Abstract It has long been suspected that sensory signal transmission is inhibited in the mammalian brain during sleep. We hypothesized that Ca v 3.1 T-type Ca2+ channel currents inhibit thalamic sensory transmission to promote sleep. We found that T-type Ca2+ channel activation caused prolonged inhibition (>9 s) of action-potential firing in thalamic projection neurons of WT but not Ca v 3.1 knockout mice. Inhibition occurred with synaptic transmission blocked and required an increase of intracellular Ca2+. Furthermore, focal deletion of the gene encoding Ca v 3.1 from the rostral–midline thalamus by using Cre/loxP recombination led to frequent and prolonged arousal, which fragmented and reduced sleep. Interestingly, sleep was not disturbed when Ca v 3.1 was deleted from cortical pyramidal neurons. These findings support the hypothesis that thalamic T-type Ca2+ channels are required to block transmission of arousal signals through the thalamus and to stabilize sleep. insomnia

α1G

thalamus

arousal

Cre recombinase

Each year, 60 million Americans experience frequent or extended periods of insomnia characterized by difficulty initiating or maintaining sleep. Understanding how sleep is produced should provide insight into insomnia and identify targets for its treatment.

Sensory perceptions and motor responses are blunted during sleep. Because sensory and motor signals are relayed to and from the cerebral cortex through the thalamus, inhibition of thalamocortical signal transmission could play an important role in achieving stable sleep. Consistent with this notion, the firing response of thalamic projection neurons to visual stimulation is suppressed during sleep in primates (1). Although not yet shown, visceral sensory signals (e.g., respiration), which relay through midline thalamus, represent a potent arousal signal and, therefore, may also be suppressed to facilitate sleep (2). The molecular and cellular basis for sensory suppression during sleep and its potential role in preventing arousal have not been well studied.

One potential mechanism for sensory inhibition is the marked increase of T-type Ca2+ channel-mediated Ca2+ transients observed in thalamus during sleep (3, 4). Thalamic neurons hyperpolarize during sleep, causing T-type Ca2+ channels to deinactivate and enabling their activation with excitatory input (4–6). Ca v 3.1 is the major T-type Ca2+ channel transcript in thalamic projection neurons, where it is required for low-threshold Ca2+ spikes and action-potential bursts (7–9). Interestingly, in animals with Ca v 3.1 T-type Ca2+ channel globally deleted pain-evoked action-potential firing in thalamic neurons of the ventrobasal nucleus is increased (9). The cellular mechanism of this Ca v 3.1 control of action-potential firing is difficult to deduce based on data from a global knockout (KO) because Ca v 3.1 is very broadly expressed throughout the brain. Nevertheless, the finding led us to hypothesize that thalamic Ca v 3.1 may inhibit action-potential firing directly and that this inhibition may serve to stabilize sleep.

To assess the role of Ca v 3.1 in thalamic projection neurons, we examined its effect on action-potential firing in vitro. To determine whether this thalamic function of Ca v 3.1 plays a role in sleep, we selectively deleted the gene from thalamic projection neurons and examined vigilance states by recording the electroencephalogram (EEG) and electromyogram (EMG). We found that Ca v 3.1 activation initiates a prolonged inhibition of action-potential firing in thalamic projection neurons in vitro. We also found that targeted deletion of Ca v 3.1 gene in thalamocortical neurons destabilizes sleep.

