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We sought to determine the mechanism by which sensory experience acted to modify the ability to induce tLTD within the visual cortex. Since tLTD observed following visual deprivation appeared to be expressed presynaptically, we hypothesized visual deprivation acted to restore a developmental-regulated form of tLTD observed at L2/3 synapses that is dependent on presynaptic, but not postsynaptic, NMDARs (Corlew et al., 2007). In addition to being required for some forms of tLTD in the developing visual cortex, presynaptic NMDARs

enhance evoked and spontaneous glutamate release at L2/3 synapses during a restricted developmental window (<P20) (Corlew et al., 2007; Larsen et al., 2011). If sensory experience restored tLTD which was dependent on presynaptic NMDARs, we hypothesized it would also restore the contribution of presynaptic NMDARs to glutamate release at L2/3 synapses.

To determine if sensory deprivation reversed the developmental loss in the contribution of presynaptic NMDARs to glutamate release, we analyzed short-term plasticity at L2/3 synapses by repetitively evoking glutamate release at variable frequencies (5-30 Hz) before and after D-AP5 application (50 µM, Fig. 3.4A). In all experiments, we included MK-801 and BAPTA in the postsynaptic recording pipette while maintaining the neuron at hyperpolarized potentials (near -75 mV) to minimize contributions of postsynaptic NMDARs. At 30 Hz, trains of

six EPSPs evoked by L4 stimulation in DR mice showed more synaptic depression and had a lower paired pulse ratio compared to their NR controls, consistent with a higher initial release probability (Fig. 3.4B-C). Additionally, D- AP5 increased the paired-pulse ratio at 30 Hz in DR mice via a reduction in the first EPSP in the train, but did not change the paired pulse ratio in NR mice. These effects of visual deprivation on glutamate release evoked at 30 Hz could be reversed by placing DR mice in normal visual environment for ten days (Fig 3.4C).

Both the initial paired-pulse ratio and whether it increased following D-AP5 was highly dependent on stimulation frequency in DR mice: the initial paired- pulse ratio at 5 Hz was not different from NR controls but was substantially lower at higher stimulation frequencies (Fig. 3.4E). In correlation with this frequency dependence, application of D-AP5 only increased the paired-pulse ratio in DR mice at frequencies above 5 Hz. Interestingly, in the presence of D-AP5 paired- pulse ratios at all frequencies were the same in recordings from NR and DR mice (Fig. 3.4F). This demonstrates that visual deprivation bidirectionally alters

presynaptic glutamate release at L2/3 synapses in a frequency- and presynaptic NMDAR-dependent manner.

Given the enhancement of evoked glutamate release at higher

frequencies following visual deprivation, we wondered if visual deprivation was accompanied by a change in contribution of presynaptic NMDARs to

excitatory postsynaptic currents (mEPSCs) in DR or NR mice while postsynaptic NMDARs were blocked by both hyperpolarization and the inclusion of MK-801 in the recording pipette. In agreement with previous results demonstrating that visual deprivation results in postsynaptic synaptic scaling of AMPAR responses (Desai, Cudmore, Nelson, & Turrigiano, 2002; Goel et al., 2006), mEPSC amplitudes were significantly larger in recording from DR mice, but were not affected by D-AP5 application (Fig. 3.5C). Additionally, while dark-rearing did not alter the baseline mEPSC frequency, D-AP5 reduced the frequency in recordings from DR mice, without affecting the frequency in NR mice (Fig. 3.5D). This

suggests that dark-rearing increases the contribution of presynaptic NMDARs to spontaneous release. However, since we did not observe a change in the

baseline mEPSC frequency following dark-rearing, this indicates that presynaptic NMDARs may only contribute to spontaneous release at a portion of L2/3

synapses or that their contribution is masked by known reductions in synapse number which occur following dark-rearing (Valverde, 1971; Wallace & Bear, 2004).

Visual deprivation beginning near birth and extending until the critical period may alter synaptic properties through the mechanisms restricted to this developmental period or duration of deprivation. To address this, we measured short-term plasticity in mice that had been normally-reared up until adulthood (P60) and which then underwent late-onset visual deprivation (LOVD) by being kept in the dark for ten days. Similar to our previous findings (Yashiro, Corlew, & Philpot, 2005), EPSP trains evoked at 30 Hz, but not 5 Hz, in recordings from

LOVD mice had lower initial paired-pulse ratios compared to normally-reared littermates (Fig. 3.6). Similarly, D-AP5 increased the initially lower 30 Hz paired- pulse ratio observed in deprived mice without affecting synaptic depression in normally-reared littermates. These results suggest that even visual deprivation that occurs for relatively brief periods during adulthood is capable of reversing the developmental loss in the contribution of presynaptic NMDARs to glutamate release at L2/3 synapses.

Given these findings, we next addressed whether visual deprivation in adulthood could also reverse the developmental loss in the ability to induce tLTD. Indeed, we were able to induce tLTD in recordings from mice which had

undergone LOVD, whereas we observed no significant tLTD in recordings from their aged-matched, normally-reared littermates (Fig. 3.6). As we observed in mice which had been visual deprived from birth until the critical period, tLTD was accompanied by increases in the paired-pulse ratio, suggesting that it resulted from a decrease in presynaptic glutamate release. Taken together with our previous findings, this suggests that sensory deprivation even in adulthood

(>P60) is capable of restoring contributions by presynaptic NMDARs to glutamate release and STDP which normally become dormant through development.

3.2.3 Presynaptic Layer 4 NMDARs are required for the effects of visual