Dynamic patterns of BDNF expression in injured sensory neurons: differential modulation by NGF and NT-3

It has been suggested that altered retrograde neurotrophin support contributes to the phenotypic switch observed in BDNF expression in injured sensory neurons. Thus, modulatory influences of NGF and NT-3 on BDNF expression in injured adult rat DRG neurons were examined using in situ hybridization and immunohistochemical approaches. Quantitative analysis reveals a biphasic response to sciatic nerve injury, whereby in the first day following injury, BDNF expression is up-regulated in approximately 83% of injured neurons including all small neurons, and a larger pool of trkB expressing neurons than in intact. By 1 week and up to 3 weeks later expression is still seen in approximately 66% of injured neurons, but the characteristic phenotypic switch in the subpopulations expressing BDNF occurs, whereby expression in the trkA population is reduced and expression in trkB- and in trkC-positive neurons is elevated. NGF infusion results in elevated levels of BDNF expression in both intact and injured trkA-positive neurons, accompanied by reduced trkB expression. NT-3 acts in an opposite fashion effecting a down-regulation in BDNF expression in intact neurons and preventing/reducing the injury-associated increases in BDNF expression in both trkC- and nontrkC-expressing subpopulations of injured neurons. These effects suggest NGF can regulate BDNF expression in trkA-expressing neurons regardless of the axonal state and that elevated levels of BDNF may contribute to the down-regulation in trkB expression associated with these states. Furthermore, the findings demonstrate that NT-3 can act in an antagonistic fashion to NGF in the regulation of BDNF expression in intact neurons, and mitigate BDNF's expression in injured neurons.     

切断周围支对背根节神经元BDNF，NT—3及其mRNA表达的影响

为探讨周围支切断对DRG神经元BDNF ,NT 3及其mRNA表达的影响 ,将 5只成年猫麻醉后暴露双侧L7DRG ,于一侧距DRG 1cm处切断外周支 ,另一侧为对照。动物存活 3d后取两侧L7DRG制作 2 0 μm厚冷冻切片。分别用BDNF、NT 3抗体及BDNFcRNA ,NT 3cRNA探针 (地高辛末端标记 )行ABC免疫组化和原位杂交染色。观察BDNF ,NT 3及其mRNA的阳性产物部位 ,计数恒定面积内BDNF ,NT 3及其mRNA的阳性神经元数量。结果BDNF及mRNA的阳性细胞主要是DRG胞体偏小的神经元 (135 7μm)。而NT 3及mRNA的阳性细胞则主要是胞体偏大的神经元 (5 710 0 μm)。恒定面积内BDNF及其mRNA和NT 3及其mRNA的阳性神经元数量实验侧分别是 16.4± 0 .7,11.1± 1.4和 2 4 .5± 2 .1,2 2 .8± 3.2 ,而对照侧则分别是 10 .3± 1.9,6.1± 0 .9和 12 .5± 2 .7,15 .3± 4 .1。比较两侧BDNF、NT 3及其mRNA的阳性神经元数量均有显著差异 (P <0 .0 5 )。表明周围支切断可致DRGBDNF ,NT 3的表达明显增多。提示BDNF ,NT 3可能在外周神经切断的早期反应中发挥作用     

Corticosterone regulates expression of BDNF and trkB but not NT-3 and trkC mRNA in the rat hippocampus

