Protein degradation by the proteasome is required for synaptic tagging and the heterosynaptic stabilization of hippocampal late-phase long-term potentiation.

Activity-dependent regulation of synaptic efficacy is believed to underlie learning and memory formation. Here we show that protein degradation by the proteasome is required for the induction of the protein synthesis-dependent late-phase of long-term potentiation (late-LTP) but not for its maintenance. Proteasome activity was also key to the polarity of heterosynaptic interactions between synapses expressing synaptic plasticity and newly activated synapses. In fact, proteasome activity was required for the consolidation of an otherwise transient potentiation (early-LTP) into late-LTP by strong tetanization of a separate afferent pathway both in the "weak-before-strong" and in the "strong-before-weak" two-pathway paradigms [Frey and Morris (1997) Nature 385:533-536; Frey and Morris (1998) Neuropharmacology 37:545-552], suggesting that proteasome activity plays a role in the synaptic tagging and capture of plasticity-related proteins at stimulated synapses. Additionally, proteasome inhibition abrogated immunity against heterosynaptic depotentiation of an established late-LTP when applied during weak tetanic stimulation in the "strong-before-weak" two-pathway paradigm. Such a heterosynaptic destabilizing effect of proteasome inhibition was abolished by concomitant inhibition of N-methyl-d-aspartate (NMDA) receptors, suggesting that it is an active process. Together, these results indicate that the proteasome plays important roles in the establishment of late-LTP and in the preservation of potentiated synapses when a subsequent synaptic plasticity is induced within the same neuronal population.

The effects of ladasten on dopaminergic neurotransmission and hippocampal synaptic plasticity in rats.

Derivatives of adamantane, like memantine, are potentially neuroprotective drugs for the favourable care of Alzheimer's and Parkinson's diseases. A further adamantane derivate is N-(2-adamantyl)-N-(para-bromophenyl)-amine (ladasten) which is capable to modulate animal performance in different learning paradigms. To clarify if some of those behavioural alterations are mediated by modulation of catecholamine syntheses we studied the effects of single administration of ladasten (50 mg/kg, per os) on catecholamines' biosynthesis in the ventral tegmental area, nucleus accumbens, hypothalamus, striatum and hippocampus. We found that ladasten differentially regulates tyrosine hydroxylase mRNA and protein as well as dopamine and L-DOPA content. We then investigated the effects of ladasten on activity-dependent hippocampal synaptic plasticity in vitro and found that application of 10 microM ladasten transforms short-term potentiation of synaptic transmission to a long-lasting form. A transformation of short-term into long-term potentiation was also observed, when ladasten was applied 40 min after a single 100 Hz 200 ms tetanization. This reinforcement was blocked by the protein synthesis inhibitor anisomycin and could be attenuated by the D1/D5 receptor antagonist SCH23390. These results suggest that ladasten induces reinforcement of short-term potentiation via protein synthesis and dopamine dependent mechanisms.

Involvement of multiple phosphatidylinositol 3-kinase-dependent pathways in the persistence of late-phase long term potentiation expression.

The mechanisms responsible for the stabilization and persistence of synaptic plasticity remain largely unknown. In this study, we investigated the time course of the dependence of late-phase long term potentiation of field excitatory post-synaptic potential on phosphatidylinositol 3-kinase and its downstream effectors mTOR and AKT. In agreement with our previous results obtained on an early-phase long-term potentiation paradigm we observed that application of a nanomolar concentration of wortmannin (100 nM) 1 h after late-phase long term potentiation induction reversed potentiation completely. However, application of wortmannin 4 h after late-phase long term potentiation induction resulted in a more limited reduction of field excitatory post-synaptic potential suggesting that the dependence of late-phase long term potentiation expression on phosphatidylinositol 3-kinase decreases over time. Application of a nanomolar concentration of rapamycin (200 nM) during the tetanization paradigm prevented the induction of late-phase long term potentiation consistent with our earlier results. Application of rapamycin 1 h after late-phase long term potentiation induction resulted in a less pronounced though significant decline of field excitatory post-synaptic potential. Immunohistological analysis demonstrated that the concentration of rapamycin used was effective in inhibiting the phosphorylation of p70S6K at Thr389, the main determinant of its pro-translational activity, and that Thr389 phosphorylation recovered after washout. Lastly, a transient application of Akt inhibitor I (10 microM) one hour after late-phase long term potentiation induction also induced a partial although significant reduction of potentiated field excitatory post-synaptic potential that stabilized at a level of approximately 114% of baseline three hours after application, suggesting that AKT also contributes to the stabilization of late-phase long term potentiation expression. These results confirm and extend previous observations that the expression of long term potentiation in the CA1 of rat hippocampus involves several elements of the phosphatidylinositol 3-kinase signaling pathway.

