Neuronal Adaptations in Rat Hippocampal CA1 Neurons during Withdrawal from Prolonged Flurazepam Exposure: Glutamatergic System Remodeling
Glutamatergic system adaptive remodeling has newly been identified as an important neural substrate of drug dependence. As extensively used anticonvulsant and hypnotic drugs, benzodiazepines act primarily as allosteric modulators of GABAA receptors; while the withdrawal symptoms are associated with enhanced AMPAR function. To investigate AMPAR remodeling during benzodiazepine withdrawal, whole-cell recordings were conducted in rat hippocampal CA1 neurons. The amplitude of AMPAR-mediated currents was enhanced 30-50% after 2-day FZP withdrawal, without changes in channel kinetic properties. Agonist-elicited AMPA currents were significantly blocked by spermine, suggesting augmented membrane incorporation of GluR2-lacking AMPARs. As GluR1-containing AMPARs are centrally involved in neuronal plasticity, we sought to determine whether changes in GluR1 subunit expression and distribution occurred during benzodiazepine withdrawal. Confocal imaging analysis revealed that FZP withdrawal promoted GluR1 subunit membrane incorporation, in the absence of GluR2 subunit alterations. Immunoblotting assays showed that GluR1, but not GluR2, subunit protein levels were enhanced in all subcellular fractions from CA1 minislices. As with LTP, FZP-withdrawal-induced GluR1 incorporation into membrane/synapse may require the GluR1-trafficking protein SAP97 and AMPAR phosphorylation by PKA may be mediated via a SAP97-AKAP interaction. Therefore, the expression and membrane/synapse redistribution of these proteins were investigated using subcellular fractionation and immunoblotting techniques. Consistent with increases in SAP97 immunoreactivity, protein levels of phospho-Ser845 GluR1 and AKAP79/150 were concomitantly elevated in membrane/synapse-associated compartments from FZP-withdrawn rats. There were no alterations in the immunoreactivity of PKA regulatory and catalytic subunits. These findings indicate that increased interaction between SAP97-AKAP-PKA, rather than enhanced PKA expression, resulted in increases in phosphorylated AMPARs and thus may be involved in AMPAR potentiation. The molecular mechanisms underlying benzodiazepine withdrawal were further explored by microarray analysis and Ingenuity Pathway Analysis. Twenty-seven known gene transcripts were differentially expressed during FZP withdrawal, including 19 significantly upregulated and 8 downregulated transcripts. Based on the transcripts identified, one network and two biological (LTP-associated and neurotransmission) pathways were constructed by IPA, illustrating the interactions among these genes products. Collectively, our findings provide evidence that during FZP withdrawal, increased expression and phosphorylation of GluR1-containing AMPARs and associated upregulation of AMPAR function in hippocampal CA1 pyramidal neurons share fundamental similarities with the mechanisms underlying activity-dependent synaptic plasticity.
School:University of Toledo Health Science Campus
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:drug dependence plasticity glutamate receptor gene profiling benzodiazepine gabaergic
Date of Publication:01/01/2007