Mice lacking metabotropic glutamate receptor 5 show impaired learning and reduced CA1 long-term potentiation (LTP) but normal CA3 LTP

Class I metabotropic glutamate receptors (mGluRs) have been postulated to play a role in synaptic plasticity. To test the involvement of one member of this class, we have recently generated mutant mice that express no mGluR5 but normal levels of other glutamate receptors. The CNS revealed normal development of gross anatomical features. To examine synaptic functions we measured evoked field EPSPs in the hippocampal slice. Measures of presynaptic function, such as paired pulse facilitation in mutant CA1 neurons, were normal. The response of mutant CA1 neurons to low concentrations of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) was missing, which suggests that mGluR5 may be the primary high affinity ACPD receptor in these neurons. Long-term potentiation (LTP) in mGluR5 mutants was significantly reduced in the NMDA receptor (NMDAR)-dependent pathways such as the CA1 region and dentate gyrus of the hippocampus, whereas LTP remained intact in the mossy fiber synapses on the CA3 region, an NMDAR-independent pathway. Some of the difference in CA1 LTP could lie at the level of expression, because the reduction of LTP in the mutants was no longer observed 20 min after tetanus in the presence of 2-amino-5-phosphonopentanoate. We propose that mGluR5 plays a key regulatory role in NMDAR-dependent LTP. These mutant mice were also impaired in the acquisition and use of spatial information in both the Morris water maze and contextual information in the fear-conditioning test. This is consistent with the hypothesis that LTP in the CA1 region may underlie spatial learning and memory.     

Protected-site phosphorylation of protein kinase C in hippocampal long-term potentiation

One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long-lasting biochemical and physiological effects in neurons. In long-term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase-reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the alpha and betaII isoforms of PKC autophosphorylated in the C-terminal domain. Using western blotting with this antiserum we observed an NMDA receptor-mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca2+, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three-dimensional structure of the PKC catalytic core, we propose a "protected site" model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long-lasting physiologic changes.     

An essential role for retinoid receptors RARbeta and RXRgamma in long-term potentiation and depression

Hippocampal long-term potentiation (LTP) and long-term depression (LTD) are the most widely studied forms of synaptic plasticity thought to underlie spatial learning and memory. We report here that RARbeta deficiency in mice virtually eliminates hippocampal CA1 LTP and LTD. It also results in substantial performance deficits in spatial learning and memory tasks. Surprisingly, RXRgamma null mice exhibit a distinct phenotype in which LTD is lost whereas LTP is normal. Thus, while retinoid receptors contribute to both LTP and LTD, they do so in different ways. These findings not only genetically uncouple LTP and LTD but also reveal a novel and unexpected role for vitamin A in higher cognitive functions.     

Calretinin expression as a critical component in the control of dentate gyrus long-term potentiation induction in mice

We have recently reported that mice homozygous (Cr-/-) for a null mutation in the calretinin gene have impaired long-term potentiation (LTP) induction in the dentate gyrus (S. Schurmans et al. (1997) Proc. Natl. Acad. Sci. USA, 94, 10415 ). Here, we investigated dentate LTP induction in mice heterozygous (Cr+/-) for the same mutation. Despite the presence of calretinin in neurons of these mice, although at reduced levels as compared with normal mice, LTP induction in dentate gyrus was totally impaired. Spatial memory and learning were found unaffected in Cr+/- mice, such as in Cr-/- mice. Altogether, our results suggest that calretinin is a critical component in the control of dentate synaptic plasticity in mice, and that levels of calretinin higher than those observed in Cr+/- mice are required to induce LTP in this area. The possible mechanisms leading to the absence of correlation between gene dosage and biological effects are discussed.     

Windup leads to characteristics of central sensitization

To elucidate the physiological role of Fyn, we analysed the properties of synaptic transmission and synaptic plasticity in hippocampal slices of mice overexpressing either wild-type Fyn (w-Fyn) or its constitutively active mutant (m-Fyn). These fyn-transgenes were driven by the calcium/calmodulin-dependent protein kinase II alpha promoter which turned on in the forebrain neurons including hippocampal pyramidal cells and in late neural development. In the hippocampal slices expressing m-Fyn the paired-pulse facilitation was reduced and the basal synaptic transmission was enhanced. A weak theta-burst stimulation, which was subthreshold for the induction of long-term potentiation (LTP) in control slices, elicited LTP in CA1 region of the slices expressing m-Fyn. When a relatively strong stimulation was applied, the magnitude of LTP in m-Fyn slices was similar to that in control slices. By contrast, the basal synaptic transmission and the threshold for the induction of LTP were not altered in the slices overexpressing wild-type Fyn. To examine the effect of expression of m-Fyn on GABAergic inhibitory system, we applied bicuculline, a GABAA receptor blocker, to the hippocampal slices. The ability of bicuculline to enhance excitatory postsynaptic potentials was attenuated in slices expressing m-Fyn, suggesting that the overexpression of m-Fyn reduced the GABAergic inhibition. The enhancement of synaptic transmission and the reduction of GABAergic inhibition may contribute to the enhanced seizure susceptibility in the mice expressing m-Fyn. Thus, these results suggest that regulation of Fyn tyrosine kinase activity is important for both synaptic transmission and plasticity.     

