Differential trafficking of AMPA and NMDA receptors by SAP102 and PSD-95 underlies synapse development.

The development of glutamatergic synapses involves changes in the number and type of receptors present at the postsynaptic density. To elucidate molecular mechanisms underlying these changes, we combine in utero electroporation of constructs that alter the molecular composition of developing synapses with dual whole-cell electrophysiology to examine synaptic transmission during two distinct developmental stages. We find that SAP102 mediates synaptic trafficking of AMPA and NMDA receptors during synaptogenesis. Surprisingly, after synaptogenesis, PSD-95 assumes the functions of SAP102 and is necessary for two aspects of synapse maturation: the developmental increase in AMPA receptor transmission and replacement of NR2B-NMDARs with NR2A-NMDARs. In PSD-95/PSD-93 double-KO mice, the maturational replacement of NR2B- with NR2A-NMDARs fails to occur, and PSD-95 expression fully rescues this deficit. This study demonstrates that SAP102 and PSD-95 regulate the synaptic trafficking of distinct glutamate receptor subtypes at different developmental stages, thereby playing necessary roles in excitatory synapse development.

Alpha1E-containing Ca2+ channels are involved in synaptic plasticity.

Long-term potentiation (LTP) is the most prominent model for the molecular and cellular mechanisms of learning and memory. Two main forms of LTP have been distinguished. The N-methyl-D-aspartate-receptor-dependent forms of LTP have been studied most extensively, whereas much less is known about N-methyl-D-aspartate-receptor-independent forms of LTP. This latter type of LTP was first described at the mossy fiber synapses in the hippocampus and subsequently at parallel fiber synapses in the cerebellum as well as at corticothalamic synapses. These presynaptic forms of LTP require a rise in the intraterminal calcium concentration, but the channel through which calcium passes has not been identified. By using pharmacological tools as well as genetic deletion, we demonstrate here that alpha1E-containing voltage-dependent calcium channels (VDCCs) shift the threshold for mossy fiber LTP. The channel is not involved in the expression mechanism, but it contributes to the calcium influx during the induction phase. Indeed, optical recordings directly show the presence and the function of alpha1E-containing VDCCs at mossy fiber terminals. Hence, a previously undescribed role for alpha1E-containing VDCCs is suggested by these results.

Metabotropic glutamate receptor activation causes a rapid redistribution of AMPA receptors.

Electrophysiology, immunostaining and time lapse imaging techniques were employed to study the mechanism of long-term depression (LTD) induced by DHPG, a specific group I metabotropic glutamate receptor (mGluR) agonist. Experiments were performed in primary hippocampal culture or in the CA1 area of acute rat hippocampal slices. In agreement with previous results by others, we show that DHPG (200 microM, 10 min) can induce LTD (DHPG-LTD) in acute slices, in the presence or absence of synaptic inhibition. In addition, in voltage clamp whole cell experiments we find that accompanying the reduction in the evoked excitatory postsynaptic current (EPSC), miniature EPSC amplitude and frequency are reduced. Similar results were obtained in cultured neurons. Immunostaining and time lapse imaging showed a long-lasting loss of AMPA receptors from the membrane surface of cultured neurons after DHPG treatment, which appears to occur in only a subset of the puncta. Further electrophysiological recordings on slices showed that blocking postsynaptic endocytosis by introducing a blocking peptide named D15 in recording pipettes abolished the DHPG-LTD. In conclusion, these data suggest that LTD induced by mGluR activation is due to a rapid removal of AMPA receptors from the postsynaptic membrane.

Presynaptic kainate receptors at hippocampal mossy fiber synapses.

Hippocampal mossy fibers, which are the axons of dentate granule cells, form powerful excitatory synapses onto the proximal dendrites of CA3 pyramidal cells. It has long been known that high-affinity binding sites for kainate, a glutamate receptor agonist, are present on mossy fibers. Here we summarize recent experiments on the role of these presynaptic kainate receptors (KARs). Application of kainate has a direct effect on the amplitude of the extracellularly recorded fiber volley, with an enhancement by low concentrations and a depression by high concentrations. These effects are mediated by KARs, because they persist in the presence of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-selective antagonist GYKI 53655, but are blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/KAR antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and the KAR antagonist SYM2081. The effects on the fiber volley are most likely caused by a depolarization of the fibers via the known ionotropic actions of KARs, because application of potassium mimics the effects. In addition to these effects on fiber excitability, low concentrations of kainate enhance transmitter release, whereas high concentrations depress transmitter release. Importantly, the synaptic release of glutamate from mossy fibers also activates these presynaptic KARs, causing an enhancement of the fiber volley and a facilitation of release that lasts for many seconds. This positive feedback contributes to the dramatic frequency facilitation that is characteristic of mossy fiber synapses. It will be interesting to determine how widespread facilitatory presynaptic KARs are at other synapses in the central nervous system.

Presynaptic specificity of endocannabinoid signaling in the hippocampus.

