Subjects were seated with their hand and forearm firmly strapped in a splint using padded Velcro bands. The splint was attached to a light-weight frame over a horizontal glass surface. A system of air jets lifted the frame supporting the arm 1 mm above the glass surface, eliminating friction during hand movements. Subjects rested their forehead above the work surface, with their hand and arm hidden from view by a mirror. Targets (green selleck inhibitor circles) and hand position (indicated, when specified by the task, by a small round cursor) were projected onto the plane of the hand and forearm using a mirror. The arrangement

of the mirror, halfway between the hand’s workspace and the image formed by the projector, made the virtual images of cursor and targets appear in the same plane as the hand. The workspace was calibrated so that the image of the cursor indicating hand position fell exactly on the unseen tip of the middle finger’s location (i.e., veridical display) (Mazzoni et al., 2007). Hand position was recorded using a pair of

6 degree of freedom magnetic sensors (Flock of Birds, Ascension Technologies, Burlington, VT) placed on the arm and forearm, which transmitted hand position and arm configuration data to the computer at 120 Hz. Custom software recorded hand and arm position in real time INCB024360 datasheet and displayed hand position as a cursor on the computer screen. The same software also controlled the display of visual targets. A total of 60 healthy, right-handed subjects participated in the study (mean age = 24.7 ± 4.9, 25 males). All subjects were naive to the purpose of the study and gave informed consent in compliance to guidelines set forth by the Columbia University Medical Center Institutional Review Board. They were randomly assigned to groups in each experiment. Subjects were asked to make fast, straight, and planar movements through a small circular target displayed veridically using a mirror

and monitor (Huang and Shadmehr, 2009 and Huang until et al., 2008). At the start of a trial, subjects were asked to move the cursor to a starting circle (2.5 mm radius) situated directly in front of them. Once the cursor was in the starting circle, a green, circular target (2.5 mm radius) appeared 6 cm away from the starting circle and the computer played a short, random-pitch tone, prompting subjects to move. If applicable for the trial, a rotation centered at the starting circle was imposed on the cursor feedback. As soon as the cursor was 6 cm away from the starting circle, a small white dot appeared at the cursor position at that time and remained there for the rest of the trial. Thus, the position of the white dot indicated the angular error the subject made in that trial. Subjects were then asked to return the overshot cursor to the target. The cursor disappeared briefly at this point.

, 2012). The inhibition of glutamate release EX 527 cost at Schaffer collaterals-CA1 synapses does not involve protein kinases but, rather, a membrane-delimited action that probably involves the direct inhibition of presynaptic Ca2+ channels by G protein γβ subunits (De Waard et al., 1997). The inhibitory

effects of KARs upon tonic activation by endogenous glutamate at these two hippocampal synaptic populations have been observed during development and involve KAR metabotropic signaling (Lauri et al., 2005, Lauri et al., 2006 and Sallert et al., 2007). Tonic activation of presynaptic KARs in the adult brain also inhibits glutamate release in the rat globus pallidus, once again mediated by a presynaptic Gi/o-protein-coupled and PKC-dependent mechanism (Jin and Smith, 2007). What now seems clear is that presynaptic KARs modulate transmitter release in a bidirectional manner: facilitation probably occurs through their ionotropic activity, while inhibition seems to involve noncanonical metabotropic signaling. It is possible that the threshold to activate one or other KAR signaling pathways would determine physiological responses. In summary, the presynaptic

modulation of both glutamate and GABA release together with the postsynaptic regulation of neuronal excitability clearly demonstrates that KARs are endowed with diverse capacities. These activities enable them to fine-tune synaptic function and regulate neuronal network activity in the adult brain, bestowing them with 3-deazaneplanocin A manufacturer a much broader role at synapses than the simple transfer of information. KARs are expressed strongly in the brain during development in a complex cell-type-specific manner. The properties of synapses during development

differ significantly from those at mature stages and it is now known that presynaptic KARs contribute to these changes. The expression of KARs, and particularly of GluK1 subunits, increases markedly and peaks during the first week of life in rodents (Bahn et al., 1994). In immature hippocampal CA1 synapses, the tonic activation of KARs by ambient glutamate keeps the probability of release low (Lauri et al., 2006). However, a burst of synaptic activity produces strongly facilitating postsynaptic responses, a facilitation that is not only abolished in the presence of a KAR antagonist nearly but also later in development. Interestingly, this change is recapitulated by inducing LTP and it seems that the tonic stimulation of KARs by ambient glutamate is no longer possible under each of these circumstances, changing the short-term synaptic dynamics. It is possible that the lower ambient glutamate contributes to the lack of tonic KAR activation after the first postnatal week. However, it does seem that the affinity of the receptor actually changes, making it less sensitive to the agonist. Indeed, it has been claimed that a change in an isoform of the GluK1 subunit in KARs is responsible for this lower affinity (Vesikansa et al., 2012).

