钠通道阻断剂在三叉神经痛药物治疗中的应用

三叉神经痛(TN)是一种神经病性疼痛综合征[1-2],其主要临床表现是面部三叉神经分布区反复发作的阵发性剧痛,性质如针刺样、电击样或刀割样等,持续时间数秒到数分钟.多数患者在面部有"扳机点",可由轻触、温度变化、面部运动或空气流动等刺激诱发疼痛.     

Effect of a neuronal sodium channel blocker on magnetic resonance derived indices of brain water content during global cerebral ischemia

Diffusion-weighted magnetic resonance imaging (DWI) with calculation of the apparent diffusion coefficient (ADC) of water is a widely used noninvasive method to measure movement of water from the extracellular to the intracellular compartment during cerebral ischemia. Lamotrigine, a neuronal Na(+) channel blocker, has been shown to attenuate the increase in extracellular concentrations of excitatory amino acids (EAA) during ischemia and to improve neurological and histological outcome. Because of its proven ability to reduce EAA levels during ischemia, lamotrigine should also minimize excitotoxic-induced increases in intracellular water content and therefore attenuate changes in the ADC. In this study, we sought to determine the effect of lamotrigine on intra- and extracellular water shifts during transient global cerebral ischemia. Fifteen New Zealand white rabbits were anesthetized and randomized to one of three groups: a control group, a lamotrigine-treated group, or a sham group. After being positioned in the bore of the magnet, a 12-min 50-s period of global cerebral ischemia was induced by inflating a neck tourniquet. During ischemia and early reperfusion there was a similar and significant decrease of the ADC in both the lamotrigine and control group. The ADC in the sham ischemia group remained at baseline throughout the experiment. Lamotrigine-mediated blockade of voltage-gated sodium channels did not prevent the intracellular movement of water during 12 min 50 s of global ischemia, as measured by the ADC, suggesting that the ADC decline may not be mediated by voltage-gated sodium influx and glutamate release.     

The use-dependent sodium channel blocker mexiletine is neuroprotective against global ischemic injury

Mechanisms responsible for anoxic/ischemic cell death in mammalian CNS grey and white matter involve an increase in intracellular Ca²⁺, however the routes of Ca²⁺ entry appear to differ. In white matter, pathological Ca²⁺ influx largely occurs as a result of reversal of Na⁺–Ca²⁺ exchange, due to increased intracellular Na⁺ and membrane depolarization. Na⁺ channel blockade has therefore been logically and successfully employed to protect white matter from ischemic injury. In grey matter ischemia, it has been traditionally presumed that activation of agonist (glutamate) operated and voltage dependent Ca²⁺ channels are the primary routes of Ca²⁺ entry. Less attention has been directed towards Na⁺–Ca²⁺ exchange and Na⁺ channel blockade as a protective strategy in grey matter. This study investigates mexiletine, a use-dependent sodium channel blocker known to provide significant ischemic neuroprotection to white matter, as a grey matter protectant. Pentobarbital (65 mg/kg) anesthetized, mechanically ventilated Sprague–Dawley rats were treated with mexiletine (80 mg/kg, i.p.). Then 25 min later the animals were subjected to 10 min of bilateral carotid occlusion plus controlled hypotension to 50 Torr by temporary partial exsanguination. Animals were sacrificed with perfusion fixation after 7 days. Ischemic and normal neurons were counted in standard H&E sections of hippocampal CA1 and the ratio of ischemic to total neurons calculated. Mexiletine pre-treatment reduced hippocampal damage by approximately half when compared to control animals receiving saline alone (45 vs. 88% damage, respectively; P<0.001). These results suggest that mexiletine (and perhaps other drugs of this class) can provide protection from ischemia to grey matter as well as white matter.     

