Mossy Fiber Terminals Research Articles

GABA and glutamate are not colocalized in mossy fiber terminals of developing rodent hippocampus

It has been hypothesized that, in the developing rodent hippocampus, mossy fiber terminals release GABA together with glutamate. Here, we used transgenic glutamic acid decarboxylase-67 (GAD67)-GFP expressing mice and multi-label immunohistochemistry to address whether glutamatergic and GABAergic markers are colocalized. We demonstrate that in the dentate gyrus, interneurons positive for GABA/GAD are sparsely distributed along the edge of the hilus, in a different pattern from that of the densely packed granule cells. Co-staining for synaptophysin and vesicular glutamate transporter1 (VGLUT1) in postnatal day 14 brain sections from both mice and rats showed mossy fiber terminals as a group of large (2–5μm in diameter) VGLUT1-positive excitatory presynaptic terminals in the stratum lucidum of area CA3a/b. Furthermore, co-staining for synaptophysin and vesicular GABA transporter (VGAT) revealed a group of small-sized (∼0.5μm in diameter) inhibitory presynaptic terminals in the same area where identified mossy fiber terminals were present. The two types of terminals appeared to be mutually exclusive, and showed no colocalization. Thus, our results do not support the hypothesis that GABA is released as a neurotransmitter from mossy fiber terminals during development.

Changes in the synaptic structure of hippocampal neurons and impairment of spatial memory in a rat model caused by chronic arsenite exposure

Many epidemiological studies and in vitro experiments have found that chronic arsenic exposure may influence memory formation. The goal of this study was to create an animal model of memory impairment induced by chronic arsenite exposure and to study the underlying mechanisms. Sixty male Sprague-Dawley (SD) male rats were randomly divided into a control group, a low-dose sodium arsenite exposure group and a high-dose sodium arsenite exposure group. Sodium arsenite was administered by adding it to drinking water for 3 months. Then, the spatial memory of the rats was examined with Morris water maze and Y maze. The concentration of arsenic in the blood and the brain was determined by an atomic fluorescence absorption spectrometer. The ultra-structure of hippocampal neurons was observed by an electron microscope. Timm staining was used for observing mossy fibers. We found that the concentration of arsenic in the blood and the brain increased in a dose–response manner (P<0.05). The performance of rats in the arsenite exposed group (15mg/kg) was significantly impaired in the Morris water maze and Y maze tasks than those in the control group (P<0.05). Sodium arsenite exposure resulted in abnormal structural changes in the myelin sheaths of nerve fibers and decreases in the terminals of mossy fibers. Together, chronic sodium arsenite exposure through drinking water results in detrimental changes in the neuronal synapses, which may contribute to the arsenite-induced impairment of spatial memory.

Remodeling of hippocampal network in pilocarpine-treated mice expressing synaptopHluorin in the mossy fiber terminals

Pilocarpine-induced seizures induce an ectopic projection of hippocampal mossy fibers (MFs). Here, the sprouting was directly examined using TV-42 mice that express synaptopHluorin (SpH) selectively in the MF boutons. The SpH was ectopically expressed in the inner molecular layer (IML) of the dentate gyrus in typical mice after seizures, but were not always accompanied by the zinc fluorescence. The expression of SpH also has a tendency to be enhanced in layers of the CA3a region. It is suggested that the abnormal connection of neurons is more widespread than expected based on the previous zinc-detecting methods.

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.

Quantitative analysis of zinc in rat hippocampal mossy fibers by nuclear microscopy

Zinc (Zn) is involved in regulating mental and motor functions of the brain. Previous approaches have determined Zn content in the brain using semi-quantitative histological methods. We present here an alternative approach to map and quantify Zn levels in the synapses from mossy fibers to CA3 region of the hippocampus. Based on the use of nuclear microscopy, which is a combination of imaging and analysis techniques encompassing scanning transmission ion microscopy (STIM), Rutherford backscattering spectrometry (RBS), and particle induced X-ray emission (PIXE), it enables quantitative elemental mapping down to the parts per million (μg/g dry weight) levels of zinc in rat hippocampal mossy fibers. Our results indicate a laminar-specific Zn concentration of 240±9μM in wet weight level (135±5μg/g dry weight) in the stratum lucidum (SL) compared to 144±6μM in wet weight level (81±3μg/g dry weight) in the stratum pyramidale (SP) and 78±10μM in wet weight level (44±5μg/g dry weight) in the stratum oriens (SO) of the hippocampus. The mossy fibers terminals in CA3 are mainly located in the SL. Hence the Zn concentration is suggested to be within this axonal presynaptic terminal system.

