A refractory period for rejuvenating GABAergic synaptic transmission and ocular dominance plasticity with dark exposure

doi:10.1523/JNEUROSCI.4384-10.2010

J Neurosci. Author manuscript; available in PMC 2012 July 11.

Published in final edited form as:

J Neurosci. 2010 December 8; 30(49): 16636–16642.

doi: 10.1523/JNEUROSCI.4384-10.2010

PMCID: PMC3394852

NIHMSID: NIHMS257118

A refractory period for rejuvenating GABAergic synaptic transmission and ocular dominance plasticity with dark exposure

Shiyong Huang,¹^† Gu Yu,²^† Elizabeth M. Quinlan,^2,³^* and Alfredo Kirkwood^1,⁴^*

¹The Mind/Brain Institute, Johns Hopkins University, Baltimore, MD

²Neuroscience and Cognitive Sciences Program, University of Maryland, College Park, MD

³Department of Biology, University of Maryland, College Park, MD

⁴Department of Neuroscience, Johns Hopkins University, Baltimore, MD

^*Corresponding authors ; Email: kirkwood/at/jhu.edu, ; Email: equinlan/at/umd.edu

^†Equally contributing authors

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Abstract

Dark exposure initiated in adulthood reactivates robust ocular dominance plasticity in the visual cortex. Here we show that a critical component of the response to dark exposure is the rejuvenation of inhibitory synaptic transmission, resulting in a decrease in functional inhibitory synaptic density, a decrease in paired-pulse depression, and a re-expression of endocannabinoid-dependent iLTD. Importantly, pharmacological acceleration of the maturation of inhibition in dark-exposed adults inhibits the re-expression of iLTD and the reactivation of ocular dominance plasticity. Surprisingly, dark exposure initiated earlier in postnatal development does not rejuvenate inhibitory synaptic transmission or facilitate rapid ocular dominance plasticity, demonstrating the presence of a refractory period for the regulation of synaptic plasticity by visual deprivation.

Introduction

In juveniles, brief monocular deprivation rapidly shifts the ocular dominance of binocular neurons away from the deprived eye. Over the course of postnatal development, brief monocular deprivation becomes increasingly ineffective. Maturation of perisomatic inhibition mediated by fast-spiking parvalbumin-positive interneurons is believed to constrain Hebbian plasticity at excitatory synapses in the visual cortex, and inhibit rapid ocular dominance plasticity (Kirkwood et al., 1995, Jiang et al., 2005, Huang et al., 1999, Rozas et al., 2001, Di Cristo et al., 2007).

In the rodent primary visual cortex, the number of perisomatic GABAergic synapses onto individual pyramidal neurons increases 2-3 fold between eye opening (postnatal day 15 (P15) and puberty (P35; Huang, et al., 1999; Morales, et al., 2002; Chattopadhyaya et al, 2004). During this time, inhibitory synapses transition from an immature state with high release probability but prone to depletion, to a mature state with a low release probability and increased fidelity of transmission (Jiang et al., 2010). The functional maturation of inhibitory synaptic transmission, mediated by an endocannabinoid-dependent long-term synaptic depression (iLTD), is complete by P35.

Acceleration of the maturation of inhibition induces a precocious critical period for ocular dominance plasticity (Huang et al., 1999; Di Cristo et al., 2007). Similarly, dark rearing decelerates the maturation of GABAergic circuits (Morales et al., 2002; Di Cristo et al., 2007; Chattopadhyaya, et al., 2004; Jiang et al., 2007; Kreczko et al., 2009; Jiang et al., 2010) and the developmental constraint on ocular dominance plasticity (Cynader, 1983; Mower and Christian, 1985; Fagiolini et al., 1994; Guire et al., 1999). However, dark exposure or suppression of GABA synthesis at P35 does not reduce the number or strength or inhibitory synapses (Morales et al., 2002; Chattopadhyaya et al., 2007; Jiang et al., 2007). This predicts that the developmental constraint on rapid ocular dominance plasticity would be equally irreversible.