The selective neuronal NO synthase inhibitor 7-nitro-indazole blocks both long-term potentiation and depotentiation of field EPSPs in rat hippocampal CA1 in vivo

The membrane-permeant gas NO is a putative intercellular messenger that has been proposed on the basis of previous in vitro studies to be involved in synaptic plasticity, especially the induction of long-term potentiation (LTP) of excitatory synaptic transmission in the hippocampus and cortex. In the present study, the role of NO in synaptic plasticity has been investigated in vivo. In particular, the action of the novel and selective neuronal NO synthase (nNOS) inhibitor 7-nitro-indazole (7-NI) has been investigated on the induction of LTP and depotentiation (DP) of field EPSPs in CA1 of the hippocampus in vivo. Unlike previously studied nonselective NOS inhibitors, 7-NI does not increase arterial blood pressure. In vehicle-injected rats, high-frequency stimulation consisting of a series of trains at 200 Hz induced LTP. However, LTP induction was strongly inhibited in 7-NI (30 mg/kg, i.p.)-treated animals. The inhibitory effect of 7-NI on the induction of LTP was prevented by pretreatment with L-arginine, the substrate amino acid used by NOS. In control animals, low-frequency stimulation consisting of 900 stimuli at 10 Hz induced DP of previously established LTP, whereas in 7-HI-treated animals only a short-term depression was induced. This effect of 7-NI also was prevented by D-arginine. The LTP and DP induced in control animals in this study were NMDA receptor-dependent, the NMDA receptor antagonist 3-(R,S)-2-carboxypiperazin-4-yl-propyl-1- phosphonic acid inhibiting the induction of both forms of synaptic plasticity. The present experiments are the first to demonstrate that an NOS inhibitor blocks the induction of the synaptic component of LTP and DP in vivo and, therefore, these results strengthen evidence that the production of NO is necessary for the induction of LTP and DP.     

G2 cell cycle arrest and apoptosis are induced in Burkitt''s lymphoma cells by the anticancer agent oracin

The gamma isoform of protein kinase C (gamma-PKC) activity is elevated and learning is superior in the inbred C57BL/6 mouse when compared to the DBA/2 mouse strain. Given the proposed link between PKC and long-term potentiation (LTP) on the one hand and PKC and learning on the other, it was predicted that LTP persistence would be greater in C57BL/6 mouse. When suprathreshold levels of tetanic stimulation were used, similar persistent LTP was observed in both C57BL/6 and DBA/2 strains. However, when tetanus was at threshold, the response in DBA/2 mice decayed to baseline in 30 min, similar to short-term potentiation (STP). Using this same paradigm with C57BL/6 mice, LTP persisted for 4 h, the longest time tested. The time course of the results parallels those observed in rat when phorbol ester, a potent PKC activator, converts STP to LTP. The present findings thus confirm the predicted difference between the two mouse strains. Moreover, the present findings are consistent with a role for gamma-PKC in LTP. Since such results call attention to the need for gamma-PKC interventive procedures, the relative utility of current PKC inhibitors, null mutants and antisense methods are discussed.     

Evidence for NMDA and mGlu receptor-dependent long-term potentiation of mossy fiber-granule cell transmission in rat cerebellum

