d-CPA 4 mg·kg −1 i.p. increased non-rapid eye movement (REM) sleep whereas 10–40 mg·kg −1 d-CPA decreased non-REM sleep at dark onset time. Nocturnal i.c.v. infusions of d-CPA (10 µmol·100 µL −1 ·10 h −1 ) increased drowsiness but not non-REM sleep, whereas the same i.c.v. infusions of cetirizine significantly increased non-REM sleep, abolished REM sleep, and decreased wakefulness for more than 10 h. The medial preoptic area contained the greatest fluorescent labelling after i.c.v. cetirizine/DBD-pz infusions. Histamine-induced Ca 2+ increases in medial preoptic neurons were blocked by d-CPA or cetirizine, whereas d-CPA, but not cetirizine, increased Ca 2+ irrespective of antihistaminergic activity at ≥100 µM.

Classic H 1 histamine receptor (H 1 R) antagonists are non-selective for H 1 R and known to produce drowsiness. Modern antihistamines are more selective for H 1 R, and are ‘non-drowsy’ presumably due to reduced permeability through the blood-brain barrier. To characterize both histaminergic sleep regulation and the central actions of antihistamines, in the present study we analysed the effect of classic and modern antihistamines on rats' sleep using continuous i.c.v. infusions.

All data are presented as means with SEM. A two-tailed unpaired t-test was used for pair-wise comparisons. One-way anova followed by Duncan's multiple range test was used for the statistical comparisons among multiple groups. One-way repeated-measures anova was used to analyse differences in hourly sleep-wake periods before and after drug infusions. A four-parameter Hill function was used for the estimation of the dose–response curve for d-CPA and cetirizine analogues. A 95% confidence level was considered to be significant.

To visualize the shape and activity of neurons, slice cultures were transfected with yellow cameleon (YC2.1) with a neuron-specific enolase promoter using a Helios gene-gun kit (Bio-Rad Laboratories, Hercules, CA, USA) 7–9 days after in vitro cultures had been established ( Ikeda et al., 2003 , 2005 ). The Ca 2+ responses in 1–4 neuron(s) were observed immediately using an upright microscope with a water immersion objective as above. Slices were perfused with ACSF containing 2 mM CaCl 2 and 1 mM MgCl 2 that was bubbled with 95% O 2 and 5% CO 2 for at least 30 min prior to the experiments. Tetrodotoxin (TTX; 0.5 µM; Wako Pure Chemicals) perfusion was initiated 15 min prior to and continued for the duration of imaging. Cameleon-expressing neurons were exposed to 440 ± 5 nm light using a high power xenon lamp with a band-pass filter (440NBD10, Omega optical) at 6 s intervals. The resultant fluorescence image was separated using a dichroic mirror (455DRLP; Omega optical) and directed into a CCD camera (CoolSnap-Ez; Photometrics) through double-view optics (Hamamatsu Photonics, Hamamatsu, Japan), in which one image was split into bilateral images using internal reflection mirrors and processed using two dichroic mirrors (515 DRLP; Omega optical) and band-pass filters (480DF30 and 535DF25 filters). The monochromator and the CCD camera were controlled using digital imaging software (MetaFluor ver 6.2, Molecular Devices).

Hypothalamic slice cultures were prepared from newborn Sprague-Dawley rats from our inbred colony. Series of coronal hypothalamic slices (380–400 µm) were prepared using a vibrating-blade microtome in ice-cold high-Mg 2+ artificial cerebrospinal fluid (ACSF) containing (mM): 138.6 NaCl, 3.35 KCl, 21 NaHCO 3 , 0.6 NaH 2 PO 4 , 9.9 d-glucose, 0.5 CaCl 2 and 3 MgCl 2 , bubbled with 95% O 2 /5% CO 2 , placed in a 0.40 µm filter cup (Millicell-CM, Millipore, Bedford, MA, USA), and incubated with 50% Eagles basal medium (Invitrogen, Carlsbad, CA, USA), 25% Earle's balanced salt solution (Invitrogen) and 25% heat-inactivated horse serum, supplemented with 5 mg·mL −1 glucose and 1:100 Glutamax (Invitrogen) in a CO 2 incubator at 35.5 ± 0.5°C and 5% CO 2 .

