameters quantifying various aspects of neuron morphology are then measured. They can be categorized as shape, magnitude, complexity, length and branching parameters. Analyzes used are neural networks, separate unifactor, cluster, principal component, discriminant and correlation-comparison analysis., Results: The external and internal border neurons differ significantly in six of the seventeen parameters investigated, mainly concerning dendritic ramification patterns, overall shape of dendritic tree and dendritic length. All six methodological approaches are in accordance showing slight clustering of data. Classification is based on six parameters: neuron (field) area, dendritic (field) area, total dendrite length, and position of maximal dendritic arborization density. Cluster analysis shows two data clusters. Separate unifactor analysis demonstrates inter-cluster differences with statistical significance (p < 0.05) for all six parameters separately. Principal component, discriminant and correlation-comparison analysis further prove the result on a more factor integrate manner and explain it, respectively. Thus, these neurons can be classified, not only according to their location but also according to some morphological features. Also, the group if internal border neurons is more homogeneous in itself than the other group of external border neurons., Conclusion: Border neurons from adult human dentate nucleus can be divided to external and internal according to its topology and based on neuromorphological computational parameters. This has potentially significant neurofunctional implications but further studies are needed to elucidate that. Multimethodological approach is shown as the best for finding the solution closest to reality. The possible functional meaning of these morphological differences for cerebellar network structure and function are discussed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The study of the clinical anatomy and functional features of the cortex, subcortical and conductive pathways of the cerebellum is necessary for clinicians for elaboration rational surgical approaches to these formations, for determination the localization of pathological processes associated with these formations. Cerebellar nucleus neurons are crucial to the olivo-cerebellar circuit as they provide the sole output of the entire cerebellum. The relationship between mobility and cognition in aging is well established, but the relationship between mobility and the structure and function of the aging brain is relatively unknown. In connection with the above, the purpose of our study was detection of the morphological characteristics of the cerebellum nuclei in aged persons. Study was performed on 48 specimens of the cerebellum from people (24 male and 24 female), who died at the age from 75 to 99 years due to diseases, which were not related to the central nervous system damaging. Formalin-fixed human hemispheres were dissected with the Ludwig and Klingler fiber dissection technique under x6 to x40 magnifications of binocular microscope Olympus BX41 (Japan). The morphological features of the human cerebellar nuclei were established. Namely, on the series of sections of the cerebellum in the horizontal, frontal and sagittal planes, as well as on the macro-microscopic preparations of the cerebellar nuclei location, their relative position, shape, linear dimensions, weight and volume were described. The features of macro-microscopic and histological structure of the nuclei of the cerebellum were made own classification of the gyri and teeth of the dentate nucleus of the cerebellum was offered. Macro-microscopic dissection of persons died after 75 years old show no significant variability of linear dimensions of cerebellar nuclei with their specific location and options. Simultaneously, reliable reducing of cellular density was detected for Purkinje, granule and basket neurons more pronounced in male for Purkinje cells.

Target recognition by developing axons is one of the fundamental steps for establishing the proper pattern of neuronal connectivity during development. However, knowledge of the mechanisms that underlie this critical event is still limited. In this study, to examine how commissural axons in vertebrates recognize their targets after crossing the midline, we analyzed in detail the behavior of postcrossing commissural axons derived from the deep cerebellar nuclei (DCN) in the developing mouse cerebellum. For this, we employed a cell-type-specific genetic labeling approach to selectively visualize DCN axons during the time when these axons project to the red nucleus (RN), one of the well-characterized targets of DCN axons. We found that, when DCN axons initially entered the RN at its caudal end, these axons continued to grow rostrally through the RN without showing noticeable morphological signs of axon branching. Interestingly, after a delay, DCN axons started forming interstitial branches from the portion of the axon shaft selectively within the RN. Because commissural axons acquire responsiveness to several guidance cues when they cross the midline, we further addressed whether midline crossing is a prerequisite for subsequent targeting by using a Robo3 knockdown strategy. We found that DCN axons were still capable of forming interstitial branches within the RN even in the absence of midline crossing. These results therefore suggest that the mechanism of RN recognition by DCN axons involves a delayed interstitial branching, and that these axons possess an intrinsic ability to respond to the target-derived cues irrespective of midline crossing., (© 2015 Wiley Periodicals, Inc.)