Materials and Methods General Animal Handling. Mice were housed in a pathogen-free environment regulated at 21–23°C under a 12:12-h light/dark schedule (lights on from 7 a.m. to 7 p.m.). All procedures relating to animal care and treatment were approved by the Massachusetts Institute of Technology and Harvard Medical School Institutional Animal Care and Use Committees, and they conformed to institutional and National Institutes of Health guidelines. Generation of Thalamic-Ca v 3.1 KO Mice. A 120-kb-long bacterial artificial chromosome (BAC) clone, containing the complete genomic sequence of mouse K v 3.2, was isolated by screening a high-density colony array of mouse C57BL/6 genomic BAC clones (Genome Systems, St. Louis) with a 339-bp cDNA fragment, which overlaps the 5′ translational start site of K v 3.2. Sequence from a 3.5-kb subcloned fragment of K v 3.2 BAC-matched mouse K v 3.2 cDNA. Using BAC recombination methods developed by Yang et al. (10), a 2.6-kb NotI fragment of pZQCRE (11) was inserted into the translational start site of K v 3.2, replacing the expression of K v 3.2 with Cre recombinase. This fragment included Cre recombinase (plasmid pBS317; a gift from Brian Sauer, Stowers Institute for Medical Research, Kansas City, MO), a nuclear-localization signal (12), and exon–intron splicing and a polyadenylation signals. Cesium-gradient-purified, supercoiled BAC DNA containing the K v 3.2 promoter and the Cre gene was injected into the pronucleus of C57BL/6 zygotes as described (13). Candidate founders were tested by PCR and Southern blot analysis. Positive lines were crossed to lacZ reporter line Rosa26 (14) or alkaline phosphatase reporter line Z/AP (15). Transgenic line, K128-Cre, recombined lacZ reporter to produce strong β-galactosidase staining in rostral and midline thalamic nuclei (xiphoid, reunions, rhomboid, centromedial, intermediodorsal, paraventricular, paracentral, centrolateral, submedius, mediodorsal, lateral dorsal, anteromedial, suprageniculate, and paratenial). Moderate staining was observed in other thalamic nuclei: anteroventral, anterodorsal, ventral lateral, ventral anterior, parafascicular, and posterior thalamic group. Weak staining was observed in lateral posterior, dorsal lateral geniculate, medial geniculate, and ventral posterior thalamic nuclei. Strong recombination was observed also in ventral medial and dorsal medial hypothalamic nuclei, pontine reticular nucleus, mesencephalic fifth, and 12th cranial nerve nuclei. Moderate recombination was also present in the superficial piriform cortex, indusium griseum, dorsal endopiriform nucleus, medial and median preoptic hypothalamic nuclei, medial vestibular nucleus, ventral cochlear nucleus, nucleus of trapezoid body, and abducens nucleus. Very small numbers of neurons expressed in the lateral septum, locus coeruleus, substantia nigra pars reticulata, and parabrachial nucleus. Construction of Floxed T-Type Calcium Channel Ca v 3.1. A BAC clone containing the Ca v 3.1 gene was isolated from a mouse C57BL/6 genomic BAC clone library (Genome Systems) and confirmed by PCR, T/A cloning, and sequencing. Restriction mapping, Southern blot hybridization, and subcloning and ligation were used to create a replacement vector for homologous recombination in ES cells. Briefly, a 7-kb EcoRV–PstI fragment containing exons 9–13 was used. A loxP sequence was placed into a SpeI site 5′ of exon 9. An LFNT cassette (containing a loxP sequence) was placed into a SpeI site 3′ of exon 12 to produce an ≈2.5 kb of loxP-flanked genomic DNA and a 1.5-kb 5′ and 3-kb 3′ flanking sequence. A diptheria toxin A gene (DT-A) was placed at a site flanking the targeting construct for negative selection (16). An LFNT cassette (K. Nakazawa and S.T., unpublished data) was modified to delete a TK cassette (LFNT-TK). A pair of FRT sites (recognition sequences for a yeast-derived, site-specific Flp recombinase) that flank PGK-Neo (for positive selection) and a 3′ loxP sequence remained after deletion of TK. The PGK-Neo cassette was deleted after germline transmission by crossing to a Flp deleter transgenic mouse (17). The “floxed” Ca v 3.1 mice (fCa v 3.1) were crossed to a global Cre deleter (K9-Cre; M.P.A. and S.T., unpublished data), a thalamic Cre deleter (K128-Cre), or a cortical Cre deleter (CW2-Cre, CAMKIIα promoter driving cortical and hippocampal CA1 pyramidal neuron expression) (18). Histocytochemistry. Mice were perfused with ice-cold 4% paraformaldehyde in PBS buffer. For X-gal staining, brains were removed and postfixed at 4°C for 30 min. Coronal sections (50 μm thick) were cut on a Vibratome in PBS. Sections were incubated in 0.1 M PBS containing 0.01% SDS, 0.02% Nonidet P-40, and 2 mM MgCl 2 at 4°C for 15 min, followed by β-galactosidase reaction in 1 PBS (pH 8.0) containing 0.5 mg/ml X-gal, 5 mM K 4 Fe(CN) 6 /3H 2 O, 5 mM K 3 Fe(CN) 6 , and 2 mM MgCl 2 at 37°C for 24 h. For alkaline phosphatase staining, sections were heat-inactivated in PBS at 70°C for 1 h, equilibrated with a reaction buffer (100 mM Tris·HCl, pH 9.5/100 mM NaCl/50 mM MgCl 2 /0.1% Tween 20/2 mM levamisole; Vector Laboratories) for 