Human antibodies neutralizing the alpha-latrotoxin of the European black widow.

Poisoning by widow-spider (genus Latrodectus) bites occurs worldwide. The illness, termed latrodectism, can cause severe and persistent pain and can lead to muscle rigidity, respiratory complications, and cardiac problems. It is a global health challenge especially in developing countries. Equine serum-derived polyclonal anti-sera are commercially available as a medication for patients with latrodectism, but the use of sera imposes potential inherent risks related to its animal origin. The treatment may cause allergic reactions in humans (serum sickness), including anaphylactic shock. Furthermore, equine-derived antivenom is observed to have batch-to-batch variability and poor specificity, as it is always an undefined mix of antibodies. Because latrodectism can be extremely painful but is rarely fatal, the use of antivenom is controversial and only a small fraction of patients is treated. In this work, recombinant human antibodies were selected against alpha-latrotoxin of the European black widow (Latrodectus tredecimguttatus) by phage display from a naïve antibody gene library. Alpha-Latrotoxin (α-LTX) binding scFv were recloned and produced as fully human IgG. A novel alamarBlue assay for venom neutralization was developed and used to select neutralizing IgGs. The human antibodies showed in vitro neutralization efficacy both as single antibodies and antibody combinations. This was also confirmed by electrophysiological measurements of neuronal activity in cell culture. The best neutralizing antibodies showed nanomolar affinities. Antibody MRU44-4-A1 showed outstanding neutralization efficacy and affinity to L. tredecimguttatus α-LTX. Interestingly, only two of the neutralizing antibodies showed cross-neutralization of the venom of the Southern black widow (Latrodectus mactans). This was unexpected, because in the current literature the alpha-latrotoxins are described as highly conserved. The here-engineered antibodies are candidates for future development as potential therapeutics and diagnostic tools, as they for the first time would provide unlimited supply of a chemically completely defined drug of constant quality and efficacy, which is also made without the use of animals.

Human iPSC-Derived Neurons with Reliable Synapses and Large Presynaptic Action Potentials.

Understanding the function of the human brain requires determining basic properties of synaptic transmission in human neurons. One of the most fundamental parameters controlling neurotransmitter release is the presynaptic action potential, but its amplitude and duration remain controversial. Presynaptic action potentials have so far been measured with high temporal resolution only in a limited number of vertebrate but not in human neurons. To uncover properties of human presynaptic action potentials, we exploited recently developed tools to generate human glutamatergic neurons by transient expression of Neurogenin 2 (Ngn2) in pluripotent stem cells. During maturation for 3 to 9 weeks of culturing in different established media, the proportion of cells with multiple axon initial segments decreased, while the amount of axonal tau protein and neuronal excitability increased. Super-resolution microscopy revealed the alignment of the pre- and postsynaptic proteins, Bassoon and Homer. Synaptic transmission was surprisingly reliable at frequencies of 20, 50, and 100 Hz. The synchronicity of synaptic transmission during high-frequency transmission increased during 9 weeks of neuronal maturation. To analyze the mechanisms of synchronous high-frequency glutamate release, we developed direct presynaptic patch-clamp recordings from human neurons. The presynaptic action potentials had large overshoots to ∼25 mV and short durations of ∼0.5 ms. Our findings show that Ngn2-induced neurons represent an elegant model system allowing for functional, structural, and molecular analyses of glutamatergic synaptic transmission with high spatiotemporal resolution in human neurons. Furthermore, our data predict that glutamatergic transmission is mediated by large and rapid presynaptic action potentials in the human brain.

Peripherally-derived LGI1-reactive monoclonal antibodies cause epileptic seizures in vivo.

