Neurobehavioural, neurochemical and electrophysiological studies in 6-hydroxydopamine lesioned and neural transplanted rats

Unilateral injection of 6-hydroxydopamine (6-OHDA) into the caudate nucleus of rat caused degeneration of dopaminergic terminals, evidenced by significant (P⊂0.05) elevation of spontaneous and drug-induced motor behaviour, enhanced DA receptor binding and significant increase in the neuronal firing rate of caudate neurons, suggesting supersensitivity of dopaminergic receptors. Eight weeks following the transplantation of embryonic cell suspensions from caudate at the lesioned site, a significant restoration of the enhanced ³H spiperone binding and neuronal activity of caudate neurons was observed in comparison with lesioned rats.These results clearly demonstrate that transplanted embryonic neuronal tissue at the lesioned site is capable of restoring the neuronal deficits caused by 6-OHDA as evidenced by significant amelioration in neurochemical, behavioral and electrophysiological alterations.     

Intrastriatal angiotensin II induces turning behaviour in 6-hydroxydopamine lesioned rats

In rats with unilateral 6-hydroxydopamine lesions in the nigrostriatal pathway, injection of Angiotensin II (2 nmol) into the unlesioned striatum elicited dose-related tight rotations ipsilateral to the lesion. This rotation was suppressed by coadministration of the Angiotensin AT₁ receptor antagonist, losartan (2 nmol), which had no significant effect when injected alone. Preadministration of the dopamine antagonist, haloperidol (2 mg/kg i.p.) completely blocked angiotensin II-induced turning at doses of 0.3–3 nmol, and partially at 10 nmol. These results further confirm the hypothesis that Ang II is intrinsically involved in modulating dopamine release in the striatum, an effect which is mediated predominantly by AT₁ receptors.     

Survival of genetically engineered, adult-derived rat astrocytes grafted into the 6-hydroxydopamine lesioned adult rat striatum

Astrocytes are potentially useful as vehicles for gene transfer into the CNS. As endogenous CNS cells, they possess secretory mechanisms and can be grown in vitro. We have developed an animal model of this system using autologous astrocyte grafts in Fischer 344 rats. Cultured cells were infected with an adenoviral vector containing the reporter gene lacZ in vitro and then grafted into the striatum of adult Fischer 344 rats previously lesioned with 6-OHDA. Survival of the cells and activity of the β-galactosidase protein were followed for up to 21 days after injection. The grafted cells were shown to survive throughout the experimental period although the expression of transgene was reduced with time. If long-term expression of therapeutically active substances can be achieved, grafts of adult-derived astrocytes genetically engineered using recombinant adenoviral vectors could be employed in the treatment of Parkinson's disease and other neurological disorders.     

High frequency stimulation of the subthalamic nucleus improves treadmill locomotion in unilateral 6-hydroxydopamine lesioned rats

This study investigated the influence of electrical stimulation of the subthalamic nucleus (STN) on motor impairment induced by unilateral 6-hydroxydopamine (6-OHDA) lesions in the medial forebrain bundle. Rats were trained to walk on a treadmill and then implanted with microelectrode arrays in and near the STN. The neurotoxin 6-OHDA was injected into the medial forebrain bundle (MFB) unilaterally to produce a targeted lesion of the dopaminergic system. Successful lesions produced impaired treadmill walking behavior. High frequency stimulation (HFS) of the STN improved treadmill walking immediately and restored normal walking patterns. The same HFS failed to evoke visible side effects such as stepping, turning, raising of the head or facial muscle contraction in the absence of treadmill movement, or to change rotational behaviors elicited by the dopamine (DA) agonist apomorphine in unilateral lesioned rats. This suggests that the stimulation did not cause movement by an activation of brainstem locomotor regions or an increase attention leading to movement. Apomorphine-induced rotation may represent an imbalance of dopaminergic activation which remains during HFS. This work may provide a rodent model for deep brain stimulation (DBS) in patients with Parkinson's disease, and be suitable for further investigation of the neural mechanisms underlying the therapeutic effects of DBS.     