噪音刺激下c—fos癌基因表达及钙拮抗剂防护作用的研究

应用机能定位的形态学方法——ｃ－ｆｏｓ癌基因表达法，对心理应激进行探讨。方法用８６ｄＢ噪音持续刺激ＳＤ大鼠１～２小时，并与受到４０ｄＢ强度生理性声音刺激和不给声音刺激的大鼠进行对比。结果接受噪音刺激的动物的脑干听觉通路各级神经元以及与情绪有关的边缘系统皮质下部位的杏仁核和下丘脑室旁核（ＰＶＮ）、视上核等处出现了明显的ｃ－ｆｏｓ癌基因表达产物Ｆｏｓ蛋白的聚集。用药与不用药组差异显著（Ｐ＜０．０５）。结论提示了心理应激的解剖基础和发病机制。     

钾通道拮抗剂IBTX对大鼠脑缺血再灌注损伤的保护作用及其机制的试验研究

目的:探讨大鼠脑缺血再灌注损伤后,海马CAl区锥体细胞caspaso-3表达及细胞凋亡的变化及使用钾通道拮抗剂IBTX(ibetiotoxin)的保护作用及其保护机制。方法:通过腹腔注射硝普钠加双侧颈总动脉夹闭建立大鼠全脑缺血一再灌模型,侧脑室注射IBTX,免疫组化法测caspase-3表达情况,TUNEL法检测细胞凋亡。结果:大鼠全脑缺血-再灌注24小时后,海马CAl区锥体细胞caspase-3阳性表达,TUNEL法检测有细胞凋亡,在用药IBTX组,caspase-3阳性表达减少,TUNEI法检测细胞凋亡亦有明显降低。结论:大鼠全脑缺血-再灌注24小时后海马CAl区锥体细胞出现细胞凋亡,使用钾通道拮抗剂IBTX可减少细胞凋亡的发生,从而发挥保护作用。     

Inhibition of electrically induced seizures by a dihydropyridine calcium channel blocker

Nimodipine, a calcium channel blocker with high affinity for central dihydropyridine Ca2+ channels, produced a dose-dependent suppression of electrically induced seizures in the rabbit. Verapamil, a diphenylalkylamine which acts at peripheral Ca2+ channels, was ineffective. Phenytoin was less effective than nimodipine. These results suggest that calcium flux into neurons may be a biochemical precipitant for seizure genesis. Centrally acting calcium channel blockers may prove to be a new class of anticonvulsants.     

Alanine to valine substitutions in the pore helix IIIP1 and linker-helix IIIL45 confer cockroach sodium channel resistance to DDT and pyrethroids

Pyrethroid insecticides exert toxic effects by prolonging the opening of voltage-gated sodium channels. More than 20 sodium channel mutations from arthropod pests and disease vectors have been confirmed to confer pyrethroid resistance. These mutations have been valuable in elucidating the molecular interaction between pyrethroids and sodium channels, including identification of two pyrethroid receptor sites. Previously, two alanine to valine substitutions, one in the pore helix IIIP1 and the other in the linker-helix connecting S4 and S5 in domain III (IIIL45), were found in Drosophila melanogaster mutants that are resistant to DDT and deltamethrin (a type II pyrethroid with an α-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids), but their role in target-site-mediated insecticide resistance has not been functionally confirmed. In this study, we functionally examined the two mutations in cockroach sodium channels expressed in Xenopus laevis oocytes. Both mutations caused depolarizing shifts in the voltage dependence of activation, conferred DDT resistance and also resistance to two Type I pyrethroids by almost abolishing the tail currents induced by Type I pyrethroids. In contrast, neither mutation reduced the amplitude of tail currents induced by the Type II pyrethroids, deltamethrin or cypermethrin. However, both mutations accelerated the decay of Type II pyrethroid-induced tail currents, which normally decay extremely slowly. These results provided new insight into the molecular basis of different actions of Type I and Type II pyrethroids on sodium channels. Computer modeling predicts that both mutations may allosterically affect pyrethroid binding.     

Calcium channel blocker influences the density of alpha-actinin labeling at the rat neuromuscular junction

Alpha-actinin is a muscle protein located along the Z-disc. Incubation of frog muscle with the calcium ionophore, A23187, can decrease the immunogold labelling of alpha-actinin. Pyridostigmine (PYR) is an inhibitor of acetylcholinesterase, which causes disruption of Z-discs only in the region of the motor endplate. This is probably due to excess influx of calcium ions, leading to activation of proteases. Pretreating animals with the calcium channel blocker diltiazem can significantly reduce damage to the Z-discs at the motor endplate caused by PYR. It was of interest to determine whether the distribution of alpha-actinin had been altered following PYR administration and whether diltiazem could prevent those changes. There was less alpha-actinin labelling at the motor endplate compared to away from this region for all treatment groups. Animals administered diltiazem showed less labelling compared to PYR, but with no disruption of Z-discs at the motor endplate following diltiazem. Pretreatment with diltiazem reduced the incidence of Z-disc damage, but the degree of alpha-actinin labeling at the endplate was less than that seen with diltiazem alone. The greater effect seen at the endplate implies that neuromuscular activity is an important factor. The drugs may be causing a reduction in alpha-actinin labelling by different mechanisms.     