HeLa cells plated on 35 mm dishes were incubated for 40 min in culture medium containing 3 µM fura-2 AM (Molecular Probes, Eugene, OR, USA) in a CO 2 incubator at 36.5 ± 0.5°C and 5% CO 2 . Cells were rinsed three times with buffered salt solution consisting of (in mM): 128 NaCl, 5 KCl, 2.7 CaCl 2 , 1.2 MgCl 2 , 1 Na 2 HPO 4 , 10 glucose and 10 HEPES/NaOH (pH 7.3). Then, the culture dish was transferred to a temperature-controlled chamber on the microscope (TC344B; Warner Instruments, Hamden, CT, USA). Fluorescence images were obtained using an upright microscope (Axioplan 2; Carl Zeiss) with a water immersion objective (Achroplan × 40 NA0.75, Carl Zeiss). The wavelength of the excitation UV light (340 nm or 380 nm pulse; 100 ms) was switched using a filter wheel (Lambda 10-2; Sutter Instruments, Novato, CA, USA). The UV light was generated by a full-spectrum 175W Xenon bulb (Lambda LS; Sutter), conducted to the microscope through a liquid light guide and reflected using a dichroic mirror (FT 395 nm; Carl Zeiss). The pair of fluorescence images was processed using a band-pass filter (BP 485–515 nm; Carl Zeiss) and captured using a multiple format cooled CCD camera (CoolSnap-FS; Photometrics, Tokyo) at 6 s intervals. The filter wheel and the CCD camera were controlled using digital imaging software (MetaFluor ver. 6.0; Japan Molecular Devices, Tokyo). The background fluorescence was also subtracted using the software. During recording, HeLa cells were perfused with buffered salt solution at a flow rate of 2.5 mL·min −1 . The histamine (30 µM) was bath-applied two times for 1 min each with a 20 min gap. H 1 R antagonists (cetirizine or cetirizine/DBD-pz) were perfused for 4 min across the second histamine application.

To estimate cerebral cetirizine distribution at a time when non-REM sleep was enhanced, cetirizine/DBD-pz (4 µmol·40 µL −1 ·4 h −1 at dark onset) was infused into the third ventricle (n= 3) at the end of the i.c.v. infusion experiments. Because cetirizine/DBD-pz at this concentration is not soluble in physiological saline, cetirizine/DBD-pz was dissolved in dimethyl sulphoxide (5% of final volume) and then diluted with saline. For the background estimation, a mixture of cetirizine/DBD-pz (4 µmol·40 µL −1 ) and cetirizine (4 µmol·40 µL −1 ) was also infused as above following the cetirizine (8 µmol·40 µL −1 ·4 h −1 ) infusion (n= 3). Subsequently, rats were deeply anaesthetized with sodium pentobarbital (100 mg·kg −1 body weight) and perfused transcardially with PBS, pH 7.4, and 4% phosphate-buffered paraformaldehyde. Brains were removed, fixed further in 4% phosphate-buffered paraformaldehyde overnight at 4°C and immersed in PBS containing 30% sucrose for 12 h at 4°C. Using a cryostat at −20°C, 50 µm coronal sections were prepared, rinsed three times with PBS, and mounted on glass slides. These samples were imaged using a cooled charge-coupled-device (CCD) camera (Ds-5mc, Nikon, Tokyo, Japan) mounted on a fluorescence stereomicroscope (SMZ-1500; Nikon). The background fluorescence was pre-averaged and subtracted using Photoshop ver. 6.0 software (Adobe Systems, San Jose, CA, USA). The field fluorescence intensity (FI) of an 8-bit depth (i.e. 128-graded) bitmap image was calculated for sleep-related brain areas.

