GABAergic actions on cholinergic laterodorsal tegmental neurons: implications for control of behavioral state

Neuroscience 171 (2010) 812– 829

GABAergic ACTIONS ON CHOLINERGIC LATERODORSAL TEGMENTAL NEURONS: IMPLICATIONS FOR CONTROL OF BEHAVIORAL STATE K. A. KOHLMEIER* AND U. KRISTIANSEN

tive agonist and antagonist, respectively, of GABAC receptors. All of these GABA-mediated actions were found to occur in histochemically-identified cholinergic neurons. Taken together, these data indicate for the first time that cholinergic neurons of the LDT exhibit functional GABAA, B and C receptors, including extrasynaptically located GABAA receptors, which may be tonically activated by synaptic overflow of GABA. Accordingly, the activity of cholinergic LDT neurons is likely to be significantly affected by GABAergic tone within the nucleus, and so, demonstrated effects of GABA on behavioral state may be mediated, in part, via direct actions on cholinergic neurons in the LDT. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Department of Pharmacology and Pharmacotherapy, The Pharmaceutical Faculty, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark

Abstract—Cholinergic neurons of the pontine laterodorsal tegmentum (LDT) play a critical role in regulation of behavioral state. Therefore, elucidation of mechanisms that control their activity is vital for understanding of how switching between wakefulness, sleep and anesthetic states is effectuated. In vivo studies suggest that GABAergic mechanisms within the pons play a critical role in behavioral state switching. However, the postsynaptic, electrophysiological actions of GABA on LDT neurons, as well as the identity of GABA receptors present in the LDT mediating these actions is virtually unexplored. Therefore, we studied the actions of GABA agonists and antagonists on cholinergic LDT cells by performing patch clamp recordings in mouse brain slices. Under conditions where detection of Clⴚ -mediated events was optimized, GABA induced gabazine (GZ)-sensitive inward currents in the majority of LDT neurons. Post-synaptic location of GABAA receptors was demonstrated by persistence of muscimol-induced inward currents in TTX and low Ca2ⴙ solutions. THIP, a selective GABAA receptor agonist with a preference for ␦-subunit containing GABAA receptors, induced inward currents, suggesting the existence of extrasynaptic GABAA receptors. LDT cells also possess GABAB receptors as baclofen-activated a TTX- and low Ca2ⴙ-resistant outward current that was attenuated by the GABAB antagonists CGP 55845 and saclofen. The tertiapin sensitivity of baclofen-induced outward currents suggests that a GIRK mediated this effect. Further, outward currents were never additive with those induced by application of carbachol, suggesting that they were mediated by activation of GABAB receptors linked to the same GIRK activated in these cells by muscarinic receptor stimulation. Activation of GABAB receptors inhibited Ca2ⴙ increases induced by a depolarizing voltage step shown previously to activate VOCCs in cholinergic LDT neurons. Baclofen-mediated reductions in depolarization-induced Ca2ⴙ were unaltered by prior emptying of intracellular Ca2ⴙ stores, but were abolished by low extracellular Ca2ⴙ and pre-application of nifedipine, indicating that activation of GABAB receptors inhibits influx of Ca2ⴙ involving L-type Ca2ⴙ channels. Presence of GABAC receptors is suggested by the induction of inward current by (E)-4- amino-2butenoic acid (TACA) and its inhibition by 1,2,5,6-tetrahydropyridine-4-ylmethylphosphinic (TPMPA), a relatively selec-

Key words: REM sleep, in vitro, mouse, patch clamp, arousal, pons.

States of arousal, wakefulness, sleep and anesthesia (Moruzzi and Magoun, 1949; Baghdoyan et al., 1984; Steriade et al., 1990) are generated by activity of the ascending reticular activating system located, in part, within the pons. From studies conducted to determine the relevant neurotransmitters which act within the brainstem to control arousal state, it emerged that a critical role in this control is played by acetylcholine (ACh) as REM sleep can be induced by application of drugs which mimic the actions of ACh or heighten cholinergic tone into a region of the brainstem called the medial pontine reticular formation (mPRF; a.k.a. REM-induction zone) (George et al., 1964; Baghdoyan et al., 1984, 1987; Vanni-Mercier et al., 1989; Yamamoto et al., 1990; Garzon et al., 1998). A major endogenous cholinergic source to the mPRF REM-induction zone is provided by cholinergic projections originating from the laterodorsal tegmentum (LDT), a nucleus rich in distinct populations of cholinergic, glutamatergic and GABAergic neurons (Jones and Beaudet, 1987; Mitani et al., 1988; Shiromani et al., 1988; Wang and Morales, 2009). Subsequent studies revealed that lesions within the dorsolateral pontomesencephalic tegmentum of cats, which included the cholinergic LDT cell group, disrupted REM sleep (Jones and Webster, 1988; Webster and Jones, 1988); elimination of REM sleep was correlated with the extent of lesion of the cholinergic neurons (Jones and Webster, 1988; Webster and Jones, 1988); a population of putatively cholinergic neurons within the LDT increase and sustain their firing rates just before the onset of and during REM sleep respectively (el Mansari et al., 1989; Steriade et al., 1990; Kayama et al., 1992); electrical stimulation of the LDT and an adjacent region, the pedunculopontine tegmentum, increases REM sleep (Thakkar et al.,

*Corresponding author. Tel: ⫹45-35-33-60-55. E-mail address: [email protected] (K. A. Kohlmeier). Abbreviations: ACh, acetylcholine; ACSF, artificial cerebrospinal fluid; GZ, gabazine; LDT, laterodorsal tegmentum; LPT, lateropontine tegmentum; mPRF, medial pontine reticular formation; nNOS, neuronal nitric oxide synthase; PAG, periaqueductal grey; SLD, sublaterodorsal tegmental nucleus; TACA, (E)-4-amino-2-butenoic acid; TPMPA, 1,2,5,6-tetrahydropyridine-4-ylmethylphosphinic.

0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.09.034

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1996); ACh levels in the mPRF in REM sleep are significantly higher than during wakefulness or non-REM sleep (Kodama et al., 1990; Leonard and Lydic, 1995); and cholinergic agonists have excitatory actions on the majority of mPRF cells recorded (Greene et al., 1989). Accordingly, current models of state control propose that activation of cholinergic LDT neurons results in ACh release within the mPRF where subsequent neuronal excitation elicits the phenomenology associated with the state of REM sleep (Mitani et al., 1988; Semba, 1993; Jones, 2004; Steriade and McCarley, 2005). A critical question, therefore, if we are to understand the mechanisms which initiate or suppress wakefulness and sleep, is what controls the activity of cholinergic LDT neurons? In vivo behavioral studies using pharmacological agents that act as GABA-mimetics or GABA receptor antagonists have provided strong evidence that GABAergic mechanisms within the pons may play a role in the switch between behavioral states. Pontine application of GABA or muscimol, a specific GABAA receptor agonist, decreases REM sleep in cats; whereas, bicuculline, a GABAA receptor antagonist, increased this state (Xi et al., 1999a,b, 2001). In rats, iontophoresis of bicuculline or gabazine (GZ), another GABAA receptor antagonist, into the rat pontine sublaterodorsal tegmental nucleus (SLD), which is immediately ventral to the LDT, induced REM sleep (Boissard et al., 2002). In a brief report, microinjections of muscimol, a GABAA receptor agonist, in the rat mPRF were found to produce wakefulness with no effect on REM sleep (Camacho-Arroyo et al., 1991); however, a more detailed study indicated that similar application of muscimol suppress REM sleep, whereas bicuculline enhances this state (Sanford et al., 2003). Such enhancement of REM sleep by bicuculline and GZ was later found to be dependent on cholinergic mechanisms as pretreatment with atropine abolished the REM promoting actions of these GABAA agonists in the rat (Marks et al., 2008). Activation and inhibition of GABAB receptors within the mPRF of cats induces wakefulness and REM sleep, respectively, (Xi et al., 2001) and GABAB receptor activation within the pedunculopontine tegmentum prevents REM sleep in rats (Ulloor et al., 2004; Datta, 2007). Taken together, these data suggest that GABAergic mechanisms, possibly by modulation of cholinergic signaling play an essential role within the pons in generating wakefulness, and/or in inhibition of REM sleep. Since one possible mechanism whereby GABAergic processes may participate in behavioral state switching between REM sleep and wakefulness is via direct actions on cholinergic REM-promoting LDT neurons, we hypothesized that GABAergic agonists have actions on cholinergic neurons of the LDT and that these actions reflect presence of different GABA receptor subtypes. Therefore, we have undertaken the present study to determine the electrophysiological actions of GABA on cholinergic LDT neurons and to assay the presence of specific GABA receptor subtypes on these cells. To this end, we performed patch clamp recordings in identified mouse LDT neurons and

