Second cistron in CACNA1A gene encodes a transcription factor mediating cerebellar development and SCA6.

The CACNA1A gene, encoding the voltage-gated calcium channel subunit α1A, is involved in pre- and postsynaptic Ca(2+) signaling, gene expression, and several genetic neurological disorders. We found that CACNA1A coordinates gene expression using a bicistronic mRNA bearing a cryptic internal ribosomal entry site (IRES). The first cistron encodes the well-characterized α1A subunit. The second expresses a transcription factor, α1ACT, which coordinates expression of a program of genes involved in neural and Purkinje cell development. α1ACT also contains the polyglutamine (polyQ) tract that, when expanded, causes spinocerebellar ataxia type 6 (SCA6). When expressed as an independent polypeptide, α1ACT-bearing an expanded polyQ tract-lacks transcription factor function and neurite outgrowth properties, causes cell death in culture, and leads to ataxia and cerebellar atrophy in transgenic mice. Suppression of CACNA1A IRES function in SCA6 may be a potential therapeutic strategy.

Intrinsic threshold plasticity: cholinergic activation and role in the neuronal recognition of incomplete input patterns.

Activity-dependent changes in membrane excitability are observed in neurons across brain areas and represent a cell-autonomous form of plasticity (intrinsic plasticity; IP) that in itself does not involve alterations in synaptic strength (synaptic plasticity; SP). Non-homeostatic IP may play an essential role in learning, e.g. by changing the action potential threshold near the soma. A computational problem, however, arises from the implication that such amplification does not discriminate between synaptic inputs and therefore may reduce the resolution of input representation. Here, we investigate consequences of IP for the performance of an artificial neural network in (a) the discrimination of unknown input patterns and (b) the recognition of known/learned patterns. While negative changes in threshold potentials in the output layer indeed reduce its ability to discriminate patterns, they benefit the recognition of known but incompletely presented patterns. An analysis of thresholds and IP-induced threshold changes in published sets of physiological data obtained from whole-cell patch-clamp recordings from L2/3 pyramidal neurons in (a) the primary visual cortex (V1) of awake macaques and (b) the primary somatosensory cortex (S1) of mice in vitro, respectively, reveals a difference between resting and threshold potentials of ∼15 mV for V1 and ∼25 mV for S1, and a total plasticity range of ∼10 mV (S1). The most efficient activity pattern to lower threshold is paired cholinergic and electric activation. Our findings show that threshold reduction promotes a shift in neural coding strategies from accurate faithful representation to interpretative assignment of input patterns to learned object categories. KEY POINTS: Intrinsic plasticity may change the action potential threshold near the soma of neurons (threshold plasticity), thus altering the input-output function for all synaptic inputs 'upstream' of the plasticity location. A potential problem arising from this shared amplification is that it may reduce the ability to discriminate between different input patterns. Here, we assess the performance of an artificial neural network in the discrimination of unknown input patterns as well as the recognition of known patterns subsequent to changes in the spike threshold. We observe that negative changes in threshold potentials do reduce discrimination performance, but at the same time improve performance in an object recognition task, in particular when patterns are incompletely presented. Analysis of whole-cell patch-clamp recordings from pyramidal neurons in the primary somatosensory cortex (S1) of mice reveals that negative threshold changes preferentially result from electric stimulation of neurons paired with the activation of muscarinic acetylcholine receptors.

SK2 channels in cerebellar Purkinje cells contribute to excitability modulation in motor-learning-specific memory traces.

Neurons store information by changing synaptic input weights. In addition, they can adjust their membrane excitability to alter spike output. Here, we demonstrate a role of such "intrinsic plasticity" in behavioral learning in a mouse model that allows us to detect specific consequences of absent excitability modulation. Mice with a Purkinje-cell-specific knockout (KO) of the calcium-activated K+ channel SK2 (L7-SK2) show intact vestibulo-ocular reflex (VOR) gain adaptation but impaired eyeblink conditioning (EBC), which relies on the ability to establish associations between stimuli, with the eyelid closure itself depending on a transient suppression of spike firing. In these mice, the intrinsic plasticity of Purkinje cells is prevented without affecting long-term depression or potentiation at their parallel fiber (PF) input. In contrast to the typical spike pattern of EBC-supporting zebrin-negative Purkinje cells, L7-SK2 neurons show reduced background spiking but enhanced excitability. Thus, SK2 plasticity and excitability modulation are essential for specific forms of motor learning.

