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Mossy cells (MCs) of the dentate gyrus (DG) are key components of an excitatory associative circuit established by reciprocal connections with dentate granule cells (GCs). MCs are implicated in place field encoding, pattern separation and novelty detection, as well as in brain disorders such as temporal lobe epilepsy and depression. Despite their functional relevance, little is known about the determinants that control MC activity. Here, we examined whether MCs express functional kainate receptors (KARs), a subtype of glutamate receptors involved in neuronal development, synaptic transmission and epilepsy. Using mouse hippocampal slices, we found that bath application of submicromolar and micromolar concentrations of the KAR agonist kainic acid induced inward currents and robust MC firing. These effects were abolished in GluK2 KO mice, indicating the presence of functional GluK2-containing KARs in MCs. In contrast to CA3 pyramidal cells, which are structurally and functionally similar to MCs, and express synaptic KARs at mossy fiber (MF) inputs (i.e., GC axons), we found no evidence for KAR-mediated transmission at MF-MC synapses, indicating that most KARs at MCs are extrasynaptic. Immunofluorescence and immunoelectron microscopy analyses confirmed the extrasynaptic localization of GluK2-containing KARs in MCs. Finally, blocking glutamate transporters, a manipulation that increases extracellular levels of endogenous glutamate, was sufficient to induce KAR-mediated inward currents in MCs, suggesting that MC-KARs can be activated by increases in ambient glutamate. Our findings provide the first direct evidence of functional extrasynaptic KARs at a critical excitatory neuron of the hippocampus.Significance StatementHilar mossy cells (MCs) are an understudied population of hippocampal neurons that form an excitatory loop with dentate granule cells. MCs have been implicated in pattern separation, spatial navigation, and epilepsy. Despite their importance in hippocampal function and disease, little is known about how MC activity is recruited. Here, we show for the first time that MCs express extrasynaptic kainate receptors (KARs), a subtype of glutamate receptors critically involved in neuronal function and epilepsy. While we found no evidence for synaptic KARs in MCs, KAR activation induced strong action potential firing of MCs, raising the possibility that extracellular KARs regulate MC excitability in vivo and may also promote dentate gyrus hyperexcitability and epileptogenesis.

Objective The pathoanatomical correlate of temporal lobe epilepsy is hippocampal sclerosis, characterized by selective neuronal death of mossy cells in the hilus and of pyramidal cells in cornu ammonis 1. Although granule cells survive, they lose mossy cells as a target and redirect their axons (mossy fibers) backward into the molecular cell layer. It has been assumed that this process results in excitatory circuits. We therefore examined whether sprouted mossy fibers form synaptic connection not only with excitatory granule cells but also with inhibitory interneurons, such as basket cells. Methods Resected hippocampal specimens of patients with hippocampal sclerosis were compared to controls of patients with extrahippocampal lesions with only mild sclerosis. Mossy fibers were traced with Neurobiotin or labeled against synaptoporin; inhibitory interneurons were labeled against parvalbumin. Synapses were examined with electron microscopy, labeled with γ-aminobutyric acid immunogold. Results Sprouted mossy fibers of epileptic hippocampi innervate not only excitatory granule cells but also inhibitory parvalbuminergic interneurons. Despite neuronal death in hippocampal sclerosis, the axonal plexus of inhibitory parvalbuminergic interneurons surrounding the granule cells is preserved. Connections of sprouted mossy fibers and inhibitory axon terminals were quantified, showing that the number of inhibitory axon terminals significantly exceeds the number of sprouted excitatory mossy fiber terminals (.03 boutons/µm vs. .11 boutons/µm; p Significance Although no definite conclusions regarding the function of our findings may be derived from this anatomical study, the observed aberrant connectivity might lead to an increased inhibition and synchronization of granule cells, because the preserved inhibitory interneurons show an additional innervation through sprouted mossy fibers. This might result in the instability of a previously balanced network.

Background Transcranial direct current stimulation (tDCS) provides a noninvasive polarity-specific constant current to treat epilepsy, through a mechanism possibly involving excitability modulation and neural oscillation. Objective To determine whether EEG oscillations underlie the interictal spike changes after tDCS in rats with chronic spontaneous seizures. Methods Rats with kainic acid-induced spontaneous seizures were subjected to cathodal tDCS or sham stimulation for 5 consecutive days. Video-EEG recordings were collected immediately pre- and post-stimulation and for the subsequent 2 weeks following stimulation. The acute pre-post stimulation and subacute follow-up changes of interictal spikes and EEG oscillations in tDCS-treated rats were compared with sham. Ictal EEG with seizure behaviors, hippocampal brain-derived neurotrophic factor (BDNF) protein expression, and mossy fiber sprouting were compared between tDCS and sham rats. Results Interictal spike counts were reduced immediately following tDCS with augmented delta and diminished beta and gamma oscillations compared with sham. Cathodal tDCS also enhanced delta oscillations in normal rats. However, increased numbers of interictal spikes with a decrease of delta and theta oscillations were observed in tDCS-treated rats compared with sham during the following 2 weeks after stimulation. Resuming tDCS suppressed the increase of interictal spike activity. In tDCS rats, hippocampal BDNF protein expression was decreased while mossy fiber sprouting did not change compared with sham. Conclusions The inverse relationship between the changes of delta oscillation and interictal spikes during tDCS on and off stimulation periods indicates that an enhanced endogenous delta oscillation underlies the tDCS inhibitory effect on epileptic excitability.

Hippocampal sclerosis (HS) in Temporal Lobe Epilepsy (TLE) shows neuronal death in cornu ammonis (CA)1, CA3, and CA4. It is known that granule cells and CA2 neurons survive and their axons, the mossy fibers (MF), lose their target cells in CA3 and CA4 and sprout to the granule cell layer and molecular layer. We examined in TLE patients and in a mouse epilepsy model, whether MF sprouting is directed to the dentate gyrus or extends to distant CA regions and whether sprouting is associated with death of target neurons in CA3 and CA4. In 319 TLE patients, HS was evaluated by Wyler grade and International League against Epilepsy (ILAE) types using immunohistochemistry against neuronal nuclei (NeuN). Synaptoporin was used to colocalize MF. In addition, transgenic Thy1-eGFP mice were intrahippocampally injected with kainate and sprouting of eGFP-positive MFs was analyzed together with immunocytochemistry for regulator of G-protein signaling 14 (RGS14). In human HS Wyler III and IV as well as in ILAE 1, 2, and 3 specimens, we found synaptoporin-positive axon terminals in CA2 and even in CA1, associated with the extent of granule cell dispersion. Sprouting was seen in cases with cell death of target neurons in CA3 and CA4 (classical severe HS ILAE type 1) but also without this cell death (atypical HS ILAE type 2). Similarly, in epileptic mice eGFP-positive MFs sprouted to CA2 and beyond. The presence of MF terminals in the CA2 pyramidal cell layer and in CA1 was also correlated with the extent of granule cell dispersion. The similarity of our findings in human specimens and in the mouse model highlights the importance and opens up new chances of using translational approaches to determine mechanisms underlying TLE.

Background. Anterior thalamic nuclei (ATN) deep brain stimulation (DBS) is an effective method of controlling epilepsy, especially temporal lobe epilepsy. Mossy fiber sprouting (MFS) plays an indispensable role in the pathogenesis and progression of epilepsy, but the effect of ATN-DBS on MFS in the chronic stage of epilepsy and the potential underlying mechanisms are unknown. This study aimed to investigate the effect of ATN-DBS on MFS, as well as potential signaling pathways by a kainic acid (KA)-induced epileptic model. Methods. Twenty-four rhesus monkeys were randomly assigned to control, epilepsy (EP), EP-sham-DBS, and EP-DBS groups. KA was injected to establish the chronic epileptic model. The left ATN was implanted with a DBS lead and stimulated for 8 weeks. Enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence staining were used to evaluate MFS and levels of potential molecular mediators in the hippocampus. One-way analysis of variance, followed by the Tukey post hoc correction, was used to analyze the statistical significance of differences among multiple groups. Results. ATN-DBS is found to significantly reduce seizure frequency in the chronic stage of epilepsy. The number of ectopic granule cells was reduced in monkeys that received ATN stimulation (P

Metabotropic glutamate receptors (mGluRs) are a group of G-protein-coupled receptors that exert a broad array of modulatory actions at excitatory synapses of the central nervous system. In the hippocampus, the selective activation of the different mGluRs modulates the intrinsic excitability, the strength of synaptic transmission, and induces multiple forms of long-term plasticity. Despite the relevance of mGluRs in the normal function of the hippocampus, we know very little about the changes that mGluRs functionality undergoes during the non-pathological aging. Here, we review data concerning the physiological actions of mGluRs, with particular emphasis on hippocampal area CA3. Later, we examine changes in the expression and functionality of mGluRs during the aging process. We complement this review with original data showing an array of electrophysiological modifications observed in the synaptic transmission and intrinsic excitability of aged CA3 pyramidal cells in response to the pharmacological stimulation of the different mGluRs.

