Deprivation-induced synaptic depression by distinct mechanisms in different layers of mouse visual cortex

Abstract

Long-term depression (LTD) induced by low-frequency synaptic stimulation (LFS) was originally introduced as a model to probe potential mechanisms of deprivation-induced synaptic depression in visual cortex. In hippocampus, LTD requires activation of postsynaptic NMDA receptors, PKA, and the clathrin-dependent endocytosis of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. It has long been assumed that LTD induced in visual cortical layer 2/3 by LFS of layer 4 uses similar mechanisms. Here we show in mouse visual cortex that this conclusion requires revision. We find that LTD induced in layer 2/3 by LFS is unaffected by inhibitors of PKA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor endocytosis but is reliably blocked by an endocannabinoid CB1 receptor antagonist. Conversely, LFS applied to synapses on layer 4 neurons produces LTD that appears mechanistically identical to that in CA1 and is insensitive to CB1 blockers. Occlusion experiments suggest that both mechanisms contribute to the loss of visual responsiveness after monocular deprivation.

Fig. 1.

Similarities in pairing-induced LTD in layers 4 and 3 of mouse visual cortex. (a and b) Displayed are grouped data time courses and representative sweeps from layers 2/3 (a) and 4 (b) recordings. (a₁ and b₁) Differential interference contrast images of typical recording configurations and confocal images of neurons filled with biocytin and subsequently detected with streptavidin. (a₁) Schematic illustration of the pairing protocol used to induce LTD: 20-mV, 100-msec postsynaptic step depolarizations paired with 1-Hz presynaptic stimulation that occur halfway (50 msec) into step depolarization. (a₂ and b₂) Control recordings from layer 2/3 (a₂) or layer 4 (b₂) demonstrate that LTD can be observed in either layer. Bath application of APV (100 μM) prevents LTD in both layers. (a₃ and b₃) Intracellular loading of the NMDAR antagonist MK801 (500 μM) by the recording pipette largely blocked LTD in both layers. The y axis is EPSC amplitude normalized to a 15-min baseline period with error bars indicating the SEM. The pairing protocol, denoted by black bar, was administered at time 0 and lasted 5 min. The dashed horizontal line indicates no change from baseline responses. Sweep numbers (1, 2) refer to averaged responses collected during the last 5 min of the baseline and postpairing periods. Stimulation artifacts were minimized for clarity. (Scale bars: a₁ and b₁; 50 μm; a₂, a₃, b₂, and b₃, 50 pA and 20 msec.) See Fig. 4 for statistical comparisons.

Fig. 2.

Spike-timing LTD is insensitive to postsynaptic NMDAR blockade. Displayed are grouped data time courses and representative sweeps from whole-cell current-clamp recordings examining STD-LTD from visual cortex slices obtained from P13–P15 mice (15). In contrast to LFS-LTD, cells loaded with MK801 manifested robust STD-LTD (EPSP amplitude, 62.4 ± 11.9%; slope, 62.4 ± 15.3%; n = 7), whereas bath application of APV prevented STD-LTD (EPSP amplitude, 95.5 ± 6.7%; slope, 99.1 ± 13.9%; n = 7). 1 and 2 indicate 5-min periods for the averaging of EPSPs just before pairing (1) or at the conclusion of the recording session (2). Stimulus artifacts were minimized for clarity. (Scale bar: 1 mV, 10 msec.) The dashed line indicates no change from baseline responses.

Fig. 3.

Differences in pairing-induced LTD in layers 4 and 3 of visual cortex. (a and b) Displayed are grouped data time courses and representative sweeps from layers 2/3 (a) and 4 (b) recordings. LTD was challenged both extracellularly and intracellularly by a battery of antagonists, which highlight differences in LTD expression between layers. (a₁ and b₁) Schematic illustrations of recording configurations for layers 2/3 (a₁) and 4 (b₁). (a₂ and b₂) Intracellular loading of a PKA inhibitor PKI (6–22 amide, 10 μM) prevented LTD in layer 4 (b₂) but not in layer 2/3 (a₂). (a₃ and b₃) Intracellular loading of G2CT (10 μM), a peptide that interferes with regulated endocytosis of GluR2, also prevents LTD in layer 4 (b₃) but not layer 2/3 (a₃). (a₄ and b₄) Bath application of the CB1 antagonist AM251 (2 μM) had no effect on LTD in layer 4 (b₄) but largely prevented LTD in layer 2/3 (a₄). The y axis is EPSC amplitude normalized to a 15-min baseline period, with error bars indicating the SEM. The pairing protocol, denoted by the black bar, was administered at time 0 and lasted 5 min. The dashed horizontal line indicates no change from baseline responses. Sweep numbers (1 and 2) refer to averaged responses collected during the last 5 min of the baseline and postpairing periods. Stimulation artifacts were minimized for clarity. (Scale bars: 50 pA, 20 msec.) See Fig. 4 for statistical comparisons.

Fig. 4.

Summary data comparing laminar differences in LTD. Filled bars represent mean ± SEM of EPSC 25–30 min after LFS (normalized to baseline) in neurons treated with the drugs indicated. Gray bars represent the effect of LFS in the interleaved vehicle-treated controls. The number of experiments in each group is indicated in the text. Note the consistency of LTD magnitude in all control experiments. APV and AM251 were bath-applied. MK801, PKI, and G2CT were loaded intracellularly. ∗, P < 0.01.

Fig. 5.

Prior MD occludes subsequent LTD ex vivo in layers 2/3 and 4. (a and b) Male mice (P21–P28) were monocularly deprived for 3 days. On the third day, slices were obtained and whole-cell voltage-clamp recordings were performed from the binocular area of the deprived hemisphere (contralateral to the lid-sutured eye) or from the control hemisphere (ipsilateral to the lid-sutured eye). LTD was saturated by doubling the LFS-pairing protocol duration to 10 min. In both layers, robust LTD was induced in the control nondeprived hemisphere (a, layer 2/3, 73.8 ± 5.4%, n = 6; b, layer 4, 72.6 ± 3.3%, n = 5). In contrast, LTD was significantly occluded in the deprived hemisphere (a, layer 2/3, 89.5 ± 3.6%, n = 7, P < 0.05; b, layer 4, 90.1 ± 3.6%, n = 6; P < 0.01). 1 and 2 indicate 5-min periods for the averaging of EPSCs just before pairing (1) or at the conclusion of the recording session (2). Stimulus artifacts were minimized for clarity. (Scale bars: 50 pA, 20 msec.) The dashed line indicates no change from baseline responses. Recordings were performed with the experimenter blind to the visual history of the animal.