One striking clinical hallmark in patients with autoantibodies to leucine-rich glioma inactivated 1 (LGI1) is the very frequent focal seizure semiologies, including faciobrachial dystonic seizures (FBDS), in addition to the amnesia. Polyclonal serum IgGs have successfully modelled the cognitive changes in vivo but not seizures. Hence, it remains unclear whether LGI1-autoantibodies are sufficient to cause seizures. We tested this with the molecularly precise monoclonal antibodies directed against LGI1 (LGI1-mAbs), derived from patient circulating B cells. These were directed towards both major domains of LGI1, LRR and EPTP and infused intracerebroventricularly over 7 days into juvenile male Wistar rats using osmotic pumps. Continuous wireless EEG was recorded from a depth electrode placed in hippocampal CA3 plus behavioural tests for memory and hyperexcitability were performed. Following infusion completion (Day 9), post-mortem brain slices were studied for antibody binding and effects on Kv1.1. The LGI1-mAbs bound most strongly in the hippocampal CA3 region and induced a significant reduction in Kv1.1 cluster number in this subfield. By comparison to control-Ab injected rats video-EEG analysis over 9 days revealed convulsive and non-convulsive seizure activity in rats infused with LGI1-mAbs, with a significant number of ictal events. Memory was not impaired in the novel object recognition test. Peripherally-derived human LGI1-mAbs infused into rodent CSF provide strong evidence of direct in vivo epileptogenesis with molecular correlations. These findings fulfill criteria for LGI1-antibodies in seizure causation.

Phosphatidylinositol 3,5-bisphosphate facilitates axonal vesicle transport and presynapse assembly.

Neurons relay information via specialized presynaptic compartments for neurotransmission. Unlike conventional organelles, the specialized apparatus characterizing the neuronal presynapse must form de novo. How the components for presynaptic neurotransmission are transported and assembled is poorly understood. Our results show that the rare late endosomal signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] directs the axonal cotransport of synaptic vesicle and active zone proteins in precursor vesicles in human neurons. Precursor vesicles are distinct from conventional secretory organelles, endosomes, and degradative lysosomes and are transported by coincident detection of PI(3,5)P2 and active ARL8 via kinesin KIF1A to the presynaptic compartment. Our findings identify a crucial mechanism that mediates the delivery of synaptic vesicle and active zone proteins to developing synapses.

Fully-primed slowly-recovering vesicles mediate presynaptic LTP at neocortical neurons.

Pre- and postsynaptic forms of long-term potentiation (LTP) are candidate synaptic mechanisms underlying learning and memory. At layer 5 pyramidal neurons, LTP increases the initial synaptic strength but also short-term depression during high-frequency transmission. This classical form of presynaptic LTP has been referred to as redistribution of synaptic efficacy. However, the underlying mechanisms remain unclear. We therefore performed whole-cell recordings from layer 5 pyramidal neurons in acute cortical slices of rats and analyzed presynaptic function before and after LTP induction by paired pre- and postsynaptic neuronal activity. LTP was successfully induced in about half of the synaptic connections tested and resulted in increased synaptic short-term depression during high-frequency transmission and a decelerated recovery from short-term depression due to an increased fraction of a slow recovery component. Analysis with a recently established sequential two-step vesicle priming model indicates an increase in the abundance of fully-primed and slowly-recovering vesicles. A systematic analysis of short-term plasticity and synapse-to-synapse variability of synaptic strength at various types of synapses revealed that stronger synapses generally recover more slowly from synaptic short-term depression. Finally, pharmacological stimulation of the cyclic adenosine monophosphate and diacylglycerol signaling pathways, which are both known to promote synaptic vesicle priming, mimicked LTP and slowed the recovery from short-term depression. Our data thus demonstrate that LTP at layer 5 pyramidal neurons increases synaptic strength primarily by enlarging a subpool of fully-primed slowly-recovering vesicles.

Human NMDAR autoantibodies disrupt excitatory-inhibitory balance, leading to hippocampal network hypersynchrony.

