NMDA application potentiates synaptic transmission in the hippocampus

The NMDA (N-methyl-D-aspartate) class of glutamate receptor plays a critical role in a variety of forms of synaptic plasticity in the vertebrate central nervous system. One extensively studied example of plasticity is long-term potentiation (LTP), a remarkably long-lasting enhancement of synaptic efficiency induced in the hippocampus by brief, high-frequency stimulation of excitatory synapses. LTP is a strong candidate for a cellular mechanism of learning and memory. The site of LTP induction appears to be the postsynaptic cell and induction requires both activation of NMDA receptors by synaptically released glutamate and depolarization of the postsynaptic membrane. It is proposed that this depolarization relieves a voltage-dependent Mg2+ block of the NMDA receptor channel, resulting in increased calcium influx which is the trigger for the induction of LTP. This model predicts that application of a large depolarizing dose of NMDA should be sufficient to evoke LTP. In agreement with a previous study, we have found that NMDA or glutamate application does potentiate synaptic transmission in the hippocampus. This agonist-induced potentiation is, however, decremental and short-lived, unlike LTP. It is occluded shortly after the induction of LTP and a similar short-term potentiation can be evoked by synaptically released glutamate. We thus propose that LTP has two components, a short-term, decremental component which can be mimicked by NMDA receptor activation, and a long-lasting, non-decremental component which, in addition to requiring activation of NMDA receptors, requires stimulation of presynaptic afferents.     

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.     

Long-term potentiation of NMDA receptor-mediated synaptic transmission in the hippocampus.

Neurotransmission at most excitatory synapses in the brain operates through two types of glutamate receptor termed alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors; these mediate the fast and slow components of excitatory postsynaptic potentials respectively. Activation of NMDA receptors can also lead to a long-lasting modification in synaptic efficiency at glutamatergic synapses; this is exemplified in the CA1 region of the hippocampus, where NMDA receptors mediate the induction of long-term potentiation (LTP). It is believed that in this region LTP is maintained by a specific increase in the AMPA receptor-mediated component of synaptic transmission. We now report, however, that a pharmacologically isolated NMDA receptor-mediated synaptic response can undergo robust, synapse-specific LTP. This finding has implications for neuropathologies such as epilepsy and neurodegeneration, in which excessive NMDA receptor activation has been implicated. It adds fundamentally to theories of synaptic plasticity because NMDA receptor activation may, in addition to causing increased synaptic efficiency, directly alter the plasticity of synapses.     

Transient activation of calcineurin is essential to initiate embryonic development in Xenopus laevis

At fertilization, an increase of cytosolic calcium ions (Ca2+) triggers various activation responses in animal eggs. In vertebrates, these responses include exit from metaphase arrest in meiosis II (MII exit) and cortical remodelling initiated by cortical granule exocytosis. Although the essential requirement of Ca2+/calmodulin-dependent protein kinase II for inducing MII exit has been documented, a role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in egg activation has not been investigated. Here we show, using cell-free extracts from unfertilized eggs of Xenopus laevis, that calcineurin is transiently activated immediately after Ca2+ addition to a concentration that induces MII exit. When calcineurin activation is inhibited, cyclin-dependent kinase 1 (Cdk1) inactivation by means of cyclin B degradation is prevented and sperm chromatin incubated in the extracts remains condensed. Similarly, if calcineurin is inhibited in intact eggs, MII exit on egg activation is prevented. In addition, the activation contraction in the cortex is suppressed whereas cortical granule exocytosis occurs. We further demonstrate that, when a high level of calcineurin activity is maintained after activation, growth of sperm asters is prevented in egg extracts and, consistently, migration of male and female pronuclei towards each other is hindered in fertilized eggs. Thus, both activation and the subsequent inactivation of calcineurin in fertilized eggs are crucial for the commencement of vertebrate embryonic development.     

Two components of long-term potentiation induced by different patterns of afferent activation

Long-term potentiation (LTP) of excitatory synaptic transmission could be a mechanism underlying memory. Induction of LTP requires Ca2+ influx into postsynaptic neurons through ion channels gated by NMDA (N-methyl-D-aspartate) receptors in hippocampus (area CA1 and dentate gyrus) and neocortex. Here we report that a component of LTP not requiring the activation of NMDA receptors can be induced in area CA1. The component is dependent on tetanus frequency, requires increases in postsynaptic intracellular Ca2+ concentrations, and is suppressed by an antagonist of voltage-dependent Ca2+ channels.     

Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors.

