Fragrant Dioxane Derivatives Identify β1-Subunit-containing GABAA Receptors*

Nineteen GABAA receptor (GABAAR) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of β1-subunit-containing GABAARs is unknown. Here we report the discovery of a new structural class of GABAAR positive modulators with unique β1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed α1βxγ2L (x-for 1,2,3) GABAAR FDD were 6 times more potent at β1- versus β2- and β3-containing receptors. Serine at position 265 was essential for the high sensitivity of the β1-subunit to FDD and the β1N286W mutation nearly abolished modulation; vice versa the mutation β3N265S shifted FDD sensitivity toward the β1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to β1-negative cerebellar Purkinje neurons. Immunostaining for the β1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by β1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of β1-containing GABAARs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABAARs.

␥-Aminobutyric acid (GABA), 4 the major inhibitory neurotransmitter in the brain, mediates inhibition via GABA A receptors (GABA A R), heteropentameric proteins constructed from subunits derived from several related gene families with six ␣-, three ␤-, three ␥-, one ␦-, one ⑀-, one -, and one -subunit in mammals. In addition 3 rho ()-subunits contribute to what have been called "GABA C receptors" (1). According to the current model of the GABA A R structure the GABA-binding pocket is formed at the ␣/␤-subunit interface, whereas the benzodiazepine (BZ)-binding pocket is located at the ␣/␥ interface (2) with the subunits arranged pseudo-symmetrically around the ion channel in the sequence ␥-␤-␣-␤-␣ anticlockwise when viewed from the synaptic cleft (3).
Functional receptor compositions are restricted in their number and delineated on the basis of several criteria such as (i) capability of selected subunits to form a heteropentamer with defined pharmacological properties, (ii) a similar pharmacological fingerprint must be found in native receptors, and (iii) immunohistochemical co-localization of these subunits must be demonstrated at synaptic or extrasynaptic sites (1). Only few subunit combinations are currently accepted as "identified" native GABA A R subtypes with ␤1-containing receptors not among them (1) mainly because subunit-selective pharmacological tools are missing.
In total, the GABA A R incorporates more than ten distinct modulatory binding sites targeted by anticonvulsive, antiepileptic, sedative, hypnotic, and anxiolytic compounds belonging to chemically different structural classes (4 -7) with some of them showing receptor type-specific actions. Benzodiazepine (BZ)-site agonists discriminate ␥2-containing GABA A Rs from recombinant ␣␤-receptor types. Moreover, incorporation of different types of ␣-subunits into the receptor influences the sensitivity to different BZ site ligands (8). Several modulators like propofol, pentobarbital, loreclezole, or etomidate are acting at the ␤-subunit of the GABA A R (8 -10). The actions of propofol and pentobarbital are independent, the actions of loreclezole and etomidate are dependent on the type of ␤-subunit present in recombinant GABA A Rs: receptors containing ␤2or ␤3-subunits are potentiated with an EC 50 of about 1 M while ␤1-subunit-containing receptors are potentiated with EC 50 values above 10 M (9, 11).
Searching for further modulators of GABA A R, we screened several libraries of odorants and report now the discovery of a new structural class of GABA A R modulators: fragrant (1, 3)-dioxane derivatives (FDDs) that enhance the action of GABA with much higher potency at the ␤1-subunit-containing compared with the ␤2or ␤3-subunit-containing GABA A R. With the help of FDDs we identify native ␤1-subunit-containing GABA A receptors in histaminergic neurons of the posterior hypothalamus that play a central role in the control of wakefulness.

