Human and Rat mGlu2 Potentiator FLIPR Assays.

AV12 cell lines stably expressing the human or rat mGlu2 receptor and cotransfected with the rat glutamate transporter excitatory amino acid transporter 1 and the Gα15 subunit were used for these studies. The expression of Gα15 allows G_i-coupled receptors to signal through the phospholipase C pathway, resulting in the ability to measure receptor activation by a fluorometric calcium response assay. The cell lines were maintained by culturing in Dulbecco's modified Eagle's medium with high glucose and pyridoxine hydrochloride supplemented with 5% heat inactivated, dialyzed fetal bovine serum, 1 mM sodium pyruvate, 10 mM HEPES, 1 mM l-glutamine, and 5 μg/ml blasticidin (all media purchased from Invitrogen, Carlsbad, CA). Confluent cultures were passaged biweekly by using an enzyme-free dissociation solution (Millipore Bioscience Research Reagents, Temecula, CA). Cells were harvested 24 h before assay and dispensed by using a Matrix Well Mate cell seeder (Thermo Fisher Scientific, Waltham, MA) at 85,000 (human) or 65,000 (rat) cells per well into 96-well, black-walled, poly-d-lysine-coated plates (BD Biosciences, San Jose, CA) in medium containing only 250 μM l-glutamine (freshly added).

Intracellular calcium levels were monitored before and after the addition of compounds by using a fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA). The assay buffer was comprised of Hank's buffered salt solution (Sigma-Aldrich, St. Louis, MO) supplemented with 20 mM HEPES. The medium was removed, and the cells were incubated with 8 μM Fluo-3AM (Invitrogen; 50 μl per well) in assay buffer for 90 min at 25°C. The dye solution was removed and replaced with fresh assay buffer (50 μl per well). A single-addition FLIPR assay generating an 11-point concentration response curve for the agonist glutamate (Thermo Fisher Scientific) was conducted before each experiment. The results were analyzed by using Prism version 4.03 (GraphPad Software Inc., San Diego, CA) to calculate the concentrations of glutamate needed to induce the EC₁₀ responses and monitor the general sensitivity of the cells for the agonist.

The compound was tested in a two-addition FLIPR assay using a 10-point concentration response profile starting at a final concentration of 25 μM (agonist mode) or 12.5 μM (potentiator mode). A 3-fold dilution series in dimethyl sulfoxide (DMSO) was followed by a single dilution into assay buffer; the final concentration of DMSO was 0.625%. After taking an initial 5-s fluorescent read on the FLIPR instrument, compound was added to the cell plate (50 μl per well). Data were collected every second for the first 30 s and then every 3 s for a total of 90 s to detect agonist activity. Immediately thereafter, the second addition consisting of 100 μl of glutamate in assay buffer (typically approximately 1 μM) was added to the cell plate, generating an EC₁₀ response. After the second addition, data were collected every second for 29 images and then every 3 s for 15 images. The maximal response was defined as that induced by EC_max (100 μM glutamate). The compound effect was measured as maximal minus minimal peak heights in relative fluorescent units corrected for basal fluorescence measured in the absence of glutamate. Determinations were carried out with single plates. Agonist effects were quantified as percentage of stimulation induced by compound alone relative to the maximal glutamate response. Potentiation effects were quantified as percentage of increase in the presence of an EC₁₀ response in glutamate relative to the EC_max response. Using calcium mobilization assays analogous to those described above, THIIC was tested for activity in agonist, potentiator, or antagonist modes at the human mGluR1, mGluR3, mGluR4, mGluR5, and mGluR8 receptor subtypes, each stably expressed in AV12 cells. Maximum concentrations were 12.5 μM (antagonist and potentiator modes) and 25 μM (agonist mode). All data were calculated as relative EC₅₀ values by using a four-parameter logistic curve-fitting program (ActivityBase version 5.3.1.22; IDBS, Surrey, U.K.).