Adult-born granule cells (abGCs) integrate into the hippocampus and form connections with dentate gyrus parvalbumin-positive (PV+) interneurons, a circuit important for modulating plasticity. Many of these interneurons are surrounded by perineuronal nets (PNNs), extracellular matrix structures known to participate in plasticity. We compared abGC projections to PV+ interneurons with negative-to-low intensity PNNs to those with high intensity PNNs using retroviral and 3R-Tau labeling in adult mice, and found that abGC mossy fibers and boutons are more frequently located near PV+ interneurons with high intensity PNNs. These results suggest that axons of new neurons preferentially stabilize near target cells with intense PNNs. Next, we asked whether the number of abGCs influences PNN formation around PV+ interneurons, and found that near complete ablation of abGCs produced a decrease in the intensity and number of PV+ neurons with PNNs, suggesting that new neuron innervation may enhance PNN formation. Experience-driven changes in adult neurogenesis did not produce consistent effects, perhaps due to widespread effects on plasticity. Our study identifies abGC projections to PV+ interneurons with PNNs, with more presumed abGC mossy fiber boutons found near the cell body of PV+ interneurons with strong PNNs.

Excitatory and inhibitory neurons are connected into microcircuits that generate circuit output. Central in the hippocampal CA3 microcircuit is the mossy fiber (MF) synapse, which provides powerful direct excitatory input and indirect feedforward inhibition to CA3 pyramidal neurons. Here, we dissect its cell-surface protein (CSP) composition to discover novel regulators of MF synaptic connectivity. Proteomic profiling of isolated MF synaptosomes uncovers a rich CSP composition, including many CSPs without synaptic function and several that are uncharacterized. Cell-surface interactome screening identifies IgSF8 as a neuronal receptor enriched in the MF pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. Consequently, IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Our results provide insight into the CSP landscape and interactome of a specific excitatory synapse and reveal IgSF8 as a critical regulator of CA3 microcircuit connectivity and function., Mossy fiber synapses are key in CA3 microcircuit function. Here, the authors profile the mossy fiber synapse proteome and cell-surface interactome. They uncover a diverse repertoire of cell-surface proteins and identify the receptor IgSF8 as a regulator of CA3 microcircuit connectivity and function.

During immature stages, adult-born neurons pass through critical periods for survival and plasticity. It is generally assumed that by 2 months of age adult-born neurons are mature and equivalent to the broader neuronal population, raising questions of how they might contribute to hippocampal function in old age when neurogenesis has declined. However, few have examined adult-born neurons beyond the critical period, or directly compared them to neurons born in infancy. Here, we used a retrovirus to visualize functionally-relevant morphological features of 2- to 24-week-old adult-born neurons in male rats. From 2-7 weeks neurons grew and attained a relatively mature phenotype. However, several features of 7-week-old neurons suggested a later wave of growth: these neurons had larger nuclei, thicker dendrites and more dendritic filopodia than all other groups. Indeed, between 7-24 weeks, adult-born neurons gained additional dendritic branches, grew a 2nd primary dendrite, acquired more mushroom spines and had enlarged mossy fiber presynaptic terminals. Compared to neonatally-born neurons, old adult-born neurons had greater spine density, larger presynaptic terminals, and more putative efferent filopodial contacts onto inhibitory neurons. By integrating rates of cell birth and growth across the lifespan, we estimate that adult neurogenesis ultimately produces half of the cells and the majority of spines in the dentate gyrus. Critically, protracted development contributes to the plasticity of the hippocampus through to the end of life, even after cell production declines. Persistent differences from neonatally-born neurons may additionally endow adult-born neurons with unique functions even after they have matured.SIGNIFICANCE STATEMENTNeurogenesis occurs in the hippocampus throughout adult life and contributes to memory and emotion. It is generally assumed that new neurons have the greatest impact on behavior when they are immature and plastic. However, since neurogenesis declines dramatically with age, it is unclear how they might contribute to behavior later in life when cell proliferation has slowed. Here we find that newborn neurons mature over many months in rats, and end up with distinct morphological features compared to neurons born in infancy. Using a mathematical model, we estimate that a large fraction of neurons is added in adulthood. Moreover, their extended growth produces a reserve of plasticity that persists even after neurogenesis has declined to low rates.

Adult hippocampal neurogenesis has been implicated in the spatial processing functions of the hippocampus but ablating neurogenesis does not consistently lead to behavioral deficits in spatial tasks. Parallel studies have shown that adult-born neurons also regulate behavioral responses to stressful and aversive stimuli. We therefore hypothesized that spatial functions of adult-born neurons may be more prominent under conditions of stress, and may differ between males and females given established sex differences in stress responding. To test this we trained intact and neurogenesis-deficient rats in the spatial water maze at temperatures that vary in their degree of aversiveness. At standard temperatures (25°C) ablating neurogenesis did not alter learning and memory in either sex, consistent with prior work. However, in cold water (16°C), ablating neurogenesis had divergent sex-dependent effects: relative to intact rats, male neurogenesis-deficient rats were slower to escape and female neurogenesis-deficient rats were faster. Neurogenesis promoted temperature-related changes in search strategy in females, but it promoted search strategy stability in males. Females displayed greater recruitment of the dorsal hippocampus than males, particularly at 16°C. However, blocking neurogenesis did not alter activity-dependent immediate-early gene expression in either sex. Finally, morphological analyses of retrovirally-labelled neurons revealed greater experience-dependent plasticity in new neurons in males. Neurons had comparable morphology in untrained rats but 16°C training increased spine density, and 25°C training caused shrinkage of mossy fiber presynaptic terminals, specifically in males. Collectively, these findings indicate that neurogenesis functions in memory are prominent under conditions of stress, they provide the first evidence for sex differences in the behavioral function of newborn neurons, and they suggest possibly distinct roles for neurogenesis in cognition and mental health in males and females.

Circuit formation in the central nervous system has been historically studied during development, after which cell-autonomous and nonautonomous wiring factors inactivate. In principle, balanced reactivation of such factors could enable further wiring in adults, but their relative contributions may be circuit dependent and are largely unknown. Here, we investigated hippocampal mossy fiber sprouting to gain insight into wiring mechanisms in mature circuits. We found that sole ectopic expression of Id2 in granule cells is capable of driving mossy fiber sprouting in healthy adult mouse and rat. Mice with the new mossy fiber circuit solved spatial problems equally well as controls but appeared to rely on local rather than global spatial cues. Our results demonstrate reprogrammed connectivity in mature neurons by one defined factor and an assembly of a new synaptic circuit in adult brain., Proceedings of the National Academy of Sciences of the United States of America, 118 (40), ISSN:0027-8424, ISSN:1091-6490

Epilepsy affects approximately 50 million people worldwide, with 60% of adult epilepsies presenting an onset of focal origin. The most common focal epilepsy is temporal lobe epilepsy (TLE). The role of astrocytes in the presentation and development of TLE has been increasingly studied and discussed within the literature. The most common histopathological diagnosis of TLE is hippocampal sclerosis. Hippocampal sclerosis is characterized by neuronal cell loss within the Cornu ammonis and reactive astrogliosis. In some cases, mossy fiber sprouting may be observed. Mossy fiber sprouting has been controversial in its contribution to epileptogenesis in TLE patients, and the mechanisms surrounding the phenomenon have yet to be elucidated. Several studies have reported that mossy fiber sprouting has an almost certain co-existence with reactive astrogliosis within the hippocampus under epileptic conditions. Astrocytes are known to play an important role in the survival and axonal outgrowth of central and peripheral nervous system neurons, pointing to a potential role of astrocytes in TLE and associated cellular alterations. Herein, we review the recent developments surrounding the role of astrocytes in the pathogenic process of TLE and mossy fiber sprouting, with a focus on proposed signaling pathways and cellular mechanisms, histological observations, and clinical correlations in human patients.

Our prior studies demonstrated that the rat hippocampal opioid system can undergo sex-specific adaptations to external stimuli that can influence opioid-associated learning processes. This opioid system extensively overlaps with the cannabinoid system. Moreover, acute administration of Δ9 Tetrahydrocannabinoid (THC), the primary psychoactive constituent of cannabis, can alter cognitive behaviors that involve the hippocampus. Here we use light and electron microscopic immunocytochemical methods to examine the effects of acute THC (5 mg/kg, i.p. 1 hour) on mossy fiber Leu-Enkephalin (LEnk) levels and the distribution and phosphorylation levels of delta and mu opioid receptors (DORs and MORs, respectively) in CA3 pyramidal cells and parvalbumin dentate hilar interneurons of adult female and male Sprague-Dawley rats. In females with elevated estrogen states (proestrus/estrus stage), acute THC altered the opioid system so that it resembled that seen in vehicle-injected females with low estrogen states (diestrus) and males: 1) mossy fiber LEnk levels in CA2/3a decreased; 2) phosphorylated-DOR levels in CA2/3a pyramidal cells increased; and 3) phosphorylated-MOR levels increased in most CA3b laminae. In males, acute THC resulted in the internalization of MORs in parvalbumin-containing interneuron dendrites which would decrease disinhibition of granule cells. In both sexes, acute THC redistributed DORs to the near plasma membrane of CA3 pyramidal cell dendrites, however, the dendritic region varied with sex. Additionally, acute THC also resulted in a sex-specific redistribution of DORs within CA3 pyramidal cell dendrites which could differentially promote synaptic plasticity and/or opioid-associated learning processes in both females and males. This article is protected by copyright. All rights reserved.