Nonetheless, several interventions have been shown to reactivate rapid ocular dominance plasticity in adulthood, and implicate the down-regulation of intracortical inhibition in this process (Pizzorusso et al., 2002; He et al., 2006; Sale et al., 2007; Maya-Vetencourt et al., 2008). Indeed, reactivation of ocular dominance plasticity in adults is induced by a GABA_A receptor antagonist (Harauzov et al., 2010) and reversed by enhancing GABA_Aergic inhibition with a benzodiazepine (Sale et al., 2007; Maya-Vetencourt et al., 2008). This work supports the view that inhibitory synaptic transmission imposes limitations on ocular dominance plasticity in adults. However, it is at odds with the view that the developmental maturation of inhibition is irreversible.

We addressed this discrepancy by examining GABAergic synaptic transmission following dark exposure, a potent and non-invasive intervention that reactivates ocular dominance plasticity in adults (He et al., 2006; 2007). Dark exposure initiated in adulthood rejuvenated inhibitory synaptic transmission, resulting in a visual cortex with immature characteristics including robust endocannabinoid-dependent iLTD and rapid ocular dominance plasticity. Surprisingly, dark exposure initiated earlier in development did not stimulate the re-expression of iLTD or facilitate ocular dominance plasticity, demonstrating a refractory period for the rejuvenation of inhibition by visual deprivation.

Materials and Methods

Long–Evans rats were raised on a 12 h light/dark cycle, with food and water available ad libitum. Subjects were moved into a dark room at the indicated age (+/− 2 days), in which case care was provided under infrared illumination. All procedures conform to the guidelines of the U.S. Department of Health and Human Services and the Institutional Animal Care and Use Committees of Johns Hopkins University and University of Maryland.

Visual cortical slices (300 μm) were prepared as described (Kirkwood and Bear, 1994) in ice-cold dissection buffer containing (in mM): 212.7 sucrose, 5 KCl, 1.25 NaH₂PO₄, 10 MgCl₂, 0.5 CaCl₂, 26 NaHCO₃, 10 dextrose, bubbled with 95% O₂/ 5% CO₂ (pH 7.4). Slices were transferred to normal artificial cerebrospinal fluid (ACSF) for at least one hour prior to recording. Normal ACSF was similar to the dissection buffer except that sucrose was replaced by 124 mM NaCl, MgCl₂ was lowered to 1 mM, and CaCl₂ was raised to 2 mM.

Visualized whole-cell voltage-clamp recordings were made from layer II/III pyramidal neurons with glass pipettes filled with intracellular solution (in mM: CsCl 140, CaCl₂ 0.2, NaCl 8, EGTA 2, NaGTP 0.5, MgATP 4, and HEPES 10, pH 7.2). Only cells with membrane potentials < -65 mV, series resistance < 20 MΩ, and input resistance > 100 MΩ were included. Cells were excluded if input resistance changed >15% over the experiment. Data were filtered at 5 kHz and digitized at 10 kHz using Igor Pro (Wave Metrics Inc., Lake Oswego, Oregon). Synaptic currents were recorded at -60 mV in the presence of 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 μM 2-amino-5-phosphonovaleric acid (APV), and evoked every 20 sec by stimulation of layer IV with 0.2 ms pulses delivered in pairs (inter-stimulus interval: i.s.i.= 100 msec) to compute paired-pulse depression (PPD=1-p2/p1, where p1 and p2 are the amplitude of the response to the first and second stimulation, respectively). Stimulation was delivered through concentric bipolar stimulating electrodes (FHC, Bowdoin, ME) with intensity adjusted to evoke 100-300 pA responses. Synaptic strength was quantified as the IPSC amplitude. 10 min of stable baseline (< 10% change) was required before any experimental manipulation. iLTD was induced with theta burst stimulation (TBS), consisting of 4 theta burst epochs delivered at 0.1 Hz. Each TBS epoch consisted of 10 trains of 4 pulses (100 Hz) delivered at 5 Hz. Statistical significance was assessed with one-tailed, unpaired t-test or two-way ANOVA followed by Tukey HSD post hoc analysis.

Monocular deprivation

Animals were anesthetized with ketamine/xylazine (50 mg/10 mg/kg, i.p.). The margins of the upper and lower lids of one eye were trimmed and sutured together. The animals were returned to their home cages for 3 days and disqualified in the event of suture opening or infection.