Survival, migration and differentiation of mouse tau-GFP embryonic stem cells transplanted into the rat auditory nerve

Stem cells have been investigated as treatment for a variety of diagnoses such as Parkinson's disease, Alzheimer's disease and spinal cord injuries. Here, we investigated the possibility of using stem cells as a replacement therapy for lesions of the auditory nerve (AN). We transplanted tau-GFP mouse embryonic stem cells into the AN either by the internal auditory meatus or via the modiolus in rats that had been previously deafened by application of β-bungarotoxin to the round window niche. We investigated the effect of brain derived neurotrophic factor (BDNF) on cell transplant survival and differentiation. Additionally chondroitinase ABC (ChABC), a digestive enzyme that cleaves the core chondroitin sulfate proteoglycans, was used in order to promote possible migration of cells and axons through the transitional zone. A bioactive isoleucine-lysine-valine-alanine-valine (IKVAV) peptide amphiphile (PA) nanofiber gel was applied around the cell injection site. This nanofiber gel has been shown to promote neural differentiation and other similar gels have been used to encapsulate and release proteins. Three weeks after injection, transplanted cells were found in the scala tympani, the modiolus, the AN trunk and the brain stem. As compared to cell transplantation and gel only, BDNF content in the PA gel increased cell survival and neuronal differentiation. In the animals treated with ChABC we observed extensive migration of cells through the transitional zone to or from the CNS.     

Correlation of apomorphine- and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats

In the unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease, controversy exists concerning the use of apomorphine- ord-amphetamine-induced rotations as reliable indicators of nigrostriatal dopamine depletion. Our objective was to evaluate which, if either, drug-induced behavior is more predictive of the extent of nigrostriatal dopamine depletion. Fischer 344 and Sprague-Dawley rats were unilaterally injected with 9μg/4μl/4 min 6-hydroxydopamine into the medial forebrain bundle. The animals were behaviorally tested with apomorphine (0.05 mg/kg, s.c.) andd-amphetamine (5.0 mg/kg, s.c.). Following testing, the brains were removed and the right and left striata, substantia nigra and ventral tegmental area were dissected free and quickly frozen at −70°C for analysis of catecholamine content by high performance liquid chromatography coupled with electrochemical detection. Our results indicate that an animal which has greater than a 90% depletion of dopamine in the striatum might not rotate substantially on apomorphine, without a concomitant depletion of 50% of the DA content in the corresponding substantia nigra. No correlations were seen involving depletions of the ventral tegmental area and the extent of the lesions to the striatum. Submaximally lesioned (75–90% depleted) rats were found to rotate ond-amphetamine but not on apomorphine. In addition, control rats that did not receive lesions were often seen to rotate extensively ond-amphetamine. We therefore conclude that maximal lesions of the striatum and substantia nigra are required to generate rotations demonstrable with low dose apomorphine but not withd-amphetamine. Apomorphine, rather thand-amphetamine, is thus a better predictor of maximal lesions of the striatum produced by 6-OHDA.     

Fate of neural stem cells grafted into injured inner ears of mice

Loss of sensory hair cells in the inner ear is a major cause of permanent hearing loss, since regeneration of hair cells rarely occurs in mammals. The aim of this study was to examine the potential of neural stem cell transplantation to restore inner ear hair cells in mice. Fetal neural stem cells were transplanted into the mouse inner ear after drug-induced injury. Histological analysis demonstrates that the majority of grafted cells differentiated into glial or neural cells in the inner ear. Strikingly, however, we show that grafted cells integrate in vestibular sensory epithelia and express specific markers for hair cells. This finding suggests that transplanted neural stem cells have the potential to differentiate and restore inner ear hair cells.     