Temporal lobe epilepsy is associated with considerable structural changes in the hippocampus. Pharmacological and electrical models of temporal lobe epilepsy in animals strongly suggest that hippocampal reorganization is based on seizure-stimulated aberrant neurogenesis but the data are often controversial and hard to interpret. The aim of the present study was to estimate neurogenesis and synaptic remodeling in the hippocampus of Krushinsky-Molodkina (KM) rats genetically prone to audiogenic seizures (AGS). In our experiments we exposed KM rats to audiogenic kindling of different durations (4, 14, and 21 AGS) to model different stages of epilepsy development. Naive KM rats were used as a control. Our results showed that even 4 AGS stimulated proliferation in the subgranular layer of the dentate gyrus (DG) accompanied with increase in number of doublecortin (DCX)-positive immature granular cells. Elevated number of proliferating cells was also observed in the hilus indicating the enhancement of abnormal migration of neural progenitors. In contrast to the DG, all DCX-positive cells in the hilus expressed VGLUT1/2 and their number was increased indicating that seizure activity accelerates glutamatergic differentiation of ectopic hilar cells. 14-day kindling further stimulated proliferation, abnormal migration, and glutamatergic differentiation of new neurons both in the DG granular and subgranular layers and in the hilus. However, after 21 AGS increased proliferation was observed only in the DG, while the numbers of immature neurons expressed VGLUT1/2 were still enhanced in both hippocampal areas. Audiogenic kindling also stimulated sprouting of mossy fibers and enhanced expression of synaptopodin in the hippocampus indicating generation of new synaptic contacts between granular cells, mossy cells, and CA3 pyramid neurons. Thus, our data suggest that epilepsy progression is associated with exacerbation of aberrant neurogenesis and reorganization of hippocampal neural circuits that contribute to the enhancement and spreading of epileptiform activity.

Neurotransmitter release is a highly controlled process by which synapses can critically regulate information transfer within neural circuits. While presynaptic receptors – typically activated by neurotransmitters and modulated by neuromodulators – provide a powerful way of fine-tuning synaptic function, their contribution to activity-dependent changes in transmitter release remains poorly understood. Here, we report that presynaptic NMDA receptors (preNMDARs) at mossy fiber boutons in the rodent hippocampus can be activated by physiologically relevant patterns of activity and selectively enhance short-term synaptic plasticity at mossy fiber inputs onto CA3 pyramidal cells and mossy cells, but not onto inhibitory interneurons. Moreover, preNMDARs facilitate brain-derived neurotrophic factor release and contribute to presynaptic calcium rise. Taken together, our results indicate that by increasing presynaptic calcium, preNMDARs fine-tune mossy fiber neurotransmission and can control information transfer during dentate granule cell burst activity that normally occur in vivo.

Increased number of newly-born neurons produced at latent stage after status epilepticus (SE) contribute to aberrant rewiring of hippocampus and are hypothesized to promote epileptogenesis. Although physical training (PT) was reported to cause further increase in neurogenesis after SE, how PT affect their integration pattern is still elusive, whether they integrate into normal circuits or increase aberrant integrations is yet to be determined. To understand this basic mechanism by which PT effects SE and to elaborate the possible role of neuronal integrations in prognosis of SE, we evaluated the effect of 4 weeks of treadmill PT in adult male mice after pilocarpine-induced SE on behavioral and aberrant integrations' parameters. Changes in BDNF gene methylation and its protein level in hippocampus was also measured at latent stage (2-weeks) to explore underlying pathways involved in increasing neurogenesis. Our results demonstrated that although PT increased proliferation and maturation of neurons in dentate gyrus, they showed reduced aberrant integrations into hippocampal circuitry assessed through a decrease in the number of ectopic granular cells, hilar basal dendrites and mossy fiber sprouting as compared to non-exercised SE mice. While SE decreased the percentage methylation of specific CpGs of BDNF gene's promoter, PT did not yield any significant difference in methylation of BDNF CpGs as compared to non-exercised SE mice. In conclusion, PT increases hippocampal neurogenesis through increasing BDNF levels by some pathways other than demethylating BDNF CpGs and causes post SE newly-born neurons to integrate into normal circuits thus resulting in decreased spontaneous recurrent seizures and enhanced spatial memory.

The deposition of tau pathology in Alzheimer’s disease (AD) may occur first in axons of neurons and then progress back into the cell bodies to form neurofibrillary tangles, however, studies have not directly analyzed this relationship in relatively discrete circuits within the human hippocampus. In the early phases of tau deposition, both AT8 phosphorylation and exposure of the amino terminus of tau occurs in tauopathies, and these modifications are linked to mechanisms of synaptic and axonal dysfunction. Here, we examined the localization of these tau pathologies in well-characterized post-mortem human tissue samples from the hippocampus of 44 cases ranging between non-demented and mild cognitively impaired to capture a time at which intrahippocampal pathways show a range in the extent of tau deposition. The tissue sections were analyzed for AT8 (AT8 antibody), amino terminus exposure (TNT2 antibody), and amyloid-β (MOAB2 antibody) pathology in hippocampal strata containing the axons and neuronal cell bodies of the CA3-Schaffer collateral and dentate granule-mossy fiber pathways. We show that tau pathology first appears in the axonal compartment of affected neurons in the absence of observable tau pathology in the corresponding cell bodies in several cases. Additionally, deposition of tau in these intrahippocampal pathways was independent of the presence of Aβ plaques. We confirmed that the majority of tau pathology positive neuropil threads were axonal in origin and not dendritic using an axonal marker (i.e. SMI312 antibody) and somatodendritic marker (i.e. MAP2 antibody). Taken together, these results support the hypothesis that AT8 phosphorylation and amino terminus exposure are early pathological events and that the deposition of tau pathology, at least in the studied pathways, occurs first in the axonal compartment prior to observable pathology in the somata. These findings highlight the importance on targeting tau deposition, ideally in the initial phases of its deposition in axons. Electronic supplementary material The online version of this article (10.1186/s40478-019-0675-9) contains supplementary material, which is available to authorized users.

Hippocampal mossy fiber axons simultaneously activate CA3 pyramidal cells and stratum lucidum interneurons (SLINs), the latter providing feedforward inhibition to control CA3 pyramidal cell excitability. Filopodial extensions of giant boutons of mossy fibers provide excitatory synaptic input to the SLIN. These filopodia undergo extraordinary structural plasticity causally linked to execution of memory tasks, leading us to seek the mechanisms by which activity regulates these synapses. High-frequency stimulation of the mossy fibers induces long-term depression (LTD) of their calcium-permeable AMPA receptor synapses with SLINs; previous work localized the site of induction to be postsynaptic and the site of expression to be presynaptic. Yet, the underlying signaling events and the identity of the retrograde signal are incompletely understood. We used whole cell recordings of SLINs in hippocampal slices from wild-type and mutant mice to explore the mechanisms. Genetic and pharmacologic perturbations revealed a requirement for both the receptor tyrosine kinase TrkB and its agonist, brain-derived neurotrophic factor (BDNF), for induction of LTD. Inclusion of inhibitors of Trk receptor kinase and PLC in the patch pipette prevented LTD. Endocannabinoid receptor antagonists and genetic deletion of the CB1 receptor prevented LTD. We propose a model whereby release of BDNF from mossy fiber filopodia activates TrkB and PLCγ1 signaling postsynaptically within SLINs, triggering synthesis and release of an endocannabinoid that serves as a retrograde signal, culminating in reduced glutamate release. Insights into the signaling pathways by which activity modifies function of these synapses will facilitate an understanding of their contribution to the local circuit and behavioral consequences of hippocampal granule cell activity.NEW & NOTEWORTHY We investigated signaling mechanisms underlying plasticity of the hippocampal mossy fiber filopodial synapse with interneurons in stratum lucidum. High-frequency stimulation of the mossy fibers induces long-term depression of this synapse. Our findings are consistent with a model in which brain-derived neurotrophic factor released from filopodia activates TrkB of a stratum lucidum interneuron; the ensuing activation of PLCγ1 induces synthesis of an endocannabinoid, which provides a retrograde signal leading to reduced release of glutamate presynaptically.