The observed ability to potentiate the glutamate activity was confirmed by using glutamate concentration-response curves generated in the presence of increasing concentrations of THIIC. Glutamate was diluted in assay buffer through 12 concentrations ranging from 100 μM to 49 nM, and the compound was three times serially diluted in DMSO for an eight-point concentration response profile starting at a final concentration of 12.5 μM. Data collection and analysis were as described previously.

GTPγ³⁵S Binding Assay.

GTPγ³⁵S binding was determined by using an antibody capture scintillation proximity technique in a 96-well plate format. In brief, 100 μl (20–40 fmol/well) of membrane preparations from AV12 cells that ectopically express either human or rat mGluR2 was incubated for 30 min with 50 μl of test compound. GDP (1 μM final concentration) was added to receptor membranes before incubation. Dose-response curves were achieved with increasing amounts of THIIC in the presence of increasing amounts of the orthosteric agonist glutamate (Tocris Bioscience, Ellisville, MO). After the incubation period, 50 μl of diluted GTP-γ-[³⁵S] (500 pM final concentration; PerkinElmer Life and Analytical Sciences, Waltham, MA) was added to each well and incubated for 30 min. The labeled membranes were then solubilized with 0.27% Nonidet P40 followed by a 3-h incubation with 20 μl (final dilution of 1:200) of anti-Gα_i-3 rabbit polyclonal antibody (Covance Research Products, Princeton, NJ) and 50 μl (1.25 mg/well) of anti-rabbit scintillation proximity assay beads (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). The plates were centrifuged for 10 min at 700g and counted for 1 min per well by using a Wallac MicroBeta TriLux scintillation counter (PerkinElmer Life and Analytical Sciences). All incubations took place at room temperature in GTP-binding assay buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgC1₂, pH 7.4). Data were analyzed by using nonlinear regression for a sigmoidal concentration response curve (Prism version 4.03). Background was subtracted from all wells, data were normalized to a full agonist response (100 μM glutamate), and efficacy was expressed as a percentage of maximal response. Mean EC₅₀ were calculated as a mean of three independent determinations ± S.E.M. K_b and cooperativity factor were calculated from EC₅₀-generated dose ratios imported into an allosteric Schild regression algorithm: Y = (K_b + α × B)/(K_b + B), where α = 10L̂ogα, K_b = 10L̂ogK_b, and B = 10X̂.

Selectivity Panel.

THIIC was tested for binding to dopamine and serotonin receptors by using receptor binding assays performed according to methods reported previously (Rorick-Kehn et al., 2007a). An additional panel of more than 30 different biogenic amine receptors, neuropeptide receptors, ion channel binding sites, and neurotransmitter transporter binding assays were run by CEREP Inc. (Celle L'Evescault, France). The targets run were: human adenosine A3 receptors; acetylcholinesterase; angiotensin II; adrenergic receptors α1 (nonselective), α2 (nonselective), β1 and β2; norepinephrine transporter; rat brain benzodiazepine receptor; dopaminergic receptors D₁ and D_2s; histaminergic receptors H₁ and H₂; muscarinic receptors M₂ and M3; neurokinin receptor 1; opioid receptor μ; serotonin receptors 5HT2B agonist site, rat Ca²⁺ channel verapamil site; rat brain voltage-gated potassium channel (K+V channel); rat brain small-conductance Ca²⁺-activated K+ channel (SK+Ca channel); rat Na⁺ channel (site 2); and rat Cl⁻ channel. All assays were run using recombinant human receptors, except where noted. The full methods and references can be found on the Cerep website (www.cerep.fr). The assays were run at 1 μM and 10 μM THIIC, and the percentage of inhibition is given as the average of three determinations. When significant displacement of radioligand was observed (>50% inhibition at 1 μM), complete concentration-dependent displacement curves (in triplicate) were constructed to generate IC₅₀ values.

Mouse Forced-Swim Test.

