Acute effects of FZ were not altered in

Acute effects of FZ were not altered in GluR-A−/− mice, thus providing a solid basis for comparing the effects of repeated drug administration. In addition, GluR-A subunit deficiency did not affect the basal behavior of saline-treated animals, despite the GluR-A−/− mice having been described as hyperactive (Zamanillo et al., 1999, Vekovischeva et al., 2001). Hyperactivity could not be detected in the present tests, which did not include any exploration tasks. The lack of GluR-A subunit-dependent neuroadaptation became evident also during the recovery from an acute dose of FZ, as the GluR-A−/− mice needed a longer time to regain their baseline performance. In fact, it is known that an AMPA receptor turnover, by trafficking of AMPA receptors to synapses, occurs quickly by mechanisms in which the GluR-A subunit has an established role (Malenka, 2003). Thus, glutamatergic compensation during the action of sedative drugs, such as benzodiazepine agonists (this study) and opioids (Vekovischeva et al., 2001), is deficient in GluR-A−/− mice. Such compensation is not, however, needed for dillution to ethanol, because ethanol effects, tolerance and dependence are unchanged in GluR-A−/− mice (Cowen et al., 2003). Although ethanol inhibits the glutamate receptor function (Lovinger et al., 1989, Möykkynen et al., 2003), it acts via multiple neurotransmitter targets (Weiss and Porrino, 2002) and may thus induce many other adaptational mechanisms to counteract its behavioral effects. Excitatory glutamate and inhibitory GABA systems are known to regulate and scale each other's functional activity during the development in vivo and in in vitro cultured neurons (Martikainen et al., 2004, Möykkynen et al., 2007). It thus seems possible that in the absence of GluR-A subunit-containing AMPA receptors, the normal reciprocal interaction is deficient when the brain is challenged by high doses of BZs. In the present study, flumazenil precipitated withdrawal symptoms after subchronic FZ treatment of mice. The present data allow one to estimate that, although below the limit of exact quantification, low nanomolar concentrations of FZ and DES may still have been present in brain tissue at 48 h after the last FZ dose. Flumazenil, acting as a selective antagonist of the allosteric BZ-site of GABAA receptors (Hunkeler et al., 1981), apparently displaced the low drug concentrations and induced the withdrawal symptoms by reducing the activity of the GABAA receptors. Although the residual FZ or DES levels at withdrawal precipitation remain unknown, significantly more withdrawal symptoms were observed in the GluR-A−/− mice than in their littermate controls. Taking into account the reduced tolerance of the knockout mice, one can suggest that a higher remaining inhibitory activity after the seven-day subchronic FZ treatment was prevailing in the knockout brains, perhaps due to the lack of down-scaling of the GABA system. This condition might then result in a more pronounced withdrawal in response to the blockade of the BZ sites. Therefore, tolerance to and withdrawal symptoms from subchronic BZ treatment may have a common neuronal substrate. Previous results using the same mouse model show that both tolerance to and withdrawal from subchronic morphine are decreased (Vekovischeva et al., 2001). Thus, the data derived from the GluR-A subunit-deficient mouse line suggest common mechanisms for opioid and BZ tolerance, probably involving GluR-A subunit-containing AMPA receptors, but different from the ones involved in the withdrawal from these compounds.
Acknowledgements
Introduction For a long time, d-amino acids were thought to be limited to microorganisms, however, recent investigations show that several free d-amino acids are widely distributed among many animals and have important roles in biological functions (Hamase et al., 2002; Radkov and Moe, 2014). In particular, the distribution and physiological functions of d-serine and d-aspartate have been studied in many works. A high level of free d-serine has been observed in mammalian brain where it acts as a positive modulators of signal transduction at the N-methyl-d-aspartate (NMDA) receptor (Mothet et al., 2000). Free d-aspartate has been found in a wide variety of cells and tissues of mammals and plays physiological roles in regulating developmental processes, hormone secretion, and steroidogenesis (Katane and Homma, 2011). Also, in the marine mollusc Aplysia californica,d-aspartate exists in central nervous system and acts as neurotransmitter (Miao et al., 2006; Patel et al., 2017). In addition, d-serine and d-aspartate were found in various non-mammalian eukaryotic phyla and probably involved several biological functions (Rosenberg and Ennor, 1961; Corrigan and Srinivasan, 1966; Saitoh et al., 2012). d-Serine and d-aspartate in animals are known to be synthesized by serine racemase (SerR) and aspartate racemase (AspR), respectively, which catalyze the interconversion of the l- and d-enantiomers. Serine racemase was first purified from rat brain (Wolosker et al., 1999b) and its cDNA was subsequently cloned from several mammals (De Miranda et al., 2000; Wolosker et al., 1999a). An aspartate racemase gene cloned from the bivalve mollusc Scapharca broughtonii showed a 44% overall amino acid sequence identity with mammalian SerR, indicating that animal SerR and AspR genes have evolved from a common ancestral gene (Abe et al., 2006; Uda et al., 2016).