Significance This study provides evidence that human induced pluripotent stem cell (hiPSC)-derived medial ganglionic eminence (MGE) cell grafting into the hippocampus after status epilepticus can greatly reduce the frequency of spontaneous seizures in the chronic phase through both antiepileptogenic and antiepileptic effects. The antiepileptogenic changes comprised reductions in host interneuron loss, abnormal neurogenesis, and aberrant mossy fiber sprouting, whereas the antiepileptic effects were evident from an increased occurrence of seizures after silencing of graft-derived interneurons. Additional curative impacts of grafting comprised improved cognitive and mood function. The results support the application of autologous human MGE cell therapy for temporal lobe epilepsy. Autologous cell therapy is advantageous as such a paradigm can avoid immune suppression and promote enduring graft–host integration., Medial ganglionic eminence (MGE)-like interneuron precursors derived from human induced pluripotent stem cells (hiPSCs) are ideal for developing patient-specific cell therapy in temporal lobe epilepsy (TLE). However, their efficacy for alleviating spontaneous recurrent seizures (SRS) or cognitive, memory, and mood impairments has never been tested in models of TLE. Through comprehensive video- electroencephalographic recordings and a battery of behavioral tests in a rat model, we demonstrate that grafting of hiPSC-derived MGE-like interneuron precursors into the hippocampus after status epilepticus (SE) greatly restrained SRS and alleviated cognitive, memory, and mood dysfunction in the chronic phase of TLE. Graft-derived cells survived well, extensively migrated into different subfields of the hippocampus, and differentiated into distinct subclasses of inhibitory interneurons expressing various calcium-binding proteins and neuropeptides. Moreover, grafting of hiPSC-MGE cells after SE mediated several neuroprotective and antiepileptogenic effects in the host hippocampus, as evidenced by reductions in host interneuron loss, abnormal neurogenesis, and aberrant mossy fiber sprouting in the dentate gyrus (DG). Furthermore, axons from graft-derived interneurons made synapses on the dendrites of host excitatory neurons in the DG and the CA1 subfield of the hippocampus, implying an excellent graft–host synaptic integration. Remarkably, seizure-suppressing effects of grafts were significantly reduced when the activity of graft-derived interneurons was silenced by a designer drug while using donor hiPSC-MGE cells expressing designer receptors exclusively activated by designer drugs (DREADDs). These results implied the direct involvement of graft-derived interneurons in seizure control likely through enhanced inhibitory synaptic transmission. Collectively, the results support a patient-specific MGE cell grafting approach for treating TLE.

More is not always better Mutations in X-linked genes usually affect male individuals more than females, but the opposite characterizes the Protocadherin-19 gene ( PCDH19 ) on the X chromosome. Mutations in the PCDH19 cell-adhesion molecule cause cognitive impairment, affecting females more than males. Hoshina et al. studied mice with PCDH19 mutations, showing that a mismatch between PCDH19 and another cell-adhesion molecule causes trouble when mossy fibers of the brain are forming synapses (see the Perspective by Shohayeb and Cooper). In the heterozygous setting, mutant PCDG19 sequesters the partner cell-adhesion molecule into dysfunctional complexes. In the hemizygous setting, as in males, enough of that partner cell-adhesion molecule roams free to make functional interactions. Science , this issue p. eaaz3893 ; see also p. 235

In the hippocampus, the excitatory synapse between dentate granule cell (GC) axons, or mossy fibers (MFs), and CA3 pyramidal cells (MF-CA3) expresses robust forms of short-term plasticity, such as frequency facilitation and post-tetanic potentiation (PTP). These forms of plasticity are due to increases in presynaptic neurotransmitter release, and can be engaged when dentate GCs fire in bursts (e.g., during exploratory behaviors) and bring CA3 pyramidal neurons above threshold. While frequency facilitation at this synapse is limited by endogenous activation of presynaptic metabotropic glutamate receptors (mGluRs), whether MF-PTP can be regulated in an activity-dependent manner is unknown. Here, using physiologically relevant patterns of MF stimulation in acute mouse hippocampal slices, we found that disrupting postsynaptic Ca2+dynamics increases MF-PTP, strongly suggesting a form of Ca2+-dependent retrograde suppression of this form of plasticity. PTP suppression requires a few seconds of MF bursting activity and Ca2+release from internal stores. Our findings raise the possibility that the powerful MF-CA3 synapse can negatively regulate its own strength not only during PTP-inducing activity typical of normal exploratory behaviors, but also during epileptic activity.

Many neuron types consist of populations with continuously varying molecular properties. Here, we show a continuum of postsynaptic molecular properties in three types of neurons and assess the functional correlates in cerebellar unipolar brush cells (UBCs). While UBCs are generally thought to form discrete functional subtypes, with mossy fiber (MF) activation increasing firing in ON-UBCs and suppressing firing in OFF-UBCs, recent work also points to a heterogeneity of response profiles. Indeed, we find a continuum of response profiles that reflect the graded and inversely correlated expression of excitatory mGluR1 and inhibitory mGluR2/3 pathways. MFs coactivate mGluR2/3 and mGluR1 in many UBCs, leading to sequential inhibition-excitation because mGluR2/3-currents are faster. Additionally, we show that DAG-kinase controls mGluR1 response duration, and that graded DAG kinase levels correlate with systematic variation of response duration over two orders of magnitude. These results demonstrate that continuous variations in metabotropic signaling can generate a stable cell-autonomous basis for temporal integration and learning over multiple time scales., The authors show that graded molecular heterogeneity in metabotropic pathways underlies a continuum of temporal responses in cerebellar unipolar brush cells (UBCs). This allows the UBC population to serve as a cell-autonomous basis for temporal integration and learning over multiple time scales.

SummaryNeurotransmitter release is a highly controlled process by which synapses can critically regulate information transfer within neural circuits. While presynaptic receptors –typically activated by neurotransmitters and modulated by neuromodulators– provide a powerful way of fine-tuning synaptic function, their contribution to activity-dependent changes in transmitter release remains poorly understood. Here, we report that presynaptic NMDA receptors (preNMDARs) at mossy fiber boutons in the rodent hippocampus can be activated by physiologically relevant patterns of activity and selectively enhance short-term synaptic plasticity at mossy fiber inputs onto CA3 pyramidal cells and mossy cells, but not onto inhibitory interneurons. Moreover, preNMDARs facilitate brain-derived neurotrophic factor (BDNF) release and contribute to presynaptic calcium rise. Taken together, our results indicate that by increasing presynaptic calcium, preNMDARs fine tune mossy fiber neurotransmission and can control information transfer during dentate granule cell burst activity that normally occur in vivo.

Nitric Oxide (NO) is an intracellular messenger whose diffusive properties enable an unconventional type of communication between neurons in the central nervous system that bypasses their anatomical connectivity. In this work, we modeled NO production and diffusion from a single source and investigated the range of action of the NO signal within a bioinspired spiking neural network. We found that a single active source will produce only a local effect on the individual synapse. While if multiple closely-located sources are active at the same time, NO will act more like a volume transmitter and influence even inactive synapses within that area. We focused our attention on the cerebellum's input layer, where NO is produced by the granule cells. In the granular layer, NO acts as a retrograde second messenger able to enhance presynaptic currents in the mossy fiber - granule cell synapses, thus potentiating them with long-term effects (LTP).

In animal models with temporal lobe epilepsy (TLE), the status epilepticus (SE) leads to a dramatic increase in number of newly born neuron in the subgranular zone (SGZ) of dentate gyrus. How the SE confers a modulation in the dentate neurogenesis is mostly unknown. Gadd45b is involved in epigenetic gene activation by DNA demethylation. This study was performed to present a novel mechanism underling SE-induced dentate neurogenesis. A transient induction (12 hrs to 3 days) of Gadd45b was observed in dentate gyrus of mice after pilocarpine-induced SE. Labeling the dividing cells with BrdU, we next found that the induction of Gadd45b was required to increase the rate of cell proliferation in the dentate gyrus at 7 and 14 days after SE. Afterward, the DNA methylation levels for candidate growth factor genes critical for the adult neurogenesis were assayed with Sequenom MassARRAY Analyzer. The results indicated that Gadd45b was necessary for SE-induced DNA demethylation of specific promoters and expression of corresponding genes in the dentate gyrus, including brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2). Using Timm staining, we further suggested that SE-induced Gadd45b might contribute to the subsequent mossy fiber sprouting (MFS) in the chronically epileptic hippocampus via epigenetic regulation of dentate neurogenesis at early stage after SE. Together, Gadd45b links pilocarpine-induced SE to epigenetic DNA modification of secreted factors in the dentate gyrus, leading to extrinsic modulation on the neurogenesis.