Long-term potentiation (LTP) is a form of synaptic plasticity that can be revealed at numerous hippocampal and neocortical synapses following high-frequency activation of N-methyl--aspartate (NMDA) receptors. However, it was not known whether LTP could be induced at the mossy fiber-granule cell relay of cerebellum. This is a particularly interesting issue because theories of the cerebellum do not consider or even explicitly negate the existence of mossy fiber-granule cell synaptic plasticity. Here we show that high-frequency mossy fiber stimulation paired with granule cell membrane depolarization (-40 mV) leads to LTP of granule cell excitatory postsynaptic currents (EPSCs). Pairing with a relatively hyperpolarized potential (-60 mV) or in the presence of NMDA receptor blockers [5-amino--phosphonovaleric acid (APV) and 7-chloro-kynurenic acid (7-Cl-Kyn)] prevented LTP, suggesting that the induction process involves a voltage-dependent NMDA receptor activation. Metabotropic glutamate receptors were also involved because blocking them with (+)-alpha-methyl-4-carboxyphenyl-glycine (MCPG) prevented potentiation. At the cytoplasmic level, EPSC potentiation required a Ca2+ increase and protein kinase C (PKC) activation. Potentiation was expressed through an increase in both the NMDA and non-NMDA receptor-mediated current and by an NMDA current slowdown, suggesting that complex mechanisms control synaptic efficacy during LTP. LTP at the mossy fiber-granule cell synapse provides the cerebellar network with a large reservoir for memory storage, which may be needed to optimize pattern recognition and, ultimately, cerebellar learning and computation.     

Regulation of hippocampal synaptic plasticity by the tyrosine kinase receptor, REK7/EphA5, and its ligand, AL-1/Ephrin-A5

The Eph-related tyrosine kinase receptor, REK7/EphA5, mediates the effects of AL-1/Ephrin-A5 and related ligands and is involved in the guidance of retinal, cortical, and hippocampal axons during development. The continued expression of REK7/EphA5 in the adult brain, in particular in areas associated with a high degree of synaptic plasticity such as the hippocampus, raises the question of its function in the mature nervous system. In this report we examined the role of REK7/EphA5 in synaptic remodeling by asking if agents that either block or activate REK7/EphA5 affect synaptic strength in hippocampal slices from adult mouse brain. We show that a REK7/EphA5 antagonist, soluble REK7/EphA5-IgG, impairs the induction of long-term potentiation (LTP) without affecting other synaptic parameters such as normal synaptic transmission or paired-pulse facilitation. In contrast, perfusion with AL-1/Ephrin-A5-IgG, an activator of REK7/EphA5, induces a sustained increase in normal synaptic transmission that partially mimics LTP. The sustained elevation of normal synaptic transmission could be attributable to a long-lasting binding of the AL-1/Ephrin-A5-IgG to the endogenous REK7/EphA5 receptor, as revealed by immunohistochemistry. Furthermore, maximal electrical induction of LTP occludes the potentiating effects of subsequent treatment with AL-1/Ephrin-A5-IgG. Taken together these results implicate REK7/EphA5 in the regulation of synaptic plasticity in the mature hippocampus and suggest that REK7/EphA5 activation is recruited in the LTP induced by tetanization.     

Impaired motor coordination correlates with persistent multiple climbing fiber innervation in PKC gamma mutant mice

It is generally believed that a smooth execution of a compound movement, or motor coordination, requires learning of component movements as well as experience-based refinement of the motor program as a whole. PKC gamma mutant mice display impaired motor coordination but intact eyeblink conditioning, a form of component movement learning. Cerebellar long-term depression, a putative cellular mechanism for component motor learning, is also unimpaired. Thus, PKC gamma mutant mice are defective in refinement of the motor program. In the accompanying paper, we demonstrate that innervation of multiple climbing fibers onto Purkinje cells persists in adulthood in these mutant mice. We propose that this defective elimination of surplus climbing fibers underlies motor discoordination.     

The characteristics of the clinico-diagnostic and therapeutic treatment procedures for workers in the marine transport shore services who are ill with circulatory encephalopathy]

Heparin-binding growth-associated molecule (HB-GAM) is an 18-kDa developmentally regulated protein, which promotes neurite outgrowth, axonal guidance and synaptogenesis through interaction with cell-surface heparan-sulphate proteoglycans. We have studied the effect of HB-GAM on synaptic transmission and long-term potentiation (LTP) in the area CA1 of rat hippocampal slices, where HB-GAM mRNA is expressed in an activity-dependent manner. Injection of recombinant HB-GAM into the dendritic area inhibited tetanus-induced LTP without affecting baseline synaptic responses or the N-methyl-D-aspartate (NMDA)-receptor mediated transmission. HB-GAM did not depotentiate tetanus-induced LTP or prevent heterosynaptic LTP induced by application of tetraethylammonium (TEA), indicating that the effect was limited to early, synapse-specific stages of LTP induction. These results suggest that HB-GAM is involved in the regulation of synaptic plasticity in hippocampus.     

