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Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of presynaptic potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed 2-photon imaging of the genetically encoded glutamate sensor iGluu that revealed an increase in the surface area used for glutamate release at potentiated terminals. Time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between P/Q-type calcium channels and release sites mapped by Munc13-1 cluster position. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: Synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements might enable long-term increase in synaptic strength., This study uses several high-resolution imaging techniques to investigate the structural correlates of presynaptic potentiation at hippocampal mossy fiber boutons, observing an increase in release sites and in release synchronicity accompanied by synaptic vesicle dispersion in the terminal and accumulation at release sites, but no modulation of the distance between calcium channel and release sites.

The Brain-Derived Neurotrophic Factor is one of the most important trophic proteins in the brain. The role of this growth factor in neuronal plasticity, in health and disease, has been extensively studied. However, mechanisms of epigenetic regulation of Bdnf gene expression in epilepsy are still elusive. In our previous work, using a rat model of neuronal activation upon kainate-induced seizures, we observed a repositioning of Bdnf alleles from the nuclear periphery towards the nuclear center. This change of Bdnf intranuclear position was associated with transcriptional gene activity. In the present study, using the same neuronal activation model, we analyzed the relation between the percentage of the Bdnf allele at the nuclear periphery and clinical and morphological traits of epilepsy. We observed that the decrease of the percentage of the Bdnf allele at the nuclear periphery correlates with stronger mossy fiber sprouting—an aberrant form of excitatory circuits formation. Moreover, using in vitro hippocampal cultures we showed that Bdnf repositioning is a consequence of transcriptional activity. Inhibition of RNA polymerase II activity in primary cultured neurons with Actinomycin D completely blocked Bdnf gene transcription and repositioning occurring after neuronal excitation. Interestingly, we observed that histone deacetylases inhibition with Trichostatin A induced a slight increase of Bdnf gene transcription and its repositioning even in the absence of neuronal excitation. Presented results provide novel insight into the role of BDNF in epileptogenesis. Moreover, they strengthen the statement that this particular gene is a good candidate to search for a new generation of antiepileptic therapies.

Presynaptic mitochondrial Ca2+ plays a critical role in the regulation of synaptic transmission and plasticity. The presynaptic bouton of the hippocampal mossy fiber (MF) is much larger in size than that of the Schaffer collateral (SC) synapse. Here we compare the structural and physiological characteristics of MF and SC presynaptic boutons to reveal functional and mechanistic differences between these two synapses. Our quantitative ultrastructural analysis using electron microscopy show many more mitochondria in MF presynaptic bouton cross-section profiles compared to SC boutons. Consistent with these results, post-tetanic potentiation (PTP), a form of presynaptic short-term plasticity dependent on mitochondrial Ca2+, is reduced by inhibition of mitochondrial Ca2+ release at MF synapses but not at SC synapses. However, blockade of mitochondrial Ca2+ release results in reduction of PTP at SC synapses by disynaptic MF stimulation. Furthermore, inhibition of mitochondrial Ca2+ release selectively decreases frequency facilitation evoked by short trains of presynaptic stimulation at MF synapses, while having no effect at SC synapses. Moreover, depletion of ER Ca2+ stores leads to reduction of PTP at MF synapses, but PTP is unaffected by ER Ca2+ depletion at SC synapses. These findings show that MF and SC synapses differ in presynaptic mitochondrial content as well as mitochondrial Ca2+ dependent synaptic plasticity, highlighting differential regulatory mechanisms of presynaptic plasticity at MF and SC synapses.

The Arf GTPase-activating protein ArfGAP1 and its brain-specific isoform ArfGAP1B play an important role in neurotransmission. Here we analyzed the distribution of ArfGAP1 in the mouse brain. We found high levels of ArfGAP1 in the mouse dentate gyrus where it displayed especially elevated level in the polymorph layer (hilus). Importantly, the ArfGAP1 signal follows the pathway of the granular cell axons so-called mossy fibers which extend from the dentate gyrus to CA3 via stratum lucidum and partially stratum oriens. Additionally, we identified differential expression of ArfGAP1 in the isocortex. Thus, staining with anti-ArfGAP1 antibodies allows distinction between cortical cell layers 1, 2, 3 and 5 from 4 and 6. Taken together, our data suggest that ArfGAP1 can be used as a specific marker of the dentate mossy fibers and as for visualization of cortical layers in immunohistochemical studies.