In the hippocampus, episodic memories are thought to be encoded by the formation of ensembles of synaptically coupled CA3 pyramidal cells driven by sparse but powerful mossy fiber inputs from dentate gyrus granule cells. The neuromodulators acetylcholine and noradrenaline are separately proposed as saliency signals that dictate memory encoding but it is not known if they represent distinct signals with separate mechanisms. Here, we show experimentally that acetylcholine, and to a lesser extent noradrenaline, suppress feed-forward inhibition and enhance Excitatory–Inhibitory ratio in the mossy fiber pathway but CA3 recurrent network properties are only altered by acetylcholine. We explore the implications of these findings on CA3 ensemble formation using a hierarchy of models. In reconstructions of CA3 pyramidal cells, mossy fiber pathway disinhibition facilitates postsynaptic dendritic depolarization known to be required for synaptic plasticity at CA3-CA3 recurrent synapses. We further show in a spiking neural network model of CA3 how acetylcholine-specific network alterations can drive rapid overlapping ensemble formation. Thus, through these distinct sets of mechanisms, acetylcholine and noradrenaline facilitate the formation of neuronal ensembles in CA3 that encode salient episodic memories in the hippocampus but acetylcholine selectively enhances the density of memory storage., Author summary How the brain decides which experiences to encode to memory and which to discard is a fundamental question in neuroscience. The neuromodulators acetylcholine and noradrenaline are believed to separately play a central role in determining what is encoded but the mechanisms by which they act are mostly unknown and there have been no direct comparisons made between these two critical neuromodulators. In this study, we investigate the effects of acetylcholine and noradrenaline on a key circuit responsible for the encoding of memories, namely, the dentate gyrus–CA3 microcircuit in the hippocampus. Using slice electrophysiology, we measure the effects of acetylcholine and noradrenaline on key synaptic and cellular nodes within this neuronal network. We then explore the network level implications of these findings on neuronal ensemble formation using a hierarchy of computational models. Based on the observed physiological effects of acetylcholine and noradrenaline, our models predict that acetylcholine facilitates efficient formation of ensembles within CA3 with a high degree of overlap whereas noradrenaline has more limited effects and no impact on the efficiency or overlap of ensemble formation.

Nectins are immunoglobulin-like cell adhesion molecules constituting a family with four members, nectin-1, nectin-2, nectin-3, and nectin-4. In the brain, nectin-2 as well as nectin-1 and nectin-3 are expressed whereas nectin-4 is hardly expressed. In the nervous system, physiological functions of nectin-1 and nectin-3, such as synapse formation, mossy fiber trajectory regulation, interneurite affinity, contextual fear memory formation, and stress-related mental disorders, have been revealed. Nectin-2 is ubiquitously expressed in non-neuronal tissues and various nectin-2 functions in non-nervous systems have been extensively investigated, but nectin-2 functions in the brain have not been revealed until recently. Recent findings have revealed that nectin-2 is expressed in the specific areas of the brain and plays important roles, such as homeostasis of astrocytes and neurons and the formation of synapses. Moreover, a single nucleotide polymorphism in the human NECTIN2 gene is associated with Alzheimer's disease. We here summarize recent progress in our understanding of nectin-2 functions in the brain.

N-methyl-D-aspartate receptors (NMDARs) are expressed in granule cell and involve in mossy fiber-granule cell (MF-GC) synaptic transmission in cerebellar cortex. In the absence GABAA receptor activity, we here studied the role of NMDARs during the facial stimulation evoked MF-GC synaptic transmission in urethane-anesthetized mice using electrophysiological recording technique and pharmacological methods. Our results showed that facial stimuli train (20 Hz, 5 pulses) evoked 5 field potential responses (N1-N5) in mouse cerebellar granular layer, which identified MF-GC synaptic transmission. Blocking NMDARs induced significant depression in the amplitude of N2 to N5, accompanied with significant decrease in pulse ratios, area under the curve (AUC) and half-width of N1. A selective GluN2A antagonist, PEAQX (10 μM) also produced significant depression in the amplitude of N2 to N5, and decreases in pulse ratios. However, a selective GluN2B antagonist, TCN-237 (10 μM) did not significantly attenuate the facial stimuli train-induced mossy fiber-granule cell synaptic transmission. Application of NMDA (1 μM) produced increases in the AUC and half-width of Ron, as well the amplitude and AUC of Roff, which was reversed by following application of PEAQX. Our present results indicated that NMDARs, especially GluN2A contribute to the facial stimulation-evoked MF-GC synaptic transmission, suggesting that the NMDARs play an important role during the lateral sensory information synaptic transmission in the cerebellar granular layer in vivo in mice.

Objective Mutations of the cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders characterized by intractable epilepsy, intellectual disability, and autism. Multiple mouse models generated for mechanistic studies have exhibited phenotypes similar to some human pathological features, but none of the models has developed one of the major symptoms affecting CDKL5 deficiency disorder (CDD) patients: intractable recurrent seizures. As disrupted neuronal excitation/inhibition balance is closely associated with the activity of glutamatergic and γ-aminobutyric acidergic (GABAergic) neurons, our aim was to study the effect of the loss of CDKL5 in different types of neurons on epilepsy. Methods Using the Cre-LoxP system, we generated conditional knockout (cKO) mouse lines allowing CDKL5 deficiency in glutamatergic or GABAergic neurons. We employed noninvasive video recording and in vivo electrophysiological approaches to study seizure activity in these Cdkl5 cKO mice. Furthermore, we conducted Timm staining to confirm a morphological alteration, mossy fiber sprouting, which occurs with limbic epilepsy in both human and mouse brains. Finally, we performed whole-cell patch clamp in dentate granule cells to investigate cell-intrinsic properties and synaptic excitatory activity. Results We demonstrate that Emx1- or CamK2α-derived Cdkl5 cKO mice manifest high-frequency spontaneous seizure activities recapitulating the epilepsy of CDD patients, which ultimately led to sudden death in mice. However, Cdkl5 deficiency in GABAergic neurons does not generate such seizures. The seizures were accompanied by typical epileptic features including higher amplitude spikes for epileptiform discharges and abnormal hippocampal mossy fiber sprouting. We also found an increase in spontaneous and miniature excitatory postsynaptic current frequencies but no change in amplitudes in the dentate granule cells of Emx1-cKO mice, indicating enhanced excitatory synaptic activity. Significance Our study demonstrates that Cdkl5 cKO mice, serving as an animal model to study recurrent spontaneous seizures, have potential value for the pathological study of CDD-related seizures and for therapeutic innovation.

Feedback inhibitory motifs are thought to be important for pattern separation across species. How feedback circuits may implement pattern separation of biologically plausible, temporally structured input in mammals is, however, poorly understood. We have quantitatively determined key properties of netfeedback inhibition in the mouse dentate gyrus, a region critically involved in pattern separation. Feedback inhibition is recruited steeply with a low dynamic range (0% to 4% of active GCs), and with a non-uniform spatial profile. Additionally, net feedback inhibition shows frequency-dependent facilitation, driven by strongly facilitating mossy fiber inputs. Computational analyses show a significant contribution of the feedback circuit to pattern separation of theta modulated inputs, even within individual theta cycles. Moreover, pattern separation was selectively boosted at gamma frequencies, in particular for highly similar inputs. This effect was highly robust, suggesting that frequency-dependent pattern separation is a key feature of the feedback inhibitory microcircuit., eLife digest You can probably recall where you left your car this morning without too much trouble. But assuming you use the same busy parking lot every day, can you remember which space you parked in yesterday? Or the day before that? Most people find this difficult not because they cannot remember what happened two or three days ago, but because it requires distinguishing between very similar memories. The car, the parking lot, and the time of day were the same on each occasion. So how do you remember where you parked this morning? This ability to distinguish between memories of similar events depends on a brain region called the hippocampus. A subregion of the hippocampus called the dentate gyrus generates different patterns of activity in response to events that are similar but distinct. This process is called pattern separation, and it helps ensure that you do not look for your car in yesterday’s parking space. Pattern separation in the dentate gyrus is thought to involve a form of negative feedback called feedback inhibition, a phenomenon where the output of a process acts to limit or stop the same process. To test this idea, Braganza et al. studied feedback inhibition in the dentate gyrus of mice, before building a computer model simulating the inhibition process and supplying the model with two types of realistic input. The first consisted of low-frequency theta brainwaves, which occur, for instance, in the dentate gyrus when animals explore their environment. The second consisted of higher frequency gamma brainwaves, which occur, for example, when animals experience something new. Testing the model showed that feedback inhibition contributes to pattern separation with both theta and gamma inputs. However, pattern separation is stronger with gamma input. This suggests that high frequency brainwaves in the hippocampus could help animals distinguish new events from old ones by promoting pattern separation. Various brain disorders, including Alzheimer’s disease, schizophrenia and epilepsy, involve changes in the dentate gyrus and altered brain rhythms. The current findings could help reveal how these changes contribute to memory impairments and to a reduced ability to distinguish similar experiences.

The Wnt signaling pathway plays key roles in various developmental processes. Wnt5a, which activates the non-canonical pathway, has been shown to be particularly important for axon guidance and outgrowth as well as dendrite morphogenesis. However, the mechanism underlying the regulation of Wnt5a remains unclear. Here, through conditional disruption of Foxg1 in hippocampal progenitors and postmitotic neurons achieved by crossing Foxg1(fl/fl) with Emx1-Cre and Nex-Cre, respectively, we found that Wnt5a rather than Wnt3a/Wnt2b was markedly upregulated. Overexpression of Foxg1 had the opposite effects along with decreased dendritic complexity and reduced mossy fibers in the hippocampus. We further demonstrated that FOXG1 directly repressed Wnt5a by binding to its promoter and one enhancer site. These results expand our knowledge of the interaction between Foxg1 and Wnt signaling and help elucidate the mechanisms underlying hippocampal development.