Expression of Recombinant GABA A Receptors in Xenopus
Oocytes and Electrophysiology-GABA A receptor subunit cDNAs and cRNAs were obtained as follows: rat ␣1 and ␤1 cDNAs were prepared using standard molecular biology procedures. Rat ␤2 receptor cDNA was kindly provided by R. Rupprecht (Munich, Germany). Mouse ␥2L, ␣2, and human ␤3 cDNA was obtained from RZPD (Berlin, Germany). All cDNAs were subcloned into pSGEM (courtesy of M. Hollmann, Bochum, Germany). Plasmids containing ␣1, ␣2, ␤1, ␤1(M286W), ␤1(S265N), ␤2, ␤3, ␤3(N265M), ␤3(N265S), and ␥2L cDNA were linearized with PacI restriction endonuclease. cRNA was synthesized using the AmpliCap T7 high-yield message marker kit (Epicenter, Madison, WI), following the manufacturer's protocol. The Xenopus oocytes expression system and screening paradigms of odorant libraries were established previously in our group and described in detail (12). 3-6 days after injection of cRNA, oocytes were screened for receptor expression by two-electrode voltage-clamp recording. Electrodes were made using a Kopf vertical micropipette puller and filled with 3 M potassium chloride, giving resistances of 0.1-0.5 M⍀. Eggs were placed in an oocyte chamber and superfused with Frog-Ringer solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl 2 , 10 mM Hepes, pH 7.2). Current signals were recorded with a two-electrode voltage-clamp amplifier (TURBO TEC-03, npi, Tamm, Germany), and analyzed using pCLAMP software (Axon Instruments, Union City, CA). The membrane potential was clamped at Ϫ40 to Ϫ60 mV. All experiments were performed at room temperature. Drugs were dissolved in Frog-Ringer and applied manually. To test for incorporation of the ␥2L-subunit, oocytes were screened with 10 M Zn 2ϩ in the presence of 5 M GABA. Whereas ␣␤-subunit combinations are highly sensitive for an inhibition by Zn 2ϩ , the ␣␤␥ isoforms are insensitive (13). For the construction of dose-response curves and potentiation experiments the GABA working concentration (close to the EC 15 ) had to be determined for each individual oocyte: for this purpose 1, 3, and 10 M of GABA as well as saturating concentrations 300 or 1000 M were applied before each experiment.
Electrophysiology in Native Neurons-Neurons acutely isolated from hypothalamus and cerebellum were prepared from the brains of adult (P28 -50) male mice (strain 129/Sv) or Wistar rats (P21-28). Transverse slices (450-m thick) were cut and incubated for 1 h in a solution containing (mM): NaCl 125, KCl 3.7, CaCl 2 1.0, MgCl 2 1.0, NaH 2 PO 4 1.3, NaHCO 3 23, D-glucose 10, phenol red 0.01%, bubbled with carbogen (pH 7.4). The tuberomamillary nucleus (TMN) was dissected from posterior hypothalamic slices after incubation with papain in crude form (0.3-0.5 mg/ml) for 40 min at 37°C. After rinsing, the tissue was placed in a small volume of recording solution with the following composition (in mM): NaCl 150, KCl 3.7, CaCl 2 2.0, MgCl 2 2.0, HEPES 10, glucose 10 (pH 7.4). Cells were separated by gentle pipetting and placed in the recording chamber. Purkinje neurons (PN) and TMN neurons used for simultaneous recordings of sIPSCs and GABA-evoked currents were isolated in the recording chamber with the help of a vibrodissociation device (14) from slices briefly (5-10 min) pre-incubated with papaine. PNs and TMN neurons were identified by the typical shape and size and with single cell RT PCR by the expression of GAD67 (GABA-synthetizing enzyme) (15) or histidine decarboxylase (HDC, the histamine-producing enzyme) (16,17), respectively.
Whole-cell patch-clamp recordings in voltage clamp mode, fast drug application, and single cell RT-PCR procedures were performed as described previously (16,18). Briefly, patch electrodes were sterilized by autoclaving and filled with the following solution (in mM): 140 KCl, 2 MgCl 2 , 0.5 CaCl 2 , 5 EGTA, 10 HEPES/KOH, adjusted to pH 7.2. The cells were voltageclamped by an EPC-9 amplifier. The holding potential was Ϫ50 mV. An acutely isolated cell was lifted into the major chute of the application system, where it was continuously perfused with the sterile control bath solution. The substances were applied through the glass capillary (application tube), 0.08 mm in diameter. All solutions flowed continuously, gravity-driven, at the same speed and lateral movements of the capillaries exposed a cell either to control or test solutions. GABA (1-10 M) responses were compared with the maximal GABA response (500 M) in the beginning of each experiment. Modulators were applied together with GABA taken at a concentration below EC 30 .