Anti-NMDA receptor autoantibodies (NMDAR-Abs) in patients with NMDAR encephalitis cause severe disease symptoms resembling psychosis and cause cognitive dysfunction. After passive transfer of patients' cerebrospinal fluid or human monoclonal anti-GluN1-autoantibodies in mice, we find a disrupted excitatory-inhibitory balance resulting from CA1 neuronal hypoexcitability, reduced AMPA receptor (AMPAR) signaling, and faster synaptic inhibition in acute hippocampal slices. Functional alterations are also reflected in widespread remodeling of the hippocampal proteome, including changes in glutamatergic and GABAergic neurotransmission. NMDAR-Abs amplify network γ oscillations and disrupt θ-γ coupling. A data-informed network model reveals that lower AMPAR strength and faster GABAA receptor current kinetics chiefly account for these abnormal oscillations. As predicted in silico and evidenced ex vivo, positive allosteric modulation of AMPARs alleviates aberrant γ activity, reinforcing the causative effects of the excitatory-inhibitory imbalance. Collectively, NMDAR-Ab-induced aberrant synaptic, cellular, and network dynamics provide conceptual insights into NMDAR-Ab-mediated pathomechanisms and reveal promising therapeutic targets that merit future in vivo validation.

What calcium channels remember.

A new mechanism involving intermediate gating states of calcium channels explains how analogue postsynaptic potentials influence neurotransmitter release.

Cav2.2 Channels Sustain Vesicle Recruitment at a Mature Glutamatergic Synapse.

The composition of voltage-gated Ca2+ channel (Cav) subtypes that gate action potential (AP)-evoked release changes during the development of mammalian CNS synapses. Cav2.2 and Cav2.3 lose their function in gating-evoked release during postnatal synapse maturation. In mature boutons, Cav2.1 currents provide the almost exclusive trigger for evoked release, and Cav2.3 currents are required for the induction of presynaptic long-term potentiation. However, the functional significance of Cav2.2 remained elusive in mature boutons, although they remain present at active zones and continue contributing significantly to presynaptic Ca2+ influx. Here, we addressed the functional significance of Cav2.2 and Cav2.3 at mature parallel-fiber (PF) to Purkinje neuron synapses of mice of either sex. These synapses are known to exhibit the corresponding developmental Cav subtype changes in gating release. We addressed two hypotheses, namely that Cav2.2 and Cav2.3 are involved in triggering spontaneous glutamate release and that they are engaged in vesicle recruitment during repetitive evoked release. We found that spontaneous miniature release is Ca2+ dependent. However, experiments with Cav subtype-specific blockers excluded the spontaneous opening of Cavs as the Ca2+ source for spontaneous glutamate release. Thus, neither Cav2.2 nor Cav2.3 controls spontaneous release from PF boutons. Furthermore, vesicle recruitment during brief bursts of APs was also independent of Ca2+ influx through Cav2.2 and Cav2.3. However, Cav2.2, but not Cav2.3, currents significantly boosted vesicle recruitment during sustained high-frequency synaptic transmission. Thus, in mature PF boutons Cav2.2 channels are specifically required to sustain synaptic transmission during prolonged neuronal activity.SIGNIFICANCE STATEMENT At young CNS synapses, action potential-evoked release is gated via three subtypes of voltage-gated Ca2+ channels: Cav2.1, Cav2.2, and Cav2.3. During postnatal maturation, Cav2.2 and Cav2.3 lose their function in gating evoked release, such that at mature synapses Cav2.1 provides the almost exclusive source for triggering evoked release. Cav2.3 currents are required for the induction of presynaptic long-term potentiation. However, the function of the still abundant Cav2.2 in mature boutons remained largely elusive. Here, we studied mature cerebellar parallel-fiber synapses and found that Cav2.2 does not control spontaneous release. However, Ca2+ influx through Cav2.2 significantly boosted vesicle recruitment during trains of action potentials. Thus, Cav2.2 in mature parallel-fiber boutons participate in sustaining synaptic transmission during prolonged activity.

Comparative Effects of Domain-Specific Human Monoclonal Antibodies Against LGI1 on Neuronal Excitability.