Male, NIH Swiss mice (Harlan, Indianapolis, IN) weighing 20 to 25 g were housed in plastic cages (40.6 × 20.3 × 15.2 cm) with 10 to 12 mice/cage in a vivarium at least 7 days before the experiments. Mice were maintained on a 12-h light/dark cycle (6:00 AM/6:00 PM), and all procedures were performed between noon and 4:00 PM. Animals were removed from the vivarium to the testing area in their home cages and allowed to adapt to the new environment for at least 1 h before testing. The forced-swim test was performed using the original method described by Porsolt et al. (1977). In brief, mice were placed individually in clear plastic cylinders (10 cm in diameter by 25 cm in height) filled to 6 cm with 22 to 25°C water for 6 min. The duration of immobility was recorded during the last 4 min of a 6-min trial. A mouse was regarded as immobile when floating motionless or making only those movements necessary to keep its head above the water. Dose-effect functions for THIIC were performed after both intraperitoneal (30 min before) and oral dosing (60 min before). Imipramine was used as a comparator standard (15 mg/kg i.p., 30 min before). Statistical analyses were performed by analysis of variance (ANOVA) followed by Dunnett's test.

DRL 72-s Behavior in Rats.

Male Sprague-Dawley rats weighing between 300 and 350 g at the beginning of the behavioral experiments (Holtzman, Madison, WI) were housed in pairs. The colony room was maintained at 20°C and 60% relative humidity. The room was illuminated 12 h/day (7:00 AM to 7:00 PM). All rats had free access to laboratory chow (Teklad 4% rat diet; Harlan Teklad, Madison, WI) except during experimental sessions. Water was available for a 20-min period after the daily behavioral session. Sixteen operant-conditioning chambers (MED Associates, St. Albans, VT) were used. The lever in these chambers was mounted on one wall with the water access next to the lever in the middle of the wall. A reinforced response caused the dipper (0.02-ml cup) to be lifted from a water trough to an opening in the floor of the access port for 4 s. The house light, which was mounted on the opposite wall, was turned on when the session began, remained on throughout the entire session, and was turned off at the end of the session. Each experimental chamber was enclosed in a separate sound-attenuating cubicle equipped with a white noise generator to provide masking noise. Rats were water deprived for 22.5 h before each session. Each rat was initially trained under an alternative fixed ratio 1, fixed time 1-min schedule for water reinforcement where each response was reinforced, and water was also provided every minute if a response did not occur. The few rats that did not acquire lever-pressing behavior after three daily 1-h sessions under this schedule were trained by the experimenter using the method of successive approximation. After the rats had acquired lever-pressing behavior, they were trained during daily DRL 18-s sessions for 2 weeks before moving directly to DRL 72-s sessions. Under the DRL schedules, a response only produced water if it occurred at least 18 or 72 s after the last water delivery; responses before the designated time period reset the timer. Responding on these sessions became stable after 8 weeks. Experimental sessions lasted for 1 h and were conducted 5 days/week during light hours. The data analyses were conducted on the raw response and reinforcement data. All behavioral data are expressed as the mean ± S.E.M. normalized to the vehicle or vehicle/vehicle condition. The effects of THIIC and imipramine were analyzed with a one-way repeated-measures ANOVA. The level of significance was set for p < 0.05 for all analyses.

Dominant-Submissive Test in Rats.