Visually evoked potentials

(VEPs) were recorded from the surface or layer IV of binocular visual cortex (V1b; ~ 7 mm posterior to Bregma and 4 mm lateral to the midline) with tungsten microelectrodes (0.5MΩ) relative to a ground screw in the frontal bone. V1b was exposed through a hole (~ 4mm diameter) in the skull following urethane anesthesia (1.4 mg/kg, i.p.). Electrode placement on the binocular region of V1 was confirmed by capturing a VEP in response to stimulation of the ipsilateral eye. Visual stimuli were full screen horizontal square wave gratings of 0.04 cycles degree⁻¹ reversing at 1 Hz, with 96.28% maximal contrast and 40 cd/m² luminosity, presented on a computer monitor 25 cm from eyes, in a darkened room. The amplitude of the primary positive (surface) or negative (layer IV) component of the VEP (~ 150 ms latency) was used to assess the cortical response to visual stimulation. VEPs were amplified (1000X), filtered (0.5 - 60Hz band pass digital filter), and averaged (100 repetitions) in synchrony with the stimulus using OpenEX software. Statistical significance was assessed with two-way ANOVA followed by Tukey HSD post hoc analysis.

Drug solutions

For systemic injections, diazepam and WIN 55212-2 {(R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo(1,2,3-de)-1,4-benzoxazin-6-yl]-1-napthalenylmethanone} were dissolved in 10% Tween 80, 20% DMSO and 70% saline to a final concentration of 1 mg/ml. Subjects received diazepam (1X/day 15 mg/kg diazepam) and WIN (2X/day 5 mg/kg) for 3 days via intraperitoneal injections. For in vitro experiments, stock solutions of AM251 [1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidy)pyrazole-3-carboxamide] or WIN were dissolved in DMSO and diluted in ACSF to the final concentration immediately before use. AM251, diazepam and WIN were purchased from Tocris. CNQX, APV, Tween 80 and DMSO were purchased from Sigma.

Results

A refractory period for the rejuvenation of GABAergic transmission

We recently described an endocannabinoid-mediated long-term depression of inhibitory synaptic transmission (iLTD) in layer II/III neurons in the visual cortex that is comparable to iLTD described in other brain regions (Jiang et al., 2010; Chevaleyre et al., 2006; McBain and Kauer, 2009). Cortical iLTD is robust in juveniles, but absent in subjects older than postnatal day 35 (P35). Importantly, dark exposure initiated at P35 does not stimulate re-expression of iLTD (Jiang et al., 2010). However, dark exposure in much older subjects can reactivate ocular dominance plasticity (He et al., 2006; 2007), which prompted us to re-examine the effect of visual deprivation on synaptic plasticity at inhibitory synapses

Pharmacologically isolated inhibitory postsynaptic currents (IPSCs) were evoked in layer II/III pyramidal neurons by stimulating layer IV in slices of rat primary visual cortex, a configuration shown to recruit inputs mediated by fast-spiking interneurons (Jiang et al 2010). iLTD was induced with theta-burst stimulation (TBS). We confirmed that iLTD was absent at P45, and that 10 days of dark exposure initiated at P35 did not stimulate iLTD re-expression (DE: 96.0±3.7% of baseline 30 min after TBS; NR: 94.0±3.4%, Figure 1A). However, if dark exposure was initiated in much older subjects (P90), robust iLTD could be subsequently induced by TBS (DE: 72.9±6.4%; NR: 98.3±4.7%; Figure 1B). In juveniles, iLTD requires the activation of type 1 endocannabinoid receptors (CB1Rs) and results in a decrease in paired-pulse depression. Similarly, the iLTD that was observed following dark exposure at P90 was blocked by the CB1R antagonist AM251 and was accompanied by a reduction in paired pulse depression (Supplemental figure 1).

Figure 1

A refractory period for the rejuvenation of inhibition in the visual cortex by 10 days of dark exposure