15 min at room temperature and incubated with 0.4 mg/ml nitroblue tetrazolium chloride/0.19 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate/100 mM Tris, pH 9.5/50 mM MgSO 4 (NBT/BCIP tablets; Roche Molecular Biochemicals) at room temperature for 10–20 min. Section were postfixed in 10% formalin for 2 h and stained with nuclear fast red (Polysciences). In Situ Hybridization. Terminal deoxyribonucleotidyl transferase (Invitrogen) and ATP [33P] (New England Nuclear) labeled the following set of 33-bp antisense oligonucleotides: 5′-AGAGCAGCCCAAGATGACGTGGAGGCCATGCCG-3′, 5′-TCTGAAAGACAGTGACAATGGCCCAGAGCAGGG-3′, 5′-GCCGA AGGGACCGTAGACGAGCAGCT TCAGCAG-3′, and 5′-ATTCTTCCGGTCTGGCAACGTGTCCCCATCCCG-3′. Probes were designed to hybridize sequences flanked by loxP sequences and to be minimally homologous to other channels. Unincorporated nucleotides were removed on Sephadex G-50 spin columns (Pharmacia). Hybridization buffers, temperatures, and wash stringency were performed as described by Talley et al. (19). Slides were exposed to film (Hyperfilm β-MAX, Amersham Biosciences) for 1 week and analyzed for relative intensity by using image-analysis software (mcid, Imaging Research, St. Catherine's, ON, Canada). For resolution of cellular labeling, slides were dipped in liquid autoradiography emulsion (NTB2, Kodak, Sigma–Aldrich), exposed for 5–9 weeks, and examined by dark-field and bright-field microscopy. Nissl counterstain was used. Implantation of EEG/EMG Electrodes. Under anesthesia with ketamine/xylazine (100/10 mg/kg, injected i.p.), screw electrodes were implanted into the skull (1-mm anterior to bregma and 1-mm anterior to lambda, 1.5-mm lateral to midline). EMG electrodes (AS633; Cooner Wire, Chatsworth, CA) were inserted into neck extensor muscles. Leads were attached to a 2 × 2-pin header secured to the skull by using dental acrylic. An activity transmitter (TA-F20; Data Sciences International, St. Paul, MN) was placed into the peritoneal cavity. Sleep Recordings. Recordings were performed under a 12:12-h light/dark schedule (lights on from 7 a.m. to 7 p.m.). Mice were habituated for 10 days to a counterbalanced, light-weight cable connected to a low-torque commutator (4-TBC-9-S; Crist Instrument, Hagerstown, MD), affixed to the center of the top of the cage. Signals from EEG/EMG electrodes were amplified and bandpass-filtered by using model 12 amplifiers (Grass Instruments, Quincy, MA). EEG signals were amplified ×5,000 and bandpass-filtered at 0.3–30 Hz. EMG signals were amplified ×5,000 and bandpass filtered at 2–100 Hz. Both signals were acquired digitally at 128 Hz by using Sleep Sign (Kissei Comtec, Matsumoto, Japan). Behavioral state was scored in 10-s epochs as wake, rapid-eye-movement (REM), or non-REM (NR) sleep by using automated methods, followed by visual screening by a blinded single examiner (20). Submerged Slice Patch Clamp Recording. Mice were anesthetized by using isoflurane, and the brains were submerged into 4°C sucrose solution (250 mM sucrose/5 mM KCl/10 mM glucose/25 mM sodium bicarbonate/5 mM KCl/1.25 mM NaH 2 PO 4 /1 mM CaCl 2 /5 mM MgSO 4 /95% O 2 /5% CO 2 (carbogen gas). Coronal sections (250 μm) were prepared on a Vibratome Plus 3000 (Vibratome, St. Louis, MO) in 4°C sucrose. Slices were incubated at 35°C for 30 min and then at room temperature for <8 h in an interface chamber containing ACSF buffer (125 mM NaCl/25 mM glucose/25 mM sodium bicarbonate/2.5 mM KCl/1.25 mM NaH 2 PO 4 /2 mM CaCl 2 /1 mM MgCl 2 /95% O 2 /5% CO 2. Recordings were performed at room temperature by using infrared-guided whole-cell patch-clamp technique. Data were acquired with an EPC10 triple patch clamp amplifier and acquisition system (HEKA, Lambrecht, Germany). Slices were recorded in rapidly flowing (1 ml/min), carbogen-perfused ACSF on the stage of an upright microscope fitted with differential interference contrast (DIC) optics and camera (Optical Analysis, Nashua, NH). T-Type Calcium Channel Current Measurements. Whole-cell, voltage clamp was performed with 1 μM tetrodotoxin/30 μM bicuculline/1 mM 4-aminopyridine/10 mM tetraethylammonium (TEA) chloride/2 mM cesium chloride in the ACSF. The pipette contained 90 mM cesium methanesulfonate, 11 mM cesium chloride, 0.5 mM magnesium dichloride, 0.5 mM CaCl 2 , 10 mM Hepes, 11 mM EGTA, 4 mM magnesium·ATP, 0.3 mM Tris·GTP, and 7 mM Tris·phosphocreatine (pH 7.3; Tris base). Ca2+ Spike and Action-Potential Measurements. Current clamp studies were performed with 10 μM CNQX and 30 μM bicuculline in ACSF. The pipette contained 135 mM potassium gluconate, 4 mM KCl, 2 mM NaCl, 10 mM Hepes, 4 mM magnesium·ATP, 0.3 mM Tris·GTP, and 7 mM Tris·phosphocreatine (pH 7.25; Tris base). In some experiments, the following drugs were added to the bath: cAMP agonist forskolin (10 μM) or TEA chloride (4 mM). BAPTA (15 mM) was substituted for equimolar potassium gluconate in the pipette. Statistical Analysis. Statistical analysis was conducted by using statview (SAS Institute, Cary, NC). Data were analyzed by two-tailed t test, ANOVA, or repeated-measures ANOVA. Values in graphs were expressed as mean ± SEM.

