In Vitro Patch-Clamp.

The patch-clamp technique is one of the most useful tools to analyze the function of electrically active cells such as neurons. This technique allows for the analysis of proteins (ion channels and receptors), cells (neurons), and synapses that are the building blocks of neuronal networks. Cortical development involves coordinated changes in functional measures at each of these levels of analysis that reflect both cellular and circuit maturation. This chapter explains the technical and theoretical basis of patch-clamp methodology and introduces several examples of how this technique can be applied in the context of cortical development.

Novel compound heterozygous ATP1A2 variants in a patient with fetal akinesia/hypokinesia sequence.

ATP1A2 encodes a subunit of sodium/potassium-transporting adenosine triphosphatase (Na+ /K+ -ATPase). Heterozygous pathogenic variants of ATP1A2 cause familial hemiplegic migraine, alternating hemiplegia of childhood, and developmental and epileptic encephalopathy. Biallelic loss-of-function variants in ATP1A2 lead to fetal akinesia, respiratory insufficiency, microcephaly, polymicrogyria, and dysmorphic facies, resulting in fetal death. Here, we describe a patient with compound heterozygous ATP1A2 variants consisting of missense and nonsense variants. He survived after birth with brain malformations and the fetal akinesia/hypokinesia sequence. We report a novel type of compound heterozygous variant that might extend the disease spectrum of ATP1A2.

Unveiling the double-peak structure of quantum oscillations in the specific heat.

Quantum oscillation phenomenon is an essential tool to understand the electronic structure of quantum matter. Here we report a systematic study of quantum oscillations in the electronic specific heat Cel in natural graphite. We show that the crossing of a single spin Landau level and the Fermi energy give rise to a double-peak structure, in striking contrast to the single peak expected from Lifshitz-Kosevich theory. Intriguingly, the double-peak structure is predicted by the kernel term for Cel/T in the free electron theory. The Cel/T represents a spectroscopic tuning fork of width 4.8kBT which can be tuned at will to resonance. Using a coincidence method, the double-peak structure can be used to accurately determine the Landé g-factors of quantum materials. More generally, the tuning fork can be used to reveal any peak in fermionic density of states tuned by magnetic field, such as Lifshitz transition in heavy-fermion compounds.

A Pathogenic Missense Mutation in Kainate Receptors Elevates Dendritic Excitability and Synaptic Integration through Dysregulation of SK Channels.

Numerous rare variants that cause neurodevelopmental disorders (NDDs) occur within genes encoding synaptic proteins, including ionotropic glutamate receptors. However, in many cases, it remains unclear how damaging missense variants affect brain function. We determined the physiological consequences of an NDD causing missense mutation in the GRIK2 kainate receptor (KAR) gene, that results in a single amino acid change p.Ala657Thr in the GluK2 receptor subunit. We engineered this mutation in the mouse Grik2 gene, yielding a GluK2(A657T) mouse, and studied mice of both sexes to determine how hippocampal neuronal function is disrupted. Synaptic KAR currents in hippocampal CA3 pyramidal neurons from heterozygous A657T mice exhibited slow decay kinetics, consistent with incorporation of the mutant subunit into functional receptors. Unexpectedly, CA3 neurons demonstrated elevated action potential spiking because of downregulation of the small-conductance Ca2+ activated K+ channel (SK), which mediates the post-spike afterhyperpolarization. The reduction in SK activity resulted in increased CA3 dendritic excitability, increased EPSP-spike coupling, and lowered the threshold for the induction of LTP of the associational-commissural synapses in CA3 neurons. Pharmacological inhibition of SK channels in WT mice increased dendritic excitability and EPSP-spike coupling, mimicking the phenotype in A657T mice and suggesting a causative role for attenuated SK activity in aberrant excitability observed in the mutant mice. These findings demonstrate that a disease-associated missense mutation in GRIK2 leads to altered signaling through neuronal KARs, pleiotropic effects on neuronal and dendritic excitability, and implicate these processes in neuropathology in patients with genetic NDDs.SIGNIFICANCE STATEMENT Damaging mutations in genes encoding synaptic proteins have been identified in various neurodevelopmental disorders, but the functional consequences at the cellular and circuit level remain elusive. By generating a novel knock-in mutant mouse, this study examined the role of a pathogenic mutation in the GluK2 kainate receptor (KAR) subunit, a subclass of ionotropic glutamate receptors. Analyses of hippocampal CA3 pyramidal neurons determined elevated action potential firing because of an increase in dendritic excitability. Increased dendritic excitability was attributable to reduced activity of a Ca2+ activated K+ channel. These results indicate that a pathogenic KAR mutation results in dysregulation of dendritic K+ channels, which leads to an increase in synaptic integration and backpropagation of action potentials into distal dendrites.