A new potent channel blocker: Effects on glutamate responses at the crayfish neuromuscular junction

The glutamate blocking action of 5-methyl-1-phenyl-2-(3-piperidinopropylamino)-hexane-1-ol (MLV-5860) was studied at the crayfish neuromuscular junction using electrophysiological techniques. The opener muscle of the dactyl in the first leg of the crayfish was used to examine the action of the drug on the glutamate response. MLV-5860 reduced the amplitude of repetitively-induced glutamate potentials in a dose-dependent manner at the crayfish neuromuscular junction and this reduction was time- and activity-dependent. The minimum effective concentration of MLV-5860 to reduce the glutamate response was estimated to be lower than 50 nM, and therefore MLV-5860 is the most powerful glutamate blocker known at the crayfish neuromuscular junction. Pretreatment of the muscle fiber with concanavalin A did not affect the action of MLV-5860. MLV-5860 reduced the amplitude of excitatory junctional potentials (EJPs) and increased the decay rate of extracellularly-recorded EJPs in a dose-dependent manner. Quisqualate responses were also reduced by this drug but the conductance increase of the muscle membrane induced by GABA was not affected. MLV-5860 did not cause a significant change in the input resistance of the opener muscle fiber at concentrations less than 10 microM. The action of the drug is possibly explained in part by the open channel block of the glutamate-activated ion channels. The forward rate constant for channel blockade was estimated from the difference between the decay rate constants of extracellular EJPs in the absence and presence of the drug and the estimated value was 6.5 +/- 1.4 X 10(7) M-1 s-1.     

Effects of the sodium channel blocker tetrodotoxin (TTX) on cellular ion homeostasis in rat brain subjected to complete ischemia

Anoxic depolarization (AD) and failure of the cellular ion homeostasis are suggested to play a key role in ischemia-induced neuronal death. Recent studies show that the blockade of Na⁺ influx significantly improved the neuronal outcome. In the present study, we investigated the effects of 10 μM tetrodotpxin (TTX) on ischemia-induced disturbances of ion homeostasis in the isolated perfused rat brain. TTX inhibited the spontaneous EEG activity, delayed the ischemia-induced tissue acidification, and significantly postponed the occurrece of AD by 65%. The [Ca²⁺]_e elevation prior to AD was attenuated from 17.8% to 6% while the increase of the [Na⁺]_e in this period was enhanced (from 2.9% to 7.3%). These findings implied that the ischemia-induced early cellular sodium load and the corresponding shrinkage of the extracellular space was counteracted by TTX. Our results suggest that the Na⁺ influx via voltage-dependent channels preceding complete breakdown of ion homeostasis is one major factor leading to cell depolarization. The massive Na⁺ influx coinciding with AD, however, may be mainly via non-selective cation channels or/and receptor-operated channels. Persistent Na⁺ influx deteriorates neuronal tissue integrity by favouring Ca²⁺ influx and edema formation. Blockade of ischemia-induced excessive Na⁺ influx is, therefore, a promising pharmacological approach for stroke treatment.     

Interaction of insecticides of the pyrethroid family with specific binding sites on the voltage-dependent sodium channel from mammalian brain

Measurement of neurotoxin binding in rat brain membranes and neurotoxin-activated 22Na+ influx in neuroblastoma cells were used to define the site and mechanism of action of pyrethroids and DDT on sodium channels. A highly potent pyrethroid, RU 39568, alone enhanced the binding of [3H]batrachotoxinin A 20-alpha-benzoate up to 30 times. This effect was amplified by the action of neurotoxins such as sea anemone toxins and brevetoxin acting at different sites of the sodium channel protein in brain membranes. The ability of various pyrethroids and DDT to enhance batrachotoxin binding was related to their capacity to activate tetrodotoxin sensitive 22Na+ uptake. These results point to an allosteric mechanism of pyrethroids and DDT action involving preferential binding to active states of sodium channels which have high affinity for neurotoxins, causing persistent activation of sodium channels. Pyrethroids do not block [3H]tetrodotoxin binding, 125I-Anemonia sulcata toxin 2 binding, 125I-Tityus serrulatus toxin gamma binding at neurotoxin receptor sites 1, 3 and 4 respectively. Pyrethroids appear to act at a new neurotoxin receptor site on the sodium channel. The distribution of pyrethroid binding sites in rat brain was determined by quantitative autoradiographic procedures using the property of pyrethroids to reveal binding sites for [3H]batrachotoxinin A 20-alpha-benzoate.     

