Prolonged Type 1 Metabotropic Glutamate Receptor Dependent Synaptic Signaling Contributes to Spino-Cerebellar Ataxia Type 1.

UNLABELLED: Type 1 metabotropic glutamate receptor (mGluR1)-dependent signaling at parallel fiber to Purkinje neuron synapses is critical for cerebellar function. In a mouse model of human spino-cerebellar ataxia type 1 (early SCA1, 12 weeks) we find prolonged parallel fiber mGluR1-dependent synaptic currents and calcium signaling. Acute treatment with a low dose of the potent and specific activity-dependent mGluR1-negative allosteric modulator JNJ16259685 shortened the prolonged mGluR1 currents and rescued the moderate ataxia. Our results provide exciting new momentum for developing mGluR1-based pharmacology to treat ataxia. SIGNIFICANCE STATEMENT: Ataxia is a progressive and devastating degenerative movement disorder commonly associated with loss of cerebellar function and with no known cure. In the early stages of a mouse model of human spinocerebellar ataxia type 1, SCA1, where mice exhibit only moderate motor impairment, we detect excess "gain of function" of metabotropic glutamate receptor signaling at an important cerebellar synapse. Because careful control of this type of signaling is critical for cerebellar function in mice and humans, we sought to remove the excess signaling with a powerful, readily available pharmacological modulator. Remarkably, this pharmacological treatment acutely restored normal motor function in the ataxic mice. Our results pave the way for exploring a new avenue for early treatment of human ataxias.

Are Type 1 metabotropic glutamate receptors a viable therapeutic target for the treatment of cerebellar ataxia?

The cerebellum is a key brain structure for accurate coordination of sensory and motor function. Compared with other brain regions, the cerebellum expresses a particularly high level of Type 1 metabotropic glutamate receptors (mGluR1). In this review we aim to explore the significance of these receptors for cerebellar synapse function and their potential for treating cerebellar ataxia, a poorly treated degenerative motor disorder that is often hereditary. We find a significant and historical literature showing pivotal mechanisms linking mGluR1 activity with healthy cerebellar synaptic function and motor coordination. This is best illustrated by the impaired motor behaviour in mGluR1 knockout mice that bears strong resemblance to human ataxias. More recent literature also indicates that an imbalance of mGluR1 signalling is as critical as its removal. Too much, as well as too little, mGluR1 activity contributes to ataxia in several clinically relevant mouse models, and perhaps also in humans. Given the availability and ongoing refinement of selective pharmacological tools to either reduce (negative allosteric modulation) or boost (positive allosteric modulation) mGluR1 activity, our findings suggest that pharmacological manipulation of these receptors should be explored as an exciting new approach for the treatment of a variety of human cerebellar ataxias.

Voltage imaging to understand connections and functions of neuronal circuits.

Understanding of the cellular mechanisms underlying brain functions such as cognition and emotions requires monitoring of membrane voltage at the cellular, circuit, and system levels. Seminal voltage-sensitive dye and calcium-sensitive dye imaging studies have demonstrated parallel detection of electrical activity across populations of interconnected neurons in a variety of preparations. A game-changing advance made in recent years has been the conceptualization and development of optogenetic tools, including genetically encoded indicators of voltage (GEVIs) or calcium (GECIs) and genetically encoded light-gated ion channels (actuators, e.g., channelrhodopsin2). Compared with low-molecular-weight calcium and voltage indicators (dyes), the optogenetic imaging approaches are 1) cell type specific, 2) less invasive, 3) able to relate activity and anatomy, and 4) facilitate long-term recordings of individual cells' activities over weeks, thereby allowing direct monitoring of the emergence of learned behaviors and underlying circuit mechanisms. We highlight the potential of novel approaches based on GEVIs and compare those to calcium imaging approaches. We also discuss how novel approaches based on GEVIs (and GECIs) coupled with genetically encoded actuators will promote progress in our knowledge of brain circuits and systems.