Modulation by pregnenolone sulfate of filtering properties in the hippocampal trisynaptic circuit

Short-term synaptic plasticity alters synaptic efficacy on a timescale that is relevant to encoding information in spike trains. The dynamics of this plasticity, combined with that of the feedback and feedforward contributions of local interneurons, impose frequency-dependent properties on neuronal networks with implications for nervous system function. The trisynaptic network of the hippocampus is especially well suited to selectively filter components of frequency-dependent signals that are transmitted from the entorhinal cortex. We measured presynaptic [Ca(2+)](i) in perforant path, mossy fiber, or Schaffer collateral terminals while simultaneously measuring field potentials of principal cells of the dentate, CA3, or CA1 synaptic fields over a range of stimulus frequencies of 2 to 77 Hz. In all three synaptic fields, the average [Ca(2+)](i) during a 500 ms stimulus train rose monotonically with stimulus frequency. The average population spike amplitude during this stimulus train, however, exhibited a non-linear relationship to frequency that was distinct for each of the three synaptic fields. The dentate synaptic field exhibited the characteristics of a low pass filter, while both CA synaptic fields had bandpass filter characteristics with a gain that was greater than 1 in the passband frequencies. Importantly, alteration of the dynamic properties of this network could significantly impact information processing performed by the hippocampus. Pregnenolone sulfate (PregS), has frequency-dependent effects on paired- and multipulse plasticity in the dentate and CA1 synaptic fields of the hippocampal formation. We investigated the PregS-dependent modulation of the dynamic properties of transmission by the principal cells of the three hippocampal synaptic fields. Importantly, PregS is capable of altering the pattern separation capabilities that may underlie hippocampal information processing.

Manganese-enhanced magnetic resonance imaging detects mossy fiber sprouting in the pilocarpine model of epilepsy.

Mossy fiber sprouting (MFS) is a frequent finding following status epilepticus (SE). The present study aimed to test the feasibility of using manganese-enhanced magnetic resonance imaging (MEMRI) to detect MFS in the chronic phase of the well-established pilocarpine (Pilo) rat model of temporal lobe epilepsy (TLE). To modulate MFS, cycloheximide (CHX), a protein synthesis inhibitor, was coadministered with Pilo in a subgroup of animals. In vivo MEMRI was performed 3 months after induction of SE and compared to the neo-Timm histologic labeling of zinc mossy fiber terminals in the dentate gyrus (DG). Chronically epileptic rats displaying MFS as detected by neo-Timm histology had a hyperintense MEMRI signal in the DG, whereas chronically epileptic animals that did not display MFS had minimal MEMRI signal enhancement compared to nonepileptic control animals. A strong correlation (r = 0.81, p < 0.001) was found between MEMRI signal enhancement and MFS. This study shows that MEMRI is an attractive noninvasive method for detection of mossy fiber sprouting in vivo and can be used as an evaluation tool in testing therapeutic approaches to manage chronic epilepsy.

Synaptic connections of hilar basal dendrites of dentate granule cells in a neonatal hypoxia model of epilepsy

Numerous animal models of epileptogenesis demonstrate neuroplastic changes in the hippocampus. These changes occur not only for the mature neurons and glia, but also for the newly generated granule cells in the dentate gyrus. One of these changes, the sprouting of mossy fiber axons, is derived predominantly from newborn granule cells in adult rats with pilocarpine-induced temporal lobe epilepsy. Newborn granule cells also mainly contribute to another neuroplastic change, hilar basal dendrites (HBDs), which are synaptically targeted by mossy fibers in the hilus. Both sprouted mossy fibers and HBDs contribute to recurrent excitatory circuitry that is hypothesized to be involved in increased seizure susceptibility and the development of spontaneous recurrent seizures (SRS) that occur following the initial pilocarpine-induced status epilepticus. Considering the putative role of these neuroplastic changes in epileptogenesis, a critical question is whether similar anatomic phenomena occur after epileptogenic insults to the immature brain, where the proportion of recently born granule cells is higher due to ongoing maturation. The current study aimed to determine if such neuroplastic changes could be observed in a standardized model of neonatal seizure-inducing hypoxia that results in development of SRS. We used immunoelectron microscopy for the immature neuronal marker doublecortin to label newborn neurons and their HBDs following neonatal hypoxia. Our goal was to determine whether synapses form on HBDs from neurons born after neonatal hypoxia. Our results show a robust synapse formation on HBDs from animals that experienced neonatal hypoxia, regardless of whether the animals experienced tonic-clonic seizures during the hypoxic event. In both cases, the axon terminals that synapse onto HBDs were identified as mossy fiber terminals, based on the appearance of dense core vesicles. No such synapses were observed on HBDs from newborn granule cells obtained from sham animals analyzed at the same time points. This aberrant circuit formation may provide an anatomic substrate for increased seizure susceptibility and the development of epilepsy.