The antihistamine activity of the conjugated forms of cetirizine remained intact. The affinity of cetirizine/DBD-pz for H 1 Rs was analysed using fura-2-based conventional Ca 2+ -imaging in HeLa cells, which intrinsically express H 1 Rs. The IC 50 value of the inhibition of histamine (30 µM, 1 min)-induced Ca 2+ rise was 951 nM for cetirizine/DBD-pz, which is equivalent to the IC 50 value of cetirizine (867 nM; ).

To synthesize fluorescent labelled cetirizine, 4-(N,N-dimethylaminosulphonyl)-7-piperazinobenzofurazan (DBD-pz; λex = 440 nm; λem = 569 nm; Tokyo Chemical Industry, Co., Tokyo, Japan) was conjugated to the carboxyl acid terminal of cetirizine ( ) as follows. Firstly, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (6.1 mg, 0.064 mmol) was added to a stirred anhydrous dichloromethane (1 mL) of cetirizine dihydrochloride (10.1 mg, 0.032 mmol; compound 1), DBD-pz (15.0 mg, 0.032 mmol; compound 2) and N,N-dimethylaminopyridine (catalytic amount). The resulting mixture was stirred at room temperature under argon atmosphere for 2 h. Subsequently, the reaction mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate buffer, and then dried over magnesium sulphate. The solvent was evaporated to leave a residue, which was subjected to silica gel column chromatography (eluent: acetone-MeOH-Et 3 N) to afford the amide compound 3 (19.6 mg, 90%) as yellow solids. Finally, the solids were dissolved in dichloromethane, and anhydrous HCl (1 M solution in Et 2 O) was added dropwise with stirring. The precipitated HCl salts were collected using filtration and dried at ambient temperature.

For i.c.v. infusions of H 1 R antagonists, the rats implanted with an i.c.v. cannula received a continuous i.c.v. infusion of sterilized saline (10 µL·h −1 ) for a week before the EEG/EMG recordings. The i.c.v. cannula was connected through the slip ring to an infusion pump via polyethylene tubing and Teflon connecting tubing (0.5 mm inner diameter). This apparatus guaranteed unrestrained movement of the rats during the study. After the rats had acclimatized to the i.c.v. saline infusion, d-CPA or cetirizine dissolved in saline (0.2 or 10 µmol in 100 µL saline) was infused for 10 h from dark onset (20h00min). After drug infusion, the saline infusion was continued for 2 days. The EEG and EMG recordings were initiated 1 day before the drug infusion. A total of 24 rats was implanted with EEG/EMG electrodes and an i.c.v. infusion cannula, and 20 of the 24 rats were successfully used for the i.c.v. infusion studies described above. Studies were terminated after three days of recording.

For i.p. injections of H 1 R antagonists, rats were habituated in recording chambers for daily i.p. injections of sterilized saline (0.2 mL) immediately before dark onset (19h 30min–19h55min). Subsequently (+)-chlorpheniramine maleate (d-CPA; F.W. 461.81) (−)-chlorpheniramine maleate (l-CPA; both purchased from Wako Pure Chemical Industries, Ltd. Tokyo), or cetirizine dihydrochloride (F.W. 390.86; CAS 83881-52-1, Sigma/RBI, St Louis, MO, USA) dissolved in 0.2 mL saline was injected instead of saline. Animals received multiple i.p. injections of d-CPA in the dose–response experiments. For example, an initial d-CPA injection of less than 1 mg·kg −1 was administered and then, after at least 1 week, a second d-CPA injection of greater than 4 mg·kg −1 was administered. The EEG and EMG recordings were initiated 1 day before the drug injection, and continued through to 1 day after the drug injection. A total of 24 rats were used for the i.p. injection studies.

One week was allowed for recovery from surgery, then rats were placed in individual sleep-recording cages in a sound-proof and electromagnetically shielded chamber. The lead wires of the EEG and EMG electrodes were connected to a multiple channel amplifier (MEG-6116; Nihon Kohden, Tokyo, Japan) via a feed-through slip ring (CAY-675; Airflyte Electronics Company, Bayonne, NJ, USA) fixed above the cage. The EEG and EMG signals were relayed to a computer via an analogue-to-digital converter (Ad 16-16U; Contec, Osaka, Japan) at a sampling rate of 128 Hz/channel and stored on a hard diskette. Sleep-wake stages were determined semi-automatically based on the amplitudes of the EEG and EMG and Fast-Fourier-Transform analysis of the EEG spectrum using SleepSign ver 2.0 software (Kissei Comtec, Nagano, Japan). After recording, the sleep-wake stages were replayed on the monitor and visually re-evaluated by two experienced investigators to correct improper scoring of sleep due to artefactual EEG/EMG patterns during active wakefulness.

