Here we studied the effects of BZ flurazepam FZ

Here we studied the effects of BZ flurazepam (FZ) at high doses in acute and chronic settings of tolerance and dependence in GluR-A−/− mice (Zamanillo et al., 1999). We used measurements in which the phenomenon of “learning while intoxicated” should play only a minor role in order to find out the significance of GluR-A subunit-containing AMPA receptors in the EX527 of the brain to the effects of high BZ levels.
Methods
Results
Discussion The deletion of the GluR-A subunit did not completely prevent the development of tolerance, indicating that also other mechanisms contributed to the BZ tolerance in the GluR-A−/− mice. Majority of the literature concerning various animal species finds the pharmacodynamic changes to be the most important factor in BZ tolerance (Loscher and Schwark, 1985, Miller et al., 1988b, Scherkl et al., 1985). However, also the metabolic induction leading to faster elimination of BZs might influence the development of tolerance to BZs, (File, 1982). FZ concentrations in blood and brain tissue were greatly reduced, i.e. to about 1/10 in blood and to about 1/2–1/4 in brain, after subchronic treatment in comparison to the concentrations after a challenge injection with the same dose. Importantly, the concentrations of DES were increased about three-fold. Subchronic FZ treatment greatly increased the DES to FZ concentration ratio in both blood and brain suggesting an induction of FZ metabolism. The elimination half-life of DES in the mouse is approximately 6 h (Miller et al., 1988a), which can lead to some cumulation of DES during the dosing of FZ at 12-h intervals. Cumulation of DES is supported by our tolerance data: only a slight further development of tolerance was observed in the latter phase of the subchronic treatment, i.e. between the fifth and seventh day, when the higher FZ dose was administered. Some further impairment was observed in the performance of the GluR-A−/− mice, e.g., in the walking beam test. The concentration of DES was lower in the females of both genotypes, indicating a gender-specificity in FZ metabolism or distribution in mice. Regardless of the gender-difference in FZ pharmacokinetics, the males and females differed in tolerance development only with respect to the body temperature reducing effect of FZ. Since the brain concentrations of DES at 2 h after FZ administration were more than 1000-fold higher than the Ki values for [3H]flunitrazepam binding to rat cortical membranes [0.9 vs. 13 nM for DES and FZ, respectively (Miller et al., 1988a)], the receptor sites were apparently saturated. Functional activity is, however, often detected in vitro only with high nanomolar or micromolar concentrations (Yu et al., 1988). In summary, the receptor sensitivity difference between DES and FZ and the difference in their brain concentrations measured in the present high-dose experiment suggest that DES was the main active compound in that study. As there were no differences in the FZ or DES concentrations between the mouse lines, pharmacokinetic differences do not explain the lesser development of tolerance in the GluR-A−/− mice. The role of the GluR-A subunit in long-term BZ treatment has previously been implicated by gene expression and protein level studies (Izzo et al., 2001, Song et al., 2007). Furthermore, BZ dependence enhances AMPA-mediated synaptic transmission in the hippocampal CA1 region (Van Sickle and Tietz, 2002), and AMPA receptor antagonists have been shown to be able to alleviate withdrawal symptoms when administered before the onset of the symptoms (Steppuhn and Turski, 1993). Our data seem to be at variance with these studies because the GluR-A−/− mice had reduced tolerance to FZ but exhibited more pronounced withdrawal symptoms. Therefore, unlike our earlier findings showing that subchronic morphine induces less tolerance and less withdrawal symptoms in the GluR-A−/− mice than in their littermates (Vekovischeva et al., 2001), the results on FZ administration suggest a divergence of the adaptation mechanisms between the development of tolerance and dependence. It is thus likely that in the absence of an upregulatable GluR-A subunit, which is an essential factor in many kinds of neuronal plasticity events (Bannerman et al., 2006, Kauer, 2004), other adaptation mechanisms, such as other glutamate and GABA mechanisms and/or e.g. the corticotropin-releasing factor mechanisms (Skelton et al., 2007), must be recruited.