Neuroanatomical clues to altered neuronal activity in epilepsy: From ultrastructure to signaling pathways of dentate granule cells

The dynamic aspects of epilepsy, in which seizures occur sporadically and are interspersed with periods of relatively normal brain function, present special challenges for neuroanatomical studies. Although numerous morphologic changes can be identified during the chronic period, the relationship of many of these changes to seizure generation and propagation remains unclear. Mossy fiber sprouting is an example of a frequently observed morphologic change for which a functional role in epilepsy continues to be debated. This review focuses on neuroanatomically identified changes that would support high levels of activity in reorganized mossy fibers and potentially associated granule cell activation. Early ultrastructural studies of reorganized mossy fiber terminals in human temporal lobe epilepsy tissue have identified morphologic substrates for highly efficacious excitatory connections among granule cells. If similar connections in animal models contribute to seizure activity, activation of granule cells would be expected. Increased labeling with two activity-related markers, Fos and phosphorylated extracellular signal-regulated kinase, has suggested increased activity of dentate granule cells at the time of spontaneous seizures in a mouse model of epilepsy. However, neuroanatomical support for a direct link between activation of reorganized mossy fiber terminals and increased granule cell activity remains elusive. As novel activity-related markers are developed, it may yet be possible to demonstrate such functional links and allow mapping of seizure activity throughout the brain. Relating patterns of neuronal activity during seizures to the underlying morphologic changes could provide important new insights into the basic mechanisms of epilepsy and seizure generation.

A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis

The development of the dentate gyrus is characterized by distinct phases establishing a durable stem-cell pool required for postnatal and adult neurogenesis. Here, we report that Bcl11b/Ctip2, a zinc finger transcription factor expressed in postmitotic neurons, plays a critical role during postnatal development of the dentate gyrus. Forebrain-specific ablation of Bcl11b uncovers dual phase-specific functions of Bcl11b demonstrated by feedback control of the progenitor cell compartment as well as regulation of granule cell differentiation, leading to impaired spatial learning and memory in mutants. Surprisingly, we identified Desmoplakin as a direct transcriptional target of Bcl11b. Similarly to Bcl11b, postnatal neurogenesis and granule cell differentiation are impaired in Desmoplakin mutants. Re-expression of Desmoplakin in Bcl11b mutants rescues impaired neurogenesis, suggesting Desmoplakin to be an essential downstream effector of Bcl11b in hippocampal development. Together, our data define an important novel regulatory pathway in hippocampal development, by linking transcriptional functions of Bcl11b to Desmoplakin, a molecule known to act on cell adhesion.

Activity maintains structural plasticity of mossy fiber terminals in the hippocampus

Neural activity plays an important role in organizing and optimizing neural circuits during development and in the mature nervous system. However, the cellular events that underlie this process still remain to be fully understood. In this study, we investigated the role of neural activity in regulating the structural plasticity of presynaptic terminals in the hippocampal formation. We designed a virus to drive the Drosophila Allatostatin receptor in individual dentate granule neurons to suppress activity of complex mossy fiber terminals 'on-demand' in organotypic slices and used time-lapse confocal imaging to determine the impact on presynaptic remodeling. We found that activity played an important role in maintaining the structural plasticity of the core region of the mossy fiber terminal (MFT) that synapses onto CA3 pyramidal cell thorny excrescences but was not essential for the motility of terminal filopodial extensions that contact local inhibitory neurons. Short-term suppression of activity did not have an impact on the size of the MFT, however, longer-term suppression reduced the overall size of the MFT. Remarkably, global blockade of activity with tetrodotoxin (TTX) interfered with the ability of single cell activity deprivation to slow down terminal dynamics suggesting that differences in activity levels among neighboring synapses promote synaptic remodeling events. The results from our studies indicate that neural activity plays an important role in maintaining structural plasticity of presynaptic compartments in the central nervous system and provide new insight into the time-frame during which activity can affect the morphology of synaptic connections.