Endocannabinoids are retrograde messengers released by neurons to modulate the strength of their synaptic inputs. Endocannabinoids are thought to mediate the suppression of GABA release that follows depolarization of a hippocampal CA1 pyramidal neuron-termed "depolarization-induced suppression of inhibition" (DSI). Here, we report that DSI is absent in mice which lack cannabinoid receptor-1 (CB1). Pharmacological and kinetic evidence suggests that CB1 activation inhibits presynaptic Ca2+ channels through direct G protein inhibition. Paired recordings show that endocannabinoids selectively inhibit a subclass of synapses distinguished by their fast kinetics and large unitary conductance. Furthermore, cannabinoid-sensitive inputs are unusual among central nervous system synapses in that they use N- but not P/Q-type Ca2+ channels for neurotransmitter release. These results indicate that endocannabinoids are highly selective, rapid modulators of hippocampal inhibition.

Kainate receptors depress excitatory synaptic transmission at CA3-->CA1 synapses in the hippocampus via a direct presynaptic action.

Kainate receptor activation depresses synaptic release of neurotransmitter at a number of synapses in the CNS. The mechanism underlying this depression is controversial, and both ionotropic and metabotropic mechanisms have been suggested. We report here that the AMPA/kainate receptor agonists domoate (DA) and kainate (KA) cause a presynaptic depression of glutamatergic transmission at CA3-->CA1 synapses in the hippocampus, which is not blocked by the AMPA receptor antagonist GYKI 53655 but is blocked by the AMPA/KA receptor antagonist CNQX. Neither a blockade of interneuronal discharge nor antagonists of several neuromodulators affect the depression, suggesting that it is not the result of indirect excitation and subsequent release of a neuromodulator. Presynaptic depolarization, achieved via increasing extracellular K(+), caused a depression of the presynaptic fiber volley and an increase in the frequency of miniature EPSCs. Neither effect was observed with DA, suggesting that DA does not depress transmission via a presynaptic depolarization. However, the effects of DA were abolished by the G-protein inhibitors N-ethylmaleimide and pertussis toxin. These results suggest that KA receptor activation depresses synaptic transmission at this synapse via a direct, presynaptic, metabotropic action.

Hippocampal synaptic transmission and plasticity are preserved in myosin Va mutant mice.

Recent studies have identified myosin Va as an organelle motor that may have important functions in neurons. Abundantly expressed at the hippocampal postsynaptic density, it interacts with protein complexes involved in synaptic plasticity. It is also located in presynaptic terminals and may function to recruit vesicles in the reserve pool to the active zone. Dilute-lethal mice are spontaneous myosin Va mutants and have severe neurological symptoms. We studied hippocampal physiology at CA3-CA1 excitatory synapses in dilute-lethal mutant mice to test the hypothesis that myosin Va plays a role in pre- or postsynaptic elements of synaptic transmission. In all assays performed, the mutant synapses appeared to be functioning normally, both pre- and postsynaptically. These data suggest that myosin Va is not essential for the synaptic release machinery, postsynaptic receptor composition, or plasticity at this synapse, but does not exclude significant roles for myosin Va in other cell types nor potential compensation by other myosin V isoforms.

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.

Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses.

Marijuana affects brain function primarily by activating the G-protein-coupled cannabinoid receptor-1 (CB1), which is expressed throughout the brain at high levels. Two endogenous lipids, anandamide and 2-arachidonylglycerol (2-AG), have been identified as CB1 ligands. Depolarized hippocampal neurons rapidly release both anandamide and 2-AG in a Ca2+-dependent manner. In the hippocampus, CB1 is expressed mainly by GABA (gamma-aminobutyric acid)-mediated inhibitory interneurons, where CB1 clusters on the axon terminal. A synthetic CB1 agonist depresses GABA release from hippocampal slices. These findings indicate that the function of endogenous cannabinoids released by depolarized hippocampal neurons might be to downregulate GABA release. Here we show that the transient suppression of GABA-mediated transmission that follows depolarization of hippocampal pyramidal neurons is mediated by retrograde signalling through release of endogenous cannabinoids. Signalling by the endocannabinoid system thus represents a mechanism by which neurons can communicate backwards across synapses to modulate their inputs.

Contribution of cytoskeleton to the internalization of AMPA receptors.

Trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) at synapses has been suggested to play an important role in the expression of synaptic plasticity. Both the regulated and the constitutive trafficking of synaptic AMPARs are thought to involve the insertion and removal of receptors by means of an exocytotic and endocytotic process, respectively. In contrast, N-methyl-d-aspartate (NMDA) receptors (NMDARs), which are colocalized with AMPARs at excitatory synapses, appear to be much less dynamic. Here, we present evidence supporting the idea that synaptic AMPARs turn over through a constitutive endocytotic process and that glutamate application greatly enhances this turnover of AMPARs. The glutamate-induced internalization of AMPARs requires a rise in postsynaptic Ca(2+). The AMPAR internalization is mimicked by latrunculin A, a drug that selectively depolymerizes actin and is blocked by jasplakinolide, a drug which stabilizes actin filaments. The rate of endocytosis is not altered by glutamate application, whereas a clear enhancement is observed with insulin application. We propose a model in which the glutamate-induced dissociation of AMPARs from their anchor on the postsynaptic membrane involves actin depolymerization, which allows the released AMPARs to segregate from the NMDARs and diffuse to a presumably perisynaptic site, where they become available to an endocytotic machinery and are selectively internalized.