BACKGROUND AND OBJECTIVES: Autoantibodies to leucine-rich glioma inactivated protein 1 (LGI1) cause an autoimmune limbic encephalitis with frequent focal seizures and anterograde memory dysfunction. LGI1 is a neuronal secreted linker protein with 2 functional domains: the leucine-rich repeat (LRR) and epitempin (EPTP) regions. LGI1 autoantibodies are known to interfere with presynaptic function and neuronal excitability; however, their epitope-specific mechanisms are incompletely understood. METHODS: We used patient-derived monoclonal autoantibodies (mAbs), which target either LRR or EPTP domains of LGI1 to investigate long-term antibody-induced alteration of neuronal function. LRR- and EPTP-specific effects were evaluated by patch-clamp recordings in cultured hippocampal neurons and compared with biophysical neuron modeling. Kv1.1 channel clustering at the axon initial segment (AIS) was quantified by immunocytochemistry and structured illumination microscopy techniques. RESULTS: Both EPTP and LRR domain-specific mAbs decreased the latency of first somatic action potential firing. However, only the LRR-specific mAbs increased the number of action potential firing together with enhanced initial instantaneous frequency and promoted spike-frequency adaptation, which were less pronounced after the EPTP mAb. This also led to an effective reduction in the slope of ramp-like depolarization in the subthreshold response, suggesting Kv1 channel dysfunction. A biophysical model of a hippocampal neuron corroborated experimental results and suggests that an isolated reduction of the conductance of Kv1-mediated K+ currents largely accounts for the antibody-induced alterations in the initial firing phase and spike-frequency adaptation. Furthermore, Kv1.1 channel density was spatially redistributed from the distal toward the proximal site of AIS under LRR mAb treatment and, to a lesser extant, under EPTP mAb. DISCUSSION: These findings indicate an epitope-specific pathophysiology of LGI1 autoantibodies. The pronounced neuronal hyperexcitability and SFA together with dropped slope of ramp-like depolarization after LRR-targeted interference suggest disruption of LGI1-dependent clustering of K+ channel complexes. Moreover, considering the effective triggering of action potentials at the distal AIS, the altered spatial distribution of Kv1.1 channel density may contribute to these effects through impairing neuronal control of action potential initiation and synaptic integration.

NMDA-receptor-Fc-fusion constructs neutralize anti-NMDA receptor antibodies.

N-methyl-D-aspartate receptor (NMDAR) encephalitis is the most common subtype of autoimmune encephalitis characterized by a complex neuropsychiatric syndrome usually including memory impairment. Patients develop an intrathecal immune response against NMDARs with antibodies that presumably bind to the amino-terminal domain of the GluN1 subunit. The therapeutic response to immunotherapy is often delayed. Therefore, new therapeutic approaches for fast neutralization of NMDAR antibodies are needed. Here, we developed fusion constructs consisting of the Fc part of immunoglobulin G and the amino-terminal domains of either GluN1 or combinations of GluN1 with GluN2A or GluN2B. Surprisingly, both GluN1 and GluN2 subunits were required to generate high-affinity epitopes. The construct with both subunits efficiently prevented NMDAR binding of patient-derived monoclonal antibodies and of patient CSF containing high-titre NMDAR antibodies. Furthermore, it inhibited the internalization of NMDARs in rodent dissociated neurons and human induced pluripotent stem cell-derived neurons. Finally, the construct stabilized NMDAR currents recorded in rodent neurons and rescued memory defects in passive-transfer mouse models using intrahippocampal injections. Our results demonstrate that both GluN1 and GluN2B subunits contribute to the main immunogenic region of the NMDAR and provide a promising strategy for fast and specific treatment of NMDAR encephalitis, which could complement immunotherapy.

Direct whole-cell patch-clamp recordings from small boutons in rodent primary neocortical neuron cultures.

Direct electrical recordings from conventional boutons in the mammalian central nervous system have proven challenging due to their small size. Here, we provide a protocol for direct whole-cell patch-clamp recordings from small presynaptic boutons of primary dissociated cultured neurons of the rodent neocortex. We describe steps to prepare primary neocortical cultures and recording pipettes, followed by identifying boutons and establishing a whole-cell bouton recording. We then provide details on precise pipette capacitance compensation required for high-resolution current-clamp recordings from boutons. For further details on the use and execution of this protocol, please refer to Ritzau-Jost et al.1.

p53-dependent c-Fos expression is a marker but not executor for motor neuron death in spinal muscular atrophy mouse models.