Single cell analysis of activity-dependent cyclic AMP-responsive element-binding protein phosphorylation during long-lasting long-term potentiation in area CA1 of mature rat hippocampal-organotypic cultures.

Phosphorylation of the transcription factor cyclic AMP (cAMP)-response element-binding protein (CREB) has been implicated in long-term synaptic plasticity and memory, and its activation has been proposed to be required for the maintenance of long-term potentiation (LTP). The previously described temporal dynamics of CREB phosphorylation during the maintenance of LTP showed differences between experimental models. In the present study the level of CREB phosphorylation was evaluated in organotypic hippocampal slices from young adult rats (P25-30) after long-lasting LTP was induced. Immunohistochemistry and confocal imaging were used to determine the ratio between non-phosphorylated and phosphorylated CREB at a single cell resolution, revealing not only the temporal dynamics but also the extent of CREB phosphorylation. The activation of CREB after LTP-induction was compared with cAMP-activation after bath application of forskolin. An increase in cAMP by forskolin resulted in a persistent, uniform increase of the phosphorylated CREB (pCREB/CREB immunofluorescence ratio) in all hippocampal principal neurons. In contrast, the induction of long-lasting LTP in CA1 was accompanied by a local increase in the pCREB/CREB ratio. Both CREB activation and LTP induction in mature cultured slices required N-methyl-D-aspartate (NMDA) receptor activation. CREB phosphorylation continued to increase for 4 h during LTP maintenance. This sustained activation is in contrast to previous observations in acutely prepared slices and supports the hypothesis that CREB plays an important role during the late phases of LTP.

A specific role for group I mGluRs in hippocampal LTP and hippocampus-dependent spatial learning.

Metabotropic glutamate receptors (mGluRs) have been implicated in long-term potentiation and in learning and memory formation. In this study, we tested the effects of group I mGluR inhibition on synaptic plasticity and learning of rats at different levels of organization (1) in the hippocampal slice preparation; (2) in freely moving animals implanted with chronic hippocampal electrodes; and (3) in different spatial learning paradigms. To allow a direct comparison of the effects obtained the same doses were used in all paradigms. Bath-application of the selective group I mGluR antagonist (S)4-carboxyphenylglycine (4-CPG) impaired a decremental long-term potentiation (LTP) induced by a weak tetanization paradigm, but failed to affect a robust LTP generated by strong tetanization. In contrast, 4-CPG impaired a robust LTP in freely moving animals if applied 30 min before tetanization. The same dose of 4-CPG only impeded spatial learning mildly in the eight-arm radial maze and had no effect on a simple configuration of the Y-maze spatial alternation task. In the more difficult configuration of this task, however, 4-CPG caused complete amnesia. The lack of state-dependent 4-CPG actions and the absence of any 4-CPG effects in the open-field test classify the obtained retention deficit as a selective impairment of memory storage. Our results indicate a specific role of group I mGluRs in certain types of synaptic plasticity and of spatial learning.

Inositol 1,3,4,5-tetrakisphosphate enhances long-term potentiation by regulating Ca2+ entry in rat hippocampus.

1. The effect of inositol 1,3,4,5-tetrakisphosphate (InsP4) on long-term potentiation (LTP) was investigated in the CA1 region of rat hippocampal slices. Intracellular application of InsP4 and EPSP recordings were carried out using the whole-cell configuration. 2. Induction of LTP in the presence of InsP4 (100 microM) resulted in a substantial enhancement of the LTP magnitude compared with control potentiation. Using an intrapipette perfusion system, it was established that application of InsP4 was required during induction of potentiation for this enhancement to occur. An enhancement of LTP was not observed if a non-metabolizable inositol 1,4,5-trisphosphate (InsP3) analogue (2,3-dideoxy-1,4,5-trisphosphate, 100 microM) was applied intracellularly. 3. Current-voltage relations of NMDA receptor-mediated EPSCs were not altered by InsP4 application. The presence of InsP4 was slightly effective in relieving a D-(-)-2-amino-5-phosphonopentanoic acid (D-APV)-induced block of LTP. 4. The peak current amplitude of voltage-gated calcium channels (VGCCs) was increased by InsP4. omega-Conotoxin GVIA inhibited the InsP4-induced LTP facilitation. 5. These data indicate that InsP4 can modify the extracellular Ca2+ entry through upregulation of VGCCs, which may in turn contribute to the observed enhancement of LTP induced by InsP4. 6. To investigate the possible involvement of intracellular Ca2+ release in the facilitatory effect of InsP4 on LTP, different inhibitors of the endoplasmic reticulum-dependent Ca2+ release were applied (heparin, ryanodine, cyclopiazonic acid). The results suggest that InsP4 activates postsynaptic InsP3-dependent Ca2+ release which normally does not contribute to the calcium-induced calcium release-dependent LTP.

