Slowly developing depression of N-methyl-D-aspartate receptor mediated responses in young rat hippocampi

BACKGROUND: Activation of N-methyl-D-aspartate (NMDA) type glutamate receptors is essential in triggering various forms of synaptic plasticity. A critical issue is to what extent such plasticity involves persistent changes of glutamate receptor subtypes and many prior studies have suggested a main role for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors in mediating the effect. Our previous work in hippocampal slices revealed that, under pharmacological unblocking of NMDA receptors, both AMPA and NMDA receptor mediated responses undergo a slowly developing depression. In the present study we have further adressed this phenomenon, focusing on the contribution via NMDA receptors. Pharmacologically isolated NMDA receptor mediated excitatory postsynaptic potentials (EPSPs) were recorded for two independent synaptic pathways in CA1 area using perfusion with low Mg(2+ )(0.1 mM) to unblock NMDA receptors. RESULTS: Following unblocking of NMDA receptors, there was a gradual decline of NMDA receptor mediated EPSPs for 2–3 hours towards a stable level of ca. 60–70 % of the maximal size. If such an experimental session was repeated twice in the same pathway with a period of NMDA receptor blockade in between, the depression attained in the first session was still evident in the second one and no further decay occurred. The persistency of the depression was also validated by comparison between pathways. It was found that the responses of a control pathway, unstimulated in the first session of receptor unblocking, behaved as novel responses when tested in association with the depressed pathway under the second session. In similar experiments, but with AP5 present during the first session, there was no subsequent difference between NMDA EPSPs. CONCLUSIONS: Our findings show that merely evoking NMDA receptor mediated responses results in a depression which is input specific, induced via NMDA receptor activation, and is maintained for several hours through periods of receptor blockade. The similarity to key features of long-term depression and long-term potentiation suggests a possible relation to these phenomena. Additionally, a short term potentiation and decay (<5 min) were observed during sudden start of NMDA receptor activation supporting the idea that NMDA receptor mediated responses are highly plastic

Spatial characterization of a multifunctional pipette for drug delivery in hippocampal brain slices

Background: Among the various fluidic control technologies, microfluidic devices are becoming powerful tools for pharmacological studies using brain slices, since these devices overcome traditional limitations of conventional submerged slice chambers, leading to better spatiotemporal control over delivery of drugs to specific regions in the slices. However, microfluidic devices are not yet fully optimized for such studies. New method: We have recently developed a multifunctional pipette (MFP), a free standing hydrodynamically confined microfluidic device, which provides improved spatiotemporal control over drug delivery to biological tissues. Results: We demonstrate herein the ability of the MFP to selectively perfuse one dendritic layer in the CA1 region of hippocampus with CNQX, an AMPA receptor antagonist, while not affecting the other layers in this region. Our experiments also illustrate the essential role of hydrodynamic confinement in sharpening the spatial selectivity in brain slice experiments. Concentration-response measurements revealed that the ability of the MFP to control local drug concentration is comparable with that of whole slice perfusion, while in comparison the required amounts of active compounds can be reduced by several orders of magnitude. Comparison with existing method: The multifunctional pipette is applied with an angle, which, compared to other hydrodynamically confined microfluidic devices, provides more accessible space for other probing and imaging techniques. Conclusions: Using the MFP it will be possible to study selected regions of brain slices, integrated with various imaging and probing techniques, without affecting the other parts of the slices

Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity-0

<p><b>Copyright information:</b></p><p>Taken from "Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity"</p><p>http://www.biomedcentral.com/1471-2202/8/55</p><p>BMC Neuroscience 2007;8():55-55.</p><p>Published online 26 Jul 2007</p><p>PMCID:PMC1959237.</p><p></p>f EPSPs, followed by recovery and stabilization (n = 15). (B) NR2B inhibitors, Ro25-6981 (0.5 μM) or Ifenprodil (3 μM), do not affect NMDA-induced plasticity (n = 8+4 = 12). (C) In presence of NR2A inhibitor, NVP-AAM077 (0.4 μM), NMDA effects are different compared with control situation, with a shorter recovery phase and much less depression (n = 10). (D) Bar diagram reveals LTD at 60 min after NMDA application as percentage of the initial baseline under the different experimental conditions in A-C. Data are given as mean ± S.E.M. Black bars in A-C indicate the duration of drug treatment. Values are shown averaged for 2 min periods. Inserts illustrate EPSP-traces taken at the indicated time points (a, b). Calibrations: 0.5 mV, 10 ms

Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity-6

<p><b>Copyright information:</b></p><p>Taken from "Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity"</p><p>http://www.biomedcentral.com/1471-2202/8/55</p><p>BMC Neuroscience 2007;8():55-55.</p><p>Published online 26 Jul 2007</p><p>PMCID:PMC1959237.</p><p></p>ing Mgconcentration (0.1 instead of 1.3 mM; n = 6). (B) Bar diagram shows NMDA-induced LTD in different Mgsolutions. Dashed bar is control data from Fig. 1D. (C) In a "slow LTD" experiment, similar to that in Fig. 3C, lowering the concentration of Mgin the perfusion solution (0.01 instead of 0.1 mM) helps with the induction of "slow LTD" in presence of NVP-AAM077 (0.4 μM; n = 7). (D) Bar diagram reveals the "slow LTD" induced in different Mgsolutions. Dashed bar is control data from Fig. 3D. (E) Similarly, using low Mgsolution (0.1 instead of 1.3 mM) in LTP experiments (compare Fig. 4C) enables a small potentiation of EPSPs under inhibition of NR2A subunits by NVP-AAM077 (n = 7). (F) Bar diagram shows LTP induced in different Mgsolutions. Dashed bar is control data from Fig. 4D. Data are shown as mean ± SEM. Inserts show the EPSP-traces taken at the indicated time points (a-d). Calibrations: 0.5 mV (A, E), 0.2 mV (C), 20 ms

Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity-2

<p><b>Copyright information:</b></p><p>Taken from "Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity"</p><p>http://www.biomedcentral.com/1471-2202/8/55</p><p>BMC Neuroscience 2007;8():55-55.</p><p>Published online 26 Jul 2007</p><p>PMCID:PMC1959237.</p><p></p>on. The experiment consists of preinduction baseline level in presence of a high concentration of AP5 (50 μM), induction period in AP5-free solution, and established LTD after reintroduction of high AP5. AMPA component (black symbols) and NMDA component (gray symbols) are plotted as functions of time, each point indicating the average in 2 min (n = 8 experiments). (B) A similar result is obtained in the same type of experiment but treated with Ro25-6981 (0.5 μM; n = 8) during the induction period. (C) The "slow LTD" is substantially blocked when NVP-AAM077 (0.4 μM; n = 7) is present under induction in AP5-free solution. (D) Bar diagram shows the "slow LTD" at 30 min after reintroducing AP5, plotted as percentage of the preinduction level under the three different experimental situations in A-C. Data are mean ± S.E.M. Black bars in A-C indicate the duration of drug treatment. Inserts show the EPSP-traces taken at the indicated time points (a-d) and the timing of the measurements for AMPA and NMDA components (0–1.5 ms after fiber volley and at 35–45 ms, respectively; see bars below traces). Calibrations: 0.2 mV, 20 ms

Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity-7

<p><b>Copyright information:</b></p><p>Taken from "Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity"</p><p>http://www.biomedcentral.com/1471-2202/8/55</p><p>BMC Neuroscience 2007;8():55-55.</p><p>Published online 26 Jul 2007</p><p>PMCID:PMC1959237.</p><p></p>f EPSPs, followed by recovery and stabilization (n = 15). (B) NR2B inhibitors, Ro25-6981 (0.5 μM) or Ifenprodil (3 μM), do not affect NMDA-induced plasticity (n = 8+4 = 12). (C) In presence of NR2A inhibitor, NVP-AAM077 (0.4 μM), NMDA effects are different compared with control situation, with a shorter recovery phase and much less depression (n = 10). (D) Bar diagram reveals LTD at 60 min after NMDA application as percentage of the initial baseline under the different experimental conditions in A-C. Data are given as mean ± S.E.M. Black bars in A-C indicate the duration of drug treatment. Values are shown averaged for 2 min periods. Inserts illustrate EPSP-traces taken at the indicated time points (a, b). Calibrations: 0.5 mV, 10 ms

Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity-1

<p><b>Copyright information:</b></p><p>Taken from "Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity"</p><p>http://www.biomedcentral.com/1471-2202/8/55</p><p>BMC Neuroscience 2007;8():55-55.</p><p>Published online 26 Jul 2007</p><p>PMCID:PMC1959237.</p><p></p>g baseline responses, defining the 100% level (1), applying NR2A inhibitor NVP-AAM077 (0.4 μM) leads to a substantial reduction of the isolated NMDA response (2). Adding NR2B inhibitor Ro25-6981 (0.5 μM) further depresses the responses down to near zero (3). Subsequent perfusion with AP5 (50 μM) fully blocks the synaptic responses and values obtained in this solution are taken as zero level (4). (ii) Traces 1–3 plotted together after subtraction of the zero level. (iii) Bar diagram quantifying the reductions of NMDA-EPSPs after sequentially adding the two subunit-specific blockers, NVP-AAM077 and Ro25-6981. (B) Similar plots for another set of experiments where the two blockers were applied in a different order (n = 6). The bar diagrams show data as mean ± S.E.M. Calibrations: 0.1 mV, 20 ms

Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity-4

<p><b>Copyright information:</b></p><p>Taken from "Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity"</p><p>http://www.biomedcentral.com/1471-2202/8/55</p><p>BMC Neuroscience 2007;8():55-55.</p><p>Published online 26 Jul 2007</p><p>PMCID:PMC1959237.</p><p></p>ear doubling of the EPSP. Subsequent application of NMDA (30 μM, 4 min) leads to persistent depression of the control pathway (gray symbols) and depotentiation of the test pathway (black symbols). Secondary tetanization of the test pathway causes a repotentiation, lifting the test responses back to a potentiated level (n = 12). (B) and (C) show similar experiments but treated with Ro25-6981 (0.5 μM; n = 4)/Ifenprodil (3 μM; n = 2) (total n = 6) or NVP-AAM077 (0.4 μM; n = 5) shortly after LTP induction. Both depotentiation and repotentiation are preferentially attenuated by NVP-AAM077 as compared to Ro25-6981/Ifenprodil. Arrows indicate the tetani. Black bars indicate the duration of drug application