Phase precession in the human hippocampus and entorhinal cortex.

Knowing where we are, where we have been, and where we are going is critical to many behaviors, including navigation and memory. One potential neuronal mechanism underlying this ability is phase precession, in which spatially tuned neurons represent sequences of positions by activating at progressively earlier phases of local network theta oscillations. Based on studies in rodents, researchers have hypothesized that phase precession may be a general neural pattern for representing sequential events for learning and memory. By recording human single-neuron activity during spatial navigation, we show that spatially tuned neurons in the human hippocampus and entorhinal cortex exhibit phase precession. Furthermore, beyond the neural representation of locations, we show evidence for phase precession related to specific goal states. Our findings thus extend theta phase precession to humans and suggest that this phenomenon has a broad functional role for the neural representation of both spatial and non-spatial information.

The temporal structure of REM sleep shows minute-scale fluctuations across brain and body in mice and humans.

Rapid eye movement sleep (REM) is believed to have a binary temporal structure with "phasic" and "tonic" microstates, characterized by motoric activity versus quiescence, respectively. However, we observed in mice that the frequency of theta activity (a marker of rodent REM) fluctuates in a nonbinary fashion, with the extremes of that fluctuation correlating with phasic-type and tonic-type facial motricity. Thus, phasic and tonic REM may instead represent ends of a continuum. These cycles of brain physiology and facial movement occurred at 0.01 to 0.06 Hz, or infraslow frequencies, and affected cross-frequency coupling and neuronal activity in the neocortex, suggesting network functional impact. We then analyzed human data and observed that humans also demonstrate nonbinary phasic/tonic microstates, with continuous 0.01 to 0.04-Hz respiratory rate cycles matching the incidence of eye movements. These fundamental properties of REM can yield insights into our understanding of sleep health.

Dynamic switching of neural oscillations in the prefrontal-amygdala circuit for naturalistic freeze-or-flight.

The medial prefrontal cortex (mPFC) and basolateral amygdala (BLA) are involved in the regulation of defensive behavior under threat, but their engagement in flexible behavior shifts remains unclear. Here, we report the oscillatory activities of mPFC-BLA circuit in reaction to a naturalistic threat, created by a predatory robot in mice. Specifically, we found dynamic frequency tuning among two different theta rhythms (~5 or ~10 Hz) was accompanied by agile changes of two different defensive behaviors (freeze-or-flight). By analyzing flight trajectories, we also found that high beta (~30 Hz) is engaged in the top-down process for goal-directed flights and accompanied by a reduction in fast gamma (60 to 120 Hz, peak near 70 Hz). The elevated beta nested the fast gamma activity by its phase more strongly. Our results suggest that the mPFC-BLA circuit has a potential role in oscillatory gear shifting allowing flexible information routing for behavior switches.

The interplay of cognitive control and feature integration: insights from theta oscillatory dynamics during conflict processing.

Adaptive behavior is fundamental to cognitive control and executive functioning. This study investigates how cognitive control mechanisms and episodic feature retrieval interact to influence adaptiveness, focusing particularly on theta (4 to 8 Hz) oscillatory dynamics. We conducted two variations of the Simon task, incorporating response-incompatible, response-compatible, and neutral trials. Experiment 1 demonstrated that cognitive adjustments-specifically, cognitive shielding following incompatible trials and cognitive relaxation following compatible ones-are reflected in midfrontal theta power modulations associated with the Simon effect. Experiment 2 showed that reducing feature overlap between trials leads to less pronounced sequential modulations in behavior and midfrontal theta activity, supporting the hypothesis that cognitive control and feature integration share a common neural mechanism. These findings highlight the interaction of cognitive control processes and episodic feature integration in modulating behavior. The results advocate for hybrid models that combine top-down and bottom-up processes as a comprehensive framework to understand cognitive control dynamics and adaptive behavior.

Hippocampal-medial prefrontal cortex network dynamics predict performance during retrieval in a context-guided object memory task.