Abnormalities of granule cell dendritic structure are a prominent feature of the intrahippocampal kainic acid model of epilepsy despite reduced postinjury neurogenesis

Aberrant plastic changes among adult-generated hippocampal dentate granule cells are hypothesized to contribute to the development of temporal lobe epilepsy. Changes include formation of basal dendrites projecting into the dentate hilus. Innervation of these processes by granule cell mossy fiber axons leads to the creation of recurrent excitatory circuits within the dentate. The destabilizing effect of these recurrent circuits may contribute to hyperexcitability and seizures. Although basal dendrites have been identified in status epilepticus models of epilepsy associated with increased neurogenesis, we do not know whether similar changes are present in the intrahippocampal kainic acid model of epilepsy, which is associated with reduced neurogenesis. In the present study, we used Thy1-YFP-expressing transgenic mice to determine whether hippocampal dentate granule cells develop hilar-projecting basal dendrites in the intrahippocampal kainic acid model. Brain sections were examined 2 weeks after treatment. Tissue was also examined using ZnT-3 immunostaining for granule cell mossy fiber terminals to assess recurrent connectivity. Adult neurogenesis was assessed using the proliferative marker Ki-67 and the immature granule cell marker calretinin. Significant numbers of cells with basal dendrites were found in this model, but their structure was distinct from basal dendrites seen in other epilepsy models, often ending in complex tufts of short branches and spines. Even more unusual, a subset of cells with basal dendrites had an inverted appearance; they completely lacked apical dendrites. Spines on basal dendrites were found to be apposed to ZnT-3 immunoreactive puncta, suggestive of recurrent mossy fiber input. Finally, YFP-expressing abnormal granule cells did not colocalize Ki-67 or calretinin, indicating that these cells were more than a few weeks old, but were found almost exclusively in proximity to the neurogenic subgranular zone, where the youngest granule cells are located. Recent studies have demonstrated in other models of epilepsy that dentate pathology develops following the aberrant integration of immature, adult-generated granule cells. Given these findings, one might predict that the intrahippocampal kainic acid model of epilepsy, which is associated with a dramatic reduction in adult neurogenesis, would not exhibit these changes. Herein we demonstrate that hilar basal dendrites are a common feature of this model, with the abnormal cells likely resulting from the disruption of juvenile granule cell born in the weeks before the insult. These studies demonstrate that postinjury neurogenesis is not required for the accumulation of large numbers of abnormal granule cells.

M2 muscarinic acetylcholine receptors regulate long-term potentiation at hippocampal CA3 pyramidal cell synapses in an input-specific fashion

Muscarinic receptors have long been known as crucial players in hippocampus-dependent learning and memory, but our understanding of the cellular underpinnings and the receptor subtypes involved lags well behind. This holds in particular for the hippocampal CA3 region, where the mechanisms of synaptic plasticity depend on the type of afferent input. Williams and Johnston (Williams S, Johnston D. Science 242: 84-87, 1988; Williams S, Johnston D. J Neurophysiol 64: 1089-1097, 1990) demonstrated muscarinic depression of mossy fiber (MF) long-term potentiation (LTP) through a presynaptic site of action and Maeda et al. (Maeda T, Kaneko S, Satoh M. Brain Res 619: 324-330, 1993) proposed a bidirectional modulation of MF LTP by muscarinic receptor subtypes. Since then, this issue, as well as muscarinic regulation of plasticity at associational/commissural (A/C) fiber-CA3 synapses has remained largely neglected, not least because of the lack of highly selective ligands for the different muscarinic receptor subtypes. In the present study, we performed field potential and whole cell recordings from the hippocampal CA3 region of M(2) receptor knockout mice to determine the role of M(2) receptors in short-term and long-term plasticity at A/C and MF inputs to CA3 pyramidal cells. At the A/C synapse, M(2) receptors promoted short-term facilitation and LTP. Unexpectedly, M(2) receptors mediated the opposite effect on LTP at the MF synapse, which was significantly reduced, most likely involving a depressant effect of M(2) receptors on adenylyl cyclase activity in MF terminals. Our data demonstrate that cholinergic projections recruit M(2) receptors to redistribute the gain of LTP in CA3 pyramidal cells in an input-specific manner.

