Hippocampal Mossy Fiber Synapses Research Articles

Coassembly of Two GluR6 Kainate Receptor Splice Variants within a Functional Protein Complex

Kainate receptors (KAR) are composed of several distinct subunits and splice variants, but the functional relevance of this diversity remains largely unclear. Here we show that two splice variants of the GluR6 subunit, GluR6a and GluR6b, which differ in their C-terminal domains, do not show distinct functional properties, but coassemble as heteromers in vitro and in vivo. Using a proteomic approach combining affinity purification and MALDI-TOF mass spectrometry, we found that GluR6a and GluR6b interact with two distinct subsets of cytosolic proteins mainly involved in Ca(2+) regulation of channel function and intracellular trafficking. Guided by these results, we provide evidence that the regulation of native KAR function by NMDA receptors depends on the heteromerization of GluR6a and GluR6b and interaction of calcineurin with GluR6b. Thus, GluR6a and GluR6b bring in close proximity two separate subsets of interacting proteins that contribute to the fine regulation of KAR trafficking and function.

Visualization of transmitter release with zinc fluorescence detection at the mouse hippocampal mossy fibre synapse

Exocytosis of synaptic vesicle contents defines the quantal nature of neurotransmitter release. Here we developed a technique to directly assess exocytosis by measuring vesicular zinc release with the zinc-sensitive dye FluoZin-3 at the hippocampal mossy fibre (MF) synapse. Using a photodiode, we were able to clearly resolve the zinc fluorescence transient ([Zn2+]t) with a train of five action potentials in mouse hippocampal brain slices. The vesicular origin of [Zn2+]t was verified by the lack of zinc signal in vesicular zinc transporter Znt3-deficient mice. Manipulating release probability with the application of neuromodulators such as DCG IV, 4-aminopyridine and forskolin as well as a paired train stimulation protocol altered both the [Zn2+]t and the field excitatory postsynaptic potential (fEPSP) coordinately, strongly indicating that zinc is co-released with glutamate during exocytosis. Since zinc ions colocalize with glutamate in small clear vesicles and modulate postsynaptic excitability at NMDA and GABA receptors, the findings establish zinc as a cotransmitter during physiological signalling at the mossy fibre synapse. The ability to directly visualize release dynamics with zinc imaging will facilitate the exploration of the molecular pharmacology and plasticity of exocytosis at MF synapses.

Assessing the Role of GLUK5and GLUK6at Hippocampal Mossy Fiber Synapses

It has been suggested recently that presynaptic kainate receptors (KARs) are involved in short-term and long-term synaptic plasticity at hippocampal mossy fiber synapses. Using genetic deletion and pharmacology, we here assess the role of GLU(K5) and GLU(K6) in synaptic plasticity at hippocampal mossy fiber synapses. We found that the kainate-induced facilitation was completely abolished in the GLU(K6)-/- mice, whereas it was unaffected in the GLU(K5)-/-. Consistent with this finding, synaptic facilitation was reduced in the GLU(K6)(-/-) and was normal in the GLU(K5)-/-. In agreement with these results and ruling out any compensatory effects in the genetic deletion models, application of the GLU(K5)-specific antagonist LY382884 [(3S,4aR,6S,8aR)-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid] did not affect short-term and long-term synaptic plasticity at the hippocampal mossy fiber synapses. We therefore conclude that the facilitatory effects of kainate on mossy fiber synaptic transmission are mediated by GLU(K6)-containing KARs.

Attenuated plasticity of postsynaptic kainate receptors in hippocampal CA3 pyramidal neurons.

