Purkinje cells (PCs) primarily project to cerebellar nuclei but also directly innervate the brainstem. Some PC-brainstem projections have been described previously, but most have not been thoroughly characterized. Here, we use a PC-specific cre line to anatomically and electrophysiologically characterize PC projections to the brainstem. PC synapses are surprisingly widespread, with the highest densities found in the vestibular and parabrachial nuclei. However, there are pronounced regional differences in synaptic densities within both the vestibular and parabrachial nuclei. Large optogenetically evoked PC-IPSCs are preferentially observed in subregions with the highest densities of putative PC boutons, suggesting that PCs selectively influence these areas and the behaviors they regulate. Unexpectedly, the pontine central gray and nearby subnuclei also contained a low density of putative PC boutons, and large PC-IPSCs are observed in a small fraction of cells. We combined electrophysiological recordings with immunohistochemistry to assess the molecular identities of two potential PC targets: PC synapses onto mesencephalic trigeminal neurons were not observed even though these cells are in close proximity to PC boutons; PC synapses onto locus coeruleus neurons are exceedingly rare or absent, even though previous studies concluded that PCs are a major input to these neurons. The availability of a highly selective cre line for PCs allowed us to study functional synapses, while avoiding complications that can accompany the use of viral approaches. We conclude that PCs directly innervate numerous brainstem nuclei, and in many nuclei they strongly inhibit a small fraction of cells. This suggests that PCs selectively target cell types with specific behavioral roles in the brainstem.

Purkinje cell (PC) synapses onto cerebellar nuclei (CbN) neurons allow signals from the cerebellar cortex to influence the rest of the brain. PCs are inhibitory neurons that spontaneously fire at high rates, and many PC inputs are thought to converge onto each CbN neuron to suppress its firing. It has been proposed that PCs convey information using a rate code, a synchrony and timing code, or both. The influence of PCs on CbN neuron firing was primarily examined for the combined effects of many PC inputs with comparable strengths, and the influence of individual PC inputs has not been extensively studied. Here, we find that single PC to CbN synapses are highly variable in size, and using dynamic clamp and modeling we reveal that this has important implications for PC-CbN transmission. Individual PC inputs regulate both the rate and timing of CbN firing. Large PC inputs strongly influence CbN firing rates and transiently eliminate CbN firing for several milliseconds. Remarkably, the refractory period of PCs leads to a brief elevation of CbN firing prior to suppression. Thus, individual PC-CbN synapses are suited to concurrently convey rate codes and generate precisely timed responses in CbN neurons. Either synchronous firing or synchronous pauses of PCs promote CbN neuron firing on rapid time scales for nonuniform inputs, but less effectively than for uniform inputs. This is a secondary consequence of variable input sizes elevating the baseline firing rates of CbN neurons by increasing the variability of the inhibitory conductance. These findings may generalize to other brain regions with highly variable inhibitory synapse sizes.

In addition to its motor functions, the cerebellum is involved in emotional regulation, anxiety and affect. We found that suppressing the firing of cerebellar Purkinje cells (PCs) rapidly excites forebrain areas that contribute to such functions (including the amygdala, basal forebrain and septum), but that the classic cerebellar outputs, the deep cerebellar nuclei, do not directly project there. We show that PCs directly inhibit parabrachial nuclei (PBN) neurons that project to numerous forebrain regions. Suppressing the PC-PBN pathway influences many regions in the forebrain and is aversive. Molecular profiling shows that PCs directly inhibit numerous types of PBN neurons that control diverse behaviors that are not involved in motor control. Therefore, the PC-PBN pathway allows the cerebellum to directly regulate activity in the forebrain, and may be an important substrate for cerebellar disorders arising from damage to the posterior vermis.

Within the cerebellar cortex, mossy fibers (MFs) excite granule cells (GCs) that excite Purkinje cells (PCs), which provide outputs to the deep cerebellar nuclei (DCNs). It is well established that PC disruption produces motor deficits such as ataxia. This could arise from either decreases in ongoing PC-DCN inhibition, increases in the variability of PC firing, or disruption of the flow of MF-evoked signals. Remarkably, it is not known whether GCs are essential for normal motor function. Here we address this issue by selectively eliminating calcium channels that mediate transmission (CaV2.1, CaV2.2, and CaV2.3) in a combinatorial manner. We observe profound motor deficits but only when all CaV2 channels are eliminated. In these mice, the baseline rate and variability of PC firing are unaltered, and locomotion-dependent increases in PC firing are eliminated. We conclude that GCs are indispensable for normal motor performance and that disruption of MF-induced signals impairs motor performance.