Intrinsic Excitability Increase in Cerebellar Purkinje Cells after Delay Eye-Blink Conditioning in Mice.

Cerebellar-based learning is thought to rely on synaptic plasticity, particularly at synaptic inputs to Purkinje cells. Recently, however, other complementary mechanisms have been identified. Intrinsic plasticity is one such mechanism, and depends in part on the downregulation of calcium-dependent SK-type K+ channels, which contribute to a medium-slow afterhyperpolarization (AHP) after spike bursts, regulating membrane excitability. In the hippocampus, intrinsic plasticity plays a role in trace eye-blink conditioning; however, corresponding excitability changes in the cerebellum in associative learning, such as in trace or delay eye-blink conditioning, are less well studied. Whole-cell patch-clamp recordings were obtained from Purkinje cells in cerebellar slices prepared from male mice ∼48 h after they learned a delay eye-blink conditioning task. Over a period of repeated training sessions, mice received either paired trials of a tone coterminating with a periorbital shock (conditioning) or trials in which these stimuli were randomly presented in an unpaired manner (pseudoconditioning). Purkinje cells from conditioned mice show a significantly reduced AHP after trains of parallel fiber stimuli and after climbing fiber evoked complex spikes. The number of spikelets in the complex spike waveform is increased after conditioning. Moreover, we find that SK-dependent intrinsic plasticity is occluded in conditioned, but not pseudoconditioned mice. These findings show that excitability is enhanced in Purkinje cells after delay eye-blink conditioning, and point toward a downregulation of SK channels as a potential underlying mechanism. The observation that this learning effect lasts at least up to 2 d after training shows that intrinsic plasticity regulates excitability in the long term.SIGNIFICANCE STATEMENT Plasticity of membrane excitability ("intrinsic plasticity") has been observed in invertebrate and vertebrate neurons, coinduced with synaptic plasticity or in isolation. Although the cellular phenomenon per se is well established, it remains unclear what role intrinsic plasticity plays in learning and if it even persists long enough to serve functions in engram physiology beyond aiding synaptic plasticity. Here, we demonstrate that cerebellar Purkinje cells upregulate excitability in delay eye-blink conditioning, a form of motor learning. This plasticity is observed 48 h after training and alters synaptically evoked spike firing and integrative properties of these neurons. These findings show that intrinsic plasticity enhances the spike firing output of Purkinje cells and persists over the course of days.

Complex spike clusters and false-positive rejection in a cerebellar supervised learning rule.

KEY POINTS: Spike doublets comprise ∼10% of in vivo complex spike events under spontaneous conditions and ∼20% (up to 50%) under evoked conditions. Under near-physiological slice conditions, single complex spikes do not induce parallel fibre long-term depression. Doublet stimulation is required to induce long-term depression with an optimal parallel-fibre to first-complex-spike timing interval of 150 ms. ABSTRACT: The classic example of biological supervised learning occurs at cerebellar parallel fibre (PF) to Purkinje cell synapses, comprising the most abundant synapse in the mammalian brain. Long-term depression (LTD) at these synapses is driven by climbing fibres (CFs), which fire continuously about once per second and therefore generate potential false-positive events. We show that pairs of complex spikes are required to induce LTD. In vivo, sensory stimuli evoked complex-spike doublets with intervals ≤150 ms in up to 50% of events. Using realistic [Ca2+ ]o and [Mg2+ ]o concentrations in slices, we determined that complex-spike doublets delivered 100-150 ms after PF stimulus onset were required to trigger PF-LTD, which is consistent with the requirements for eyeblink conditioning. Inter-complex spike intervals of 50-150 ms provided optimal decoding. This stimulus pattern prolonged evoked spine calcium signals and promoted CaMKII activation. Doublet activity may provide a means for CF instructive signals to stand out from background firing.