Experimentally naive male Sprague-Dawley rats (Harlan), weighing 160 to 180 g at the start of the study, were assigned randomly to pairs, and the paired animals remained separated through the course of the experiment, except during the 5-min testing periods. Rats were maintained on a 12 h light/dark cycle (6:00 AM/6:00 PM), and all procedures were performed between 12:00 PM and 4:00 PM in ambient room light. The testing apparatus was constructed from transparent plastic material and consisted of two identical chambers (24 × 17 × 14 cm) connected by a round tunnel (4.5 cm diameter × 52 cm long). A container (10-ml beaker) of sweetened milk (9% sucrose) was placed in an opening in the floor at the midpoint of the tunnel. Animals were food-deprived overnight before the first test session. During the testing period, each member of a pair was placed in the different chambers of the testing apparatus, the gates were opened, and the time spent drinking was recorded for each animal for a 5-min period. At the end of the 5-min testing period, animals were returned to their home cages and given free access to food for 1 h. The animals were also given free access to food from Friday afternoon (after the test session) to Sunday morning, when they were once again food-deprived. During the first week of the testing (acclimation week), drinking time was not scored. During the second week of testing (selection week), the time spent drinking was recorded. Pairs of animals that passed the following criteria continued into the drug administration phase of the experiment: 1) the difference between the average daily drinking scores of the two animals was significant (two-tailed t test, p < 0.05), and 2) the dominant animal's score was at least 25% greater than the submissive animal's score. Any pairs not passing the selection criteria were dropped from the study. The animal of a pair with the higher drinking score was labeled as “dominant,” and the animal of a pair with lower drinking score was labeled as “submissive”. The submissive rat of each pair was treated with the THIIC (3 or 30 mg/kg) or imipramine (10 mg/kg), whereas the dominant rat of each pair was treated with vehicle for the next 21 days. The 5-min test was repeated once daily, except weekends, for 21 days after the beginning of the drug treatment. Dominance levels were calculated as the difference in daily drinking scores between paired rats. The daily dominance level values were averaged over each week, and statistical comparisons were made by analysis of repeated measurements coupled Tukey's post hoc test.

Marble Burying and Rotorod Performances.

Male CD1 mice were used as described previously (Li et al.,2006). In brief, mice were dosed with either THIIC (intraperitoneally) or chlordiazepoxide (intraperitoneally) and tested on a rotating rod without prior training. Mice were scored as failing if they fell off the rotorod on two occasions during a 2-min test. Marble-burying behavior was assessed in the same mice immediately after rotorod testing. The number of marbles buried out of 20 by sawdust bedding by at least 2/3 was measured. The number of marbles buried was evaluated by ANOVA followed by Dunnett's test; rotorod performances were evaluated by Fisher's exact probability test.

Rat Stress-Induced Hyperthermia.

Male Fischer F-344 rats (Harlan), weighing between 275 and 350 g, were individually housed with food and water available ad libitum and maintained on a 12-h light/dark cycle (lights on at 6:00 AM). The rats were fasted for approximately 12 to 18 h before the experiment. Rats were transported from the colony room in groups of 10 to a procedure room for dosing. Rats were dosed orally in a dose volume of 2 ml/kg with THIIC (1, 3, 10, and 30 mg/kg). The mGlu5 receptor antagonist MTEP, which has demonstrated robust anxiolytic-like activity in preclinical models, was used as a positive control (10 mg/kg p.o.). Immediately after dosing, rats were returned to their home cage, and the dosing room (room A) was darkened for the remainder of the 4-h pretreatment period. After the pretreatment period, rats were taken individually to a brightly lit adjacent room (room B) where baseline body temperatures were determined by insertion of a rectal probe lubricated with mineral oil. Core body temperature was assessed using a Physitemp BAT-12 Microprobe Thermometer with a Physitemp RET-2 rat rectal probe (Physitemp Instruments Inc., Clifton, NJ) inserted approximately 2 cm into the rectum and is designated as the baseline body temperature, T1, in °C. The rat was then placed back in the home cage and remained in room B. Ten minutes later, a second body temperature measurement was recorded (T2). The difference between the first and second body temperature measurements (T2 − T1) was used as an index of stress-induced hyperthermia. Data represent means and S.E.M. and were subjected to one-way ANOVA followed by Dunnett's test.

Reversal of Stress-Induced Increase in Cerebellar cGMP.