Purkinje cells (PCs) are spontaneously active neurons of the cerebellar cortex that inhibit glutamatergic projection neurons within the deep cerebellar nuclei (DCN) that provide the primary cerebellar output. Brief reductions of PC firing rapidly increase DCN neuron firing. However, prolonged reductions of PC inhibition, as seen in some disease states, certain types of transgenic mice, during optogenetic suppression of PC firing, and in acute slices of the cerebellum, do not lead to large, sustained increases in DCN firing. Here we test whether DCN neurons undergo spike frequency adaptation that could account for these properties. We perform current-clamp recordings at near physiological temperature in acute brain slices from mice of both sexes to examine how DCN neurons respond to prolonged depolarizations. DCN neuron adaptation is exceptionally slow and bidirectional. A depolarizing current step evokes large initial increases in firing that decay to ∼20% of the initial increase within ∼10 s. We find that spike frequency adaptation in DCN neurons is mediated by a novel mechanism that is independent of the most promising candidates, including calcium entry and Na+-activated potassium channels mediated by Slo2.1 and Slo2.2 Slow adaptation allows DCN neurons to gradually and bidirectionally adapt to prolonged currents but to respond linearly to current injection on rapid timescales. This suggests that an important consequence of slow adaptation is that DCN neurons respond linearly to the rate of PC firing on rapid timescales but adapt to slow firing rate changes of PCs on long timescales.SIGNIFICANCE STATEMENT Excitatory neurons in the cerebellar nuclei provide the primary output from the cerebellum. This study finds that these neurons exhibit very slow bidirectional spike frequency adaptation that has important implications for cerebellar function. This mechanism allows neurons in the cerebellar nuclei to adapt to long-lasting changes in synaptic drive while also remaining responsive to short-term changes in excitatory or inhibitory drive.

Stress is the most common trigger among episodic neurologic disorders. In episodic ataxia type 2 (EA2), physical or emotional stress causes episodes of severe motor dysfunction that manifest as ataxia and dystonia. We used the tottering (tg/tg) mouse, a faithful animal model of EA2, to dissect the mechanisms underlying stress-induced motor attacks. We find that in response to acute stress, activation of α 1 -adrenergic receptors (α1-Rs) on Purkinje cells by norepinephrine leads to their erratic firing and consequently motor attacks. We show that norepinephrine induces erratic firing of Purkinje cells by disrupting their spontaneous intrinsic pacemaking via a casein kinase 2 (CK2)–dependent signaling pathway, which likely reduces the activity of calcium-dependent potassium channels. Moreover, we report that disruption of this signaling cascade at a number of nodes prevents stress-induced attacks in the tottering mouse. Together, our results suggest that norepinephrine and CK2 are required for the initiation of stress-induced attacks in EA2 and provide previously unidentified targets for therapeutic intervention.

Climbing fibers (CFs) from the inferior olive (IO) provide strong excitatory inputs onto the dendrites of cerebellar Purkinje cells (PC), and trigger distinctive responses known as complex spikes (CSs). We find that in awake, behaving mice, a CS in one PC suppresses conventional simple spikes (SSs) in neighboring PCs for several milliseconds. This involves a novel form of ephaptic coupling, in which an excitatory synapse nonsynaptically inhibits neighboring cells by generating large negative extracellular signals near their dendrites. The distance dependence of CS-SS ephaptic signaling, combined with the known divergence of CF synapses made by IO neurons, allows a single IO neuron to influence the output of the cerebellum by synchronously suppressing the firing of potentially over one hundred PCs. Optogenetic studies in vivo and dynamic clamp studies in slice indicate that such brief PC suppression can effectively promote firing in neurons in the deep cerebellar nuclei and motor thalamus.

Although the cerebellum is traditionally associated with balance and motor function, it also plays wider roles in affective and cognitive behaviors. Evidence suggests that the cerebellar vermis may regulate aggressive behavior, though the cerebellar circuits and patterns of activity that influence aggression remain unclear. We used optogenetic methods to bidirectionally modulate the activity of spatially-delineated cerebellar Purkinje cells to evaluate the impact on aggression in mice. Increasing Purkinje cell activity in the vermis significantly reduced the frequency of attacks in a resident-intruder assay. Reduced aggression was not a consequence of impaired motor function, because optogenetic stimulation did not alter motor performance. In complementary experiments, optogenetic inhibition of Purkinje cells in the vermis increased the frequency of attacks. These results suggest Purkinje cell activity in the cerebellar vermis regulates aggression, and further support the importance of the cerebellum in driving affective behaviors that could contribute to neurological disorders.