To study the olivary input to the cerebellar nuclei (CN) we used optogenetic stimulation in transgenic mice expressing channelrhodopsin-2 (ChR2) in olivary neurons. We obtained in vivo extracellular Purkinje cell (PC) and CN recordings in anesthetized mice while stimulating the contralateral inferior olive (IO) with a blue laser (single pulse, 10-50 ms duration). Peri-stimulus histograms (PSTHs) were constructed to show the spike rate changes after optical stimulation. Among 29 CN neurons recorded, 15 showed a decrease in spike rate of variable strength and duration, and only 1 showed a transient spiking response. These results suggest that direct olivary input to CN neurons is usually overridden by stronger PC inhibition triggered by climbing fiber responses. To further investigate the direct input from the climbing fiber collaterals we also conducted whole cell recordings in brain slices, where we used local stimulation with blue light. Due to the expression of ChR2 in PC axons as well as the IO in our transgenic line, strong inhibitory responses could be readily triggered with optical stimulation (13 of 15 neurons). After blocking this inhibition with GABAzine, only in 5 of 13 CN neurons weak excitatory responses were revealed. Therefore our in vitro results support the in vivo findings that the excitatory input to CN neurons from climbing fiber collaterals in adult mice is masked by the inhibition under normal conditions.

The olivo-cerebellar system has an essential role in the detection and adaptive correction of movement errors. While there is evidence of an error signal in the cerebellar cortex and inferior olivary nucleus, the deep cerebellar nuclei have been less thoroughly investigated. Here, we recorded local field potential activity in the rodent lateral cerebellar nucleus during a skilled reaching task and compared event-related changes in neural activity between unsuccessful and successful attempts. Increased low gamma (40-50 Hz) band power was present throughout the reach and grasp behavior, with no difference between successful and unsuccessful trials. Beta band (12-30 Hz) power, however, was significantly increased in unsuccessful reaches, compared to successful, throughout the trial, including during the epoch preceding knowledge of the trial's outcome. This beta band activity was greater in unsuccessful trials of high-performing days, compared to unsuccessful trials of low-performing days, indicating that this activity may reflect an error prediction signal, developed over the course of motor learning. These findings suggest an error-related discriminatory oscillatory hallmark of movement in the deep cerebellar nuclei., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In this study mathematical analyses such as the analysis of area and length, fractal analysis and modified Sholl analysis were applied on two dimensional (2D) images of neurons from adult human dentate nucleus (DN). Using mathematical analyses main morphological properties were obtained including the size of neuron and soma, the length of all dendrites, the density of dendritic arborization, the position of the maximum density and the irregularity of dendrites. Response surface methodology (RSM) was used for modeling the size of neurons and the length of all dendrites. However, the RSM model based on the second-order polynomial equation was only possible to apply to correlate changes in the size of the neuron with other properties of its morphology. Modeling data provided evidence that the size of DN neurons statistically depended on the size of the soma, the density of dendritic arborization and the irregularity of dendrites. The low value of mean relative percent deviation (MRPD) between the experimental data and the predicted neuron size obtained by RSM model showed that model was suitable for modeling the size of DN neurons. Therefore, RSM can be generally used for modeling neuron size from 2D images., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The control of deep cerebellar nuclear (DCN) neuronal firing is central to cerebellar function but is not well understood. The large majority of synapses onto DCN neurons derive from Purkinje cells (PCs), suggesting that PC activity is an important determinant of DCN firing; however, PCs fire both simple and complex spikes (CSs), and little is known about how the latter's action affects DCN activity. Thus, here, we explored the effects of CSs on DCN activity. CSs were recorded from PC arrays along with individual DCN neurons. Presumed synaptically connected PC-DCN cell pairs were identified using CS-triggered correlograms of DCN activity, which also showed that CS activity was associated with a predominantly inhibitory effect on DCN activity. The strength of the CS effect varied as a function of synchrony, such that isolated CSs produced only weak inhibition of DCN activity, whereas highly synchronous CSs caused a larger drop in firing levels. Although the present findings were obtained in anesthetized animals, similar CS synchrony levels exist in awake animals, and changes in synchrony level have been observed in association with movements in awake animals. Thus, the present data suggest that synchronous CS activity may be a mechanism for shaping DCN output related to motor commands.