Remembering life episodes is a complex process that requires interaction among multiple brain areas. It is thought that contextual information provided by the hippocampus (HPC) can trigger the recall of a past event through the activation of medial prefrontal cortex (mPFC) neuronal ensembles, but the underlying mechanisms remain poorly understood. However, little is known about the coordinated activity between these structures during recall. We performed electrophysiological recordings in behaving rats during the retrieval phase of the object-in-context (OIC) memory task. Context-guided recognition of objects in this task requires the activity of both the mPFC and the ventral HPC (vHPC). Coherence, phase locking, and theta amplitude correlation analysis showed an increase in vHPC-mPFC LFP synchronization in the theta range when animals explore contextually mismatched objects. Moreover, we identified ensembles of putative pyramidal cells in the mPFC that encode specific object­context associations. Interestingly, the increase of vHPC-mPFC synchronization during exploration of the contextually mismatched object and the preference of mPFC incongruent object neurons predicts the animals' performance during the resolution of the OIC task. Altogether, these results identify changes in vHPC-mPFC synchronization and mPFC ensembles encoding specific object­context associations likely involved in the recall of past events.

Action-driven remapping of hippocampal neuronal populations in jumping rats.

The current dominant view of the hippocampus is that it is a navigation "device" guided by environmental inputs. Yet, a critical aspect of navigation is a sequence of planned, coordinated actions. We examined the role of action in the neuronal organization of the hippocampus by training rats to jump a gap on a linear track. Recording local field potentials and ensembles of single units in the hippocampus, we found that jumping produced a stereotypic behavior associated with consistent electrophysiological patterns, including phase reset of theta oscillations, predictable global firing-rate changes, and population vector shifts of hippocampal neurons. A subset of neurons ("jump cells") were systematically affected by the gap but only in one direction of travel. Novel place fields emerged and others were either boosted or attenuated by jumping, yet the theta spike phase versus animal position relationship remained unaltered. Thus, jumping involves an action plan for the animal to traverse the same route as without jumping, which is faithfully tracked by hippocampal neuronal activity.

Stimulus-Induced Theta-Band LFP Oscillations Format Neuronal Representations of Social Chemosignals in the Mouse Accessory Olfactory Bulb.

Social communication is crucial for the survival of many species. In most vertebrates, a dedicated chemosensory system, the vomeronasal system (VNS), evolved to process ethologically relevant chemosensory cues. The first central processing stage of the VNS is the accessory olfactory bulb (AOB), which sends information to downstream brain regions via AOB mitral cells (AMCs). Recent studies provided important insights about the functional properties of AMCs, but little is known about the principles that govern their coordinated activity. Here, we recorded local field potentials (LFPs) and single-unit activity in the AOB of adult male and female mice during presentation of natural stimuli. Our recordings reveal prominent LFP theta-band oscillatory episodes with a characteristic spatial pattern across the AOB. Throughout an experiment, the AOB network shows varying degrees of similarity to this pattern, in a manner that depends on the sensory stimulus. Analysis of LFP signal polarity and single-unit activity indicates that oscillatory episodes are generated locally within the AOB, likely representing a reciprocal interaction between AMCs and granule cells. Notably, spike times of many AMCs are constrained to the negative LFP oscillation phase in a manner that can drastically affect integration by downstream processing stages. Based on these observations, we propose that LFP oscillations may gate, bind, and organize outgoing signals from individual AOB neurons to downstream processing stages. Our findings suggest that, as in other neuronal systems and brain regions, population-level oscillations play a key role in organizing and enhancing transmission of socially relevant chemosensory information.SIGNIFICANCE STATEMENT The accessory olfactory bulb (AOB) is the first central stage of the vomeronasal system, a chemosensory system dedicated to processing cues from other organisms. Information from the AOB is conveyed to other brain regions via activity of its principal neurons, AOB mitral cells (AMCs). Here, we show that socially relevant sensory stimulation of the mouse vomeronasal system leads not only to changes in AMC activity, but also to distinct theta-band (∼5 Hz) oscillatory episodes in the local field potential. Notably AMCs favor the negative phase of these oscillatory events. Our findings suggest a novel mechanism for the temporal coordination of distributed patterns of neuronal activity, which can serve to efficiently activate downstream processing stages.

Cross-strata co-occurrence of ripples with theta-frequency oscillations in the hippocampus of foraging rats.