Experiments were conducted and analyzed with commercially available software (TIDA for Windows, HEKA, Lambrecht, Germany). Fitting of dose-response data points in experiments with fragrant dioxane derivatives was performed with Equation 1, where R is the relative potentiation as a fraction of maximal potentiation R max , EC 50 is the modulator concentration producing a half-maximal potentiation of the control response, [modulator] is modulator (odorant) concentration, and n is the Hill slope. Data are presented as the mean Ϯ S.E.
Peak amplitude, the time to peak (rise time), time to decay, area, and frequency of spontaneous IPSCs were analyzed with MiniAnalysis 4.2 (Synaptosoft, Leonia, NJ). The detection threshold was set to 5 pA amplitude and 20 pA ϫ ms area. The frequency of sIPSCs was determined from all automatically detected events after false positives were removed during visual inspection of the recording traces. Experiments were done in the presence of the AMPA receptor antagonist CNQX (10 M). Previous studies using the same preparation (16,18) demonstrated that under these conditions all recorded sIPSCs are GABAergic as they can be completely blocked by the selective GABA A receptor antagonist gabazine (10 M). Detection parameters were set in a way that time to decay (to 30% of peak in a 100 ms window) and amplitude of selected single events on average did not differ by more than 10% (except for the maximal concentrations of modulator) from corresponding values obtained after "curve fitting" of the same events after their alignment followed by their averaging (decay time constants were obtained in this case by fitting a double exponential to the falling phase of the averaged events). Times to decay and amplitudes were plotted as cumulative histograms and compared with the Kolmogorov-Smirnov 2 sample test in each cell between control (before and washout periods together) conditions versus presence of modulator (each of three testing periods lasted 60 -90 s, 23-256 single events/60 s recording period were selected for the analysis). The significance level was set at p Ͻ 0.05. GABA A R Expression Analysis in Native Cells (Single Cell RT-PCR)-Mouse GABA A R cDNAs were amplified in a first amplification round with degenerate subfamily-specific primers, followed by the subunit-specific amplification in the second round. Magnesium concentration used in all reactions was 2.5 mM, annealing temperature was 50°C in most of the reactions if not indicated otherwise. The ␣-subunit-subfamily was ampli-  (16). Thin-walled PCR tubes contained a mixture of first strand cDNA template (1 l), 10ϫ PCR buffer, 10 pM each of sense and antisense primer, 200 M of each dNTP, and 2.5 units of Taq polymerase. The final reaction volume was adjusted to 10 l with nuclease-free water (Promega, Mannheim, Germany). The magnesium was taken at 2.5 mM. The Taq enzyme, PCR buffer, Mg 2ϩ solution, and four dNTPs were all purchased from Qiagen (Erkrath, Germany). All oligonucleotides were synthesized by MWG-Biotech (Ebersberg, Germany). Amplification was performed on a thermal cycler (Mastercycler, Eppendorf, Germany). A two round amplification strategy was used in each protocol. In each round 35 cycles of the following thermal programs were used: denaturation at 94°C for 48 s, annealing at 50°C for 48 s, and extension at 72°C for 1 min. For the second amplification round 1 l of the product of the first PCR was used as a template. Products were visualized by staining with ethidium bromide and analyzed by electrophoresis in 2% agarose gels. Randomly selected PCR products obtained after two amplification rounds were purified in water and sequenced. The obtained sequences corresponded to the known one for the mouse (GenBank TM , accession number): Primary Dissociated Cultures, Electrophysiological Recordings, and siRNA-based Knock-down Technique-Primary cultures from posterior hypothalamus were prepared as previously described (18). Whole-cell voltage clamp recordings were performed from non-identified hypothalamic neurons on days 10 -21 after plating using an application system adapted for adherent cells (18). Multielectrode array (MEA) recordings were performed from cultured neurons as previously described (19). In knock-down experiments, the culture medium was changed on day 10 either to transfection medium alone or to transfection medium with 4 siRNAs (100 M, Accell SMART pool, Thermo Scientific) directed toward the following target sequences on mouse GABA A R ␤1-subunit (NM_008069): GCAGCAUGCAUGAUGGAUC; CCCUGGAGA-UUGAAA-GUUA; GGAUCUUACUGGAUUA-UUU, CCACCAAUUG-CUUUGUUUA. A non-targeting siRNA pool was used as a negative control (no difference from control in GABA response sensitivity to FDD in cultured hypothalamic neurons was observed in three experiments). On day 15, patch clamp recordings were done from cultures treated in a parallel way, some cultures were used for the mRNA isolation and semiquantitative real-time RT-PCR analysis of the receptor expression (for methods see Ref. 19).