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monitored the response of the membrane to application of specific GABA receptor agonists and antagonists. We report here that GABA has profound inhibitory actions on cholinergic LDT neurons and more excitingly, we provide for the first time pharmacological evidence for the existence of three distinct subtypes of GABA receptors within the LDT. Our findings demonstrate that GABAergic mechanisms within the LDT can control excitability of cholinergic LDT neurons and in addition to synaptic presence within the LDT, GABAA receptors are also located extrasynaptically. Accordingly, we conclude that volume transmission of GABA, as well as classical synaptic transmission, could play a role in controlling tonic and phasic inhibition of LDT neurons and thereby, behavioral state.

EXPERIMENTAL PROCEDURES Preparation of brain slices Animal use studies were permitted by the Animal Welfare Committee, appointed by the Danish Ministry of Justice, and all studies complied with the EC Directive 86/609/EEC and with the Danish law regulating experiments on animals. All attempts were made to minimize number of animals used in this study and their suffering. The methods for preparing mouse brain slices, whole-cell recording, immunocytochemistry (Burlet et al., 2002) and calcium imaging (Kohlmeier et al., 2004, 2008) are similar to those utilized previously, and are briefly summarized here. Brain slices for whole-cell recordings and bis-fura 2 calcium imaging were obtained from 14 to 32-day old NMRI mice (Taconic, Denmark). Brain slices (250 ␮m) were prepared in ice-cold artificial cerebrospinal fluid (ACSF) which contained (in mM): 121 NaCl, 5 KCl, 1.2 NaH2PO4, 2.7 CaCl2, 1.2 MgSO4, 26 NaHCO3, and 20 dextrose, and was oxygenated by bubbling with carbogen (95% O2 and 5% CO2). Slices containing the LDT were incubated at 35 °C for 15 min in oxygenated ACSF, and were then allowed to equilibrate to room temperature for 1 h. Following this period, the slice was transferred to a recording chamber and immersed in 21 °C ACSF, which was perfused through the chamber at a flow rate of 4 – 6 ml/min. In those cases where low Ca2⫹ ACSF was utilized to reduce terminal release, Ca2⫹ was buffered by inclusion of EGTA to approximately 20 ␮M as calculated using Patcher’s Powers tools (Dr. Francisco Mendez, currently administered by Frank Würriehausen, Department of Membrane Biophysics, MPI Biophysical Chemistry, Germany).

Whole-cell patch clamp recordings Electrodes for whole cell patch clamp recordings were prepared from borosilicate thin walled glass (6 –11 M⍀, item number TW150F-4, WPI) with a Sutter P-97 horizontal puller (Sutter Instruments, CA, USA). The electrode was filled with an internal solution of (in mM): 144 K-gluconate, 0.2 EGTA, 3 MgCl2, 10 HEPES, 0.3 NaGTP, 4 Na2ATP or in those cases where calcium imaging was being conducted with a gluconate containing patch solution, pipettes contained the potassium salt of Fura-2 (bis-fura 2, 50 ␮M, Molecular Probes, OR, USA) dissolved in the patch pipette solution of, (in mM): 144 K-gluconate, 3 MgCl2, 10 HEPES, 0.3 NaGTP, and 4 Na2ATP. As the reversal potential of chloride with this solution (⫺79 mV) was not very different from the holding potential (⫺60 mV), recordings conducted to examine GABA A and C mediated currents were performed with a high chloride recording solution to optimize detection of inhibitory events by increasing the driving force, and reversing the direction, of chloride-mediated currents (reversal potential: ⫺2 mV) which contained (in mM) 140

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KCl, 10 mM EGTA, 1 CaCl2, 1 MgCl2, 10 HEPES, 2 Mg2⫹ATP. In all cases, ATP was added right before use of the patch solution and biotinylated Alexa-594 (25 ␮M, Molecular Probes, OR, USA) was also included in the solution so cells could be histochemically identified. Whole cell patch clamp recordings were made with an EPC-9 patch-clamp amplifier (HEKA, Lambrecht/Pfalz, Germany) and regions for recording were chosen from within the boundary of the LDT determined using a 4⫻ objective on an Olympus BX51WI microscope with brightfield illumination. Neurons were then visualized using a Sensicam VGA camera (Sensicam; PCO Imaging, Germany) under DIC optics with a 40⫻ water immersion objective (Olympus; NA 0.8, FL, USA). Images were displayed using TILLvision software (version 4.2; Till Photonics, Germany) on a personal computer. Although size is not a reliable indicator of presence of ACh in LDT neurons, cholinergic LDT neurons tend to be larger on average than GABA cells in this nucleus (Maloney et al., 1999; Boucetta and Jones, 2009) and more polygonal in shape than the more round GABAergic and putative glutamatergic cells also present (Boucetta and Jones, 2009). Accordingly, the largest (⬎18 ␮m), polygonal neurons in the field were selected for gigaseal recording in voltage clamp or current clamp mode to preferentially record ACh-containing neurons, although transmitter content was directly examined using immunohistochemical methods post hoc. Voltage measurements were not corrected for the junction potential which was measured and calculated to be approximately ⫺13 mV. Series resistances were in the range of 15–25 MOhm and were compensated by 50 –70%. If the series resistance changed by more than 20% during the recording, the data were discarded. Signals were filtered at 10 kHz and analyzed with IgorPro version 6 (Wavemetrics, OR, USA).

Calcium imaging Fluorescence related to intracellular Ca2⫹ concentration was measured in slices from neurons that had been filled with bis-fura2 from patch pipettes. Ca2⫹-transients were monitored by measuring the emission at 515 nm resulting from excitation of fura-2 with 380 nm (F380; 71000 Chroma fura 2 filter set, USA) from a shuttered 75 W xenon light source (Till Polychrome IV, TillPhotonics, Germany). Optical recordings were made using a 12-bit, frametransfer, VGA camera (Sensicam, PCO Imaging, Germany) controlled with TILLvision software (Till Photonics, version 4.2) running on a personal computer. Images were acquired continuously (⬃50 ms/frame), with the shutter open for the entire epoch. Changes in intracellular Ca2⫹ concentration were inferred from changes in delta F/F (df/F) where F is the fluorescence at rest within a region of interest (ROI) following subtraction of background fluorescence. df is the change in fluorescence following subtraction of the average F before stimulation. df/F was corrected for photobleaching where appropriate. Since increases in Ca2⫹ produce a decrease in F380 of fura2, all df/F measures are inverted such that positive-going traces indicate elevation of [Ca2⫹]i.

Immunohistochemistry Recorded neurons were putatively- and definitively-identified as cholinergic by two methods. It has been previously reported that cholinergic LDT neurons respond to cholinergic agonists, such as carbachol, with hyperpolarization caused by activation of GIRK channels (Luebke et al., 1993; Leonard and Llinas, 1994). The majority of non-cholinergic neurons within the LDT respond to carbachol with depolarization (Kohlmeier and Reiner, 1999). Therefore, electrophysiological response to carbachol, in combination with relative large size within the nucleus, serves as a good, first pass, marker of cholinergic phenotype (Kristensen and Leonard, unpublished observations). Accordingly, carbachol, a mixed muscarinic and nicotinic agonist was applied to every cell following determination of GABA actions and the electrophysiological response was monitored.