Expression of the developmental transcription factor Fezf2 identifies a distinct subpopulation of layer 5 intratelencephalic-projection neurons in mature mouse motor cortex.

The transcription factor encoded by Fez family zinc finger 2 (Fezf2) is necessary for normal development of the cerebral cortex. However, Fezf2 continues to be expressed in the mature brain, indicating that it might also be necessary for cortical function throughout life. Here, we show a unique identity of Fezf2-expressing intratelencephalic-projection neurons (IT-PNs) in layer 5 of the mature mouse motor cortex, using a Fezf2-Gfp reporter mouse, in vivo retrograde labeling, whole-cell electrophysiology with morphology reconstruction, and cluster analysis. Fezf2-expressing IT-PNs occupy layer 5A and display an apical dendritic tuft; functionally, they fire broad, adapting action potentials and exhibit an Ih-mediated voltage sag that influences their synaptic properties. In contrast, IT-PNs without Fezf2 expression mainly occupy layer 5B, do not display a tuft, and exhibit regular action potential firing and little sag. Both groups of IT-PNs demonstrated distinct frequency-selective synaptic responses to commissural inputs, indicating unique contributions within the cortical microcircuitry. Our findings establish a new, distinct physiological identity of Fezf2-expressing neurons within mature motor cortex.

Functional contributions of the plasma membrane calcium ATPase and the sodium-calcium exchanger at mouse parallel fibre to Purkinje neuron synapses.

We investigated how two calcium clearance mechanisms, the sodium-calcium exchanger-NCX, and the plasma membrane calcium ATPase-PMCA2, function at the facilitating cerebellar parallel fibre to Purkinje neuron (PF-PN) synapse. Forward mode NCX helped recover PF presynaptic calcium elevations when the PFs received a double stimulation and the calcium load was sufficiently high. A smaller presynaptic calcium load evoked by a single PF stimulation failed to recruit NCX in wild-type mice but did so when PMCA2 was absent in PFs from PMCA2 knockout mice. Simulated calcium dynamics using a simple single-compartment model reported qualitatively similar effects. Functionally, reduced NCX activity in the absence of PMCA also prolonged the recovery of facilitation at the PF-PN synapse, beyond that seen by reduced NCX activity alone. We conclude that PMCA and NCX work in parallel to accurately shape residual presynaptic calcium recovery dynamics and fine-tune facilitation at this important cerebellar synapse.

Transient reversal of the sodium/calcium exchanger boosts presynaptic calcium and synaptic transmission at a cerebellar synapse.

The sodium/calcium exchanger (NCX) is a widespread transporter that exchanges sodium and calcium ions across excitable membranes. Normally, NCX mainly operates in its "forward" mode, harnessing the electrochemical gradient of sodium ions to expel calcium. During membrane depolarization or elevated internal sodium levels, NCX can instead switch the direction of net flux to expel sodium and allow calcium entry. Such "reverse"-mode NCX operation is frequently implicated during pathological or artificially extended periods of depolarization, not during normal activity. We have used fast calcium imaging, mathematical simulation, and whole cell electrophysiology to study the role of NCX at the parallel fiber-to-Purkinje neuron synapse in the mouse cerebellum. We show that nontraditional, reverse-mode NCX activity boosts the amplitude and duration of parallel fiber calcium transients during short bursts of high-frequency action potentials typical of their behavior in vivo. Simulations, supported by experimental manipulations, showed that accumulation of intracellular sodium drove NCX into reverse mode. This mechanism fueled additional calcium influx into the parallel fibers that promoted synaptic transmission to Purkinje neurons for up to 400 ms after the burst. Thus we provide the first functional demonstration of transient and fast NCX-mediated calcium entry at a major central synapse. This unexpected contribution from reverse-mode NCX appears critical for shaping presynaptic calcium dynamics and transiently boosting synaptic transmission, and is likely to optimize the accuracy of cerebellar information transfer.

Enhanced synaptic inhibition in the cerebellar cortex of the ataxic PMCA2(-/-) knockout mouse.