Long-term potentiation (LTP) in the hippocampus is thought to contribute to memory formation. In the Ca1 region, LTP requires the NMDA (N-methyl-D-aspartate) receptor-dependent influx of Ca2+ and activation of serine and threonine protein kinases. Because of the high amount of protein tyrosine kinases in hippocampus and cerebellum, two regions implicated in learning and memory, we examined the possible additional requirement of tyrosine kinase activity in LTP. We first examined the specificity in brain of five inhibitors of tyrosine kinase and found that two of them, lavendustin A and genistein, showed substantially greater specificity for tyrosine kinase from hippocampus than for three serine-threonine kinases: protein kinase A, protein kinase C, and Ca2+/calmodulin kinase II. Lavendustin A and genistein selectively blocked the induction of LTP when applied in the bath or injected into the postsynaptic cell. By contrast, the inhibitors had no effect on the established LTP, on normal synaptic transmission, or on the neurotransmitter actions attributable to the actions of protein kinase A or protein kinase C. These data suggest that tyrosine kinase activity could be required postsynaptically for long-term synaptic plasticity in the hippocampus. As Ca2+ calmodulin kinase II or protein kinase C seem also to be required, the tyrosine kinases could participate postsynaptically in a kinase network together with serine and threonine kinases.     

An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation

The phenomenon of long-term potentiation (LTP), a long lasting increase in the strength of synaptic transmission which is due to brief, repetitive activation of excitatory afferent fibres, is one of the most striking examples of synaptic plasticity in the mammalian brain. In the CA1 region of the hippocampus, the induction of LTP requires activation of NMDA (N-methyl-D-aspartate) receptors by synaptically released glutamate with concomitant postsynaptic membrane depolarization. This relieves the voltage-dependent magnesium block of the NMDA-receptor ion channel, allowing calcium to flow into the dendritic spine. Although calcium has been shown to be a necessary trigger for LTP (refs 11, 12), little is known about the immediate biochemical processes that are activated by calcium and are responsible for LTP. The most attractive candidates have been calcium/calmodulin-dependent protein kinase II (CaM-KII) (refs 13-16), protein kinase C (refs 17-19), and the calcium-dependent protease, calpain. Extracellular application of protein kinase inhibitors to the hippocampal slice preparation blocks the induction of LTP (refs 21-23) but it is unclear whether this is due to a pre- and/or postsynaptic action. We have found that intracellular injection into CA1 pyramidal cells of the protein kinase inhibitor H-7, or of the calmodulin antagonist calmidazolium, blocks LTP. Furthermore, LTP is blocked by the injection of synthetic peptides that are potent calmodulin antagonists and inhibit CaM-KII auto- and substrate phosphorylation. These findings demonstrate that in the postsynaptic cell both activation of calmodulin and kinase activity are required for the generation of LTP, and focus further attention on the potential role of CaM-KII in LTP.     

Presynaptic enhancement shown by whole-cell recordings of long-term potentiation in hippocampal slices

Long-term potentiation (LTP) of synaptic transmission in the hippocampus is a widely studied model system for understanding the cellular mechanisms of memory. In region CA1, LTP is triggered postsynaptically by Ca2(+)-dependent activation of protein kinases, but the locus of persistent modification remains controversial. Statistical analysis of synaptic variability has been proposed as a means of settling this debate, although a major obstacle has been the poor signal-to-noise ratio of conventional intracellular recordings. We have applied the whole-cell voltage clamp technique to study synaptic transmission in conventional hippocampal slices (compare refs 28-30). Here we report that robust LTP can be recorded with much improved signal resolution and biochemical access to the postsynaptic cell. Prolonged dialysis of the postsynaptic cell blocks the triggering of LTP, with no effect on expression of LTP. The improved signal resolution unmasks a large trial-to-trial variability, reflecting the probabilistic nature of transmitter release. Changes in the synaptic variability, and a decrease in the proportion of synaptic failures during LTP, suggest that transmitter release is significantly enhanced.     

Different voltage-dependent thresholds for inducing long-term depression and long-term potentiation in slices of rat visual cortex

In the hippocampus and neocortex, high-frequency (tetanic) stimulation of an afferent pathway leads to long-term potentiation (LTP) of synaptic transmission. In the hippocampus it has recently been shown that long-term depression (LTD) of excitatory transmission can also be induced by certain combinations of synaptic activation. In most hippocampal and all neocortical pathways studied so far, the induction of LTP requires the activation of N-methyl-D-aspartate (NMDA) receptor-gated conductances. Here we report that LTD can occur in neurons of slices of the rat visual cortex and that the same tetanic stimulation can induce either LTP or LTD depending on the level of depolarization of the postsynaptic neuron. By applying intracellular current injections or pharmacological disinhibition to modify the depolarizing response of the postsynaptic neuron to tetanic stimulation, we show that the mechanisms of induction of LTD and LTP are both postsynaptic. LTD is obtained if postsynaptic depolarization exceeds a critical level but remains below a threshold related to NMDA receptor-gated conductances, whereas LTP is induced if this second threshold is reached.     

