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Cessation of one-week oral administration of the benzodiazepine flurazepam (FZP) to rats results in withdrawal anxiety after 1 day of withdrawal. FZP withdrawal is correlated with synaptic incorporation of homomeric GluA1-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) in the proximal stratum radiatum of CA1 neurons. After 2 days of withdrawal, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylates GluA1 subunits at Ser(831), increasing channel conductance. Secondary to AMPAR potentiation, GluN2B-containing N-methyl-D-aspartate receptors (NMDARs), known binding partners of CaMKII, are selectively removed from the postsynaptic density (PSD). While activation of synaptic CaMKII is known to involve translocation to the PSD, CaMKII bound to NMDARs may be removed from the PSD. To distinguish these possibilities, the current studies used postembedding immunogold electron microscopy to investigate alterations in CaMKII signaling at CA1 stratum radiatum synapses after 2 days of FZP withdrawal. These studies revealed decreased total, but not autophosphorylated (Thr(286)) CaMKIIα expression in CA1 PSDs. The removal of CaMKII-GluN2B complexes from the PSD during drug withdrawal may serve as a homeostatic mechanism to limit AMPAR-mediated CA1 neuron hyperexcitability and benzodiazepine withdrawal anxiety.

Prolonged benzodiazepine treatment leads to tolerance and increases the risk of dependence. Flurazepam (FZP) withdrawal is associated with increased anxiety correlated with increased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor (AMPAR)-mediated synaptic function and AMPAR binding in CA1 pyramidal neurons. Enhanced AMPAR synaptic strength is also associated with a shift toward inward rectification of synaptic currents and increased expression of GluR1, but not GluR2, subunits, suggesting augmented membrane incorporation of GluR1-containing, GluR2-lacking AMPARs. To test this hypothesis, the postsynaptic incorporation of GluR1 and GluR2 subunits in CA1 neurons after FZP withdrawal was examined by postembedding immunogold quantitative electron microscopy. The percentage of GluR1 positively labeled stratum radiatum (SR) synapses was significantly increased in FZP-withdrawn rats (88.2% +/- 2.2%) compared with controls (74.4% +/- 1.9%). In addition, GluR1 immunogold density was significantly increased by 30% in SR synapses in CA1 neurons from FZP-withdrawn rats compared with control rats (FZP: 14.1 +/- 0.3 gold particles/mum; CON: 10.8 +/- 0.4 gold particles/mum). In contrast, GluR2 immunogold density was not significantly different between groups. Taken together with recent functional data from our laboratory, the current study suggests that the enhanced glutamatergic strength at CA1 neuron synapses during benzodiazepine withdrawal is mediated by increased incorporation of GluR1-containing AMPARs. Mechanisms underlying synaptic plasticity in this model of drug dependence are therefore fundamentally similar to those that operate during activity-dependent plasticity.