Kainate receptor-mediated components of postsynaptic currents at hippocampal mossy fiber synapses have markedly slower kinetics than currents arising from AMPA receptors. Here, we demonstrate that other aspects of kainate and AMPA receptor function at this synapse are distinct; in particular, kainate receptor currents are less sensitive to short- and long-term increases in presynaptic strength. EPSCs arising predominantly from AMPA receptors exhibited well characterized paired-pulse facilitation, frequency facilitation, and NMDA receptor-independent long-term potentiation, whereas isolated kainate receptor synaptic currents (KA-EPSCs) exhibited attenuated facilitation and long-term potentiation. In addition, KA-EPSCs varied in their sensitivity to a low-affinity competitive antagonist, suggestive of a synaptic heterogeneity greater than that of EPSCs comprised predominantly of AMPA receptors. These data suggest that the proportional contribution of AMPA and kainate receptors to ensemble synaptic currents will vary depending on the firing frequency of mossy fiber afferents. These synaptic features may be a mechanism for limiting activation of kainate receptors at mossy fiber synapses, which has been shown to be involved in seizurogenic firing of the CA3 network.

Glutamate escape from a tortuous synaptic cleft of the hippocampal mossy fibre synapse

The time course of neurotransmitter in the synaptic cleft contributes substantially to the fast kinetics of synaptic signalling. Hippocampal mossy fibres (MFs), a well-characterised excitatory pathway from dentate granule cells to the hippocampus proper, form large glutamatergic synapses at branched spiny structures in CA3 pyramidal cell dendrites. To what extent transmission at these synapses is affected by retarded glutamate clearance from the large tortuous synaptic cleft is not known. Here, we propose a simple geometrical approximation representing the ‘typical’ geometry of thorny excrescences that form the tortuous cleft interface at a MF synapse. We then employ Monte Carlo simulations to monitor movements of 3000 individual glutamate molecules released within the cleft. The results predict that, in the absence of neuronal glutamate transporters, it should take approximately 10 ms for 50% and 60–70 ms for 90% of glutamate molecules to escape the MF synapse.

Of Plasticity and Purinergic Suppression

Most synapses display activity-dependent changes in synaptic efficacy that influence the likelihood that a particular neuronal pathway will fire; different types of synapses, however, can show marked differences in their responses to repetitive firing, and these differences depend on mechanisms that are incompletely understood. Zhang et al. uncovered an intriguing bidirectional conversation between neurons and astrocytes that mediates a novel form of use-dependent heterosynaptic inhibition (see Preview by Pascual and Haydon). The authors used pharmacologic manipulation in combination with electrophysiological analysis of cocultures of hippocampal neurons and astrocytes to show that endogenous adenosine triphosphate (ATP), acting through presynaptic purinergic P2Y receptors, tonically suppressed glutamatergic transmission. Repetitive stimulation of glutamatergic neurons led to heterosynaptic suppression of nearby neurons, which depended on the sequential activation of P2Y and non- N -methyl-D-aspartate glutamate receptors. Using pharmacologic analysis, separate neuronal and astrocyte cultures, and bioluminescence detection of released ATP, the authors demonstrated that this suppression depended on the release of ATP from glutamate-stimulated astrocytes. The ATP metabolite adenosine mediated a similar heterosynaptic suppression of glutamatergic synapses on CA1 pyramidal neurons in rat hippocampal slices. Pharmacologic analysis, combined with calcium imaging and measurement of ATP release, indicated that this adenosine derived at least in part from ATP released from glia. In a separate study, Moore et al. demonstrated that the dramatic paired-pulse facilitation, frequency facilitation, and an unusual form of long-term potentiation characteristic of hippocampal mossy fiber synapses depended on tonic inhibition through presynaptic adenosine A1 receptors of basal transmitter release. Pharmacologic blockade of A1 receptors or degradation of extracellular adenosine in rat or mouse hippocampal slices enhanced the basal synaptic response and suppressed these use-dependent responses, as did A1 knockout. Both studies emphasize the importance of the local environment--and of adenosine in particular--in modulating activity-dependent changes in synaptic function. J. Zhang, H. Wang, C. Ye, W. Ge, Y. Chen, Z. Jiang, C. Wu, M. Poo, S. Duan, ATP released by astrocytes mediates glutamatergic activity-dependent heterosynaptic suppression. Neuron 40 , 971-982 (2003). [Online Journal] K. A. Moore, R. A. Nicoll, D. Schmitz, Adenosine gates synaptic plasticity at hippocampal mossy fiber synapses. Proc. Natl. Acad. Sci. U.S.A. 100 , 14397-14402 (2003). [Abstract] [Full Text] O. Pascual, P. G. Haydon, Synaptic inhibition mediated by glia. Neuron 40 , 873-875 (2003). [Online Journal]

Adenosine gates synaptic plasticity at hippocampal mossy fiber synapses.