We previously reported that an air-puff stimulation on the ipsilateral whisker pad evoked responses in molecular layer (ML) and Purkinje cell (PC) layer in cerebellar cortex folium Crus II. We used anterograde tracing and electrophysiological methods to investigate the anatomical and functional relationships between the trigeminal tactile response area in the cerebellar cortex Crus II and deep cerebellar nuclei (DCN) in living mice. We found that the axons of tactile activated PCs projected in anterior part (IntA) and posterior part (IntP), and dorsolateral hump (IntDL) of ipsilateral interposed cerebellar nucleus (ICN). In ICN, the tactile stimulus evoked-field potential expressed a sequence of two negative components N1 and N2, while extracellular recordings from ICN neurons revealed that an increase in spike frequency in response to tactile stimulus. When the duration of facial air-puff stimulus were ≥ 30 ms, stimulation off response (Roff) were observed in the ICN, but an increase in the duration of facial air-puff stimulation did not significantly affect the amplitude of Ron (N1 and N2) and Roff. The latency and time to peak of N1 in ICN were significantly shorter than that of N1 in the ML, but the latency and time to peak of N2 in ICN were significantly later than that of P1 in the ML. The present results suggest that the facial sensory information, at least in part, is transferred to ICN by PC axons from Crus II, which evokes excitation in ICN neurons., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Neurons of the cerebellar nuclei convey the final output of the cerebellum to their targets in various parts of the brain. Within the cerebellum their direct upstream connections originate from inhibitory Purkinje neurons. Purkinje neurons have a complex firing pattern of regular spikes interrupted by intermittent pauses of variable length. How can the cerebellar nucleus process this complex input pattern? In this modeling study, we investigate different forms of Purkinje neuron simple spike pause synchrony and its influence on candidate coding strategies in the cerebellar nuclei. That is, we investigate how different alignments of synchronous pauses in synthetic Purkinje neuron spike trains affect either time-locking or rate-changes in the downstream nuclei. We find that Purkinje neuron synchrony is mainly represented by changes in the firing rate of cerebellar nuclei neurons. Pause beginning synchronization produced a unique effect on nuclei neuron firing, while the effect of pause ending and pause overlapping synchronization could not be distinguished from each other. Pause beginning synchronization produced better time-locking of nuclear neurons for short length pauses. We also characterize the effect of pause length and spike jitter on the nuclear neuron firing. Additionally, we find that the rate of rebound responses in nuclear neurons after a synchronous pause is controlled by the firing rate of Purkinje neurons preceding it.

Execution of accurate eye movements depends critically on the cerebellum, suggesting that the major output neurons of the cerebellum, Purkinje cells, may predict motion of the eye. However, this encoding of action for rapid eye movements (saccades) has remained unclear: Purkinje cells show little consistent modulation with respect to saccade amplitude or direction, and critically, their discharge lasts longer than the duration of a saccade. Here we analysed Purkinje-cell discharge in the oculomotor vermis of behaving rhesus monkeys (Macaca mulatta) and found neurons that increased or decreased their activity during saccades. We estimated the combined effect of these two populations via their projections to the caudal fastigial nucleus, and uncovered a simple-spike population response that precisely predicted the real-time motion of the eye. When we organized the Purkinje cells according to each cell's complex-spike directional tuning, the simple-spike population response predicted both the real-time speed and direction of saccade multiplicatively via a gain field. This suggests that the cerebellum predicts the real-time motion of the eye during saccades via the combined inputs of Purkinje cells onto individual nucleus neurons. A gain-field encoding of simple spikes emerges if the Purkinje cells that project onto a nucleus neuron are not selected at random but share a common complex-spike property.

