Essential role for a long-term depression mechanism in ocular dominance plasticity

Abstract

The classic example of experience-dependent cortical plasticity is the ocular dominance (OD) shift in visual cortex after monocular deprivation (MD). The experimental model of homosynaptic long-term depression (LTD) was originally introduced to study the mechanisms that could account for deprivation-induced loss of visual responsiveness. One established LTD mechanism is a loss of sensitivity to the neurotransmitter glutamate caused by internalization of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Although it has been shown that MD similarly causes a loss of AMPARs from visual cortical synapses, the contribution of this change to the OD shift has not been established. Using an herpes simplex virus (HSV) vector, we expressed in visual cortical neurons a peptide (G2CT) designed to block AMPAR internalization by hindering the association of the C-terminal tail of the AMPAR GluR2 subunit with the AP2 clathrin adaptor complex. We found that G2CT expression interferes with NMDA receptor (NMDAR)-dependent AMPAR endocytosis and LTD, without affecting baseline synaptic transmission. When expressed in vivo, G2CT completely blocked the OD shift and depression of deprived-eye responses after MD without affecting baseline visual responsiveness or experience-dependent response potentiation in layer 4 of visual cortex. These data suggest that AMPAR internalization is essential for the loss of synaptic strength caused by sensory deprivation in visual cortex.

Fig. 1.

Viral expression of G2CT blocks NMDA-stimulated AMPAR internalization in visual cortical cultures and slices. (A) Experimental design for cortical culture experiments. (B) HSV-G2CT infection prevents NMDA-stimulated AMPAR internalization. NMDA treatment produced a significant reduction of surface-expressed GluR1 in noninfected cultures (mean ± SEM of untreated control: 76.2 ± 6.1%, n = 8, P < 0.05) and HSV-GFP infected cultures (82.4 ± 5.0%, n = 8, P < 0.05), but not in HSV-G2CT-infected cultures (98.6 ± 2.6%, n = 8, P > 0.5). Note that surface GluR1 levels under control conditions (no infection, n = 8; HSV-GFP, n = 12; HSV-G2CT, n = 6) were comparable between all groups, indicating that the G2CT peptide does not affect basal AMPAR trafficking (T, total; S, surface). (C) Viral expression of G2CT does not affect transferrin receptor (TfR) endocytosis. To rule out a nonspecific effect on clathrin-mediated endocytosis, the level of surfaced expressed TfR was measured after transferrin application. Levels of internalized TfR at 37 °C were comparable across cultures, and nonspecific GluR1 internalization was not observed. (D) GFP expression in neurons after injection of HSV-G2CT into layer 4 of visual cortex is robust and sustained (Scale bars, 200 μm.) (E) No colocalization of GFP+ (green cells) and GABA+ (red cells, marked with asterisks) indicates HSV infection is restricted primarily to excitatory neurons (Scale bar, 25 μm.) (F) Experimental design for slice preparation experiments. Visual cortical slices were obtained 48–72 h after in vivo infection, and a region in the middle of the cortical thickness, corresponding to layer 4, was microdissected from both the HSV-infected and contralateral (noninfected) visual cortices. After recovery, slices were treated with NMDA (100 μM, 15 min), and 30 min later, surface proteins were biotinylated and immunoblot analysis performed. (G) NMDA treatment produced a significant reduction of surface-expressed AMPARs in noninfected visual cortex (GluR1: 77.3 ± 6.6% of control, n = 5, P < 0.05; GluR2/3: 84.7 ± 3.5% of control, n = 5, P < 0.05), whereas internalization was blocked in HSV-G2CT infected cortex (GluR1: 91.0 ± 9.4% of control, n = 5, P > 0.40; GluR2/3: 105 ± 12.4% of control, n = 5, P > 0.69).

Fig. 2.