Intraocular injection of folate antagonist methotrexate induces neuronal differentiation of embryonic stem cells transplanted in the adult mouse retina

Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuronal cells. However, the integrated ES cells also have a tumorigenic effect just because they have the ability for multipotential differentiation to various types of tissues. Thus, control of neoplastic potentials of ES cells is very important for the treatment of degenerative or injured diseases. Mouse ES cells carrying the sequence for the green fluorescent protein (GFP) gene were transplanted into adult mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-d-aspartate (NMDA) administration. One week after the ES cell injection, folate antagonist methotrexate (MTX) was injected intravitreally. Eyes were retrieved 4 weeks after ES cell transplantation for histologic analyses. Conventional histological analysis was performed by hematoxylin and eosin staining with the use of paraffin-embedded sections. Neuronal differentiation and teratogenic potential of ES cells were demonstrated by immunohistochemistry. The proliferative activity of transplanted cells was detected by mitotic index, proliferating cell nuclear antigen index and AgNOR count. The incorporation of transplanted ES cells in MTX-treated and non-treated retinas at 4 weeks after transplantation was observed in 8/16 eyes (50%) and 8/16 eyes (50%), respectively. Transplanted ES cells in MTX-treated retina showed increased neuronal differentiation and decreased expression of teratogenic markers, compared with ES cells in non-treated retina. The proliferative activity of transplanted ES cells in MTX-treated retina was lower than that in non-treated retina. These results suggest that intravitreal MTX treatment following transplantation can induce neuronal differentiation in the transplanted ES cells and decrease their proliferative activity.     

Dopaminergic mRNA expression in the intact sebstantia nigra of unilaterally 6-OHDA-lesioned and grafted rats: an in situ hybridization study

Summary. The present study was performed to investigate the influence of intrastriatal fetal mesencephalic grafts on dopaminergic mRNA expression in the non-lesioned substantia nigra pars compacta of unilaterally 6- hydroxydopamine-lesioned rats. The expression of dopamine transporter mRNA, synaptic vesicular monoamine transporter mRNA and tyrosine hydroxylase mRNA was assessed in adjacent cryostat sections using in situ hybridization. Rotational behavior induced by apomorphine and amphetamine as well as hybridization of striatal sections cut at the grafting coordinates were used to prove the functional recovery and the presence of grafted cells, respectively. After grafting, the number of rotations was decreased and hybridization signals overlying cells in the grafted striatum were detected. Mean grain densities overlying labeled neurons in the substantia nigra pars compacta of grafted rats were compared to those of shamgrafted rats and revealed differential expression of dopamine transporter mRNA, whereas synaptic vesicular monoamine transporter mRNA and tyrosine hydroxylase mRNA expression showed no difference. The results will be discussed in relation to previous in vitro and in vivo studies suggesting a reduction of functional dopamine transporter molecules in the contralateral striatum. Received April 25, 2000; accepted August 17, 2000     

NGF-producing transfected 3T3 cells: Behavioral and histological assessment of transplants in nigral lesioned rats

The rodent fibroblast clonal cell line, 3T3, was retrovirally transfected with the rat nerve growth factor (NGF) gene and selected for NGF synthesis. This study tested the hypothesis that transplanted 3T3 cells, transfected to secrete nerve growth factor (3T3^NGF+), change motor behavioral indices created by striatal denervation in a dose-dependent fashion. 3T3^NGF+ cells were transplanted into the lateral ventricle of rats following ipsilateral lesions of the substantia nigra pars compacta by stereotaxic injections of 6-hydroxydopamine (10 μg), an established lesion model. Control groups included vehicle injections and transplanted untransfected cells. The extent of the lesions was measured by determining rotational behavior before and two weeks after transplantation. Immediately prior to transplantation, cells were incubated with the fluorescent dye marker, Dil. To assess cell viability, whole brains were cryosectioned and examined for Dil-labeled 3T3 cells using fluorescent microscopy. The number Uf Dil-labeled profiles in five animals per group were counted in at least five noncontiguous sections per animal. From these data a statistically derived estimate of viable, transplanted 3T3 cells was obtained. The number of surviving transplanted cells correlated with the behavioral changes measured. The 3T3NGF + transplants reduced rotational behavior, while control 3T3 transplants exacerbated rotational behavior. Thus, while NGF delivery was found to be beneficial, it was apparent that naive 3T3 had detrimental effects. These results underscore the importance of making doseresponse measurements when attempting transplantbased modifications of CNS behavior. © 1995 Wiley-Liss, Inc.     

Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

Parthenogenetic embryonic stem cells have pluripotent differentiation potentials,akin to fertilized embryo-derived embryonic stem cells.The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells.Before differentiation,karyotype analysis was performed,with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells.Sex chromosomes were identified as XX.Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene,Oct4,at both the mRNA and protein levels,indicating pluripotent differentiation potential of the two embryonic stem cell subtypes.Embryonic stem cells were induced with retinoic acid to form embryoid bodies,and then dispersed into single cells.Single cells were differentiated in N2 differentiation medium for 9 days.Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin,βIII-tubulin and myelin basic protein.Quantitative real-time PCR found expression of neurogenesis related genes(Sox-1,Nestin,GABA,Pax6,Zic5 and Pitx1) in both types of embryonic stem cells,and Oct4 expression was significantly decreased.Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells.Thus,our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.     

An assessment of the validity of densitometric measures of striatal tyrosine hydroxylase-positive fibers: relationship to apomorphine-induced rotations in 6-hydroxydopamine lesioned rats

The power of immunohistochemical staining as a tool for the study of the neurochemical anatomy of the brain would be greatly enhanced if quantitative measures of staining were to be developed. We have here assessed the reliability and validity of two population measures of extent of fiber innervation: percent area occupied by staining, and average optical density (AOD) of staining. We have evaluated these measures for tyrosine hydroxylase-positive staining of the striatum in relation to apomorphine-induced rotational behavior in 6-hydroxydopamine lesioned rats. We have found that inter-operator reliability for the area measure is high (r = 0.98). Apomorphine-induced rotations were observed when the area measured was reduced to 2% or less of the control side, and when the density measure was reduced to 15% or less. These results are similar to those obtained previously for biochemical assay of TH activity, which showed rotations at reductions to 10% or less. We conclude that these density measures provide valid relative indices of extent of fiber innervation on the same section. The AOD measure appears to be more sensitive at lower levels of innervation.     

小鼠胚胎干细胞诱导的神经前体细胞纹状体移植对PD大鼠的治疗作用

目的 观察来源于小鼠胚胎干细胞的神经前体细胞移植PD大鼠纹状体后的存活、分化以及细胞移植对PD大鼠的治疗作用。方法 采用无血清方法将小鼠胚胎干细胞定向诱导为神经前体细胞，免疫组化技术观察移植细胞的存活、分化。结果 胚胎体在N2选择性培养基选择生长5d后，85％以上的小鼠胚胎干细胞分化为nestin阳性的神经前体细胞。移植到PD大鼠纹状体后大部分神经前体细胞存活良好，移植细胞分别保持为未分化的nestin阳性的神经前体细胞和TH阳性的神经元。移植后3周，PD大鼠的旋转次数明显减少。结论 胚胎干细胞来源的神经前体细胞移植PD大鼠纹状体后能分化为TH阳性的神经细胞，对PD有治疗作用。     

Role of striatal acetylcholine on dopamine D1 receptor agonist-induced turning behavior in 6-hydroxydopamine lesioned rats: a microdialysis-behavioral study

The effects of MK-801, a non-competitive N-methyl D-aspartate (NMDA) receptor antagonist, of quinpirole, a dopamine (DA) D₂ receptor agonist, and of SCH 58261, an A_2A adenosine antagonist, were studied on acetylcholine (ACh) release in the striatum of 6-hydroxydopamine (60HDA) lesioned rats and on turning behavior induced by the administration of the DA D₁ agonist CY 208-243. Administration of CY 208-243 to 60HDA lesioned rats induced turning behavior and dose-dependently stimulated ACh release. At the dose of 50 μg/kg, MK-801 failed to affect basal ACh, while at 100 μg/kg MK-801 reduced it; however, MK-801 (50 and 100 μg/kg) potentiated the turning behavior elicited by CY 208-243, but failed to affect the CY 208-243-induced increase of striatal ACh release. The administration of quinpirole induced low-intensity turning behavior and decreased basal ACh release; on the other hand, quinpirole potentiated the turning behavior induced by CY 208-243, but failed to affect the CY 208-243-elicited increase of ACh release. Finally, intravenous administration of SCH 58261 stimulated basal ACh release but not turning behavior, SCH 58261, however, potentiated turning behavior induced by CY 208-243, while failing to affect the D₁-elicited increase of ACh release. These results indicate that potentiation of D₁-dependent turning behavior by MK-801, quinpirole and SCH 58261 is not mediated by a reduced ability of D₁-agonists to stimulate ACh release from the denervated striatum.     