Emergent anisotropy in the Fulde-Ferrell-Larkin-Ovchinnikov state.

Exotic superconductivity is formed by unconventional electron pairing and exhibits various unique properties that cannot be explained by the basic theory. The Fulde-Ferrell-Larkin-Ovchinnikov (FFLO) state is known as an exotic superconducting state in that the electron pairs have a finite center-of-mass momentum leading to a spatially modulated pattern of superconductivity. The spatial modulation endows the FFLO state with emergent anisotropy. However, the anisotropy has never been experimentally verified despite numerous efforts over the years. Here, we report detection of anisotropic acoustic responses depending on the sound propagation direction appearing above the Pauli limit. This anisotropy reveals that the two-dimensional FFLO state has a center-of-mass momentum parallel to the nesting vector on the Fermi surface. The present findings will facilitate our understanding of not only superconductivity in solids but also exotic pairings of various particles.

Interneuron Dysfunction and Inhibitory Deficits in Autism and Fragile X Syndrome.

The alteration of excitatory-inhibitory (E-I) balance has been implicated in various neurological and psychiatric diseases, including autism spectrum disorder (ASD). Fragile X syndrome (FXS) is a single-gene disorder that is the most common known cause of ASD. Understanding the molecular and physiological features of FXS is thought to enhance our knowledge of the pathophysiology of ASD. Accumulated evidence implicates deficits in the inhibitory circuits in FXS that tips E-I balance toward excitation. Deficits in interneurons, the main source of an inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), have been reported in FXS, including a reduced number of cells, reduction in intrinsic cellular excitability, or weaker synaptic connectivity. Manipulating the interneuron activity ameliorated the symptoms in the FXS mouse model, which makes it reasonable to conceptualize FXS as an interneuronopathy. While it is still poorly understood how the developmental profiles of the inhibitory circuit go awry in FXS, recent works have uncovered several developmental alterations in the functional properties of interneurons. Correcting disrupted E-I balance by potentiating the inhibitory circuit by targeting interneurons may have a therapeutic potential in FXS. I will review the recent evidence about the inhibitory alterations and interneuron dysfunction in ASD and FXS and will discuss the future directions of this field.

Gabra2 is a genetic modifier of Dravet syndrome in mice.

Pathogenic variants in epilepsy genes result in a spectrum of clinical severity. One source of phenotypic heterogeneity is modifier genes that affect expressivity of a primary pathogenic variant. Mouse epilepsy models also display varying degrees of clinical severity on different genetic backgrounds. Mice with heterozygous deletion of Scn1a (Scn1a+/-) model Dravet syndrome, a severe epilepsy most often caused by SCN1A haploinsufficiency. Scn1a+/- mice recapitulate features of Dravet syndrome, including spontaneous seizures, sudden death, and cognitive/behavioral deficits. Scn1a+/- mice maintained on the 129S6/SvEvTac (129) strain have normal lifespan and no spontaneous seizures. In contrast, admixture with C57BL/6J (B6) results in epilepsy and premature lethality. We previously mapped Dravet Survival Modifier loci (Dsm1-Dsm5) responsible for strain-dependent differences in survival. Gabra2, encoding the GABAA α2 subunit, was nominated as a candidate modifier at Dsm1. Direct measurement of GABAA receptors found lower abundance of α2-containing receptors in hippocampal synapses of B6 mice relative to 129. We also identified a B6-specific single nucleotide deletion within Gabra2 that lowers mRNA and protein by nearly 50%. Repair of this deletion reestablished normal levels of Gabra2 expression. In this study, we used B6 mice with a repaired Gabra2 allele to evaluate Gabra2 as a genetic modifier of severity in Scn1a+/- mice. Gabra2 repair restored transcript and protein expression, increased abundance of α2-containing GABAA receptors in hippocampal synapses, and rescued epilepsy phenotypes of Scn1a+/- mice. These findings validate Gabra2 as a genetic modifier of Dravet syndrome, and support enhancing function of α2-containing GABAA receptors as treatment strategy for Dravet syndrome.