Recorded neurons in the LDT were also, in those cases where conditions of tissue preservation allowed recovery of the recorded cell, identified as cholinergic by immunohistochemistry. Cholinergic neurons in the LDT selectively co-localize the enzyme, neuronal nitric oxide synthase (nNOS; Vincent et al., 1983); and therefore, labeling for nNOS in recorded cells detected using immunohistochemical methods served as identification of cells in this study as cholinergic. For immunohistochemical processing, cryoprotected brain slices were re-sectioned on a Leica crysostat CM 3050S (Leica, Germany) at 40 ␮m. Filled neurons were visualized with Alexa-594 biocytin and nNOS was visualized using a FITC-labeled secondary antibody (Alexa Fluor 488, goat antirabbit IgG, Item #A11008, Molecular Probes, Invitrogen, Denmark) following incubation with a polyclonal primary directed against nNOS (antibNOS, rabbit polyclonal, Cat # N7280, Lot # 115K4854, Sigma-Aldrich, Denmark A/S). Mounted sections were imaged using appropriate filter sets with a monochrome digital camera (Axiocam MRM, Zeiss) mounted on an epi-fluorescence microscope (Axioskop 2, Zeiss). Images were collected using Axiovision 4.6 (Zeiss, Germany) software and post-hoc image processing consisted in changes of contrast applied equally across all images by use of Adobe Photoshop CS3 (Version 10.0.1).

Drugs All drugs were applied with a seven barrel perfusion pipette positioned in the ACSF just above the LDT. Drugs were gravity fed with application time controlled by a solenoid valve system (Lee Valves, The Lee Company, USA). Tissue was pre-incubated in antagonists at least 10 min before, and continuously exposed during, application of agonists. GABA, carbachol and nifedipine were obtained from Sigma and applied at a final concentration of 100 ␮M, 10 ␮M and 10 ␮M, respectively. Muscimol (30 ␮M, Sigma) and SR-95531 (GZ, 10 ␮M, Sigma) were used to selectively activate and inhibit GABAA receptors, respectively. Stock solutions of GZ and light-protected nifedipine were prepared in ACSF containing 50% and 100% DMSO, respectively, making the final ratio of dilution of DMSO in ACSF 1:2000 or 1:1000. This vehicle in preliminary experiments had no actions of its own on cholinergic LDT neurons. Baclofen (10 ␮M, Sigma) and CGP 55845 (10 ␮M, Tocris) or saclofen (30 ␮M, Tocris) were used to selectively activate and inhibit GABAB receptors, respectively. (E)-4-amino-2-butenoic acid (TACA, 10 ␮M, Sigma) was used to activate GABAC receptors; however, because this drug can have agonistic actions at the GABAA receptor, for this series of experiments, GZ was also included to limit responses to activation of the GABAC receptor. A selective antagonist of GABAC receptors, 1,2,5,6-tetrahydropyridine-4-ylmethylphosphinic (TPMPA, 20 ␮M, Tocris), was used to confirm actions of TACA were restricted to GABAC receptors. THIP (Gaboxadol) was a gift from Bente Frølund (University of Copenhagen, Denmark) and prepared at a final concentration of 1 ␮M, which has been reported to be selective for activation of ␦-subunit containing GABAA receptors (Jia et al., 2005; Drasbek and Jensen, 2006). To rule out a role of diazepam sensitivity of the THIP response, potentiation studies were conducted with 1 ␮M diazapam (DMSO, Sigma). TTX, which blocks voltage dependent sodium channels, was obtained from Alamone and applied at a final concentration of 500 nM. Tertiapin Q (Tocris) a high affinity blocker of inwardly rectifying K⫹ channels (GIRK) was applied at a final concentration of 10 nM. In some experiments, glutamatergic ionotropic synaptic activity was suppressed by inclusion of L-2-amino-5-phosphonovaleric acid (APV, 50 ␮M, Sigma) and 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 10 ␮M, Sigma) or kynurenic acid (1 mM, Sigma). Glycinergic responses were blocked by 2.5 ␮M strychnine (Sigma).

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Data analysis Changes in holding current elicited by application of drugs were measured between the holding current at baseline and the holding current required at the peak of the drug induced inward or outward current. Detection of PSCs and some analyses were done using Mini Analysis software (Synaptosoft, Decatur, GA, USA). To compare the effects of diazepam on the number and amplitude of PSCs, epochs of 30 s before and following application of drugs were used. Cumulative distributions of IPSCs for each cell were compared in control and drug conditions using Kolmogorov–Smirnov statistics (K–S test) with Mini Analysis software to determine significance with the level of significance set at values below 0.05. Analysis of the decay phase of IPSCs were conducted from 10% to 90% of the peak amplitude and were best fit by the twoexponential function, suggesting presence of a fast and a slow component. Analysis, and figure preparation of electrophysiological and df/F signals were performed using Igor Pro 6 software (Wavemetrics). Differences between means were determined by utilizing of a paired/unpaired Student’s t-test or a one-way, repeated measures ANOVA, as required. Non-parametric comparisons were conducted utilizing Chi-Square analysis. The value for statistical significance was set at P⬍0.05. Numerical results are reported as mean⫾SEM.

RESULTS GABAA receptors in the LDT In all cells recorded with normal chloride internal solution, GABA (100 ␮M, 5 s) induced an outward current (8.05⫾1.1 pA, n⫽14/14). Application of GABA induced inward currents (⫺71.8⫾18.1 pA n⫽22/27; Fig. 1A, B) in the majority of LDT neurons when recorded with pipettes high in chloride ions to shift the chloride gradient and thereby increase the drive for chloride. The reversal of GABA-induced currents coincident with alteration of the chloride gradient indicates that this current was mediated primarily by a chloride conductance consistent with opening of ionotropic GABAA receptors. In four of the remaining five cells recorded with high chloride pipettes, a net outward current of 16.7⫾13.6 pA was induced, consistent with stimulation of a GABAB receptor, which was examined in closer detail (below). GABA failed to elicit a detectable membrane response in the remaining cell (n⫽1/27). The induction of inward or outward current by GABA was unaffected by including TTX in the ACSF (500 nM; P⬎0.05, n⫽10) to block voltage-gated sodium channels or by low Ca2⫹ ACSF solutions (20 ␮M; P⬎0.05, n⫽8), indicating responses were mediated via activation of postsynaptic receptors. As part of the selection criterion for inclusion in this study, all of these cells exhibited outward currents on application of carbachol (average outward current with high chloride pipettes: 10.3⫾1.2 pA, n⫽27; average outward current with normal patch: 20.1⫾9.3 pA, n⫽14; Fig. 1A, B). To investigate the GABA receptor subtype(s) present on LDT neurons in more detail, specific GABA receptor agonists and antagonists were utilized. Muscimol, a specific GABAA receptor agonist, when applied in ACSF with TTX to a group of cells recorded with high chloride internal solution, induced large amplitude, inward currents (⫺148⫾42.1 pA, n⫽11/11; Fig. 1D, E). Immunohistochem-

