Requirement of appropriate glutamate concentrations in the synaptic cleft for hippocampal LTP induction

Although glutamate transporters maintain low extracellular levels of the excitatory neurotransmitter glutamate in the nervous system, little is known about their roles in synaptic plasticity. Here, using knockout mice lacking GLT-1, that is the most abundant glial subtype of glutamate transporters, we showed that long-term potentiation (LTP) induced by tetanic stimulation in mutant mice was impaired in the hippocampal CA1 region. When tetanic stimulation was applied in the presence of low concentrations of an N-methyl-D-aspartate (NMDA) receptor antagonist, the impairment was overcome. Consistent with these results, the increased glutamate in the synaptic cleft of mutant mice preferentially activated NMDA receptors. Furthermore, analyses of mutant mice revealed that the magnitude of NMDA receptor-dependent transient synaptic potentiation during low-frequency stimulation depended on the concentration of glutamate in the synaptic cleft. These findings suggest that GLT-1 plays critical roles in LTP induction, as well as in short-term potentiation, through regulation of extracellular levels of glutamate, which enables appropriate NMDA receptor activation.     

Repetitive febrile seizures in rat pups cause long-lasting deficits in synaptic plasticity and NR2A tyrosine phosphorylation

Adult rats with early-life frequently repetitive febrile seizures (FRFS), but not single febrile seizure (SFS), exhibited impaired performance in inhibitory avoidance tasks but without significant hippocampal neuronal loss. The mechanisms of long-term memory impairment in the hippocampus of adult rats with early-life FRFS remain unknown. Using a heated-air febrile seizures (FS) paradigm, male rat pups were subjected to single or nine episodes of brief FS at days 10 to 12 postpartum. We found that early-life FRFS led to long-term bidirectional modulation in hippocampal synaptic plasticity, i.e., impaired long-term potentiation and facilitated long-term depression. Three hours after inhibitory avoidance training, phosphorylation of hippocampal extracellular signal-regulated kinase (ERK) 1/2 was significantly less in the FRFS group than in controls. Furthermore, there was a selective alteration in NMDA receptor-mediated ERK1/2 phosphorylation in the hippocampus of the FRFS group. Although the expression levels of NMDA receptor subunits and interaction of NMDA receptor and postsynaptic density 95 did not alter quantitatively, there was a specific alteration in NR2A, but not NR2B, subunit tyrosine phosphorylation after NMDA stimulation in the FRFS group. These data offer a potential molecular explanation for the hippocampus-dependent memory deficits observed in the rats with early-life FRFS.     

Extracellular Alpha-Synuclein Oligomers Modulate Synaptic Transmission and Impair LTP Via NMDA-Receptor Activation

Parkinson's disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of α-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.     

Plasticity, hippocampal place cells, and cognitive maps

Memory of even the briefest event can last a lifetime. Thus, learning and memory require neuronal mechanisms that allow rapid, yet persistent, changes to brain circuits. Hippocampal neuropsychology, synaptic and cellular electrophysiology, pharmacology, and molecular genetics converge and begin to reveal these mechanisms. Lesions of the hippocampus profoundly impair memory for recent events in humans and rodents. Circuits within the hippocampus are remarkably plastic, and this plasticity is mediated in part through changes in synaptic strength and revealed by long-term potentiation (LTP) and long-term depression (LTD). N-methyl D-aspartate (NMDA) receptors, a subtype of glutamate receptor, are crucial for inducing these plastic changes, and blocking these receptors reduces plasticity and impairs learning in tasks that require the hippocampus. Molecular genetic alterations that disrupt signaling mechanisms downstream of the NMDA receptor also prevent LTP induction and impair hippocampus-dependent learning. N-methyl D-aspartate receptor mechanisms have also been linked to information coding by hippocampal neurons. Hippocampal cells fire selectively in specific and restricted locations (place fields) as rodents move through open environments. Place fields form within minutes and persist for months. N-methyl D-aspartate receptor antagonists prevent the establishment of stable place fields. The same molecular genetic manipulations that interfere with hippocampal NMDA receptor function, prevent LTP induction, and impair spatial learning also disrupt the formation of stable hippocampal place fields. Finally, learning has been improved in mice with genetically modified NMDA receptors that enhance LTP induction. Thus, hippocampal cells "learn" to encode the salient features of experience through NMDA receptor-dependent synaptic plasticity mechanisms, and this rapid and persistent neuronal encoding is a crucial step toward the formation of long-term memory. Disruption of these plasticity mechanisms may underlie age-related memory deficits.     