LGI1 (Leucine-Rich Glioma-Inactivated 1) is a secreted trans-synaptic protein that interacts presynaptically with Kv1.1 potassium channels and ADAM23, and postsynaptically, influencing AMPA receptors through a direct link with the ADAM22 cell adhesion protein. Haploinsufficiency of LGI1 or autoantibodies directed against LGI1 are associated with human epilepsy, generating the hypothesis that a subacute reduction of LGI1 is sufficient to increase network excitability. We tested this hypothesis inex vivohippocampal slices and in neuronal cultures, by subacutely reducing LGI1 expression with shRNA. Injection of shRNA-LGI1 in the hippocampus increased dentate granule cell excitability and low frequency facilitation of mossy fibers to CA3 pyramidal cell neurotransmission. Application of the Kv1 family blocker, alpha-dendrotoxin, occluded this effect, implicating the involvement of Kv1.1. This subacute reduction of LGI1 was also sufficient to increase neuronal network activity in neuronal primary culture. These results indicate that a subacute reduction in LGI1 potentiates neuronal excitability and short-term synaptic plasticity, and increases neuronal network excitability, opening new avenues for the treatment of limbic encephalitis and temporal lobe epilepsies.Significant StatementDown-regulation of LGI1 protein increases excitability at mossy fiber to CA3 pyramidal cell synapses and in hippocampal primary cultures. These alterations alter network function and so could contribute to symptoms observed in patients affected by limbic encephalitis.

In temporal lobe epilepsy (TLE), abnormal axon guidance and synapse formation lead to sprouting of mossy fibers in the hippocampus, which is one of the most consistent pathological findings in patients and animal models with TLE. Glypican 4 (Gpc4) belongs to the heparan sulfate proteoglycan family, which play an important role in axon guidance and excitatory synapse formation. However, the role of Gpc4 in the development of mossy fibers sprouting (MFS) and its underlying mechanism remain unknown. Using a pilocarpine-induced mice model of epilepsy, we showed that Gpc4 expression was significantly increased in the stratum granulosum of the dentate gyrus at 1 week after status epilepticus (SE). Using Gpc4 overexpression or Gpc4 shRNA lentivirus to regulate the Gpc4 level in the dentate gyrus, increased or decreased levels of netrin-1, SynI, PSD-95, and Timm score were observed in the dentate gyrus, indicating a crucial role of Gpc4 in modulating the development of functional MFS. The observed effects of Gpc4 on MFS were significantly antagonized when mice were treated with L-leucine or rapamycin, an agonist or antagonist of the mammalian target of rapamycin (mTOR) signal, respectively, demonstrating that mTOR pathway is an essential requirement for Gpc4-regulated MFS. Additionally, the attenuated spontaneous recurrent seizures (SRSs) were observed during chronic stage of the disease by suppressing the Gpc4 expression after SE. Altogether, our findings demonstrate a novel control of neuronal Gpc4 on the development of MFS through the mTOR pathway after pilocarpine-induced SE. Our results also strongly suggest that Gpc4 may serve as a promising target for antiepileptic studies.

Angelman syndrome (AS) is a neurodevelopmental disorder in which epilepsy is common (~90%) and often refractory to antiepileptics. AS is caused by mutation of the maternal allele encoding the ubiquitin protein ligase E3A (UBE3A), but it is unclear how this genetic insult confers vulnerability to seizure development and progression (i.e., epileptogenesis). Here, we implemented the flurothyl kindling and retest paradigm in AS model mice to assess epileptogenesis and to gain mechanistic insights owed to loss of maternal Ube3a. AS model mice kindled similarly to wild-type mice, but they displayed a markedly increased sensitivity to flurothyl-, kainic acid-, and hyperthermia-induced seizures measured a month later during retest. Pathological characterization revealed enhanced deposition of perineuronal nets in the dentate gyrus of the hippocampus of AS mice in the absence of overt neuronal loss or mossy fiber sprouting. This pro-epileptogenic phenotype resulted from Ube3a deletion in GABAergic but not glutamatergic neurons, and it was rescued by pancellular reinstatement of Ube3a at postnatal day 21 (P21), but not during adulthood. Our results suggest that epileptogenic susceptibility in AS patients is a consequence of the dysfunctional development of GABAergic circuits, which may be amenable to therapies leveraging juvenile reinstatement of UBE3A.

Mossy fiber sprouting (MFS) is a pathological phenomenon that is commonly observed in epilepsy, and plentiful data reveal that abnormal phosphorylated modification of tau protein plays a critical role in MSF by the regulation of microtubule dynamics and axonal transport. Ubiquitin C-terminal hydrolase L1 (UCH-L1), a proteasomal deubiquitinating enzyme, has been proved to be associated with tau aggregation through mediating degradation of ubiquitinated and hyperphosphorylated tau. Thus, this study aimed to determine the expression of UCH-L1 in the rat hippocampus during the pentylenetetrazole (PTZ)-induced process and to demonstrate the possible correlation with MFS in epileptogenesis. Seizures were established by intraperitoneal injection of PTZ and LDN-57444 was used to inhibit the hydrolase activity of UCH-L1. We used western blot, immunofluorescence, immunoprecipitation, and timm staining to detect phosphorylated modification of tau and MSF. The results presented that LDN-57444 induced the deteriorated severity of seizures, increased phosphorylation of tau and increased distribution of Timm granules in both the supragranular region of the dentate gyrus (DG) and the stratum pyramidale of CA3 subfield. Our results suggest that UCH-L1 may be associated with hippocampal MSF followed the epileptogenesis through mediating phosphorylation of tau. UCH-L1 may be a potential and novel therapeutic target to limit epileptogenesis.

The current electrophysiological study investigated the functional roles of high- and low-voltage-activated Ca2+ channel subtypes on glutamatergic small mossy fiber nerve terminals (SMFTs) that synapse onto rat hippocampal CA3 neurons. Experiments combining both the “synapse bouton” preparation and single-pulse focal stimulation technique were performed using the conventional whole cell patch configuration under voltage-clamp conditions. Nifedipine, at a high concentration, and BAY K 8644 inhibited and facilitated the glutamatergic excitatory postsynaptic currents (eEPSCs) that were evoked by 0.2-Hz stimulation, respectively. However, these drugs had no effects on spontaneous EPSCs (sEPSCs). Following the use of a high stimulation frequency of 3 Hz, however, nifedipine markedly inhibited eEPSCs at the low concentration of 0.3 µM. Moreover, ω-conotoxin GVIA and ω-agatoxin IVA significantly inhibited both sEPSCs and eEPSCs. Furthermore, SNX-482 slightly inhibited eEPSCs. R(−)-efonidipine had no effects on either sEPSCs or eEPSCs. It was concluded that glutamate release from SMFTs depends largely on Ca2+ entry through N- and P/Q-type Ca2+ channels and, to a lesser extent, on R-type Ca2+ channels. The contribution of L-type Ca2+ channels to eEPSCs was small at low-firing SMFTs but more significant at high-firing SMFTs. T-type Ca2+ channels did not appear to be involved in neurotransmission at SMFTs. NEW & NOTEWORTHY Action potential-evoked glutamate release from small mossy fiber nerve terminals (SMFTs) that synapse onto rat hippocampal CA3 neurons is regulated by high-threshold but not low-threshold Ca2+ channel subtypes. The functional contribution mainly depends on N- and P/Q-type Ca2+ channels and, to a lesser extent, on R-type Ca2+ channels. However, in SMFTs stimulated at a high 3-Hz frequency, L-type Ca2+ channels contributed significantly to the currents. The present results are consistent with previous findings from fluorometric studies of large mossy fiber boutons.

The Golgi cells, together with granule cells and mossy fibers, form a neuronal microcircuit regulating information transfer at the cerebellum input stage. Despite theoretical predictions, little was known about long-term synaptic plasticity at Golgi cell synapses. Here we have used whole-cell patch-clamp recordings and calcium imaging to investigate long-term synaptic plasticity at excitatory synapses impinging on Golgi cells. In acute mouse cerebellar slices, mossy fiber theta-burst stimulation (TBS) could induce either long-term potentiation (LTP) or long-term depression (LTD) at mossy fiber-Golgi cell and granule cell-Golgi cell synapses. This synaptic plasticity showed a peculiar voltage-dependence, with LTD or LTP being favored when TBS induction occurred at depolarized or hyperpolarized potentials, respectively. LTP required, in addition to NMDA channels, activation of T-type Ca2+ channels, while LTD required uniquely activation of L-type Ca2+ channels. Notably, the voltage-dependence of plasticity at the mossy fiber-Golgi cell synapses was inverted with respect to pure NMDA receptor-dependent plasticity at the neighboring mossy fiber-granule cell synapse, implying that the mossy fiber presynaptic terminal can activate different induction mechanisms depending on the target cell. In aggregate, this result shows that Golgi cells show cell-specific forms of long-term plasticity at their excitatory synapses, that could play a crucial role in sculpting the response patterns of the cerebellar granular layer.Significance statementThis paper shows for the first time a novel form of Ca2+ channel-dependent synaptic plasticity at the excitatory synapses impinging on cerebellar Golgi cells. This plasticity is bidirectional and inverted with respect to NMDA receptor-dependent paradigms, with LTD and LTP being favored at depolarized and hyperpolarized potentials, respectively. Furthermore, LTP and LTD induction requires differential involvement of T-ype and L-type voltage-gated Ca2+channels rather than the NMDA receptors alone. These results, along with recent computational predictions, support the idea that Golgi cell plasticity could play a crucial role in controlling information flow through the granular layer along with cerebellar learning and memory.