Behavioral studies have established a role for adult-born dentate granule cells in discriminating between similar memories. However, it is unclear how these cells mediate memory discrimination. Excitability is enhanced in maturing adult-born neurons, spurring the hypothesis that the activity of these cells "directly" encodes and stores memories. An alternative hypothesis posits that maturing neurons "indirectly" contribute to memory encoding by regulating excitation-inhibition balance. We evaluated these alternatives by using dentate-sensitive active place avoidance tasks to assess experience-dependent changes in dentate field potentials in the presence and absence of neurogenesis. Before training, X-ray ablation of adult neurogenesis-reduced dentate responses to perforant-path stimulation and shifted EPSP-spike coupling leftward. These differences were unchanged after place avoidance training with the shock zone in the initial location, which both groups learned to avoid equally well. In contrast, sham-treated mice decreased dentate responses and shifted EPSP-spike coupling leftward after the shock zone was relocated, whereas X-irradiated mice failed to show these changes in dentate function and were impaired on this test of memory discrimination. During place avoidance, excitation-inhibition coupled neural synchrony in dentate local field potentials was reduced in X-irradiated mice, especially in the θ band. The difference was most prominent during conflict learning, which is impaired in the X-irradiated mice. These findings indicate that maturing adult-born neurons regulate both functional network plasticity in response to memory discrimination and dentate excitation-inhibition coordination. The most parsimonious interpretation of these results is that adult neurogenesis indirectly regulates hippocampal information processing., Significance Statement: Adult-born neurons in the hippocampal dentate gyrus are important for flexibly using memories, but the mechanism is controversial. Using tests of hippocampus-dependent place avoidance learning and dentate electrophysiology in mice with normal or ablated neurogenesis, we find that maturing adult-born neurons are crucial only when memory must be used flexibly, and that these neurons regulate dentate gyrus synaptic and spiking responses to neocortical input rather than directly storing information, as has been proposed. A day after learning to avoid the initial or changed locations of shock, the dentate synaptic responses are enhanced or suppressed, respectively, unlike mice lacking adult neurogenesis, which did not change. The contribution of adult neurogenesis to memory is indirect, by regulating dentate excitation-inhibition coupling., (Copyright © 2015 the authors 0270-6474/15/3511656-11$15.00/0.)

The cerebellum, a crucial center for motor coordination, is composed of a cortex and several nuclei. The main mode of interaction between these two parts is considered to be formed by the inhibitory control of the nuclei by cortical Purkinje neurons. We now amend this view by showing that inhibitory GABA-glycinergic neurons of the cerebellar nuclei (CN) project profusely into the cerebellar cortex, where they make synaptic contacts on a GABAergic subpopulation of cerebellar Golgi cells. These spontaneously firing Golgi cells are inhibited by optogenetic activation of the inhibitory nucleo-cortical fibers both in vitro and in vivo. Our data suggest that the CN may contribute to the functional recruitment of the cerebellar cortex by decreasing Golgi cell inhibition onto granule cells.

We previously have shown that cerebellar fastigial nucleus (FN) modulates immune function, but pathways or mechanisms underlying this immunomodulation require clarification. Herein, an anterograde and retrograde tracing of nerve tracts between the cerebellar FN and hypothalamus/thalamus was performed in rats. After demonstrating a direct cerebellar FN-hypothalamic/thalamic glutamatergic projection, 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase that catalyzes glutamate synthesis, was injected bilaterally in the cerebellar FN and simultaneously, D,L-threo-β-hydroxyaspartic acid (THA), an inhibitor of glutamate transporters on cell membrane, was bilaterally injected in the lateral hypothalamic area (LHA) or the ventrolateral (VL) thalamic nucleus. DON treatment in the FN alone decreased number of glutamatergic neurons that projected axons to the LHA and also diminished glutamate content in both the hypothalamus and the thalamus. These effects of DON were reduced by combined treatment with THA in the LHA or in the VL. Importantly, DON treatment in the FN alone attenuated percentage and cytotoxicity of natural killer (NK) cells and also lowered percentage and cytokine production of T lymphocytes. These DON-caused immune effects were reduced or abolished by combined treatment with THA in the LHA, but not in the VL. Simultaneously, DON treatment elevated level of norepinephrine (NE) in the spleen and mesenteric lymphoid nodes, and THA treatment in the LHA, rather than in the VL, antagonized the DON-caused NE elevation. These findings suggest that glutamatergic neurons in the cerebellar FN regulate innate and adaptive immune functions and the immunomodulation is conveyed by FN-hypothalamic glutamatergic projections and sympathetic nerves that innervate lymphoid tissues.