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istry revealed that 6/6 of the recovered cells that responded in this manner were bNOS⫹ (Fig. 1F). Gabazine is a competitive antagonist at the GABAA receptor, and when bound, antagonizes the ability of GABA or muscimol to bind. Application of GZ revealed tonic inhibitory current by shifting the holding current outward in some of the cells tested (11.1⫾2.3 pA, n⫽5, P⬍0.5, Fig. 1C). Consistent with the interpretation that GABA and muscimol were acting at postsynaptic GABAA receptors, we found that presence of 10 ␮M GZ produced a reversible block of muscimol-induced inward currents in the same cells in which muscimol elicited inward current in control conditions (control inward current: ⫺145.9⫾80.4 pA; muscimol/GZ ⫺0.7⫾0.7 pA; n⫽5/5; Fig. 1D, E). GABA-mediated inward currents were attenuated in the same cells if GABA (100 ␮M) was applied following pre-incubation with GZ, but GZ failed to completely block the inward current or in some cases, outward current was revealed (control GABA: ⫺123⫾63.7 pA, GABA/GZ ⫺10.8⫾4.8 pA, n⫽4/6; GABA/GZ 15.3⫾2.3 pA, n⫽2/6; Fig. 1D1a). These data indicate that GABA and muscimol are acting predominantly at GABAA receptors, but suggest that GABA also stimulates non-GABAA receptors, which result in activation of a chloride conductance or an outward current, presumably mediated by a GABAB receptor. Gaboxodol (THIP) at low concentrations preferentially activates GABAA receptors containing the ␦ subunit in those regions where this has been studied (Brown et al., 2002; Jia et al., 2005; Shen et al., 2005; Drasbek and Jensen, 2006). Although THIP has been shown to be more potent and efficacious at ␦ subunit containing receptors (Storustovu and Ebert, 2006), it has been shown to activate a putative ␣5␤␥2 receptor in hippocampus (Lindquist et al., 2003). ␦ subunits are typically incorporated in GABAA receptors located away from the synapse allowing activation by ambient levels of GABA resulting from spillover following synaptic release (Nusser et al., 1998). The ␣5␤␥2 putatively activated by THIP in hippocampal cells is also extrasynaptic in location (Lindquist et al., 2003). We wished to test whether GABAA receptors exhibiting THIP sensitivity, and therefore probably extrasynaptic in location were functionally present in the LDT. In some brain regions, expression of the ␦ subunit was found to exhibit a developmentally-dependent pattern, although the majority of regions examined were found to express ␦-containing subunits by P12 (Laurie et al., 1992). As the age-dependency of ␦ subunit presence within the LDT was not reported, recordings for this examination were conducted in brain slices obtained from older animals between P19 and P23 days of age. THIP at low concentrations used therapeutically and previously reported to be specific to activation of extrasynaptic GABAA receptors (1 ␮M, 5 s; (Jia et al., 2005; Drasbek and Jensen, 2006)) was found to induce a TTX-resistant inward current in the majority of LDT cells recorded (⫺7.4⫾1.5 pA, n⫽19, P⬍0.05, Fig. 2A, B, D). Gabazine (10 ␮M) was able to fully block responses to THIP (n⫽4/4) indicating actions were limited to GABAA receptors (Fig. 2B2, C). Two of the four cells were recovered and found to be bNOS⫹. THIP responses in pres-

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Fig. 1. GABA (100 ␮M, 5 s) acting at GABAA receptors induces inward currents in the majority of cells in the LDT in which carbachol (carb) induces outward currents. (A) Representative voltage clamp recording of 1 cell in the LDT recorded with a high chloride pipette in which GABA induced an inward current. Subsequent application of carbachol induced an outward current. (B) Histogram of the mean amplitude of inward current induced by GABA and outward current induced by carbachol in the population of cells (n⫽22). (C) Gabazine (GZ, 10 ␮M), an antagonist of GABAA receptors induced outward current in some cells, indicating presence of tonic inhibitory current and was effective in reducing inward currents induced by both GABA (D1a) and those elicited by the specific GABAA receptor agonist, muscimol (musc, 30 ␮M) (D1b). Control responses to GABA and muscimol are shown with overlaid responses in presence of antagonist demonstrating attenuation of agonist-induced inward currents. (D2) Recovery of the GABA and muscimol responses was possible after washout of gabazine. Response to carbachol is also shown in the same cell. (E) Summary histogram showing the complete block of muscimol-induced inward currents in presence of gabazine (n⫽5). * Indicates P⬍0.05 in this, and subsequent figures. (F) Representative example of the histochemical identification of LDT cells as bNOS⫹ and classification of recorded cells as cholinergic. Left panel viewed under fluorescent optics (488 nM) to show bNOS⫹ cells, right panel is of the same field viewed under optics allowing visualization of Alexa-594 (594 nM), demonstrating that the recorded cell (arrow right) is bNOS⫹ (arrow left) and hence, cholinergic. Scale: 20 ␮M.

ence of 1 ␮M diazapam were not found to be different (P⬍0.05, n⫽3), indicating that the ␣5␤␥2, diazepam-sensitive extrasynaptic receptor reported in CA1 hippocampal

cells does not play a role in THIP responses in LDT cells (Fig. 2D). Interestingly, we found that diazepam significantly increased the frequency, amplitude and the decay

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Fig. 2. Extrasynaptic GABAA receptors are present on cholinergic LDT neurons. (A) THIP (1 ␮M) induced an inward current in a cell (right trace) in which GABA induced a larger inward current (left trace). (B) Voltage clamp recordings conducted in another cell in which responses to THIP (left traces) and GABA (right traces) during blockade of GABAB and GABAC receptors with CGP 55845 (10 ␮M) and TPMPA (20 ␮M), respectively, are shown. Recordings were conducted in absence of (B1), presence of (B2), and recovery from (B3) blockade of GABAA receptors with gabazine (10 ␮M). A response to muscimol was also recorded in this cell after recovery from all blockers, except TTX (B4). (C) Voltage clamp recording showing the effect of diazepam on increasing the frequency and amplitude of phasic inhibitory events. This action of diazepam extended to all six cells examined (K–S test, P⬍0.05). (D) Summary histogram of inward currents induced by GABA and THIP in the same group of cells (n⫽15), THIP-induced inward currents in presence of gabazine in four of these cells and THIP-induced inward currents in presence of diazepam in three cells indicating blockade of the putative THIP sensitive, diazepam insensitive extrasynaptic GABA receptor response by the GABAA antagonist, gabazine.

time constant of phasic inhibitory events in all cholinergic LDT cells tested (IPSC frequency increase from control: 280⫾60%, IPSC amplitude increase from control, 150⫾ 23%, fast decay time constant increase from control,

21.3⫾9.2%, slow decay time constant increase from control, 41.2⫾5.3% n⫽6, P⬍0.05, Fig. 2C). One caveat of our study is that before diazepam, as amplitudes of PSCs were small, they may have been below detection threshold in

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presence of tonic current. Consequently, the number of inhibitory events in control conditions may have been undercounted. Regardless, our findings that GZ reveals a tonic inhibitory current and that diazepam has strong actions on the amplitude and decay of the time constant of phasic inhibitory events in cholinergic LDT cells suggests presence of a functionally active level of endogenous GABAergic tone in our slices. Taken together, these data provide the first functional evidence that cholinergic LDT neurons possess functional extrasynaptic GABAA receptors suggesting volume transmission could play a role in controlling tonic inhibition of LDT neurons. Likely inclusion of the ␦ subunit within these extrasynaptic receptors suggests one more potential therapeutic target of THIP. GABAB receptors are present in the LDT and mediate two types of inhibition In addition to ionotropic receptors, GABA also activates metabotropic GABAB receptors (Sivilotti and Nistri, 1991). Baclofen is a selective agonist for the GABAB receptor. Application of 10 ␮M baclofen induced an outward current in all LDT neurons tested regardless of whether the intracellular patch solution contained high Cl⫺ or normal K⫹/ gluconate (Fig. 3A, B). The average amplitude of the baclofen-induced outward current was 24.8⫾4.02 pA in 20/20 cells in which the amplitude of the average carbachol-induced outward current was 17.4⫾3.7 pA (n⫽20). It was possible to recover 10 of the 20 cells and all of them proved to be bNOS⫹. Baclofen-induced outward currents exhibited a slow onset and a long duration (Fig. 3A, B). Effects were repeatable following a recovery time of 20 min. TTX and low Ca2⫹ solutions did not block this response (control 25.2⫾8.2 pA, TTX 38.0⫾12.3 pA, n⫽5 P⬎0.05; control 23.4⫾38.0 pA, low Ca2⫹ 23.7⫾4.2 pA, n⫽3 P⬎0.05), indicating that GABAB receptors were located on postsynaptic sites. CGP 55845 (n⫽4) and saclofen (n⫽3), antagonists of the GABAB receptor, blocked this response (bac 24.0⫾8.6 pA, bac/ CGP55845 2.16⫾2.7 pA, P⬍0.05. Fig. 3A, C1; bac 20.9⫾5.2 pA, bac/sac 2.8⫾1.5 pA, P⬍0.05, Fig. 3B, C2). Taken together, our data demonstrate presence of postsynaptic GABAB receptors on cholinergic LDT neurons. In other cell types, GABAB receptors can activate an inwardly-rectifying K⫹ (GIRK) current. In cholinergic LDT neurons, GIRK can be activated by muscarinic cholinergic agonists demonstrating presence of this channel in these cells (Luebke et al., 1993; Leonard and Llinas, 1994). We reasoned that if GABAB mediates its effects via this channel, then actions of carbachol, a cholinergic agonist and baclofen should not be additive. In seven cells, baclofen induced outward currents of 18.9⫾5.7 pA, whereas, carbachol induced outward currents of 14.2⫾1.5 pA (Fig. 4A). In these same cells, application of baclofen together with carbachol induced an outward current of 15.4⫾2.5 pA, which was not significantly different than the current induced by baclofen alone (n⫽7, P⬎0.05, Fig. 4A). Coapplication of carbachol and baclofen to the same cells induced a current that was significantly less than the current predicted assuming additive effects (n⫽7; P⬍0.05,

