Thomas Behnisch

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+]imeasurements 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)-α-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+]iresponse. 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.

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 Alzheimers disease, our finding suggests that changes in the sensitivity to apamin may result in memory disorders.

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.

LTP in cultured hippocampal-entorhinal cortex slices from young adult (P25-30) rats.

Cultured hippocampal neurons and immature organotypic slice cultures overcome temporal limitations of acute hippocampal slices and have been useful for investigating long-lasting plasticity. Difficulties with culturing adult neurons have restricted such studies to preparations from embryonic, perinatal, and juvenile tissue. By improving the methods for culturing and maintaining hippocampal-entorhinal cortex slices obtained from mature rats (P25-30), we show that their use in long-term electrophysiological investigations is feasible. Our cultured slices maintained an intact and functional trisynaptic cascade, normal synaptic function, and reliable long-term recording stability for at least 14 days in vitro. The electrophysiological properties and, in particular, the induction of long-term potentiation (LTP) in our mature organotypic slices were highly sensitive to dissection and tissue culture techniques. We present data describing the extracellular stimulation requirements for LTP-induction and its long-lasting maintenance (>4 h) at the Schaffer-collateral-CA1 synapse, and show that such changes in synaptic efficiency are NMDA receptor dependent. Our hippocampal-entorhinal cortex cultures from mature tissue can retain the electrophysiological properties required for long-term plasticity for several weeks in vitro.

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 μM) 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 μM) 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. ω‐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.

HIV secreted protein Tat prevents long-term potentiation in the hippocampal CA1 region

HIV-associated dementia (HAD) is a complication of advanced HIV disease. Both viral products and host cytokines are believed to be involved in the pathogenesis of HIV-associated neurological manifestations. Among the viral products released by HIV-infected cells is the soluble protein Tat. We investigated the effect of exposure of organotypic hippocampal slices to 100 nM recombinant Tat 1-86 on long-term potentiation (LTP) of field excitatory postsynaptic potential (fEPSP) at Schaffer collateral/commissural fiber-CA1 synapses. Exposure to Tat 1-86 prevented the induction of LTP without affecting post-tetanic potentiation. Tat 1-72delta31-61, which lacks the neurotoxic domain of Tat, had no significant effect on LTP. Tats ability to disrupt synaptic plasticity may be relevant to the pathogenesis of the cognitive impairments seen in patients with HIV disease.

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.

Hippocampal long-term potentiation: transient increase but no persistent translocation of protein kinase C isoenzymes α and β

Abstract Using a monoclonal antibody the translocation of the Ca 2+ -dependent protein kinase C (PKC) isoenzymes α/β 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 α/β 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 α/β 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 α/β 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 α/β 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.

High frequency stimulation-induced dendritic calcium waves in rat hippocampal neurons

An activity-dependent intracellular Ca(2+) increase represents a key signal for the activation of mechanisms involved in synaptic long-term plasticity. Here we present data from confocal microscopic imaging in conjunction with electrophysiological studies, that local 100 Hz stimulations of the hippocampal Schaffer collateral fibers, usually used for the induction of synaptic long-term potentiation (LTP), elicits an intradendritic Ca(2+) rise, that propagates as a short-distance wave within dendrites of CA1-neurons, which could be involved in triggering the dendritic (local signal cascade) machinery of LTP generation. Pharmacological investigations elucidated the coincidental involvement of N-methyl-D-aspartate- and of group I metabotropic glutamate receptors in the generation of the Ca(2+) wave.

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.