Memory-related activity in the Dentate Gyrus (DG) is characterized by sparsity. Memory representations are seen as activated neuronal populations of granule cells, the main encoding cells in DG, which are estimated to engage 2–4% of the total population. This sparsity is assumed to enhance the ability of DG to perform pattern separation, one of the most valuable contributions of DG during memory formation. In this work, we investigate how features of the DG such as its excitatory and inhibitory connectivity diagram can be used to develop theoretical algorithms performing Sparse Approximation, a widely used strategy in the Signal Processing field. Sparse approximation stands for the algorithmic identification of few components from a dictionary that approximate a certain signal. The ability of DG to achieve pattern separation by sparsifing its representations is exploited here to improve the performance of the state of the art sparse approximation algorithm “Iterative Soft Thresholding” (IST) by adding new algorithmic features inspired by the DG circuitry. Lateral inhibition of granule cells, either direct or indirect, via mossy cells, is shown to enhance the performance of the IST. Apart from revealing the potential of DG-inspired theoretical algorithms, this work presents new insights regarding the function of particular cell types in the pattern separation task of the DG.

The formation and remodeling of mossy fiber-CA3 pyramidal cell synapses in the stratum lucidum of the hippocampus are implicated in the cellular basis of learning and memory. Afadin and its binding cell adhesion molecules, nectin-1 and nectin-3, together with N-cadherin, are concentrated at puncta adherentia junctions (PAJs) in these synapses. Here, we investigated the roles of afadin in PAJ formation and presynaptic differentiation in mossy fiber-CA3 pyramidal cell synapses. At these synapses in the mice in which the afadin gene was conditionally inactivated before synaptogenesis by using nestin-Cre mice, the immunofluorescence signals for the PAJ components, nectin-1, nectin-3 and N-cadherin, disappeared almost completely, while those for the presynaptic components, VGLUT1 and bassoon, were markedly decreased. In addition, these signals were significantly decreased in cultured afadin-deficient hippocampal neurons. Furthermore, the interevent interval of miniature excitatory postsynaptic currents was prolonged in the cultured afadin-deficient hippocampal neurons compared with control neurons, indicating that presynaptic functions were suppressed or a number of synapse was reduced in the afadin-deficient neurons. Analyses of presynaptic vesicle recycling and paired recordings revealed that the cultured afadin-deficient neurons showed impaired presynaptic functions. These results indicate that afadin regulates both PAJ formation and presynaptic differentiation in most mossy fiber-CA3 pyramidal cell synapses, while in a considerable population of these neurons, afadin regulates only PAJ formation but not presynaptic differentiation.

Beta-amyloid precursor protein cleaving enzyme 1 (BACE1), a major neuronal β-secretase critical for the formation of β-amyloid (Aβ) peptide, is considered one of the key therapeutic targets that can prevent the progression of Alzheimer’s disease (AD). Although a complete ablation of BACE1 gene prevents Aβ formation, we previously reported that BACE1 knockouts (KOs) display presynaptic deficits, especially at the mossy fiber (MF) to CA3 synapses. Whether the defect is specific to certain inputs or postsynaptic targets in CA3 is unknown. To determine this, we performed whole-cell recording from pyramidal cells (PYR) and the stratum lucidum (SL) interneurons in the CA3, both of which receive excitatory MF terminals with high levels of BACE1 expression. BACE1 KOs displayed an enhancement of paired-pulse facilitation at the MF inputs to CA3 PYRs without changes at the MF inputs to SL interneurons, which suggests postsynaptic target specific regulation. The synaptic dysfunction in CA3 PYRs was not restricted to excitatory synapses, as seen by an increase in the paired-pulse ratio of evoked inhibitory postsynaptic currents from SL to CA3 PYRs. In addition to the changes in evoked synaptic transmission, BACE1 KOs displayed a reduction in the frequency of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) in CA3 PYRs without alteration in mEPSCs recorded from SL interneurons. This suggests that the impairment may be more global across diverse inputs to CA3 PYRs. Our results indicate that the synaptic dysfunctions seen in BACE1 KOs are specific to the postsynaptic target, the CA3 PYRs, independent of the input type.