Animals were anaesthetized with an i.p. injection of sodium pentobarbital (50 mg·kg −1 body weight) and placed in a stereotaxic apparatus. For EEG recording, rats were implanted with four gold-plated screw-electrodes (Unique Medical Co., Tokyo, Japan) placed through the skull on the frontal and occipital cortex, according to standard methods ( Ikeda et al., 2001 ; 2005 ;). Briefly, three recording electrodes were placed over the right frontal cortex (mm) 1.8-, 4.2- and 7.4 rostral and 1.8 lateral to bregma, and one reference electrode was placed over the left frontal cortex 4.2 rostral and 1.8 lateral to bregma. To achieve continuous third ventricular infusion, a stainless steel cannula (0.35 mm inner diameter) was inserted into the third ventricle, 2.3 lateral and 0.9 caudal to bregma at a 15° angle. In addition, two stainless steel hook-electrodes were inserted into the cervical portion of the trapezius muscle for EMG recording. All electrodes and the cannulae were permanently affixed to the skull using dental acrylic resin. During and at the end of surgery, a total of 40 000 U of penicillin G potassium (Meiji Pharmaceutical Company, Tokyo, Japan) was injected s.c. and locally applied to the incision. Further details of the surgery have been described previously ( Ikeda et al., 2001 ; 2005 ;).

Adult male Sprague-Dawley rats (300–450 g), 60–70 days old, purchased from Ninox Labo Supply Inc., Ishikawa, Japan) were maintained on a 12:12 light : dark cycle at a constant ambient temperature (25 ± 1°C) and housed with our inbred colony at the Gofuku campus of the University of Toyama. Food and water were available ad libitum. All animal care and experimental procedures complied with international guidelines and were approved by the Animal Care and Use Committee at the University of Toyama.

To further analyse possible non-histaminergic effects of CPA compounds on sleep regulatory circuits, we also applied millimolar concentrations of d-CPA to cultured POAH neurons ( ). In about 42% (five out of 12 neurons in seven slices) of medial POAH neurons, there was an increase in cytosolic Ca 2+ in response to d-CPA at 100 µM. Cytosolic Ca 2+ was increased in half the neurons in response to d-CPA at 1 mM (eight out of 16 neurons in eight slices) and most neurons responsed to d-CPA 5 mM (20 out of 21 neurons in 14 slices). Because the slice culture used did not contain histaminergic neurons and their axon fragments are completely lost during the culturing process, the d-CPA-induced increase in cytosolic Ca 2+ is not explained by antihistaminergic activity. Indeed, cetirizine 100 µM–5 mM did not induce Ca 2+ mobilization in cultured POAH neurons (13 out of 13 neurons in five slices; ).

To analyse the effects of histamine and H 1 R antagonists on cultured POAH neurons, the yellow cameleon gene was randomly transfected into cultured POAH neurons ( ). When added to circulating ACSF, histamine (100 µM for 45 s) evoked Ca 2+ transients in 41% of medial POAH neurons (11 out of 27 neurons in eight slices). These histamine-induced cytosolic Ca 2+ responses were completely abolished by d-CPA (10 µM) and recovered after washout of d-CPA ( ). Cetirizine (10 µM) also completely blocked histamine-induced cytosolic Ca 2+ responses in POAH neurons (number of neurons = eight in three slices). Histamine-induced Ca 2+ transients were also observed in periventricular neurons (four out of six neurons in two slices) but not in neurons located in the VLPO, although the number of recordings in this area was limited (four neurons in two slices; ). To visualize the net histamine response without synaptic interactions, the same histamine stimulation was examined with perfusion of ACSF containing TTX (0.5 µM). The proportion of medial POAH neurons that responded to histamine (10 out of 26 neurons in six slices; 38%) was similar to that without TTX (41%; ).