Calcineurin is required to release Xenopus egg extracts from meiotic M phase

Fertilization induces a transient increase in cytoplasmic Ca2+ concentration in animal eggs that releases them from cell cycle arrest in the second meiotic metaphase. In frog eggs, Ca2+ activates Ca2+/calmodulin-activated kinase, which inactivates cytostatic factor, allowing the anaphase-promoting factor to turn on and ubiquitinate cyclins and securin, which returns the cell cycle to interphase. Here we show that the calcium-activated protein phosphatase calcineurin is also important in this process. Calcineurin is transiently activated after adding Ca2+ to egg extracts, and inhibitors of calcineurin such as cyclosporin A (ref. 8) delay the destruction of cyclins, the global dephosphorylation of M-phase-specific phosphoproteins and the re-formation of a fully functional nuclear envelope. We found that a second wave of phosphatase activity directed at mitotic phosphoproteins appears after the spike of calcineurin activity. This activity disappeared the next time the extract entered M phase and reappeared at the end of mitosis. We surmise that inhibition of this second phosphatase activity is important in allowing cells to enter mitosis, and, conversely, that its activation is required for a timely return to interphase. Calcineurin is required to break the deep cell cycle arrest imposed by the Mos-MAP (mitogen-activated protein) kinase pathway, and we show that Fizzy/Cdc20, a key regulator of the anaphase-promoting factor, is an excellent substrate for this phosphatase.     

Protein kinase C injection into hippocampal pyramidal cells elicits features of long term potentiation

Long-term potentiation (LTP) in the hippocampus is an interesting example of synaptic plasticity because of its induction by physiological discharge rates and its long duration. Of the possible biochemical mechanisms that regulate prolonged changes in cell function, protein phosphorylation is a particularly attractive candidate. We have therefore examined the effect of intracellular injection of calcium/diacylglycerol-dependent protein kinase (protein kinase C (PKC] in CA1 pyramidal neurones in hippocampal slices. Injection of the active enzyme elicited long-lasting enhancement of synaptic transmission, similar to LTP, whereas inactivated kinase failed to do so. The observed changes included an increased amplitude of the excitatory post-synaptic potential (e.p.s.p.) and an increased probability of firing and a reduced latency of the associated actin potential.     

Long-term potentiation and NMDA receptors in rat visual cortex

In the hippocampus, which is phylogenetically older than the cerebral neocortex, high frequency stimulation of afferent pathways leads to long-term potentiation (LTP) of synaptic transmission. This use-dependent malleability is of considerable interest because it may serve as a substrate for memory processes. However, in the neocortex, whose involvement in learning is undisputed, attempts to demonstrate LTP have remained inconclusive. Here we use intracellular recording techniques to show that LTP can be induced by high frequency stimulation of the optic radiation in slices of the visual cortex of adult rats. We identify as a necessary prerequisite for the induction of LTP the activation of the membrane channel that is associated with the NMDA (N-methyl-D-aspartate) receptor. Selective blockade of this receptor system with DL-2-amino-5-phosphonovalerate consistently prevents LTP as in most hippocampal pathways. In most cortical neurons the activation of the NMDA mechanism and hence the induction of LTP in these experiments requires a concomitant reduction of GABAergic inhibition by low doses of the GABAA antagonist bicuculline. This indicates that in the neocortex the activation threshold of the NMDA-mechanism and consequently the susceptibility to LTP, are strongly influenced by inhibitory processes.     

Novel form of long-term potentiation produced by a K+ channel blocker in the hippocampus.

Long-term potentiation (LTP) of synaptic transmission in the hippocampus is a widely studied model of memory processes. In the CA1 region, LTP is triggered by the entry of Ca2+ through N-methyl-D-aspartate (NMDA) receptor channels and maintained by the activation of Ca2(+)-sensitive intracellular messengers. We now report that in CA1, a transient block by tetraethylammonium of IC, IM and the delayed rectifier (IK) produces a Ca2(+)-dependent NMDA-independent form of LTP. Our results suggest that this new form of LTP (referred as to LTPK) is induced by a transient enhanced release of glutamate which generates a depolarization by way of the non-NMDA receptors and the consequent activation of voltage-dependent Ca2+ channels.     