TABLE 1 Comparison of the GABA-modulatory and GABA-mimetic activities of PI 24513 across WT and point-mutated GABA A receptors expressed in Xenopus oocytes
The GABA-mimetic action is expressed relative to the maximal response to GABA. Modulatory efficacy was calculated as the potentiation (I(GABAϩ PI24513)/ I(GABA)) of a GABA (EC x ) evoked current by 1 mM PI24513. Data are means of 3-10 experiments Ϯ S.E.
The presence of methyl groups in R4,5,6, hydrogen at R7 and hydrogen or methyl group in R3 correlated with high activity.
Replacing the methyl groups either by hydrogen or at the positions R4 and R5 by ethyl groups (PI24514) reduced FDD activity (supplemental Table S1). Our screening data allow a clear activity ranking of the FDD but leave open the question whether weak GABA A R modulation by 100 M of FDD (groups B and C) may be a result of low potency, low efficacy, or both.
The following experiments demonstrated that the ␤-subunit is necessary and sufficient for the modulatory action of FDDs. In Xenopus oocytes expressing homomeric ␤1 GABA A R, 100 M PI24513 potentiated the response to 10 M GABA 5.4 Ϯ 0.49-fold whereas the direct activation by 100 M PI24513 was only 33 Ϯ 64% of the maximal GABA-response (n ϭ 4) (Fig.  1G). Previous mutational studies have identified two sites on the ␤-subunit involved in the action of propofol and etomidate: 1) the asparagine (N265) residue in the transmembrane domain 2 (TM2) region (20)) and 2) the methionine (M286) residue in the TM3 region (5) (for more details see location of the aforementioned mutations on aligned rat ␤ and 1-subunits of the GABA A R and the RDL receptor from Drosophila in Fig. 1 of the study by Siegwart et al. (10)). In contrast to ␤2and ␤3-subunits, the ␤1-subunit contains a serine residue (instead of asparagine) at position 265, which underlies the relative insensitivity of ␤1-containing GABA A Rs to loreclezole and etomidate (11). Therefore, we investigated the action of FDDs after introduction of the mutation M286W in the TM3 region of the ␤1-subunit. The GABA-evoked currents in oocytes expressing ␣1␤1 M286W ␥2L GABA A R were only weakly potentiated by 100 M PI24513 (1,6 Ϯ 0.16-fold), whereas wild-type receptors were potentiated under the same conditions by a factor of 6.4 Ϯ 1.2 (Fig. 1G, see also difference in potentiation by 1 mM PI24513 in Table 1). The mutation ␤1S265N in the TM2 region generated receptors with FDD sensitivity of the ␤3-type (EC 50 155 Ϯ 34 M versus GABA A R of the same composition with a wildtype ␤1-subunit EC 50 32.5 Ϯ 5.6 M). The mutation ␤3N265S shifted FDD potency toward the ␤1 receptor-type (EC 50 47 Ϯ 7 M versus 196 Ϯ 42 M in WT receptors, Table 1), while the mutation ␤3N265M nearly abolished modulation by FDD: the averaged potentiation by PI24513 (1 mM) was 72 Ϯ 19% over control, while corresponding WT receptors were potentiated to 850 Ϯ 280% of control ( Table 1). Presence of the ␥-subunit in the receptor did not affect the potency of modulation by FDD ( Table 1).
As the action of propofol slightly differs between ␣1and ␣2-containing recombinant GABA A Rs (21), we compared the potencies of PI24513 at ␣1␤1␥2L and ␣2␤1␥2L receptors in the modulation of GABA-evoked currents. The difference in EC 50 values was not significant between the two receptor types (n ϭ 5 for each, p ϭ 0.61, t test) when expressed in Xenopus oocytes from the same batch (parallel experiments).