Fig. 4B, C). Thus, the currents partially occluded one another, which is consistent with convergence of the signaling resulting from activation of these two receptors onto the same G protein and/or channel population. Although the data from the occlusion experiments suggested that outward currents mediated by GABAB activation are mediated by the same GIRK shown to be responsible for carbachol-mediated outward currents in these cells, we wished to more directly examine this issue. Accordingly, we applied baclofen following pretreatment of the cells with 10 nM tertiapin, which we have previously shown inhibits the GIRK activated by carbachol in cholinergic LDT neurons (Kohlmeier et al., 2004). In six cells in which baclofen had induced an outward current of 11.5⫾1.9 pA, tertiapin reduced the outward current induced by baclofen to 1.85⫾1.2 pA (P⬍0.05, Fig. 4D, E). These data strongly suggest that activation of GABAB receptors on cholinergic LDT neurons activates the same GIRK linked to muscarinic receptors in these cells. GABAB receptors have been reported to reduce Ca2⫹ via inhibition of L-type Ca2⫹ channels. We previously reported that L-type Ca2⫹ channels can be activated in cholinergic LDT neurons by stepping the membrane potential from a holding potential of ⫺60 mV to ⫺30 mV (5 s duration) (Kohlmeier et al., 2008). Using high speed calcium imaging to monitor bis-fura 2 changes in fluorescence, in accordance with this report, in the present study, we found that a voltage step to ⫺30 mV in control conditions induced a change in df/F of 5.2⫾0.68% (n⫽12) in cells loaded with this Ca2⫹ binding dye. Baclofen was then applied and following establishment that baclofen induced an outward current, we found that changes in df/F induced by the step to ⫺30 mV, were significantly reduced by 32.6⫾4.5% from the pre-baclofen values (n⫽12, P⬍0.05, Fig. 3D). In the presence of CGP 55845, baclofen failed to reduce df/F (n⫽3). To further investigate the role that L-type Ca2⫹ channels play in GABAB mediated reduction of Ca2⫹, we pre-treated cells with nifedipine (10 ␮M), an inhibitor of L-type Ca2⫹ channels. Nifedipine itself reduced the df/F transient induced by the step to ⫺30 mV by 33.1⫾9.4% (n⫽4) which is in accord with previous reports (Kohlmeier and Leonard, 2006). In the presence of nifedipine, baclofen failed to significantly reduce df/F further (4.07⫾1.28%, n⫽4, P⬎0.05, Fig. 3D). Taken together, our data indicate that reduction of Ca2⫹ transients induced by baclofen in cholinergic LDT neurons is mediated by GABAB mediated inhibition of L-type Ca2⫹ channels. GABAC receptors are functionally present in the LDT The GABA receptor ␳ subunits are thought to form GABA receptor channels which belong to a pharmacologically distinct receptor class called GABAC (Drew et al., 1984). With utilization of reverse-transcriptase PCR techniques, ␳ expression was noted in the rat brainstem, although more precise localization was not presented (Wegelius et al., 1998). We wished to examine whether GABAC receptors are functionally present in the LDT. Accordingly, we applied a relatively selective GABAC agonist, TACA to LDT neurons. Because TACA may have some weak agonist

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Fig. 3. GABAB receptors are located postsynaptically on cholinergic LDT cells and mediate outward currents and a reduction in Ca2⫹ via inhibition of the L-type Ca2⫹ channel. (A) Voltage clamp recording of 1 cell in which baclofen (10 ␮M), a specific agonist of GABAB receptors, induced an outward current, which could be antagonized by CGP 55845 (10 ␮M), an inhibitor of GABAB receptors. Recovery of the response after wash-out of CGP was possible. (B) Another antagonist of GABAB receptors, saclofen (30 ␮M), also inhibited outward currents induced by baclofen. Baclofen responses recovered following wash out of saclofen (C) Summary histograms of inhibition of baclofen induced outward currents by CGP (C1, n⫽4) and saclofen (C2, n⫽3) in the population of cells examined with the two GABAB. receptor antagonists. (D) Recordings with the calcium indicator dye, bis Fura-2 (panel on left shows fluorescence image of representative cell filled with bis fura-2; inset shows same cell under bright field optics), revealed that stimulation of GABAB receptors reduced Ca2⫹ induced by a voltage step to ⫺30 mV for 500 msec, a protocol previously demonstrated to activate L-type Ca2⫹ channels in these cells. Application of nifedipine (10 ␮M) reduced this change in df/F and baclofen in nifedipine failed to reduce this transient further, indicating a role of L-type Ca2⫹ channels in this action of baclofen.

actions at GABAA receptors, TACA was applied in the presence of GZ to block contributions from GABAA receptor activation. TACA induced an inward current in the majority of cells when recorded with pipettes containing high chloride solution (⫺16.9⫾4.5, n⫽16/29, Fig. 5A, B), with repeatable effects at 20 min intervals. Five of these 16 cells were recovered and found to be bNOS⫹. Unexpectedly, TACA induced an outward current in some of the cells (14.6⫾2.3, n⫽5/29, Fig. 5C), while no apparent mem-

brane action of TACA was elicited on the membrane of the remaining eight cells, this outward current had a very slow onset and decay and thereby resembled that induced by both carbachol and baclofen. However, because TTX abolished this outward current, revealing an inward current in the same cells (control: 13.0⫾3.3 pA; TTX: ⫺16.17⫾3.46 pA, n⫽5/5, Fig. 5C), we did not further pursue the mechanism of this presumably pre-synaptic action of TACA. We did test whether TTX affected cells in which in its absence

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Fig. 4. The same inwardly rectifying current activated by carbachol in cholinergic LDT cells is stimulated by GABAB receptors. (A) Current traces of responses of 1 cell to baclofen (1), then following recovery, to an application of carbachol (2). Baclofen and carbachol were then added together (3); the resultant current was smaller than the summation of the currents induced by these agonists when applied alone, suggesting that the same current is activated by both. After washout of both agonists, baclofen was re-applied, but in low Ca2⫹ ACSF (4). These data not only provide evidence that actions of baclofen are repeatable but also, when taken in combination with our data showing presence of TTX-insensitive baclofen-induced currents, that functional GABAB receptors are located on postsynaptic sites. (B) Actual responses of individual cells to co-application of baclofen and carbachol are plotted (Actual) and contrast to responses predicted if individual currents activated by baclofen and carbachol were summed (predicted). Each cell is shown to demonstrate that occlusion extended to all cells tested. (C) Summary histogram of occlusion experiments with baclofen and carbachol are shown. Tertiapin (10 nM), which has been shown to block the inward rectifier in cholinergic LDT cells was successful in inhibiting the membrane response to baclofen, as shown in one representative cell in current traces (D) and in the summary data shown in the histogram from a population of cells (E). TTX and kynurenic acid (1 mM) were present in these experiments and arrow in top trace of panels (A, D) indicates time at which agonist was applied under each condition.