ACPD-mediated slow-onset potentiation is associated with cell death in the rat CA1 region in vivo.

Slow-onset potentiation of synaptic transmission in the hippocampus in vitro and in vivo is induced by application of the metabotropic glutamate receptor (mGluR) agonist, 1S,3R-amino cyclopentane 2,3-dicarboxylic acid (ACPD). This study investigated the cellular response in the CA1 region of freely moving rats to ACPD application. Drugs were applied via the lateral cerebral ventricle, and measurements were obtained from the CA1 region via permanently implanted electrodes. ACPD (20 nmol/5 microl) produced a dose-dependent slow-onset potentiation in the CA1 region which lasted over 4 h. Histological evaluation at either 4 h or 7 days following ACPD-injection indicated that slow-onset potentiation was associated with gradual but marked cell death in the CA1 region. Whereas 20 nmol ACPD produced significant CAI neurotoxicity, concentrations which did not induce potentiation had little or no neurotoxic effect. Both the general mGluR antagonist R,S-alpha-methyl-carboxyphenylglycine (1 micromol/5 microl), and the group 1 mGluR antagonist (S)-4-carboxyphenylglycine (4CPG, 100 nmol/5 microl) significantly inhibited ACPD-induced neuropathology. In addition, 4CPG inhibited the expression of ACPD-mediated slow-onset potentiation. These results confirm previous findings that in the CA1 region in vivo, slow-onset potentiation is mediated group 1 mGluRs, and indicate that slow-onset potentiation may involve pathological processes.

Inhibition of apamin-sensitive calcium dependent potassium channels facilitate the induction of long-term potentiation in the CA1 region of rat hippocampus in vitro.

Using field potential recording in the CA1-region of rat hippocampal slices we investigated the effect of apamin; a specific antagonist of small conductive calcium activated potassium channels on long-term potentiation (LTP). The experiments revealed that LTP of excitatory postsynaptic potentials induced by a single 100 Hz tetanization was intensified by extracellular application of apamin in a concentration range of 1-200 nM. No effects of apamin on LTP induced by triple 100 Hz tetanization were seen. We conclude that the positive modulation of LTP by apamin is effective in a nanomolar concentration range and dependent upon the employed tetanization. Because it has been shown that apamin-binding sites are affected by learning disorders including Alzheimer's disease, our finding suggests that changes in the sensitivity to apamin may result in memory disorders.

The role of group II metabotropic glutamate receptors in hippocampal CA1 long-term potentiation in vitro.

The role of group II metabotropic glutamate receptors (mGlu receptors) in mechanisms of long-term potentiation was investigated by analysis of excitatory postsynaptic field potentials of the CA1 region in rat hippocampal slices. The application of the group II agonists (2S,1'S,2'S)-2-(carboxycyclopropyl) glycine (L-CCG-I) and (2S,1'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl) glycine (DCG IV) resulted in a dose-dependent reduction of long term potentiation in the concentration range 3-50 microM. In contrast to the effects of group II agonists on long-term potentiation, the group II antagonists (RS)-alpha-methyl-3-carboxy-4-hydroxy-phenylglycine (M3C4HPG) and (RS)-alpha-methylserine-O-phosphate monophenyl ester (MSOPPE) elicited a dose-dependent enhancement of long-term potentiation (50-100 microM or 20-50 microM, respectively). We conclude that group II mGlu receptors are not essential for the induction of long-term potentiation; however, they may be involved in feedback mechanisms in long-term potentiation.

When are class I metabotropic glutamate receptors necessary for long-term potentiation?