Corticosterone has profound effects on growth, differentiation, and synaptic transmission of hippocampal neurons by activation of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs). In the present study we tested if neurotrophins can be implicated in these effects. For this purpose we injected 30, 300, and 1,000 μg corticosterone s.c. (per kg body weight) in adrenalectomized rats and measured the mRNA levels of brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase (trk)B, neurotrophin (NT)-3, and trkC in hippocampal cell fields at 6 hr after steroid administration by in situ hybridization. NT-3 and trkC mRNA did not show significant changes in any hippocampal region after the various doses of conticosterone. BDNF mRNA decreased after corticosterone administration dose dependently, resulting in a maximal suppression of 35, 20, and 50% in dentate gyrus, CA3, CA1, respectively. Interestingly, trkB responded to corticosterone in an inverted U-shaped fashion in CA3 and dentate gyrus: the low dose of corticosterone increased trkB mRNA expression in both regions by approximately 30%, while the effect of the two higher doses was not different from the vehicle injected controls. In conclusion, we found differential effects of low and high doses of corticosterone on BDNF and trkB expression in hippocampus, which suggests involvement of a coordinated MR- and GR-mediated action. J. Neurosci. Res. 48:334–341, 1997. © 1997 Wiley-Liss, Inc.     

BDNF和NT-3在糖尿病周围神经病大鼠肌肉中的表达

目的观察链脲佐菌素(Streptozotocin STZ)诱导的糖尿病周围神经病大鼠腓肠肌脑源性神经生长因子(BDNF)、神经营养素-3(NT-3)的mRNA表达。方法用STZ复制Wistar大鼠糖尿病周围神经病模型。成模8周、12周麻醉后处死大鼠,提取腓肠肌组织RNA,应用RT-PCR半定量方法检测其BDNF及NT-3mRNA含量。结果糖尿病周围神经病组大鼠腓肠肌BDNF的mRNA含量明显高于正常对照组(P<0.01),12周糖尿病周围神经病大鼠组明显高于8周糖尿病周围神经病大鼠组(P<0.01);腓肠肌NT-3的mRNA含量各组间无显著性差异(P>0.05)。结论糖尿病周围神经病大鼠腓肠肌组织BDNF的mRNA含量随着病程逐渐增加,而NT-3的mRNA含量无明显变化,二者在糖尿病周围神经病中发挥不同作用。     

Differential mRNA expression of neurotrophic factors GDNF, BDNF, and NT-3 in experimental herpes simplex virus encephalitis

Glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) mRNA levels were studied in the course of murine herpes simplex virus encephalitis. Induction of GNDF and NT-3 (both P < 0.05) was found during acute encephalitis. Despite absence of clinical impairment, both neurotrophic factors were overexpressed 2 months (NT-3) and 6 months (GDNF) following infection (both P < 0.05). Neurotrophic factors play an important role in neuronal survival and recovery after acute injury to the central nervous system (CNS) and may represent an additional therapeutic target for treatment of viral encephalitis.     

Distribution of BDNF, NT-3 and NT-4 in the developing auditory brainstem

This study describes the developmental expression of three neurotrophins, brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin (NT-4) in the rat auditory brain-stem using immunohistochemistry. At postnatal day 0 (PND 0), neurotrophins expression was virtually absent from all auditory nuclei in the brainstem, even though some positive neurons were observed in the mesencephalic trigeminal nucleus at this age. However, BDNF, NT-3 and NT-4 positive neurons were observed in most brainstem auditory nuclei by PND 6. At the following stages, there was a general increase in the intensity of the neurotrophins immunoreactivity and BDNF labeling was particularly prominent in most cochlear nucleus neurons. A differential pattern of staining emerged in cochlear nucleus subdivisions, with more intense staining present in the ventral part. The superior olivary complex nuclei followed a similar pattern of BDNF staining compared to the cochlear nucleus. In the adult, BDNF heavily labeled most neurons of the superior olivary nuclei and moderately labeled neurons of the inferior colliculus (IC). NT-3 and NT-4 showed a similar pattern of staining in most auditory brainstem nuclei. The first staining was observed by PND 6 in some neuronal cell bodies. NT-3 and NT-4 immunoreactivity increased in the following stages and in the adult moderate labelings were observed in most neurons of the cochlear nucleus, the superior olivary nuclei and the IC. These results show that neurotrophins are expressed 1 week before the onset of hearing and the increase of their expressions correlate with the appearance of sound-evoked activity in the system. The temporal distribution of neurotrophins does not correlate with neuronal birth, axonal outgrowth or the formation of connection in the auditory structures, suggesting a role primarily in the maintenance and/ or modulation of postnatal and adult functions.     