Genetic disruption of Grm5 causes complex alterations in motor activity, anxiety and social behaviors.

Autism is a neurodevelopmental disorder characterized by impaired social interactions and restricted and repetitive behaviors. Although group 1 metabotropic glutamate receptors (mGluRs), and in particular mGluR5, have been extensively proposed as potential targets for intervention in autism and other neurodevelopmental disorders, there has not been a comprehensive analysis of the effect of mGluR5 loss on behaviors typically assessed in autism mouse models thought to be correlates of behavioral symptoms of human disorders. Here we present a behavioral characterization of mice with complete or partial loss of mGluR5 (homozygous or heterozygous null mutations in Grm5 gene). We tested several autism related behaviors including social interaction, repetitive grooming, digging and locomotor behaviors. We found that digging and marble burying behaviors were almost completely abolished in mGluR5 ko mice, although self-grooming was not altered. Social interaction was impaired in ko but not in heterozygote (het) mice. In tests of locomotor activity and anxiety related behaviors, mGluR5 ko mice exhibited hyperactivity and reduced anxiety in the open field test but unexpectedly, showed hypoactivity in the elevated zero-maze test. There was no impairment in motor learning in the accelerating rotarod in both ko and het mutant. Together these results provide support for the importance of mGluR5 in motor and social behaviors that are specifically affected in autism disorders.

Pulse-Chase Proteomics of the App Knockin Mouse Models of Alzheimer's Disease Reveals that Synaptic Dysfunction Originates in Presynaptic Terminals.

Compromised protein homeostasis underlies accumulation of plaques and tangles in Alzheimer's disease (AD). To observe protein turnover at early stages of amyloid beta (Aß) proteotoxicity, we performed pulse-chase proteomics on mouse brains in three genetic models of AD that knock in alleles of amyloid precursor protein (APP) prior to the accumulation of plaques and during disease progression. At initial stages of Aß accumulation, the turnover of proteins associated with presynaptic terminals is selectively impaired. Presynaptic proteins with impaired turnover, particularly synaptic vesicle (SV)-associated proteins, have elevated levels, misfold in both a plaque-dependent and -independent manner, and interact with APP and Aß. Concurrent with elevated levels of SV-associated proteins, we found an enlargement of the SV pool as well as enhancement of presynaptic potentiation. Together, our findings reveal that the presynaptic terminal is particularly vulnerable and represents a critical site for manifestation of initial AD etiology. A record of this paper's transparent peer review process is included in the Supplemental Information.

Neuronal BIN1 Regulates Presynaptic Neurotransmitter Release and Memory Consolidation.

BIN1, a member of the BAR adaptor protein family, is a significant late-onset Alzheimer disease risk factor. Here, we investigate BIN1 function in the brain using conditional knockout (cKO) models. Loss of neuronal Bin1 expression results in the select impairment of spatial learning and memory. Examination of hippocampal CA1 excitatory synapses reveals a deficit in presynaptic release probability and slower depletion of neurotransmitters during repetitive stimulation, suggesting altered vesicle dynamics in Bin1 cKO mice. Super-resolution and immunoelectron microscopy localizes BIN1 to presynaptic sites in excitatory synapses. Bin1 cKO significantly reduces synapse density and alters presynaptic active zone protein cluster formation. Finally, 3D electron microscopy reconstruction analysis uncovers a significant increase in docked and reserve pools of synaptic vesicles at hippocampal synapses in Bin1 cKO mice. Our results demonstrate a non-redundant role for BIN1 in presynaptic regulation, thus providing significant insights into the fundamental function of BIN1 in synaptic physiology relevant to Alzheimer disease.

Potentiating α2 subunit containing perisomatic GABAA receptors protects against seizures in a mouse model of Dravet syndrome.