We next asked whether CaCC also affects spike duration in the axon terminals. If so, applying CaCC blocker should increase transmitter release from CA3 axon terminals that form synapses with CA1 neurons. First, we performed field recording of the pharmacologically isolated AMPA-fEPSP in the CA1 dendritic field, while stimulating Schaffer collaterals ten times at 10 Hz (Figure 5C). NFA did not alter the AMPA-fEPSPs (106% ± 8.4%, n = 7,

p = 0.8). 100 μM NFA also did not alter the pharmacologically isolated NMDA-EPSCs IOX1 supplier recorded from individual CA1 pyramidal neurons (101% ± 3.1%, n = 5, p = 0.2; Figure 5D) (external Mg2+ was removed to facilitate NMDA receptor activation, and 10 mM internal Cl− was used to minimize the driving force for Cl− ions when ECl is −64.4 mV and holding potential is −65 mV). Thus, blocking CaCC alters the action potential

waveform in the soma without altering transmitter release, indicating that functional CaCCs reside in somatodendritic regions find more but not the nerve terminals of CA3 pyramidal neurons. Next, we asked whether CaCCs are near NMDA-Rs to be activated during synaptic responses. To maximize the chance of detecting CaCC activation during voltage-clamp recording of isolated NMDA-EPSC in the presence of 20 μM CNQX, we replaced external Mg2+ with Ca2+ and increased the Cl− driving force by including 130 mM Cl− in the whole-cell patch pipette solution (ECl ∼0 mV) and holding the cell at −65 mV (65 mV driving force), so that CaCC activation would result in Cl− efflux thus enhancing the NMDA-EPSCs elicited from CA1 pyramidal neurons in acute almost slices (P14–21) by stimulating Schaffer collaterals every 20 s. As shown in Figure 5E, blocking CaCC with NFA reduced the NMDA-EPSC by 28% ± 4.3% (n = 10, p < 0.05), indicating that CaCC is in the vicinity of NMDA-Rs to be activated

by the Ca2+ influx through NMDA-Rs. Importantly, when 10 mM of BAPTA was included in the 130 mM Cl− internal solution, NFA no longer had effect on NMDA-EPSC (100% ± 1.4%, n = 10, p = 0.13; Figure 5F), providing further evidence that NFA has no presynaptic effect on transmitter release. In contrast, when we included 10 mM EGTA in the patch pipette solution with 130 mM Cl−, 100 μM NFA still reduced NMDA-EPSC by 32 ± 9% (n = 5, p < 0.01; Figure 5G). As summarized in the histogram (Figure 5H), Ca2+ influx through NMDA-Rs is capable of activating CaCCs that are in close proximity so that the slower Ca2+ chelator EGTA, but not the fast Ca2+ chelator BAPTA, allows CaCC activation for feedback modulation of NMDA-EPSCs. To explore the physiological contribution of CaCCs, first we performed field recording of the pharmacologically isolated NMDA-fEPSP in the CA1 dendritic field in the presence of 2.5 mM Ca2+ and 1.3 mM Mg2. During a 10 Hz stimulation of Schaffer collaterals, the NMDA-fEPSPs gradually increased (Figure S5, left). Bath application of NFA enhanced the third, sixth, and tenth (n = 8; third, 138.

Here we demonstrate that the N-type voltage-gated calcium channel, a major presynaptic calcium channel, is a Cdk5 substrate. Phosphorylation of the CaV2.2 pore-forming

