Benzodiazepine Withdrawal-Induced Glutamatergic Plasticity Involves Up-Regulation of GluR1-Containing α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid Receptors in Hippocampal CA1 Neurons

Abstract

Modification of glutamatergic synaptic function, a mechanism central to neuronal plasticity, may also mediate long-term drug effects, including dependence and addiction. Benzodiazepine withdrawal results in increased glutamatergic strength, but whether ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs) are functionally and structurally remodeled during benzodiazepine withdrawal is uncertain. Whole-cell recordings of rat hippocampal CA1 neurons, either acutely dissociated or in hippocampal slices, revealed that AMPAR function was enhanced up to 50% during flurazepam (FZP) withdrawal, without changes in whole-cell channel kinetic properties. Agonist-elicited AMPA currents showed a negative shift in rectification in the presence of spermine, suggesting augmented membrane incorporation of glutamate receptor (GluR) 2-lacking AMPARs. As GluR1-containing AMPARs are critical for activity-dependent alterations in excitatory strength, we sought to determine whether changes in GluR1 subunit distribution in CA1 neurons occurred during benzodiazepine withdrawal. Confocal image analysis revealed that FZP withdrawal promoted GluR1 subunit incorporation into somatic and proximal dendritic membranes of CA1 neurons without GluR2 subunit alterations. Findings of immunoblot studies were consistent with immunofluorescent studies indicating increased GluR1, but not GluR2, subunit protein levels in cytosolic, crude membrane and postsynaptic density-enriched fractions from CA1 minislices. As with long-term potentiation (LTP), the FZPwithdrawal-induced GluR1 incorporation into CA1 neuron membranes may require the GluR1-trafficking protein, synapse-associated protein 97, which was also elevated in membrane-associated fractions. Together, our findings provide evidence that during FZP withdrawal, increased membrane incorporation of GluR1-containing AMPARs and associated upregulation of AMPAR functions in hippocampal CA1 pyramidal neurons share fundamental similarities with the mechanisms underlying LTP. This implies that glutamatergic neuronal remodeling observed in LTP also subserves physiological adaptations to drug withdrawal.Remodeling of glutamatergic neurotransmission has recently been associated with dependence on and addiction to various drugs of abuse (Nestler, 2005), including the benzodiazepines (Izzo et al., 2001;Van Sickle et al., 2004;Xiang and Tietz, 2007). Benzodiazepines are widely prescribed anxiolytics and hypnotics but have more limited clinical use as anticonvulsants due to the development of tolerance, largely mediated by dysfunction of GABA A receptors, the site of benzodiazepine allosteric actions (Zeng and Tietz, 1999;Bateson, 2002). Abrupt benzodiazepine withdrawal causes anxiety and insomnia, which can lead to drug self-administration and contribute to benzodiazepine misuse and abuse This work was supported by Department of Health and Human Services Grants R01-DA-04075-15 and R01-DA18342-02 from the National Institute on Drug Abuse (to E.I.T.) and predoctoral fellowships from t...

Regulation of GABAAReceptor Subunit Expression by Pharmacological Agents

“…In rats withdrawn from flurazepam, amplitudes of AMPA receptor-mediated miniature excitatory postsynaptic currents were increased in hippocampal CA1 neurons (Van Sickle et al, 2004;Xiang and Tietz, 2007). The 50% enhancement in AMPA receptor function was attributed to an increase in GluA1 polypeptide trafficking from the endoplasmic reticulum and its subsequent incorporation into membranes (Song et al, 2007;Das et al, 2008), whereas NMDA receptor-mediated currents were reduced in this brain region (Van Sickle et al, 2004;Xiang and Tietz, 2007). Mice deficient in GluA1 subunits have reduced short-term tolerance (i.e., prolonged short-term impairment to high doses of flurazepam) and develop less tolerance but show increased withdrawal signs after a 7-day flurazepam treatment and challenge with flumazenil (Aitta-Aho 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

“…Although NMDAR currents were not downregulated after 1 day of drug withdrawal, AMPAR currents were already potentiated and linked to withdrawal anxiety at that time point (Song et al, 2007;Xiang and Tietz, 2007). Systemic AMPAR antagonist injection eliminated both AMPAR current enhancement and NMDAR current reduction (Van Sickle et al, 2004;Xiang and Tietz 2007).…”

“…PSD-enriched subcellular fractions of CA1 minislices were prepared at 0 to 4°C as described previously (Song et al, 2007). In brief, rats were decapitated and the brains were removed to bubbled ice-cold artificial cerebrospinal fluid (ACSF).…”

“…Therefore, NMDAR subunit expression levels were hypothesized to parallel modulation of NMDAR function across the drug-withdrawal period. PSD-enriched subcellular fractions were collected from CA1 minislices derived from 1-and 2-day FZP-withdrawn rats (Song et al, 2007;Shen et al, 2010) and probed for the expression of NMDAR subunits. Because most NMDARs are GluN1/GluN2 assemblies (Lau and Zukin 2007), GluN2C subunits are restricted largely to cerebellum, and GluN2D subunits are more prominently expressed early in development (Wenthold et al, 2003), only GluN1, GluN2A.…”

“…We have previously shown that the molecular mechanisms associated with benzodiazepine-induced withdrawal anxiety involves membrane insertion of GluA1 homomeric receptors into CA1 neuron asymmetric synapses, increased AMPAR current amplitude followed by Ca 2ϩ /calmodulin-dependent protein kinase type II (CaMKII)-mediated Ser831 GluA1 phosphorylation, and enhanced AMPAR single-channel conductance (Song et al, 2007;Das et al, 2008;Shen et al, 2009Shen et al, , 2010, in common with AMPAR potentiation during activitydependent plasticity (Derkach et al, 2007). However, Ca 2ϩ influx in benzodiazepine-withdrawn rats was mediated via L-type voltage-gated calcium channels (Xiang et al, 2008) and CaMKII was not autophosphorylated (Shen et al, 2010).…”