The activation of the p53 pathway has been associated with neuronal degeneration in different neurological disorders, including spinal muscular atrophy (SMA) where aberrant expression of p53 drives selective death of motor neurons destined to degenerate. Since direct p53 inhibition is an unsound therapeutic approach due carcinogenic effects, we investigated the expression of the cell death-associated p53 downstream targets c-fos, perp and fas in vulnerable motor neurons of SMA mice. Fluorescence in situ hybridization (FISH) of SMA motor neurons revealed c-fos RNA as a promising candidate. Accordingly, we identified p53-dependent nuclear upregulation of c-Fos protein in degenerating motor neurons from the severe SMNΔ7 and intermediate Smn2B/- SMA mouse models. Although motor neuron-specific c-fos genetic deletion in SMA mice did not improve motor neuron survival or motor behavior, p53-dependent c-Fos upregulation marks vulnerable motor neurons in different mouse models. Thus, nuclear c-Fos accumulation may serve as a readout for therapeutic approaches targeting neuronal death in SMA and possibly other p53-dependent neurodegenerative diseases.

An action potential initiation mechanism in distal axons for the control of dopamine release.

Information flow in neurons proceeds by integrating inputs in dendrites, generating action potentials near the soma, and releasing neurotransmitters from nerve terminals in the axon. We found that in the striatum, acetylcholine-releasing neurons induce action potential firing in distal dopamine axons. Spontaneous activity of cholinergic neurons produced dopamine release that extended beyond acetylcholine-signaling domains, and traveling action potentials were readily recorded from dopamine axons in response to cholinergic activation. In freely moving mice, dopamine and acetylcholine covaried with movement direction. Local inhibition of nicotinic acetylcholine receptors impaired dopamine dynamics and affected movement. Our findings uncover an endogenous mechanism for action potential initiation independent of somatodendritic integration and establish that this mechanism segregates the control of dopamine signaling between axons and somata.

Laser microscopy acquisition and analysis of premotor synapses in the murine spinal cord.

Loss of synapses on spinal motor neurons is a major feature of several neurodegenerative diseases; however, analyzing these premotor synapses is challenging because of their small size and high density. This protocol describes confocal and Stimulated Emission Depletion (STED) imaging of murine spinal premotor synapses and their segment-specific quantification by confocal microscopy. We detail the preparation of spinal cord segments, followed by image acquisition and analysis. This protocol enables in-depth analysis of pathological changes in spinal premotor synapses during neurodegeneration. For complete details on the use and execution of this protocol, please refer to Buettner et al. (2021).

Calcium dependence of neurotransmitter release at a high fidelity synapse.

The Ca2+-dependence of the priming, fusion, and replenishment of synaptic vesicles are fundamental parameters controlling neurotransmitter release and synaptic plasticity. Despite intense efforts, these important steps in the synaptic vesicles' cycle remain poorly understood due to the technical challenge in disentangling vesicle priming, fusion, and replenishment. Here, we investigated the Ca2+-sensitivity of these steps at mossy fiber synapses in the rodent cerebellum, which are characterized by fast vesicle replenishment mediating high-frequency signaling. We found that the basal free Ca2+ concentration (<200 nM) critically controls action potential-evoked release, indicating a high-affinity Ca2+ sensor for vesicle priming. Ca2+ uncaging experiments revealed a surprisingly shallow and non-saturating relationship between release rate and intracellular Ca2+ concentration up to 50 µM. The rate of vesicle replenishment during sustained elevated intracellular Ca2+ concentration exhibited little Ca2+-dependence. Finally, quantitative mechanistic release schemes with five Ca2+ binding steps incorporating rapid vesicle replenishment via parallel or sequential vesicle pools could explain our data. We thus show that co-existing high- and low-affinity Ca2+ sensors mediate priming, fusion, and replenishment of synaptic vesicles at a high-fidelity synapse.

Large, Stable Spikes Exhibit Differential Broadening in Excitatory and Inhibitory Neocortical Boutons.