Striatal NTS1 , dopamine D2 and NMDA receptor regulation of pallidal GABA and glutamate release--a dual-probe microdialysis study in the intranigral 6-hydroxydopamine unilaterally lesioned rat

The current microdialysis study elucidates a functional interaction between the striatal neurotensin NTS(1) receptor and the striatal dopamine D(2) and N-methyl-d-aspartic acid (NMDA) receptors in the regulation of striatopallidal gamma-aminobutyric acid (GABA) and glutamate levels after an ipsilateral intranigral 6-hydroxydopamine-induced lesion of the ascending dopamine pathways to the striatum. Lateral globus pallidus GABA levels were higher in the lesioned group while no change was observed in striatal GABA and glutamate levels. The 6-hydroxydopamine-induced lesion did not alter the ability of intrastriatal NT (10 nm) to counteract the decrease in pallidal GABA and glutamate levels induced by the dopamine D(2) -like receptor agonist quinpirole (10 μm). A more pronounced increase in the intrastriatal NMDA- (10 μm) induced increase in pallidal GABA levels was observed in the lesioned group while it attenuated the increase in striatal glutamate levels and amplified the increase in pallidal glutamate levels compared with that observed in the controls. NT enhanced the NMDA-induced increase in pallidal GABA and glutamate and striatal glutamate levels; these effects were counteracted by the NTS(1) antagonist SR48692 (100 nm) in both groups. These findings demonstrate an inhibitory striatal dopamine D(2) and an excitatory striatal NMDA receptor regulation of striatopallidal GABA transmission in both groups. These actions are modulated by NT via antagonistic NTS(1) /D(2) and facilitatory NTS(1) /NMDA receptor-receptor interactions, leading to enhanced glutamate drive of the striatopallidal GABA neurons associated with motor inhibition, effects which all are counteracted by SR48692. Thus, NTS(1) antagonists in combination with conventional treatments may provide a novel therapeutic strategy in Parkinson's disease.     

Zolpidem ameliorates motor impairments in the unilaterally 6‐hydroxydopamine‐lesioned rat

Nuclei within the basal ganglia—such as the globus pallidus external segment, subthalamic nucleus, and substantia nigra pars reticulata—have been shown to exhibit synchronous bursting activity entrained to excessive cortical beta oscillations following dopamine depletion. Zolpidem binds to GABA_A receptors with selectivity for those expressing the α₁ subunit, potentiating inhibitory postsynaptic currents and increasing the time decay of channel opening. Interestingly, zolpidem‐sensitive nuclei within the basal ganglia circuitry are also those that have been shown to exhibit hyperexcitation in a dopamine‐depleted state. We hypothesized that a drug with selectivity for these nuclei may improve motor impairments associated with Parkinson's disease. In order to determine the threshold dose at which zolpidem might encumber motor behavior, a dose‐response experiment was performed in intact rats using rotarod. Next, we tested whether subthreshold doses (0.1, 0.25, 0.5 mg/kg; i.p.) of zolpidem improved volitional motor behavior/coordination using the rotarod balance beam and cylinder/paw preference tests in unilaterally 6‐hydroxydopamine‐lesioned rats. It was found that 0.1 mg/kg zolpidem significantly improved rotarod performance and significantly reduced forelimb use asymmetry compared to undrugged post‐lesion conditions. Here, we present the first translational evidence for a role of zolpidem‐sensitive GABA_A receptors in the treatment of PD motor symptoms. Our data show that zolpidem improves both motor coordination and volitional forelimb use in the unilateral 6‐hydroxydopamine lesion model of PD, and thus suggest that zolpidem‐sensitive GABA_A receptors may represent a novel therapeutic target for the treatment of motor symptoms of Parkinson's disease.     