Posttraumatic epilepsy is a major source of disability following traumatic brain injury (TBI) and a common cause of medically-intractable epilepsy. Previous attempts to prevent the development of posttraumatic epilepsy with treatments administered immediately following TBI have failed. Recently, the mammalian target of rapamycin complex 1 (mTORC1) pathway has been implicated in mechanisms of epileptogenesis and the mTORC1 inhibitor, rapamycin, has been proposed to have antiepileptogenic effects in preventing some types of epilepsy. In this study, we have tested the hypothesis that rapamycin has antiepileptogenic actions in preventing the development of posttraumatic epilepsy in an animal model of TBI. A detailed characterization of posttraumatic epilepsy in the mouse controlled cortical impact model was first performed using continuous video-EEG monitoring for 16 weeks following TBI. Controlled cortical impact injury caused immediate hyperactivation of the mTORC1 pathway lasting at least one week, which was reversed by rapamycin treatment. Rapamycin decreased neuronal degeneration and mossy fiber sprouting, although the effect on mossy fiber sprouting was reversible after stopping rapamycin and did not directly correlate with inhibition of epileptogenesis. Most posttraumatic seizures occurred greater than 10 weeks after TBI, and rapamycin treatment for one month after TBI decreased the seizure frequency and rate of developing posttraumatic epilepsy during the entire 16 week monitoring session. These results suggest that rapamycin may represent a rational treatment for preventing posttraumatic epilepsy in patients with TBI.

Down syndrome (DS) is a high-incidence genetic pathology characterized by severe impairment of cognitive functions, including declarative memory. Impairment of hippocampus-dependent long-term memory in DS appears to be related to anatomo-functional alterations of the hippocampal trisynaptic circuit formed by the dentate gyrus (DG) granule cells - CA3 pyramidal neurons - CA1 pyramidal neurons. No therapies exist to improve cognitive disability in individuals with DS. In previous studies we demonstrated that pharmacotherapy with fluoxetine restores neurogenesis, granule cell number and dendritic morphology in the DG of the Ts65Dn mouse model of DS. The goal of the current study was to establish whether treatment rescues the impairment of synaptic connectivity between the DG and CA3 that characterizes the trisomic condition. Euploid and Ts65Dn mice were treated with fluoxetine during the first two postnatal weeks and examined 45-60 days after treatment cessation. Untreated Ts65Dn mice had a hypotrophyc mossy fiber bundle, fewer synaptic contacts, fewer glutamatergic contacts, and fewer dendritic spines in the stratum lucidum of CA3, the terminal field of the granule cell projections. Electrophysiological recordings from CA3 pyramidal neurons showed that in Ts65Dn mice the frequency of both mEPSCs and mIPSCs was reduced, indicating an overall impairment of excitatory and inhibitory inputs to CA3 pyramidal neurons. In treated Ts65Dn mice all these aberrant features were fully normalized, indicating that fluoxetine can rescue functional connectivity between the DG and CA3. The positive effects of fluoxetine on the DG-CA3 system suggest that early treatment with this drug could be a suitable therapy, possibly usable in humans, to restore the physiology of the hippocampal networks and, hence, memory functions.

"We have previously shown that double deletion of the genes for Rac1 and Rac3 GTPases during neuronal development affects late developmental events that perturb the circuitry of the hippocampus, with ensuing epileptic phenotype. These effects include a defect in mossy cells, the major class of excitatory neurons of the hilus. Here, we have addressed the mechanisms that affect the loss of hilar mossy cells in the dorsal hippocampus of mice depleted of the two Rac GTPases. Quantification showed that the loss of mossy cells was evident already at postnatal day 8, soon after these cells become identifiable by a specific marker in the dorsal hilus. Comparative analysis of the hilar region from control and double mutant mice revealed that synaptogenesis was affected in the double mutants, with strongly reduced presynaptic input from dentate granule cells. We found that apoptosis was equally low in the hippocampus of both control and double knockout mice. Labelling with bromodeoxyuridine at embryonic day 12.5 showed no evident difference in the proliferation of neuronal precursors in the hippocampal primordium, while differences in the number of bromodeoxyuridine-labelled cells in the developing hilus revealed a defect in the migration of immature, developing mossy cells in the brain of double knockout mice. Overall, our data show that Rac1 and Rac3 GTPases participate in the normal development of hilar mossy cells, and indicate that they are involved in the regulation of the migration of the mossy cell precursor by preventing their arrival to the dorsal hilus.. "