Presynaptic action potential spikes control neurotransmitter release and thus interneuronal communication. However, the properties and the dynamics of presynaptic spikes in the neocortex remain enigmatic because boutons in the neocortex are small and direct patch-clamp recordings have not been performed. Here, we report direct recordings from boutons of neocortical pyramidal neurons and interneurons. Our data reveal rapid and large presynaptic action potentials in layer 5 neurons and fast-spiking interneurons reliably propagating into axon collaterals. For in-depth analyses, we establish boutons of mature cultured neurons as models for excitatory neocortical boutons, demonstrating that the presynaptic spike amplitude is unaffected by potassium channels, homeostatic long-term plasticity, and high-frequency firing. In contrast to the stable amplitude, presynaptic spikes profoundly broaden during high-frequency firing in layer 5 pyramidal neurons, but not in fast-spiking interneurons. Thus, our data demonstrate large presynaptic spikes and fundamental differences between excitatory and inhibitory boutons in the neocortex.

Autoimmune encephalitis: novel therapeutic targets at the preclinical level.

INTRODUCTION: Antibody-mediated encephalitides (AE) with pathogenic autoantibodies (aAB) against neuronal surface antigens are a growing group of diseases characterized by antineuronal autoimmunity in the brain. AE patients typically present with rapidly progressive encephalitis and characteristic disease symptoms dependent on the target antigen. Current treatment consists of an escalating immunotherapy strategy including plasma exchange, steroid application, and B cell depletion. AREAS COVERED: For this review, we searched Medline database and google scholar with inclusive dates from 2000. We summarize current treatment strategies and present novel therapeutic approaches of target-specific interventions at the pre-clinical level as well as immunotherapy directed at antibody-induced pathology. Treatment options include modulation of target proteins, intervention with downstream pathways, antibody modification, and depletion of antibody-secreting cells. EXPERT OPINION: Although current therapies in AE are effective in many patients, recovery is often prolonged and relapses as well as persistent deficits can occur. Specific immunotherapy together with supportive target-specific therapy may provide faster control of severe symptoms, shorten the disease course, and lead to long-lasting disease stability. Among the various novel therapeutic approaches, modulation of targeted receptors by small molecules crossing the blood-brain barrier as well as prevention of aAB binding is of particular interest.

Loss of Piccolo Function in Rats Induces Cerebellar Network Dysfunction and Pontocerebellar Hypoplasia Type 3-like Phenotypes.

Piccolo, a presynaptic active zone protein, is best known for its role in the regulated assembly and function of vertebrate synapses. Genetic studies suggest a further link to several psychiatric disorders as well as Pontocerebellar Hypoplasia type 3 (PCH3). We have characterized recently generated Piccolo KO (Pclogt/gt ) rats. Analysis of rats of both sexes revealed a dramatic reduction in brain size compared with WT (Pclowt/wt ) animals, attributed to a decrease in the size of the cerebral cortical, cerebellar, and pontine regions. Analysis of the cerebellum and brainstem revealed a reduced granule cell layer and a reduction in size of pontine nuclei. Moreover, the maturation of mossy fiber afferents from pontine neurons and the expression of the α6 GABAA receptor subunit at the mossy fiber-granule cell synapse are perturbed, as well as the innervation of Purkinje cells by cerebellar climbing fibers. Ultrastructural and functional studies revealed a reduced size of mossy fiber boutons, with fewer synaptic vesicles and altered synaptic transmission. These data imply that Piccolo is required for the normal development, maturation, and function of neuronal networks formed between the brainstem and cerebellum. Consistently, behavioral studies demonstrated that adult Pclogt/gt rats display impaired motor coordination, despite adequate performance in tasks that reflect muscle strength and locomotion. Together, these data suggest that loss of Piccolo function in patients with PCH3 could be involved in many of the observed anatomical and behavioral symptoms, and that the further analysis of these animals could provide fundamental mechanistic insights into this devastating disorder.SIGNIFICANCE STATEMENT Pontocerebellar Hypoplasia Type 3 is a devastating developmental disorder associated with severe developmental delay, progressive microcephaly with brachycephaly, optic atrophy, seizures, and hypertonia with hyperreflexia. Recent genetic studies have identified non-sense mutations in the coding region of the PCLO gene, suggesting a functional link between this disorder and the presynaptic active zone. Our analysis of Piccolo KO rats supports this hypothesis, formally demonstrating that anatomical and behavioral phenotypes seen in patients with Pontocerebellar Hypoplasia Type 3 are also exhibited by these Piccolo deficient animals.

