Immunogold electron microscopic evidence of differential regulation of GluN1, GluN2A, and GluN2B, NMDA‐type glutamate receptor subunits in rat hippocampal CA1 synapses during benzodiazepine withdrawal

Abstract

Benzodiazepine withdrawal-anxiety is associated with enhanced AMPA receptor (AMPAR)-mediated glutamatergic transmission in rat hippocampal CA1 synapses due to enhanced synaptic insertion and phosphorylation of GluA1 homomers. Interestingly, attenuation of withdrawalanxiety is associated with a reduction in NMDA receptor (NMDAR)-mediated currents and subunit expression, secondary to AMPA receptor potentiation. Therefore, in this study ultrastructural evidence for possible reductions in NMDAR GluN1, GluN2A and GluN2B subunits was sought at CA1 stratum radiatum synapses in proximal dendrites using postembedding immunogold labeling of tissues from rats withdrawn for 2-days from 1-week daily oral administration of the benzodiazepine, flurazepam (FZP). GluN1-immunogold density and the percentage of immunopositive synapses were significantly decreased in tissues from FZPwithdrawn rats. Similar decreases were observed for GluN2B subunits, however the relative lateral distribution of GluN2B-immunolabeling within the postsynaptic density did not change after BZ withdrawal. In contrast to the GluN2B subunit, the percentage of synapses labeled with the GluN2A subunit antibody and the density of immunogold labeling for this subunit was unchanged. The spatial localization of immunogold particles asssociated with each NMDAR subunit was consistent with a predominantly postsynaptic localization. The data therefore provide direct evidence for reduced synaptic GluN1/GluN2B receptors and preservation of GluN1/GluN2A receptors in the CA1 stratum radiatum region during BZ withdrawal. Based on collective findings in this benzodiazepine withdrawal-anxiety model, we propose a functional model illustrating the changes in glutamate receptor populations at excitatory synapses during benzodiazepine withdrawal.

Calcium/Calmodulin-Dependent Protein Kinase II Mediates Hippocampal Glutamatergic Plasticity During Benzodiazepine Withdrawal

“…Notably, expression of withdrawal-anxiety can be modulated in 2-day FZP-withdrawn rats via depression of NMDAR function (Van Sickle et al , 2002; Van Sickle et al , 2004). Moreover, preliminary immunoblot and EM findings indicate that depression of NMDAR function involves a reduction in GluN1/GluN2B receptors at CA1 neuron synapses (Shen et al , 2008; Das et al , 2009). Distinct from NMDAR-dependent LTP, in which Ca 2+ influx primarily through NMDAR initiates CaMKII activation and AMPAR potentiation (Barria et al , 1997b, Collingridge et al , 2004), Ca 2+ entry during benzodiazepine withdrawal may primarily occur via an increase in high voltage-activated Ca 2+ channel current density (Katsura et al , 2007; Van Sickle et al , 2004; Xiang et al , 2008; Xiang and Tietz, 2007) and perhaps subsequently, via the increased density of Ca 2+ -permeable, GluA1 homomeric AMPARs (Das et al , 2008; Song et al , 2007).…”

Unraveling glutamate–opioid receptor interactions using high-resolution electron microscopy: Implications for addiction-related processes

“…The role of glutamate in cocaine seeking has received attention from numerous groups [for selected publications, see (Anderson, et al, 2008, Famous, et al, 2008, Famous, et al, 2007, Ghasemzadeh, et al, 2009, Ghasemzadeh, et al, 2009, Hearing, et al, 2010, Knackstedt and Kalivas, 2009, Kumaresan, et al, 2009, Schmidt, et al, 2005)]. Important glutamatergic mechanisms have been identified in the neural processes that occur in the context of benzodiazepine withdrawal (Das, et al, 2008, Das, et al, 2010), nicotine dependence and reward (Berrendero, et al, 2010), and cannabinoid exposure (Good and Lupica, 2010). Glutamatergic-opioid interactions have already been demonstrated in studies of the delta-opioid receptor function in the amygdala following ethanol exposure (Bie, et al, 2009, Bie, et al, 2009).…”

Down-Regulation of Synaptic GluN2B Subunit-Containing N-methyl-d-aspartate Receptors: A Physiological Brake on CA1 Neuron α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Hyperexcitability during Benzodiazepine Withdrawal

“…The smaller reduction in GluN1 subunit expression level herein probably was related to its obligatory role in the formation of all functional receptors (Cull-Candy et al, 2001). Therefore, the removal of functional heteromeric GluN1/GluN2B receptors might serve as a molecular basis for the decreased NMDAR eEPSC amplitude during FZP withdrawal at CA1 neuron synapses, a finding extended by ultrastructural analyses (Das et al, 2010). Unlike at early developmental stages in which the GluN2B subunits are replaced by GluN2A subunits (Cull-Candy et al, 2001), no change in GluN2A subunit expression was observed during FZP withdrawal.…”