Male CF-1 mice (18–20 g) (Harlan) were housed in an enriched environment in groups of eight mice per cage with food and automated water available ad libitum. Animals were kept under a 12-h light/dark cycle with lights on at 6:00 AM in a temperature- and humidity-controlled environment for 5 to 7 days before their use. The mice were kept in their home cages to avoid stress and received an intraperitoneal injection of either vehicle, THIIC (3, 10, and 30 mg/kg), or alprazolam (1 mg/kg) 30 min before stressor (foot shock). To evaluate the effects of the test compounds on basal cGMP levels, vehicle control “no stress” mice were sacrificed without being subjected to a foot shock. Vehicle stress, THIIC-treated, and alprazolam-treated mice were subjected to a stressor of inescapable electric foot shock (1-mA intensity for 10 s) in a metal cage with a stainless-steel grid floor (Coulbourn Instruments, Allentown, PA), and immediately sacrificed for estimation of basal cGMP levels. All mice were euthanized by using a beam of microwave radiation focused on the skull for 0.5 s (Thermax Thermatron, Louisville, KY). The cerebellum was quickly removed from the skull, and the weight was recorded. The tissue was then placed in a polystyrene tube containing 2.0 ml of 1% perchloric acid on ice. Tissues were then homogenized and incubated on ice for 30 min after which the samples were boiled for 5 min. The tubes were then centrifuged at 11,700g for 20 min at room temperature. The supernatant was acetylated using a 2:1 ratio of triethylamine (EM Science, Gibbstown, NJ) and acetic anhydride. The samples were immediately vortexed and then centrifuged at 13,000g for 20 min at 4°C. The cGMP assay buffer included 0.05 M sodium acetate, trihydrate (6.8 g/liter), and 0.1% sodium azide adjusted to pH 6.2 with acetic acid. For the radioligand tracer, a separate tracer buffer was prepared using the cGMP assay buffer and adding 0.002 M EDTA disodium salt dihydrate (0.7 g/liter), with 1% normal rabbit serum (PerkinElmer Life and Analytical Sciences) added just before use.

[¹²⁵I]cGMP, obtained from PerkinElmer Life and Analytical Sciences (SA 2200 Ci/mmol, 32.3 μCi/ml), was prepared in the tracer diluent, pH 6.2, at a concentration of 0.75 μCi/10-ml tracer buffer per FlashPlate (PerkinElmer Life and Analytical Sciences). A cGMP standard curve was prepared by using guanosine-3′:5′-cyclic monophosphate lyophilized standard (PerkinElmer Life and Analytical Sciences) reconstituted with 2 ml of distilled water to make a 2000 pm/ml solution (stored at 4°C). This stock was diluted with assay buffer to generate a nine-point standard curve. A 1-ml aliquot of each standard, including a blank, were acetylated and then vortexed immediately. One hundred microliters of each standard was added, in duplicate, to flashplate wells, including two 100-μl aliquots directly from the stock cGMP standard bottle for nonspecific binding determination. Forty microliters of each sample, in duplicate, was added to flashplate wells containing 60 μl of cGMP assay buffer for a total sample volume of 100 μl (yields a 1:50 final dilution). One hundred microliters of [¹²⁵I]cGMP was added to all flashplate wells, and plates were covered before being shaken overnight (16–24 h) at 2 to 8°C using a titer plate shaker. Contents of the wells were aspirated and the plates were resealed. Plates were then counted for 1 min/well in a Packard TopCount radioactive counter (PerkinElmer Life and Analytical Sciences).

cGMP data calculations were analyzed by using Excel (Microsoft, Redmond, WA) and GraphPad Prism. Mean cpm from duplicate samples were averaged, and the average nonspecific binding cpm was subtracted to get specific cpm. Specific cpm were used to determine the normalized percentage bound. GraphPad Prism was used to determine the nonlinear regression curve fit from the normalized percentage bound for the standard curve and interpolated the values of the unknown samples as log picomole of cGMP. Results were normalized to tissue weights for each sample to calculate picomoles of cGMP/g tissue. Group averages were calculated as percentage of vehicle control and percentage of vehicle stress control, and the S.E.M. was determined. Statistical analyses were carried out by GraphPad Prism using one-factor (dose group) ANOVA followed by Dunnett's multiple comparison test versus vehicle and vehicle stress groups indicating a p < 0.05 value for significance.