α1 subunit by Cdk5 increases calcium influx by enhancing channel open probability and also facilitates neurotransmitter release. These events are mediated by an interaction between CaV2.2 and RIM1, which impacts vesicle docking at the active zone. Our results outline a mechanism by which Cdk5 regulates N-type calcium channels and affects presynaptic function. To investigate whether the N-type calcium channel is a Cdk5 substrate, click here we cloned the intracellular domains of the CaV2.2 α1 subunit into glutathione S-transferase (GST) fusion protein constructs for in vitro kinase assays (Figure 1A). Each UMI-77 cell line purified GST-CaV2.2 protein fragment was incubated with an activated Cdk5/p25 protein complex along

with radioactive [γ32-P]ATP to assay the level of Cdk5 kinase activity (Figure 1B). Two GST-CaV2.2 fusion protein fragments, the C-terminal 3 (CT 3, amino acids 1981–2120) and C-terminal 4 fragments (CT 4, amino acids 2121–2240) were consistently phosphorylated by Cdk5 (Figure 1C). Mutagenesis of serine 2013 (S2013), a consensus Cdk5 site on the CT 3 fragment, to alanine abolished Cdk5 phosphorylation. However, several combinations of point mutations on the CT 4 fragment were insufficient to reduce Cdk5/p25 phosphorylation (Figure S1 available online). Only mutagenesis of all seven putative Cdk5 phosphorylation sites on the CT 4 fragment resulted in undetectable phosphorylation levels (Figure 1D). These kinase assays identify the N-type calcium channel as a Cdk5 substrate. To confirm phosphorylation of the Sitaxentan N-type calcium channel, we generated and purified a phosphorylation-state-specific

antibody to S2013, a well-conserved residue (Figure 2A). The phospho-CaV2.2 antibody (pCaV2.2) signal was robust when the CT 3 fragment, but not the CT 3 (S2013A) fragment, was coincubated with Cdk5/p25, indicating that the antibody was specific to S2013-phosphorylated CaV2.2 in vitro (Figure S2). Furthermore, the pCaV2.2 antibody signal was observed only in the presence of Cdk5/p35 in a cell line stably expressing the rat isoform of CaV2.2 (Lin et al., 2004), and alkaline phosphatase (CIP) treatment abolished the signal (Figure 2B). Since S2013 is also conserved in P/Q-type calcium channels, we tested the specificity of the Cdk5-dependent S2013 phosphorylation by immunoprecipitation of brain lysates with an anti-CaV2.2 antibody, followed by immunoblotting for pCaV2.2 in lysates of control and Cdk5 conditional knockout (cKO) mice (Guan et al., 2011). We noted that pCaV2.

In contrast, in individuals with ASD, one MET risk allele was sufficient to give rise to the atypical pattern of functional activity, showing less deactivation than the nonrisk group. In fact, when comparing those with one risk allele, individuals with ASD exhibited

significantly less deactivation in these regions compared to TD subjects, indicative of an even more atypical phenotype in the clinical population with the GABA activation same MET risk genotype. Consistent with the ROI analysis, a whole-brain comparison of TD versus ASD subgroups within the heterozygous risk group found stronger and more widespread differences than those observed when comparing the TD and ASD groups across genotype ( Figure S1B; Table S3). Based on prior reports of altered DMN function TGF-beta inhibitor in ASD (Kennedy et al., 2006; Kennedy and Courchesne, 2008) and MET’s high expression in the PCC (Judson et al., 2011a), as well as our finding of atypical DMN deactivation in MET risk carriers, we next examined the extent to which the MET functional risk variant modulates intrinsic DMN functional connectivity. We used a seed centered in the PCC ( Fox et al., 2005) for whole-brain functional connectivity analyses in rs-fcMRI

data in a matched sample of 33 TD and 38 children and adolescents diagnosed with ASD. The results were remarkably consistent with the functional activation findings: the MET risk genotype significantly modulated functional connectivity, such that those in the highest risk group (CC; n = 16) had reduced intrinsic connectivity between the PCC and MPFC as well as other nearby regions in the PCC compared to the nonrisk group (n = 16; Figure 2A; Table S4). In agreement with the functional activation analyses, the heterozygous risk group diagnosed with ASD (n = 24) showed a pattern of functional connectivity similar

to that observed in the homozygous risk group, whereas functional connectivity before in the TD heterozygous risk group (n = 15) was no different than the homozygous nonrisk group. Collapsed across genotype, the ASD group exhibited reduced PCC-MPFC connectivity relative to the TD group ( Figure 2B). A whole-brain analysis comparing TD and ASD groups independent of genotype revealed similar, and even more extensive, reductions in DMN connectivity as a function of ASD diagnosis ( Figure S2B). This diagnostic effect appeared to be partially driven by a stronger penetrance of the MET risk allele in the ASD group, as significant differences between TD and ASD subgroups were only observed in risk carriers ( Figure 2B); indeed, MET genotype explained 1.7 times as much variance in DMN connectivity in autistic relative to neurotypical individuals. Using an additional seed within the MPFC, we confirmed that both short- and long-range intrinsic DMN functional connectivity was reduced as a function of both MET risk genotype and ASD diagnosis ( Figure S2D; Table S5).