Long-term potentiation of synaptic transmission in the avian hippocampus

The avian hippocampus plays a pivotal role in memory required for spatial navigation and food storing. Here we have examined synaptic transmission and plasticity within the hippocampal formation of the domestic chicken using an in vitro slice preparation. With the use of sharp microelectrodes we have shown that excitatory synaptic inputs in this structure are glutamatergic and activate both NMDA- and AMPA-type receptors on the postsynaptic membrane. In response to tetanic stimulation, the EPSP displayed a robust long-term potentiation (LTP) lasting >1 hr. This LTP was unaffected by blockade of NMDA receptors or chelation of postsynaptic calcium. Application of forskolin increased the EPSP and reduced paired-pulse facilitation (PPF), indicating an increase in release probability. In contrast, LTP was not associated with a change in the PPF ratio. Induction of LTP did not occlude the effects of forskolin. Thus, in contrast to NMDA receptor-independent LTP in the mammalian brain, LTP in the chicken hippocampus is not attributable to a change in the probability of transmitter release and does not require activation of adenylyl cyclase. These findings indicate that a novel form of synaptic plasticity might underlie learning in the avian hippocampus.     

Increase in syntaxin 1B and glutamate release in mossy fibre terminals following induction of LTP in the dentate gyrus: a candidate molecular mechanism underlying transsynaptic plasticity

A growing body of evidence suggests that modulation of certain proteins of the exocytotic machinery is, in part, involved in the biochemical changes that underlie long-term synaptic plasticity. We have previously shown that the induction of long-term potentiation (LTP) at perforant path to dentate granule cell synapses in the rat hippocampus induces changes in the mRNA levels of syntaxin 1B and synapsin I, known to be involved in neurotransmitter release. Immunohistochemical staining suggested that concomitant changes in these proteins occurred at mossy fibre synapses, downstream of those synapses at which LTP was induced, leading us to postulate that such a mechanism might underlie a form of transsynaptic plasticity. Here we have used a specific mossy-fibre synaptosome preparation to quantify levels of proteins and measure, using a chemiluminescent glutamate assay, depolarization-induced glutamate release from these synaptosomes after induction of LTP in the dentate gyrus in vivo. We show that 5 h after the induction of LTP, there is an increase in the protein levels of syntaxin 1B and, although to a lesser extent, the synapsins I and II, associated with an increase in depolarization-induced release of glutamate within these terminals. Increases in both the protein levels and glutamate release were not observed when dentate gyrus LTP was blocked by an NMDA receptor antagonist. From these results we propose a molecular mechanism for the propagation of synaptic plasticity through hippocampal circuits.     

Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation

Although classically studied as regulators of cell proliferation and differentiation, mitogen-activated protein kinases (MAPKs) are highly expressed in post-mitotic neurons of the adult nervous system. We have begun investigating the potential role of MAPKs in the regulation of synaptic plasticity in mature neurons. In particular, we have studied the regulation of two MAPK isoforms, p44 and p42 MAPK, in hippocampal long term potentiation (LTP), a system widely studied as a model for the cellular basis of learning and memory. We have found that p42 MAPK, but not p44 MAPK, is activated in area CA1 following direct stimulation of two required components of the LTP induction cascades: protein kinase C and the N-methyl--aspartate (NMDA) subtype of glutamate receptor. Furthermore, we have demonstrated that p42 MAPK, but not p44 MAPK, is activated in area CA1 in response to LTP-inducing high frequency stimulation and that this activation requires NMDA receptor stimulation. These data demonstrate that p42 MAPK can be regulated in an activity-dependent manner in the hippocampus and identify it as a potential component of the LTP induction cascades in area CA1. Such observations suggest that p42 MAPK might be an important regulator of synaptic plasticity in post-mitotic neurons.     

Morphological changes associated with long-term potentiation

Long-term potentiation (LTP) is a long-lasting form of synaptic plasticity induced by brief repetitive afferent stimulation that is thought to be associated with learning and memory. It is most commonly studied in the hippocampus where it may last for several weeks, and involves the synthesis of new proteins that might play a structural role. In this review we summarize the evidence in favor of modifications of neuronal architecture during LTP. We focus our attention on changes occurring at the level of single synapses, including components of postsynaptic dendrites (dendritic spines, the postsynaptic density, and synaptic curvature), of presynaptic terminals, and the formation of new synapses. We conclude that although many morphological changes at various sites have been observed during LTP, there is no definitive proof in favor of structural changes associated with LTP. However, morphological modifications remain a valid candidate for mechanisms of learning and memory.     

