Double dissociation of spike timing-dependent potentiation and depression by subunit-preferring NMDA receptor antagonists in mouse barrel cortex

Abstract

Spike timing-dependent plasticity (STDP) is a strong candidate for an N-methyl-D-aspartate (NMDA) receptor-dependent form of synaptic plasticity that could underlie the development of receptive field properties in sensory neocortices. Whilst induction of timing-dependent long-term potentiation (t-LTP) requires postsynaptic NMDA receptors, timing-dependent long-term depression (t-LTD) requires the activation of presynaptic NMDA receptors at layer 4-to-layer 2/3 synapses in barrel cortex. Here we investigated the developmental profile of t-LTD at layer 4-to-layer 2/3 synapses of mouse barrel cortex and studied their NMDA receptor subunit dependence. Timing-dependent LTD emerged in the first postnatal week, was present during the second week and disappeared in the adult, whereas t-LTP persisted in adulthood. An antagonist at GluN2C/D subunit-containing NMDA receptors blocked t-LTD but not t-LTP. Conversely, a GluN2A subunit-preferring antagonist blocked t-LTP but not t-LTD. The GluN2C/D subunit requirement for t-LTD appears to be synapse specific, as GluN2C/D antagonists did not block t-LTD at horizontal cross-columnar layer 2/3-to-layer 2/3 synapses, which was blocked by a GluN2B antagonist instead. These data demonstrate an NMDA receptor subunit-dependent double dissociation of t-LTD and t-LTP mechanisms at layer 4-to-layer 2/3 synapses, and suggest that t-LTD is mediated by distinct molecular mechanisms at different synapses on the same postsynaptic neuron.

Figure 1.

Input-specific timing-dependent plasticity in mouse barrel cortex. (A) Light-microscopic view of thalamocortical slice showing positioning of stimulation electrode (S) and recording pipette (R). Scale bar, 100 μm. (B) Cytochrome oxidase-stained thalamocortical slice showing barrels (*) in layer 4. Scale bar, 150 μm. (C) Biocytin-filled pyramidal neuron in layer 2 stained with 1:1000 dilution of Streptavidin Alexa Fluor 594. Scale bar, 20 μm. (D) Schematic diagram of a layer 2/3 pyramidal neuron with patch pipette at the soma and an extracellular stimulation electrode in layer 4. (E) Diagram of pairing paradigm. (Ei) Post-before-pre pairing protocol induces t-LTD. Δt is the time between peak of spike and EPSP onset. (Eii) Pre-before-post pairing protocol induces t-LTP. Δt is the time between EPSP onset and peak of spike. (F) A post-before-pre pairing protocol induces t-LTD. EPSP slope monitored in paired experimental (downward black triangles) and unpaired control pathway (open circles). Inset, Traces show EPSP amplitude from a sample cell before 1) and 30 min after 2) post–pre pairing. (G) A pre-before-post pairing protocol induces robust t-LTP. EPSP slope monitored in paired experimental (upward black triangles) and unpaired control pathway (open circles). Stimulation electrode of unpaired pathway was placed in the same barrel column. Inset, Traces show EPSP amplitude from a sample cell before 1) and 30 min after 2) pre–post pairing. (H) Summary of results. Error bars are SEM. *P < 0.05, **P < 0.01, Student's t-test. The number of slices used for each protocol is indicated in parentheses at the top of each error bar.

Figure 2.

Developmental profile of timing-dependent LTD and timing-dependent LTP at layer 4-to-layer 2/3 synapses in mouse barrel cortex. Synaptic efficacy was monitored over time following post-before-pre single-spike pairing protocol in (A) P6–8, (B) P11–15, (C) P19–25 (black triangles), and (D) P25–42 animals (black circles). Developmental profile of timing-dependent LTP at layer 4-to-layer 2/3 synapses in mouse barrel cortex was observed using pre-before-post protocol. Synaptic efficacy was monitored over time following pre-before-post single-spike pairing protocol in (E) P19–45, and (F) P81–102 animals (black triangles). (G) Summary of results. Error bars are SEM. *P < 0.05, Student's t-test. The number of slices used for age group is indicated in parentheses at the top of each error bar.

Figure 3.

NMDA receptor dependence of timing-dependent plasticity in mouse barrel cortex. Control t-LTD and t-LTP (black triangles) were induced using a post-before-pre and a pre-before-post protocol, respectively, in P11–15 mice. Induction of both t-LTD (A) and t-LTP (B) was completely blocked following bath application of 50 μM D-AP5 (gray squares). (C) Summary of results. Error bars are SEM. *P < 0.05, **P < 0.01, Student's t-test. The number of slices for each condition is indicated in parentheses at the top of each error bar.

Figure 4.

GluN2A subunit dependence of timing-dependent LTP. (A–C) t-LTD induction following a post-before-pre pairing paradigm (A; black triangles) was unaffected by 100 nM NVP-AAM077 (gray triangles) in P11–15 mice, whereas t-LTP induction was blocked (B; gray squares). NVP-AAM077 also did not block t-LTD in P6–8 mice (C; gray triangles). (D) Summary of results. Error bars are SEM. *P < 0.05, Student's t-test. The number of slices used for each condition is indicated in parentheses at the top of each error bar.

Figure 5.

GluN2B subunit in timing-dependent plasticity. (A, B) Ro 25-6981 (0.5 μM) did not affect t-LTD (A; gray triangles) or t-LTP (B; gray triangles) in P11–15 mice. (C) Summary of results. Error bars are SEM. The number of slices used for each condition is indicated in parentheses at the top of each error bar.

Figure 6.

GluN2C/D subunit dependence of timing-dependent LTD. (A) PPDA (10 μM) blocked t-LTD following post-before-pre pairing in P11–15 mice (gray squares). (B) PPDA (10 μM) did not block t-LTP following pre-before-post pairing in P11–15 mice (gray triangles). (C, D) A more selective GluN2C/D blocker, UBP141, also blocked t-LTD (C; gray squares) in layer 4-to-layer 2/3 synapses but had no effect on t-LTP (D; gray triangles). (E) PPDA also blocked t-LTD in young, immature synapses (gray squares). (F) Summary of results. Error bars are SEM. **P < 0.01, Student's t-test. The number of slices used for each condition is indicated in parentheses at the top of each error bar.

Figure 7.

Different induction requirements for timing-dependent LTD at vertical layer 4-to-layer 2/3 and horizontal layer 2/3-to-layer 2/3 synapses. (A) t-LTD is blocked by 500 nM PPDA at layer 4-to-layer 2/3 synapses in P11–15 mice (gray squares). (B) t-LTD induction at layer 2/3-to-layer 2/3 synapses is unaffected by 10 μM PPDA (gray triangles) but is blocked by 0.5 μM Ro 25-6981 (dark gray squares). (C) Summary of results. Error bars are SEM. *P < 0.05, Student's t-test. The number of slices used for each condition is indicated in parentheses at the top of each error bar. Control t-LTD at layer 4-to-layer 2/3 synapses is the same data as presented in Figure 6A,F. (D, E) Preincubation and bath application of CB1 receptor antagonist AM251 did not block t-LTD at layer 4-to-layer 2/3 synapses (D; gray triangles), but blocked t-LTD at layer 2/3-to-layer 2/3 synapses (E; gray squares). (F) Summary of results. Error bars are SEM. **P < 0.01, Student's t-test. The number of slices used for each condition is indicated in parentheses at the top of each error bar.