Comparative aspects of hippocampal and neocortical long-term potentiation

Long-term potentiation (LTP) is a candidate for the synaptic alternations underlying memory storage in the mammalian CNS. In this chapter LTP in hippocampus and in visual neocortex are compared. Comparisons of the optimal tetanus parameters revealed that 2-3 trains of high-frequency stimulation (100-400 Hz) delivered within a brief period of time (minutes) results in maximal potentiation in hippocampal synapses. In contrast, the parameters most effective in neocortex were either low-frequency (2 Hz for 60 min) or high-frequency bursts (100 Hz, 100 ms train at 1/5 s for 10 min), both of which deliver at least an order of magnitude more afferent activation than that required for hippocampus. Hippocampal population spike potentiation averages 250% and the population excitatory postsynaptic potential (EPSP) potentiation averages 50%. Neocortical LTP also averages about 50%. The expression of LTP requires about 5 min in CA1 hippocampus, whereas about 30 min are required for expression of neocortical potentiation. Both hippocampus and visual neocortex display an enhanced potentiation early in development, with a later stabilization at lower adult levels. Centering at postnatal day 15, hippocampal CA1 displays an LTP magnitude that is over twice that seen at day 60. Neocortical responses display a similar peak at postnatal day 15 and a subsequent adult stabilization at approximately half of the day 15 maximum. Both tissues first display LTP during the early stages of synapse formation between postnatal days 6-10. The role of the NMDA receptor is implicated in aspects of both hippocampal and neocortical LTP.     

JNK1 contributes to metabotropic glutamate receptor-dependent long-term depression and short-term synaptic plasticity in the mice area hippocampal CA1

Several recent reports implicate an important role played by c-Jun N-terminal kinases (JNKs) in long-term potentiation (LTP). However, little is known about how the isoforms of JNKs participate in synaptic plasticity. Here we showed that short-term synaptic plasticity was impaired in the hippocampal area CA1 of JNK1-deficient (JNK1-/-) mice; these mice showed normal LTP in response to a strong tetanus and no alteration of N-methyl-D-aspartate receptor-dependent long-term depression (LTD) in the hippocampus. However, LTD induced either by group I metabotropic glutamate receptors (mGluRs) agonist dihydroxyphenylglycine or by paired-pulse low-frequency stimulation was absent in both the JNK1-/- slices and in JNK inhibitor anthrax [1, 9-cd] pyrazol-6(2H)-1 (SP600125)-pretreated slices. Induction of mGluR-dependent LTD resulted in an increase in phosphorylation of JNK1 substrates, including p-c-Jun and p-ATF2 in wild-type (WT) mice, and these increases failed to occur in the JNK1-/- or SP600125-pretreated mice. These results demonstrated that JNK1 played a crucial role in the short-term synaptic plasticity and mGluR-dependent LTD, whereas hippocampus LTP was not affected by JNK1 deficiency.     

Electrical stimulation of the human hippocampus produces verbal intrusions during memory testing

Verbal memory testing was conducted during electrical stimulation of the human hippocampus in 12 epilepsy surgery candidates with unilateral temporal lobe seizure onset. Performance was assessed during baseline, left hippocampal stimulation and right hippocampal stimulation. Verbal intrusion errors were greater during electrical stimulation of the hippocampus contralateral to the seizure focus. These findings suggest that verbal intrusions are related to memory deficits, and that patients with cerebral disease who intrude words from an earlier portion of a learning test are likely to have bilateral cerebral dysfunction.     

