We discovered a wake-promoting action of UDCA in mice at a dose used for the standard treatment of primary biliary cirrhosis. Cortical EEG and sleep-wake monitoring showed a difference between wild type and histamine-deficient mice in response to UDCA, indicating that recruitment of histamine is necessary in the wake-promoting effect. In vitro patch-clamp recordings from histaminergic neurons revealed no increase in firing rate by UDCA, but inhibition of GABAergic currents. Hypothalamic network activity recorded with MEAs was synchronized by UDCA through a reduction of GABAergic inhibition. Synchronization of neuronal firing is expected to increase histamine release from varicosities.The GABAAR was blocked by UDCA also in HEK293 cells carrying recombinant receptors of different subunit combinations. We conclude that the UDCA action at the GABAAR in the central nervous system and the periphery (lymphocytes, liver, lung and pancreas) takes place at a new specific site.
The wake-promoting action of UDCA is in keeping with GABAA
R antagonism and the well known sedative effect of GABAA
R agonists like muscimol applied either systemically or locally into the posterior hypothalamus. In contrast to cholate and chenodeoxycholate (all found in the brain) 
UDCA did not suppress neuronal activity and did not block NMDA receptors. UDCA and other BS 
shorten the kinetics of spontaneous GABAergic synaptic potentials with potencies similar to those obtained from macroscopic GABA-evoked currents on acutely isolated histaminergic neurons. Investigation of the mechanisms of GABAA
R block by BS in our previous study 
showed rapid BS binding to the open, but not to the closed (unliganded) state of the receptor. No inhibitory action of BS on the closed channel was observed. In order to explore further the possibility of an open channel block we compared the action of UDCA with the action of a classical open channel blocker. In contrast to the picrotoxin-block, the UDCA-block was not use-dependent. In spite of the non-additive actions of UDCA and picrotoxin at homopentameric β3 receptors expressed in HEK293 cells, mutational analysis convincingly demonstrated that the picrotoxin-block involves mechanisms different from those employed by BS. The UDCA- block was impaired in α1β2γ2L receptors containing a mutated α-subunit (α1V256S). This mutation is known to reduce the inhibition by pregnenolone sulphate 
but it does not affect the potency of picrotoxin 
. Another mutation at the GABAA
R α-subunit (α1Q241L) which is known to abolish the potentiating action of neurosteroids 
had no influence on GABAA
R inhibition by UDCA. The similarity of GABAA
R block by UDCA in TMN neurons and in HEK293 cells and dependence of this block on the α1V256 site in the α-subunit thus provided evidence for the direct interaction of BS with the GABAA
receptor. Unfortunately the mutation α1V256S causes a dramatic change in receptor sensitivity to GABA (the same is true for the mutation α1Q241L), a situation that excludes the generation of mutant mice carrying these mutations. Therefore it is not possible at present to elucidate whether conditional (HDC/cre driven) expression of the α1V256S mutation in TMN neurons would impair the action of UDCA on waking stages with EEG recordings.
The wake-promoting action of UDCA in wild type but not in HDC knockout mice was evident at 32 mg/kg. This dose is nearly 20 times below the one necessary to combat retinal degeneration in the mouse (500 mg/kg) 
. We found significant changes in cortical EEG starting from the third hour after oral UDCA application, in keeping with the serum peak time of the UDCA concentration (~18±10 µM) in volunteers (men) taking a single oral dose of UDCA (~12 mg/kg) 
. Previous studies have shown that UDCA reaches the CNS 
. We also found that UDCA enhances arousal during the active period of the day, an effect that was reversed in histamine-deficient mice. This clearly suggests involvement of the histaminergic system. It is known that histaminergic neurons are spontaneously active during waking and silent during sleep 
and that GABAergic inputs play a major role in inactivating histaminergic neurons and thus promoting sleep, although the sleep-wake balance is also controlled by potent circadian mechanisms, other excitatory and inhibitory inputs and arousal systems 
. As UDCA enhanced wakefulness during the active period but not during the sleepy period in wild type mice, it appears as if the GABAA
R-mediated disinhibition could effectively change the activity of histamine neurons and lead to wake enhancement only when excitatory inputs to histamine neurons are enhanced as a consequence of behavioral activation. When the excitatory inputs decrease during the sleepy period, such a disinhibition alone would be insufficient to impact TMN neuronal firing.
Effects described here on the sleep-wake patterns in wild type mice were missing in mice lacking histamine (HDC−/− mice). Whereas these data clearly demonstrate the histamine-dependent nature of the wake-promoting effect of UCDA, it remains to determine why, intriguingly, UCDA caused opposite effects in HDC KO mice, i.e., decrease in waking and increase in total sleep (SWS+PS). It is possible that, without histamine in TMN neurons, the GABAAR-mediated disinhibition would enhance or facilitate the release of other neurotransmitters defined in wild type animals as co-transmitters of histamine: GABA or galanin for instance are inhibitory. We did not detect any significant difference in GABAAR expression or function in the posterior hypothalamus of HDC−/− mice. The possible circadian variation in GABAergic systems between wild type and knockout mice also remains to be investigated.
Besides the GABAA
R other central and peripheral sites of UDCA action cannot be ignored; among those nuclear receptors 
, the BS receptor TGR5 (which is rather insensitive to UDCA 
), BS transporters and intracellular signal cascades 
We show that central histamine is necessary for the wake-promoting action of UDCA. The hypothalamic histamine system is involved in the control of energy balance, endocrine functions and sleep-wake regulation 
. Ionotropic receptors for histamine (with yet elusive structures) control the excitability of oxytocin neurons in the supraoptic nucleus 
. One likely candidate is a GABAA
receptor devoid of GABA-binding site 
. We show here that β3 homopentameric GABAA
receptors gated by histamine represent the most sensitive target for BS (IC50
7.4 µM). Synaptic GABAA
Rs (like γ2-subunit-containing recombinant receptors) were less sensitive to UDCA: thus 10 µM of UDCA significantly accelerated the decay kinetics of sIPSCs in some and 30 µM in all TMN neurons. Hypothalamic wake-on neurons, such as histaminergic and orexinergic (hypocretinergic) neurons are under tonic GABAergic inhibition. Local injection in rat of a GABAA
R antagonist in the perifornical area containing orexin-neurons induces cortical arousal 
. These and other wake-on neurons expressing UDCA-sensitive GABAA
receptors may represent further important sites of UDCA action.
Our study expands the list of extrahepatic BS-targets and suggests that BS may play regulatory roles in the brain and at peripheral sites interacting with the GABAA
R. This finding is particularly relevant for the development of new therapeutics to treat neurological abnormalities in hepatic encephalopathy, accompanied by an increased GABAergic tone. It was suggested recently that reduced locomotor activity in hyperammonemic rats can be recovered by the GABAA
R antagonist bicuculline 
. Rats with liver cirrhosis show decreased wakefulness 
. The wake-promoting potential of UDCA awaits to be tested in these models of hepatic encephalopathy.
In conclusion, the clinical benefit of UDCA is complemented by its wake-promoting action during the active period of the day. This involves a GABAAR-mediated disinhibition of the histaminergic system.