Sleep EEG in the Rat.

Male Wistar rats were surgically prepared with a cranial implant that permitted chronic EEG and electromyogram (EMG) recording, in addition to an abdominally implanted telemeter to measure temperature and locomotor activity. After recovery, animals were individually housed and connected to low torque commutator via a flexible tether allowing access to all areas of the cage. Animals were maintained on a 24-h light/dark schedule (12:12) with ad libitum food and water. Animals were undisturbed for 48 h before and after each treatment. Sleep and wakefulness were determined by using SCORE2004 (Hypnion, Inc., Lexington, MA), a microcomputer-based sleep-wake and physiological monitoring system. In the present study, the system monitored amplified EEG [×10,000, bandpass 1–30 Hz; initial digitization rate 400 Hz (Grass Instruments Corp., Quincy, MA)], integrated EMG (bandpass 10–100 Hz, RMS integration), telemetered body temperature, nonspecific locomotor activity, and drinking activity simultaneously. Arousal states were classified on-line as NREM sleep, REM sleep, wake, or θ-dominated wake every 10 s by using EEG period and amplitude feature extraction and ranked membership algorithms. Individually taught EEG-arousal-state templates and EMG criteria differentiated states of arousal. Drug was administered 6 h after lights off (circadian time denoted CT-18) to look for sleep-promoting effects of the drug at 10 or 30 mg/kg or vehicle. Experiments were run as simultaneous parallel groups, and all doses including vehicle were tested in parallel on the same day in approximately equal-sized dose groups of 10 to 14 rats. Total NREM sleep and total REM sleep were computed for each animal. The primary outcomes were the total NREM sleep and REM sleep during the first 12 h and during the first 18 h after treatment. To assess sleep continuity, the single longest bout of the six hourly longest sleep bouts during the first 6 h after treatment was calculated, as was the average sleep bout over this period. Each outcome was analyzed by analysis of covariance using treatment group as the factor and the pretreatment period as the covariate. Sleep bouts were analyzed on the log scale to stabilize the variation. Adjusted means and the change from vehicle means and their corresponding standard errors were summarized for each treatment group. For longest sleep bouts results were transformed back to the linear scale and represent fold changes over vehicle. Unadjusted and Dunnett's multiple-comparison adjusted P values are shown for each outcome in each period.

In Vivo Microdialysis in the Rat.

Implantation of a BAS guide cannula into the mPFC [anterior (A) 3.2 mm; lateral (L) 0.8 mm; and ventral (V), -2 mm] was carried out by Taconic Farms (Germantown, NY) 5 to 7 days before the experiment as described previously (Fell et al., 2010). A concentric type probe (BR-4) from BAS Bioanalytical Systems (West Lafayette, IN) to match the implanted cannula with a 4-mm membrane tip extending below the cannula was flushed with water and carefully inserted through the cannula 16 h before the experiment began. Immediately after insertion of the probe the rat was returned to the home cage. On the morning of the experiment the rat was moved to the cage where the experiment was to be conducted and allowed to acclimate for a period of 4 h. After the acclimation period the rat was connected to a fraction collection system for freely moving animals (BAS Bioanalytical Systems). The input tube of the dialysis probe was connected to a syringe pump (BeeHive and BabyBee; BAS Bioanalytical Systems), which delivered an artificial cerebrospinal fluid containing 150 mM NaCl, 3 mM KCl, 1.7 mM CaCl₂, and 0.9 mM MgCl₂, pH 6.0, to the probe at a rate of 1 μl/min. The output tubes from the rats were attached to a refrigerated fraction collector (BAS Bioanalytical Systems). After a period of 2 h for equilibration of the probe and establishment of stable histamine baseline levels, collection of 60-min fractions was started. The flow from the output lines was collected in 400-μl plastic tubes that contained 15 μl of an antioxidant solution (3 mM l-cysteine, 10 nM EDTA, and 50 nM isoproterenol as an internal standard, in 0.5 M acetic acid). The antioxidant serves to acidify the dialysate and prevents degradation of the amines and metabolites during the 24-h period that it takes to collect the samples and complete the HPLC assays. Typically the total sample volume was 60 μl. Three baseline samples were collected before injection of any drugs. At 6:00 PM the lights were turned out in the experimental room and not turned back on until 6:00 AM the next morning. THIIC (10 and 30 mg/kg) or vehicle were dosed orally 60 min before the onset of the dark phase (6:00 PM). All microdialysis data were calculated as percentage change from dialysate basal concentrations with 100% defined as the average of the three drug preinjection values. Each group had five to eight rats.