an inward current had been elicited and found no difference in inward current when TTX was present (control: ⫺13.5⫾2.04 pA, TTX: ⫺9.8⫾1.8 pA, n⫽3/3, P⬎0.05). We also examined the ability of TPMPA, a highly selective GABAC receptor antagonist to inhibit both TACA and GABA-induced inward currents. In a sub-population of cells in which TACA and GABA induced an inward current (TACA: ⫺11.1⫾1.2 pA GABA: ⫺21.6⫾3.7, pA, n⫽5), TACA/TTX was re-applied in presence of TPMPA. In the presence of TPMPA, TACA induced inward current was

significantly decreased (TACA/TPMPC: 1.3⫾2.2 pA, n⫽5, Fig. 5B, D). GABA-induced inward currents were also significantly decreased by TPMPA (38.2⫾3.8%; P⬍0.05 n⫽5, Fig. 5B), indicating that GABA acts at GABAC receptors. Taken together, our data show that GABAC receptors are present in a sub-population of LDT cholinergic neurons and suggest the possibility that GABAC receptors are present on neurons pre-synaptic to cholinergic LDT cells. An interesting possibility is that GABAC receptors exist on cholinergic LDT neurons sending cholinergic projections to

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Fig. 5. Functional GABAC receptors are present on cholinergic LDT neurons. (A) TACA (10 ␮M), an agonist of GABAC receptors, in presence of gabazine and TTX induces an inward current, which is blocked by the specific GABAC receptor antagonist, TPMPA (20 ␮M). (B) GABA induces an inward current which can be partially blocked by TPMPA, suggesting this portion of the current is mediated by GABAC receptors. (C) In some cells, TACA induced an outward current, which was blocked by application of TTX revealing an inward current, suggesting an action of this agonist on GABAC receptors located presynaptic to the recorded cholinergic cell. Arrow in top trace of panels (A–C) indicates time at which agonist was applied under each condition. (D) Summary histogram of the inward currents induced by TACA, and their inhibition by TPMPA (GZ/TTX conditions, n⫽5).

neighboring or contralateral LDT cholinergic cells (Semba and Fibiger, 1992); however, this connectivity requires explicit experimental examination.

DISCUSSION Cholinergic neurons of the LDT play a critical role in behavioral state control (Mitani et al., 1988; Webster and Jones, 1988; Jones, 1990, 2004; Semba, 1993). One of the most recently, experimentally-addressed mechanisms that may control activity of these cells is inhibition by GABA (Xi et al., 1999a,b; Boissard et al., 2002; Sanford et al., 2003; Vazquez and Baghdoyan, 2004; Marks et al., 2008). An important tenant of the hypothesis that GABAergic activity controls cholinergic LDT neurons is that GABA must have actions on cholinergic LDT neurons, which has not been systematically studied. Accordingly, we have undertaken this study to examine whether electrical changes

and alterations in intracellular Ca2⫹ levels are induced in these cells by GABAergic stimulation. Additionally, as the LDT is a site important in regulation of states of consciousness (Lydic and Baghdoyan, 2005; Van Dort et al., 2008), and a plethora of subtype-specific, GABAergic pharmacological agents are in clinical use in management of sleeprelated disorders, as well as anesthetic agents that alter consciousness by acting at specific GABA receptors, characterization of the GABA receptor subtypes functionally present in the LDT was also conducted. GABA and GABAmimetics were found to have strong actions on virtually every cholinergic LDT neuron examined. Further, actions of specific GABAergic receptor agonists and antagonists indicates presence on cholinergic LDT neurons of functional GABAA, GABAB and GABAC receptors. Based on our data, we conclude (1) endogenous GABA could play a role in control of cholinergic LDT neurons and thereby,

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behavioral state control; (2) exogenous application of GABA receptor acting agents, such as benzodiazepines or anesthetics, likely exert direct effects on cholinergic LDT neurons; (3) GABAA receptors containing ␦-subunits, and therefore likely to be extrasynaptic in location, are also present on cholinergic LDT neurons, suggesting that volume transmission of GABA may be a method of neuronal communication within the LDT. Functional GABAA receptors are present on cholinergic LDT neurons GABA activates three major classes of GABA receptors: GABAA, GABAB and GABAC. They are grouped based on differences in subunit composition, gating properties and pharmacological profile. GABAA receptors are ligandgated chloride channels. These receptors are hetero-oligomeric pentamers and made up predominantly of 2␣, 2␤ and either the ␥, ⑀ or ␦ subunit (Johnston, 1996; Davies et al., 1997); in addition, several classes of these subunits exist, some of which exhibit splice variants. Classical synaptic GABA transmission which exerts phasic postsynaptic inhibition, is mediated by GABAA receptors containing the ␥ subunit (Ebert et al., 2006; Rudolph and Mohler, 2006; Olson and Nestler, 2007). These receptors are activated by the specific agonist, muscimol and inhibited by GZ (Fig. 1). Although not specifically examined within the LDT, immunohistochemical evidence has been presented that the ␣, ␤, and ␥ subunits are located in the pontine region wherewith the LDT is located, which suggests that GABAA receptors mediating classic synaptic phasic inhibition are present (Fritschy et al., 1992; Pirker et al., 2000). Indeed, a recent report presents functional evidence that multiple GABAA receptors are present in rat pons (HambrechtWiedbusch et al., 2010). Consistent with these studies, we found that muscimol induced a GZ-sensitive inward current in cholinergic LDT neurons. Accordingly, our data provide further evidence that synaptic GABAA receptors are located within the LDT on cholinergic neurons and suggest that on GABAergic input, activation of these receptors would lead to phasic inhibition of cholinergic LDT cells. Functional extrasynaptic GABAA receptors are present on cholinergic LDT neurons In contrast to GABAA receptors which contain the ␥ subunit and are located within synapses and activated by release events, GABAA receptors containing the ␦-subunit are located extrasynaptically (Nusser et al., 1998), and, are therefore activated endogenously by GABA resulting from synaptic outflow, or non-synaptic, ectopic release. These extrasynaptic GABAA receptors display a high affinity for GABA and are weakly desensitizing (Saxena and Macdonald, 1994, 1996), which confers reactivity to low levels of extracellular GABA leading to a tonic inhibitory conductance in postsynaptic neurons. Low concentrations (1 ␮M) of the agonist, THIP (a.k.a. gaboxodol) have been shown to act in a selective manner on GABAA receptors containing the ␦ subunit within the mouse cortex (Drasbek and

Jensen, 2006) and the thalamus (Jia et al., 2005). Immunohistochemical studies in the mouse brain have demonstrated presence in the pontine reticular formation of ␦ subunit (Fritschy et al., 1992; Pirker et al., 2000) and our data provide the first functional evidence suggesting that GABA receptors containing the ␦ subunit are present on cholinergic LDT cells (Fig. 2). While low concentrations of THIP have been shown to activate a diazepam-sensitive putative ␣5␤␥2-containing extrasynaptic GABAA receptor in rat hippocampal CA1 cells (Lindquist et al., 2003), our data showing a lack of potentiation of THIP responses in presence of diazapam do not support a role of this GABAA receptor subunit configuration in mediating THIP-elicited responses within the LDT. We therefore conclude that the GABAA receptor activated by THIP in LDT cells contains the ␦-subunit and based on findings from other cell types, we conclude that these receptors are extrasynaptic in location. Accordingly, taken together, our data provide the first direct evidence that cholinergic LDT neurons express functionally active intrasynaptic as well as extrasynaptic GABAA receptors. Functional extrasynaptic GABAB receptors are present on cholinergic LDT neurons and modulate voltage-activated calcium channels GABAB receptors are seven transmembrane domain receptors coupled to second messenger systems and Ca2⫹ and K⫹ channels via G-proteins. These receptors are activated by the specific agonist, baclofen and blocked by saclofen and newer antagonist compounds such as CGP 55845. GABAB receptors consist of GABAB1 and GABAB2 subunits (Jones et al., 1998; Benke et al., 1999; Filippov et al., 2000; Duthey et al., 2002), with the recent characterization of a novel subunit GABABL in rat brain (Calver et al., 2003; Charles et al., 2003). GABAB receptor subunit presence has been detected in the rat pons (Liang et al., 2000); however, detailed localization within individual pontine nuclei was not presented. Our data therefore provide the first evidence that GABAB receptors are present postsynaptically on cholinergic LDT neurons (Figs. 3 and 4). Stimulation of these receptors on cholinergic LDT neurons by the high affinity agonist, baclofen, resulted in outward currents mediated by the same tertiapin-sensitive inwardly rectifying potassium (GIRK) conductance activated in these cells by muscarinic stimulation. Moreover, GABAB activation in hippocampal pyramidal neurons also results in a potassium conductance that is sensitive to barium, which is a putative blocker of G-protein-activated GIRK channels (Newberry and Nicoll, 1984; Russo et al., 1998). Stimulation of GABAB receptors also modulates voltage-activated Ca2⫹ channels (Maguire et al., 1989; Heidelberger and Matthews, 1991; Tatebayashi and Ogata, 1992; Bussieres and El Manira, 1999). We show here that stimulation of GABAB receptors reduces Ca2⫹ entry through L-type Ca2⫹ channels, as previously demonstrated in other neuronal types (Sah, 1990; Scholz and Miller, 1991; Matsushima et al., 1993; Russo et al., 1998).