Ontogenetic changes in the distribution of the vesicular GABA transporter VGAT correlate with the excitation/inhibition shift of GABA action

GABA is the major inhibitory neurotransmitter in the adult CNS and is among others involved in the synchronization of large neuronal networks. During development, GABA acts as a morphogenetic factor and has transient excitatory actions in many brain regions. One distinct protein, the vesicular GABA transporter (VGAT), has been identified accumulating GABA into presynaptic vesicles prior to its exocytotic release. The function of VGAT and its distribution is well defined in the adult, but its contribution to the transient excitatory action at putative GABAergic nerve terminals in the immature brain and its potential roles in putative glutamatergic nerve terminals remain elusive. We have studied VGAT expression in the brain from late embryonic stages through several postnatal stages until adulthood. Quantitative immunoblotting and immunolabeling of tissue sections at the light microscope and the electron microscope levels show an abrupt augmentation in VGAT staining in the cerebral cortex during the first three postnatal weeks, resembling the increase in other proteins involved in GABA synthesis and recycling in the same time frame – such as GAD65, GAD67, GAT1 (Slc6a1) and SN1 (Slc38a3) – and coincides with the synaptogenetic spurt. Dynamic changes in the expression of VGAT are seen in many cellular populations and in several layers in different brain regions. However, mossy fiber terminals (MFT) elude staining for VGAT. We also demonstrate that VGAT+ nerve terminals undergo a developmental reorganization so that from targeting primarily the dendrites of the principal neurons in several brain regions in the immature brain, they target the soma of the same cells in the adult. This shift in the targeted subcellular compartment coincides with the conversion of the chloride gradient across neuronal membranes and suggests that it may be important for the shift of GABA action from excitation to inhibition and for the establishment of the potent synchronization of neuronal networks.

Mixed Electrical–Chemical Synapses in Adult Rat Hippocampus are Primarily Glutamatergic and Coupled by Connexin-36

Dendrodendritic electrical signaling via gap junctions is now an accepted feature of neuronal communication in mammalian brain, whereas axodendritic and axosomatic gap junctions have rarely been described. We present ultrastructural, immunocytochemical, and dye-coupling evidence for “mixed” (electrical/chemical) synapses on both principal cells and interneurons in adult rat hippocampus. Thin-section electron microscopic images of small gap junction-like appositions were found at mossy fiber (MF) terminals on thorny excrescences of CA3 pyramidal neurons (CA3pyr), apparently forming glutamatergic mixed synapses. Lucifer Yellow injected into weakly fixed CA3pyr was detected in MF axons that contacted four injected CA3pyr, supporting gap junction-mediated coupling between those two types of principal cells. Freeze-fracture replica immunogold labeling revealed diverse sizes and morphologies of connexin-36-containing gap junctions throughout hippocampus. Of 20 immunogold-labeled gap junctions, seven were large (328–1140 connexons), three of which were consistent with electrical synapses between interneurons; but nine were at axon terminal synapses, three of which were immediately adjacent to distinctive glutamate receptor-containing postsynaptic densities, forming mixed glutamatergic synapses. Four others were adjacent to small clusters of immunogold-labeled 10-nm E-face intramembrane particles, apparently representing extrasynaptic glutamate receptor particles. Gap junctions also were on spines in stratum lucidum, stratum oriens, dentate gyrus, and hilus, on both interneurons and unidentified neurons. In addition, one putative GABAergic mixed synapse was found in thin-section images of a CA3pyr, but none were found by immunogold labeling, suggesting the rarity of GABAergic mixed synapses. Cx36-containing gap junctions throughout hippocampus suggest the possibility of reciprocal modulation of electrical and chemical signals in diverse hippocampal neurons.