, 2011). In conclusion, we show that the interplay

between different senses can occur by means of interareal synaptic inhibition. The elucidation of the synaptic basis of multimodal interactions in primary sensory areas could pave the way for further exploration of how a complete sensory deprivation of one modality during development affects interareal connectivity and the local microcircuitry (Bavelier and Neville, 2002). Intriguingly, such sensory deprivations cause anatomically detectable changes of the GABAergic system in the affected primary cortices (Sanchez-Vives et al., 2006). Four to six weeks C57BL/6J mice were used throughout all experiments adhering to the Italian Health Ministry Guidelines and Permissions. Mice were lightly anaesthetized under urethane (ca 0.9 g/kg i.p.) and anesthesia depth was monitored using ALK inhibitor FPs and membrane potential spectra, together with physiological signs. Recordings in awake, head-fixed mice were done after implantation of a recording chamber and habituation to the setup. Craniotomies in V1, A1, and S1 were see more guided by ISI through the thinned skull. Injections of muscimol (both normal and fluorescent) in A1 and in V1 were done

with a pressure-injection device (Picospritzer, General Valve, UK). Transections were done rostrocaudally based on ISI of V1 and A1 and were done with a 30 gauge blade: the depth and coronal height of the transection were verified postmortem in Nissl-counterstained sections. Cannulae for acute pharmacology were implanted in the center of V1 5–6 days before

experiments (done within 10–15 min from infusion of 0.7 μl of drugs). Single-, multiunit, and FP recordings were done using 1–3 MΩ borosilicate or tungsten electrodes for acute or chronic recordings in freely moving animals, respectively. In vivo whole-cell recordings were done in current clamp using an EPC 10 double-plus amplifier (HEKA, Germany) using 5–9 MΩ borosilicate pipettes. Series resistance, spike height and resting Vm were stable throughout recordings too (duration: 15–120 min). No holding currents were used unless for excitatory and inhibitory conductances estimates. At the end of the experiments, animals were perfused with fixative and biocytin-filled cells were revealed together with layering for cell recovery. For PSP measurements, sweeps have been averaged after spike removal, whereas for AP counts, 50 ms binning was applied. Unless otherwise stated, PSP amplitudes have been measured in the 0–300 ms poststimulus time window, whereas onset latencies were taken when the Vm was larger than 2 standard deviations above baseline. For conductance measurements and extracellular data analysis, see Supplemental Experimental Procedures.

These data suggest

that retinotopically organized projections from V1 to RL are a determinant of visual responsiveness of RL and hence also of its multimodal character. We made four main findings concerning MI in the mouse visuotactile area RL. (1) ME is more pronounced at the level of spike outputs compared to synaptic inputs; (2) ME is pronounced in supragranular pyramids but scarce among the deep infragranular pyramids and in the main interneuron population—Pv-INs; (3) the scarce ME of Pv-INs permits ME in neighboring pyramids; (4) there is a precise spatial distribution of uni- and bimodal cells at the microscale level. Whole-cell recordings combined with anatomical tracings suggest that RL neurons receive tactile and visual synaptic inputs from S1 and V1, respectively. However, fewer neurons in RL were bimodal al the level of APs than PSPs, and ME was stronger Angiogenesis inhibitor for APs compared to PSPs. This difference is presumably due to the nonlinear threshold mechanism underlying AP generation (see also Allman and Meredith, 2007 and Schroeder and Foxe, 2002). The same threshold mechanism may account for the sublinear summation of PSPs on one hand, and for the (supra)linear summation of APs on the other hand. The multisensory synaptic integration we observed in RL differs from the integration of two different unisensory stimuli in primary cortices. In

primary cortices concurrent presentation of two unisensory stimuli typically suppresses responses, both in S1 ( Higley and Contreras, 2005) selleck kinase inhibitor and V1 ( Priebe and Ferster, 2006),

whereas in RL the interaction was largely additive. Interestingly, a similar difference between unimodal integration (suppression) and bimodal integration (enhancement) has been described in the cat colliculus ( Alvarado et al., 2007). It would be interesting to investigate whether different cellular circuitries are responsible for these distinct computations. While bimodal cells were more abundant in layer 5 compared to layer 2/3, ME was scarce in layer 5 pyramids, already for synaptic inputs. Resminostat However, layer 5 is innervated by layer 2/3 neurons (Thomson and Bannister, 1998), and ME was common in the AP output of layer 2/3 pyramids. Why then do the two cortical layers have different ME, given this connection? A number of mechanisms can be hypothesized. First, many layer 5 cells do not receive inputs from layer 2/3 (Thomson and Bannister, 1998) but instead receive inputs from the thalamus (Ferster and Lindström, 1983) and layer 4 (Feldmeyer et al., 2005). Second, temporal integration properties in the cortex are layer specific. For example, the lower expression of HCN channels in layer 2/3 compared with layer 5 pyramids (Spruston, 2008) could enable stronger ME in layer 2/3, because HCN currents reduce temporal integration (Williams and Stuart, 2000).

