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

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 GABAA 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 the University of Toledo College of Medicine (to J.S. and G.S). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.121798. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: AMPA, -amino-3-hydroxy-5-methylisoxazole-4-propionic acid; AMPAR, AMPA receptor; mEPSCs, miniature excitatory postsynaptic currents; FZP, flurazepam; LTP, long-term potentiation; GluR, glutamate receptor; SAP97, synapse-associated protein 97; PIPES, 1,4-piperazinediethanesulfonic acid; TTX, tetrodotoxin; APV, DL-2-amino-5-phosphopentanoic acid; GYKI53655, 7H-1,3-dioxolo(4,5-H)(2,3)benzodiazepine-7-carboxamide-8,9-dihydro-5-(4-aminophenyl)-8-dimethyl-monohydrochloride; NAS, 1-naphthylacetyl spermine; QX-314, N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide; CGP-35348, (3-aminopropyl)(diethoxymethyl) phosphinic acid; sp, stratum pyramidale; sr, stratum radiatum; PSD, postsynaptic density; CON, control; so, stratum oriens; PDZ, PSD-95, Dlg, and ZO-1; PKA, cAMPdependent protein kinase A. 0022-3565/07/3222-569–581$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 322, No. 2 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 121798/3233968 JPET 322:569–581, 2007 Printed in U.S.A.