Hippocampal Mossy Fiber Terminals Research Articles

RIM-BP2 regulates Ca2+ channel abundance and neurotransmitter release at hippocampal mossy fiber terminals

Synaptic vesicles dock and fuse at the presynaptic active zone (AZ), the specialized site for transmitter release. AZ proteins play multiple roles such as recruitment of Ca2+ channels as well as synaptic vesicle docking, priming, and fusion. However, the precise role of each AZ protein type remains unknown. In order to dissect the role of RIM-BP2 at mammalian cortical synapses having low release probability, we applied direct electrophysiological recording and super-resolution imaging to hippocampal mossy fiber terminals of RIM-BP2 knockout (KO) mice. By using direct presynaptic recording, we found the reduced Ca2+ currents. The measurements of excitatory postsynaptic currents (EPSCs) and presynaptic capacitance suggested that the initial release probability was lowered because of the reduced Ca2+ influx and impaired fusion competence in RIM-BP2 KO. Nevertheless, larger Ca2+ influx restored release partially. Consistent with presynaptic recording, STED microscopy suggested less abundance of P/Q-type Ca2+ channels at AZs deficient in RIM-BP2. Our results suggest that the RIM-BP2 regulates both Ca2+ channel abundance and transmitter release at mossy fiber synapses.

RIM-BP2 regulates Ca2+ channel abundance and neurotransmitter release at hippocampal mossy fiber terminals.

Synaptic vesicles dock and fuse at the presynaptic active zone (AZ), the specialized site for transmitter release. AZ proteins play multiple roles such as recruitment of Ca2+ channels as well as synaptic vesicle docking, priming, and fusion. However, the precise role of each AZ protein type remains unknown. In order to dissect the role of RIM-BP2 at mammalian cortical synapses having low release probability, we applied direct electrophysiological recording and super-resolution imaging to hippocampal mossy fiber terminals of RIM-BP2 knockout (KO) mice. By using direct presynaptic recording, we found the reduced Ca2+ currents. The measurements of excitatory postsynaptic currents (EPSCs) and presynaptic capacitance suggested that the initial release probability was lowered because of the reduced Ca2+ influx and impaired fusion competence in RIM-BP2 KO. Nevertheless, larger Ca2+ influx restored release partially. Consistent with presynaptic recording, STED microscopy suggested less abundance of P/Q-type Ca2+ channels at AZs deficient in RIM-BP2. Our results suggest that the RIM-BP2 regulates both Ca2+ channel abundance and transmitter release at mossy fiber synapses.

Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy

Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy

Intracellular Zn2+ Signaling Facilitates Mossy Fiber Input-Induced Heterosynaptic Potentiation of Direct Cortical Inputs in Hippocampal CA3 Pyramidal Cells.

Repetitive action potentials (APs) in hippocampal CA3 pyramidal cells (CA3-PCs) backpropagate to distal apical dendrites, and induce calcium and protein tyrosine kinase (PTK)-dependent downregulation of Kv1.2, resulting in long-term potentiation of direct cortical inputs and intrinsic excitability (LTP-IE). When APs were elicited by direct somatic stimulation of CA3-PCs from rodents of either sex, only a narrow window of distal dendritic [Ca2+] allowed LTP-IE because of Ca2+-dependent coactivation of PTK and protein tyrosine phosphatase (PTP), which renders non-mossy fiber (MF) inputs incompetent in LTP-IE induction. High-frequency MF inputs, however, could induce LTP-IE at high dendritic [Ca2+] of the window. We show that MF input-induced Zn2+ signaling inhibits postsynaptic PTP, and thus enables MF inputs to induce LTP-IE at a wide range of [Ca2+]i values. Extracellular chelation of Zn2+ or genetic deletion of vesicular zinc transporter abrogated the privilege of MF inputs for LTP-IE induction. Moreover, the incompetence of somatic stimulation was rescued by the inhibition of PTP or a supplement of extracellular zinc, indicating that MF input-induced increase in dendritic [Zn2+] facilitates the induction of LTP-IE by inhibiting PTP. Consistently, high-frequency MF stimulation induced immediate and delayed elevations of [Zn2+] at proximal and distal dendrites, respectively. These results indicate that MF inputs are uniquely linked to the regulation of direct cortical inputs owing to synaptic Zn2+ signaling.SIGNIFICANCE STATEMENT Zn2+ has been mostly implicated in pathological processes, and the physiological roles of synaptically released Zn2+ in intracellular signaling are little known. We show here that Zn2+ released from hippocampal mossy fiber (MF) terminals enters postsynaptic CA3 pyramidal cells, and plays a facilitating role in MF input-induced heterosynaptic potentiation of perforant path (PP) synaptic inputs through long-term potentiation of intrinsic excitability (LTP-IE). We show that the window of cytosolic [Ca2+] that induces LTP-IE is normally very narrow because of the Ca2+-dependent coactivation of antagonistic signaling pairs, whereby non-MF inputs become ineffective in inducing excitability change. The MF-induced Zn2+ signaling, however, biases toward facilitating the induction of LTP-IE. The present study elucidates why MF inputs are more privileged for the regulation of PP synapses.

