The Journal of Neuroscience, January 26, 2011 • 31(4):1213–1218 • 1213 Brief Communications Thalamic Ryanodine Receptors Are Involved in Controlling the Tonic Firing of Thalamocortical Neurons and Inflammatory Pain Signal Processing Eunji Cheong,1,2 Chanki Kim,1 B. Jiwon Choi,1 Minjeong Sun,1 and Hee-Sup Shin1,2 1Center for Neural Science, Korea Institute of Science and Technology, Seoul, 136-791, Republic of Korea, and 2Department of Neuroscience, University of Science and Technology, Daejon 305-333, Republic of Korea Ryanodine receptors (RyRs) are highly conductive intracellular Ca 2ϩ release channels which are widely expressed in the CNS. They rapidly increase the intracellular Ca 2ϩ concentrations in neuronal cells in response to Ca 2ϩ influx through voltage-gated Ca 2ϩ channels. A previous study reported that RyRs were expressed in thalamocortical (TC) neurons, but their physiological function has remained elusive. Here, we show that the activation of RyRs in TC neurons in mice decreases their tonic firing rate while blocking them induces the opposite response. Furthermore, activation of RyRs in ventroposteriomedial/ventroposteriolateral nuclei reduces the behavioral re- sponses to inflammatory pain and blocking them increases the responses. This study highlights the importance of the intracellular Ca 2ϩ release via RyRs in controlling the excitability of TC neurons and in inflammatory pain signal processing in the thalamus. Introduction which are the main relay nuclei for innocuous tactile information The two major Ca 2ϩ sources in neurons are the Ca 2ϩ influx from but also contain neurons which respond to noxious stimuli and the extracellular space and the Ca 2ϩ release from internal storage relay pain information to the cortex (Peschanski et al., 1983; (Berridge et al., 2003). The latter occurs through ryanodine re- Willis and Westlund, 1997). Therefore, we examined a possible role for RyRs in the control of TC firing and their potential func- ceptors (RyRs) or inositol-1,4,5-triphosphate receptors (IP3Rs). RyRs, triggered by the Ca 2ϩ influx through voltage gated Ca 2ϩ tion in the pain sensory relay. The results suggest that RyRs in the channels on the plasma membrane, rapidly release Ca 2ϩ into the VPM/VPL nuclei play a substantial role in controlling the excit- cytosolic matrix with a large conductance. Therefore, this Ca 2ϩ- ability of TC neurons and affecting pain signal processing. induced Ca 2ϩ release (CICR) by RyRs acts to boost the intracel- lular Ca 2ϩ increase in an immediate response to external stimuli. Materials and Methods Ryanodine receptors are widely expressed in neuronal cells in the Animals. All experiments used male F1 hybrid mice generated by the CNS (Furuichi et al., 1994). Recently, evidence is accumulating mating of two inbred strains, 129/Sv and C57BL/6J. Animal care and all that the thalamocortical (TC) neurons in rodents also express experiments were conducted under the ethical guidelines of the Institu- ϩ tional Animal Care and Use Committee of the Korea Institute of Science both types of intracellular Ca 2 channels, RyRs and IP Rs 3 and Technology. (Budde et al., 2000; Pape et al., 2004). However, the physiological Preparation of brain slices and acutely dissociated neuronal cells. The function of RyRs in TC neurons has not yet been determined. firing patterns of TC neurons were recorded in acutely isolated brain The thalamus plays a pivotal role in sensory signal processing, slices from ϳ4-week-old mice. The brain was sectioned in the coronal integrating sensory inputs from the periphery and inputs from plane in ice-cold slicing solution (in mM: 124 NaCl, 26 NaHCO3, 1.25 other brain regions, and then sending the integrated information NaH2PO4, 5 MgCl2, 1 CaCl2, 3 KCl, and 10 glucose) and incubated for 1 h ϩ to the cortex (Sherman and Guillery, 1996). The firing modes of at room temperature before use. Ca 2 imaging experiments were per- TC neurons have been implicated in reflecting the status of signal formed using acutely dissociated cells from 14- to 21-d-old mice. Brain transmission from the thalamus to the cortex (McCormick and slices were treated at 37°C in an oxygenated HEPES-buffered solution (in mM: 150 NaCl, 3 KCl, 2 CaCl , 10 HEPES, 2 MgCl , and 10 glucose, pH von Krosigk, 1992). In addition, it has been reported that RyRs 2 2 ϳ7.4 and 320–330 mOsm) containing 2 mg/ml protease XIV (Sigma) for are highly expressed in ventroposteriomedial/ventroposteriolat- 10 min, followed by an incubation at room temperature for 5 min. After eral (VPM/VPL) nuclei in the thalamus (Furuichi et al., 1994), the enzymatic treatment, slices containing the VPM/VPL nuclei were dissected by scalpel cuts, and cells were triturated with fire-polished Pas- teur pipettes. Received June 22, 2010; revised Nov. 12, 2010; accepted Nov. 21, 2010. Intracellular Ca2ϩ imaging. fura-2AM (Invitrogen) was used as the