Activation of NMDA receptors in hippocampal area CA1 by low and high frequency orthodromic stimulation and their contribution to induction of long-term potentiation

N-methyl-D-aspartate (NMDA) receptors are important in many instances of synaptic plasticity. In hippocampal area CA1, long-term potentiation (LTP) can be induced by both NMDA receptor-dependent and -independent mechanisms. Using intracellular recordings and single-electrode voltage clamp, we isolated and characterized NMDA receptor-mediated synaptic responses. NMDA receptor-mediated responses evoked by low frequency orthodromic stimulation were inhibited in a dose-dependent manner by the competitive antagonist D,L-2-amino-5-phosphonovaleric acid (APV). High frequency (tetanic) stimulation, which facilitates synaptic release of glutamate, failed to overcome the blockade of NMDA receptors by APV. Using extracellular recordings of field potentials, we studied the contribution of NMDA receptors to LTP induced by different patterns of tetanic stimulation. LTP was inhibited in a dose-dependent manner by APV, but was more sensitive to APV than were NMDA receptor-mediated synaptic responses. This most likely reflects a threshold for NMDA receptor activation in LTP induction. A component of LTP that resisted blockade by APV was induced by high (200 Hz), but not low (25 Hz), frequency tetanization. This NMDA receptor-independent component of LTP persisted for > 4 hours and accounted for approximately half the potentiation induced by 200 Hz tetanization. Procedures necessary to induce LTP at the Schaffer collateral/ commissural synapses in area CA1 by both NMDA receptor-dependent and -independent mechanisms are now well characterized. Using the same neuronal population, it will be possible to ask if processes involved in the maintenance of LTP are shared even when LTP is induced through two different mechanisms. © 1994 Wiley-Liss, Inc.     

Distinct mechanisms of bidirectional activity-dependent synaptic plasticity in superficial and deep layers of rat entorhinal cortex

The entorhinal cortex plays a key role in processing memory information in the brain; superficial layers relay information to, and deep layers receive information from, the hippocampus. The cellular mechanisms of memory are thought to include a number that produce long-term potentiation (LTP) and depression (LTD) of synaptic strength. Our work presents evidence that LTP and LTD occur simultaneously at memory-relevant synapses. We report here that low frequency stimulation generates NMDA receptor-dependent LTD in Wistar rat superficial (layers II and III), and LTP in the deep entorhinal cortex layers (layers V and VI). LTP in deep layers is masked by simultaneously occurring voltage-gated calcium channel-dependent LTD. Our data support a novel mechanism for the sliding-threshold (BCM) model of synaptic plasticity: The sliding thresholds for induction of LTP and LTD in entorhinal cortex deep layers will be driven by the relative activation state of NMDA receptors and voltage-gated calcium channels. The co-expression of LTD and LTP at presynaptic sites in the entorhinal cortex deep layers reveals an intriguing mechanism for differential processing of synaptic information, which may underlie the vast dynamic capacity for information storage by this cortical structure.     

Developmental switch from LTD to LTP in low frequency-induced plasticity

The stimulation of the Schaffer collateral/commissural fibers at low frequency (1 Hz) for 3-5 min can trigger a slow-onset form of low-frequency stimulation (LFS)-long-term potentiation (LTP) (LFS-LTP) in the CA1 area of the adult rat hippocampus. Here we have examined the developmental profile of this plasticity. In 9-15 day-old rats, the application of 1 Hz for 5 min induced long-term depression (LFS-LTD). In 17-21 day-old rats, 1 Hz stimulation had no effect when applied for 5 min but mediated LTD when stimulus duration was increased to 15 min. Over 25 day-old, 1 Hz stimulation mediated LFS-LTP. LFS-LTD was dependent on both N-methyl-D-aspartate (NMDA) and mGlu5 receptor activation. Antagonists of mGlu1alpha and cannabinoid type 1 receptor were ineffective to block LTD induction. LFS-LTD was not associated with a change in paired-pulse facilitation ratio, suggesting a postsynaptic locus of expression of this plasticity. Next, we examined whether LFS-LTD was related to 'chemical' LTDs obtained by the direct stimulation of mGlu5 and NMDA receptors. The saturation of LFS-LTD completely occluded NMDA- and (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG)-induced LTD. CHPG-LTD and NMDA-LTD occluded each other. In addition, we observed that NMDA-LTD was dependent on mGlu5 receptor activation in 9-12 day old rats while it was not in animals older than 15 day-old. Therefore we postulate that during LFS application, NMDA and mGlu5 receptor could interact to trigger LTD. Low-frequency-mediated synaptic plasticity is subject to a developmental switch from NMDA- and mGlu5 receptor-dependent LTD to mGlu5 receptor-dependent LTP with a transient period (17-21 day-old) during which LFS is ineffective.     