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Functional extrasynaptic GABAC receptors are present on cholinergic LDT neurons The most recently recognized member of the GABA receptor family, the GABAC receptor, is thought to be composed primarily of ␳ subunits (Zhang et al., 1995; Lukasiewicz, 1996) and multiple ␳ subunits have been cloned from various species (Cutting et al., 1991, 1992; Enz and Bormann, 1995; Lukasiewicz, 1996). Expression of mRNA transcripts for the ␳1 subunit in rat revealed presence in the brainstem, albeit at low levels (Wegelius et al., 1998) consistent with presence of GABAC receptors outside the retina, where they have been extensively characterized (Feigenspan et al., 1993; Qian and Dowling, 1993, 1995; Lukasiewicz and Werblin, 1994; Lukasiewicz and Wong, 1997), although the exact nature of subunits that comprise brainstem GABAC receptors in human beings remains an open question (Enz and Cutting, 1999). Our data provide the first evidence of the presence of functional GABAC receptors specifically on cholinergic LDT neurons (Fig. 5). For our characterization, we used TACA, which has been reported to be the most potent agonist of the GABAC receptor (Chebib et al., 1997); however, it is not completely selective. Accordingly, we applied TACA concurrently with GZ to antagonize the GABAA receptor and thus limit actions to agonism of GABAC receptors. Under these conditions, we found that TACA elicited small, chloride-mediated, inward currents in cholinergic LDT neurons. TPMPA, a selective antagonist of GABAC receptors (Murata et al., 1996; Ragozzino et al., 1996) antagonized TACA- and some portion of GABA-induced inward currents, indicating activation of GABAC receptors. Although it was noted that the amplitude of the GABAC mediated current was, on average, much smaller than that induced by activation of GABAA receptors, a careful characterization of the affinity, activation and desensitization kinetics of these receptors was not conducted in the present study. However, in other cell types where it has been studied, GABAC receptors show a higher sensitivity for GABA, do not desensitize and exhibit a smaller conductance than classic GABAA receptors (Enz, 2001), suggesting that the presence of this receptor in a subpopulation of cholinergic LDT neurons might afford a longer lasting, chloride-mediated inhibition of these cells at lower concentrations of GABA, compared to that induced following activation of synaptic GABAA receptors. Pontine applications of GABAergic agents result in alterations in behavioral state Demonstrating a site-specific role within the pons for GABA in state control are multiple findings that focal application of agonists and antagonists alter behavioral state. GABAA receptor agonists and antagonists induce wakefulness and promote REM sleep, respectively, when injected into the medial pontine reticular formation (a.k.a. NPO), a site in which injection of cholinergic agents induces REM sleep or within a region of the pons in the rodent dorsomedial tegmentum, corresponding to the SLD delineated by Swanson (Swanson, 1998; Xi et al., 1999b, 2001; Bois-

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sard et al., 2002; Torterolo et al., 2002; Pollock and Mistlberger, 2003; Sanford et al., 2003). Muscimol microinjections into the periaqueductal grey (PAG), a region of the pons located slightly rostral to the LDT resulted in hypersomnia in cats (Sastre et al., 1996). Muscimol induced similar effects when injected into the ventrolateral PAG and the dorsal mPRF of guinea pigs (Vanini et al., 2007) as well as the cat peduculopontine tegmentum (PPT), a cholinergic cell group continuous with the LDT (Torterolo et al., 2002), although only slight effects on behavioral state were noted in a different study (Sanford et al., 1998). GABAB receptor agonists and antagonists applied into the NPO (mPRF) also induced wakefulness and REM sleep, respectively, albeit to a lesser degree than GABAA acting compounds (Xi et al., 2001). GABAB receptor agonists induce wakefulness in rats when injected into the PPT (Ulloor et al., 2004). Further evidence of GABAergic mechanisms involved in state control are findings that bilateral injections within the cat NPO (mPRF) of phosphorothioated antisense oligonucleotides against the GABA synthetic enzyme, GAD, induced a decrease in wakefulness with an increase in REM sleep (Xi et al., 1999b). Additionally, loss of consciousness induced by isoflurane anesthesia was found to be associated with a decrease of GABA within the cat pons (Vanini et al., 2008). Taken together, these behavioral studies strongly implicate a role of GABA mechanisms located in or near to the LDT in endogenous control of sleep or wakefulness state. Do GABAergic mechanisms gate the cholinergic mechanisms? The cholinergic hypothesis of state control is based on multiple reports that injection of cholinergic agonists into the mPRF (NPO) of cats induces a REM like state and it is widely believed that a large endogenous source of input to the mPRF (NPO) of this ACh derives from cholinergic LDT neurons. Several studies in the cat suggest that mPRF GABAergic actions on behavioral state involve interactions with pontine cholinergic systems (Maloney et al., 1999; Vazquez and Baghdoyan, 2004; Marks et al., 2008; Fort et al., 2009). Pre-incubation of the cat mPRF (NPO) with muscimol blocked the ability of microinjection of carbachol, a cholinergic agonist, to induce a REM-like state (Xi et al., 2004). Pre-incubation of the mPRF with atropine, which blocks muscarinic receptors, prevented promotion of REM sleep by GZ, although, scopolamine was unable to block bicuculline-induced REM sleep (Xi et al., 2004). Bicuculline infusion into the mPRF caused a concentration-dependent increase in ACh, an effect blocked by co-administration of muscimol (Vazquez and Baghdoyan, 2004). Complicating the understanding of the neurochemical control of behavioral state is the emergence of evidence indicating that while GABA mechanisms within the pontine tegmentum are importantly involved in state control in rodents and cats, cholinergic processes may not play as strong a role in gating behavioral state in rodents as they do in cat. Only moderate actions or no effects of cholinergic agents on behavioral state have been noted when injected into a region in rats equivalent to the feline REM

