General anesthetics are among the most widely used and important therapeutic agents. The molecular targets mediating different endpoints of the anesthetic state in vivo are currently largely unknown. The analysis of mice carrying point mutations in neurotransmitter receptor subunits is a powerful tool to assess the contribution of the respective receptor subtype to the pharmacological actions of clinically used general anesthetics. We examined the involvement of beta3-containing GABA(A) receptors in the respiratory, cardiovascular, hypothermic, and sedative actions of etomidate and propofol using beta3(N265M) knock-in mice carrying etomidate- and propofol-insensitive beta3-containing GABA(A) receptors. Although the respiratory depressant action of etomidate and propofol, as determined by blood gas analysis, was almost absent in beta3(N265M) mice, the cardiac depressant and hypothermic effects, as determined by radiotelemetry, and the sedative effect, as determined by decrease of motor activity, were still present. Taken together with previous findings, our results show that both immobilization and respiratory depression are mediated by beta3-containing GABA(A) receptors, hypnosis by both beta3- and beta2-containing GABA(A) receptors, while the hypothermic, cardiac depressant, and sedative actions are largely independent of beta3-containing GABA(A) receptors.

The early detection of cognitive impairment or dementia is in the focus of current research as the amount of cognitively impaired individuals will rise intensely in the next decades due to aging population worldwide. Currently available diagnostic tools to detect mild cognitive impairment (MCI) or dementia are time-consuming, invasive or expensive and not suitable for wide application as required by the high number of people at risk. Thus, a fast, simple and sensitive test is urgently needed to enable an accurate detection of people with cognitive dysfunction and dementia in the earlier stages to initiate specific diagnostic and therapeutic interventions. We examined digital Clock Drawing Test (dCDT) kinematics for their clinical utility in differentiating patients with amnestic MCI (aMCI) or mild Alzheimer’s dementia (mAD) from healthy controls (HCs) and compared it with the diagnostic value of the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) neuropsychological battery total score. Data of 381 participants (138 patients with aMCI, 106 patients with mAD and 137 HCs) was analyzed in the present study. All participants performed the clock drawing test (CDT) on a tablet computer and underwent the CERAD test battery and depression screening. CERAD total scores were calculated by subtest summation, excluding MMSE scores. All tablet variables (i.e. time in air, time on surface, total time, velocity, pressure, pressure/velocity relation, strokes per minute, time not painting, pen-up stroke length, pen-up/pen-down relation, and CDT score) during dCDT performance were entered in a forward stepwise logistic regression model to assess, which parameters best discriminated between aMCI or mAD and HC. Receiver operating characteristics (ROC) curves were constructed to visualize the specificity in relation to the sensitivity of dCDT variables against CERAD total scores in categorizing the diagnostic groups. dCDT variables provided a slightly better diagnostic accuracy of 81.5% for discrimination of aMCI from HCs than using CERAD total score (accuracy 77.5%). In aMCI patients with normal CDT scores, both dCDT (accuracy 78.0%) and CERAD total scores (accuracy 76.0%) were equally accurate in discriminating against HCs. Finally, in differentiating patients with mAD from healthy individuals, accuracy of both dCDT (93.0%) and CERAD total scores (92.3%) was excellent. Our findings suggest that dCDT is a suitable screening tool to identify early cognitive dysfunction. Its performance is comparable with the time-consuming established psychometric measure (CERAD test battery).

Barbiturates were introduced into medical practice in 1934. They are widely used today as general anesthetics. Although in vitro studies revealed that the activity of a variety of ligandgated channels is modulated by barbiturates, the target(s) mediating the anesthetic actions of barbiturates in vivo are unknown. Studying pentobarbital action in ␤3(N265M) mice harboring ␤3-containing GABA A receptors insensitive to a variety of general anesthetic agents, we found that the immobilizing action of pentobarbital is mediated fully, and the hypnotic action is mediated in part by this receptor subtype. It was surprising that the respiratory depressant action of pentobarbital is indistinguishable between ␤3(N265M) and wild-type mice and thus is mediated by other as-yet-unidentified targets. Whereas the target for the immobilizing and hypnotic actions of pentobarbital seems to be the same as for etomidate and propofol, these latter agents' respiratory depressant actions are mediated by ␤3-containing GABA A receptors. Thus, in contrast to etomidate and propofol, pentobarbital can elicit respiratory depression by a ␤3-independent pathway. Pentobarbital reduced heart rate and body temperature to a slightly smaller extent in ␤3(N265M) mice compared with wild-type mice, indicating that these actions are largely mediated by other targets. Pentobarbital-induced increase of heart rate variability and prolongation of ECG intervals are seen in both ␤3(N265M) mice and wild-type mice, suggesting that they are not dependent on ␤3-containing GABA A receptors. In summary, we show a clear pharmacological dissociation of the immobilizing/hypnotic and respiratory/cardiovascular actions of pentobarbital.The introduction of general anesthetics into medical practice 160 years ago has revolutionized surgery. However, the mechanisms of action of this class of drugs are still only poorly understood. Although general anesthetics have been shown to modulate the activity of a number of proteins (e.g., ligand-gated ion channels; Krasowski and Harrison, 1999) and two-pore domain potassium channels in vitro (Franks and Honore, 2004), the identification of targets mediating specific actions of general anesthetics in vivo has only just begun.GABA A receptors are pentameric ligand-gated ion channels, the majority of them containing two ␣, two ␤, and one ␥ subunit (Backus et al., 1993;Chang et al., 1996). Mutagenesis studies have identified amino acid residues in GABA A receptor ␤ subunits that are crucial for the actions of the general anesthetics etomidate and propofol in vitro (Belelli et al., 1997;Mihic et al., 1997;Krasowski et al., 1998;Siegwart et al., 2002Siegwart et al., , 2003.It has been shown that ␤3(N265M) mice are insensitive to the immobilizing and respiratory depressant action of etomidate and propofol and have a reduced sensitivity to the hypnotic action of these drugs, suggesting that ␤3-containing GABA A receptors mediate these actions, whereas etomidate retains its sedative (motor depressant) action at subanesthetic doses (Jur...