钙通道阻滞剂对大鼠坐骨神经损伤后c-fos表达及神经功能的影响

目的探讨钙通道阻滞剂(CCB)对周围神经损伤后c-fos表达及神经功能的影响。方法制作坐骨神经嵌压性损伤大鼠模型,给予模型大鼠分别腹腔注射氟桂利嗪1mg/kg(低剂量组)、2mg/kg(高剂量组),或生理盐水10ml/kg(模型组)。在坐骨神经嵌压后第1周、第4周时取大鼠坐骨神经,采用免疫组织化学、行为医学和电生理学的方法测定c-fos阳性细胞数及第4周时足趾间距、神经传导速度(NCV);并与正常大鼠比较。结果(1)损伤后1周时,模型组、氟桂利嗪低剂量组坐骨神经c-fos阳性细胞数显著多于正常对照组(均P<0.01);氟桂利嗪高剂量组c-fos阳性细胞数轻度增加,也显著多于正常对照组(P<0.05),但明显少于模型组和氟桂利嗪低剂量组(均P<0.01);损伤4周时各组c-fos阳性细胞数均无明显增高。(2)损伤后4周时,模型组和氟桂利嗪低剂量组、高剂量组坐骨神经NCV显著慢于正常对照组(均P<0.01),氟桂利嗪低剂量组、高剂量组的NCV快于模型组(P<0.05,P<0.01)。(3)损伤后4周时,模型组大鼠右后肢足趾间距明显小于其他3组(均P<0.01);氟桂利嗪高剂量组、低剂量组与正常对照组比较差异无统计学意义(均P>0.05)。结论CCB使周围神经损伤后早期c-fos表达下调,并使神经功能受损减轻。     

Neuroprotective effects of the sodium channel blocker RS100642 and attenuation of ischemia-induced brain seizures in the rat

Seizurogenic activity develops in many patients following brain injury and may be involved in the pathophysiological effects of brain trauma and stroke. We have evaluated the effects of the use-dependent sodium channel blocker RS100642, an analog of mexiletine, as a neuroprotectant and anti-seizure agent in a rat model of transient middle cerebral artery occlusion (MCAo). Post-injury treatment with RS100642 (0.01-5.0 mg/kg) dose-dependently reduced brain infarction, improved functional recovery of electroencephalographic (EEG) power, and improved neurological outcome following 2 h of MCAo and 24 h recovery. This effect was more potent and offered a larger reduction of brain infarct volume than a maximal neuroprotective dose of mexiletine (10.0 mg/kg). Furthermore, brain seizure activity recorded following 1 h MCAo and 72 h of recovery in injured rats was either completely blocked (30 min pre-MCAo treatment) or significantly reduced (30 min post-MCAo treatment) with RS100642 (1.0 mg/kg) treatment resulting in greater than 60% reduction of core brain infarct. These results indicate that brain seizure activity during MCAo likely contributes to the pathophysiology of brain injury and that RS100642 may be an effective neuroprotective treatment not only to decrease brain injury but also to reduce the pathological EEG associated with focal ischemia.     