NT-4/5 and LIF,but not NT-3 and BDNF,promote NPY mRNA expression in cortical neurons in the absence of spontaneous bioelectrical activity

Epigenetic factors are known to influence the differentiation of neocortical neurons. The present study analyses the role of spontaneous bioelectrical activity (SBA) and neurotrophic factors on the expression of neuropeptide Y (NPY) in rat visual cortical neurons using organotypic monocultures prepared from newborn animals and in situ hybridization to detect the NPY messenger ribonucleic acid (mRNA). Spontaneously active cortex cultures display NPY mRNA expression in about 7% of all cortical neurons from 10 days in vitro (DIV) on. Blocking the SBA by chronic application of 10 mm Mg²⁺ for 3–30 DIV reduces the percentage of NPY neurons to about 2%. Allowing an initial phase of SBA (1–20 DIV) followed by an SBA blockade (for 21–50 DIV) results in 2% labelled neurons, indicating a dramatic reduction of NPY mRNA expression in the absence of SBA. Surprisingly, the reverse experiment (a period of SBA blockade for 1–20 DIV followed by a period of SBA recovery for 21–40 DIV) does not cause an upregulation of NPY mRNA expression. However, allowing cultures to differentiate as spontaneously active cultures, then applying a transient period of SBA blockade which is followed by a second period of SBA, does rescue the NPY mRNA expression in 7% of the cortical neurons. We conclude that SBA is a main trigger for NPY mRNA expression and it is particularly important during an early postnatal period of differentiation. We then analysed whether neurotrophic factors known to modulate cortical neuropeptide expression are able to do so in the absence of SBA. Supplementing chronically blocked cultures with the neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and the cytokine, leukaemia inhibitory factor (LIF), reveals that BDNF and NT-3 are unable to increase the percentage of NPY neurons. In contrast, LIF and NT-4/5 increase the percentage of NPY neurons to 4 and 6–7%, respectively. Moreover, neurons treated with NT-4/5 display a very high level of NPY mRNA expression in somata and in the dendritic trees. The data suggest a complex interplay and a hierarchy of epigenetic factors in regulating the neurochemical architecture of the developing neocortex.     

Old-onset Parkinson's disease compared with young-onset disease: clinical differences and similarities

Of 261 patients with clinically diagnosed Parkinson's disease (PD), whose age at the onset was 58.2 ± 11.3, 46 patients with the onset age above 70 (the mean for the whole group + ISD) were compared to 44 patients with onset age below 47 (the mean for the whole group – ISD). Old-onset PD patient were more susceptible to develop psychotic complications of levodopa treatment. More often had they tremor both as presenting and dominant symptom of their disease. Among young-onset PD bradykinesia was more often the dominant clinical feature, and susceptibility to levodopa induced dyskinesia was higher. In 9 cases of young-onset PD (20.5% of this group) paraesthesia was a presenting symptom, compared to only 1 patient (2%) in the group of old-onset PD.     

Hippocampal CA3 and CA2 have distinct bilateral innervation patterns to CA1 in rodents

Ipsilateral and contralateral hippocampal CA3-CA1 and CA2-CA1 projections were investigated in adult male Long-Evans rats by retrograde tracing. Injection of the retrograde tracer cholera toxin subunit B in the strata oriens and radiatum of dorsal CA1 resulted in labeling of predominantly pyramidal cells in ipsilateral and contralateral CA3 and CA2. The contralateral and ipsilateral anterior-posterior extents of CA3 innervation to CA1 were similar. Fifteen to twenty per cent of the hippocampus proper cells that give rise to CA1 stratum oriens innervation were CA2 pyramidal cells, whereas CA2 cells were a mere 3% for CA1 stratum radiatum innervation. The preferred projection of CA2 pyramidal cells to the CA1 stratum oriens was also manifested in transgenic mice that express GFP under the control of the CACNG5 promoter, in which CA2 cells express high amounts of GFP. The ratios of ipsilateral to contralateral projections were compared. For the CA3-CA1 connection, we found that dorsal CA1 stratum radiatum received more ipsilateral projections whereas CA1 stratum oriens received more contralateral innervation. Interestingly, ipsilateral connections dominated for both CA2-CA1 stratum oriens and CA2-CA1 stratum radiatum. These results demonstrate that the primary intrahippocampal target of CA2 pyramidal cells is the ipsilateral CA1 stratum oriens, in contrast to CA3 cells which project more diversely to bilateral CA1 regions. Such innervation patterns may suggest differential dendritic information processing in apical and basal dendrites of CA1 pyramidal cells.     