Cetirizine/DBD-pz (40 µmol·4 h −1 ) was infused into the third ventricle using the same experimental procedures for sleep recordings, and brain sections were prepared from rats killed at the approximate time of cetirizine-initiated non-REM sleep enhancement (24:00–24:30; ). The tip of the infusion cannula was located in the third ventricle, −0.6 ± 0.4 mm from bregma on the midline in these animals. Cetirizine/DBD-pz fluorescence was observed most significantly in the medial preoptic area (FI = 33.6 ± 6.0), near the coronal plane of pipette placement. The sleep-active neuronal nucleus, the median preoptic nucleus (MnPO; FI = 29.7 ± 6.4), but not VLPO (FI = 0.7 ± 0.5; P < 0.01 compared with MnPO levels), was significantly labelled ( ). This difference in labelling was due to the limited lateral diffusion of cetirizine/DBD-pz from the third ventricle, as there was also no fluorescent signal detected in the substantia innominata. In contrast, cetirizine/DBD-pz diffused into the bilateral lateral ventricles, as evidenced by the fibrous staining patterns detected in the septum ( ; FI = 25.4 ± 4.9 for the lateral septum and 24.0 ± 4.1 for the medial septum). In addition, cetirizine/DBD-pz diffused caudally through the mesencephalic duct and permeated the periaqueductus parenchyma, including the ventrolateral periaqueductal grey matter (vlPAG; FI = 28.1 ± 6.3; ). A signal was also detected in the dorsal raphe nucleus (FI = 6.2 ± 0.8), whereas it was almost absent in the dorsal end of the laterodorsal tegmental nuclei (LDTg; FI = 0.8 ± 0.2) 4 h after i.c.v. infusions. These findings indicate that there are cetirizine concentration gradients in the brain during continuous i.c.v. infusion.

d-CPA 10 µmol evoked an unusual drowsy state ( ), which coincided with higher EMG tones than those during non-REM sleep ( ) and the predominant EEG spectrum in the slow-wave range (1–3.5 Hz) was a lower amplitude than that during normal non-REM sleep ( ). After review of the video of the rats' behaviour during the drowsy state, this state was judged to be a part of wakefulness rather than non-REM sleep. The EEG spectrum during cetirizine-induced non-REM sleep had a peak at 1.75 Hz with a power larger than that during the light and dark under control conditions or during the dark period with d-CPA infusion (F 3,16 = 75.28, P < 0.01 by Duncan's multiple range test following one-way anova; ).

Infusion of 10 µmol cetirizine produced similar but stronger effects on REM sleep. REM sleep during the dark period of drug infusion (F 4,35 = 13.7, P < 0.01 by Duncan's multiple range test following one-way anova) and REM sleep during the subsequent light period (F 4,35 = 37.1, P < 0.01 by Duncan's multiple range test following one-way anova; and ) were almost completely suppressed. Non-REM sleep during the dark period during cetirizine infusion was significantly increased above the amount of sleep during the light period (F 4,35 = 19.46, P < 0.01 in comparison with saline-infused control by Duncan's multiple range test following one-way anova; and ). The increase in amount of non-REM sleep coincided with prolongation of the non-REM sleep episode and the maximum duration was 47.8 ± 18.6 min (n= 5) during cetirizine infusion. Infusion of d-CPA or cetirizine at 0.2 µmol had no effects on daily sleep-wake cycles (n= 5 for each group; ).

The effect of cerebral H 1 R blockade on sleep-wake cycles was further examined using continuous, 10 h i.c.v. infusions of d-CPA ( ) or cetirizine ( ) into the vicinity of the POAH in unrestrained rats at two different doses (0.2 or 10 µmol·100 µL −1 ·10 h −1 ). While 10 µmol d-CPA failed to evoke a consistent increase or decrease in the amount of non-REM sleep ( and ), infusion of 10 µmol d-CPA resulted in a long-term reduction in REM sleep during the dark period of drug infusion (F 4,35 = 13.7, P < 0.05 by Duncan's multiple range test following one-way anova) and during the subsequent light period (F 4,35 = 37.1, P < 0.01 by Duncan's multiple range test following one-way anova; and ).