Calcium threshold shift enables frequency-independent control of plasticity by an instructive signal.

At glutamatergic synapses, both long-term potentiation (LTP) and long-term depression (LTD) can be induced at the same synaptic activation frequency. Instructive signals determine whether LTP or LTD is induced, by modulating local calcium transients. Synapses maintain the ability to potentiate or depress over a wide frequency range, but it remains unknown how calcium-controlled plasticity operates when frequency variations alone cause differences in calcium amplitudes. We addressed this problem at cerebellar parallel fiber-Purkinje cell synapses, which can undergo LTD or LTP in response to 1-Hz and 100-Hz stimulation. We observed that high-frequency activation elicits larger spine calcium transients than low-frequency stimulation under all stimulus conditions, but, regardless of activation frequency, climbing fiber (CF) coactivation provides an instructive signal that further enhances calcium transients and promotes LTD. At both frequencies, buffering calcium prevents LTD induction and LTP results instead, identifying the enhanced calcium signal amplitude as the critical parameter contributed by the instructive CF signal. These observations show that it is not absolute calcium amplitudes that determine whether LTD or LTP is evoked but, instead, the LTD threshold slides, thus preserving the requirement for relatively larger calcium transients for LTD than for LTP induction at any given stimulus frequency. Cerebellar LTD depends on the activation of calcium/calmodulin-dependent kinase II (CaMKII). Using genetically modified (TT305/6VA and T305D) mice, we identified α-CaMKII inhibition upon autophosphorylation at Thr305/306 as a molecular event underlying the threshold shift. This mechanism enables frequency-independent plasticity control by the instructive CF signal based on relative, not absolute, calcium thresholds.

Asymmetries in Cerebellar Plasticity and Motor Learning.

Synaptic plasticity at the parallel fiber to Purkinje cell synapse has long been considered a cellular correlate for cerebellar motor learning. Functionally, long-term depression and long-term potentiation at these synapses seem to be the reverse of each other, with both pre- and post-synaptic expression occurring in both. However, different cerebellar motor learning paradigms have been shown to be asymmetric and not equally reversible. Here, we discuss the asymmetric reversibility shown in the vestibulo-ocular reflex and eyeblink conditioning and suggest that different cellular plasticity mechanisms might be recruited under different conditions leading to unequal reversibility.

Muscarinic acetylcholine receptor activation blocks long-term potentiation at cerebellar parallel fiber-Purkinje cell synapses via cannabinoid signaling.

Muscarinic acetylcholine receptors (mAChRs) are known to modulate synaptic plasticity in various brain areas. A signaling pathway triggered by mAChR activation is the production and release of endocannabinoids that bind to type 1 cannabinoid receptors (CB1R) located on synaptic terminals. Using whole-cell patch-clamp recordings from rat cerebellar slices, we have demonstrated that the muscarinic agonist oxotremorine-m (oxo-m) blocks the induction of presynaptic long-term potentiation (LTP) at parallel fiber (PF)-Purkinje cell synapses in a CB1R-dependent manner. Under control conditions, LTP was induced by delivering 120 PF stimuli at 8 Hz. In contrast, no LTP was observed when oxo-m was present during tetanization. PF-LTP was restored when the CB1R antagonist N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251) was coapplied with oxo-m. Furthermore, the suppressive effect of oxo-m on PF-LTP was abrogated by the GDP analog GDP-ß-S (applied intracellularly), the phospholipase C inhibitor U-73122, and the diacylglycerol lipase inhibitor tetrahydrolipstatin (THL), suggesting that cannabinoid synthesis results from the activation of Gq-coupled mAChRs present on Purkinje cells. The oxo-m-mediated suppression of LTP was also prevented in the presence of the M3 receptor antagonist DAU 5884, and was absent in M1/M3 receptor double-KO mice, identifying M3 receptors as primary oxo-m targets. Our findings allow for the possibility that cholinergic signaling in the cerebellum--which may result from long-term depression (LTD)-related disinhibition of cholinergic neurons in the vestibular nuclei--suppresses presynaptic LTP to prevent an up-regulation of transmitter release that opposes the reduction of postsynaptic responsiveness. This modulatory capacity of mAChR signaling could promote the functional penetrance of LTD.