Calcium-dependent activator protein for secretion 2 (CADPS2) deficiency causes abnormal synapse development in hippocampal mossy fiber terminals

Hippocampal mossy fibers (MFs) project from dentate gyrus granule cells onto the CA2–CA3 region. MF-mediated synaptic transmission plays an important role in hippocampal learning and memory. However, the molecular mechanisms underlying MF synaptic development and subsequent functional organization are not fully understood. We previously reported that calcium-dependent activator protein for secretion 2 (CADPS2, also known as CAPS2) regulates the secretion of dense-core vesicles (DCVs). Because CADPS2 is strongly expressed in MF terminals, we hypothesized that CADPS2 regulates the development and functional organization of MF synapses by controlling the secretion of DCVs and their contents. To test this, we compared the synaptic microstructures of hippocampal MF terminals in Cadps2 knockout (KO) mice and wild-type (WT) mice by electron microscopy (EM). On postnatal day 15 (P15), KO mice exhibited morphological abnormalities in MF boutons, including smaller bouton size, a larger number of DCVs and a smaller number of post-synaptic densities (PSDs), compared with WT mice. In adults (P56), MF boutons were larger in KO mice. Synaptic vesicles (SVs) were increased but with a lower density compared with the WT. Furthermore, the number of SVs was decreased near the active zone. Moreover, MF-innervated CA3 postsynapses in KO mice displayed aberrant structures at the postsynaptic density (PSD), with an increased number of PSDs (likely because of a larger number of perforated PSDs), compared with WT mice. Taken together, our findings suggest that CADPS2 plays a critical role in MF synaptic development and functional organization.

The cancer chemotherapeutic agent paclitaxel (Taxol) reduces hippocampal neurogenesis via down-regulation of vesicular zinc

Chemotherapy-induced cognitive impairment (CICI) is increasingly recognized as a major unwanted side effect of an otherwise highly valuable life-saving technology. In part, this awareness is a result of increased cancer survival rates following chemotherapy. Altered hippocampal neurogenesis may play a role in mediating CICI. In particular, zinc could act as a key regulator of this process. To test this hypothesis, we administered paclitaxel (Px) to male C57BL/6 mice for set time periods and then evaluated the effects of Px treatment on hippocampal neurogenesis and vesicular zinc. We found that vesicular zinc levels and expression of zinc transporter 3 (ZnT3) were reduced in Px-treated mice, compared to vehicle-treated mice. Moreover, Px-treated mice demonstrated a significant decrease in the number of neuroblasts present. However, no difference in the number of progenitor cells were observed. In addition, zinc supplementation by treatment with ZnCl2 ameliorated the Px-induced decrease in hippocampal neurogenesis and cognitive impairment. These results suggest that via disruption of vesicular zinc stores in hippocampal mossy fiber terminals, chemotherapy may impinge upon one or more of the sequential stages involved in the maturation of new neurons derived via adult neurogenesis and thereby leads to the progressive cognitive decline associated with CICI.

Short-Term Facilitation at a Detonator Synapse Requires the Distinct Contribution of Multiple Types of Voltage-Gated Calcium Channels.

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 STATEMENT In 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.

Protein-energy malnutrition developing after global brain ischemia induces an atypical acute-phase response and hinders expression of GAP-43.