Alterations of BDNF and NT-3 genes expression in the nucleus paragigantocellularis during morphine dependency and withdrawal

Locus coeruleus (LC) plays a key role in opioid dependence and withdrawal. Chronic morphine administration induces neurochemical adaptations in the noradrenergic system. The nature of signal responsible for opiate-induced adaptations of noradrenergic neurons in LC is not well defined. Neurotrophins-signaling pathways such as brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3) play a key role for regulating the noradrenergic response of LC neurons to opiates. The nucleus paragigantocellularis (PGi) is one of the two major afferents to LC. The present study was designed to evaluate the expression of BDNF and NT-3 in the context of opiate dependence and withdrawal in PGi. Such data are important because they could reveal the role of PGi as an additional source of BDNF and NT-3 in the neurochemical plasticity of LC neurons. Opiate dependence was induced by a progressive intraperitoneal treatment of morphine. In morphine dependent group PGi nucleus was extracted for gene expression assay 6h after the last injection of morphine. In spontaneous withdrawal, rats received the same chronic treatment as morphine group. PGi was extracted for gene expression assay 24, 48 and 72 h after the last injection of morphine. PGi nucleus was assayed for the expression of BDNF and NT-3 using semi-quantitative RT-PCR normalized to beta-actin gene expression. Results showed that chronic administration of morphine significantly increased BDNF and NT-3 gene expression in PGi. In spontaneous withdrawal, BDNF/NT-3 genes expression were high in comparison to control group. It seems that BDNF/NT-3 -signaling pathway originating from PGi is essential for opiate-induced adaptations of the LC neurons.     

Effects of antiepileptic drugs on mRNA levels of BDNF and NT-3 and cell neogenesis in the developing rat brain

Epilepsy is a common neurological disorder that occurs more frequently in childhood than in adulthood. Antiepileptic drugs (AEDs) which are used to treat seizures in pregnant women, infants, and young children may cause cognitive impairment or other uncertain injury. However, the exact mechanisms responsible for adverse effects of AEDs in the developing brain are still not clear. In the present study, we investigate the effects of AEDs on mRNA levels of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), cell neogenesis and mossy fiber sprouting (MFS) in the developing rat brain. Long-term treatment with Phenobarbital (40 mg/kg), valproate (100 mg/kg) and topiramate (40 mg/kg) reduces BDNF and NT-3 mRNA expression in the developing brain, while lamotrigine reduces mRNA expression only at high dose level (80 mg/kg). Cell neogenesis only increases in the rats treated with valproate and lamotrigine. And no differences are observed between the control group and the AEDs-treated groups in the Timm scores of the CA3 region and supragranular region. Our findings present some possible mechanisms to explain why different AEDs cause different cognitive impairment.     

