Cognitive and social capacities require postnatal experience, yet the pathways by which experience guides development are unknown. Here we show that the normal development of motor and nonmotor capacities requires cerebellar activity. Using chemogenetic perturbation of molecular layer interneurons to attenuate cerebellar output in mice, we found that activity of posterior regions in juvenile life modulates adult expression of eyeblink conditioning (paravermal lobule VI, crus I), reversal learning (lobule VI), persistive behavior and novelty-seeking (lobule VII), and social preference (crus I/II). Perturbation in adult life altered only a subset of phenotypes. Both adult and juvenile disruption left gait metrics largely unaffected. Contributions to phenotypes increased with the amount of lobule inactivated. Using an anterograde transsynaptic tracer, we found that posterior cerebellum made strong connections with prelimbic, orbitofrontal, and anterior cingulate cortex. These findings provide anatomical substrates for the clinical observation that cerebellar injury increases the risk of autism.
Although feedforward inhibition onto Purkinje cells was first documented 40 years ago, we understand little of how inhibitory interneurons contribute to cerebellar function in behaving animals. Using a mouse line (PC-Deltagamma2) in which GABA(A) receptor-mediated synaptic inhibition is selectively removed from Purkinje cells, we examined how feedforward inhibition from molecular layer interneurons regulates adaptation of the vestibulo-ocular reflex. Although impairment of baseline motor performance was relatively mild, the ability to adapt the phase of the vestibulo-ocular reflex and to consolidate gain adaptations was strongly compromised. Purkinje cells showed abnormal patterns of simple spikes, both during and in the absence of evoked compensatory eye movements. On the basis of modeling our experimental data, we propose that feedforward inhibition, by controlling the fine-scale patterns of Purkinje cell activity, enables the induction of plasticity in neurons of the cerebellar and vestibular nuclei.
Translating neuronal activity to measurable behavioral changes has been a long-standing goal of systems neuroscience. Recently, we have developed a model of phase-reversal learning of the vestibulo-ocular reflex, a well-established, cerebellar-dependent task. The model, comprising both the cerebellar cortex and vestibular nuclei, reproduces behavioral data and accounts for the changes in neural activity during learning in wild type mice. Here, we used our model to predict Purkinje cell spiking as well as behavior before and after learning of five different lines of mutant mice with distinct cell-specific alterations of the cerebellar cortical circuitry. We tested these predictions by obtaining electrophysiological data depicting changes in neuronal spiking. We show that our data is largely consistent with the model predictions for simple spike modulation of Purkinje cells and concomitant behavioral learning in four of the mutants. In addition, our model accurately predicts a shift in simple spike activity in a mutant mouse with a brainstem specific mutation. This combination of electrophysiological and computational techniques opens a possibility of predicting behavioral impairments from neural activity.
Sensory integration difficulties have been reported in autism, but their underlying brain-circuit mechanisms are underexplored. Using five autism-related mouse models, Shank3+/ΔC, Mecp2(R308/Y), Cntnap2-/-, L7-Tsc1 (L7/Pcp2(Cre)::Tsc1(flox/+)), and patDp(15q11-13)/+, we report specific perturbations in delay eyeblink conditioning, a form of associative sensory learning requiring cerebellar plasticity. By distinguishing perturbations in the probability and characteristics of learned responses, we found that probability was reduced in Cntnap2-/-, patDp(15q11-13)/+, and L7/Pcp2(Cre)::Tsc1(flox/+), which are associated with Purkinje-cell/deep-nuclear gene expression, along with Shank3+/ΔC. Amplitudes were smaller in L7/Pcp2(Cre)::Tsc1(flox/+) as well as Shank3+/ΔC and Mecp2(R308/Y), which are associated with granule cell pathway expression. Shank3+/ΔC and Mecp2(R308/Y) also showed aberrant response timing and reduced Purkinje-cell dendritic spine density. Overall, our observations are potentially accounted for by defects in instructed learning in the olivocerebellar loop and response representation in the granule cell pathway. Our findings indicate that defects in associative temporal binding of sensory events are widespread in autism mouse models.