Protein-energy malnutrition (PEM) is a common post-stroke problem. PEM can independently induce a systemic acute-phase response, and pre-existing malnutrition can exacerbate neuroinflammation induced by brain ischemia. In contrast, the effects of PEM developing in the post-ischemic period have not been studied. Since excessive inflammation can impede brain remodeling, we investigated the effects of post-ischemic malnutrition on neuroinflammation, the acute-phase reaction, and neuroplasticity-related proteins. Male, Sprague-Dawley rats were exposed to global forebrain ischemia using the 2-vessel occlusion model or sham surgery. The sham rats were assigned to control diet (18% protein) on day 3 after surgery, whereas the rats exposed to global ischemia were assigned to either control diet or a low protein (PEM, 2% protein) diet. Post-ischemic PEM decreased growth associated protein-43, synaptophysin and synaptosomal-associated protein-25 immunofluorescence within the hippocampal CA3 mossy fiber terminals on day 21, whereas the glial response in the hippocampal CA1 and CA3 subregions was unaltered by PEM. No systemic acute-phase reaction attributable to global ischemia was detected in control diet-fed rats, as reflected by serum concentrations of alpha-2-macroglobulin, alpha-1-acid glycoprotein, haptoglobin, and albumin. Acute exposure to the PEM regimen after global brain ischemia caused an atypical acute-phase response. PEM decreased the serum concentrations of albumin and haptoglobin on day 5, with the decreases sustained to day 21. Serum alpha-2-macroglobulin concentrations were significantly higher in malnourished rats on day 21. This provides the first direct evidence that PEM developing after brain ischemia exerts wide-ranging effects on mechanisms important to stroke recovery.

Information processing and synaptic plasticity at hippocampal mossy fiber terminals

Granule cells of the dentate gyrus receive cortical information and they transform and transmit this code to the CA3 area via their axons, the mossy fibers (MFs). Structural and functional complexity of this network has been extensively studied at various organizational levels. This review is focused on the anatomical and physiological properties of the MF system. We will discuss the mechanism by which dentate granule cells process signals from single action potentials (APs), short bursts and longer stimuli. Various parameters of synaptic interactions at different target cells such as quantal transmission, short- and long-term plasticity (LTP) will be summarized. Different types of synaptic contacts formed by MFs have unique sets of rules for information processing during different rates of granule cell activity. We will investigate the complex interactions between key determinants of information transfer between the dentate gyrus and the CA3 area of the hippocampus.

A Function for EHD Family Proteins in Unidirectional Retrograde Dendritic Transport of BACE1 and Alzheimer’s Disease Aβ Production

Abnormal accumulation of β-secretase (BACE1) in dystrophic neurites and presynaptic β-amyloid (Aβ) production contribute to Alzheimer's disease pathogenesis. Little, however, is known about BACE1 sorting and dynamic transport in neurons. We investigated BACE1 trafficking in hippocampal neurons using live-cell imaging and selective labeling. We report that transport vesicles containing internalized BACE1 in dendrites undergo exclusive retrograde transport toward the soma, whereas they undergo bidirectional transport in axons. Unidirectional dendritic transport requires Eps15-homology-domain-containing (EHD) 1 and 3 protein function. Furthermore, loss of EHD function compromises dynamic axonal transport and overall BACE1 levels in axons. EHD1/3 colocalize with BACE1 and APP β-C-terminal fragments in hippocampal mossy fiber terminals, and their depletion in neurons significantly attenuates Aβ levels. These results demonstrate unidirectional endocytic transport of a dendritic cargo and reveal a role for EHD proteins in neuronal BACE1 transcytosis and Aβ production, processes that are highly relevant for Alzheimer's disease.

The Alzheimer’s β-secretase BACE1 localizes to normal presynaptic terminals and to dystrophic presynaptic terminals surrounding amyloid plaques

β-Site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) is the β-secretase that initiates Aβ production in Alzheimer’s disease (AD). BACE1 levels are increased in AD, which could contribute to pathogenesis, yet the mechanism of BACE1 elevation is unclear. Furthermore, the normal function of BACE1 is poorly understood. We localized BACE1 in the brain at both the light and electron microscopic levels to gain insight into normal and pathophysiologic roles of BACE1 in health and AD, respectively. Our findings provide the first ultrastructural evidence that BACE1 localizes to vesicles (likely endosomes) in normal hippocampal mossy fiber terminals of both non-transgenic and APP transgenic (5XFAD) mouse brains. In some instances, BACE1-positive vesicles were located near active zones, implying a function for BACE1 at the synapse. In addition, BACE1 accumulated in swollen dystrophic autophagosome-poor presynaptic terminals surrounding amyloid plaques in 5XFAD cortex and hippocampus. Importantly, accumulations of BACE1 and APP co-localized in presynaptic dystrophies, implying increased BACE1 processing of APP in peri-plaque regions. In primary cortical neuron cultures, treatment with the lysosomal protease inhibitor leupeptin caused BACE1 levels to increase; however, exposure of neurons to the autophagy inducer trehalose did not reduce BACE1 levels. This suggests that BACE1 is degraded by lysosomes but not by autophagy. Our results imply that BACE1 elevation in AD could be linked to decreased lysosomal degradation of BACE1 within dystrophic presynaptic terminals. Elevated BACE1 and APP levels in plaque-associated presynaptic dystrophies could increase local peri-plaque Aβ generation and accelerate amyloid plaque growth in AD.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1152-3) contains supplementary material, which is available to authorized users.