The effects of i.p. injections of cetirizine on sleep were also tested at 4 mg·kg −1 and 40 mg·kg −1 . Lower doses (4 mg·kg −1 ) of cetirizine failed to produce significant effects on non-REM and REM sleep when compared with saline-injected controls ( ). On the other hand, cetirizine 40 mg·kg −1 significantly increased non-REM sleep (146% of saline-injected controls; F 2,29 = 7.4, P < 0.01 by Duncan's multiple range test following one-way anova; ) and decreased REM sleep (34% of saline-injected controls; F 2,29 = 4.9, P < 0.05 by Duncan's multiple range test following one-way anova; ).

The effects of peripheral administration of d-CPA on sleep were analysed using a conventional i.p. injection strategy with a detailed dose–response curve. Rats injected with 4 mg·kg −1 d-CPA at the onset of dark exhibited a transient increase in non-REM sleep (148% of saline-injected controls; F 6,45 = 17.7, P < 0.05 by Duncan's multiple range test following one-way anova) and a decrease in REM sleep (34% of saline-injected controls; F 6,45 = 11.4, P < 0.01 by Duncan's multiple range test following one-way anova) during the 3 h following the injection ( and ). The peak non-REM sleep increase and decrease in wakefulness were observed 2 h after the injection, whereas the reduction in REM sleep was observed immediately (≤1 h) after the injection ( ). There was no consistent increase or decrease in sleep or wakefulness during the subsequent dark period (data not shown). The effects of d-CPA on non-REM sleep were dose-dependent up to 10 mg·kg −1 , with an EC 50 of 0.68 mg·kg −1 ( ). The inhibitory effects of d-CPA on REM sleep were also dose- dependent, with an IC 50 of 1.09 mg·kg −1 (excluding data points greater than 10 mg·kg −1 ) and 2.68 mg·kg −1 (including data points greater than 10 mg·kg −1 ; ). On the other hand, higher doses of d-CPA (10 mg·kg −1 or 40 mg·kg −1 ) strongly decreased non-REM sleep (F 6,45 = 17.7, P < 0.01 by Duncan's multiple range test following one-way anova, ) and produced complete insomnia for an hour after injection ( ). Similarly, 40 mg·kg −1 l-CPA reduced non-REM sleep (F 2,29 = 14.6, P < 0.01 by Duncan's multiple range test following one-way anova, ) and REM sleep (F 2,29 = 7.8, P < 0.01 by Duncan's multiple range test following one-way anova, ).

Discussion

The affinities of d-CPA (pK i = 8.2) and cetirizine (pK i = 8.6) for H 1 R were previously reported in CHO cells over-expressing human H 1 receptors (Gillard et al., 2002), but their pharmacokinetics and specificity for producing the desired actions in vivo remained to be analysed. In the present study the dose-dependent effects of d-CPA and cetirizine were compared on REM sleep, non-REM sleep and wakefulness in rats. d-CPA facilitated non-REM sleep and suppressed REM sleep within a narrow dose window (≤4 mg·kg−1, i.p.). Inconsistent decreases in non-REM sleep and total REM sleep suppression (i.e. arousal effects) were observed with higher doses of d-CPA (10 or 40 mg·kg−1, i.p.). The present results also demonstrated that (i) l-CPA, which has less than 10% H 1 R affinity of d-CPA (Tanda et al., 2008), similarly suppressed sleep at 40 mg·kg−1, i.p., and (ii) continuous i.c.v. infusion of d-CPA (10 µmol·100 µL−1·10 h−1) induced unusual drowsiness rather than non-REM sleep. These sleep suppressive effects of d-CPA at high concentrations were consistent with the psychomotor stimulant actions reported for CPA compounds (Bergman, 1990; Tanda et al., 2008). d-CPA but not cetirizine mobilized intracellular Ca2+ in cultured POAH neurons irrespective of antihistaminergic activity at ≥100 µM; therefore, the bimodal effects of d-CPA on sleep can be explained by this non-specific pharmacological action. However, the increase in non-REM sleep and the reduction in REM sleep with i.p. injections of d-CPA at 4 mg·kg−1 may be the result of blocking cerebral H 1 R, because similar but more prolonged effects were observed with higher i.p. doses of cetirizine (40 mg·kg−1, i.p.) or with a continuous i.c.v. infusion of cetirizine (10 µmol·100 µL−1·10 h−1). Taken together, these results indicate that blocking cerebral H 1 Rs induces prolonged, continual non-REM sleep in rats.