Dysbindin-1 (dystrobrevin-binding protein 1, DTNBP1) is one of the promising schizophrenia susceptibility genes. Dysbindin protein is abundantly expressed in synaptic regions of the hippocampus, including the terminal field of the mossy fibers, and this hippocampal expression of dysbindin is strongly reduced in patients with schizophrenia. In the present study, we examined the functional role of dysbindin in hippocampal mossy fiber-CA3 synaptic transmission and its modulation using the sandy mouse, a spontaneous mutant with deletion in the dysbindin gene. Electrophysiological recordings were made in hippocampal slices prepared from adult male sandy mice and their wild-type littermates. Basic properties of the mossy fiber synaptic transmission in the mutant mice were generally normal except for slightly reduced frequency facilitation. Serotonin and dopamine, two major neuromodulators implicated in the pathophysiology of schizophrenia, can potentiate mossy fiber synaptic transmission probably via an increase in cAMP levels. Synaptic potentiation induced by serotonin and dopamine was very variable in magnitude in the mutant mice, with some mice showing prominent enhancement as compared with the wild-type mice. In addition, the magnitude of potentiation induced by these monoamines significantly correlated with each other in the mutant mice, indicating that a subpopulation of sandy mice has marked hypersensitivity to both serotonin and dopamine. While direct activation of the cAMP cascade by forskolin induced robust synaptic potentiation in both wild-type and mutant mice, this forskolin-induced potentaition correlated in magnitude with the serotonin-induced potentiation only in the mutant mice, suggesting a possible change in coupling of receptor activation to downstream signaling. These results suggest that the dysbindin deficiency could be an essential genetic factor that causes synaptic hypersensitivity to dopamine and serotonin. The altered monoaminergic modulation at the mossy fiber synapse could be a candidate pathophysiological basis for impairment of hippocampus-dependent brain functions in schizophrenia.

In this study, we examined the effect of chronic administration of simvastatin immediately after status epilepticus (SE) on rat brain with temporal lobe epilepsy (TLE). First, we evaluated cytokines expression at 3 days post KA-lesion in hippocampus and found that simvastatin-treatment suppressed lesion-induced expression of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α). Further, we quantified reactive astrocytosis using glial fibrillary acidic protein (GFAP) staining and neuron loss using Nissl staining in hippocampus at 4-6 months after KA-lesion. We found that simvastatin suppressed reactive astrocytosis demonstrated by a significant decrease in GFAP-positive cells, and attenuated loss of pyramidal neurons in CA3 and interneurons in dentate hilar (DH). We next assessed aberrant mossy fiber sprouting (MFS) that is known to contribute to recurrence of spontaneous seizure in epileptic brain. In contrast to the robust MFS observed in saline-treated animals, the extent of MFS was restrained by simvastatin in epileptic rats. Attenuated MFS was related to decreased neuronal loss in CA3 and DH, which is possibly a mechanism underlying decreased hippocampal susceptibility in animal treated with simvastatin. Electronic encephalography (EEG) was recorded during 4 to 6 months after KA-lesion. The frequency of abnormal spikes in rats with simvastatin-treatment decreased significantly compared to the saline group. In summary, simvastatin treatment suppressed cytokines expression and reactive astrocytosis and decreased the frequency of discharges of epileptic brain, which might be due to the inhibition of MFS in DH. Our study suggests that simvastatin administration might be a possible intervention and promising strategy for preventing SE exacerbating to chronic epilepsy.

Background Synapses exhibit strikingly different forms of plasticity over a wide range of time scales, from milliseconds to hours. Studies on synaptic plasticity typically use constant-frequency stimulation to activate synapses, whereas in vivo activity of neurons is irregular. Methodology/Principal Findings Using extracellular and whole-cell electrophysiological recordings, we have here studied the synaptic responses at hippocampal mossy fiber synapses in vitro to stimulus patterns obtained from in vivo recordings of place cell firing of dentate gyrus granule cells in behaving rodents. We find that synaptic strength is strongly modulated on short- and long-lasting time scales during the presentation of the natural stimulus trains. Conclusions/Significance We conclude that dynamic short- and long-term synaptic plasticity at the hippocampal mossy fiber synapse plays a prominent role in normal synaptic function.