Background: Agents belonging to diverse chemical classes are used clinically as general anesthetics. The molecular targets mediating their actions are however still only poorly defined. Both chemical diversity and substantial differences in the clinical actions of general anesthetics suggest that general anesthetic agents may have distinct pharmacological targets. It was demonstrated previously that the immobilizing action of etomidate and propofol is completely, and the immobilizing action of isoflurane partly mediated, by β3-containing GABA A receptors. This was determined by using the β3(N265M) mice, which carry a point mutation known to decrease the actions of general anesthetics at recombinant GABA A receptors. In this communication, we analyzed the contribution of β3-containing GABA A receptors to the pharmacological actions of isoflurane, etomidate and propofol by means of β3(N265M) mice.

The neuronal circuits mediating the sedative action of diazepam are unknown. Although the motor-depressant action of diazepam is suppressed in ␣1(H101R) homozygous knockin mice expressing diazepam-insensitive ␣1-GABA A receptors, global ␣1-knockout mice show greater motor sedation with diazepam. To clarify this paradox, attributed to compensatory up-regulation of the ␣2 and ␣3 subunits, and to further identify the neuronal circuits supporting diazepam-induced sedation, we generated Emx1-cre-recombinase-mediated conditional mutant mice, selectively lacking the ␣1 subunit (forebrain-specific ␣1 Ϫ/Ϫ ) or expressing either a single wild-type (H) or a single point-mutated (R) ␣1 allele (forebrain-specific ␣1Ϫ/H and ␣1 Ϫ/R mice, respectively) in forebrain glutamatergic neurons. In the rest of the brain, ␣1Ϫ/R mutants are heterozygous ␣1(H101R) mice. Forebrain-specific ␣1 Ϫ/Ϫ mice showed enhanced diazepam-induced motor depression and increased expression of the ␣2 and ␣3 subunits in the neocortex and hippocampus, in comparison with their pseudo-wild-type littermates. Forebrain-specific ␣1 Ϫ/R mice were less sensitive than ␣1Ϫ/H mice to the motor-depressing action of diazepam, but each of these conditional mutants had a similar behavioral response as their corresponding control littermates. Unexpectedly, expression of the ␣1 subunit was reduced in forebrain, notably in ␣1 Ϫ/R mice, and the ␣3 subunit was up-regulated in neocortex, indicating that proper ␣1 subunit expression requires both alleles. In conclusion, conditional manipulation of GABA A receptor ␣1 subunit expression can induce compensatory changes in the affected areas. Specifically, alterations in GABA A receptor expression restricted to forebrain glutamatergic neurons reproduce the behavioral effects seen after a global alteration, thereby implicating these neurons in the motor-sedative effect of diazepam.

General anesthetics have been in clinical use for more than 160 years. Nevertheless, their mechanism of action is still only poorly understood. In this review, we describe studies suggesting that inhibitory ligand-gated ion channels are potential targets for general anesthetics in vitro and describe how the involvement of y-aminobutyric acid (GABA)(A) receptor subtypes in anesthetic actions could be demonstrated by genetic studies in vivo.

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Reduction in the expression or function of α5-subunit-containing GABA A receptors (α5GABA A Rs) leads to improvement in several hippocampus-dependent memory domains. However, studies thus far mostly lack anatomical specificity in terms of neuronal circuits and populations. We demonstrate that mice with a selective knockdown of α5GABA A Rs in CA1 pyramidal neurons (α5CA1KO mice) show improved spatial and trace fear-conditioning memory. Unexpectedly, α5CA1KO mice were comparable to controls in contextual fear-conditioning but showed an impairment in context discrimination, suggesting fine-tuning of activity in CA1 pyramidal cell dendrites through α5-mediated inhibition might be necessary for distinguishing highly similar contexts.