Rapid critical period induction by tonic inhibition in visual cortex

Abstract

Mice lacking a synaptic isoform of glutamic acid decarboxylase (GAD65) do not exhibit ocular dominance plasticity unless an appropriate level of GABAergic transmission is restored by direct infusion of benzodiazepines into the brain. To better understand how intracortical inhibition triggers experience-dependent changes, we dissected the precise timing requirement for GABA function in the monocular deprivation (MD) paradigm. Diazepam (DZ) or vehicle solution was infused daily before and/or during 4 d of MD in GAD65 knock-out mice. Extracellular single-unit recordings from the binocular zone of visual cortex were performed at the end of deprivation. We found that a minimum treatment of 2 d near the beginning of MD was sufficient to fully activate plasticity but did not need to overlap the deprivation per se. Extended delay after DZ infusion eventually led to loss of plasticity accompanied by improved intrinsic inhibitory circuit function. Two day DZ treatment just after eye opening similarly closed the critical period prematurely in wild-type mice. Raising wild-type mice in complete darkness from birth delayed the peak sensitivity to MD as in other mammals. Interestingly, 2 d DZ infusion in the dark also closed the critical period, whereas equally brief light exposure during dark-rearing had no such effect. Thus, enhanced tonic signaling through GABA(A) receptors rapidly creates a milieu for plasticity within neocortex capable of triggering a critical period for ocular dominance independent of visual experience itself.

Figure 1.

Brief DZ infusion is sufficient to rescue MD effects in GAD65 KO mice. A, OD histogram of nondeprived GAD65 KO mice exhibits a typical contralateral (cont.) eye bias. Number of animals and cells as indicated. B, DZ infusion the day before and on the first day of a 4 d MD shift OD toward the open, ipsilateral (ipsi.) eye in GAD65 KO mice, yielding a balanced distribution (p < 0.0001 vs no MD; χ² test). C, D, WT mice during the critical period (C) and GAD65 KO mice infused for 5 d (D) display similar OD distributions in response to 4 d MD. Neither is significantly different from that in B (p > 0.2 and p > 0.08, respectively; χ² test). CBI value in top right corner of each cumulative histogram indicates bias in favor of the contralateral eye.

Figure 2.

Initial 2 d DZ treatment at MD onset is minimum effective requirement. A, Each symbol represents CBI per animal. Filled and open symbols indicate GAD65 KO and WT mice, respectively. Whereas DZ infusion just the day before 4 d MD (Pre 1d) slightly reduces the CBI (0.63 ± 0.04; p > 0.08 vs no MD; t test), a further 1, 2, or 4 d of treatment into brief MD shifts the OD to a similarly significant extent (0.55 ± 0.03, 0.54 ± 0.02, 0.51 ± 0.03 for first 2 d, 3 d, full 5 d, respectively; p < 0.002 vs no MD; t test). These values are identical to WT mice deprived during the critical period (0.54 ± 0.01; p > 0.4; t test). B, DZ infusion for the last 2 d during a 4 d MD (Last 2 d) is ineffective (0.66 ± 0.02, n = 5; p < 0.04 vs first 2 d DZ; t test). Shaded region indicates range of nondeprived CBIs for GAD65 KO mice. ^*p < 0.05, ^**p < 0.01, t test.

Figure 3.

Two day DZ pretreatment produces a plastic milieu independent of MD. A, Vehicle or DZ infusion for 2 d was completed 1 d before brief MD in GAD65 KO mice (Pre 2d Veh; Pre 2d DZ). B, Pre 2d DZ induces a significant reduction of CBI values (0.48 ± 0.03, n = 6 vs 0.71 ± 0.02, n = 3 for Pre 2d Veh; p < 0.003; t test). C, D, GAD65 KO mice with Pre 2d DZ (D) exhibit prominent OD shifts toward the open, ipsilateral eye [p < 0.0001 vs Pre 2d Veh (C); χ² test]. ^**p < 0.01, t test.

Figure 6.