Brain rhythms have been postulated to play central roles in animal cognition. A prominently reported dichotomy of hippocampal rhythms links theta-frequency oscillations (4-12 Hz) and ripples (120-250 Hz) exclusively to preparatory and consummatory behaviours, respectively. However, because of the differential power expression of these two signals across hippocampal strata, such exclusivity requires validation through analyses of simultaneous multi-strata recordings. We assessed co-occurrence of theta-frequency oscillations with ripples in multi-channel recordings of extracellular potentials across hippocampal strata from foraging rats. We detected all ripple events from an identified stratum pyramidale (SP) channel. We then defined theta epochs based on theta oscillations detected from the stratum lacunosum-moleculare (SLM) or the stratum radiatum (SR). We found ∼20% of ripple events (in SP) to co-occur with theta epochs identified from SR/SLM channels, defined here as theta ripples. Strikingly, when theta epochs were instead identified from the SP channel, such co-occurrences were significantly reduced because of a progressive reduction in theta power along the SLM-SR-SP axis. Behaviourally, we found most theta ripples to occur during immobile periods, with comparable theta power during exploratory and immobile theta epochs. Furthermore, the progressive reduction in theta power along the SLM-SR-SP axis was common to exploratory and immobile periods. Finally, we found a strong theta-phase preference of theta ripples within the fourth quadrant [3π/2 - 2π] of the associated theta oscillation. The prevalence of theta ripples expands the potential roles of ripple-frequency oscillations to span the continuum of encoding, retrieval and consolidation, achieved through interactions with theta oscillations. KEY POINTS: The brain manifests oscillations in recorded electrical potentials, with different frequencies of oscillation associated with distinct behavioural states. A prominently reported dichotomy assigns theta-frequency oscillations (4-12 Hz) and ripples (120-250 Hz) recorded in the hippocampus to be exclusively associated with preparatory and consummatory behaviours, respectively. Our multi-strata recordings from the rodent hippocampus coupled with cross-strata analyses provide direct quantitative evidence for the occurrence of ripple events nested within theta oscillations. These results highlight the need for an analysis pipeline that explicitly accounts for the specific strata where individual oscillatory power is high, in analysing simultaneously recorded data from multiple strata. Our observations open avenues for investigations involving cross-strata interactions between theta oscillations and ripples across different behavioural states.

State-dependent dynamics of cuttlefish mantle activity.

Cuttlefish skin is a powerful rendering device, capable of producing extraordinary changes in visual appearance over a broad range of temporal scales. This unique ability is typically associated with camouflage; however, cuttlefish often produce skin patterns that do not appear connected with the surrounding environment, such as fast large-scale fluctuations with wave-like characteristics. Little is known about the functional significance of these dynamic patterns. In this study, we developed novel tools for analyzing pattern dynamics, and demonstrate their utility for detecting changes in feeding state that occur without concomitant changes in sensory stimulation. Under these conditions, we found that the dynamic properties of specific pattern components differ for different feeding states, despite no measurable change in the overall expression of those components. Therefore, these dynamic changes are not detectable by conventional analyses focusing on pattern expression, requiring analytical tools specifically targeted to pattern dynamics.

Hippocampal theta activity during encoding promotes subsequent associative memory in humans.

Hippocampal theta oscillations have been implicated in associative memory in humans. However, findings from electrophysiological studies using scalp electroencephalography or magnetoencephalography, and those using intracranial electroencephalography are mixed. Here we asked 10 pre-surgical epilepsy patients undergoing intracranial electroencephalography recording, along with 21 participants undergoing magnetoencephalography recordings, to perform an associative memory task, and examined whether hippocampal theta activity during encoding was predictive of subsequent associative memory performance. Across the intracranial electroencephalography and magnetoencephalography studies, we observed that theta power in the hippocampus increased during encoding, and that this increase differed as a function of subsequent memory, with greater theta activity for pairs that were successfully retrieved in their entirety compared with those that were not remembered. This helps to clarify the role of theta oscillations in associative memory formation in humans, and further, demonstrates that findings in epilepsy patients undergoing intracranial electroencephalography recordings can be extended to healthy participants undergoing magnetoencephalography recordings.

Volitional learning promotes theta phase coding in the human hippocampus.

Electrophysiological studies in rodents show that active navigation enhances hippocampal theta oscillations (4-12 Hz), providing a temporal framework for stimulus-related neural codes. Here we show that active learning promotes a similar phase coding regime in humans, although in a lower frequency range (3-8 Hz). We analyzed intracranial electroencephalography (iEEG) from epilepsy patients who studied images under either volitional or passive learning conditions. Active learning increased memory performance and hippocampal theta oscillations and promoted a more accurate reactivation of stimulus-specific information during memory retrieval. Representational signals were clustered to opposite phases of the theta cycle during encoding and retrieval. Critically, during active but not passive learning, the temporal structure of intracycle reactivations in theta reflected the semantic similarity of stimuli, segregating conceptually similar items into more distant theta phases. Taken together, these results demonstrate a multilayered mechanism by which active learning improves memory via a phylogenetically old phase coding scheme.