In the mammalian cerebellum, deep cerebellar nuclear (DCN) cells convey all information from cortical Purkinje cells (PCs) to premotor nuclei and other brain regions. However, how DCN cells integrate inhibitory input from PCs with excitatory inputs from other sources has been difficult to assess, in part due to the large spatial separation between cortical PCs and their target cells in the nuclei. To circumvent this problem we have used a Cre-mediated genetic approach to generate mice in which channelrhodopsin-2 (ChR2), fused with a fluorescent reporter, is selectively expressed by GABAergic neurons, including PCs. In recordings from brain slice preparations from this model, mammalian PCs can be robustly depolarized and discharged by brief photostimulation. In recordings of postsynaptic DCN cells, photostimulation of PC axons induces a strong inhibition that resembles these cells' responses to focal electrical stimulation, but without a requirement for the glutamate receptor blockers typically applied in such experiments. In this optogenetic model, laser pulses as brief as 1 ms can reliably induce an inhibition that shuts down the spontaneous spiking of a DCN cell for ∼50 ms. If bursts of such brief light pulses are delivered, a fixed pattern of bistable bursting emerges. If these pulses are delivered continuously to a spontaneously bistable cell, the immediate response to such photostimulation is inhibitory in the cell's depolarized state and excitatory when the membrane has repolarized; a less regular burst pattern then persists after stimulation has been terminated. These results indicate that the spiking activity of DCN cells can be bidirectionally modulated by the optically activated synaptic inhibition of cortical PCs., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

A common view of the architecture of different brain regions is that, despite their heterogeneity, they have optimized their wiring schemes to make maximal use of space. Based on experimental findings, computational models have delineated how about two-thirds of the neuropil is filled out with dendrites and axons optimizing cable costs and conduction time while keeping the connectivity at the highest level. However, whether this assumption can be generalized to all brain regions has not yet been tested. Here we quantified and charted the components of the neuropil in the four deep cerebellar nuclei (DCN) of the rat's brain. We segmented and traced the neuropil stained with one of two antibodies, one antibody against dendritic microtubule-associated proteins (MAP2a,b) and the second against the Purkinje cell axons (PCP2). We compared fiber length density, average fiber diameter, and volume fraction within different components of the DCN in a random, systematic fashion. We observed differences in dendritic and axonal fiber length density, average fiber diameters, and volume fraction within the four different nuclei that make up the DCN. We observe a relative increase in the length density of dendrites and Purkinje cell axons in two of the DCN, namely, the posterior interposed nucleus and the lateral nucleus. Furthermore, the DCN have a surprisingly low volume fraction of their dendritic length density, which we propose is related to their special circuitry. In summary, our results show previously unappreciated functional adaptations among these nuclei., (© 2014 Wiley Periodicals, Inc.)

The cerebellum is necessary and sufficient for the acquisition and execution of adaptively timed conditioned motor responses following repeated paired presentations of a conditioned stimulus and an unconditioned stimulus. The underlying plasticity depends on the convergence of conditioned and unconditioned stimuli signals relayed to the cerebellum by the pontine nucleus and the inferior olive (IO), respectively. Adaptive timing of conditioned responses relies on the correctly predicted onset of the unconditioned stimulus, usually a noxious somatosensory stimulus. We addressed two questions: First, does the IO relay information regarding the duration of somatosensory stimuli to the cerebellum? Multiple-unit recordings from the IO of anesthetized rats that received periorbital airpuffs of various durations revealed that sustained somatosensory stimuli are invariably transformed into phasic IO outputs. The phasic response was followed by a post-peak depression in IO activity as compared to baseline, providing the cerebellum with a highly synchronous signal, time-locked to the stimulus' onset. Second, we sought to examine the involvement of olivocerebellar interactions in this signal transformation. Cerebello-olivary inhibition was interrupted using temporary pharmacological inactivation of cerebellar output nuclei, resulting in more sustained (i.e., less synchronous) IO responses to sustained somatosensory stimuli, in which the post-peak depression was substituted with elevated activity as compared to baseline. We discuss the possible roles of olivocerebellar negative-feedback loops and baseline cerebello-olivary inhibition levels in shaping the temporal dynamics of the IO's response to somatosensory stimuli and the consequences of this shaping for cerebellar plasticity and its ability to adapt to varying contexts.