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

The graceful, purposeful motion of our body is an engineering feat that remains unparalleled in robotic devices using advanced artificial intelligence. Much of the information required for complex movements is generated by the cerebellum and the basal ganglia in conjunction with the cortex. Cerebellum and basal ganglia have been thought to communicate with each other only through slow, multi-synaptic cortical loops, begging the question as to how they coordinate their outputs in real time. We found that the cerebellum rapidly modulates the activity of the striatum via a disynaptic pathway in mice. Under physiological conditions, this short latency pathway was capable of facilitating optimal motor control by allowing the basal ganglia to incorporate time-sensitive cerebellar information and by guiding the sign of cortico-striatal plasticity. Conversely, under pathological condition, this pathway relayed aberrant cerebellar activity to the basal ganglia to cause dystonia.

The cerebellum has been implicated in a number of nonmotor mental disorders such as autism spectrum disorder, schizophrenia, and addiction. However, its contribution to these disorders is not well understood. In mice, we found that the cerebellum sends direct excitatory projections to the ventral tegmental area (VTA), one of the brain regions that processes and encodes reward. Optogenetic activation of the cerebello-VTA projections was rewarding and, in a three-chamber social task, these projections were more active when the animal explored the social chamber. Intriguingly, activity in the cerebello-VTA pathway was required for the mice to show social preference in this task. Our data delineate a major, previously unappreciated role for the cerebellum in controlling the reward circuitry and social behavior.

Correlated neuronal activity at various timescales plays an important role in information transfer and processing. We find that in awake-behaving mice, an unexpectedly large fraction of neighboring Purkinje cells (PCs) exhibit sub-millisecond synchrony. Correlated firing usually arises from chemical or electrical synapses, but, surprisingly, neither is required to generate PC synchrony. We therefore assessed ephaptic coupling, a mechanism in which neurons communicate via extracellular electrical signals. In the neocortex, ephaptic signals from many neurons summate to entrain spiking on slow timescales, but extracellular signals from individual cells are thought to be too small to synchronize firing. Here we find that a single PC generates sufficiently large extracellular potentials to open sodium channels in nearby PC axons. Rapid synchronization is made possible because ephaptic signals generated by PCs peak during the rising phase of action potentials. These findings show that ephaptic coupling contributes to the prevalent synchronization of nearby PCs.

Optical methods of interrogating neural circuits have emerged as powerful tools for understanding how the brain drives behaviors. Optogenetic proteins are widely used to control neuronal activity, while genetically encoded fluorescent reporters are used to monitor activity. These proteins are often expressed by injecting viruses, which frequently leads to inconsistent experiments due to misalignment of expression and optical components. Here, we describe how silk fibroin films simplify optogenetic experiments by providing targeted delivery of viruses. Films composed of silk fibroin and virus are applied to the surface of implantable optical components. After surgery, silk releases the virus to transduce nearby cells and provide localized expression around optical fibers and endoscopes. Silk films can also be used to express genetically encoded sensors in large cortical regions by using cranial windows coated with a silk/virus mixture. The ease of use and improved performance provided by silk make this a promising approach for optogenetic studies.

The cerebellum and the basal ganglia are critically important for motor control, and their cooperation is crucial to generate the motor signals necessary for proper motor execution and coordination. For decades, direct and functionally relevant communication between these structures was thought to be unlikely due to the lack of corroborating anatomical or functional data. More recent novel methodologies have uncovered the presence of a pathway connecting the output of the basal ganglia to the cerebellum and a disynaptic connection from the cerebellum to the input of the basal ganglia via the thalamus in both rodents and primates. In particular, the disynaptic connection allows for a rapid communication between the cerebellum and the basal ganglia and is capable of modulating synaptic plasticity between the basal ganglia and the motor cortex. These mechanistic insights have helped determine how aberrant activity in the cerebellum can dynamically affect the basal ganglia. Cerebellar-induced dystonia is a clear example in which erratic cerebellar burst firing significantly alters normal basal ganglia activity causing dystonia. Further understanding of this impaired interaction will promote the development of novel therapeutic approaches to target defective networks in multiple pathologies.

Recent studies have identified impairments in neural induction and in striatal and cortical neurogenesis in Huntington's disease (HD) knock-in mouse models and associated embryonic stem cell lines. However, the potential role of these developmental alterations for HD pathogenesis and progression is currently unknown. To address this issue, we used BACHD:CAG-Cre(ERT2) mice, which carry mutant huntingtin (mHtt) modified to harbor a floxed exon 1 containing the pathogenic polyglutamine expansion (Q97). Upon tamoxifen administration at postnatal day 21, the floxed mHtt-exon1 was removed and mHtt expression was terminated (Q97(CRE)). These conditional mice displayed similar profiles of impairments to those mice expressing mHtt throughout life: (i) striatal neurodegeneration, (ii) early vulnerability to NMDA-mediated excitotoxicity, (iii) impairments in motor coordination, (iv) temporally distinct abnormalities in striatal electrophysiological activity, and (v) altered corticostriatal functional connectivity and plasticity. These findings strongly suggest that developmental aberrations may play important roles in HD pathogenesis and progression.