The involvement of metabotropic glutamate receptors (mGluRs) in hippocampal long-term potentiation (LTP) is a matter of controversial debate. Using [Ca2+]i measurements by confocal laser scanning microscopy and field recordings of EPSPs (fEPSPs) in the hippocampal CA1-region, we found that the efficacy of the broad-spectrum mGluR-antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG) and of (S)-4-carboxy-phenylglycine (4-CPG), a selective antagonist at class I mGluRs, in LTP is contingent on the tetanization strength and the resulting [Ca2+]i response. As indicated by experiments in which we blocked voltage-dependent calcium channels (VDCCs) and intracellular Ca2+ stores (ICSs), the functional significance of class I mGluRs in LTP is confined to certain types of potentiation, which are induced by weak tetanization protocols and require the release of Ca2+ from ICSs for induction. During strong tetanic stimulation, this Ca2+ source is functionally bypassed by activating VDCCs.

Pharmacological characterisation of metabotropic glutamatergic and purinergic receptors linked to Ca2+ signalling in hippocampal astrocytes.

Intracellular Ca2+ ([Ca2+]i) signals induced by metabotropic glutamate receptor (mGluR) agonists and by purinergic agonists in cultured hippocampal astrocytes were investigated using [Ca2+]-sensitive fluorophores. The mGluR agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) and (R,S)-3,5-dihydroxyphenylglycine (DHPG) induced [Ca2+]i responses in 76 and 93% of the cells, respectively. The broad-spectrum mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) and the mGluR1 antagonists (S)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) and (S)-4-carboxyphenylglycine (4CPG) suppressed the agonist-evoked [Ca2+]i response in about 25% of the cells completely and in about 60% partially, depending on the agonist concentration employed. Together with immunohistochemical receptor localisations these results suggest the presence of at least two subpopulations of class I mGluRs recruited from the truncated splice variants of mGluR1 (mGluR 1b, 1c, 1d) and/or hitherto unknown glial-specific class I mGluRs. Of the hippocampal astrocytes 88, 92 or 83% of the cells responded with a [Ca2+]i elevation (mostly oscillations) to application of ATP, ADP, or 2-methylthio-ATP (2-MeS-ATP), respectively, whereas only 14 and 5% responded to AMP and adenosine, respectively, indicating the predominance of P2 receptors. The ATP-induced [Ca2+]i signal was suppressed by suramin. Release of Ca2+ from intracellular stores was involved in the response to ATP because the cells also exhibited [Ca2+]i elevations in Ca2+-free medium. Cells did not respond to 10 microM UTP. We conclude that the P2Y subtype represents the main [Ca2+]i-linked purinoceptor in hippocampal astrocytes. Sequential application of ATP and DHPG in Ca-free medium showed that metabotropic glutamate and purinergic receptors initiate release of Ca2+ from subsets of cyclopiazonic acid-sensitive Ca2+ stores which are partly independent.

Interaction between paired-pulse facilitation and long-term potentiation of minimal excitatory postsynaptic potentials in rat hippocampal slices: a patch-clamp study.

Long-term potentiation is an experimental paradigm used to study synaptic plasticity and memory mechanisms. One similarity between long-term potentiation and memory is the existence of several distinct phases. However, our preliminary quantal analysis did not reveal essential differences in expression mechanisms of the early (< 1 h) and later (up to 3 h) phases of long-term potentiation. The data were compatible with presynaptic mechanisms of both phases. Another approach to distinguish between presynaptic and postsynaptic mechanisms is analysis of interaction between long-term potentiation and presynaptic paired-pulse facilitation. Such analysis had been previously done mainly with recordings of field potentials reflecting the activity of large neuronal populations. Only the early potentiation phase had been previously analysed with recordings from single neurons. The results from different groups were contradictory. In the present study, minimal excitatory postsynaptic potentials were recorded from CA1 pyramidal neurons of rat hippocampal slices. Paired-pulse facilitation ratios were calculated for various periods (up to 2-3 h) following induction of long-term potentiation. The ratio persistently decreased in the majority of neurons following long-term potentiation induction. The decrease in the paired-pulse facilitation ratio correlated with the magnitude of long-term potentiation and with the initial (pretetanic) facilitation ratio. Therefore, the general results of the present analysis was similar with the results of the quantal analysis: it is consistent with a strong involvement of presynaptic mechanisms in maintenance of both early and late phases of long-term potentiation. However, individual neurons could show variable changes in the paired-pulse facilitation, e.g., increases at late (> 0.5-1 h) periods after tetanus. Calculations of partial correlations and regression analysis indicated that positive correlation between potentiation magnitude and initial (pretetanic) paired-pulse facilitation tended to increase in the late potentiation phase (1.5-2.5 h post-tetanus) indicating that different mechanisms are involved in the early (0.5 h post-tetanus) and the late phase of long-term potentiation. The findings are compatible with involvement of presynaptic mechanisms in both the early and late phases of long-term potentiation. However, the results suggest that contribution of changes in release probability and in effective number of transmitter release sites may differ during the two phases. It is suggested that activation of silent synapses and increases in the number of transmission zones due to pre- and postsynaptic structural rearrangements represent important mechanisms of the late phase of long-term potentiation.