Mice with genetic deletion of a calcium extrusion pump, the plasma membrane calcium ATPase isoform 2, PMCA2, exhibit overt cerebellar ataxia, but the cellular mechanisms are only partially understood. Here, we report an enhanced synaptic GABAergic inhibition within the molecular layer of cerebellar cortex slices from PMCA2 knockout (PMCA2(-/-)) mice, a finding that could contribute to the observed ataxia. Purkinje neurons from PMCA2(-/-) mice exhibited an increased frequency and amplitude of spontaneous inhibitory post-synaptic currents that was accompanied by an enhanced spontaneous firing frequency of molecular layer interneurons (both basket cells and stellate cells). The elevated inhibition was sufficient to reduce the frequency and regularity of spike firing by PMCA2(-/-) Purkinje neurons. Acute pharmacological inhibition of PMCA recapitulated some of these features in wild-type mice indicating that the changes were in part a direct result of PMCA2 removal. However, additional compensatory mechanisms within the PMCA2(-/-) mouse were also a major factor. Indeed, morphological studies revealed an abnormally large number of molecular layer interneurons (basket cells and stellate cells) and GABAergic synapses within the PMCA2(-/-) cerebellar cortex. We conclude that loss of PMCA2 adversely influences the function and organisation of Purkinje neuron synaptic inhibition as a major contributory mechanism to the ataxic phenotype of the PMCA2(-/-) mouse.

The contribution of the sodium-calcium exchanger (NCX) and plasma membrane Ca(2+) ATPase (PMCA) to cerebellar synapse function.

The cerebellum, a part of the brain critically involved in motor learning and sensory adaptation, expresses high levels of the sodium-calcium exchanger (NCX) and the plasma membrane calcium ATPase (PMCA). Both these transporters control calcium dynamics at a variety of synapses, and here, we draw upon the available literature to discuss how NCX and PMCA work together to shape pre-synaptic calcium dynamics at cerebellar synapses.

Functional integration of calcium regulatory mechanisms at Purkinje neuron synapses.

Cerebellar Purkinje neurons receive synaptic inputs from three different sources: the excitatory parallel fibre and climbing fibre synapses as well as the inhibitory synapses from molecular layer stellate and basket cells. These three synaptic systems use distinct mechanisms in order to generate Ca(2+) signals that are specialized for specific modes of neurotransmitter release and post-synaptic signal integration. In this review, we first describe the repertoire of Ca(2+) regulatory mechanisms that generate and regulate the amplitude and timing of Ca(2+) fluxes during synaptic transmission and then explore how these mechanisms interact to generate the unique functional properties of each of the Purkinje neuron synapses.

Altered membrane properties but unchanged intrinsic excitability and spontaneous postsynaptic currents in an aged APP swe /PS1dE9 model of Alzheimer's disease.

Neuronal hyperexcitability in Alzheimer's disease (AD) models is thought to either contribute to the formation of amyloid beta plaques or result from their formation. Neuronal hyperexcitability has been shown in the cerebral cortex of the widely used young APPswe/PS1dE9 mice, which have accelerated plaque formation. However, it is currently unclear if hyperexcitability also occurs in CA1 hippocampal neurons of aged animals in this model. In the present work, we have compared intrinsic excitability and spontaneous synaptic inputs from CA1 pyramidal cells of 8-month-old APPswe/PS1dE9 and wildtype control mice. We find no change in intrinsic excitability or spontaneous postsynaptic currents (PSCs) between groups. We did, however, find a reduced input resistance and an increase in hyperpolarization-activated sag current. These results are consistent with findings from other aged AD model mice, including the widely used 5xFAD and 3xTg. Together these results suggest that neuronal hyperexcitability is not a consistent feature of all AD mouse models, particularly at advanced ages.

Essential Tremor - A Cerebellar Driven Disorder?

Abnormal tremors are the most common of all movement disorders. In this review we focus on the role of the cerebellum in Essential Tremor, a highly debilitating but poorly treated movement disorder. We propose a variety of mechanisms driving abnormal burst firing of deep cerebellar nuclei neurons as a key initiator of tremorgenesis in Essential Tremor. Targetting these mechanisms may generate more effective treatments for Essential Tremor.