β-Adrenergic activation enhances NMDA-induced current in pyramidal cells of the basolateral nucleus of amygdala

NMDA receptor （NMDA-R） in the amygdala complex is critical for both long-term potentiation （LTP） and formation of conditioned fear memory. It is reported that activation of β-adrenoceptors （β-AR） in the amygdala facilitates LTP and enhances memory consolidation. The present study examined the regulatory effect of β-AR activation on NMDA-R mediated current in pyramidal cells of the basolateral nucleus of amygdala （BLA）, using whole-cell recording technique. Bath application of the β-AR agonist isoproterenol enhanced NMDA-induced current, and this facilitatory effect was blocked by co-administered propranolol, a β-AR antagonist. The facilitatory effect of isoproterenol on NMDA-induced current could not be induced when the protein kinase A （PKA） inhibitor Rp-cAMPs was added in electrode internal solution.The present results suggest that β-AR activation in the BLA could modulate NMDA-R activity directly and positively, probably via PKA.     

Temporally distinct pre- and post-synaptic mechanisms maintain long-term potentiation

Long-term potentiation (LTP) in the hippocampus is widely studied as the mechanisms involved in its induction and maintenance are believed to underlie fundamental properties of learning and memory in vertebrates. Most synapses that exhibit LTP use an excitatory amino-acid neurotransmitter that acts on two types of receptor, the N-methyl-D-aspartate (NMDA) and quisqualate receptors. The quisqualate receptor mediates the fast synaptic response evoked by low-frequency stimulation, whereas the NMDA receptor system is activated transiently by tetanic stimulation, leading to the induction of LTP. The events responsible for maintaining LTP once it is established are not known. We now demonstrate that the sensitivity of CA1 neurons in hippocampal slices to ionophoretically-applied quisqualate receptor ligands slowly increases following the induction of LTP. This provides direct evidence for a functional post-synaptic change and suggests that pre-synaptic mechanisms also contribute, but in a temporally distinct manner, to the maintenance of LTP.     

Persistent protein kinase activity underlying long-term potentiation

Long-term potentiation (LTP) of synaptic transmission in the hippocampus is a much-studied example of synaptic plasticity. Although the role of N-methyl-D-aspartate (NMDA) receptors in the induction of LTP is well established, the nature of the persistent signal underlying this synaptic enhancement is unclear. Involvement of protein phosphorylation in LTP has been widely proposed, with protein kinase C (PKC) and calcium-calmodulin kinase type II (CaMKII) as leading candidates. Here we test whether the persistent signal in LTP is an enduring phosphoester bond, a long-lived kinase activator, or a constitutively active protein kinase by using H-7, which inhibits activated protein kinases and sphingosine, which competes with activators of PKC (ref. 17) and CaMKII (ref. 18). H-7 suppressed established LTP, indicating that the synaptic potentiation is sustained by persistent protein kinase activity rather than a stably phosphorylated substrate. In contrast, sphingosine did not inhibit established LTP, although it was effective when applied before tetanic stimulation. This suggests that persistent kinase activity is not maintained by a long-lived activator, but is effectively constitutive. Surprisingly, the H-7 block of LTP was reversible; evidently, the kinase directly underlying LTP remains activated even though its catalytic activity is interrupted indicating that such kinase activity does not sustain itself simply through continual autophosphorylation (see refs 9, 13, 15).     

Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5

Recent work has shown that the hippocampus contains a class of receptors for the excitatory amino acid glutamate that are activated by N-methyl-D-aspartate (NMDA) and that exhibit a peculiar dependency on membrane voltage in becoming active only on depolarization. Blockade of these sites with the drug aminophosphonovaleric acid (AP5) does not detectably affect synaptic transmission in the hippocampus, but prevents the induction of hippocampal long-term potentiation (LTP) following brief high-frequency stimulation. We now report that chronic intraventricular infusion of D,L-AP5 causes a selective impairment of place learning, which is highly sensitive to hippocampal damage, without affecting visual discrimination learning, which is not. The L-isomer of AP5 did not produce behavioural effects. AP5 treatment also suppressed LTP in vivo. These results suggest that NMDA receptors are involved in spatial learning, and add support to the hypothesis that LTP is involved in some, but not all, forms of learning.     

GABA autoreceptors regulate the induction of LTP.

Understanding the mechanisms involved in long-term potentiation (LTP) should provide insights into the cellular and molecular basis of learning and memory in vertebrates. It has been established that in the CA1 region of the hippocampus the induction of LTP requires the transient activation of the N-methyl-D-aspartate (NMDA) receptor system. During low-frequency transmission, significant activation of this system is prevented by gamma-aminobutyric acid (GABA) mediated synaptic inhibition which hyperpolarizes neurons into a region where NMDA receptor-operated channels are substantially blocked by Mg2+ (refs. 5, 6). But during high-frequency transmission, mechanisms are evoked that provide sufficient depolarization of the postsynaptic membrane to reduce this block and thereby permit the induction of LTP. We now report that this critical depolarization is enabled because during high-frequency transmission GABA depresses its own release by an action on GABAB autoreceptors, which permits sufficient NMDA receptor activation for the induction of LTP. These findings demonstrate a role for GABAB receptors in synaptic plasticity.