To further explore the unexpected interaction between visual deprivation and age, we examined the effects of 10 days of dark exposure initiated at ages ranging from P21 to P90 on the magnitude of iLTD and paired-pulse depression. Dark exposure enhanced iLTD if initiated at P21 (iLTD: 22.4±5.3 % depression from baseline), when cortical inhibitory circuitry is immature (Huang, et al., 1999; Morales, et al., 2002; Chattopadhyaya et al, 2004), but had little effect if initiated at P35 – P49 (4.0±3.7%; 1.0±6.6%). Nevertheless, dark exposure enabled robust iLTD if initiated at P70 – P90 (18.5±6.2%; 28.7±6.5%; Figure 1C). The U-shaped relationship between age at initiation of dark exposure (DE) and magnitude of iLTD contrasts with the uniformly small iLTD evoked in normal-reared (NR) age-matched P31 – P100 controls (Figure 1C). A parallel U-shaped relationship exists between the age at initiation of dark exposure and the magnitude of paired pulse depression (Figure 1D). The loss of iLTD observed over development is accompanied by a reduction in CB1R levels and a loss of response to CB1R activation (Jiang et al., 2010). We therefore asked if adult dark exposure reversed this process. We found that dark exposure initiated at P90, but not P45, restored the sensitivity of the IPSC to depression by the CB1R agonist WIN (10 M, 10 min; DE P45: 92.7±7.1%; NR P45: 98.0±6.6%; Figure 1E; DE P90: 57.4±5.1%; NR P90: 88.7±4.7%; Figure 1F). Similarly, quantitative PCR confirmed that DE at P90, but not P45 induced in a significant increase in the level of CB1R mRNA in the visual cortex (Supplemental figure 2). Thus, dark exposure rejuvenates many aspects of inhibitory synaptic transmission, but is only effective if initiated before or after a refractory period.

The anatomical and functional maturation of inhibition can be decelerated by dark rearing from birth, but is unaffected by dark exposure initiated at P35 (Morales et al., 2002; Chattopadhyaya et al., 2007; Jiang et al., 2007). We therefore tracked changes in the maximal evoked IPSC to ask if the density of functional inhibitory synapses onto individual pyramidal neurons could be regulated by visual experience in adulthood (Morales et al., 2002; Choi et al., 2002; Goldberg et al., 2005). Dark exposure initiated at P35-38 did not decrease IPSC magnitude (DE: 4.32±0.27 nA; NR: 4.22±0.32 nA: Figure 2A) or the input/output relationship across a range of stimulus intensities (5-100 mA, Two way ANOVA F1,225=0.71, p=0.79). In contrast, dark exposure initiated at P90-93 induced a significant decrease in the maximal IPSC amplitude (DE: 2.97±0.37 nA; NR: 4.53±0.36 nA; Figure 2B) and a reduction in the input/out relationship (Two way ANOVA F1,145=12.59, p=0.0013). Together this demonstrates a parallel refractory period for the rejuvenation of many aspects of inhibitory synaptic transmission, including endocannabinoid-dependent synaptic depression, regulation of presynaptic neurotransmitter release and functional connectivity.

Figure 2

Refractory period for the reduction of maximal IPSC amplitude in the visual cortex by 10 days of dark exposure

A refractory period for the reactivation of ocular dominance plasticity

The maturation of perisomatic inhibition is widely believed to constrain Hebbian synaptic plasticity at excitatory synapses in the visual cortex and consequently reduce rapid ocular dominance plasticity (Kirkwood et al., 1995; Huang et al., 1999; Rozas et al., 2001; Di Cristo et al., 2007). Dark exposure initiated in adulthood induces a reactivation of juvenile-like ocular dominance plasticity (He et al., 2006; He et al., 2007). We therefore used visually evoked potentials (VEPs) in response to high contrast gratings (0.04 cycles/degree reversing at 1 Hz) to ask if there is a refractory period for the reactivation of ocular dominance plasticity by dark exposure. The rodent visual system has an innate contralateral bias, resulting in 2-fold larger VEP amplitudes in response to stimulation of the contralateral eye relative to the ipsilateral eye (VEP average ± SEM: 2.33±0.22, n=4, grey bar, Figure 3A). Brief monocular deprivation induces a significant shift in ocular dominance if initiated before P65 (VEP amplitude C/I average±SEM: P38 0.96±0.04; P45 1.03±0.05; P55 0.93±0.18; P65 1.94±0.05; P100 2.33±0.22; Figure 3A). The decrease in the VEP contralateral bias was mediated by a decrease in the deprived eye VEP and an increase in the non-deprived eye VEP (shown for P45 subjects in supplemental figure 3C,D). However, when brief monocular deprivation is preceded by 10 days of dark exposure (DE), a significant shift in ocular dominance was observed in all ages up to P100 (DE P38 0.74±0.06; DE P45 1.00±0.04; DE P55 1.15±0.07; DE P65 0.89±0.08; DE P100 0.86±0.08, Figure 3A). To separate the effects of dark exposure from baseline ocular dominance plasticity, we normalized the VEP amplitudes from dark exposed subjects to age-matched normal reared controls. A U shape is observed in the relationship between the age at initiation of dark exposure and the deprived eye depression and the non-deprived eye potentiation induced by monocular deprivation (Figure 3B). Thus, dark exposure enhances ocular dominance plasticity, but is only effective if initiated before or after a refractory period.