Differential needs of zinc in the CA3 area of dorsal hippocampus for the consolidation of contextual fear and spatial memories

One peculiarity of the hippocampal CA3 mossy fiber terminals is the co-release of zinc and glutamate upon synaptic transmission. How these two players act on hippocampal-dependent memories is still unclear. To decipher their respective involvement in memory consolidation, a pharmacological approach was chosen. Using two hippocampal-dependent behavioral paradigms (water maze and contextual fear conditioning) we now report that glutamate at CA3 synapses is necessary and sufficient for the spatial learning consolidation process, whereas glutamate and zinc released by mossy fibers are both mandatory and exert cumulative effects on contextual fear consolidation, a form of learning with a strong emotional component.

Is GABA Co-Released with Glutamate from Hippocampal Mossy Fiber Terminals?

The coexistence and co-release of different neurotransmitters from the same fiber has been well documented in several brain structures, including the retina, the spinal cord, and the auditory system ([Hnasko and Edwards, 2012][1]). Although mossy fibers (MFs), the axons of dentate gyrus granule

Evidence for connexin36 localization at hippocampal mossy fiber terminals suggesting mixed chemical/electrical transmission by granule cells.

Electrical synaptic transmission via gap junctions has become an accepted feature of neuronal communication in the mammalian brain, and occurs often between dendrites of interneurons in major brain structures, including the hippocampus. Electrical and dye-coupling has also been reported to occur between pyramidal cells in the hippocampus, but ultrastructurally-identified gap junctions between these cells have so far eluded detection. Gap junctions can be formed by nerve terminals, where they contribute the electrical component of mixed chemical/electrical synaptic transmission, but mixed synapses have only rarely been described in mammalian CNS. Here, we used immunofluorescence localization of the major gap junction forming protein connexin36 to examine its possible association with hippocampal pyramidal cells. In addition to labeling associated with gap junctions between dendrites of parvalbumin-positive interneurons, a high density of fine, punctate immunolabeling for Cx36, non-overlapping with parvalbumin, was found in subregions of the stratum lucidum in the ventral hippocampus of rat brain. A high percentage of Cx36-positive puncta in the stratum lucidum was localized to mossy fiber terminals, as indicated by co-localization of Cx36-puncta with the mossy terminal marker vesicular glutamate transporter-1, as well as with other proteins that are highly concentrated in, and diagnostic markers of, these terminals. These results suggest that mossy fiber terminals abundantly form mixed chemical/electrical synapses with pyramidal cells, where they may serve as intermediaries for the reported electrical and dye-coupling between ensembles of these principal cells. This article is part of a Special Issue entitled Electrical Synapses.

Vesicular Zinc Regulates the Ca2+Sensitivity of a Subpopulation of Presynaptic Vesicles at Hippocampal Mossy Fiber Terminals

Synaptic vesicles segregate into functionally diverse subpopulations within presynaptic terminals, yet there is no information about how this may occur. Here we demonstrate that a distinct subgroup of vesicles within individual glutamatergic, mossy fiber terminals contain vesicular zinc that is critical for the rapid release of a subgroup of synaptic vesicles during increased activity in mice. In particular, vesicular zinc dictates the Ca(2+) sensitivity of release during high-frequency firing. Intense synaptic activity alters the subcellular distribution of zinc in presynaptic terminals and decreases the number of zinc-containing vesicles. Zinc staining also appears in endosomes, an observation that is consistent with the preferential replenishment of zinc-enriched vesicles by bulk endocytosis. We propose that functionally diverse vesicle pools with unique membrane protein composition support different modes of transmission and are generated via distinct recycling pathways.