Developmental regulation of BDNF and NT-3 expression by quinolinic acid in the striatum and its main connections

Interactions between neurotrophic factors and neurotransmitters participate in the formation and maintenance of appropriate connections, as well as in neurodegenerative processes. Here we have measured changes in the developmental expression pattern of BDNF and NT-3 in the striatum, cortex, and substantia nigra induced by intrastriatal injection of the N-methyl-d-aspartate glutamate receptor agonist quinolinic acid (QUIN). Animals were injected at different postnatal ages, and BDNF and NT-3 mRNA levels were determined 6 h after lesion using a ribonuclease protection assay. Our results show a biphasic increase in BDNF mRNA levels in striatum and in the ipsilateral cortex at postnatal day (P)5 and P21. In contrast, although NT-3 expression did not change in the striatum, it was down-regulated in the ipsilateral cortex at P5 and P30. Intrastriatal QUIN injection did not induce changes in either BDNF or NT-3 expression in the ipsilateral substantia nigra. These findings show that neurotrophin expression is developmentally regulated after excitotoxic injury, which suggests that this endogenous response may be involved in different neuronal maturation and vulnerability during development.     

Dopaminergic innervation of the cerebral cortex: unexpected differences between rodents and primates.

Until recently, views on the organization and role of the mesotelencephalic dopaminergic (DA) systems were mostly based on studies of rodents, and it was assumed that homology existed across mammalian species. However, recent studies of both human and non-human primates indicate that this might not be so. The mesocortical DA system in primates, which is directly involved in the pathophysiology of severe illnesses such as Parkinson's disease and psychoses, shows substantial differences from that of rodents. These differences include much larger, re-organized terminal fields, a different phenotype for the co-localization of neuropeptides and a very early prenatal development.     

Changes in spinal GDNF, BDNF, and NT-3 expression after transient spinal cord ischemia in the rat

Previous studies have demonstrated that the expression of several growth factors including glial cell-derived neurotrophic factor (GDNF), brain-derived growth factor (BDNF), and neurotrophin-3 (NT-3) play an important role in defining neuronal survival after brain ischemia. In the present study, using a well-defined model of transient spinal ischemia in rat, we characterized the changes in spinal GDNF, BDNF, and NT-3 expression as defined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence coupled with deconvolution microscopy. In control animals, baseline levels of GDNF, BDNF, and NT-3 (74 +/- 22, 3,600 +/- 270, 593 +/- 176 pg/g tissue, respectively) were measured. In the ischemic group, 6 min of spinal ischemia resulted in a biphasic response with increases in tissue GDNF and BDNF concentrations at the 2-hr and 72-hr points after ischemia. No significant differences in NT-3 concentration were detected. Deconvolution analysis revealed that the initial increase in tissue GDNF concentration corresponded to a neuronal upregulation whereas the late peak seen at 72 hr corresponded with increased astrocyte-derived GDNF synthesis. Increased expression of BDNF was seen in neurons, astrocytes, and oligodendrocytes. These data suggest that the early increase in neuronal GDNF/BDNF expression likely modulates neuronal resistance/recovery during the initial period of postischemic reflow. Increased astrocyte-derived BDNF/GDNF expression corresponds with transient activation of astrocytes and may play an active role in neuronal plasticity after non-injurious intervals of spinal ischemia.     

Granule cell mRNA levels for BDNF, NGF, and NT-3 correlate with neuron losses or supragranular mossy fiber sprouting in the chronically damaged and epileptic human hippocampus

This study determined in temporal lobe epilepsy patients if there were correlations among hippocampal granule cell expression of neurotrophin mRNAs, aberrant supragranular mossy fiber sprouting, and neuron losses. Consecutive surgically resected hippocampi (n=9) and comparison tissue from autopsies (n=3) were studied for:

1. Granule cell mRNA levels usingin situ hybridization for brainderived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3);
2. neo-Timm supragranular mossy fiber sprouting; and
3. Ammon’s horn neuron densities.