Figure 3

Refractory period for the reactivation of ocular dominance plasticity by 10 days of dark exposure

Endocannabinoid-mediated regulation of inhibitory synapse maturation and ocular dominance plasticity

We hypothesized that the rejuvenation GABAergic circuits was necessary for the reactivation of ocular dominance plasticity in adults. Therefore, we used two complementary methods to ask if enhancement of GABAergic inhibition would reduce the reactivation of ocular dominance plasticity by dark exposure at P90. In the juvenile visual system, activation of CB1 receptors with the agonist WIN accelerates the maturation of GABAergic inhibition (Jiang et al., 2010). To ask if the rejuvenation of GABAergic inhibition by dark exposure at P90 is reversed by CB1R activation, we administered WIN during the last 3 days of a 10-day period of dark exposure (2x/day for 3 days, 5 mg/kg, i.p.). Administration of WIN induced a loss of iLTD (WIN: 95.5±7.0% of baseline; vehicle: 75.6±5.11%; Figure 4A) and an increase in the maximal IPSC amplitude in P90 DE subjects (WIN: 3.08±0.20nA; vehicle: 2.40±0.22nA; Figure 4B), suggesting that WIN stimulated the maturation of GABAergic inhibition. Similarly, the reactivation of ocular dominance plasticity by dark-exposure at P90 was significantly reduced by systemic injection of WIN (2x/day for 3 days, 5 mg/kg, i.p.). Importantly, enhancement of GABA_AR function with diazepam (1x/day for the last 3 days of a 10 day period of dark exposure, 15 mg/kg, i.p.) completely blocked the reactivation of ocular dominance plasticity at P100 (layer IV VEP amplitude C/I: P90 no MD, 2.06±0.26; P90 MD, 2.41±0.20; P90 DE+Veh, 1.11±0.05; P90 DE+WIN, 1.67±0.13, p=0.0072 versus MD+DE+Veh; P90 DE+DZ, 2.04±0.04, p<0.0001 versus MD+DE+Veh in Tukey-Kramer post hoc; Figure 5A). WIN significantly reduced and diazepam completely eliminated the decrease in deprived eye VEP and the increase in non-deprived eye VEP induced by monocular deprivation in P90 DE subjects, and these changes were equally reflected in VEPs recorded from layer IV and the surface of the binocular region of the primary visual cortex (Supplemental figure 3A,B).

Figure 4

Systemic administration of the CB1R agonist WIN (open circles) but not vehicle (filled circles) reverses the effect of adult dark exposure

Figure 5

Reversal of the effects of dark exposure on ocular dominance plasticity in adults

This suggests that an immature state of inhibitory synaptic transmission, induced by dark exposure in adults, permits the reactivation of ocular dominance plasticity. Curiously however, we observed significant ocular dominance plasticity at P45, despite the evidence that perisomatic inhibition is mature at this age (Kirkwood et al., 1995; Hensch et al., 1998; Huang et al., 1999; Rozas et al., 2001; Di Cristo et al., 2007; Jiang et al., 2010). To ask if the persistence of ocular dominance plasticity persists at P45 is due to submaximal inhibition, GABA_AR function was enhanced with diazepam. Systemic diazepam (1x/day for 3 days, 15 mg/kg, i.p. prior to the initiation of recording) inhibited ocular dominance plasticity in P45 subjects. As expected, neither dark exposure of WIN administration (2x/day for 3 days, 5 mg/kg, i.p. during the last 3 days of dark exposure) regulated ocular dominance plasticity in P45 subjects (layer IV VEP amplitude C/I P45 No MD, 1.96±0.18; P45 MD, 1.00±0.05; P45 DE+Veh, 1.12±0.11; P45 DE+WIN, 0.94±0.14; P45 DZ, 2.03±0.03; Figure 5B). Diazepam, but not dark exposure or WIN, inhibited the decrease in deprived eye VEP and the increase in non-deprived eye VEP induced by monocular deprivation in P45 subjects, which was equally reflected in VEPs recorded from layer IV and the surface of the binocular region of the primary visual cortex (Supplemental figure 3C,D). This suggests that at P45, inhibitory circuitry has developed both functionally and anatomically, but is not maximally recruited by visual experience.

