Persistent alterations in network activity trigger compensatory changes in excitation and inhibition that restore neuronal firing rate to an optimal range. One example of such synaptic homeostasis is the downregulation of inhibitory transmission by chronic inactivity, in part through the reduction of vesicular transmitter content. The enzyme glutamic acid decarboxylase 67 (GAD67) is critical for GABA synthesis, but its involvement in homeostatic plasticity is unclear. We explored the role of GAD67 in activity-dependent synaptic plasticity using a mouse line (Gad1^−/−) in which GAD67 expression is disrupted by genomic insertion of the green fluorescent protein (GFP). Homozygous deletion of Gad1 significantly reduced miniature inhibitory postsynaptic current (mIPSC) amplitudes and GABA levels in cultured hippocampal neurons. The fractional block of mIPSC amplitude by a low affinity, competitive GABA_A receptor antagonist was higher in GAD67-lacking neurons, suggesting that GABA concentration in the synaptic cleft is lower in knockout animals. Chronic suppression of activity by the application of tetrodotoxin (TTX) reduced mIPSC amplitudes and the levels of GAD67 and GABA. Moreover, TTX reduced GFP levels in interneurons, suggesting that GAD67 gene expression is a key regulatory target of activity. These in vitro experiments were corroborated by in vivo studies in which olfactory deprivation reduced mIPSC amplitudes and GFP levels in glomerular neurons in the olfactory bulb. Importantly, TTX-induced downregulation of mIPSC was attenuated in Gad1^−/− neurons. Altogether, these findings indicate that activity-driven expression of GAD67 critically controls GABA synthesis and, thus, vesicular filling of the transmitter.

NMDARs (N-methyl-D-aspartate receptors) are critical for synaptic function throughout the CNS (central nervous system). NMDAR-mediated Ca²⁺ influx is implicated in neuronal differentiation, neuronal migration, synaptogenesis, structural remodelling, long-lasting forms of synaptic plasticity and higher cognitive functions. NMDAR-mediated Ca²⁺ signalling in dendritic spines is not static, but can be remodelled in a cell- and synapse-specific manner by NMDAR subunit composition, protein kinases and neuronal activity during development and in response to sensory experience. Recent evidence indicates that Ca²⁺ permeability of neuronal NMDARs, NMDAR-mediated Ca²⁺ signalling in spines and induction of NMDAR-dependent LTP (long-term potentiation) at hippocampal Schaffer collateral–CA1 synapses are under control of the cAMP/PKA (protein kinase A) signalling cascade. Thus, by enhancing Ca²⁺ influx through NMDARs in spines, PKA can regulate the induction of LTP. An emerging concept is that activity-dependent regulation of NMDAR-mediated Ca²⁺ signalling by PKA and by extracellular signals that modulate cAMP or protein phosphatases at synaptic sites provides a dynamic and potentially powerful mechanism for bi-directional regulation of synaptic efficacy and remodelling.

The NMDA-type glutamate receptor (NMDAR) is essential for synaptogenesis, synaptic plasticity, and higher cognitive function. Emerging evidence indicates that NMDAR Ca²⁺permeability is under the control of cAMP/protein kinase A (PKA) signaling. Whereas the functional impact of PKA on NMDAR-dependent Ca²⁺signaling is well established, the molecular target remains unknown. Here we identify serine residue 1166 (Ser1166) in the carboxy-terminal tail of the NMDAR subunit GluN2B to be a direct molecular and functional target of PKA phosphorylation critical to NMDAR-dependent Ca²⁺permeation and Ca²⁺signaling in spines. Activation of β-adrenergic and D₁/D₅-dopamine receptors induces Ser1166 phosphorylation. Loss of this single phosphorylation site abolishes PKA-dependent potentiation of NMDAR Ca²⁺permeation, synaptic currents, and Ca²⁺rises in dendritic spines. We further show that adverse experience in the form of forced swim, but not exposure to fox urine, elicits striking phosphorylation of Ser1166in vivo, indicating differential impact of different forms of stress. Our data identify a novel molecular and functional target of PKA essential to NMDAR-mediated Ca²⁺signaling at synapses and regulated by the emotional response to stress.

Protein kinase C (PKC) enhances NMDA receptor (NMDAR)-mediated currents and promotes NMDAR delivery to the cell surface via SNARE-dependent exocytosis. Although the mechanisms of PKC potentiation are established, the molecular target of PKC is unclear. Here we show that synaptosomal-associated protein of 25 kDa (SNAP-25), a SNARE protein, is functionally relevant to PKC-dependent NMDAR insertion, and identify serine residue-187 as the molecular target of PKC phosphorylation. Constitutively active PKC delivered via the patch pipette potentiated NMDA (but not AMPA) whole-cell currents in hippocampal neurons. Expression of RNAi targeting SNAP-25 or mutant SNAP-25(S187A) and/or acute disruption of the SNARE complex by treatment with BoNT A, BoNT B or SNAP-25 C-terminal blocking peptide abolished NMDAR potentiation. A SNAP-25 peptide and function-blocking antibody suppressed PKC potentiation of NMDA EPSCs at mossy fiber-CA3 synapses. These findings identify SNAP-25 as the target of PKC phosphorylation critical to PKC-dependent incorporation of synaptic NMDARs and document a postsynaptic action of this major SNARE protein relevant to synaptic plasticity.