The Spontaneous Ataxic Mouse Mutant Tippy is Characterized by a Novel Purkinje Cell Morphogenesis and Degeneration Phenotype.

This study represents the first detailed analysis of the spontaneous neurological mouse mutant, tippy, uncovering its unique cerebellar phenotype. Homozygous tippy mutant mice are small, ataxic, and die around weaning. Although the cerebellum shows grossly normal foliation, tippy mutants display a complex cerebellar Purkinje cell phenotype consisting of abnormal dendritic branching with immature spine features and patchy, non-apoptotic cell death that is associated with widespread dystrophy and degeneration of the Purkinje cell axons throughout the white matter, the cerebellar nuclei, and the vestibular nuclei. Moderate anatomical abnormalities of climbing fiber innervation of tippy mutant Purkinje cells were not associated with changes in climbing fiber-EPSC amplitudes. However, decreased ESPC amplitudes were observed in response to parallel fiber stimulation and correlated well with anatomical evidence for patchy dark cell degeneration of Purkinje cell dendrites in the molecular layer. The data suggest that the Purkinje neurons are a primary target of the tippy mutation. Furthermore, we hypothesize that the Purkinje cell axonal pathology together with disruptions in the balance of climbing fiber and parallel fiber-Purkinje cell input in the cerebellar cortex underlie the ataxic phenotype in these mice. The constellation of Purkinje cell dendritic malformation and degeneration phenotypes in tippy mutants is unique and has not been reported in any other neurologic mutant. Fine mapping of the tippy mutation to a 2.1 MB region of distal chromosome 9, which does not encompass any gene previously implicated in cerebellar development or neuronal degeneration, confirms that the tippy mutation identifies novel biology and gene function.

Enhanced AMPA receptor function promotes cerebellar long-term depression rather than potentiation.

Ampakines are allosteric modulators of AMPA receptors that facilitate hippocampal long-term potentiation (LTP) and learning, and have been considered for the treatment of cognition and memory deficits. Here, we show that the ampakine CX546 raises the amplitude and slows the decay time of excitatory postsynaptic currents (EPSCs) at cerebellar parallel fiber (PF) to Purkinje cell synapses, thus resembling CX546 effects described at hippocampal synapses. Using the fluorescent calcium indicator dye Oregon Green BAPTA-2 and an ultra-high-speed CCD camera, we also monitored calcium transients in Purkinje cell dendrites. In the presence of CX546 in the bath, PF-evoked calcium transients were enhanced and prolonged, suggesting that CX546 not only enhances synaptic transmission, but also boosts dendritic calcium signaling at cerebellar synapses. In contrast to previous observations in the hippocampus, however, CX546 applied during cerebellar recordings facilitates long-term depression (LTD) rather than LTP at PF synapses. These findings show that ampakines selectively modify the LTP-LTD balance depending on the brain area and type of synapse, and may provide tools for the targeted regulation of synaptic memories.

Contiguity in perception: origins in cellular associative computations.

Our brains are good at detecting and learning associative structures; according to some linguistic theories, this capacity even constitutes a prerequisite for the development of syntax and compositionality in language and verbalized thought. I will argue that the search for associative motifs in input patterns is an evolutionary old brain function that enables contiguity in sensory perception and orientation in time and space. It has its origins in an elementary material property of cells that is particularly evident at chemical synapses: input-assigned calcium influx that activates calcium sensor proteins involved in memory storage. This machinery for the detection and learning of associative motifs generates knowledge about input relationships and integrates this knowledge into existing networks through updates in connectivity patterns.