Developmental pyrethroid exposure causes long-term decreases of neuronal sodium channel expression

Pyrethroid insecticide use has increased over recent years because of their low to moderate acute toxicity in mammals. However, there is increasing concern over the potential detrimental effects of pyrethroids on developing animals. Most recently, we have shown that developmental exposure to deltamethrin results in long-term neurobehavioral effects. Pyrethroids exert their toxicity by acting on the voltage-gated sodium channel (Na_v), delaying channel inactivation and causing hyperexcitability in the nervous system. Previous in vitro studies found that exposure to agents that increase Na⁺ influx, including deltamethrin decreased Na_v mRNA expression. However, it is unknown whether this occurs in vivo. To determine whether developmental pyrethroid exposure decreases Na_v mRNA expression, pregnant mice were exposed to the pyrethroid deltamethrin (0 or 3 mg/kg) every three days throughout gestation and lactation. Na_v mRNA expression was measured in the striatum and cortex of the offspring at 10–11 months of age, a time at which behavioral abnormalities were still observed. Developmental exposure to deltamethrin decreased expression of Na_v mRNA in a region- and isoform-specific fashion by 24–50%. Deltamethrin exposure also resulted in the persistent down-regulation of brain-derived neurotrophic factor (Bdnf) in the striatum by 66% but not in the cortex, suggesting a plausible mechanism for some of the associated behavioral effects observed previously. Taken together these data suggest that developmental deltamethrin exposure results in persistent deficits in Na_v and BDNF mRNA expression that may contribute to long-term behavioral deficits.     

Scn2a sodium channel mutation results in hyperexcitability in the hippocampus in vitro

PURPOSE: To investigate in vitro, the cellular network activity of the hippocampus in Q54 mice that display spontaneous seizures because of a gain-of-function mutation of the Scn2a sodium channel gene. METHODS: Extacellular recordings were obtained from CA1 and CA3 pyramidal neurons in hippocampal slices prepared from Q54 transgenic and nontransgenic littermates (WT) under physiologic conditions as well as during periods of orthodromic stimulation of the Schaffer collaterals. Cerebral spinal fluid samples were analyzed and cresyl violet histology of the hippocampus was conducted. RESULTS: Increased spontaneous extracellular activity was found in both CA1 and CA3 regions of Q54 hippocampal slices. Q54 slices also demonstrated significantly greater spontaneous and afterdischarge activity as well as population spike amplitude and duration following tetanic stimulus in comparison to WT slices. Frequency analysis of tetanically stimulated recordings indicated high-frequency components (100 and 200 Hz) unique to Q45 slices. Analysis of cresyl violet histology supports healthy Q54 slices up to 10 weeks, while Q54 cerebral spinal fluid shows elevated osmolarity. CONCLUSION: Evidence for hyperexcitability and increased synaptic efficacy in Q54 mice was found by observing spontaneous activity as well as evoked activity. Response to tetanic stimulation included unique high-frequency oscillations, and resulted in an increased population spike amplitude and duration. Histological assessment shows equivalent neuronal development in both experimental groups. The data support the hypothesis that modified Scn2a channels in Q54 mice result in network hyperexcitability of the hippocampus necessary for the development and maintenance of temporal lobe seizures.     

A unique ion channel clustering domain on the axon initial segment of mammalian neurons

The axon initial segment (AIS) plays a key role in initiation of action potentials and neuronal output. The plasma membrane of the AIS contains high densities of voltage‐gated ion channels required for these electrical events, and much recent work has focused on defining the mechanisms for generating and maintaining this unique neuronal plasma membrane domain. The Kv2.1 voltage‐gated potassium channel is abundantly present in large clusters on the soma and proximal dendrites of mammalian brain neurons. Kv2.1 is also a component of the ion channel repertoire at the AIS. Here we show that Kv2.1 clusters on the AIS of brain neurons across diverse mammalian species including humans define a noncanonical ion channel clustering domain deficient in Ankyrin‐G. The sites of Kv2.1 clustering on the AIS are sites where cisternal organelles, specialized intracellular calcium release membranes, come into close apposition with the plasma membrane, and are also sites of clustering of γ‐aminobutyric acid (GABA)ergic synapses. Using an antibody specific for a single Kv2.1 phosphorylation site, we find that the phosphorylation state differs between Kv2.1 clusters on the proximal and distal portions of the AIS. Together, these studies show that the sites of Kv2.1 clustering on the AIS represent specialized domains containing components of diverse neuronal signaling pathways that may contribute to local regulation of Kv2.1 function and AIS membrane excitability. J. Comp. Neurol. 522:2594–2608, 2014. © 2014 Wiley Periodicals, Inc.     