Selective Impairment of Some Forms of Synaptic Plasticity by Oligomeric Amyloid-β Peptide in the Mouse Hippocampus: Implication of Extrasynaptic NMDA Receptors

Alzheimer's disease is characterized by the loss of memory and synaptic damage. Evidence is accumulating for a causal role of soluble oligomeric species of amyloid-β peptide (Aβo) in the impairment of synaptic plasticity and cognition but the precise mechanisms underlying these effects are still not clear. Synaptic plasticity such as long-term potentiation is thought to underlie learning and memory. While the effect of Aβ on long-term potentiation is well documented, a more general understanding of Aβ action on various aspects of plasticity involving synaptic and extrasynaptic receptors and the nature of the mechanisms involved in its effects are lacking. Using a combination of electrophysiological and biochemical techniques in mouse hippocampal slices, we show here that Aβo drastically affects synaptic plasticities induced by high stimulation frequencies through the involvement of extrasynaptic glutamate receptors. Experiments on hippocampal slices as well as on cultured cortical neurons show that Aβo potentiates extrasynaptic NMDA receptors-mediated responses. Pharmacological characterization indicates that GluN2B-containing NMDARs are involved in these responses. When synaptic and extrasynaptic glutamate receptor-mediated effects are dissociated using cortical neurons in culture, it appears that Aβo has differential effects on these two receptors types. We conclude that the pool of extrasynaptic GluN2B-containing NMDARs is a major target of Aβo in the hippocampus. During high frequency stimulation, Aβo dramatically impairs long-term neuronal responses.     

Differential expression of NMDA receptor subunits and splice variants among the CA1, CA3 and dentate gyrus of the adult rat

N-Methyl-d-aspartate (NMDA)-type glutamate receptors in the hippocampus are important mediators of both memory formation and excitotoxicity. It is thought that glutamatergic neurons of the CA1, CA3 and dentate gyrus regions of the hippocampus contribute differentially to memory formation and are differentially sensitive to excitotoxicity. The subunit and/or splice variant composition of the NMDA receptor controls many aspects of receptor function such as ligand affinity, calcium permeability and channel kinetics, as well as interactions with intracellular anchoring and regulatory proteins. Thus, one possible explanation of the differences in NMDA receptor-dependent processes, such as synaptic plasticity and excitotoxicity, among the hippocampal sub-regions is that they differ in subunit and/or splice variant expression. Here we report that the NMDA receptor subunits NR1 and NR2B, along with the four splice variant cassettes of the NR1 subunit are differentially expressed in the CA1, CA3 and dentate gyrus of the hippocampus. Expression of the AMPA receptor subunits GluR1 and GluR2 also differ. These differences may contribute to functional differences, such as with excitotoxicity and synaptic plasticity, that exist between the sub-regions of the hippocampus.     

Bidirectional synaptic plasticity induced by conditioned stimulations with different number of pulse at hippocampal CA1 synapses: roles of N-methyl-D-aspartate and metabotropic glutamate receptors