Histamine was separated on an HPLC column and quantified by fluorimetric detection after a postcolumn derivatization with an o-phthaldialdehyde (OPA)-containing reagent as follows. The mobile phase consisted of 0.16 M KH₂PO₄, 0.1 mM sodium octanesulfonic acid, and 0.1 mM EDTA; the pH was adjusted to 4.6. The flow rate of the mobile phase was 0.7 ml/min. The eluent line was connected by a T-piece for mixing with a reagent line through which a 0.02% solution of OPA in 0.15 M NaOH was delivered at 0.6 ml/min. The OPA reagent was mixed with the eluent in a mixing coil of Teflon tubing (o.d. 1.1 mm; i.d. 0.55 mm; length 1 m), which was insulated to allow the derivatization reaction to proceed at ambient temperature. The OPA reagent solution was prepared fresh daily, protected from light, and kept cooled in ice. Histamine was separated on a reverse-phase column, a BDS Hypersil, 3 μm, C18 analytical column (4.6 × 100 mm from ThermoFisher Scientific). The fluorescence of the reaction product was measured by a fluorimeter (Jasco, Tokyo, Japan; excitation: 350 nm; emission: 450 nm) set at the highest sensitivity. The sensitivity for histamine was approximately 20 fmol per sample (20 μl). All dialysis probe placements were checked by perfusing a tetra red solution through the dialysis probe for subsequent histological examination. Only results derived from rats with correctly positioned probes were included in the data analysis. Time course data represent the percentage change from baseline and were analyzed by two-way ANOVA followed by post hoc Bonferroni corrected t test. The level of significance was set at p < 0.05. All analyses were performed using the GraphPad PRISM statistical program.

Analysis of Tele-Methylhistamine in Rat CSF.

Male Harlan-Sprague-Dawley rats (200 g) were dosed acutely or for 5 days with THIIC at doses of 1 to 10 mg/kg orally. Six hours after dosing the rats were sacrificed with CO₂ and CSF samples were collected from the cisterna magna. Concentrations of histamine and tele-methylhistamine (t-MeHA) were determined by using HPLC separation and detected by tandem mass spectrometry. Cerebrospinal fluid (50–100 μl) samples were spiked with stable label internal standards for both analytes. The CSF was then passed though an anion exchange solid-phase extraction cartridge to remove protein and interferences. The extracts for CSF were injected onto a Varian Inc. (Palo Alto, CA) monochrome 100 × 2 mm HPLC column for separation. The separation was achieved by using a mobile phase of 0.5% heptafluorobutyric acid and 10% acetic acid/0.5% heptafluorobutyric acid in acetonitrile. A linear gradient was used for separation of histamine and its metabolite. Histamine, its tele-methyl metabolite, and the stable label internal standards were detected by electrospray ionization with the specific tandem mass spectrometry transitions. Standard curves were analyzed from 0.150 to 50 ng/ml, and unknown concentrations were calculated by using an internal standard method. Detection limit for t-MeHA was 0.3 ng/ml. Data are expressed as mean ± S.E.M. and were subjected to a one-way ANOVA followed by post hoc analyses using Dunnett's corrected t test.