Expression of G2CT blocks pairing-induced LTD in layer 4 of visual cortex. (A) Recordings were made from infected GFP positive cells in layer 4 of visual cortex. (Top) IR-DIC (Left) and GFP (Right) images showing target cell before seal formation with recording pipet outlined in white. (Bottom Left) Nissl stained section indicating laminar borders. A biocytin filled and stained neuron is shown overlying the Nissl stain in yellow, and alone to the right. (Bottom Right) Higher magnification image of cell shown in bottom left. Note prominent apical dendrite and descending basal dendrites, features indicative of a layer 4 star pyramidal neuron (Scale bars, 25, 25, 200, 50 μM for top left to bottom right, respectively.) (B) Pairing-induced LTD is blocked in neurons infected with HSV-G2CT. Traces are representative EPSCs averaged over 6 consecutive responses recorded at times shown in summary graph below (Scale bars, 10 ms, 100 pA.) Lower graph shows the time course of average EPSC amplitude in control and G2CT-expressing cells. A 1 Hz pairing protocol was delivered at time indicated by arrow. LFS pairing resulted in significant LTD in control (71.8 ± 2.2% of baseline, n = 5, P < 0.05, paired t test) but not G2CT expressing (96.7 ± 10.1% of baseline, n = 5, P > 0.2, paired t test) cells. A significant between group difference in LTD magnitude is also observed (P < 0.05, unpaired t test). (C) G2CT expression does not affect basal synaptic transmission from white matter to layer 4 assayed via input-output relationships. (D) AMPA/NMDA ratios in G2CT-expressing neurons are comparable to noninfected controls. Traces at right show representative EPSCs recorded at +40 mV (Scale bars, 50 ms, 400 pA.) Histogram presents average AMPA/NMDA ratios, normalized to control. No significant difference was observed between groups (control: 100 ± 12.5%, n = 7; G2CT: 82.8 ± 24.5%, n = 6; P > 0.5).

Fig. 3.

Selective disruption of OD plasticity in layer 4 by HSV-G2CT. (A) Experimental design for monocular deprivation and VEP recordings. (B) Histological example verifying placement of recording electrode tip (indicated with asterisk) within the HSV infected cortical area (Scale bar, 200 μm.) (C) HSV-G2CT does not alter baseline VEP amplitudes. Animals were infected with HSV-GFP (n = 14) or HSV-G2CT (n = 17), and baseline VEPs were measured before random assignment into MD or SRP groups. No significant differences were observed between groups (contralateral eye response, 174.7 ± 13.5 μV vs HSV-G2CT 175.1 ± 10.9 μV, t test: P > 0.9; ipsilateral eye response, HSV-GFP 98.7 ± 8.4 μV vs HSV-G2CT 98.7 ± 7.7 μV, t test: P > 0.9). (D) HSV-G2CT infection blocks MD-induced synaptic depression. A significant decrease in contralateral eye VEP amplitude is observed after 3 d of MD in control (HSV-GFP) animals (baseline 184.9 ± 23.2 vs post-3 d MD 119.3 ± 15.3 μV, P < 0.01), whereas this decrease is blocked in animals infected with HSV-G2CT (baseline 168 ± 15 μV vs post-3 d MD 156 ± 21.7 μV, P > 0.5). (E) Experimental design for single unit recordings. (F–H) Infection with HSV-G2CT blocks OD plasticity in layer 4 but not layer 2/3 neurons. (Left) Schematics showing position of multichannel recording electrode in layer 4 (F and G) or layer 2/3 (H) and the targeted site of infection with HSV-GFP (F) or HSV-G2CT (G and H). (Right) Distribution of OD scores for neurons recorded either pre- or post-MD for the corresponding condition. (F) A significant OD shift is observed in layer 4 single units from animals infected with HSV-GFP (n = 6 animals, n = 18 neurons pre-MD, 14 post-MD, Mann–Whitney U test, P = 0.01). (G) The distribution of OD scores of neurons recorded from HSV-G2CT infected animals in layer 4 before and after 3 days of MD is not significantly different (n = 3 animals, n = 10 neurons pre-MD, 14 post-MD, Mann–Whitney U test, P > 0.8). (H) MD produces a significant shift in the distribution of OD scores of layer 2/3 neurons recorded after infection with HSV-G2CT (n = 5 animals, n = 15 neurons pre-MD, 10 post-MD, Mann–Whitney U test, P < 0.05).