In the mammalian brain, the hippocampus has been established as a principle structure for learning and memory processes, which involve synaptic plasticity. Although a relationship between synaptic plasticity and stimulation frequency has been reported in numerous studies, little is known about the importance of pulse number on synaptic plasticity. Here we investigated whether the pulse number can modulate bidirectional plasticity in hippocampal CA1 areas. When a CA1 area was induced by a paired-pulse (PP) with a 10-ms interval, the strength of the synapse was altered to form a long-term depression (LTD), with a 68 ± 4% decrease in expression. The PP-induced LTD (PP-LTD) was blocked by the metabotropic glutamate receptors subtype 5 (mGluR5) antagonist MPEP, suggesting that the PP-LTD relied on the activation of GluR5. In addition, this modulation of LTD was protein kinase C (PKC)- and Group II mGluR-independent. However, when increasing the pulse number to 4 and 6, potentiated synaptic strength was observed, which was N-methyl-D-aspartate receptor (NMDAR)-dependent but mGluR5-independent. Surprisingly, when blocking mGluR, the synaptic efficacy induced by triple-pulse stimulation was altered to form a long-term potentiation (LTP) with a 142 ± 7% enhancement, and was further blocked by NMDA antagonist APV. Following treatment with APV and PKC blocker chelerythrine, the LTP expression induced by 4- and 6-pulse stimulation was switched to LTD. We suggest that CA1 synaptic plasticity is regulated by the result of competition between NMDA and mGluR5 receptors. We suggest that the pulse number can bidirectionally modulate synaptic plasticity through the activation of NMDA and mGluR5 in hippocampal CA1 areas.     

Altered hippocampal synaptic transmission in transgenic mice with astrocyte-targeted enhanced CCL2 expression

Elevated expression of neuroinflammatory factors in the central nervous system (CNS) contributes to the cognitive impairment in CNS disorders such as injury, disease and neurodegenerative disorders. However, information on the role of specific neuroimmune factors in normal and abnormal CNS function is limited. In this study, we investigated the effects of chronic exposure to the chemokine CCL2 on hippocampal synaptic function at the Schaffer collateral-CA1 synapse, a synapse that is known to play an important role in cognitive functions such as memory and learning. Synaptic function was measured in vitro using hippocampal slices obtained from transgenic mice that express elevated levels of CCL2 in the CNS through astrocyte expression and their non-transgenic littermate controls. Extracellular field potential electrophysiological recordings showed a significant reduction in the magnitude of synaptic responses in hippocampal slices from the CCL2 transgenic mice compared with slices from non-transgenic littermate controls. Two forms of short-term synaptic plasticity (post-tetanic potentiation and short-term potentiation) thought to be important cellular mechanisms of short-term memory were enhanced in hippocampal slices from CCL2 transgenic mice compared to non-transgenic hippocampal slices, whereas long-term synaptic plasticity (LTP), which is critical to long-term memory formation, was not altered. Western blot analysis of hippocampus from the CCL2 transgenic mice and non-transgenic mice showed no change in level of neuronal specific enolase, a neuronal specific protein, GFAP, an astrocyte specific protein, and several synaptic proteins compared with non-transgenic littermate controls. These results show that CCL2, which is known to be chronically produced at elevated levels within the CNS in a number of CNS disorders, can significantly alter hippocampal function and implicate a role for CCL2 in the cognitive dysfunction associated with these CNS disorders.     

Plasticity-dependent changes in metabotropic glutamate receptor expression at excitatory hippocampal synapses

Group I metabotropic glutamate receptors, mGluR1 and mGluR5, modulate NMDA receptor-mediated synaptic transmission and plasticity and mediate mGluR-dependent plasticity. Here we report that the synaptic expression of mGluRs can be regulated by NMDA receptor-dependent synaptic plasticity, but that this is dependent on the subtype of mGluR. Silent synapses, but not active synapses, were found to lack Group I mGluRs showing that mGluRs must be inserted into synapses after they are unsilenced. The induction of LTP resulted in an increased synaptic expression of mGluR1 in an NMDA receptor-dependent manner. mGluR1 is internalized from synapses via NMDA receptor-dependent LTD. Interestingly we found no evidence for the regulation of mGluR5 by NMDA receptor-dependent plasticity. This regulation of Group I mGluRs will determine the ability of synapses to undergo mGluR-dependent modulation of synaptic transmission and plasticity, providing a mechanism for metaplasticity and state-dependent plasticity at hippocampal synapses.     