Previous studies demonstrated that ingestion of the emetic compound copper sulfate (CuSO4) alters the responses to vestibular stimulation of a large fraction of neurons in brainstem regions that mediate nausea and vomiting, thereby affecting motion sickness susceptibility. Other studies suggested that the processing of vestibular inputs by cerebellar neurons plays a critical role in generating motion sickness and that neurons in the cerebellar fastigial nucleus receive visceral inputs. These findings raised the hypothesis that stimulation of gastrointestinal receptors by a nauseogenic compound affects the processing of labyrinthine signals by fastigial nucleus neurons. We tested this hypothesis in decerebrate cats by determining the effects of intragastric injection of CuSO4 on the responses of rostral fastigial nucleus to whole-body rotations that activate labyrinthine receptors. Responses to vestibular stimulation of fastigial nucleus neurons were more complex in decerebrate cats than reported previously in conscious felines. In particular, spatiotemporal convergence responses, which reflect the convergence of vestibular inputs with different spatial and temporal properties, were more common in decerebrate than in conscious felines. The firing rate of a small percentage of fastigial nucleus neurons (15%) was altered over 50% by the administration of CuSO4; the firing rate of the majority of these cells decreased. The responses to vestibular stimulation of a majority of these cells were attenuated after the compound was provided. Although these data support our hypothesis, the low fraction of fastigial nucleus neurons whose firing rate and responses to vestibular stimulation were affected by the administration of CuSO4 casts doubt on the notion that nauseogenic visceral inputs modulate motion sickness susceptibility principally through neural pathways that include the cerebellar fastigial nucleus. Instead, it appears that convergence of gastrointestinal and vestibular inputs occurs mainly in the brainstem.

The principal neurons of the cerebellar nuclei (CN), the sole output of the olivo-cerebellar system, receive a massive inhibitory input from Purkinje cells (PCs) of the cerebellar cortex. Morphological evidence suggests that CN principal cells are also contacted by inhibitory interneurons, but the properties of this connection are unknown. Using transgenic, tracing, and immunohistochemical approaches in mice, we show that CN interneurons form a large heterogeneous population with GABA/glycinergic phenotypes, distinct from GABAergic olive-projecting neurons. CN interneurons are found to contact principal output neurons, via glycine receptor (GlyR)-enriched synapses, virtually devoid of the main GABA receptor (GABAR) subunits α1 and γ2. Those clusters account for 5% of the total number of inhibitory receptor clusters on principal neurons. Brief optogenetic stimulations of CN interneurons, through selective expression of channelrhodopsin 2 after viral-mediated transfection of the flexed gene in GlyT2-Cre transgenic mice, evoked fast IPSCs in principal cells. GlyR activation accounted for 15% of interneuron IPSC amplitude, while the remaining current was mediated by activation of GABAR. Surprisingly, small GlyR clusters were also found at PC synapses onto principal CN neurons in addition to α1 and γ2 GABAR subunits. However, GlyR activation was found to account for <3% of the PC inhibitory synaptic currents evoked by electrical stimulation. This work establishes CN glycinergic neurons as a significant source of inhibition to CN principal cells, forming contacts molecularly distinct from, but functionally similar to, Purkinje cell synapses. Their impact on CN output, motor learning, and motor execution deserves further investigation., (Copyright © 2014 the authors 0270-6474/14/349418-14$15.00/0.)

It has long been known that serotonergic afferent inputs are the third largest afferent population in the cerebellum after mossy fibers and climbing fibers. However, the role of serotonergic inputs in cerebellar-mediated motor behaviors is still largely unknown. Here, we show that only 5-HT2A receptors among the 5-HT2 receptor subfamily are expressed and localized in the rat cerebellar fastigial nucleus (FN), one of the ultimate outputs of the spinocerebellum precisely regulating trunk and limb movements. Remarkably, selective activation of 5-HT2A receptors evokes a postsynaptic excitatory effect on FN neurons in a concentration-dependent manner in vitro, which is in accord with the 5-HT-elicited excitation on the same tested neurons. Furthermore, selective 5-HT2A receptor antagonist M100907 concentration-dependently blocks the excitatory effects of 5-HT and TCB-2, a 5-HT2A receptor agonist, on FN neurons. Consequently, microinjection of 5-HT into bilateral FNs significantly promotes rat motor performances on accelerating rota-rod and balance beam and narrows stride width rather than stride length in locomotion gait. All these motor behavioral effects are highly consistent with those of selective activation of 5-HT2A receptors in FNs, and blockage of the component of 5-HT2A receptor-mediated endogenous serotonergic inputs in FNs markedly attenuates these motor performances. All these results demonstrate that postsynaptic 5-HT2A receptors greatly contribute to the 5-HT-mediated excitatory effect on cerebellar FN neurons and promotion of the FN-related motor behaviors, suggesting that serotonergic afferent inputs may actively participate in cerebellar motor control through their direct modulation on the final output of the spinocerebellum.