Brief DZ prematurely closes, and DR delays critical period onset in WT mice. A-C, MD 10 d after 2 d of DZ infusion at P16 (A, P16 DZ + 10d) produces little or no ocular dominance shift, compared with MD during the same period without pretreatment (B, P28 LR; p < 0.002, χ² test). Complete darkness from birth to the peak of the natural critical period (C, P28 DR) fails to activate plasticity (p < 0.0001 vs P28 LR; χ²test). Data for P28 LR (B) adapted from Fagiolini et al. (2003). D, Both P28 DR and P16 DZ + 10d abolish the prominent plasticity seen at P28 with LR. CBI in response to MD for P28 LR (0.50 ± 0.01; n = 10) was significantly different from that of P28 DR (0.66 ± 0.01; n = 4; p < 0.001) and P16 DZ + 10d (0.63 ± 0.02; n = 3; p < 0.001). ^***p < 0.001, t test.

Figure 7.

Brief DZ (but not light exposure) closes the critical period during DR in WT mice. A, WT mice reared in complete darkness from birth, except for 2-4 d light exposure at P30, underwent MD as adults (>P50). B, Whereas WT mice reared normally hardly respond to brief MD in adulthood (Adult+MD, CBI = 0.69 ± 0.02; n = 3), DR mice shift significantly (Adult DR+MD, CBI = 0.57 ± 0.03; n = 9; p < 0.03 vs Adult+MD; t test). Light exposure for 2-4 d during DR has little effect on the delayed OD plasticity (2d light+MD, CBI = 0.60 ± 0.02; n = 9; p < 0.03 vs Adult+MD; t test). C, Vehicle (Veh) or DZ-treated (2 d at P30) DR mice were later subjected to MD as adults (>P50). D, DZ pretreatment in the dark eliminates the typical delay of OD plasticity (2d DZ+MD, CBI = 0.71 ± 0.01; n = 8), whereas vehicle infusion does not (2d Veh+MD, 0.61 ± 0.02, n = 6, p < 0.004 vs 2d DZ+MD; t test; Welch's correction). Shaded region indicates range of nondeprived CBIs for DR mice (0.75 ± 0.02; n = 6) that was significantly different from that of DR+MD, 2d light+MD, or 2d Veh+MD (p < 0.001) but not from Adult+MD or 2d DZ+MD (p > 0.09). ^**p < 0.03.

Figure 4.

Early 2 d DZ treatment abolishes adult plasticity in GAD65 KO mice. A, GAD65 KO mice were preinfused with DZ or vehicle for 2 d, followed 14 or >60 d later by 4 d MD concurrent with DZ infusion. B, Whereas vehicle infusion leaves the mutant sensitive to MD into adulthood even 60 d later (Veh + 60d, CBI = 0.58 ± 0.01; n = 3), DZ pretreatment abolishes this plastic potential (DZ + 60d, CBI = 0.71 ± 0.02, n = 3, p < 0.01 vs Veh + 60d; t test), identical to that of nondeprived adult mice (no MD, CBI = 0.70 ± 0.02; n = 4). Just 14 d after DZ treatment, plasticity partially remains (DZ + 14d, CBI = 0.61 ± 0.01, n= 3, p < 0.03 vs DZ + 60d, t test) but is already significantly reduced compared with the maximal shift at 1 d after DZ (p < 0.03 vs DZ + 1d; t test). ^*p < 0.05, ^**p < 0.01, t test.

Figure 5.

Brief DZ treatment progressively reduces prolonged discharge in GAD65 KO. A, Examples of neuronal responses showing normal (top trace) or prolonged discharge (bottom trace). Excess spikes in bottom trace continue even after light-bar stimuli have exited the receptive field (RF). B, Visual cortical neurons in GAD65 KO mice exhibit prolonged discharge throughout life (∼P30: 69 ± 3%, 135 cells, 6 mice; adult: 66 ± 5%, 59 cells, 3 mice). Immediately (55 ± 4%, 124 cells, 5 mice, p < 0.03, t test) or 5 d after 2 d DZ infusion at approximately P30 (43 ± 4%, 122 cells, 6 mice, p < 0.001), hyperexcitability is significantly reduced compared with naive GAD65 KO mice. In addition to such early effects, 60 d after DZ infusion, further reduction (23 ± 5%, 62 cells, 3 mice, p < 0.05 vs DZ + 5d) is comparable to mature WT levels (24 ± 4%, 150 cells, 6 mice P28-P67 combined) and correlates with a gradual end to the critical period. ^*p < 0.05, t test.