Cholinergic modulation of sensory processing in awake mouse cortex.

Cholinergic modulation of brain activity is fundamental for awareness and conscious sensorimotor behaviours, but deciphering the timing and significance of acetylcholine actions for these behaviours is challenging. The widespread nature of cholinergic projections to the cortex means that new insights require access to specific neuronal populations, and on a time-scale that matches behaviourally relevant cholinergic actions. Here, we use fast, voltage imaging of L2/3 cortical pyramidal neurons exclusively expressing the genetically-encoded voltage indicator Butterfly 1.2, in awake, head-fixed mice, receiving sensory stimulation, whilst manipulating the cholinergic system. Altering muscarinic acetylcholine function re-shaped sensory-evoked fast depolarisation and subsequent slow hyperpolarisation of L2/3 pyramidal neurons. A consequence of this re-shaping was disrupted adaptation of the sensory-evoked responses, suggesting a critical role for acetylcholine during sensory discrimination behaviour. Our findings provide new insights into how the cortex processes sensory information and how loss of acetylcholine, for example in Alzheimer's Disease, disrupts sensory behaviours.

Reduced expression of the Ca(2+) transporter protein PMCA2 slows Ca(2+) dynamics in mouse cerebellar Purkinje neurones and alters the precision of motor coordination.

Cerebellar Purkinje neurones (PNs) express high levels of the plasma membrane calcium ATPase, PMCA2, a transporter protein critical for the clearance of calcium from excitable cells. Genetic deletion of one PMCA2 encoding gene in heterozygous PMCA2 knock-out (PMCA2(+/-) mice enabled us to determine how PMCA2 influences PN calcium regulation without the complication of the severe morphological changes associated with complete PMCA2 knock-out (PMCA2(-/-) in these cells. The PMCA2(+/-) cerebellum expressed half the normal levels of PMCA2 and this nearly doubled the time taken for PN dendritic calcium transients to recover (mean fast and slow recovery times increased from 70 ms to 110 ms and from 600 ms to 1100 ms). The slower calcium recovery had distinct consequences for PMCA2(+/-) PN physiology. The PNs exhibited weaker climbing fibre responses, prolonged outward Ca(2+)-dependent K(+) current (mean fast and slow recovery times increased from 136 ms to 192 ms and from 595 ms to 1423 ms) and a slower mean frequency of action potential firing (7.4 Hz compared with 15.8 Hz). Our findings were consistent with prolonged calcium accumulation in the cytosol of PMCA2(+/-) Purkinje neurones. Although PMCA2(+/-) mice exhibited outwardly normal behaviour and little change in their gait pattern, when challenged to run on a narrow beam they exhibited clear deficits in hindlimb coordination. Training improved the motor performance of both PMCA2(+/-) and wild-type mice, although PMCA2(+/-) mice were always impaired. We conclude that reduced calcium clearance perturbs calcium dynamics in PN dendrites and that this is sufficient to disrupt the accuracy of cerebellar processing and motor coordination.

Reduced expression of the "fast" calcium transporter PMCA2a during homeostatic plasticity.

In a model of homeostatic plasticity, hippocampal slice culture CA3 pyramidal neurons responded to excitatory synapse inactivity by enhancing glutamate release through an increased number of miniature excitatory post-synaptic currents, mEPSCs and excitatory pre-synaptic terminals. Also accompanying these changes was a specific reduction in the expression of a "fast" calcium transporter, the plasma membrane calcium ATPase, PMCA2a. This transporter normally influences glutamate release from excitatory terminals where it helps control calcium levels. The reduction in PMCA2a expression occurred within 2 days of synapse inactivity; it was specific and reversible in young and mature hippocampal slice cultures and required removal of NMDA receptor mediated activity. Furthermore, the enhanced mEPSCs in the model were resistant to pharmacological inhibition of PMCA transporter activity. Reduced expression of PMCA2a during homeostatic plasticity therefore provides a mechanism to remodel pre-synaptic Ca2+ dynamics as a flexible way to alter glutamate release.

