Next we checked whether the suppression occurs at the end of the cascade, at the level of AMPA receptor trafficking in and out of the synapse. To that end we exploited the facts that LTP and LTD can be both reversed by activity. The

reversal of LTP (termed de-potentiation) and LTD (termed de-depression) share common downstream mechanism of expression with LTD and LTP, as they involve changes in AMPA receptor function; yet they differ in induction mechanisms, as they involve different kinase and phosphatase pathways (Hardingham et al., 2008 and Lee and Huganir, 2008). We reasoned that if the GPCR-mediated suppression occurs at the expression level (AMPAR trafficking), de-potentiation and de-depression should also be affected. The experiments were carried out in a two independent inputs setting, to allow internal controls, and using pairing PD-1/PD-L1 inhibitor conditioning (to 0mV or –40mV) to induce LTP and LTD as well as to reverse them (Figure 5). First LTD was induced in both inputs, and 20 min later one input was de-depressed by pairing with 0mV while the other input was not stimulated. The second pairing effectively reversed LTD in either control Wnt inhibitor conditions (de-depressed versus nonstimulated; paired t test: p = 0.0086) (Figure 5A), and in the presence of methoxamine (paired

t test: p = 0.0368. Figure 5B), indicating that α1 adrenergic receptors do not suppress de-depression. A similar strategy was used to test the role of β-adrenergic receptors on de-potentiation: LTP induction in both pathways, followed by pairing with –40mV in one input (Figures 5E and 5F). The second pairing reversed LTP either in control conditions (p = 0.0343. Figure 5E) or in the presence

of isoproterenol (p = 0.0007) (Figure 5F). Next we compared the effects of methoxamine Unoprostone on LTD and de-potentiation simultaneously by first inducing LTD in one input and then applying the 0mV pairing to both inputs. In control experiments (Figure 5C) the second pairing potentiated both the depressed input (p = 0.0008) and the naive (p = 0.0038); in the presence of methoxamine (Figure 5D) the depressed inputs potentiated (p = 0.0236), but not the naive inputs (p = 0.2054), confirming that α1-adrenergic receptors prevent LTP but they do not affect de-potentiation. The effects of β-adrenergic receptors on LTD and depotentiation were compared with a similar strategy: first LTP induction of one input, followed by simultaneous pairing with −40mV of both potentiated and naive inputs. Under normal conditions both inputs became depressed (potentiated inputs: p = 0.001; naive inputs: p = 0.0006) (Figure 5G). In contrast, in the presence of isoproterenol only the previously potentiated input became depressed (potentiated inputs: p = 0.048; naive inputs: p = 0.604) (Figure 5H). These results confirmed that β-adrenergic receptors prevent LTD but do not affect de-depression.