Synapses are precisely established, maintained, and modified throughout life by molecules called synaptic organizers, which include neurexins and neuroligins (Nlgn). Despite the importance of synaptic organizers in defining functions of neuronal circuits, the cellular and subcellular localization of many synaptic organizers has remained largely elusive because of the paucity of specific antibodies for immunohistochemical studies. In the present study, rather than raising specific antibodies, we generated knock-in mice in which a hemagglutinin (HA) epitope was inserted in the Nlgn1 gene. We have achieved high-throughput and precise gene editing by delivering the CRISPR/Cas9 system into zygotes. Using HA-Nlgn1 mice, we found that HA-Nlgn1 was enriched at synapses between parallel fibers and molecular layer interneurons (MLIs) and the glomeruli, in which mossy fiber terminals synapse onto granule cell dendrites. HA immunoreactivity was colocalized with postsynaptic density 95 at these synapses, indicating that endogenous Nlgn1 is localized at excitatory postsynaptic sites. In contrast, HA-Nlgn1 signals were very weak in dendrites and somata of Purkinje cells. Interestingly, HA-immunoreactivities were also observed in the pinceau, a specialized structure formed by MLI axons and astrocytes. HA-immunoreactivities in the pinceau were significantly reduced by knockdown of Nlgn1 in MLIs, indicating that in addition to postsynaptic sites, Nlgn1 is also localized at MLI axons. Our results indicate that epitope-tagging by electroporation-based gene editing with CRISPR/Cas9 is a viable and powerful method for mapping endogenous synaptic organizers with subcellular resolution, without the need for specific antibodies for each protein.

Mesial temporal lobe epilepsy (MTLE) is the most frequent focal epileptic syndrome in adults, and the majority of seizures originate primarily from the hippocampus. The resected hippocampal tissue often shows severe neuronal loss, a condition referred to as hippocampal sclerosis (HS). In order to understand hippocampal epileptogenesis in MTLE, it seems important to clarify any discrepancies between the clinical and pathological features of affected patients. Here we investigated epileptiform activities ex vivo using living hippocampal tissue taken from patients with MTLE. Flavoprotein fluorescence imaging and local field potential recordings revealed that epileptiform activities developed from the subiculum. Moreover, physiological and morphological experiments revealed possible impairment of K+ clearance in the subiculum affected by HS. Stimulation of mossy fibers induced recurrent trans-synaptic activity in the granule cell layer of the dentate gyrus, suggesting that mossy fiber sprouting in HS also contributes to the epileptogenic mechanism. These results indicate that pathophysiological alterations involving the subiculum and dentate gyrus could be responsible for epileptogenesis in patients with MTLE., Highlights • Enhanced activity including HFO was detected in subiculum samples from patients with MTLE. • Decreased Kir4.1 channel activity in astrocytes was considered to induce hyperexcitability in the subiculum in HS. • MFS was observed in granule cells of the DG only in HS patients and was correlated with the degree of dispersion. In most cases of mesial temporal lobe epilepsy (MTLE), seizure onset can be detected accurately in the hippocampus and the prognosis after surgical resection is good, suggesting that epileptogenesis originates in the hippocampus. We physiologically investigated surgically resected human hippocampal tissue ex vivo, and found enhanced activities including HFO in the subiculum. Failure of the potassium buffering system in astrocytes was considered to contribute to the epileptic activity in the hippocampus affected by severe sclerosis. Our results suggest that different pathophysiological mechanisms may underlie MTLE depending on the degree of hippocampal sclerosis.

SUMMARY Dentate gyrus (DG) development requires specification of granule cell (GC) progenitors in the hippocampal neuroepithelium, as well as their proliferation and migration into the primordial DG. We identify the Plexin family members Plxna2 and Plxna4 as important regulators of DG development. Distribution of immature GCs is regulated by Sema5A signaling through PlxnA2 and requires a functional PlxnA2 GTPase-activating protein (GAP) domain and Rap1 small GTPases. In adult Plxna2−/− but not Plxna2-GAP-deficient mice, the dentate GC layer is severely malformed, neurogenesis is compromised, and mossy fibers form aberrant synaptic boutons within CA3. Behavioral studies with Plxna2−/− mice revealed deficits in associative learning, sociability, and sensorimotor gating—traits commonly observed in neuropsychiatric disorder. Remarkably, while morphological defects are minimal in Plxna2-GAP-deficient brains, defects in fear memory and sensorimotor gating persist. Since allelic variants of human PLXNA2 and RAP1 associate with schizophrenia, our studies identify a biochemical pathway important for brain development and mental health., In Brief Zhao et al. find that Sema5A-PlexinA2 forward signaling through Rap1 GTPases is required for progenitor distribution in the developing mouse dentate gyrus. Adult Plxna2−/−, but not Plxna2-GAP-deficient, mice show defects in dentate morphology, neurogenesis, and mossy fiber connectivity. Plxna2−/− and Plxna2-GAP mice exhibit behavioral defects suggestive of neuropsychiatric illness.

Appropriate axonal pathfinding is a necessary step for the function of neuronal circuits. The mossy fibers (MFs) in the hippocampus of CaMKIIα heterozygous knockout (CaMKIIα-hKO) psychiatric model mice project onto not only the stratum lucidum but also the stratum oriens region in the CA3, which is a projection pattern distinct from that in normal mice. Thus, we examined the electrophysiological properties of the MF-CA3 connection in this mutant mouse on field recordings and found a lower synaptic connection. This study suggested that the phenotype of abnormal MF pathfindings could induce aberrant neuronal functions, which may link to cognition and memory.

The application of tetraethylammonium (TEA), a blocker of voltage-dependent potassium channels, can induce long-term potentiation (LTP) in the synaptic systems CA3–CA1 and mossy fiber-CA3 pyramidal cells of the hippocampus. In the mossy fibers, the depolarization evoked by extracellular TEA induces a large amount of glutamate and also of zinc release. It is considered that zinc has a neuromodulatory role at the mossy fiber synapses, which can, at least in part, be due to the activation of presynaptic ATP-dependent potassium (KATP) channels. The aim of this work was to study properties of TEA-induced zinc signals, detected at the mossy fiber region, using the permeant form of the zinc indicator Newport Green. The application of TEA caused a depression of those signals that was partially blocked by the KATP channel inhibitor tolbutamide. After the removal of TEA, the signals usually increased to a level above baseline. These results are in agreement with the idea that intense zinc release during strong synaptic events triggers a negative feedback action. The zinc depression, caused by the LTP-evoking chemical stimulation, turns into potentiation after TEA washout, suggesting the existence of a correspondence between the observed zinc potentiation and TEA-evoked mossy fiber LTP.

Accumulating data have revealed that abnormal activity of the mTOR (mammalian target of rapamycin) pathway plays an important role in epileptogenesis triggered by various factors. We previously reported that pretreatment with perifosine, an inhibitor of Akt (also called protein kinase B), abolishes the rapamycin-induced paradoxical increase of S6 phosphorylation in a rat model induced by kainic acid (KA). Since Akt is an upstream target in the mTOR signaling pathway, we set out to determine whether perifosine has a preventive effect on epileptogenesis. Here, we explored the effect of perifosine on the model of temporal epilepsy induced by KA in rats and found that pretreatment with perifosine had no effect on the severity or duration of the KA-induced status epilepticus. However, perifosine almost completely inhibited the activation of p-Akt and p-S6 both acutely and chronically following the KA-induced status epilepticus. Perifosine pretreatment suppressed the KA-induced neuronal death and mossy fiber sprouting. The frequency of spontaneous seizures was markedly decreased in rats pretreated with perifosine. Accordingly, rats pretreated with perifosine showed mild impairment in cognitive functions. Collectively, this study provides novel evidence in a KA seizure model that perifosine may be a potential drug for use in anti-epileptogenic therapy.

Although it has been established that recurrent or prolonged clinical seizures during infancy may cause lifelong brain damage, the underlying molecular mechanism is still not well elucidated. The present study, to the best of our knowledge, is the first to investigate the expression of twenty zinc (Zn)/lipid metabolism‑associated genes in the hippocampus and cerebral cortex of rats following recurrent neonatal seizures. In the current study, 6‑day‑old Sprague‑Dawley rats were randomly divided into control (CONT) and recurrent neonatal seizure (RS) groups. On postnatal day 35 (P35), mossy fiber sprouting and gene expression were assessed by Timm staining and reverse transcription‑quantitative polymerase chain reaction, respectively. Of the twenty genes investigated, seven were significantly downregulated, while four were significantly upregulated in the RS group compared with CONT rats, which was observed in the hippocampus but not in the cerebral cortex. Meanwhile, aberrant mossy fiber sprouting was observed in the supragranular region of the dentate gyrus and Cornu Ammonis 3 subfield of the hippocampus in the RS group. In addition, linear correlation analysis identified significant associations between the expression of certain genes in the hippocampus, which accounted for 40% of the total fifty‑five gene pairs among the eleven regulated genes. However, only eight gene pairs in the cerebral cortex exhibited significant positive associations, which accounted for 14.5% of the total. The results of the present study indicated the importance of hippocampal Zn/lipid metabolism‑associated genes in recurrent neonatal seizure‑induced aberrant mossy fiber sprouting, which may aid the identification of novel potential targets during epileptogenesis.