The release properties of synapses in the central nervous system vary greatly, not only across anatomically distinct types of synapses but also among the same class of synapse. This variation manifests itself in large part by differences in the probability of transmitter release, which affects such activity-dependent presynaptic forms of plasticity as paired-pulse facilitation and frequency facilitation. This heterogeneity in presynaptic function reflects differences in the intrinsic properties of the synaptic terminal and the activation of presynaptic neurotransmitter receptors. Here we show that the unique presynaptic properties of the hippocampal mossy fiber synapse are largely imparted onto the synapse by the continuous local action of extracellular adenosine at presynaptic A1 adenosine receptors, which maintains a low basal probability of transmitter release.

Developmental decrease in synaptic facilitation at the mouse hippocampal mossy fibre synapse.

Transmission at the hippocampal mossy fibre (MF)-CA3 pyramidal cell synapse is characterized by prominent activity-dependent facilitation, which is thought to provide a wide dynamic range in hippocampal informational flow. At this synapse in mice the magnitude of paired-pulse facilitation and frequency-dependent facilitation markedly decreased with postnatal development from 3 weeks (3W) to 9 weeks (9W). Throughout this period the mean amplitude and variance of unitary EPSCs stayed constant. By altering extracellular Ca2+/Mg2+ concentrations the paired-pulse ratio could be changed to a similar extent as observed during development. However, this was accompanied by an over 30-fold change in EPSC amplitude, suggesting that the developmental change in facilitation ratio cannot simply be explained by a change in release probability. With paired-pulse stimulation the Ca2+ transients at MF terminals, monitored using mag-fura-5, showed a small facilitation, but its magnitude remained similar between 3W and 9W mice. Pharmacological tests using CNQX, adenosine, LY341495, H-7 or KN-62 suggested that neither presynaptic receptors (kainate, adenosine and metabotropic glutamate) nor protein kinases are responsible for the developmental change in facilitation. Nevertheless, loading the membrane-permeable form of BAPTA attenuated the paired-pulse facilitation in 3W mice to a much greater extent than in 9W mice, resulting in a marked reduction in age difference. These results suggest that the developmental decrease in the MF synaptic facilitation arises from a change associated with residual Ca2+, a decrease in residual Ca2+ itself or a change in Ca2+-binding sites involved in the facilitation. A developmental decline in facilitation ratio reduces the dynamic range of MF transmission, possibly contributing to the stabilization of hippocampal circuitry.

Release and the Resistant Channel

Several subtypes of voltage-gated calcium channels that differ in their biophysical properties and pharmacological sensitivities have been identified. The N-, P/Q-, and R- subtypes can all mediate calcium influx into presynaptic nerve terminals. N- and P/Q-type channels play key roles in mediating evoked neurotransmitter release; the function of presynaptic R-type channels, however, has remained obscure. Dietrich et al. used a combination of pharmacological, electrophysiological, and genetic techniques to show that, at adult mouse hippocampal mossy fiber synapses, Ca v 2.3 channels (molecularly identified channels that contribute to R-type calcium current) selectively contribute to specific forms of calcium-dependent plasticity of neurotransmitter release. Electrophysiological analysis of evoked release to solitary action potentials, or in response to various stimulus paradigms, showed that Ca v 2.3 knockout or pharmacological blockade of R-type channels (using SNX-482 or low concentrations of nickel) selectively inhibited long-term potentiation and posttetanic potentiation of transmitter release, which depend on high-frequency stimulation. Baseline release, paired-pulse facilitation (which occurs after a single conditioning stimulus), and frequency facilitation (which occurs during low-frequency stimulation) were not inhibited. Calcium imaging confirmed that Ca v 2.3 channels contributed to R-type current and showed that calcium influx through R-type channels during individual action potentials was greater during high-frequency stimulation. These data identify a distinct role for presynaptic R-type channels at mossy fiber synapses and suggest a plausible mechanism that may contribute to certain forms of synaptic plasticity. Brenowitz and Regehr provide useful background information and discuss some of the implications of this research. D. Dietrich, T. Kirschstein, M. Kukley, A. Pereverzev, C. von der Brelie, T. Schneider, H. Beck, Functional specialization of presynaptic Ca v 2.3 Ca 2+ channels. Neuron 39 , 483-496 (2003). [Online Journal] S. D. Brenowitz, W. G. Regehr, "Resistant" channels reluctantly reveal their roles. Neuron 39 , 391-394 (2003). [Online Journal]