Discussion

Dark exposure initiated in adulthood reactivates robust ocular dominance plasticity in the mammalian visual cortex. Here we show that a critical step in the reactivation of ocular dominance plasticity in adults is the rejuvenation of inhibitory synaptic transmission, resulting in a decrease in functional inhibitory synaptic density, a decrease in paired-pulse depression, and a re-expression of endocannabinoid-dependent iLTD. Pharmacological acceleration of the maturation of inhibition, through activation of CB1Rs, reverses the re-expression of iLTD and the reactivation of ocular dominance plasticity in dark exposed adults. However, dark exposure initiated earlier in postnatal development does not rejuvenate inhibitory synaptic transmission or facilitate rapid ocular dominance plasticity, demonstrating the presence of a refractory period for the regulation of synaptic plasticity by visual deprivation. The refractory period for the rejuvenation of inhibitory synaptic transmission demonstrates the existence of constraints on the regulation of synaptic function by visual deprivation. In addition, it suggests that the efficacy of therapeutic interventions used to promote ocular dominance plasticity may increase with age.

An important outstanding issue is the location of the synapses that underlie the ocular dominance plasticity observed in dark exposed adults. Several lines of evidence implicate a role of layer II/III circuits in the reactivation of ocular dominance plasticity. The activation of CB1Rs, which has been implicated in the plasticity of neuronal circuits in cortical layer III, but not layer IV, (Li et al., 2009; Yoon et al., 2009; Crozier et al., 2007; Liu et al., 2008) significantly reduces the reactivation of ocular dominance plasticity by dark exposure in adults. Similarly, pyramidal neurons in layer III, which receive substantial excitation from thalamic afferents, exhibit a rejuvenation of inhibitory synaptic transmission following dark exposure in adulthood. Importantly, excitatory synapses onto layer II/III pyramidal neurons retain the capacity to express Hebbian plasticity in adulthood (Jiang et al., 2007), but induction is constrained by GABAergic inhibition (Kirkwood and Bear, 1995; Jang et al, 2009). Together this suggests that the reduction of inhibition onto layer II/III pyramidal contributes to the reactivation of ocular dominance plasticity.

The rejuvenation of inhibitory synaptic transmission

The experience-dependent maturation of inhibition was previously thought to be irreversible, as visual deprivation during postnatal development retards, rather than reverses the developmental progression at inhibitory synapses (Morales et al., 2002; Di Cristo et al., 2007; Chattopadhyaya et al., 2004; Jiang et al., 2007). However, here we show that dark exposure in adulthood rejuvenates inhibitory circuits. Immature inhibition, characterized by sparse synapses with a high density of presynaptic CB1Rs and a high probability of GABA release, is seen in the visual cortex of juveniles and dark-exposed adults. Postnatal visual experience stimulates the maturation of inhibition through an endocannabinoid-dependent long-term depression of GABA release. In contrast, mature inhibition is characterized by dense synapses that no longer express CB1Rs and have a low probability of GABA release (Jiang et al., 2010).

The mechanism by which dark exposure induces the rejuvenation of inhibitory synaptic transmission and the reactivation of ocular dominance plasticity in adults is not yet known. In response to the reduction in synaptic activity induced by dark exposure, pre-existing inhibitory synapses in the adult visual cortex may revert to a more immature phenotype, accompanied by an increase in CB1R expression and an increase in presynaptic release probability. Alternatively, dark exposure may increase the turnover of GABAergic terminals by promoting the degradation of factors that restrict axonal outgrowth, such as myelin-derived signaling molecules (McGee et al., 2005) or chondroitin sulfate proteoglycans (Pizzorusso et al., 2002). Interestingly, a significant increase in the condensation of extracellular matrix components into perineuronal nets occurs between P45 – P65 (Pizzorusso et al., 2002), which may impart increased resistance to experience-dependent degradation of a restrictive extracellular environment and contribute to the refractory period.