Gradients in the mammalian cerebellar cortex enable Fourier-like transformation and improve storing capacity.

Cerebellar granule cells (GCs) make up the majority of all neurons in the vertebrate brain, but heterogeneities among GCs and potential functional consequences are poorly understood. Here, we identified unexpected gradients in the biophysical properties of GCs in mice. GCs closer to the white matter (inner-zone GCs) had higher firing thresholds and could sustain firing with larger current inputs than GCs closer to the Purkinje cell layer (outer-zone GCs). Dynamic Clamp experiments showed that inner- and outer-zone GCs preferentially respond to high- and low-frequency mossy fiber inputs, respectively, enabling dispersion of the mossy fiber input into its frequency components as performed by a Fourier transformation. Furthermore, inner-zone GCs have faster axonal conduction velocity and elicit faster synaptic potentials in Purkinje cells. Neuronal network modeling revealed that these gradients improve spike-timing precision of Purkinje cells and decrease the number of GCs required to learn spike-sequences. Thus, our study uncovers biophysical gradients in the cerebellar cortex enabling a Fourier-like transformation of mossy fiber inputs.

The timing of movements such as posture, balance and speech are coordinated by a region of the brain called the cerebellum. Although this part of the brain is small, it contains a huge number of tiny nerve cells known as granule cells. These cells make up more than half the nerve cells in the human brain. But why there are so many is not well understood.The cerebellum receives signals from sensory organs, such as the ears and eyes, which are passed on as electrical pulses from nerve to nerve until they reach the granule cells. These electrical pulses can have very different repetition rates, ranging from one pulse to a thousand pulses per second. Previous studies have suggested that granule cells are a uniform population that can detect specific patterns within these electrical pulses. However, this would require granule cells to identify patterns in signals that have a range of different repetition rates, which is difficult for individual nerve cells to do.To investigate if granule cells are indeed a uniform population, Straub, Witter, Eshra, Hoidis et al. measured the electrical properties of granule cells from the cerebellum of mice. This revealed that granule cells have different electrical properties depending on how deep they are within the cerebellum. These differences enabled the granule cells to detect sensory signals that had specific repetition rates: signals that contained lots of repeats per second were relayed by granule cells in the lower layers of the cerebellum, while signals that contained fewer repeats were relayed by granule cells in the outer layers.This ability to separate signals based on their rate of repetition is similar to how digital audio files are compressed into an MP3. Computer simulations suggested that having granule cells that can detect specific rates of repetition improves the storage capacity of the brain.These findings further our understanding of how the cerebellum works and the cellular mechanisms that underlie how humans learn and memorize the timing of movement. This mechanism of separating signals to improve storage capacity may apply to other regions of the brain, such as the hippocampus, where differences between nerve cells have also recently been reported.

HCN channel-mediated neuromodulation can control action potential velocity and fidelity in central axons.

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels control electrical rhythmicity and excitability in the heart and brain, but the function of HCN channels at the subcellular level in axons remains poorly understood. Here, we show that the action potential conduction velocity in both myelinated and unmyelinated central axons can be bidirectionally modulated by a HCN channel blocker, cyclic adenosine monophosphate (cAMP), and neuromodulators. Recordings from mouse cerebellar mossy fiber boutons show that HCN channels ensure reliable high-frequency firing and are strongly modulated by cAMP (EC50 40 µM; estimated endogenous cAMP concentration 13 µM). In addition, immunogold-electron microscopy revealed HCN2 as the dominating subunit in cerebellar mossy fibers. Computational modeling indicated that HCN2 channels control conduction velocity primarily by altering the resting membrane potential and are associated with significant metabolic costs. These results suggest that the cAMP-HCN pathway provides neuromodulators with an opportunity to finely tune energy consumption and temporal delays across axons in the brain.