Principal component analysis of minimal excitatory postsynaptic potentials.

'Minimal' excitatory postsynaptic potentials (EPSPs) are often recorded from central neurones, specifically for quantal analysis. However the EPSPs may emerge from activation of several fibres or transmission sites so that formal quantal analysis may give false results. Here we extended application of the principal component analysis (PCA) to minimal EPSPs. We tested a PCA algorithm and a new graphical 'alignment' procedure against both simulated data and hippocampal EPSPs. Minimal EPSPs were recorded before and up to 3.5 h following induction of long-term potentiation (LTP) in CA1 neurones. In 29 out of 45 EPSPs, two (N=22) or three (N=7) components were detected which differed in latencies, rise time (Trise) or both. The detected differences ranged from 0.6 to 7.8 ms for the latency and from 1.6-9 ms for Trise. Different components behaved differently following LTP induction. Cases were found when one component was potentiated immediately after tetanus whereas the other with a delay of 15-60 min. The immediately potentiated component could decline in 1-2 h so that the two components contributed differently into early (< 1 h) LTP1 and later (1-4 h) LTP2 phases. The noise deconvolution techniques was applied to both conventional EPSP amplitudes and scores of separate components. Cases are illustrated when quantal size (upsilon) estimated from the EPSP amplitudes increased whereas upsilon estimated from the component scores was stable during LTP1. Analysis of component scores could show apparent double-fold increases in upsilon which are interpreted as reflections of synchronized quantal releases. In general, the results demonstrate PCA applicability to separate EPSPs into different components and its usefulness for precise analysis of synaptic transmission.

Analysis of the tetanic and post-tetanic components of intradendritic Ca2+ signals in hippocampal CA1 neurons.

The importance of both the activation of second messenger cascades and an increase of the intracellular Ca2+ concentration for the induction of synaptic plasticity in hippocampal CA1 neurons is well established. Using the dye Calcium Green-1, we analysed the Ca2+ increases evoked by different 100-Hz tetanization paradigms commonly used to induce long-term potentiation. We found that the normalized total area of fluorescence intensity changes (F/F0) was correlated with both the strength and the duration of tetanization. Furthermore, the normalized area of fluorescence intensity changes during the time of tetanization (tetanic component) correlated strongly in a linear manner with the tetanization duration. Moreover, the tetanic component strongly determined the area of the post-tetanic Ca2+ signal. Interestingly, the normalized relationship of the post-tetanic Ca2+ signal to the total Ca2+ change decreased with prolonged tetanizations. In contrast, with an increased stimulation strength, a positive correlation of the relationship of the post-tetanic component to the total amount Ca2+ could be obtained. The time constants of the Ca2+ extrusion depend linearly on both the tetanization duration and the amount of the tetanic Ca2+ signal. Our data demonstrate that augmenting the stimulation strength leads to a respective increase of the post-tetanic Ca2+ portion within the total Ca2+ signal, whereas a prolongation of the tetanization duration does not. Thus, no further significant prolongation of the Ca2+ signal occurs during increased durations of tetanization.

Semi-automated analysis of NMDA-mediated toxicity in digitised colour images from rat hippocampus.

The evaluation of neuronal cell survival after, for example, mechanical, hypoxic or drug-mediated injury requires the analysis of a high number of histological specimens. Since this is a time-consuming occupation, we have developed a semi-automated analysis routine for the determination of the distribution of live and dead cells. After digitalization of the histological preparations, 8-bit colour bitmaps were assessed using a compiled image-analysis programme of the software package Khoros. In the current study a detailed example of the application of this image-processing approach is described for the investigation of the cell survival after intraventricular application of N-methyl-D-aspartate (NMDA). The samples were prepared as fuchsin acid/toluidine blue stained hippocampal thin slices. The calculated areas of the live and dead cells were highly correlated with manual counts of live and dead cells in the 100 samples examined in this study. Twenty-four hours following NMDA-treatment animals (n = 5) were found to have significantly fewer live and more dead hippocampal cells than the saline-treated animals (n = 5), using either automated or manual examination techniques. The automated technique also revealed that NMDA treatment resulted in a reduction in the density of live cell distribution.

