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.

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.

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.

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.

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.

Upregulation of SYNGAP1 expression in mice and human neurons by redirecting alternative splicing.

The Ras GTPase-activating protein SYNGAP1 plays a central role in synaptic plasticity, and de novo SYNGAP1 mutations are among the most frequent causes of autism and intellectual disability. How SYNGAP1 is regulated during development and how to treat SYNGAP1-associated haploinsufficiency remain challenging questions. Here, we characterize an alternative 3' splice site (A3SS) of SYNGAP1 that induces nonsense-mediated mRNA decay (A3SS-NMD) in mouse and human neural development. We demonstrate that PTBP1/2 directly bind to and promote SYNGAP1 A3SS inclusion. Genetic deletion of the Syngap1 A3SS in mice upregulates Syngap1 protein and alleviates the long-term potentiation and membrane excitability deficits caused by a Syngap1 knockout allele. We further report a splice-switching oligonucleotide (SSO) that converts SYNGAP1 unproductive isoform to the functional form in human iPSC-derived neurons. This study describes the regulation and function of SYNGAP1 A3SS-NMD, the genetic rescue of heterozygous Syngap1 knockout mice, and the development of an SSO to potentially alleviate SYNGAP1-associated haploinsufficiency.

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.

Sensory Over-responsivity and Aberrant Plasticity in Cerebellar Cortex in a Mouse Model of Syndromic Autism.

Background: Patients with autism spectrum disorder often show altered responses to sensory stimuli as well as motor deficits, including an impairment of delay eyeblink conditioning, which involves integration of sensory signals in the cerebellum. Here, we identify abnormalities in parallel fiber (PF) and climbing fiber (CF) signaling in the mouse cerebellar cortex that may contribute to these pathologies. Methods: We used a mouse model for the human 15q11-13 duplication (patDp/+) and studied responses to sensory stimuli in Purkinje cells from awake mice using two-photon imaging of GCaMP6f signals. Moreover, we examined synaptic transmission and plasticity using in vitro electrophysiological, immunohistochemical, and confocal microscopic techniques. Results: We found that spontaneous and sensory-evoked CF-calcium transients are enhanced in patDp/+ Purkinje cells, and aversive movements are more severe across sensory modalities. We observed increased expression of the synaptic organizer NRXN1 at CF synapses and ectopic spread of these synapses to fine dendrites. CF-excitatory postsynaptic currents recorded from Purkinje cells are enlarged in patDp/+ mice, while responses to PF stimulation are reduced. Confocal measurements show reduced PF+CF-evoked spine calcium transients, a key trigger for PF long-term depression, one of several plasticity types required for eyeblink conditioning learning. Long-term depression is impaired in patDp/+ mice but is rescued on pharmacological enhancement of calcium signaling. Conclusions: Our findings suggest that this genetic abnormality causes a pathological inflation of CF signaling, possibly resulting from enhanced NRXN1 expression, with consequences for the representation of sensory stimuli by the CF input and for PF synaptic organization and plasticity.

Behavioral Tests for Mouse Models of Autism: An Argument for the Inclusion of Cerebellum-Controlled Motor Behaviors.

Mouse models of Autism Spectrum Disorder (ASD) have been interrogated using a variety of behavioral tests in order to understand the symptoms of ASD. However, the hallmark behaviors that are classically affected in ASD - deficits in social interaction and communication as well as the occurrence of repetitive behaviors - do not have direct murine equivalents. Thus, it is critical to identify the caveats that come with modeling a human disorder in mice. The most commonly used behavioral tests represent complex cognitive processes based on largely unknown brain circuitry. Motor impairments provide an alternative, scientifically rigorous approach to understanding ASD symptoms. Difficulties with motor coordination and learning - seen in both patients and mice - point to an involvement of the cerebellum in ASD pathology. This brain area supports types of motor learning that are conserved throughout vertebrate evolution, allowing for direct comparisons of functional abnormalities between humans with autism and ASD mouse models. Studying simple motor behaviors provides researchers with clearly interpretable results. We describe and evaluate methods used on mouse behavioral assays designed to test for social, communicative, perseverative, anxious, nociceptive, and motor learning abnormalities. We comment on the effectiveness and validity of each test based on how much information its results give, as well as its relevance to ASD, and will argue for an inclusion of cerebellum-supported motor behaviors in the phenotypic description of ASD mouse models. LAY SUMMARY: Mouse models of Autism Spectrum Disorder help us gain insight about ASD symptoms in human patients. However, there are many differences between mice and humans, which makes interpreting behaviors challenging. Here, we discuss a battery of behavioral tests for specific mouse behaviors to explore whether each test does indeed evaluate the intended measure, and whether these tests are useful in learning about ASD.

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.

Muscarinic Modulation of SK2-Type K+ Channels Promotes Intrinsic Plasticity in L2/3 Pyramidal Neurons of the Mouse Primary Somatosensory Cortex.