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their non-junctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses. This article is protected by copyright. All rights reserved.

Objective: Our primary objective is to verify whether 5-HTR6 is involved in the development of mossy fiber sprouting (MFS), and to determine how the progression of MFS is affected by 5-HTR6. Methods: A total of 90 male adult Sprague-Dawley rats were allocated into either the control group (n=36) or the epileptic group (n=54). Status epilepticus (SE) of rats was induced by the intraperitoneal (i.p.) injection of LiCl-pilocarpine. We conducted our experiments in two stages. The first stage involves equally dividing 36 epileptic rats into three groups with treatments of none, 5-HTR6 antagonist SB-27104 (SB) and vehicle DMSO. Then behavior and electroencephalogram (EEG) of rats were monitored by video-EEG. The second stage involves dividing 126 epileptic rats into seven groups with treatments of none, 10% DMSO, SB (100 µg/kg), Fyn antagonist PP2 (50 µg/kg), p-ERK1/2 antagonist PD-98059 (30 µg/kg), SB (100 µg/ kg) + PP2 (50 µg/kg); SB (100 µg/kg) + PD-98059 (30 µg/kg). We also treated 18 rats in the control group of the first stage with 100 µg/kg 5-HTR6 agonist WAY-181187 (WAY). MFS of rats was detected through the approach of Timm’s staining. Finally, expressions of 5-HTR6, Fyn, p-ERK1/2 and GAP-3 were qualified and semi-quantified via western blotting or RT-PCR. Results: Induction of SE could stimulate formation of MFS and increased GAP-43 expressions. Expressions of 5-HTR6, Fyn and p-ERK1/2 were also up-regulated with increasing time after establishment of SE models. The development of MFS was remarkably inhibited by SB, PP2 and PD. Compared to the single antagonist, such an inhibitory effect was enhanced by SB+PD or SB+PP. Moreover, treatment of healthy rats with WAY would contribute to up-regulated Fyn and p-ERK1/2 expressions, as well as development of MFS (P < 0.05). Suppression of Fyn triggered a down-regulating trend of p-ERK1/2 (P < 0.05), however, suppressed p-ERK1/2 did not have such a significant effect on Fyn expression. Conclusion: HTR6 may affect the progression of MFS by activating both p-ERK1/2 and Fyn, which further modulate the expression of GAP-43.

Neuronal calcium elevations are shaped by several key parameters, including the properties, density, and the spatial location of voltage-gated calcium channels (VGCCs). These features allow presynaptic terminals to translate complex firing frequencies and tune the amount of neurotransmitter released. Although synchronous neurotransmitter release relies on both P/Q- and N-type VGCCs at hippocampal mossy fiber–CA3 synapses, the specific contribution of VGCCs to calcium dynamics, neurotransmitter release, and short-term facilitation remains unknown. Here, we used random-access two-photon calcium imaging together with electrophysiology in acute mouse hippocampal slices to dissect the roles of P/Q- and N-type VGCCs. Our results show that N-type VGCCs control glutamate release at a limited number of release sites through highly localized Ca2+elevations and support short-term facilitation by enhancing multivesicular release. In contrast, Ca2+entry via P/Q-type VGCCs promotes the recruitment of additional release sites through spatially homogeneous Ca2+elevations. Altogether, our results highlight the specialized contribution of P/Q- and N-types VGCCs to neurotransmitter release.SIGNIFICANCE STATEMENTIn presynaptic terminals, neurotransmitter release is dynamically regulated by the transient opening of different types of voltage-gated calcium channels. Hippocampal giant mossy fiber terminals display extensive short-term facilitation during repetitive activity, with a large several fold postsynaptic response increase. Though, how giant mossy fiber terminals leverage distinct types of voltage-gated calcium channels to mediate short-term facilitation remains unexplored. Here, we find that P/Q- and N-type VGCCs generate different spatial patterns of calcium elevations in giant mossy fiber terminals and support short-term facilitation through specific participation in two mechanisms. Whereas N-type VGCCs contribute only to the synchronization of multivesicular release, P/Q-type VGCCs act through microdomain signaling to recruit additional release sites.

CaMKII has been proposed as a molecular substrate for long-term memory storage due to its capacity to maintain an active autophosporylated state even after the decay of the external stimuli. The hippocampal mossy fiber-CA3 pathway (MF-CA3) is considered as a relevant area for acquisition and storage of different learning tasks. MF-CA3 pathway exhibits a form of LTP characterized by a slow initial increase in the EPSP slope that is independent of NMDA receptors activation. Our previous studies show that application of high frequency stimulation sufficient to elicit MF-CA3 LTP produces structural reorganization, in a manner independent of LTP induction, at the stratum oriens of hippocampal CA3 area 7days after stimulation. However, the molecular mechanisms that underlie the maintenance of MF-CA3 LTP as well as the concomitant structural reorganization in this area remain to be elucidated. Here we show that acute microinfusion of myr-CaMKIINtide, a noncompetitive inhibitor of CaMKII, in the hippocampal CA3 area of adult rats during the late-phase of in vivo MF-CA3 LTP blocked its maintenance and prevented the accompanying morphological reorganization in CA3 area. These findings support the idea that CaMKII is a key molecular substrate for the long-term hippocampal synaptic plasticity maintenance.

Objective: Rewiring of excitatory glutamatergic neuronal circuits is a major abnormality in epilepsy. Besides the rewiring of excitatory circuit, an abnormal depolarizing GABAergic drive has been hypothesized to participate in the epileptogenic processes. However, a remaining clinically relevant question is whether early post Status Epilepticus (SE) evoked chloride dysregulation is important for the remodeling of aberrant glutamatergic neuronal circuit. Methods: Osmotic mini-pumps were used to infuse intracerebrally a specific inhibitor of depolarizing GABAergic transmission as well as a functionally blocking antibody towards the pan-neurotrophin receptor p75 (p75NTR). The compounds were infused between 2 and 5 days after pilocarpine-induced SE. Immunohistochemistry for NKCC1, KCC2 and ectopic recurrent mossy fiber (rMF) sprouting as well as telemetric EEG and electrophysiological recordings were performed at day 5 and 2 months post-SE. Results: Blockade of NKCC1 after SE with the specific inhibitor bumetanide restored NKCC1 and KCC2 expression, normalized chloride homeostasis and significantly reduced the glutamatergic recurrent mossy fiber sprouting (rMF) within the dentate gyrus. This mechanism partially involves p75NTR signaling as bumetanide application reduced SE-induced p75NTR expression and functional blockade of p75NTR decreased rMF sprouting. The early transient (3days) post-SE infusion of bumetanide reduced rMF sprouting and recurrent seizures in the chronic epileptic phase. Interpretation: Our findings show that early post-SE abnormal depolarizing GABA and p75NTR signalings foster a long lasting rearrangement of glutamatergic network that contributes to the epileptogenic process. This finding defines promising and novel targets to constrain reactive glutamatergic network rewiring in adult epilepsy. This article is protected by copyright. All rights reserved.

Electroconvulsive therapy (ECT) is an established effective treatment for medication-resistant depression with the rapid onset of action. However, its cellular mechanism of action has not been revealed. We have previously shown that chronic antidepressant drug treatments enhance dopamine D1-like receptor-dependent synaptic potentiation at the hippocampal mossy fiber (MF)-CA3 excitatory synapse. In this study we show that ECT-like treatments in mice also have marked effects on the dopaminergic synaptic modulation. Repeated electroconvulsive stimulation (ECS), an animal model of ECT, strongly enhanced the dopamine-induced synaptic potentiation at the MF synapse in hippocampal slices. Significant enhancement was detectable after the second ECS, and further repetition of ECS up to 11 times monotonously increased the magnitude of enhancement. After repeated ECS, the dopamine-induced synaptic potentiation remained enhanced for more than 4 wk. These synaptic effects of ECS were accompanied by increased expression of the dopamine D1 receptor gene. Our results demonstrate that robust neuronal activation by ECS induces rapid and long-lasting enhancement of dopamine-induced synaptic potentiation at the MF synapse, likely via increased expression of the D1 receptor, at least in part. This rapid enhancement of dopamine-induced potentiation at the excitatory synapse may be relevant to the fast-acting antidepressant effect of ECT. NEW & NOTEWORTHY We show that electroconvulsive therapy (ECT)-like stimulation greatly enhances synaptic potentiation induced by dopamine at the excitatory synapse formed by the hippocampal mossy fiber in mice. The effect of ECT-like stimulation on the dopaminergic modulation was rapidly induced, maintained for more than 4 wk after repeated treatments, and most likely mediated by increased expression of the dopamine D1 receptor. These effects may be relevant to fast-acting strong antidepressant action of ECT.