GABA and GABAA receptors at hippocampal mossy fibre synapses.

Anatomical and electrophysiological evidence has raised the possibility that corelease of GABA and glutamate occurs at hippocampal mossy fibre synapses which, however, lack the vesicular GABA transporter VGAT. Here, we apply immunogold cytochemistry to show that GABA, like glutamate, has a close spatial relation to synaptic vesicles in rat mossy fibre terminals, implying that a mechanism exists to package GABA in synaptic vesicles. We also show that GABAA and AMPA receptors are colocalized at mossy fibre synapses. The expression of GABA and GABAA receptors is, however, weaker than in inhibitory synapses. Electrical stimuli that recruit mossy fibres evoke monosynaptic GABAA receptor-mediated signals in post-synaptic targets that show marked frequency-dependent facilitation and sensitivity to group II metabotropic receptors, two features that are characteristic of mossy fibre transmission. These results provide further evidence for GABA and glutamate cotransmission at mossy fibre synapses, although paired pre- and post-synaptic recordings will be required to determine the role of GABA at this unusual synapse.

A large pool of releasable vesicles in a cortical glutamatergic synapse.

To probe exocytosis at a cortical glutamatergic synapse, we made capacitance measurements in whole-cell recorded hippocampal mossy fiber terminals. Evaluation of different methods by using a morphology-based equivalent electrical model revealed that quantitative capacitance measurements are possible in this presynaptic structure. Voltage pulses leading to presynaptic Ca2+ inflow evoked large capacitance signals that showed saturation with increasing pulse duration. The mean peak capacitance increase was 100 fF, corresponding to a pool of approximately 1,400 releasable vesicles. Thus hippocampal mossy fiber synapses have a vesicular "maxipool." Large pool size and rapid vesicle recycling may underlie the uniquely large extent of activity-dependent plasticity in this synapse.

Reexamination of the role of hyperpolarization-activated cation channels in short- and long-term plasticity at hippocampal mossy fiber synapses

We tested a proposal that the hyperpolarization-activated cation channel ( I h channel) is involved in the induction of short- and long-term plasticity at the hippocampal mossy fiber–CA3 synapses. Bath application of a specific I h channel blocker ZD 7288, at a concentration at which it blocked I h channels, substantially depressed mossy fiber synaptic transmission, and this inhibition was occluded by previous blockade of these channels by CsCl. In addition, ZD 7288 attenuated the amplitude of both AMPA and NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) equally and caused a coincident increase in the failure rate of single-fiber EPSCs and paired-pulse facilitation (PPF). It also blocked long-term potentiation (LTP) induction when applied before high-frequency tetanic stimulation (TS), and reversed LTP when applied afterwards. Continuous application of CsCl, which efficiently blocks I h channels, mimicked ZD 7288 in inhibiting LTP. Furthermore, ZD 7288 blocked both forskolin- and Sp-8-CPT-cAMPS-mediated enhancements of synaptic transmission. However, it did not affect the frequency facilitation induced by increasing the stimulus frequency from 0.05–1 Hz and the expression of the long-term depression (LTD) induced by low-frequency stimulation (LFS) or DCG-IV. Perforated patch-clamp recordings from granule cells revealed that the voltage for half-maximal activation ( V 1/2) of I h was significantly shifted towards the depolarizing direction following forskolin or Sp-8-CPT-cAMPS treatment. This enhanced I h current was not due to persistent activation of protein kinase A (PKA), because PKA inhibitor KT5720 did not abolish the difference between the activation curves. Therefore, we conclude that I h channels may contribute to the development and regulation of short- and long-term plasticity at the mossy fiber–CA3 synapses.