The FDD VC potentiated submaximal GABA-evoked currents (EC 15 Ϯ 4 ) in TMN neurons with an EC 50 ϭ 23 Ϯ 2. Localization of ␤1 immunoreactivity was investigated in rat brain slices. Antibodies were proven to be specific on recombinant GABA A Rs expressed in HEK293 cells (Fig. 2D). Co-localization analysis of ␤1 immunoreactivity within histaminergic (histidine decarboxylase-positive) neurons revealed that virtually all TMN neurons carry ␤1-protein on the membrane surface and to the lesser extent and infrequent within the cytoplasm. Some non-identified neuropil elements were ␤1-positive. In addition, ␤1-antisera stained the nuclear envelope in many HDC-positive neurons (Fig. 2E).
FDDs Reveal Synaptic Localization of ␤1-Subunit-containing GABA A Rs in TMN Neurons-We used native neurons to examine the physiological effect of FDDs on synaptic GABA A R-mediated currents (Fig. 3). Spontaneous inhibitory postsynaptic currents (sIPSCs) occur as a result of GABA release from attached synaptic boutons (24). Their kinetics but not frequency or amplitude was affected by the FDDs. In the presence of VC (20 M) the decay time constant was significantly prolonged from 18 Ϯ 1.5 ms in control to 47 Ϯ 5 ms (n ϭ 6) in rat TMN neurons. Similar effects were observed for PI24513 (n ϭ 7, Fig. 3A). Different concentrations of FDD were applied in the next experiments to mouse PN and TMN neurons in order to determine threshold and EC 50 concentrations for the prolongation of sIPSC decay kinetics. In each cell, decay times calculated for individual spontaneous synaptic events collected within 60 -90 s during control and FDD periods were compared (see Fig. 3B). VC at 1 M prolonged sIPSCs recorded from TMN neurons (n ϭ 7) from 22.5 Ϯ 3.6 ms to 29.63 Ϯ 5.7 ms (132% of control). In 6 cells the difference in decay kinetics between control and FDD period was significant (Kolmogorov-Smirnov 2 sample test). At further concentrations tested (VC 5, 25, and 100 M) decay kinetics were prolonged by 89% (n ϭ 9), 141% (n ϭ 9), and 241% (n ϭ 7) over control values, respectively (Fig. 3, C and D). At concentrations higher than 1 M VC significantly prolonged sIPSCs in all cells tested. Concentrations larger than 100 M were not tested in TMN neurons as they produced large amplitude direct inward currents, which shunted sIPSCs. In PN neurons VC at 20 M increased the decay kinetics significantly only in one cell out of 4 tested (by 25%) and at 125, 250, and 1000 M produced significant effects in all PN tested (prolonged sIPSC decay time by 106, 176, and 215% over control, respectively) (Fig.  3C). Calculated EC 50 values for the sIPSC prolongation by FDD were 14 Ϯ 8.0 M and 100 Ϯ 10.5 M for TMN and Purkinje neurons, respectively. Thus, ␤1-subunit-rich synaptic GABA A receptors can be identified with FDD in TMN neurons by their high sensitivity for FDD modulation.
Knockdown of ␤1 mRNA and Protein in Posterior Hypothalamic Cultures Changes FDD Modulation-Somatic membranes and synaptic clusters on MAP2-positive dendrites were stained with a ␤1-specific antibody in hypothalamic cultures (Fig. 4A). In addition, ␤1 immunoreactivity was also found in the nuclei of neurons and glia. Five days treatment with ␤1-specific siRNA drastically reduced the immunoreactivity (Fig. 4B).