Classic H 1 R blocker actions on sleep Monti et al. (1986) examined the effects of a single i.c.v. injection of the H 1 R agonist 2-thiazolylethylamine (2-TEA) into the lateral ventricle of rats, and observed a dose-dependent (0.5–2 µmol dissolved in 5 µL saline) and transient (<1 h) increase in wakefulness. The 2-TEA effect was blocked by i.p. injections of mepyramine (1–2 mg·kg−1), thus it was hypothesized that the arousal-inducing effects of 2-TEA are mediated via H 1 R. Lin et al. (1994) further examined the effects of bilateral micro-injections of mepyramine (120 µg·1 µL−1; 420 mM) in the POAH of cats and observed an increase in non-REM sleep and prolongation of REM sleep latency (i.e. delayed onset of REM sleep after injections). These results strongly suggest that direct H 1 R antagonist actions in the POAH facilitate non-REM sleep, whereas it should be noted that most classic H 1 R antagonists cause non-histaminergic effects when applied at high concentrations or in large volumes. For example, mepyramine greater than 30 µM is known to reduce a potassium current (M-current) in dissociated cortical neurons (Sato et al., 2005). In addition, l-CPA as well as d-CPA injections at 5.6 mg·kg−1 induce the release of dopamine in the nucleus accumbens of rats, suggesting that higher doses of these CPA compounds have non-histaminergic effects (Tanda et al., 2008). For this reason, in the present study we re-evaluated the net central actions of H 1 R antagonists on sleep-wake activity. In the present study we demonstrated that 10 h infusions of 10 µmol d-CPA (c.a. 6 mg·kg−1, i.c.v. per night) had little effect on non-REM sleep, but instead, induced an unusual state of drowsiness. According to our previous study with a synthetic peroxide reagent (tert-butyl hydroperoxide), applied using the same i.c.v. infusion technique, the concentrations of drugs in the brain parenchyma nearby the infusion pipette were approximately 1000–10 000 times less than that in the infusion pipette (Ikeda et al., 2005). Although the permeability of drugs from the third ventricle to the brain parenchyma may depend on their chemical formula, 10 µmol·100 µL−1·10 h−1d-CPA (i.e. 100 mM in the infusion pipette) probably diffuses into the brain parenchyma at levels similar to those observed in the previous tert-butyl hydroperoxide studies (i.e. 10–100 µM), because d-CPA is highly membrane permeable. This is supported by the present results showing that i.c.v. infusion of 0.2 µmol·100 µL−1·10 h−1d-CPA or cetirizine (i.e. 2 mM in the infusion pipette) failed to modulate sleep, probably because the levels diffused into the brain parenchyma were too low. We also demonstrated that d-CPA at 100 µM or higher induced a histamine-independent mobilization of cytosolic Ca2+ in cultured POAH neurons. Therefore, it is likely that non-specific and excitatory actions of high concentrations of d-CPA may influence many neuronal circuits in the vicinity of the i.c.v. infusion pipette, which would mask its inhibitory actions on H 1 R. The results of the present study confirmed that i.p. injections of d-CPA increase non-REM sleep, although the effective dose window was quite limited (4 mg·kg−1). Taken together with the fact that most classic H 1 antagonists produce non-histaminergic effects at high concentrations, we suggest careful use of classic H 1 antagonists as sleeping pills.