Rapid and Differential Regulation of AMPA and Kainate Receptors at Hippocampal Mossy Fibre Synapses by PICK1 and GRIP

We identified four PDZ domain-containing proteins, syntenin, PICK1, GRIP, and PSD95, as interactors with the kainate receptor (KAR) subunits GluR5 2b, GluR5 2c, and GluR6. Of these, we show that both GRIP and PICK1 interactions are required to maintain KAR-mediated synaptic function at mossy fiber-CA3 synapses. In addition, PKCα can phosphorylate ct-GluR5 2b at residues S880 and S886, and PKC activity is required to maintain KAR-mediated synaptic responses. We propose that PICK1 targets PKCα to phosphorylate KARs, causing their stabilization at the synapse by an interaction with GRIP. Importantly, this mechanism is not involved in the constitutive recycling of AMPA receptors since blockade of PDZ interactions can simultaneously increase AMPAR- and decrease KAR-mediated synaptic transmission at the same population of synapses.

Molecular Basis of Mossy Fiber LTP

Long-term potentiation (LTP) at hippocampal mossy fiber synapses is distinct from most other forms of LTP. Mellor et al . found that the hyperpolarization-activated mixed cation current I h unexpectedly plays an important role. Calcium entry into the hippocampal granule cells during repetitive stimulation activates a calcium-calmodulin-sensitive adenylate cyclase. The subsequent rise in adenosine 3′,5′-monophosphate (cAMP) activates protein kinase A, which leads to increases in I h . The resulting depolarization causes an enhancement of mossy fiber synaptic transmission. J. Mellor, R. A. Nicoll, D. Schmitz, Mediation of hippocampal mossy fiber long-term potentiation by presynaptic I h channels. Science 295 , 143-147 (2002). [Abstract] [Full Text]

Molecular Basis of Mossy Fiber LTP

Long-term potentiation (LTP) at hippocampal mossy fiber synapses is distinct from most other forms of LTP. Mellor et al. found that the hyperpolarization-activated mixed cation current Ih unexpectedly plays an important role. Calcium entry into the hippocampal granule cells during repetitive stimulation activates a calcium-calmodulin-sensitive adenylate cyclase. The subsequent rise in adenosine 3′,5′-monophosphate (cAMP) activates protein kinase A, which leads to increases in Ih. The resulting depolarization causes an enhancement of mossy fiber synaptic transmission.J. Mellor, R. A. Nicoll, D. Schmitz, Mediation of hippocampal mossy fiber long-term potentiation by presynaptic Ih channels. Science 295, 143-147 (2002). [Abstract] [Full Text]

RIM1alpha is required for presynaptic long-term potentiation.

Two main forms of long-term potentiation (LTP)-a prominent model for the cellular mechanism of learning and memory-have been distinguished in the mammalian brain. One requires activation of postsynaptic NMDA (N-methyl d-aspartate) receptors, whereas the other, called mossy fibre LTP, has a principal presynaptic component. Mossy fibre LTP is expressed in hippocampal mossy fibre synapses, cerebellar parallel fibre synapses and corticothalamic synapses, where it apparently operates by a mechanism that requires activation of protein kinase A. Thus, presynaptic substrates of protein kinase A are probably essential in mediating this form of long-term synaptic plasticity. Studies of knockout mice have shown that the synaptic vesicle protein Rab3A is required for mossy fibre LTP, but the protein kinase A substrates rabphilin, synapsin I and synapsin II are dispensable. Here we report that mossy fibre LTP in the hippocampus and the cerebellum is abolished in mice lacking RIM1alpha, an active zone protein that binds to Rab3A and that is also a protein kinase A substrate. Our results indicate that the long-term increase in neurotransmitter release during mossy fibre LTP may be mediated by a unitary mechanism that involves the GTP-dependent interaction of Rab3A with RIM1alpha at the interface of synaptic vesicles and the active zone.