KEY POINTS: Dravet syndrome mice (Scn1a+/- ) demonstrate a marked strain dependence for the severity of seizures which is correlated with GABAA receptor α2 subunit expression. The α2 /α3 subunit selective positive allosteric modulator (PAM) AZD7325 potentiates inhibitory postsynaptic currents (IPSCs) specifically in perisomatic synapses. AZD7325 demonstrates stronger effects on IPSCs in the seizure resistant mouse strain, consistent with higher α2 subunit expression. AZD7325 demonstrates seizure protective effects in Scn1a+/- mice without apparent sedative effects in vivo. ABSTRACT: GABAA receptor potentiators are commonly used for the treatment of epilepsy, but it is not clear whether targeting distinct GABAA receptor subtypes will have disproportionate benefits over adverse effects. Here we demonstrate that the α2 /α3 selective positive allosteric modulator (PAM) AZD7325 preferentially potentiates hippocampal inhibitory responses at synapses proximal to the soma of CA1 neurons. The effect of AZD7325 on synaptic responses was more prominent in mice on the 129S6/SvEvTac background strain, which have been demonstrated to be seizure resistant in the model of Dravet syndrome (Scn1a+/- ), and in which the α2 GABAA receptor subunits are expressed at higher levels relative to in the seizure prone C57BL/6J background strain. Consistent with this, treatment of Scn1a+/- mice with AZD7325 elevated the temperature threshold for hyperthermia-induced seizures without apparent sedative effects. Our results in a model system indicate that selectively targeting α2 is a potential therapeutic option for Dravet syndrome.

Infantile-onset spinocerebellar ataxia type 5 associated with a novel SPTBN2 mutation: A case report.

BACKGROUND: Spinocerebellar ataxia type 5 (SCA5), a dominant spinocerebellar ataxia is caused by spectrin beta nonerythrocytic 2 gene (SPTBN2) mutation. It typically consists of a slow progressive cerebellar ataxia with an onset principally in adulthood. Here, we report on the first Japanese patient with infantile-onset SCA5 associated with a novel heterozygous SPTBN2 mutation. CASE REPORT: The patient, a 6-year-old girl, developed delayed motor development and unsteady arm movement during infancy. She also showed gaze-evoked nystagmus, saccadic eye pursuit, dysarthria, dysmetria, intention tremor and mild intellectual disability. Brain MRI revealed moderate cerebellar atrophy and mild pontine atrophy. Comprehensive target capture sequencing to identify the causative gene identified a novel missense mutation in SPTBN2 (c.1309C

Genetic analysis of undiagnosed ataxia-telangiectasia-like disorders.

OBJECTIVES: Defects in DNA damage responses or repair mechanisms cause numerous rare inherited diseases, referred to as "DNA-repair defects" or "DNA damage deficiency", characterized by neurodegeneration, immunodeficiency, and/or cancer predisposition. Early accurate diagnosis is important for informing appropriate clinical management; however, diagnosis is frequently challenging and can be delayed, due to phenotypic heterogeneity. Comprehensive genomic analysis could overcome this disadvantage. The objectives of this study were to determine the prevalence of ataxia-telangiectasia (A-T) and A-T-like DNA-repair defects in Japan and to determine the utility of comprehensive genetic testing of presumptively diagnosed patients in facilitating early diagnosis. METHODS: A nationwide survey of diseases presumably caused by DNA-repair defects, including A-T, was performed. Additionally, comprehensive next-generation sequencing (NGS) analysis, targeting known disease-causing genes, was conducted. RESULTS: Sixty-three patients with A-T or other diseases with characteristics of DNA-repair defects were identified. Thirty-four patients were genetically or clinically definitively diagnosed with A-T (n = 22) or other DNA-repair defects (n = 12). Genetic analysis of 17 presumptively diagnosed patients revealed one case of ataxia with oculomotor apraxia type 1 (AOA1); one ataxia with oculomotor apraxia type 2 (AOA2); two types of autosomal dominant spinocerebellar ataxia (SCA5, SCA29); two CACNA1A-related ataxias; one microcephaly with or without chorioretinopathy, lymphedema, or mental retardation (MCLMR); and one autosomal dominant KIF1A-related disorder with intellectual deficit, cerebellar atrophy, spastic paraparesis, and optic nerve atrophy. The diagnostic yield was 58.8%. CONCLUSION: Comprehensive genetic analysis of targeted known disease-causing genes by NGS is a powerful diagnostic tool for subjects with indistinguishable neurological phenotypes resembling DNA-repair defects.