Distribution of the voltage gated sodium channel Na(v)1.3-like immunoreactivity in the adult rat central nervous system

Voltage-gated sodium channels are transmembrane proteins responsible for the initiation and propagation of action potentials. One subtype, Na_v1.3 (brain type III) is tetrodotoxin sensitive and fast inactivated. Na_v1.3 has been shown to be expressed at low levels in the adult rat, but to be upregulated after sciatic nerve axotomy in the dorsal root ganglia. In the present study, we used immunohistochemistry to look at the distribution of Na_v1.3 in the adult rat central nervous system. We used a polyclonal antibody, raised against residues 511–524. This epitope corresponds to the sequence located in the intracellular loop between domains I and II of Na_v1.3 and is specific for this sodium channel subtype. We found Na_v1.3-like immunoreactivity (-LI) neurons in the cerebral cortex, hippocampal formation, colliculi, and mesencephalic reticular formation. Na_v1.3-LI was observed in fiber tracts such as the corpus callosum, anterior commissure, corticofugal fibers, lateral lemniscus, and cerebellar peduncles. Na_v1.3-LI was particularly intense in sensory nerve tracts such as the mesencephalic trigeminal tract, vestibulospinal tract, or spinal trigeminal tract. In the spinal cord, Na_v1.3-LI was intense throughout the white matter and the dorsal roots. In the spinal cord grey matter, Na_v1.3-LI fibers terminate in the deep laminae of the dorsal horn and in the ventral horn. Na_v1.3-LI was also found in motoneurons as well as in ventral roots. This study shows that Na_v1.3 is present at the protein level in the adult rat.     

Rapid sodium channel augmentation in response to inflammation induced by complete Freund's adjuvant

The mechanisms by which inflammation induces a chronic pain state are poorly understood. Following the induction of many painful conditions, an increase in the spontaneous firing rate of neurons is often observed in peripheral sensory ganglia. Since ion channels are essential mediators of spike generation and impulse conduction, it is reasonable to postulate that local changes in ion channel expression might underlie the changes in membrane excitability. Such alterations may serve to enhance the efficiency by which painful stimuli are transduced and then conducted to the central nervous system. In these studies, we employed immunocytochemical methods to investigate the changes in sodium channel expression in dorsal root ganglia of rats following a subcutaneous injection of complete Freund's adjuvant, an inducer of chronic inflammation. We find that sodium channel immunoreactivity within primary sensory neurons is dramatically increased within 24 h of the complete Freund's adjuvant injection. These changes persist in small neurons for at least 2 months and roughly parallel the time course of behaviorally measured changes in pain thresholds. Thus, the regulation of sodium channel synthesis may play a role in the generation and maintenance of the hyperesthetic state seen in chronic inflammation.     

Ⅰ型电压门控钠离子通道蛋白在大鼠海马区不同类型神经元上的表达差异

目的 研究Ⅰ型电压门控钠离子通道(Nay1.1)蛋白在大鼠海马区不同类型神经元上的表达情况.方法 采用成年SD大鼠脑组织切片.通过免疫组织化学方法和荧光标记激光共聚焦显微镜法观察比较海马区不同类型神经元上Nay1.1蛋向的表达.结果 在SD大鼠的海马中,Nay1.1蛋白在各区的锥体神经元和齿状回的颗粒细胞胞体上的表达均很微弱.而散在分布的中间神经元上则有很强的阳性表达.结论 SD大鼠海马区散在分布的中间神经元上Nav1.1蛋白的表达较强,而投射神经元CA1-4各区的锥体神经元和齿状回的颗粒细胞则表达微弱:推测不同类型的神经元上可能存在不同亚型的钠离子通道优势分布.     

Heterozygous CLCN1 mutations can modulate phenotype in sodium channel myotonia

Nondystrophic myotonias are characterized by muscle stiffness triggered by voluntary movement. They are caused by mutations in either the CLCN1 gene in myotonia congenita or in the SCN4A gene in paramyotonia congenita and sodium channel myotonias. Clinical and electrophysiological phenotypes of these disorders have been well described. No concomitant mutations in both genes have been reported yet. We report five patients from three families showing myotonia with both chloride and sodium channel mutations. Their clinical and electrophysiological phenotypes did not fit with the phenotype known to be associated with the mutation initially found in SCN4A gene, which led us to screen and find an additional mutation in CLCN1 gene. Our electrophysiological and clinical observations suggest that heterozygous CLCN1 mutations can modify the clinical and electrophysiological expression of SCN4A mutation.