The neuronal calcium-sensor protein VILIP modulates cyclic AMP accumulation in stably transfected C6 glioma cells: amino-terminal myristoylation determines functional activity.

VILIP (visinin-like protein) is a member of the neuronal subfamily of EF-hand calcium sensor proteins. Members of this family are involved in the calcium-dependent regulation of the desensitization of signal cascades in retinal photoreceptors. To gain insight into the function of VILIP in cell signaling, we have transfected wild-type VILIP and mutant VILIP lacking the myristoylation consensus sequence into C6 glioma cells. Expression of wild-type VILIP did not significantly influence the desensitization of beta-adrenergic receptors, which are coupled to adenylyl cyclase in C6 cells. However, VILIP expression increased the beta-adrenergic receptor-stimulated cyclic AMP (cAMP) level in these cells severalfold. The stimulatory effect was also observed after direct stimulation of the adenylyl cyclase with forskolin, indicating that VILIP acts downstream of receptor and G protein in the beta-adrenergic signaling pathway in C6 cells. In contrast, the nonmyristoylated mutant of VILIP reduced cellular cAMP levels in C6 cells. Myristoylated wild-type VILIP was associated in a calcium-dependent manner with membrane fractions during subcellular fractionation, presumably owing to a calcium-myristoyl switch. In contrast, association of nonmyristoylated mutant VILIP with membranes was strongly reduced. Thus, myristoylation and most likely the calcium-dependent membrane association of VILIP are important prerequisites for the activating effect of wild-type VILIP on cAMP accumulation in C6 cells. These results suggest that VILIP acts as a calcium sensor molecule that modulates cell signaling cascades, possibly by direct or indirect regulation of adenylyl cyclase activity.

Noise deconvolution based on the L1-metric and decomposition of discrete distributions of postsynaptic responses.

A statistical approach to analysis of amplitude fluctuations of postsynaptic responses is described. This includes (1) using a L1-metric in the space of distribution functions for minimisation with application of linear programming methods to decompose amplitude distributions into a convolution of Gaussian and discrete distributions; (2) deconvolution of the resulting discrete distribution with determination of the release probabilities and the quantal amplitude for cases with a small number (< 5) of discrete components. The methods were tested against simulated data over a range of sample sizes and signal-to-noise ratios which mimicked those observed in physiological experiments. In computer simulation experiments, comparisons were made with other methods of 'unconstrained' (generalized) and constrained reconstruction of discrete components from convolutions. The simulation results provided additional criteria for improving the solutions to overcome 'over-fitting phenomena' and to constrain the number of components with small probabilities. Application of the programme to recordings from hippocampal neurones demonstrated its usefulness for the analysis of amplitude distributions of postsynaptic responses.

Metabotropic glutamate receptors are involved in TEA-induced long-term potentiation in area CA1 of the hippocampus.

A brief application of the K+ channel blocker tetraethylammonium induces a long-lasting potentiation in the CA1 region of hippocampal slices (TEA LTP). We report here that metabotropic glutamate receptors (mGluRs) contribute to this kind of synaptic enhancement, since the mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+) MCPG) inhibits TEA LTP with a concentration-dependent component.

Hippocampal long-term potentiation: transient increase but no persistent translocation of protein kinase C isoenzymes alpha and beta.

Using a monoclonal antibody the translocation of the Ca(2+)-dependent protein kinase C (PKC) isoenzymes alpha/beta was studied in hippocampal slices after stimulation of glutamate receptors or induction of long-term potentiation. In submerged slices preincubated for 60 min in a medium usually used in electrophysiological studies, cytosolic PKC was not detectable and the amount of membrane-associated enzyme was increased. The treatment of these slices with 10(-6) M phorbol-12,13-dibutyrate induced a time-dependent translocation of alpha/beta PKC from the membrane-associated into the membrane-inserted state. The glutamatergic agonists N-methyl-D-aspartate, quisqualate and trans-ACPD did not cause a membrane insertion of alpha/beta PKC as observed for the phorbol ester when applied alone or in combination. Furthermore, 2 min and 15 min after induction of LTP in the Schaffer collateral-CA1 pathway the distribution of alpha/beta PKC between the two membrane fractions remained unchanged. An increase in the total amount of PKC immunoreactivity was measured immediately after tetanization (142.6% of controls). The data suggest that a membrane insertion of alpha/beta PKC is not a prerequisite for the LTP-induced increased phosphorylation of PKC substrates and that the enzyme might be recruited from a previously inactive pool.