Intrinsic and synaptic determinants of receptive field plasticity in Purkinje cells of the mouse cerebellum.

Non-synaptic (intrinsic) plasticity of membrane excitability contributes to aspects of memory formation, but it remains unclear whether it merely facilitates synaptic long-term potentiation or plays a permissive role in determining the impact of synaptic weight increase. We use tactile stimulation and electrical activation of parallel fibers to probe intrinsic and synaptic contributions to receptive field plasticity in awake mice during two-photon calcium imaging of cerebellar Purkinje cells. Repetitive activation of both stimuli induced response potentiation that is impaired in mice with selective deficits in either synaptic or intrinsic plasticity. Spatial analysis of calcium signals demonstrated that intrinsic, but not synaptic plasticity, enhances the spread of dendritic parallel fiber response potentiation. Simultaneous dendrite and axon initial segment recordings confirm these dendritic events affect axonal output. Our findings support the hypothesis that intrinsic plasticity provides an amplification mechanism that exerts a permissive control over the impact of long-term potentiation on neuronal responsiveness.

Pinceau organization in the cerebellum requires distinct functions of neurofascin in Purkinje and basket neurons during postnatal development.

Basket axon collaterals synapse onto the Purkinje soma/axon initial segment (AIS) area to form specialized structures, the pinceau, which are critical for normal cerebellar function. Mechanistic details of how the pinceau become organized during cerebellar development are poorly understood. Loss of cytoskeletal adaptor protein Ankyrin G (AnkG) results in mislocalization of the cell adhesion molecule Neurofascin (Nfasc) at the Purkinje AIS and abnormal organization of the pinceau. Loss of Nfasc in adult Purkinje neurons leads to slow disorganization of the Purkinje AIS and pinceau morphology. Here, we used mouse conditional knock-out techniques to show that selective loss of Nfasc, specifically in Purkinje neurons during early development, prevented maturation of the AIS and resulted in loss of Purkinje neuron spontaneous activity and pinceau disorganization. Loss of Nfasc in both Purkinje and basket neurons caused abnormal basket axon collateral branching and targeting to Purkinje soma/AIS, leading to extensive pinceau disorganization, Purkinje neuron degeneration, and severe ataxia. Our studies reveal that the Purkinje Nfasc is required for AIS maturation and for maintaining stable contacts between basket axon terminals and the Purkinje AIS during pinceau organization, while the basket neuron Nfasc in combination with Purkinje Nfasc is required for proper basket axon collateral outgrowth and targeting to Purkinje soma/AIS. Thus, cerebellar pinceau organization requires coordinated mechanisms involving specific Nfasc functions in both Purkinje and basket neurons.

Ethanol affects NMDA receptor signaling at climbing fiber-Purkinje cell synapses in mice and impairs cerebellar LTD.

Ethanol profoundly influences cerebellar circuit function and motor control. It has recently been demonstrated that functional N-methyl-(D)-aspartate (NMDA) receptors are postsynaptically expressed at climbing fiber (CF) to Purkinje cell synapses in the adult cerebellum. Using whole cell patch-clamp recordings from mouse cerebellar slices, we examined whether ethanol can affect NMDA receptor signaling in mature Purkinje cells. NMDA receptor-mediated currents were isolated by bath application of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzol[f]quinoxaline (NBQX). The remaining (D)-2-amino-5-phosphonovaleric acid ((D)-APV)-sensitive current was reduced by ethanol at concentrations as low as 10 mM. At a concentration of 50 mM ethanol, the blockade of (D)-APV-sensitive CF-excitatory postsynaptic currents was significantly stronger. Ethanol also altered the waveform of CF-evoked complex spikes by reducing the afterdepolarization. This effect was not seen when NMDA receptors were blocked by (D)-APV before ethanol wash-in. In contrast to CF synaptic transmission, parallel fiber (PF) synaptic inputs were not affected by ethanol. Finally, ethanol (10 mM) impaired long-term depression (LTD) at PF to Purkinje cell synapses as induced under control conditions by paired PF and CF activity. However, LTD induced by pairing PF stimulation with depolarizing voltage steps (substituting for CF activation) was not blocked by ethanol. These observations suggest that the sensitivity of cerebellar circuit function and plasticity to low concentrations of ethanol may be caused by an ethanol-mediated impairment of NMDA receptor signaling at CF synapses onto cerebellar Purkinje cells.