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induction zone, a site in which application of cholinergic agonists robustly induce a REM like state (Gnadt and Pegram, 1986; Shiromani and Fishbein, 1986; VelazquezMoctezuma et al., 1989; Bourgin et al., 1995; Deurveilher et al., 1997). While injections of GABAergic antagonists have been effective in inducing REM sleep when injected into the SLD, a site slightly rostral to the mPRF, injections of carbachol into this site increase wakefulness (Boissard et al., 2002). Nevertheless, some studies indicate that cholinergic processes within the rodent pontine tegmentum are in some way involved in state control. If carbachol injections in the SLD are performed during anesthesia, while GABAergic neurons may be inhibited, motor atonia similar to that occurring naturally in REM sleep is induced in rats (Taguchi et al., 1992; Fenik et al., 1999). Mice lacking the muscarinic cholinergic M3 receptor show a reduction in REM sleep (Goutagny et al., 2005) and local injection studies showed a clear role of mPRF M2 receptors in state control in mice (Coleman et al., 2004). Further, increases in cFOS expression in rat cholinergic LDT neurons were noted during REM sleep recovery following sleep deprivation, indicating activity of these cells during REM (Maloney et al., 1999), although the degree of cFOS staining was not as dense in the LDT as that seen by another group using the same behavioral paradigm (Verret et al., 2005). The ability of bicuculline to induce a REM-like state when injected into the mPRF of rats was blocked by pretreatment with atropine (Marks et al., 2008). Lastly, pontine administration of eszopiclone, a non benzodiazapine GABA receptor agonist, increased ACh release within the pons (Hambrecht-Wiedbusch et al., 2010). Taken together, these data suggest that cholinergic processes within the pontine tegmentum of both cats and rodents are involved in state control and could be influenced by GABAergic mechanisms; and, indeed that behavioral state is controlled, in part, by an interplay of these two transmitter system. Several circuitry sites where the interplay may be effectuated have been proposed that are not mutually exclusive. One possibility that has been suggested is the presence of presynaptic GABAergic inhibition of cholinergic terminals within the mPRF, which may regulate ACh release within the mPRF (Vazquez and Baghdoyan, 2004; Marks et al., 2008); although direct post synaptic GABA actions on NPO neurons was suggested as another likely possibility (Xi et al., 2004). What is the source of endogenous GABAergic input? If endogenous GABA activity is involved in natural behavioral state control via inhibitory actions on REM-on cholinergic LDT cells, the source of this GABA input is an important question. GABAergic neurons are densely present in brainstem regions controlling REM sleep in the rat (Maloney et al., 1999, 2000; Gervasoni et al., 2000; Boissard et al., 2002; de la Roza and Reinoso-Suarez, 2006; Lu et al., 2008). In mice expressing green fluorescent protein (GFP) under the control of the GAD67 promoter, the distribution of GFP-positive neurons paralleled the distribution of GAD-positive neurons reported in the rat using GAD immunohistochemistry (Brown et al., 2008; Sapin et al.,

2009). GABAergic neurons are intermingled with the cholinergic neurons in the LDT (Jones, 1990). If these local GABA cells silence their neighboring REM-on cholinergic LDT cells, their activity should be highest during wakefulness; however, a study using cFOS as a marker of activity indicated that GAD⫹ cells co-distributed with the cholinergic cells of the pons showed high levels of cFOS during recovery from REM sleep deprivation (Maloney et al., 1999). These data, taken together with microdialysis findings that GABA levels are highest in the LC and DR during REM, when spontaneous activity of these two monoaminergic cell groups is lowest, led to the suggestion that GAD⫹ cells that demonstrated high cFOS⫹ during rebound REM sleep serve to inhibit monoaminergic REM off cells, without ruling out a role of these cells in control of the cholinergic LDT cells (Nitz and Siegel, 1997a,b). Other populations of GABA neurons exhibiting behavioral state specific firing are located in the ventrolateral part of the PAG and the lateropontine tegmentum (LPT), a site dorsal to the LDT (Lu et al., 2006). These neurons stain positive for GAD⫹mRNA and exhibit REM-off firing patterns. Ibotenic acid-induced lesion of the vlPAG and LPT increase REM sleep with the LPT showing greater effects on behavioral state (Lu et al., 2006). GABAergic neurons have also been shown to be present within the SLD (Boissard et al., 2003). While the role of these pontine GABA cells in behavioral state control was hypothesized to be caused by inhibition of REM-on cells in the SLD (Boissard et al., 2003; Lu et al., 2008; Fort et al., 2009; Sapin et al., 2009), these data do not rule out a role for their inhibition of closely proximal cholinergic LDT cells. Finally, two cFOS studies have suggested that local GABAergic neurons of the mPRF may control behavioral state. Activity of a population of GABAergic neurons of the lateral mPRF was high during wakefulness (Xi et al., 1999b) and activity of GABAergic neurons decreased in the mPRF during recovery from REM sleep deprivation (Maloney et al., 1999). The location of the terminals of this population remains an open question, however, projections from these cells could terminate within the LDT. Functional significance Although currently the source of GABAergic input directed to cholinergic LDT neurons is unknown, it is highly likely that GABA is released onto synaptically-located receptors and/or via mechanisms of volume transmission, available to activate extrasynaptic receptors within this nucleus. This conclusion is supported by our findings that application of diazepam and GZ had actions on LDT neurons, suggesting presence of GABA tone in the slice. By demonstrating the diversity of GABA receptor subtypes on cholinergic LDT neurons, our data provide a functional basis on which to make predictions of the postsynaptic actions of this inhibitory input, when it is naturally present. Our speculations on the actions of GABA at specific receptor subtypes within the LDT must however be tempered by the limitation of studies using pharmacological agents. Actions of GABA receptor subtype agonists and antagonists utilized in this study may not be specific to one receptor subtype. With

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this caveat in mind, we predict that stimulation of rapidlydesensitizing GABAA receptors would cause a phasic inhibition of the majority of cholinergic cells. In some cholinergic LDT neurons, GABA would also produce a longerlived inhibition by increasing a K⫹ conductance following stimulation of postsynaptic GABAB receptors. Stimulation of the GABAB receptor in cholinergic neurons would also be expected to reduce the Ca2⫹ flux through activated L-type Ca2⫹ channels. The slowly deactivating GABAC receptors in cholinergic LDT neurons are suitable for eliciting a more longer-term inhibition. Tonic inhibitory currents such as those mediated by the extrasynaptic, THIP-sensitive GABAA receptor, are important regulators of cellular excitability as blocking extrasynaptic GABAA receptor-mediated tonic inhibition significantly in all cases enhanced neuronal excitability (Stell and Mody, 2002). Presence of this receptor on cholinergic LDT neurons suggests that tonic inhibition may inhibit release of ACh to rostral and caudal target regions of these cells (Hamann et al., 2002; Stell and Mody, 2002; Semyanov et al., 2003, 2004; Stell et al., 2003; Caraiscos et al., 2004; Mody, 2005). Relevant to our data are the reports that THIP, a non-traditional sleep aid, the clinical development of which was abandoned due to side effects, efficiently activates GABAA receptors, and is highly potent at ␦-containing receptors which are believed to be extrasynaptic in location (Adkins et al., 2001; Brown et al., 2002; Winsky-Sommerer et al., 2007). Although we report that the cholinergic LDT neurons exhibited responses to low concentrations of THIP, it is unlikely that the sleep-promoting actions of THIP result from actions on these cells as systemic THIP does not appear to have actions on REM sleep (Lancel et al., 1996; Lancel and Langebartels, 2000). Moreover, belying electrophysiological studies of THIP using mouse brain slices suggesting that THIP could promote sleep via actions on thalamic cells (Belelli et al., 2005; Jia et al., 2005), recent in vivo studies in mice revealed that systemic administration of THIP does not promote sleep in this species (Vyazovskiy et al., 2005; Winsky-Sommerer et al., 2007), even though it does promote sleep when administered in rats and human beings (Faulhaber et al., 1997; Lancel et al., 1997). Nevertheless, our data do not rule out a role of LDT extrasynaptic GABA receptors in mediation of the effects of anesthetics/hypnotics as ␦-containing GABAA receptors have been demonstrated to be exquisitely sensitive to several classes of sedative-hypnotic compounds.

CONCLUSION By acting at a diversity of receptor subtypes, which we show are functionally present on cholinergic LDT neurons, GABA would be expected to counteract the depolarizing action of excitatory processes directed to these neurons. GABAergic tone within the LDT cholinergic neurons is therefore expected to play a significant role in control of outflow of their signature neurotransmitter to target regions thereby influencing the physiological process in which principal neurons of this nucleus play a role, such as in the control of naturally-occurring behav-

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ioral state, as well as pharmacologically-induced altered states of consciousness. Acknowledgments—The authors would like to gratefully acknowledge Mirja Hansen Andersen for her technical assistance and Dr. Morten P. Kristensen for comments on previous versions of this manuscript. Research described in this article was supported by an Alfred Benzon Fellowship (KAK), Philip Morris USA Inc. (KAK) and The Carlsberg Foundation (UK).

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(Accepted 17 September 2010) (Available online 25 September 2010)