, 2008a;

Grahn and Rowe, 2009), and during playing on a silent piano keyboard (Baumann et al., 2007). There is a large literature on the acquisition of motor skills through training, suggesting different contributions of parts of the motor network in different phases of Selleckchem NVP-AUY922 learning (Doyon et al., 2009; Hikosaka et al., 2002). Models of motor skill learning suggest that M1 and premotor cortices are particularly important for learning and storage of the representation of a specific motor sequence, whereas the basal ganglia are more strongly involved in initial stimulus-response associations, and the cerebellum is engaged in online error correction mechanisms, and in optimization of acquired motor sequences (Penhune and Steele, 2012). These models fit well with short- and long-term musical training effects, which have mostly been found for the cortical and cerebellar parts of this network, possibly related to the fact that in music learning fine-tuning of complex Rigosertib molecular weight motor sequences is most relevant. In a cross-sectional study of highly trained pianists, anatomical changes to motor-related pathways were seen in white matter micro-organization as measured with diffusion imaging (Bengtsson et al., 2005), such that amount of musical practice during childhood was associated with greater integrity of corticospinal

tracts. Other parts of the motor network that differ anatomically between trained musicians and nonmusicians include the anterior corpus callosum (Schlaug et al., 1995), motor and premotor cortex (Bermudez et al., 2009; Gaser and Schlaug, 2003), and the cerebellum

(Hutchinson et al., 2003). White-matter connections between auditory and anterior regions also appear to be anatomically more well-organized in musicians (Halwani et al., 2011), a finding which fits well with the more focal cortical thickness intercorrelations reported between temporal and frontal cortices among musicians (Bermudez et al., 2009). Changes in the cortical Sitaxentan representations within the motor network can also be related to the specific type of instrumental practice. Bangert and Schlaug (2006) showed that pianists’ and violinists’ brains can be distinguished even on the gross macroscopic level by examining the shape and size of the part of the motor cortex that contains the representations of the hands. Moreover, pianists and violinists differ regarding lateralization, with a left- and right-hemispheric enlargement, respectively, in line with the fine motor control required for their instruments. Elbert et al. (1995) showed that the cortical representations of the fingers of violinists’ left hands, which are engaged in fine-tuned fingering of the strings during playing, are expanded as assessed by the amplitude and source location of tactile evoked responses measured in MEG, compared to their right hands’ representations or to controls.

Additional evidence for the importance of spike timing is found in development of visual motion tuning in Xenopus, sensory prediction in electric fish, map plasticity in sensory cortex, and olfactory learning in insects. In the Xenopus visual system, spikes in retinal ganglion cells evoke EPSCs in tectal neurons. When a subthreshold retinal input is stimulated before a second, suprathreshold

input that evokes a postsynaptic spike, the subthreshold response is potentiated (0 < Δt < 20 ms). When order is reversed, the subthreshold input is weakened (−20 < Δt < 0 ms) in a Hebbian STDP rule ( Zhang et al., 1998). Identical STDP of visual-evoked synaptic currents occurs after pairing visual stimuli at precise times relative to postsynaptic spikes elicited Androgen Receptor Antagonist by intracellular current injection ( Mu and Poo, 2006). Such sensory-spike pairing within specific receptive field subregions increases or decreases visual responses to those subregions as predicted by STDP, thereby shifting tectal neuron receptive fields in vivo ( Vislay-Meltzer et al., 2006). STDP is also observed with single, suprathreshold visual stimuli, which naturally elicit pre-leading-post spiking in tectal neurons, thus driving LTP of visual responses ( Zhang et al., 2000). Sensory-spike pairing also induces Hebbian STDP in cortical pyramidal cells in anesthetized rats. In primary visual

cortex (V1), visual-evoked EPSCs recorded in L2/3 pyramidal cells are potentiated by pairing visual responses prior to intracellularly evoked postsynaptic learn more spikes (0 < Δt < 20 ms) and are depressed by pairing after evoked spikes (−50 < Δt < 0 ms). For temporally extended visual responses, sensory-spike pairing