Climbing fiber multi-innervation of mouse Purkinje dendrites with arborization common to human.

Canonically, each Purkinje cell (PC) in the adult cerebellum receives only one climbing fiber (CF) from the inferior olive. Underlying current theories of cerebellar function is the notion that this highly conserved one-to-one relationship renders Purkinje dendrites into a single computational compartment. However, we discovered that multiple primary dendrites are a near-universal morphological feature in humans. Using tract tracing, immunolabeling, and in vitro electrophysiology, we found that in mice ~25% of mature multibranched cells receive more than one CF input. Two-photon calcium imaging in vivo revealed that separate dendrites can exhibit distinct response properties to sensory stimulation, indicating that some multibranched cells integrate functionally independent CF-receptive fields. These findings indicate that PCs are morphologically and functionally more diverse than previously thought.

Intrinsic and synaptic determinants of receptive field plasticity in Purkinje cells of the mouse cerebellum.

Non-synaptic ('intrinsic') plasticity of membrane excitability contributes to aspects of memory formation, but it remains unclear whether it merely facilitates synaptic long-term potentiation (LTP), or whether it plays a permissive role in determining the impact of synaptic weight increase. We use tactile stimulation and electrical activation of parallel fibers to probe intrinsic and synaptic contributions to receptive field (RF) plasticity in awake mice during two-photon calcium imaging of cerebellar Purkinje cells. Repetitive activation of both stimuli induced response potentiation that is impaired in mice with selective deficits in either intrinsic plasticity (SK2 KO) or LTP (CaMKII TT305/6VA). Intrinsic, but not synaptic, plasticity expands the local, dendritic RF representation. Simultaneous dendrite and axon initial segment recordings confirm that these dendritic events affect axonal output. Our findings support the hypothesis that intrinsic plasticity provides an amplification mechanism that exerts a permissive control over the impact of LTP on neuronal responsiveness.

Overexpression of the autism candidate gene Cyfip1 pathologically enhances olivo-cerebellar signaling in mice.

Cyfip1, the gene encoding cytoplasmic FMR1 interacting protein 1, has been of interest as an autism candidate gene for years. A potential role in autism spectrum disorder (ASD) is suggested by its location on human chromosome 15q11-13, an instable region that gives rise to a variety of copy number variations associated with syndromic autism. In addition, the CYFIP1 protein acts as a binding partner to Fragile X Messenger Ribonucleoprotein (FMRP) in the regulation of translation initiation. Mutation of FMR1, the gene encoding FMRP, causes Fragile X syndrome, another form of syndromic autism. Here, in mice overexpressing CYFIP1, we study response properties of cerebellar Purkinje cells to activity of the climbing fiber input that originates from the inferior olive and provides an instructive signal in sensorimotor input analysis and plasticity. We find that CYFIP1 overexpression results in enhanced localization of the synaptic organizer neurexin 1 (NRXN1) at climbing fiber synaptic input sites on Purkinje cell primary dendrites and concomitant enhanced climbing fiber synaptic transmission (CF-EPSCs) measured using whole-cell patch-clamp recordings from Purkinje cells in vitro. Moreover, using two-photon measurements of GCaMP6f-encoded climbing fiber signals in Purkinje cells of intact mice, we observe enhanced responses to air puff stimuli applied to the whisker field. These findings resemble our previous phenotypic observations in a mouse model for the human 15q11-13 duplication, which does not extend to the Cyfip1 locus. Thus, our study demonstrates that CYFIP1 overexpression shares a limited set of olivo-cerebellar phenotypes as those resulting from an increased number of copies of non-overlapping genes located on chromosome 15q11-13.