ThisworkwassupportedbytheNationalHonorScientistProgramofKorea,andtheCenterofExcellenceProgram fluorescent Ca 2ϩ indicator. Briefly, acutely dissociated cells were loaded of the Korea Institute of Science and Technology. ␮ ␮ Correspondence should be addressed to Dr. Eunji Cheong, Center for Neural Science, Korea Institute of Science with fura-2 AM (5 g) mixed with 5 l of pluronic acid in HEPES- and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Republic of Korea. E-mail: eunjicheong@ buffered solution and plated onto poly-D-lysine-coated coverslips in a kist.re.kr. chamber placed on an inverted microscope (TE3000, Nikon). Then cells DOI:10.1523/JNEUROSCI.3203-10.2011 were perfused continuously with the buffer at a rate of 2 ml/min. Cells Copyright © 2011 the authors 0270-6474/11/311213-06$15.00/0 were illuminated with excitation wavelengths (340/380 nm) provided by 1214 • J. Neurosci., January 26, 2011 • 31(4):1213–1218 Cheong et al. • Thalamic RyRs Control Pain Signal Processing the IonOptix chopper equipped with a xenon light source and a computer-controlled elec- tronic shutter (Photon Technology Interna- tional). The intracellular Ca 2ϩ concentration was measured by an intensified CCD camera (PTI IC-200, Photon Technology International) coupled to a microscope and a computer. NMDA, ryanodine, dantrolene and caffeine were purchased from Sigma. Reverse-transcriptase-PCR analysis. Total RNA was isolated from both the whole brain and the VPM/VPL nuclei using RNA isolation reagents (TRI Reagent, Molecular Research Center). To produce cDNA, one microgram of total RNA was reverse-transcribed with M-MLV reverse transcriptase (Invitrogen) us- ing oligo(dT) priming. The PCR primers and PCR conditions are shown in supplemental method (available at www.jneurosci.org as supplemental material). Whole-cell patch clamping. The firing pat- terns of TC neurons in brain slices were re- corded under the current-clamp mode in ACSF solution (in mM: 124 NaCl, 26 NaHCO3, 1.25 NaH2PO4, 1.3 MgSO4, 2.4 CaCl2, 3 KCl, and 10 glucose) bubbled with 95% O2/5% ⍀ CO2. Patch electrodes (4–6 M ) fabricated from borosilicate glass (GC150F-10, Warner Instruments) were filled with an intrapipette Figure 1. Caffeine evokes intracellular calcium transients via Ca 2ϩ release through RyRs in TC neurons. A, A representative solution (in mM: 140 K-gluconate, 10 KCl, 1 traceoffura-2ratiometricCa 2ϩ imagingonTCneuronsacutelydissociatedfromVPM/VPLnuclei.Ca 2ϩ transientswereevokedby 2ϩ 2ϩ 2ϩ MgCl2, 10 HEPES, 0.02 EGTA, 4 Mg-ATP, an application of 10 mM caffeine either in 2 mM Ca buffer or Ca -free buffer, which indicates that a Ca transient was via 2ϩ 2ϩ and 0.3 Na2-GTP, with pH 7.35). Signals Ca release from internal storage. B, Caffeine-induced Ca transients were blocked by pretreatment with 50 ␮M ryanodine. 2ϩ were amplified with a Multiclamp 700-A am- NMDA (60 ␮M) evoked Ca transients in the same sets of neurons. C, A representative trace from an individual TC neuron ϩ plifier (Molecular Devices) and analyzed us- obtainedbysubsequentapplicationofhighK solution(30mM KCl)undercontrolconditions,inthepresenceofcaffeine(0.5mM), ing pCLAMP 9.2 and MiniAnalysis software ␮ 2ϩ and during the coapplication of caffeine (0.5 mM) and ryanodine (50 M). D, Comparison of the depolarization-induced [Ca ]i (Synaptosoft). transients in the presence of caffeine (Caf, n ϭ 22, *p Ͻ 0.001 by paired t test) and the coapplication of caffeine and ryanodine Drug infusion via micro-osmotic pump im- ϭ Ͻ 2ϩ ϩ (Caf/Ry, n 22, *p 0.001 by paired t test). Values were normalized to the average [Ca ]i transient area evoked by high K plantation. For drug infusion into the brain, solution under the control conditions. E, The RT-PCR data show that the RyR2 and three subtypes are abundantly expressed in micro-osmotic pumps (Alzet) were placed in thalamic neurons in VPM/VPL nuclei, while the RyR1 subtype is barely expressed. M, DNA size marker; N, negative control; B, the mouse brain with the tips targeting the tha- whole-brain cDNA; Th, thalamic VPM/VPL nuclei cDNA. lamic VPM/VPL nuclei (anteroposterior, 1.7; lateral, Ϯ1.7; ventral, 3.5 mm) using a stereo- Pain test and data analysis. Pain tests and data analysis were performed taxic device (Kopf Instruments) as previously described (Kim et al., in a double-blinded manner. To prevent any bias, information about the 2003). The infusion implant placements were confirmed by postmortem infused drugs was not exposed to either the investigator who performed histology. the pain tests or to the evaluators. Pain responses were recorded with a Visceral pain tests and formalin-induced pain test. Visceral pain tests CCD camera, and the results were independently analyzed by at least two were performed as previously described (Kim et al., 2003). Mice were evaluators. A two-tailed t test was used for statistical analysis unless oth- placed in an acrylic cylinder positioned over a mirror angled at 45° to erwise specified.