potentiates components of the response occurring prior old to the postsynaptic spike, and depresses components after the spike, consistent with STDP (Meliza and Dan, 2006). Orientation tuning can be modified by STDP, as shown by repeatedly pairing an oriented visual stimulus with extracellularly evoked spikes in V1 neurons. When visual responses precede spikes (Δt ≈20 ms), orientation tuning shifts toward the paired stimulus, but when the order is reversed (Δt ≈−10 ms), tuning shifts away from the paired orientation, consistent with Hebbian STDP at intracortical synapses (Schuett et al., 2001). Similar plasticity occurs in L2/3 pyramidal cells in rat somatosensory cortex. Pairing whisker-evoked postsynaptic potentials (wPSPs) following intracellularly evoked postsynaptic spikes (−30 ms < Δt < 0 ms) weakens wPSPs, but evokes no depression, and sometimes potentiation, when wPSPs lead spikes (Δt ≈20 ms) (Jacob et al., 2007). This is reminiscent of Hebbian STDP at L4-L2/3 synapses in vitro, but with reduced LTP (Feldman, 2000). Significant LTP has been observed with this pairing protocol in older mice (F. Gambino and A. Holtmaat, 2011, Soc. Neorosci., abstract).

Mice were administered either a vehicle control (medium chain triglyceride; The Nisshin Oillio Group, Tokyo, Japan) (MCT), eldecalcitol (0.2 μg/kg), or calcitriol (2 μg/kg) by once-a-day oral gavage for 14 days (n = 5). selleck chemicals Blood, kidney, and intestine samples were collected 6 h after the last dosing. Six-week-old male Sprague-Dawley rats were purchased from CLEA Japan. Animals were fed with normal rodent chow and tap water and acclimated to the above conditions for 1 week. Rats were divided into 11 groups based on body weight. Various doses of eldecalcitol (0.025, 0.05, 0.1, 0.25, and 0.5 μg/kg), calcitriol (0.25, 0.5, 1, 2.5, and

5 μg/kg), or MCT vehicle were administered by once-a-day oral gavage for 14 days (n = 6). On the 13th day, rats were transferred to and kept in metabolic cages for 24 h to collect urine samples. Blood, bone, kidney, and intestine samples were collected at 6 h after the last dosing on the 14th day. Both animal studies were carried out in accordance with Chugai Pharmaceutical’s ethical guidelines of animal care, and the experimental protocols were approved by the animal care committee of the institution. Levels of calcium, phosphorus, and creatinine in serum and urine were determined by using an automatic analyzer (TBA-120FR; Toshiba Medical Systems, Tochigi, Japan). PTH in plasma was measured by

rat intact PTH ELISA kit (Immutopics International, San Clemente, CA, USA). FGF-23 in serum was measured by FGF-23 ELISA kit (Kainos Laboratories, DNA Synthesis inhibitor Tokyo, Japan). Calcitriol in serum was measured by 1,25(OH)2D RIA kit (TFB, Tokyo, Japan). Measurement of eldecalcitol in plasma was performed at the BoZo Research Center (Tokyo, Japan). The right femur, intestine, and kidneys of mice, and the right femur, intestine, and kidneys of rats were excised and immediately frozen in liquid nitrogen. A small portion of each of the frozen tissues was soaked in TRIzol (Invitrogen, Carlsbad, CA, USA) and crushed in a homogenizer (Physcotron NS-310E; Microtec, Chiba, Japan). Total RNA was extracted

PD184352 (CI-1040) with an RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized from 200 ng of total RNA by reverse transcription PCR using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA). The reaction was performed at 37 °C for 1 h. Expression of mRNA in the tissues was detected using TaqMan Gene Expression Assays (Applied Biosystems). Target cDNA was amplified by 40 cycles (1 cycle: 95 °C for 15 s, 60 °C for 1 min) of PCR in an ABI PRISM 7000 Sequence Detector System (Applied Biosystems). The TaqMan probes used in this study were TRPV5, TRPV6, calbindin-D28k, and calbindin-D9k of mice and TRPV5, TRPV6, calbindin-D28k, calbindin-D9k, receptor activator of NF-κB ligand (RANKL), FGF-23, CYP27B1, CYP24A1, and VDR of rats. 18S rRNA was used as a control. Data are represented as expression relative to that in rats treated with vehicle control.