Axonal organization defects in the hippocampus of adult conditional BACE1 knockout mice.

ß-Site APP (amyloid precursor protein) cleaving enzyme 1 (BACE1) is the ß-secretase enzyme that initiates production of the toxic amyloid-ß peptide that accumulates in the brains of patients with Alzheimer's disease (AD). Hence, BACE1 is a prime therapeutic target, and several BACE1 inhibitor drugs are currently being tested in clinical trials for AD. However, the safety of BACE1 inhibition is unclear. Germline BACE1 knockout mice have multiple neurological phenotypes, although these could arise from BACE1 deficiency during development. To address this question, we report that tamoxifen-inducible conditional BACE1 knockout mice in which the Bace1 gene was ablated in the adult largely lacked the phenotypes observed in germline BACE1 knockout mice. However, one BACE1-null phenotype was induced after Bace1 gene deletion in the adult mouse brain. This phenotype showed reduced length and disorganization of the hippocampal mossy fiber infrapyramidal bundle, the axonal pathway of dentate gyrus granule cells that is maintained by neurogenesis in the mouse brain. This defect in axonal organization correlated with reduced BACE1-mediated cleavage of the neural cell adhesion protein close homolog of L1 (CHL1), which has previously been associated with axon guidance. Although our results indicate that BACE1 inhibition in the adult mouse brain may avoid phenotypes associated with BACE1 deficiency during embryonic and postnatal development, they also suggest that BACE1 inhibitor drugs developed for treating AD may disrupt the organization of an axonal pathway in the hippocampus, an important structure for learning and memory.

Delayed Maturation of Fast-Spiking Interneurons Is Rectified by Activation of the TrkB Receptor in the Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder that is a leading cause of inherited intellectual disability, and the most common known cause of autism spectrum disorder. FXS is broadly characterized by sensory hypersensitivity and several developmental alterations in synaptic and circuit function have been uncovered in the sensory cortex of the mouse model of FXS (Fmr1 KO). GABA-mediated neurotransmission and fast-spiking (FS) GABAergic interneurons are central to cortical circuit development in the neonate. Here we demonstrate that there is a delay in the maturation of the intrinsic properties of FS interneurons in the sensory cortex, and a deficit in the formation of excitatory synaptic inputs on to these neurons in neonatal Fmr1 KO mice. Both these delays in neuronal and synaptic maturation were rectified by chronic administration of a TrkB receptor agonist. These results demonstrate that the maturation of the GABAergic circuit in the sensory cortex is altered during a critical developmental period due in part to a perturbation in BDNF-TrkB signaling, and could contribute to the alterations in cortical development underlying the sensory pathophysiology of FXS.SIGNIFICANCE STATEMENT Fragile X (FXS) individuals have a range of sensory related phenotypes, and there is growing evidence of alterations in neuronal circuits in the sensory cortex of the mouse model of FXS (Fmr1 KO). GABAergic interneurons are central to the correct formation of circuits during cortical critical periods. Here we demonstrate a delay in the maturation of the properties and synaptic connectivity of interneurons in Fmr1 KO mice during a critical period of cortical development. The delays both in cellular and synaptic maturation were rectified by administration of a TrkB receptor agonist, suggesting reduced BDNF-TrkB signaling as a contributing factor. These results provide evidence that the function of fast-spiking interneurons is disrupted due to a deficiency in neurotrophin signaling during early development in FXS.

High-speed 100 MHz strain monitor using fiber Bragg grating and optical filter for magnetostriction measurements under ultrahigh magnetic fields.

A high-speed 100 MHz strain monitor using a fiber Bragg grating, an optical filter, and a mode-locked optical fiber laser has been devised, whose resolution is ΔL/L∼10-4. The strain monitor is sufficiently fast and robust for the magnetostriction measurements of materials under ultrahigh magnetic fields generated with destructive pulse magnets, where the sweep rate of the magnetic field is in the range of 10-100 T/µs. As a working example, the magnetostriction of LaCoO3 was measured at room temperature, 115 K, and 7 ∼ 4.2 K up to a maximum magnetic field of 150 T. The smooth dependence on the squared magnetic field and the first-order transition were observed at 115 K and 7 ∼ 4.2 K, respectively, reflecting the field-induced spin state evolution.