The role of inhibition in the developmental constraint of ocular dominance plasticity

The late maturation of inhibitory circuitry relative to excitatory circuitry creates a window in postnatal development that is permissive for experience-dependent synaptic plasticity in the visual system. Perisomatic inhibition mediated by fast spiking interneurons exerts a powerful influence on the excitability and plasticity of postsynaptic targets. While a threshold level of inhibition is necessary to initiate the critical period for ocular dominance plasticity early in postnatal development (Hensch et al., 1998; Huang et al., 1999), a subsequent increase in the strength of perisomatic inhibition has been implicated in the constraint of ocular dominance plasticity later in development (Kirkwood et al., 1995; Rozas et al., 2001; Di Cristo et al., 2007). However, here we show that ocular dominance plasticity is robust after postnatal day 35, the time point at which perisomatic inhibition reaches maturity in the rodent cortex (Morales et al., 2002; Rozas et al., 2001; Huang et al., 1999; Chattopadhyaya et al., 2004). A similar persistence of rapid ocular dominance plasticity beyond the expected age of the maturation of inhibition has been observed in cats and mice (Mower et al., 2001; Pham et al., 2004; Lehmann and Lowell, 2008). Interestingly, ocular dominance plasticity at P45 could be inhibited by enhancing GABA_A receptor function with diazepam, but not by accelerating the maturation of inhibitory synaptic transmission with the CB1R agonist WIN. This suggests that at P45, inhibitory synapses are present and mature, but ocular dominance plasticity persists because activity at inhibitory synapses is not maximally recruited by visual experience at this age.

Supplementary Material

Supp1

Supplemental figure 1. The iLTD reactivated by dark exposure at P90 is endocannabinoid-dependent. (A) IPSC amplitudes recorded in layer II/III pyramidal neurons following TBS (arrow) delivered to layer IV. AM251 (filled circles) inhibits the iLTD reactivated by dark exposure at P90 (open circles). (B) Superimposed average of 6 consecutive IPSCs evoked in response to paired stimulation pulses (100 ISI) recorded before (red) and 20 minutes after TBS (black). (C) Average paired pulse depression (PPD = 1-the response to second pulse/response to first pulse) recorded before and after TBS in P90 normal reared (NR) and P90 dark-exposed (DE) rats. *p<0.05 one tail t-test.

Supplemental figure 2. Increase in CB1R mRNA in the visual cortex following dark exposure at P90.

CB1R mRNA levels were normalized to average within-experiment control (white histogram) run in parallel QT-PCR reactions. CB1R mRNA level from P90 frontal cortex (FCtx) was normalized to average P90 NR visual cortex run in parallel QT-PCR reactions. *p<0.05 one-tail t-test.

Supplemental figure 3. Parallel regulation of adult ocular dominance plasticity by the CB1R agonist WIN and diazepam revealed by layer IV and surface VEPs (A,B) VEPs recorded from layer IV (A) and the surface of the binocular visual cortex (B). The deprived eye depression (closed circles) and non-deprived eye potentiation (open circles) induced by monocular deprivation in P90 DE subjects are significantly reduced by WIN and completely eliminated by diazepam (DZ; One way ANOVA (F_4,24=16.91, p<0.0001). Inset: Representative VEP waveforms recorded from layer IV (A) and the surface (B) of the contralateral binocular visual cortex in response to stimulation of the deprived and non-deprived eyes. (C,D) VEPs recorded from layer IV (C) and the surface of the binocular visual cortex (D). The deprived eye depression and the non-deprived eye potentiation induced by monocular deprivation in P45 subjects are inhibited by diazepam, but unaffected by dark exposure or WIN (One way ANOVA (F_4,21=10.75, p=0.0003). Inset: Representative VEP waveforms recorded from layer IV (C) and the surface (D) of the contralateral binocular visual cortex in response to stimulation of the deprived and non-deprived eyes. Scale bars: horizontal 50ms, vertical 50μV. *p<0.05 versus age-matched control contralateral eye, #p<0.05 versus age-matched control ipsilateral eye in Tukey HSD post hoc.

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Acknowledgment:s

NIH R01EY016431 to EMQ and NIH R01EY012124 to AK

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