Leptin promotes rapid dynamic changes in hippocampal dendritic morphology

Recent studies have implicated the hormone leptin in synaptic plasticity associated with neuronal development and learning and memory. Indeed, leptin facilitates hippocampal long-term potentiation and leptin-insensitive rodents display impaired hippocampal synaptic plasticity suggesting a role for endogenous leptin. Structural changes are also thought to underlie activity-dependent synaptic plasticity and this may be regulated by specific growth factors. As leptin is reported to have neurotrophic actions, we have examined the effects of leptin on the morphology and filopodial outgrowth in hippocampal neurons. Here, we demonstrate that leptin rapidly enhances the motility and density of dendritic filopodia and subsequently increases the density of hippocampal synapses. This process is dependent on the synaptic activation of NR2A-containing NMDA receptors and is mediated by the MAPK (ERK) signaling pathway. As dendritic morphogenesis is associated with activity-dependent changes in synaptic strength, the rapid structural remodeling of dendrites by leptin has important implications for its role in regulating hippocampal synaptic plasticity and neuronal development.     

Electrical stimulation protocols for hippocampal synaptic plasticity and neuronal hyper-excitability: are they effective or relevant?

Long-term potentiation (LTP) of synaptic transmission is a widely accepted model that attempts to link synaptic plasticity with memory. LTP models are also now used in order to test how a variety of neurological disorders might affect synaptic plasticity. Interestingly, electrical stimulation protocols that induce LTP appear to display different efficiencies and importantly, some may not be as physiologically relevant as others. In spite of advancements in our understanding of these differences, many types of LTP inducing protocols are still widely used. In addition, in some cases electrical stimulation leads to normal biological phenomena, such as putative memory encoding and in other cases electrical stimulation triggers pathological phenomena, such as epileptic seizures. Kindling, a model of epileptogenesis involving repeated electrical stimulation, leads to seizure activity and has also been thought of, and studied as, a form of long-term neural plasticity and memory. Furthermore, some investigators now use electrical stimulation in order to reduce aspects of seizure activity. In this review, we compare in vitro and in vivo electrical stimulation protocols employed in the hippocampal formation that are utilized in models of synaptic plasticity or neuronal hyperexcitability. Here the effectiveness and physiological relevance of these electrical stimulation protocols are examined in situations involving memory encoding (e.g., LTP/LTD) and epileptiform activity.     

Target-cell-specific bidirectional synaptic plasticity at hippocampal output synapses

It is commonly accepted that the hippocampus is critically involved in the explicit memory formation of mammals. The subiculum is the principal target of CA1 pyramidal cells and thus serves as the major relay station for the outgoing hippocampal information. Pyramidal cells in the subiculum can be classified according to their firing properties into burst-spiking and regular-spiking cells. In the present study we demonstrate that burst-spiking and regular-spiking cells show fundamentally different forms of low frequency-induced synaptic plasticity in rats. In burst-spiking cells, low-frequency stimulation (at 0.5–5 Hz) induces frequency-dependent long-term depression (LTD) with a maximum at 1 Hz. This LTD is dependent on the activation of NMDAR and masks an mGluR-dependent long-term potentiation (LTP). In contrast, in regular-spiking cells low-frequency stimulation induces an mGluR-dependent LTP that masks an NMDAR-dependent LTD. Both processes depend on postsynaptic Ca²⁺-signaling as BAPTA prevents the induction of synaptic plasticity in both cell types. Thus, mGluR-dependent LTP and NMDAR-dependent LTD occur simultaneously at CA1-subiculum synapses and the predominant direction of synaptic plasticity relies on the cell type investigated. Our data indicate a novel mechanism for the sliding-threshold model of synaptic plasticity, in which induction of LTP and LTD seems to be driven by the relative activation state of NMDAR and mGluR. Our observation that the direction of synaptic plasticity correlates with the discharge properties of the postsynaptic cell reveals a novel and intriguing mechanism of target specificity that may serve in tuning the significance of neuronal information by trafficking hippocampal output onto either subicular burst-spiking or regular-spiking cells.