Differential effect of MK 801 and scopolamine on c-fos expression induced by L-dopa in the striatum of 6-hydroxydopamine lesioned rats

In rats with a unilateral 6-hydroxydopamine lesion of the dopaminergic nigro-striatal pathway, striatal D_l-receptor-stimulated c-fos expression and turning behavior are positively modulated by D₂ receptor stimulation and by blockade of N-methyl-D-aspartate (NMDA) or muscarinic receptors. Combined D₁/D₂ receptor stimulation by L-dopa activates c-fos in a manner not additive with muscarinic receptor blockade by scopolamine. On the other hand, blockade of NMDA receptors by MK 801 reduced c-fos expression induced by L-dopa while, depending on the dose of L-dopa, differentially affecting contralateral turning behavior. The results are interpreted to suggest that D₂ receptor stimulation amplifies D₁-receptor-mediated c-fos expression by two mechanisms differentially related to muscarinic and NMDA receptors. ©1994 Wilev-Lisa. Inc.     

The novel type B MAO inhibitor PF9601N enhances the duration of L-DOPA-induced contralateral turning in 6-hydroxydopamine lesioned rats

Summary. The present study examined the effect of the highly potent and selective MAO B inhibitor PF9601N on L-DOPA-induced rotational behavior in unilateral nigrostriatal 6-hydroxydopamine lesioned rats. Three doses of PF9601N (20, 40 and 60 mg/kg) were administered 30 min before an injection of L-DOPA (25 mg/kg), and both contralateral and ipsilateral rotational behavior was measured. In addition, we also studied the effect produced by another MAO B inhibitor, deprenyl (20 mg/kg), the MAO A inhibitor, clorgyline (20 mg/kg), and the dopamine reuptake inhibitor, GBR2909 (7.5 mg/kg) on L-DOPA-induced rotational behavior. The results showed that PF9601N plus L-DOPA significantly enhanced the duration of contralateral rotational behavior with respect to L-DOPA plus vehicle in a dose-related manner. At the dose of 40 and 60 mg/kg, PF9601N produced significantly more overall contralateral turning than L-DOPA plus vehicle, and at the dose of 60 mg/kg, PF9601N produced significantly more turning behavior than L-DOPA plus deprenyl. These results suggest that PF9601N may be used as a novel tool in the treatment of Parkinson's disease. Received March 18, 1999; accepted September 23, 1999     

Fate of multipotent neural precursor cells transplanted into mouse retina selectively depleted of retinal ganglion cells

In some parts of the CNS, depletion of a particular class of neuron might induce changes in the microenvironment that influence the differentiation of newly grafted neural precursor cells. This hypothesis was tested in the retina by inducing apoptotic retinal ganglion cell (RGC) death in neonatal and adult female mice and examining whether intravitreally grafted male neural precursor cells (C17.2), a neural stem cell (NSC)-like clonal line, become incorporated into these selectively depleted retinae. In neonates, rapid RGC death was induced by removal of the contralateral superior colliculus (SC), in adults, delayed RGC death was induced by unilateral optic nerve (ON) transection. Cells were injected intravitreally 6-48 h after SC ablation (neonates) or 0-7 days after ON injury (adults). Cells were also injected into non-RGC depleted neonatal and adult retinae. At 4 or 8 weeks, transplanted cells were identified using a Y-chromosome marker and in situ hybridisation or by their expression of the lacZ reporter gene product Escherichia coli beta-galactosidase (beta-gal). No C17.2 cells were identified in axotomised adult-injected eyes undergoing delayed RGC apoptosis (n = 16). Donor cells were however stably integrated within the retina in 29% (15/55) of mice that received C17.2 cell injections 24 h after neonatal SC ablation; 6-31% of surviving cells were found in the RGC layer (GCL). These NSC-like cells were also present in intact retinae, but on average, there were fewer cells in GCL. In SC-ablated mice, most grafted cells did not express retinal-specific markers, although occasional donor cells in the GCL were immunopositive for beta-III tubulin, a protein highly expressed by, but not specific to, developing RGCs. Targeted rapid RGC depletion thus increased cell incorporation into the GCL, but grafted C17.2 cells did not appear to differentiate into an RGC phenotype.