Clinically, patients were classified into those with hippocampal sclerosis (HS;n=7) and non-HS cases (i.e., mass lesions and autopsies;n=5). Results showed that compared to non-HS cases, HS patients showed increased granule cell mRNA levels for BDNF, NGF, and NT-3 (p=0.035,p=0.04,p=0.045 respectively; one-tail directional test). Moreover, granule cell BDNF mRNA levels correlated inversely with Ammon’s horn neuron densities (p=0.02) and correlated positively with greater supragranular mossy fiber sprouting (p=0.02). NGF mRNA levels correlated inversely with Ammon’s horn neuron densities (p=0.02), and TN-3 mRNA levels correlated inversely with age at surgery (p=0.04) and correlated positively with greater mossy fiber sprouting (p=0.026). These results indicate in the chronically damaged human hippocampus that granule cells express neurotrophin mRNAs, and mRNA levels correlate with either hippocampal neuron losses or aberrant supragranular mossy fiber sprouting. These data support the hypothesis that in the epileptic human hippocampus, there may be pathophysiologic associations among mossy fiber synaptic plasticity, hippocampal neuron damage, and granule cell mRNA neurotrophin levels.     

Developmentally Regulated Expression of HDNF/NT-3 mRNA in Rat Spinal Cord Motoneurons and Expression of BDNF mRNA in Embryonic Dorsal Root Ganglion

Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13 - 14 and 15 - 16 spinal cord. The level decreased at E18 - 19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18 - 19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia.     

Differential expression of GDNF, BDNF, and NT-3 in the aging nigrostriatal system following a neurotoxic lesion

Protein levels for brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial cell line-derived neurotrophic factor (GDNF) were measured in the striatum and ventral midbrain of young and aged Brown Norway/F344 F1 (F344BNF(1)) hybrid rats following a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway. At 2 weeks post-lesion, protein levels of BDNF and GDNF were higher in the denervated striatum when compared to the intact striatum for young (4-5 months old) but not old (31-33 months old) rats. Interestingly, in old rats BDNF protein in the denervated striatum was significantly lower than that measured in the intact striatum. At the same time point BDNF protein levels in the ventral midbrain were higher on the lesioned versus intact side for both young and old rats while no significant side differences were detected for GDNF protein in the ventral midbrain of young or old rats. No significant differences in NT-3 protein levels were detected between the lesioned and intact sides for striatal or ventral midbrain regions in either young or old brain. While no significant age effects were detected for BDNF or NT-3 protein, young rats showed higher GDNF protein levels in both the striatum (lesioned or intact) and ventral midbrain (lesioned or intact) than old rats. These data show that two endogenous neurotrophic factors, BDNF and GDNF, are differentially affected by a 6-OHDA lesion in the aging nigrostriatal system with young brain showing a significant compensatory increase of these two factors in the denervated striatum while no compensatory increase is observed in aged brain.     

Estrogen-receptor-beta distribution in the human hypothalamus: similarities and differences with ER alpha distribution

This study reports the first systematic rostrocaudal distribution of estrogen receptor beta immunoreactivity (ER beta-ir) in the human hypothalamus and adjacent areas in five males and five females between 20-39 years of age and compares its distribution to previously reported ER alpha in the same patients. ER beta-ir was generally observed more frequently in the cytoplasm than in the nucleus and appeared to be stronger in women. Basket-like fiber stainings, suggestive for ER beta-ir in synaptic terminals, were additionally observed in various areas. Men showed more robust nuclear ER beta-ir than women in the medial part of the bed nucleus of the stria terminalis, paraventricular and paratenial nucleus of the thalamus, while less intense, but more nuclear, ER beta-ir appeared to be present in, e.g., the BSTc, sexually dimorphic nucleus of the medial preoptic area, diagonal band of Broca and ventromedial nucleus. Women revealed more nuclear ER beta-ir than men of a low to intermediate level, e.g., in the suprachiasmatic, supraoptic, paraventricular, infundibular, and medial mamillary nucleus. These data indicate potential sex differences in ER beta expression. ER beta-ir expression patterns in subjects with abnormal hormone levels suggests that there may be sex differences in ER beta-ir that are "activational" rather than "organizational" in nature. Similarities, differences, potential functional, and clinical implications of the observed ER alpha and ER beta distributions are discussed in relation to reproduction, autonomic-function, mood, cognition, and neuroprotection in health and disease.