Muscarinic acetylcholine receptors (mAChRs) inhibit small-conductance calcium-activated K+ channels (SK channels) and enhance synaptic weight via this mechanism. SK channels are also involved in activity-dependent plasticity of membrane excitability ("intrinsic plasticity"). Here, we investigate whether mAChR activation can drive SK channel-dependent intrinsic plasticity in L2/3 cortical pyramidal neurons. Using whole-cell patch-clamp recordings from these neurons in slices prepared from mouse primary somatosensory cortex (S1), we find that brief bath application of the mAChR agonist oxotremorine-m (oxo-m) causes long-term enhancement of excitability in wild-type mice that is not observed in mice deficient of SK channels of the SK2 isoform. Similarly, repeated injection of depolarizing current pulses into the soma triggers intrinsic plasticity that is absent from SK2 null mice. Intrinsic plasticity lowers spike frequency adaptation and attenuation of spike firing upon prolonged activation, consistent with SK channel modulation. Depolarization-induced plasticity is prevented by bath application of the protein kinase A (PKA) inhibitor H89, and the casein kinase 2 (CK2) inhibitor TBB, respectively. These findings point toward a recruitment of two known signaling pathways in SK2 regulation: SK channel trafficking (PKA) and reduction of the calcium sensitivity (CK2). Using mice with an inactivation of CaMKII (T305D mice), we show that intrinsic plasticity does not require CaMKII. Finally, we demonstrate that repeated injection of depolarizing pulses in the presence of oxo-m causes intrinsic plasticity that surpasses the plasticity amplitude reached by either manipulation alone. Our findings show that muscarinic activation enhances membrane excitability in L2/3 pyramidal neurons via a downregulation of SK2 channels.

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.

Development of Hierarchically Porous Ionomer Membranes for Versatile and Fast Metal Ion Conduction.

Innovative design concepts can play a key role in the realization of high-performance ionomer membranes that are capable of exclusive metal ion conduction and potentially applicable in electrochemical devices including sensors, fuel cells, and high-energy batteries. Herein, we report on the development of new ionomers, based on sulfonated poly(ether ether ketone) (SPEEK), engineered to conduct a variety of ions, namely, Li+, Na+, K+, Zn2+, and Mg2+, when soaked with nonaqueous solvents. Application of a facile phase-inversion method results in M-SPEEK (M = Li/Na/K/Zn/Mg) membranes with a hierarchical porous network, facilitating organic solvent infusion that is necessary to promote dissociation and rapid transport of cations between anionic sulfonate groups on the polymer chains. This strategy leads to membranes with alkali ion conductivities approaching 10-4 S cm-1 at room temperature, and near unity cation transference numbers (t M+ ≥ 0.9). Furthermore, an exceptionally high Zn-ion conductivity of 10-2 S cm-1 is obtained for the water-infused Zn-SPEEK membrane. In comparison, the dense membranes demonstrate 2-3 orders of magnitude lower conductivities because of insufficient solvent infusion. Preliminary electrochemical studies with solvent-infused ionomer membranes as the electrolyte look promising.

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.

α1ACT Is Essential for Survival and Early Cerebellar Programming in a Critical Neonatal Window.

Postnatal cerebellar development is a precisely regulated process involving well-orchestrated expression of neural genes. Neurological phenotypes associated with CACNA1A gene defects have been increasingly recognized, yet the molecular principles underlying this association remain elusive. By characterizing a dose-dependent CACNA1A gene deficiency mouse model, we discovered that α1ACT, as a transcription factor and secondary protein of CACNA1A mRNA, drives dynamic gene expression networks within cerebellar Purkinje cells and is indispensable for neonatal survival. Perinatal loss of α1ACT leads to motor dysfunction through disruption of neurogenesis and synaptic regulatory networks. However, its elimination in adulthood has minimal effect on the cerebellum. These findings shed light on the critical role of α1ACT in facilitating neuronal development in both mice and humans and support a rationale for gene therapies for calcium-channel-associated cerebellar disorders. Finally, we show that bicistronic expression may be common to the voltage-gated calcium channel (VGCC) gene family and may help explain complex genetic syndromes.

Synaptic Potential and Plasticity of an SK2 Channel Gate Regulate Spike Burst Activity in Cerebellar Purkinje Cells.

Neurons store information and participate in memory engrams as a result of experience-dependent changes in synaptic weights and in membrane excitability. Here, we examine excitatory postsynaptic potential (EPSP) amplitude and neuronal excitability in relation to these two mechanisms of plasticity. We analyze somato-dendritic double-patch recordings from cerebellar Purkinje cells while inducing intrinsic, SK2 channel-dependent plasticity or blocking SK channels with bath application of apamin. Both manipulations increase the build-up of EPSP amplitudes during an EPSP train and enhance the number of EPSP-evoked spikes, yielding insights into the mechanistic contribution of EPSP amplitude to single spikes and spike bursts. EPSP amplitude has an impact on whether spikes are fired or not, but direct measures of excitability (spike threshold/AHP) are better predictors of whether individual spikes or spike bursts are fired. Our findings show that Purkinje cell spiking is synaptically driven but that burst firing is gated by SK2 channel modulation and plasticity.

Toward a Neurocentric View of Learning.

Synaptic plasticity (e.g., long-term potentiation [LTP]) is considered the cellular correlate of learning. Recent optogenetic studies on memory engram formation assign a critical role in learning to suprathreshold activation of neurons and their integration into active engrams ("engram cells"). Here we review evidence that ensemble integration may result from LTP but also from cell-autonomous changes in membrane excitability. We propose that synaptic plasticity determines synaptic connectivity maps, whereas intrinsic plasticity-possibly separated in time-amplifies neuronal responsiveness and acutely drives engram integration. Our proposal marks a move away from an exclusively synaptocentric toward a non-exclusive, neurocentric view of learning.