Morphine-6beta-glucuronide-induced hyperphagia: characterization of opioid action by selective antagonists and antisense mapping in rats

Copper/zinc superoxide dismutase (CuZn-SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene encoding CuZn-SOD resides on human chromosome 21 and is overexpressed in Down syndrome (DS) patients. Overexpression of CuZn-SOD in transgenic (Tg) mice and cultured cells creates chronic oxidative stress leading to enhanced susceptibility to degeneration and apoptotic cell death. We have now found that three lines of Tg-CuZn-SOD mice, one of which also overexpresses S100beta, a glial calcium binding protein, are deficient in spatial memory. Furthermore, hippocampal slices taken from these mice have an apparently normal synaptic physiology, but are impaired in the ability to express long-term potentiation (LTP). This effect on hippocampal LTP was abrogated by treatment of slices with the H2O2 scavenger catalase or the antioxidant N-t-butyl-phenylnitrone (BPN). It is proposed that elevated CuZnSOD causes an increase in tetanic stimulation-evoked formation of H2O2 which leads to diminished LTP and cognitive deficits in these mice.     

Amygdala beta-noradrenergic influence on hippocampal long-term potentiation in vivo

We have previously shown that hippocampal long-term potentiation (LTP), one form of synaptic plasticity that may underlie learning and memory, is attenuated by blocking neuron activity of the basolateral amygdala (BLA). In the present study we investigated the amygdala noradrenergic or cholinergic contribution to hippocampal LTP formation. When propranolol, a beta-adrenoceptor antagonist, was injected into the BLA 10 min before tetanus, the formation of LTP in the perforant path-dentate granule cell synapses was significantly impaired. Scopolamine, a muscarinic cholinergic receptor antagonist, did not affect the formation of LTP. These results suggest that amygdala beta-noradrenergic activity plays a critical role in modulation of hippocampal LTP.     

The involvement of brain-derived neurotrophic factor in hippocampal long-term potentiation revealed by gene targeting experiments

Brain-derived neurotrophic factor (BDNF) is a member of the NGF gene family, which has been shown to influence the survival and differentiation of specific classes of neurons in vitro and in vivo. The possibility that neurotrophins are also involved in processes of neuronal plasticity has only recently begun to receive attention. To determine whether BDNF has a function in processes like long-term potentiation (LTP), we produced a strain of mice with a deletion in the coding sequence of the BDNF-gene. We then used hippocampal slices from these mice to investigate whether LTP is affected by this mutation. Mutant mice showed significantly weaker LTP in the CA1 region. The magnitude of the potentiation as well as the percentage of cases in which LTP could be induced successfully was clearly reduced whereas important pharmacological and morphological control parameters in the hippocampus of these animals were unaffected. Adenoviral vectors were used to re-express BDNF in acute slices of BDNF-knock-out mice. In most cases LTP could be rescued with this approach. These results suggest that BDNF has an important functional role in the expression of LTP in the hippocampus.     

Differing mechanisms of expression for short- and long-term potentiation

Long-term potentiation (LTP) is a use-dependent form of synaptic plasticity that is of great interest as a cellular mechanism that may contribute to memory storage. It is the sustained phase of population excitatory postsynaptic potential induced by high-frequency stimulation (HFS). HFS can also induce short-term potentiation (STP), a decremental potentiation lasting approximately 15 min. It has been unclear whether STP is simply a reversible form of LTP elicited by subthreshold stimuli or whether it is an independently expressed form of synaptic plasticity. We have attempted to clarify the relationship between LTP and STP in the extracellular recording technique in area CA1 of the adult rat hippocampal slice preparation to test four predictions of the hypothesis that LTP and STP are expressed via the same mechanism. First, occluding LTP expression should block STP expression. Saturating LTP under six different conditions, however, did not occlude STP expression. Second, occluding STP expression should occlude LTP expression. The partial or full occlusion of STP by two maneuvers (increasing the stimulus intensity used for HFS or applying 3-isobutyl-1-methylxanthine), however, did not occlude LTP expression. Third, LTP increases and decreases paired-pulse facilitation (PPF), and STP should have the same effect. STP did not change PFF, however. The first three results, then, suggest that STP and LTP are expressed via different mechanisms. Fourth, STP should be maximal near the LTP induction threshold, and then decrease above it. Surprisingly, STP was maximal at or very close to the LTP induction threshold, but it did not decrease above this threshold. This relationship suggests the possibility that STP and LTP share an induction step(s). What is the function of the independently expressed STP? We find that LTP can be induced by two HFSs, each of which is subthreshold for LTP, if the second is given during STP from the first. This suggests that STP can temporarily lower the LTP induction threshold. Three lines of evidence, then, suggest that STP and LTP may be expressed via different mechanisms; however, the proximity of STP saturation to LTP induction suggests that they may share an induction step(s). STP may also have the very important function of temporarily lowering the LTP induction threshold. Finally, these data suggestion caution in interpreting LTP data obtained <20-30 min after HFS, because they may be contaminated by STP, which appears to have different underlying mechanisms.