Selective presynaptic terminal remodeling induced by spatial, but not cued, learning: A quantitative confocal study

The hippocampal mossy fibers (MFs) are capable of behaviorally selective, use-dependent structural remodeling. Indeed, we previously observed a new layer of Timm's staining induced in the stratum oriens (SO) in CA3 after spatial but not cued water maze learning (Rekart et al., (2007) Learn Mem 14:416-421). This led to the prediction that there is a learning-specific induction of presynaptic terminal plasticity of MF axons. This study confirms this prediction demonstrating, at the confocal level of analysis, terminal-specific, and behavior-selective presynaptic structural plasticity linked to long-term memory. Male adult Wistar rats were trained for 5 days to locate a hidden or visible platform in a water maze and a retention test was performed 7 days later. MF terminal subtypes, specifically identified by an antibody to zinc transporter 3 (ZnT3), were counted from confocal z-stacks in the stratum lucidum (SL) and the SO. In hidden platform trained rats, there was a significant increase in the number of large MF terminals (LMTs, 2.5-10 μm diameter, >2 μm(2) area) compared to controls both in the proximal SL (P < 0.05) and in the SO (P < 0.01). Surprisingly, there was no detectable increase in small MF terminals (SMTs, 0.5-2 μm diameter, <2 μm(2) area) in either SL or SO as a consequence of training. This distinction of the two MF terminal types is functionally important as LMTs synapse on CA3 pyramidal neurons, while SMTs are known to target inhibitory interneurons. The present findings highlight the pivotal role in memory of presynaptic structural plasticity. Because the "sprouting" observed is specific to the LMT, with no detectable change in the number of the SMT, learning may enhance net excitatory input to CA3 pyramidal neurons. Given the sparse coding of the MF-CA3 connection, and the role that granule cells play in pattern separation, the remodeling observed here may be expected to have a major impact on the long-term integration of spatial context into memory.

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.

Mixed electrical–chemical transmission between hippocampal mossy fibers and pyramidal cells

Morphological and electrophysiological studies have shown that granule cell axons, the mossy fibers (MFs), establish gap junctions and therefore electrical communication among them. That granule cells express gap junctional proteins in their axons suggests the possibility that their terminals also express them. If this were to be the case, mixed electrical-chemical communication could be supported, as MF terminals normally use glutamate for fast communication with their target cells. Here we present electrophysiological studies in the rat and modeling studies consistent with this hypothesis. We show that MF activation produced fast spikelets followed by excitatory postsynaptic potentials in pyramidal cells (PCs), which, unlike the spikelets, underwent frequency potentiation and were strongly depressed by activation of metabotropic glutamate receptors, as expected from transmission of MF origin. The spikelets, which persisted during blockade of chemical transmission, were potentiated by dopamine and suppressed by the gap junction blocker carbenoxolone. The various waveforms evoked by MF stimulation were replicated in a multi-compartment model of a PC by brief current-pulse injections into the proximal apical dendritic compartment, where MFs are known to contact PCs. Mixed electrical and glutamatergic communication between granule cells and some PCs in CA3 may ensure the activation of sets of PCs, bypassing the strong action of concurrent feed-forward inhibition that granule cells activate. Importantly, MF-to-PC electrical coupling may allow bidirectional, possibly graded, communication that can be faster than chemical synapses and subject to different forms of modulation.

Synaptically evoked Ca2+release from intracellular stores is not influenced by vesicular zinc in CA3 hippocampal pyramidal neurones

The co-release of neuromodulatory substances in combination with classic neurotransmitters such as glutamate and GABA from individual presynaptic nerve terminals has the capacity to dramatically influence synaptic efficacy and plasticity. At hippocampal mossy fibre synapses vesicular zinc is suggested to serve as a cotransmitter capable of regulating calcium release from internal stores in postsynaptic CA3 pyramidal cells. Here we investigated this possibility using combined intracellular ratiometric calcium imaging and patch-clamp recording techniques. In acute hippocampal slices a brief train of mossy fibre stimulation produced a large, delayed postsynaptic Ca(2+) wave that was spatially restricted to the proximal apical dendrites of CA3 pyramidal cells within stratum lucidum. This calcium increase was sensitive to intracellularly applied heparin indicating reliance upon release from internal stores and was triggered by activation of both group I metabotropic glutamate and NMDA receptors. Importantly, treatment of slices with the membrane-impermeant zinc chelator CaEDTA did not influence the synaptically evoked postsynaptic Ca(2+) waves. Moreover, mossy fibre stimulus evoked postsynaptic Ca(2+) signals were not significantly different between wild-type and zinc transporter 3 (ZnT3) knock-out animals. Considered together our data do not support a role for vesicular zinc in regulating mossy fibre evoked Ca(2+) release from CA3 pyramidal cell internal stores.

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