A major goal of the BRAIN Initiative is the development of technologies to monitor neuronal network activity during active information processing. Toward this goal, genetically encoded calcium indicator proteins have become widely used for reporting activity in preparations ranging from invertebrates to awake mammals. However, slow response times, the narrow sensitivity range of Ca and in some cases, poor signal-to-noise ratio still limit their usefulness. Here, we review recent improvements in the field of neural activity-sensitive probe design with a focus on the GCaMP family of calcium indicator proteins. In this context, we present our newly developed Fast-GCaMPs, which have up to 4-fold accelerated off-responses compared with the next-fastest GCaMP, GCaMP6f. Fast-GCaMPs were designed by destabilizing the association of the hydrophobic pocket of calcium-bound calmodulin with the RS20 binding domain, an intramolecular interaction that protects the green fluorescent protein chromophore. Fast-GCaMP6f-RS06 and Fast-GCaMP6f-RS09 have rapid off-responses in stopped-flow fluorimetry, in neocortical brain slices, and in the intact cerebellum . Fast-GCaMP6f variants should be useful for tracking action potentials closely spaced in time, and for following neural activity in fast-changing compartments, such as axons and dendrites. Finally, we discuss strategies that may allow tracking of a wider range of neuronal firing rates and improve spike detection.
Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions.
Cerebellar research has focused principally on adult motor function. However, the cerebellum also maintains abundant connections with nonmotor brain regions throughout postnatal life. Here we review evidence that the cerebellum may guide the maturation of remote nonmotor neural circuitry and influence cognitive development, with a focus on its relationship with autism. Specific cerebellar zones influence neocortical substrates for social interaction, and we propose that sensitive-period disruption of such internal brain communication can account for autism's key features.
The cerebellum fine-tunes motor activity via its Purkinje cell output. Purkinje cells produce two different types of spikes, complex spikes and simple spikes, which often show reciprocal activity: a periodical increase in complex spikes is associated with a decrease in simple spikes, and vice versa. This reciprocal firing is thought to be essential for coordinated motor behavior, yet how it is accomplished is debated. Here, we show in Ptf1a::cre;Robo3(lox/lox) mice that selectively rerouting the climbing fibers from a contralateral to an ipsilateral projection reversed the complex-spike modulation during sensory stimulation. Strikingly, modulation of simple spikes, which is supposed to be controlled by mossy fibers, reversed as well. Climbing fibers enforce this reciprocity in part by influencing activity of inhibitory interneurons, because the phase of their activity was also converted. Ptf1a::cre;Robo3(lox/lox) mice showed severe ataxia highlighting that climbing fiber input and its impact on reciprocity of Purkinje cell firing play an important role in motor coordination.
Cerebellar cortical throughput involved in motor control comprises granule cells (GCs) and Purkinje cells (PCs), both of which receive inhibitory GABAergic input from interneurons. The GABAergic input to PCs is essential for learning and consolidation of the vestibulo-ocular reflex, but the role of GC excitability remains unclear. We now disrupted the Kcc2 K-Cl cotransporter specifically in either cell type to manipulate their excitability and inhibition by GABA(A)-receptor Cl(-) channels. Although Kcc2 may have a morphogenic role in synapse development, Kcc2 disruption neither changed synapse density nor spine morphology. In both GCs and PCs, disruption of Kcc2, but not Kcc3, increased [Cl(-)](i) roughly two-fold. The reduced Cl(-) gradient nearly abolished GABA-induced hyperpolarization in PCs, but in GCs it merely affected excitability by membrane depolarization. Ablation of Kcc2 from GCs impaired consolidation of long-term phase learning of the vestibulo-ocular reflex, whereas baseline performance, short-term gain-decrease learning and gain consolidation remained intact. These functions, however, were affected by disruption of Kcc2 in PCs. GC excitability plays a previously unknown, but specific role in consolidation of phase learning.
For half a century it was assumed that granule cells use ultra-sparse encoding, but now in vivo calcium-imaging studies have shown that large ensembles of granule cells provide dense signals, which themselves evolve and adapt during training.
Cerebellar granule cells, which constitute half the brain's neurons, supply Purkinje cells with contextual information necessary for motor learning, but how they encode this information is unknown. Here we show, using two-photon microscopy to track neural activity over multiple days of cerebellum-dependent eyeblink conditioning in mice, that granule cell populations acquire a dense representation of the anticipatory eyelid movement. Initially, granule cells responded to neutral visual and somatosensory stimuli as well as periorbital airpuffs used for training. As learning progressed, two-thirds of monitored granule cells acquired a conditional response whose timing matched or preceded the learned eyelid movements. Granule cell activity covaried trial by trial to form a redundant code. Many granule cells were also active during movements of nearby body structures. Thus, a predictive signal about the upcoming movement is widely available at the input stage of the cerebellar cortex, as required by forward models of cerebellar control.