Contrasting localizations of MALS/LIN-7 PDZ proteins in brain and molecular compensation in knockout mice.

Proteins containing PDZ (postsynaptic density-95, discs large, zonula occludens) domains play a general role in recruiting receptors and enzymes to specific synaptic sites. In Caenorhabditis elegans, a complex of three PDZ proteins, LIN-2/7/10, mediates basolateral targeting of a receptor tyrosine kinase. Homologs of these LIN proteins have also been identified in higher organisms, and here we analyze the MALS/Veli (mammalian LIN-7/vertebrate homolog of LIN-7) proteins in brain. Immunohistochemical staining and in situ hybridization show that MALS occur differentially in discrete populations of neurons throughout the brain. Most neurons express only one MALS protein, although some cells contain two or even all three MALS isoforms. At the subcellular level, MALS proteins are found in both dendritic and axonal locations, suggesting that they may regulate processes at both pre- and postsynaptic sites. Targeted disruption of MALS-1 and MALS-2 does not yield a detectable phenotype, and hippocampal synaptic function and plasticity are intact in the MALS-1/2 double knockouts. Interestingly, MALS-3 protein is dramatically induced in the MALS-1/2 double knockouts, implying that dynamic changes in protein expression may play an important regulatory role for this family of synaptic PDZ proteins.

PSD-95 involvement in maturation of excitatory synapses.

PSD-95 is a neuronal PDZ protein that associates with receptors and cytoskeletal elements at synapses, but whose function is uncertain. We found that overexpression of PSD-95 in hippocampal neurons can drive maturation of glutamatergic synapses. PSD-95 expression enhanced postsynaptic clustering and activity of glutamate receptors. Postsynaptic expression of PSD-95 also enhanced maturation of the presynaptic terminal. These effects required synaptic clustering of PSD-95 but did not rely on its guanylate kinase domain. PSD-95 expression also increased the number and size of dendritic spines. These results demonstrate that PSD-95 can orchestrate synaptic development and are suggestive of roles for PSD-95 in synapse stabilization and plasticity.

Synaptic activation of presynaptic kainate receptors on hippocampal mossy fiber synapses.

Kainate receptors (KARs) are a poorly understood family of ionotropic glutamate receptors. A role for these receptors in the presynaptic control of transmitter release has been proposed but remains controversial. Here, KAR agonists are shown to enhance fiber excitability, and a number of experiments show that this is a direct effect of KARs on the presynaptic fibers. In addition, KAR activation inhibits evoked transmitter release from mossy fiber synapses. Synaptic release of glutamate from either neighboring mossy fiber synapses or associational/commisural (A/C) synapses results in the activation of these presynaptic ionotropic KARs. These results, along with previous studies, indicate that KARs, through the endogenous release of glutamate, mediate excitatory postsynaptic potentials (EPSPs), alter presynaptic excitability, and modulate transmitter release.

The role of brain-derived neurotrophic factor receptors in the mature hippocampus: modulation of long-term potentiation through a presynaptic mechanism involving TrkB.

The neurotrophin BDNF has been shown to modulate long-term potentiation (LTP) at Schaffer collateral-CA1 hippocampal synapses. Mutants in the BDNF receptor gene trkB and antibodies to its second receptor p75NTR have been used to determine the receptors and cells involved in this response. Inhibition of p75NTR does not detectably reduce LTP or affect presynaptic function, but analyses of newly generated trkB mutants implicate TrkB. One mutant has reduced expression in a normal pattern of TrkB throughout the brain. The second mutant was created by cre-loxP-mediated removal of TrkB in CA1 pyramidal neurons of this mouse. Neither mutant detectably impacts survival or morphology of hippocampal neurons. TrkB reduction, however, affects presynaptic function and reduces the ability of tetanic stimulation to induce LTP. Postsynaptic glutamate receptors are not affected by TrkB reduction, indicating that BDNF does not modulate plasticity through postsynaptic TrkB. Consistent with this, elimination of TrkB in postsynaptic neurons does not affect LTP. Moreover, normal LTP is generated in the mutant with reduced TrkB by a depolarization-low-frequency stimulation pairing protocol that puts minimal demands on presynaptic terminal function. Thus, BDNF appears to act through TrkB presynaptically, but not postsynaptically, to modulate LTP.

Synaptic kainate receptors.

Kainate receptors are a family of ionotropic glutamate receptors with poorly understood functions. Recent evidence firmly establishes kainate receptors as postsynaptic mediators of synaptic transmission. A second, presynaptic, modulatory role of kainate receptors has also been suggested, although the mechanism(s) involved remain controversial.

Synaptic plasticity and dynamic modulation of the postsynaptic membrane.

The biochemical composition of the postsynaptic membrane and the structure of dendritic spines may be rapidly modulated by synaptic activity. Here we review these findings, discuss their implications for long-term potentiation (LTP) and long-term depression (LTD) and propose a model of sequentially occurring expression mechanisms.