Plasma membrane Ca2+ ATPase 2 contributes to short-term synapse plasticity at the parallel fiber to Purkinje neuron synapse.

Plasma membrane Ca2+ ATPase 2 (PMCA2) is a fast, highly effective mechanism to control resting cytosolic Ca2+ and Ca2+ excursions in neurons and other excitable cells. The strong expression of PMCA2 in the cerebellum and the cerebellar behavioral deficits presented by PMCA2-/- knock-out mice all point to its importance for cerebellar circuit dynamics. Here, we provide direct functional evidence for the influence of presynaptic PMCA2-mediated Ca2+ extrusion for short-term plasticity at cerebellar parallel fiber to Purkinje neuron synapses. Dramatic structural alterations to the Purkinje neurons in the absence of PMCA2 also suggest a strong influence of this fast PMCA2 isoform for development and maintenance of cerebellar function.

Survival strategies for mouse cerebellar Purkinje neurons lacking PMCA2.

Expression of the fast calcium extrusion protein, PMCA2, in the cerebellum is amongst the highest found throughout the central nervous system, and unsurprisingly PMCA2 knockout mice exhibit cerebellar ataxia or loss of controlled movement. The sole output neurons of the cerebellar cortex, Purkinje neurons, are functionally compromised in these knockout mice, yet remarkably these neurons survive. In this mini-review we review and speculate on the importance of multiple PMCA2 dependent actions at cellular and synaptic sites within the cerebellar Purkinje neuron network. We also explore how loss of PMCA2-/- can lead to the ataxic phenotype, but can paradoxically also minimise calcium rises in cerebellar Purkinje neurons, thereby ensuring their resilience and survival.

Transcriptome Profiling of Layer 5 Intratelencephalic Projection Neurons From the Mature Mouse Motor Cortex.

The mature cortex contains hugely diverse populations of pyramidal projection neurons (PNs), critical to normal forebrain circuits. In order to understand the healthy cortex, it is essential to characterize this neuronal complexity. We recently demonstrated different identities for Fezf2-positive (Fezf2+ve) and Fezf2-negative (Fezf2-ve) intratelencephalic-PNs (IT-PNs) from layer 5 of the motor cortex (M1). Comparatively, each IT-PN type has a distinct electrophysiological phenotype and the Fezf2+ve IT-PNs display a unique apical dendritic tuft. Here, we aimed to expand our understanding of the molecular underpinnings defining these unique IT-PN types. Using a validated Fezf2-GFP reporter mouse, retrograde labeling techniques and fluorescence activated cell sorting (FACS), combined with a novel approach for low-input RNA-sequencing, we isolated mature Fezf2+ve and Fezf2-ve IT-PNs for transcriptome profiling. Through the comparison of Fezf2+ve and Fezf2-ve IT-PN gene expression profiles, we identified significant enrichment of 81 genes in the Fezf2+ve IT-PNs and 119 genes in the Fezf2-ve IT-PNs. Term enrichment analysis of these enriched genes demonstrated significant overrepresentation of the calcium-binding EF-hand domain in Fezf2+ve IT-PNs, suggesting a greater importance for calcium handling in these neurons. Of the Fezf2-ve IT-PN enriched genes an unexpected and unique enrichment of genes, previously associated with microglia were identified. Our dataset identifies the molecular profiles of two unique IT-PN types in the mature M1, providing important targets to investigate for their maintenance in the healthy mature brain.

RNA-Sequencing Analysis Reveals a Regulatory Role for Transcription Factor Fezf2 in the Mature Motor Cortex.