DISCUSSION
We describe here a new class of positive GABA A R modulators with a unique specificity for receptors containing the ␤1-subunit. Following its detection in recombinant receptors, the ␤1-subunit selectivity of FDD is characterized in recordings from native neurons, providing first time evidence for the synaptic localization of ␤1-containing GABA A Rs in hypothalamic neurons and their role in somatic GABA responses. Pharmacological detection of native ␤1-containing GABA A Rs in hypothalamic neurons was supported further by immunohistochemistry and by an in vitro knockdown technique, demonstrating superior performance of FDD for the detection of ␤1-containing GABA A Rs in comparison to previously available pharmacological tools such as salicylidene salicylhydrazide (23). Recombinant receptors containing ␤1-subunits were nearly 6ϫ more sensitive to FDD compared with receptors composed of ␤2or ␤3-subunits. Macroscopic GABA-evoked currents recorded from Purkinje neurons lacking expression of the ␤1-subunit were 4.5 times less sensitive to FDD compared with the TMN neurons, while FDD potency in synaptic current modulation differed 7.1 times between these neurons. Although an ideal selectivity would be more than 10 times difference in potency, in all our different experimental approaches ␤1-containing GABA A receptors showed significantly higher modulation compared with the ␤1-lacking receptors on the whole concentration scale. The action of FDD was independent of ␥-subunit and totally relied on the type of ␤-subunit present in the GABA A R. Two different ␣-subunits tested in recombinant GABA A Rs (␣1 and ␣2) did not differ in their FDD sensitivity, however we cannot exclude that GABA-binding sites formed by other ␣-subunits may carry different properties. In keeping with the block of etomidate-evoked current by bicuculline in a study by Belelli et al. (20) and the block of pentobarbital-evoked current by bicuculline and gabazine (25), FDD-evoked currents in our study were inhibited by gabazine, leaving open the possibility that the GABA-binding site is involved. However, an "allosteric model" suggests that gabazine inhibits the pentobar-bital-current by reducing the probability of channel opening acting as an "inverse agonist" (25).
Decay kinetics of sIPSCs recorded from acutely isolated Purkinje and TMN neurons differed significantly in their modulation by FDD. While in most of the TMN neurons (86%) decay kinetics of sIPSCs were significantly prolonged by FDD 1 M, and half-maximal prolongation was achieved at 14 M, in Purkinje neurons minimal and half-effective concentrations were 20 and 100 M, respectively. Thus, FDD reveal a synaptic localization of the ␤1-subunit in hypothalamic TMN neurons. As the ␤1-subunit was never expressed alone in TMN neurons (single cell RT-PCR data), it is likely, that ␤3and ␤1-subunits are either co-assembled in the same receptors (26) or present as separate populations carrying different functions. A recent study demonstrated reduced modulation by propofol (1.5 M) of sIPSCs in TMN neurons recorded from ␤3N265M mice (27), supporting functional presence of ␤3-containing GABA A Rs (28); however, propofol effects on neuronal firing of TMN neurons were not investigated.
Future studies employing mice with mutated GABA A Rs will answer the question about the relative contributions of all three ␤-subunits in controlling the firing of wake active hypothalamic neurons. Lesions in the posterior hypothalamus, which contains wake-on pacemaker neurons, are responsible for the encephalitis lethargica von Economo (29). GABA released from axons of ventrolateral preoptic area (VLPO) neurons during sleep (30,31) inhibits two major groups of posterior hypothalamic wake-promoting neurons, the orexin-and histamine-producing neurons, which grow and can be recorded in posterior hypothalamic cultures (18).
We demonstrate a high sensitivity of the firing of posterior hypothalamic neurons to FDD, only 3ϫ lower than the propofol sensitivity. Moreover, incubation with ␤1-siRNA significantly decreased inhibition of neuronal firing by FDD at a large concentration range, but did not affect modulation by propofol. As a result, FDD became 11 times less potent in inhibiting neuronal firing compared with propofol after ␤1-siRNA treatment. Thus, the ␤1-containing GABA A R population controls not only synaptic integration but also the firing of hypothalamic neurons. Asterisks indicate staining of the nuclei of glial (MAP2-negative) cell. B, distribution of ␤1 immunoreactivity (AF488) in neurons after patch-clamp recordings (filled with biocytin, in red). Ten-day-old cultures were grown further either in transfection medium without siRNA (control) or with ␤1-siRNA for 5 days. Left: ␤1-subunit immunoreactivity, right: co-localization of biocytin and ␤1 immunoreactivities. Scale bars in A and B, 20 m. C, dose-response curves for PI 24513-modulation of GABA responses (EC 10 -20 ) differ between control and siRNA-treated hypothalamic neurons. Averaged (4 -9 cells) EC 50 values are indicated. D, firing frequency of total population of hypothalamic neurons, normalized to the corresponding control value, measured with MEAs as total number of spikes (NoS) per min, is dose-dependently reduced by FDD or propofol. Reduced sensitivity to FDD is seen after ␤1-siRNA treatment. Open symbols indicate control measurements, filled symbols the measurements in cultures treated with ␤1-siRNA. Significance levels for the difference in modulatory action of FDD are indicated with stars. *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.005.