Synaptic activation of a presynaptic kainate receptor facilitates AMPA receptor-mediated synaptic transmission at hippocampal mossy fibre synapses.

The development of GluR5-selective kainate receptor ligands is helping to elucidate the functions of kainate receptors in the CNS. Here we have further characterised the actions of a GluR5 selective agonist, ATPA, and a GluR5 selective antagonist, LY382884, in the CA3 region of rat hippocampal slices. In addition, we have used LY382884 to study a novel synaptic mechanism. This antagonist substantially reduces frequency facilitation of mossy fibre synaptic transmission, monitored as either AMPA or NMDA receptor-mediated EPSCs. This suggests that GluR5-containing kainate receptors on mossy fibres function as autoreceptors to facilitate the synaptic release of L-glutamate, in a frequency-dependent manner.

Presynaptic kainate receptor mediation of frequency facilitation at hippocampal mossy fiber synapses.

Inhibition of transmitter release by presynaptic receptors is widespread in the central nervous system and is typically mediated via metabotropic receptors. In contrast, very little is known about facilitatory receptors, and synaptic activation of a facilitatory autoreceptor has not been established. Here we show that activation of presynaptic kainate receptors can facilitate transmitter release from hippocampal mossy fiber synapses. Synaptic activation of these presumed ionotropic kainate receptors is very fast (<10 ms) and lasts for seconds. Thus, these presynaptic kainate receptors contribute to the short-term plasticity characteristics of mossy fiber synapses, which were previously thought to be an intrinsic property of the synapse.

The actions of synaptically released zinc at hippocampal mossy fiber synapses.

Zn2+ is present at high concentrations in the synaptic vesicles of hippocampal mossy fibers. We have used Zn2+ chelators and the mocha mutant mouse to address the physiological role of Zn2+ in this pathway. Zn2+ is not involved in the unique presynaptic plasticities observed at mossy fiber synapses but is coreleased with glutamate from these synapses, both spontaneously and with electrical stimulation, where it exerts a strong modulatory effect on the NMDA receptors. Zn2+ tonically occupies the high-affinity binding site of NMDA receptors at mossy fiber synapses, whereas the lower affinity voltage-dependent Zn2+ binding site is occupied during action potential driven-release. We conclude that Zn2+ is a modulatory neurotransmitter released from mossy fiber synapses and plays an important role in shaping the NMDA receptor response at these synapses.

PSA-NCAM: an important regulator of hippocampal plasticity

The Neural Cell Adhesion Molecule (NCAM) serves as a temporally and spatially regulated modulator of a variety of cell-cell interactions. This review summarizes recent results of studies aimed at understanding its regulation of expression and biological function, thereby focussing on its polysialylated isoforms (PSA-NCAM). The detailed analysis of the expression of PSA and NCAM in the hippocampal mossy fiber system and the morphological consequences of PSA-NCAM deficiency in mice support the notion that the levels of expression of NCAM are important not only for the regulation and maintenance of structural changes, such as migration, axonal growth and fasciculation, but also for activity-induced plasticity. There is evidence that PSA-NCAM can specifically contribute to a presynaptic form of plasticity, namely long-term potentiation at hippocampal mossy fiber synapses. This is consistent with previous observations that NCAM-deficient mice show deficits in spatial learning and exploratory behavior. Furthermore, our data points to an important role of the hypothalamic–pituitary–adrenal axis, which is the principle adaptive response of the organism to environmental challenges, in the control of PSA-NCAM expression in the hippocampal formation. In particular, we evidence an inhibitory influence of corticosterone on PSA-NCAM expression.