To date, single neuron recordings remain the gold standard for monitoring the activity of neuronal populations. Since obtaining single neuron recordings is not always possible, high frequency or 'multiunit activity' (MUA) is often used as a surrogate. Although MUA recordings allow one to monitor the activity of a large number of neurons, they do not allow identification of specific neuronal subtypes, the knowledge of which is often critical for understanding electrophysiological processes. Here, we explored whether prior knowledge of the single unit waveform of specific neuron types is sufficient to permit the use of MUA to monitor and distinguish differential activity of individual neuron types. We used an experimental and modeling approach to determine if components of the MUA can monitor medium spiny neurons (MSNs) and fast-spiking interneurons (FSIs) in the mouse dorsal striatum. We demonstrate that when well-isolated spikes are recorded, the MUA at frequencies greater than 100Hz is correlated with single unit spiking, highly dependent on the waveform of each neuron type, and accurately reflects the timing and spectral signature of each neuron. However, in the absence of well-isolated spikes (the norm in most MUA recordings), the MUA did not typically contain sufficient information to permit accurate prediction of the respective population activity of MSNs and FSIs. Thus, even under ideal conditions for the MUA to reliably predict the moment-to-moment activity of specific local neuronal ensembles, knowledge of the spike waveform of the underlying neuronal populations is necessary, but not sufficient.

Fn14, the sole known signaling receptor for the TNF family member TWEAK, is inducibly expressed in the central nervous system (CNS) in endothelial cells, astrocytes, microglia, and neurons. There is increasing recognition of the importance of the TWEAK/Fn14 pathway in autoimmune neurologic conditions, including experimental autoimmune encephalomyelitis and neuropsychiatric lupus. Previously, we had found that Fn14 knockout lupus-prone MRL/lpr mice display significantly attenuated neuropsychiatric manifestations. To investigate whether this improvement in disease is secondary to inhibition of TWEAK/Fn14 signaling within the CNS or the periphery, and determine whether TWEAK-mediated neuropsychiatric effects are strain dependent, we performed intracerebroventricular (ICV) injection of Fc-TWEAK or an isotype matched control protein to C57Bl6/J non-autoimmune mice. We found that Fc-TWEAK injected C57Bl6/J mice developed significant depression-like behavior and cognitive dysfunction. Inflammatory mediators associated with lupus brain disease, including CCL2, C3, and iNOS, were significantly elevated in the brains of Fc-TWEAK treated mice. Furthermore, Fc-TWEAK directly increased blood brain barrier (BBB) permeability, as demonstrated by increased IgG deposition in the brain and reduced aquaporin-4 expression. Finally, Fc-TWEAK increased apoptotic cell death in the cortex and hippocampus. In conclusion, TWEAK can contribute to lupus-associated neurobehavioral deficits including depression and cognitive dysfunction by acting within the CNS to enhance production of inflammatory mediators, promote disruption of the BBB, and induce apoptosis in resident brain cells. Our study provides further support that the TWEAK/Fn14 signaling pathway may be a potential therapeutic target for inflammatory diseases involving the CNS.

Although the neurobiology of rodent facial whiskers has been studied intensively, little is known about sensing in other vibrissae. Here we describe the under-investigated submandibular "whisker trident" on the rat's chin. In this three-whisker array, a unique unpaired midline whisker is laterally flanked by two slightly shorter whiskers. All three whiskers point to the ground and are curved backwards. Unlike other whiskers, the trident is not located on an exposed body part. Trident vibrissae are not whisked and do not touch anything over long stretches of time. However, trident whiskers engage in sustained ground contact during head-down running while the animal is exploring or foraging. In biomechanical experiments, trident whiskers follow caudal ground movement more smoothly than facial whiskers. Remarkably, deflection angles decrease with increasing ground velocity. We identified one putative trident barrel in the left somatosensory cortex and two barrels in the right somatosensory cortex. The elongated putative trident-midline barrel is the longest and largest whisker barrel, suggesting that the midline trident whisker is of great functional significance. Cortical postsynaptic air-puff responses in the trident representation show much less temporal precision than facial whisker responses. Trident whiskers do not provide as much high-resolution information about object contacts as facial whiskers. Instead, our observations suggest an idiothetic function: their biomechanics allow trident whiskers to derive continuous measurements about ego motion from ground contacts. The midline position offers unique advantages in sensing heading direction in a laterally symmetric manner. The changes in trident deflection angle with velocity suggest that trident whiskers might function as a tactile speedometer.