Discussion In conclusion, the results suggest Ca v 3.1 is required in the thalamus, but not the cortex, to stabilize sleep. Despite their reduced activity and possible increased homeostatic sleep drive, Ca v 3.1 KO mice have difficulty initiating and maintaining sleep. Sensory gating may help ensure that sleep is not disrupted. We have demonstrated that Ca v 3.1 T-type Ca2+ channel activation inhibits action potentials through an intracellular pathway involving increases of intracellular Ca2+. This gating of thalamic action potentials is defective in global Ca v 3.1 KO mice. We have also shown that Ca v 3.1 T-type Ca2+ channel function is required in the thalamus, but not in the cerebral cortex to achieve normal patterns of sleep. Mice with thalamic Ca v 3.1 deletion experience frequent arousals from sleep. During the dark period, these mice also experience sleep loss because of a delayed sleep onset. Based on these findings, we suggest Ca v 3.1 T-type Ca2+ channels inhibit the transmission of thalamic action potentials to prevent feedforward excitation, which could destabilize activity within the neural circuitry maintaining the sleep state (28). Inhibition of thalamic transmission may occur during various conditions that activate Ca v 3.1. A feedback-inhibitory pathway has been identified (23, 24) that interconnects neurons of the thalamic relay and reticular nuclei. Based on the >9 s inhibition that we observed, GABAergic synaptic inputs from the reticular nuclei could be transformed into a prolonged inhibition by Ca v 3.1. Thalamic projection neurons also display various spontaneous rhythmic activities that are thought to involve T-type Ca2+ channels (29, 30). One purpose of these rhythmic activities may be to activate Ca v 3.1 and, thereby, provide a continuous inhibition of transmission independent of GABAergic signaling. Ca v 3.1 inhibits action potentials through a TEA-sensitive, Ca2+-dependent and cAMP-inhibited intracellular pathway. The sensitivity to a potassium channel blocker, TEA, the slow kinetics of inhibition, and the dependence on Ca2+ suggest the slow after hyperpolarization current (sAHP) may be responsible. This potassium current is activated slowly by Ca2+ and inhibited by the cAMP-dependent protein kinase, consistent with our observations (31). Although some recombination occurred outside the thalamus, the intense pattern of Ca v 3.1 deletion observed in rostral–midline thalamus in K128-Cre suggests that this region may normally carry arousal signals that disrupt sleep (2, 32). Alternatively, the midline (intralaminar) thalamic nuclei may produce Ca v 3.1-mediated action-potential bursts during sleep that excite reticular thalamic neurons to provide feedforward inhibition to other thalamic nuclei (33). Some researchers have speculated that thalamic bursts produced by the T-type Ca2+ channel may underlie the cortical EEG waves observed during sleep (4, 5). We were surprised to find that cortical EEG power within the delta frequency range (1–4 Hz) was not significantly reduced but, rather, was moderately increased (data not shown). The observation suggests that other systems (e.g., intracortical circuits or basal forebrain GABAergic projections; ref. 34) drive the delta waves of the cortical EEG during sleep. We speculate that this delta-wave-generating system may be critical to the maintenance of sleep when thalamic sensory filtering by Ca v 3.1 is defective. The pattern of brief arousals in Ca v 3.1 KO mice resembles the sleep-microstructure alterations recently recognized in patients with insomnia and other sleep disorders of arousal (35). It remains to be established whether these mice rouse from sleep because of inappropriate transmission of normal sensations or because of pathologic sensations like those in sleep apnea or periodic limb movement disorder. If these brief arousals from sleep in Ca v 3.1 KO mice were caused by inadequate gating of sensory stimuli, it would possibly provide a perspective on human insomnia in which relatively mild sensations can disrupt sleep.

Acknowledgments We thank Lorene Leiter, Xiaoning Zhou, and Dennis King for technical assistance. M.P.A. is supported by a Howard Hughes Fellowship for Physicians, a Mentored Clinical Scientist Development Award from the National Institute of Mental Health, and a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund. This research was also supported by National Institutes of Health Grants R01-NS32925 and P50-MH58880 (to S.T.) and R01-MH62589 and P01-HL60292 (to T.E.S.), as well as a grant from the RIKEN Brain Research Institute (to S.T.).