Forebrain embryonic zinc finger (Fezf2) encodes a transcription factor essential for the specification of layer 5 projection neurons (PNs) in the developing cerebral cortex. As with many developmental transcription factors, Fezf2 continues to be expressed into adulthood, suggesting it remains crucial to the maintenance of neuronal phenotypes. Despite the continued expression, a function has yet to be explored for Fezf2 in the PNs of the developed cortex. Here, we investigated the role of Fezf2 in mature neurons, using lentiviral-mediated delivery of a shRNA to conditionally knockdown the expression of Fezf2 in the mouse primary motor cortex (M1). RNA-sequencing analysis of Fezf2-reduced M1 revealed significant changes to the transcriptome, identifying a regulatory role for Fezf2 in the mature M1. Kyoto Encyclopedia Genes and Genomes (KEGG) pathway analyses of Fezf2-regulated genes indicated a role in neuronal signaling and plasticity, with significant enrichment of neuroactive ligand-receptor interaction, cell adhesion molecules and calcium signaling pathways. Gene Ontology analysis supported a functional role for Fezf2-regulated genes in neuronal transmission and additionally indicated an importance in the regulation of behavior. Using the mammalian phenotype ontology database, we identified a significant overrepresentation of Fezf2-regulated genes associated with specific behavior phenotypes, including associative learning, social interaction, locomotor activation and hyperactivity. These roles were distinct from that of Fezf2-regulated genes identified in development, indicating a dynamic transition in Fezf2 function. Together our findings demonstrate a regulatory role for Fezf2 in the mature brain, with Fezf2-regulated genes having functional roles in sustaining normal neuronal and behavioral phenotypes. These results support the hypothesis that developmental transcription factors are important for maintaining neuron transcriptomes and that disruption of their expression could contribute to the progression of disease phenotypes.

Motor and Cerebellar Architectural Abnormalities during the Early Progression of Ataxia in a Mouse Model of SCA1 and How Early Prevention Leads to a Better Outcome Later in Life.

Exposing developing cerebellar Purkinje neurons (PNs) to mutant Ataxin1 (ATXN1) in 82Q spinocerebellar ataxia type 1 (SCA1) mice disrupts motor behavior and cerebellar climbing fiber (CF) architecture from as early as 4 weeks of age. In contrast, if mutant ATXN1 expression is silenced until after cerebellar development is complete, then its impact on motor behavior and cerebellar architecture is greatly reduced. Under these conditions even 6 month old SCA1 mice exhibit largely intact motor behavior and molecular layer (ML) and CF architecture but show a modest reduction in PN soma area as a first sign of cerebellar disruption. Our results contrast the sensitivity of the developing cerebellum and remarkable resilience of the adult cerebellum to mutant ATXN1 and imply that SCA1 in this mouse model is both a developmental and neurodegenerative disorder.

Fezf2 expression in layer 5 projection neurons of mature mouse motor cortex.

The mature cerebral cortex contains a wide diversity of neuron phenotypes. This diversity is specified during development by neuron-specific expression of key transcription factors, some of which are retained for the life of the animal. One of these key developmental transcription factors that is also retained in the adult is Fezf2, but the neuron types expressing it in the mature cortex are unknown. With a validated Fezf2-Gfp reporter mouse, whole-cell electrophysiology with morphology reconstruction, cluster analysis, in vivo retrograde labeling, and immunohistochemistry, we identify a heterogeneous population of Fezf2(+) neurons in both layer 5A and layer 5B of the mature motor cortex. Functional electrophysiology identified two distinct subtypes of Fezf2(+) neurons that resembled pyramidal tract projection neurons (PT-PNs) and intratelencephalic projection neurons (IT-PNs). Retrograde labeling confirmed the former type to include corticospinal projection neurons (CSpPNs) and corticothalamic projection neurons (CThPNs), whereas the latter type included crossed corticostriatal projection neurons (cCStrPNs) and crossed-corticocortical projection neurons (cCCPNs). The two Fezf2(+) subtypes expressed either CTIP2 or SATB2 to distinguish their physiological identity and confirmed that specific expression combinations of key transcription factors persist in the mature motor cortex. Our findings indicate a wider role for Fezf2 within gene expression networks that underpin the diversity of layer 5 cortical projection neurons.