Complete Disruption of the Kainate Receptor Gene Family Results in Corticostriatal Dysfunction in Mice.

Kainate receptors are members of the glutamate receptor family that regulate synaptic function in the brain. They modulate synaptic transmission and the excitability of neurons; however, their contributions to neural circuits that underlie behavior are unclear. To understand the net impact of kainate receptor signaling, we generated knockout mice in which all five kainate receptor subunits were ablated (5ko). These mice displayed compulsive and perseverative behaviors, including over-grooming, as well as motor problems, indicative of alterations in striatal circuits. There were deficits in corticostriatal input to spiny projection neurons (SPNs) in the dorsal striatum and correlated reductions in spine density. The behavioral alterations were not present in mice only lacking the primary receptor subunit expressed in adult striatum (GluK2 KO), suggesting that signaling through multiple receptor types is required for proper striatal function. This demonstrates that alterations in striatal function dominate the behavioral phenotype in mice without kainate receptors.

Distinct Subunit Domains Govern Synaptic Stability and Specificity of the Kainate Receptor.

Synaptic communication between neurons requires the precise localization of neurotransmitter receptors to the correct synapse type. Kainate-type glutamate receptors restrict synaptic localization that is determined by the afferent presynaptic connection. The mechanisms that govern this input-specific synaptic localization remain unclear. Here, we examine how subunit composition and specific subunit domains contribute to synaptic localization of kainate receptors. The cytoplasmic domain of the GluK2 low-affinity subunit stabilizes kainate receptors at synapses. In contrast, the extracellular domain of the GluK4/5 high-affinity subunit synergistically controls the synaptic specificity of kainate receptors through interaction with C1q-like proteins. Thus, the input-specific synaptic localization of the native kainate receptor complex involves two mechanisms that underlie specificity and stabilization of the receptor at synapses.

Subchronic phencyclidine treatment in adult mice increases GABAergic transmission and LTP threshold in the hippocampus.

Repeated administration of non-competitive N-methyl-d-aspartate (NMDA) receptor antagonists such as phencyclidine (PCP) to rodents causes long-lasting deficits in cognition and memory, and has effects on behaviors that have been suggested to be models of the cognitive impairment associated with schizophrenia (CIAS). Despite this being a widely studied animal model, little is known about the long lasting changes in synapses and circuits that underlie the altered behaviors. Here we examined synaptic transmission ex-vivo in the hippocampus of mice after a subchronic PCP (scPCP) administration regime. We found that after at least one week of drug free washout period when mice have impaired cognitive function, the threshold for long-term potentiation (LTP) of CA1 excitatory synapses was elevated. This elevated LTP threshold was directly related to increased inhibitory input to CA1 pyramidal cells through increased activity of GABAergic neurons. These results suggest repeated PCP administration causes a long-lasting metaplastic change in the inhibitory circuits in the hippocampus that results in impaired LTP, and could contribute to the deficits in hippocampal-dependent memory in PCP-treated mice. Changes in GABA signaling have been described in patients with schizophrenia, therefore our results support using scPCP as a model of CIAS. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

Epac2 Mediates cAMP-Dependent Potentiation of Neurotransmission in the Hippocampus.

Presynaptic terminal cAMP elevation plays a central role in plasticity at the mossy fiber-CA3 synapse of the hippocampus. Prior studies have identified protein kinase A as a downstream effector of cAMP that contributes to mossy fiber LTP (MF-LTP), but the potential contribution of Epac2, another cAMP effector expressed in the MF synapse, has not been considered. We investigated the role of Epac2 in MF-CA3 neurotransmission using Epac2(-/-) mice. The deletion of Epac2 did not cause gross alterations in hippocampal neuroanatomy or basal synaptic transmission. Synaptic facilitation during short trains was not affected by loss of Epac2 activity; however, both long-term plasticity and forskolin-mediated potentiation of MFs were impaired, demonstrating that Epac2 contributes to cAMP-dependent potentiation of transmitter release. Examination of synaptic transmission during long sustained trains of activity suggested that the readily releasable pool of vesicles is reduced in Epac2(-/-) mice. These data suggest that cAMP elevation uses an Epac2-dependent pathway to promote transmitter release, and that Epac2 is required to maintain the readily releasable pool at MF synapses in the hippocampus.