In the mammalian hippocampus, the dentate gyrus (DG) is characterized by sparse and powerful unidirectional projections to CA3 pyramidal cells, the so-called mossy fibers. Mossy fiber synapses appear to duplicate, in terms of the information they convey, what CA3 cells already receive from entorhinal cortex layer II cells, which project both to the dentate gyrus and to CA3. Computational models of episodic memory have hypothesized that the function of the mossy fibers is to enforce a new, well separated pattern of activity onto CA3 cells, to represent a new memory, prevailing over the interference produced by the traces of older memories already stored on CA3 recurrent collateral connections. Can this hypothesis apply also to spatial representations, as described by recent neurophysiological recordings in rats? To address this issue quantitatively, we estimate the amount of information DG can impart on a new CA3 pattern of spatial activity, using both mathematical analysis and computer simulations of a simplified model. We confirm that, also in the spatial case, the observed sparse connectivity and level of activity are most appropriate for driving memory storage – and not to initiate retrieval. Surprisingly, the model also indicates that even when DG codes just for space, much of the information it passes on to CA3 acquires a non-spatial and episodic character, akin to that of a random number generator. It is suggested that further hippocampal processing is required to make full spatial use of DG inputs., Author Summary The CA3 region at the core of the hippocampus, a structure crucial to memory formation, presents one striking anatomical feature. Its neurons receive many thousands of weak inputs from other sources, but only a few tens of very strong inputs from the neurons in the directly preceding region, the dentate gyrus. It had been proposed that such sparse connectivity helps the dentate gyrus to drive CA3 activity during the storage of new memories, but why it needs to be so sparse had remained unclear. Recent recordings of neuronal activity in the dentate gyrus (Leutgeb, et al. 2007) show the firing maps of granule cells of rodents engaged in exploration: the few cells active in a given environment, about 3% of the total, present multiple firing fields. Following these findings, we could now construct a network model that addresses the question quantitatively. Both mathematical analysis and computer simulations of the model show that, while the memory system would function also otherwise, connections as sparse as those observed make it function optimally, in terms of the bits of information new memories contain. Much of this information, we show, is encoded however in a difficult format, suggesting that other regions of the hippocampus, until now with no clear role, may contribute to decode it.

Zinc homeostasis in the brain is associated with the etiology and manifestation of epileptic seizures. Adult Noda epileptic rats (NER, >12-week-old) exhibit spontaneously generalized tonic-clonic convulsion about once a day. To pursue the involvement of synaptic Zn(2+) signal in susceptibility to spontaneous seizures, in the present study, the effect of zinc chelators on epileptogenesis was examined using adult NER. Clioquinol (CQ) and TPEN are lipophilic zinc chelotors, transported into the brain and reduce the levels of synaptic Zn(2+). The incidence of tonic-clonic convulsion was markedly increased after i.p. injection of CQ (30-100 mg/kg) and TPEN (1 mg/kg). The basal levels of extracellular Zn(2+) measured by ZnAF-2 were decreased before tonic-clonic convulsion was induced with zinc chelators. The hippocampal electroencephalograms during CQ (30 mg/kg)-induced convulsions were similar to those during sound-induced convulsions in NER reported previously. Exocytosis of hippocampal mossy fibers, which was measured with FM4-64, was significantly increased in hippocampal slices from CQ-injected NER that did not show tonic-clonic convulsion yet. These results indicate that the abnormal excitability of mossy fibers is induced prior to epileptic seizures by injection of zinc chelators into NER. The incidence of tonic-clonic convulsion induced with CQ (30 mg/kg) was significantly reduced by co-injection with aminooxyacetic acid (5-10 mg/kg), an anticonvulsant drug enhancing GABAergic activity, which did not affect locomotor activity. The present paper demonstrates that the abnormal excitability in the brain, especially in mossy fibers, which is potentially associated with the insufficient GABAergic neuron activity, may be a factor to reduce the threshold for epileptogenesis in NER.

Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting.

Temporal lobe epilepsy (TLE) is a neurological condition associated with neuron loss, axon sprouting, and hippocampal sclerosis, which results in modified synaptic circuitry. Cannabinoids appear to be anti-convulsive in patients and animal models of TLE, but the mechanisms of this effect are not known. A pilocarpine-induced status epilepticus mouse model of TLE was used to study the effect of cannabinoid agonists on recurrent excitatory circuits of the dentate gyrus using electrophysiological recordings in hippocampal slices isolated from control mice and mice with TLE. Cannabinoid agonists WIN 55,212-2, anandamide (AEA), or 2-arachydonoylglycerol (2-AG) reduced the frequency of spontaneous and tetrodotoxin-resistant excitatory postsynaptic currents (EPSCs) in mice with TLE, but not in controls. WIN 55,212-2 also reduced the frequency of EPSCs evoked by glutamate-photolysis activation of other granule cells in epileptic mice. Secondary population discharges evoked after antidromic electrical stimulation of mossy fibers in the hilus were also attenuated by cannabinoid agonists. Agonist effects were blocked by the cannabinoid type 1 receptor (CB1R) antagonist AM251. No change in glutamate release was observed in slices from mice that did not undergo status epilepticus. Western blot analysis suggested an up-regulation of CB1R in the dentate gyrus of animals with TLE. These findings indicate that activation of CB1R present on nerve terminals can suppress recurrent excitation in the dentate gyrus of mice with TLE. This suggests a mechanism for the anti-convulsive role of cannabinoids aimed at modulating receptors on synaptic terminals expressed de novo after epileptogenesis.