Integration of cortical Purkinje cell inputs and brain stem inputs is essential in generating cerebellar outputs to the cerebellar nuclei (CN). Currently, collaterals of climbing and mossy fiber axons, noradrenergic, serotoninergic and cholinergic axons, and collaterals of rubrospinal axons are known to innervate the CN from the brain stem. We investigated whether other afferents to the CN from the medulla exist in the rat. Retrograde labeling revealed the presence of neurons that project to the CN but not to the cerebellar cortex in the median reticular formation in the rostrodorsal medulla (tentatively named 'caudal raphe interpositus area', CRI). Anterograde tracer injection into the CRI labeled abundant axonal terminals in the CN, mainly in the ventral parvocellular part of the posterior interposed and lateral nucleus. Axonal reconstruction showed that a single CRI axon projected to the CN with 170-1086 varicosities, more broadly and densely than collaterals of a mossy or climbing fiber axon. CRI axons had no or a few collaterals that projected to the granular and Purkinje cell layers of the cerebellar cortex with some small terminals, indicating that these axons are different from mossy fiber axons. CRI axons also had collaterals that projected to the medial vestibular nucleus and an ascending branch that was not reconstructed. The location of the CRI, electron microscopic observations, and immunostaining results all indicated that CRI axons are not monoaminergic. We conclude that CRI axons form a type of afferent projection to the CN that is different from mossy, climbing or monoaminergic fibers., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The caudal fastigial nucleus (FN) is known to be related to the control of eye movements and projects mainly to the contralateral reticular nuclei where excitatory and inhibitory burst neurons for saccades exist [the caudal portion of the nucleus reticularis pontis caudalis (NRPc), and the rostral portion of the nucleus reticularis gigantocellularis (NRG) respectively]. However, the exact reticular neurons targeted by caudal fastigioreticular cells remain unknown. We tried to determine the target reticular neurons of the caudal FN and superior colliculus (SC) by recording intracellular potentials from neurons in the NRPc and NRG of anesthetized cats. Neurons in the rostral NRG received bilateral, monosynaptic excitation from the caudal FNs, with contralateral predominance. They also received strong monosynaptic excitation from the rostral and caudal contralateral SC, and disynaptic excitation from the rostral ipsilateral SC. These reticular neurons with caudal fastigial monosynaptic excitation were not activated antidromically from the contralateral abducens nucleus, but most of them were reticulospinal neurons (RSNs) that were activated antidromically from the cervical cord. RSNs in the caudal NRPc received very weak monosynaptic excitation from only the contralateral caudal FN, and received either monosynaptic excitation only from the contralateral caudal SC, or monosynaptic and disynaptic excitation from the contralateral caudal and ipsilateral rostral SC, respectively. These results suggest that the caudal FN helps to control also head movements via RSNs targeted by the SC, and these RSNs with SC topographic input play different functional roles in head movements.

Many mossy fiber pathways to the neurons of the deep cerebellar nucleus (DCN) originate from the spinal motor circuitry. For cutaneously activated spinal neurons, the receptive field is a tag indicating the specific motor function the spinal neuron has. Similarly, the climbing fiber receptive field of the DCN neuron reflects the specific motor output function of the DCN neuron. To explore the relationship between the motor information the DCN neuron receives and the output it issues, we made patch clamp recordings of DCN cell responses to tactile skin stimulation in the forelimb region of the anterior interposed nucleus in vivo. The excitatory responses were organized according to a general principle, in which the DCN cell responses became stronger the closer the skin site was located to its climbing fiber receptive field. The findings represent a novel functional principle of cerebellar connectivity, with crucial importance for our understanding of the function of the cerebellum in movement coordination.