Observation of hippocampal mossy fiber axons and terminals by single-terminal electroporation of fluorescent dye

Observation of hippocampal mossy fiber axons and terminals by single-terminal electroporation of fluorescent dye

Assessing the roles of presynaptic ryanodine receptors and adenosine receptors in caffeine-induced enhancement of hippocampal mossy fiber transmission

Caffeine robustly enhances transmitter release from the hippocampal mossy fiber terminals, although it remains uncertain whether calcium mobilization through presynaptic ryanodine receptors mediates this enhancement. In this study, we adopted a selective adenosine A1 blocker to assess relative contribution of A1 receptors and ryanodine receptors in caffeine-induced synaptic enhancement. Application of caffeine further enhanced transmission at the hippocampal mossy fiber synapse even after full blockade of adenosine A1 receptors. This result suggests that caffeine enhances mossy fiber synaptic transmission by two distinct presynaptic mechanisms, i.e., removal of A1 receptor-mediated tonic inhibition and ryanodine receptor-mediated calcium release from intracellular stores.

Zinc transporter ZnT-3 regulates presynaptic Erk1/2 signaling and hippocampus-dependent memory

The physiological role of vesicular zinc at central glutamatergic synapses remains poorly understood. Here we show that mice lacking the synapse-specific vesicular zinc transporter ZnT3 (ZnT3KO mice) have reduced activation of the Erk1/2 MAPK in hippocampal mossy fiber terminals, disinhibition of zinc-sensitive MAPK tyrosine phosphatase activity, and impaired MAPK signaling during hippocampus-dependent learning. Activity-dependent exocytosis is required for the effect of zinc on presynaptic MAPK and phosphatase activity. ZnT3KO mice have complete deficits in contextual discrimination and spatial working memory. Local blockade of zinc or MAPK in the mossy fiber pathway of wild-type mice impairs contextual discrimination. We conclude that ZnT3 is important for zinc homeostasis modulating presynaptic MAPK signaling and is required for hippocampus-dependent memory.

Ultrastructural GABA immunocytochemistry in the mossy fiber terminals of Wistar and genetic absence epileptic rats receiving amygdaloid kindling stimulations

The existence of absence epilepsy and temporal lobe epilepsy in the same patient is not common in clinical practice. The reason why both types of seizures are rarely seen in the same patient is not well understood. Therefore, we aimed to investigate kindling in a well known model of human absence epilepsy, genetic absence epilepsy rats from Strasbourg (GAERS). In the present study, we analyzed whether the GABA content of GAERS that received kindling stimulations was altered in the hippocampal mossy fiber terminals compared to non-epileptic control (NEC) Wistar rats. For this purpose, we used an immunocytochemical technique at the ultrastructural level. Ultrathin sections were immunolabeled with anti-GABA antibody and transmission electron microscopy was used for the ultrastructural examination. The number of gold particles per nerve terminal was counted and the area of the nerve terminal was determined using NIH image analysis program. The GABA density was found to be higher in sham-operated GAERS than sham-operated Wistar rats. The density was increased in kindling Wistar group compared to sham-operated Wistar and kindling GAERS groups. No statistical difference was observed between sham-operated GAERS and kindling GAERS groups. The increase in GABA levels in stimulated Wistar rats may be a result of a protective mechanism. Furthermore, there may be strain differences between Wistar rats and GAERS and our findings addressing different epileptogenesis mechanisms in these strains might be a basis for future experimental studies.

Activity-dependent α-Cleavage of Nectin-1 Is Mediated by A Disintegrin and Metalloprotease 10 (ADAM10)

Nectin-1 is known to undergo ectodomain shedding by alpha-secretase and subsequent proteolytic processing by gamma-secretase. How secretase-mediated cleavage of nectin-1 is regulated in neuronal cells and how nectin-1 cleavage affects synaptic adhesion is poorly understood. We have investigated alpha-and gamma-secretase-mediated processing of nectin-1 in primary cortical neurons and identified which protease acts as a alpha-secretase. We report here that NMDA receptor activation, but not stimulation of AMPA or metabotropic glutamate receptors, resulted in robust alpha- and gamma-secretase cleavage of nectin-1 in mature cortical neurons. Cleavage of nectin-1 required influx of Ca(2+) through the NMDA receptor, and activation of calmodulin, but was not dependent on calcium/calmodulin-dependent protein kinase II (CaMKII) activation. We found that ADAM10 is the major secretase responsible for nectin-1 ectodomain cleavage in neurons and the brain. These observations suggest that alpha- and gamma-secretase processing of nectin-1 is a Ca(2+)/calmodulin-regulated event that occurs under conditions of activity-dependent synaptic plasticity and ADAM10 and gamma-secretase are responsible for these cleavage events.