Median Raphe Nonserotonergic Neurons Modulate Hippocampal Theta Oscillations.

Hippocampal theta oscillations (HTOs) during rapid eye movement (REM) sleep play an important role in mnemonic processes by coordinating hippocampal and cortical activities. However, it is not fully understood how HTOs are modulated by subcortical regions, including the median raphe nucleus (MnR). The MnR is thought to suppress HTO through its serotonergic outputs. Here, our study on male mice revealed a more complex framework indicating roles of nonserotonergic MnR outputs in regulating HTO. We found that nonselective optogenetic activation of MnR neurons at theta frequency increased HTO amplitude. Granger causality analysis indicated that MnR theta oscillations during REM sleep influence HTO. By using three transgenic mouse lines, we found that MnR serotonergic neurons exhibited little or no theta-correlated activity during HTO. Instead, most MnR GABAergic neurons and Vglut3 neurons respectively increased and decreased activities during HTO and exhibited hippocampal theta phase-locked activities. Although MnR GABAergic neurons do not directly project to the hippocampus, they could modulate HTO through local Vglut3 and serotonergic neurons as we found that MnR GABAergic neurons monosynaptically targeted Vglut3 and serotonergic neurons. Additionally, pontine wave recorded from the MnR during REM sleep accompanied nonserotonergic activity increase and HTO acceleration. These results suggest that MnR nonserotonergic neurons modulate hippocampal theta activity during REM sleep, which regulates memory processes.SIGNIFICANCE STATEMENT The MnR is the major source of serotonergic inputs to multiple brain regions including the hippocampus and medial septal area. It has long been thought that those serotonergic outputs suppress HTOs. However, our results revealed that MnR serotoninergic neurons displayed little firing changes during HTO. Instead, MnR Vglut3 neurons were largely silent during HTO associated with REM sleep. Additionally, many MnR GABAergic neurons fired rhythmically phase-locked to HTO. These results indicate an important role of MnR nonserotonergic neurons in modulating HTO.

Measurement of Frontal Midline Theta Oscillations using OPM-MEG.

Optically pumped magnetometers (OPMs) are an emerging lightweight and compact sensor that can measure magnetic fields generated by the human brain. OPMs enable construction of wearable magnetoencephalography (MEG) systems, which offer advantages over conventional instrumentation. However, when trying to measure signals at low frequency, higher levels of inherent sensor noise, magnetic interference and movement artefact introduce a significant challenge. Accurate characterisation of low frequency brain signals is important for neuroscientific, clinical, and paediatric MEG applications and consequently, demonstrating the viability of OPMs in this area is critical. Here, we undertake measurement of theta band (4-8 Hz) neural oscillations and contrast a newly developed 174 channel triaxial wearable OPM-MEG system with conventional (cryogenic-MEG) instrumentation. Our results show that visual steady state responses at 4 Hz, 6 Hz and 8 Hz can be recorded using OPM-MEG with a signal-to-noise ratio (SNR) that is not significantly different to conventional MEG. Moreover, we measure frontal midline theta oscillations during a 2-back working memory task, again demonstrating comparable SNR for both systems. We show that individual differences in both the amplitude and spatial signature of induced frontal-midline theta responses are maintained across systems. Finally, we show that our OPM-MEG results could not have been achieved without a triaxial sensor array, or the use of postprocessing techniques. Our results demonstrate the viability of OPMs for characterising theta oscillations and add weight to the argument that OPMs can replace cryogenic sensors as the fundamental building block of MEG systems.

Gated transformations from egocentric to allocentric reference frames involving retrosplenial cortex, entorhinal cortex, and hippocampus.

This paper reviews the recent experimental finding that neurons in behaving rodents show egocentric coding of the environment in a number of structures associated with the hippocampus. Many animals generating behavior on the basis of sensory input must deal with the transformation of coordinates from the egocentric position of sensory input relative to the animal, into an allocentric framework concerning the position of multiple goals and objects relative to each other in the environment. Neurons in retrosplenial cortex show egocentric coding of the position of boundaries in relation to an animal. These neuronal responses are discussed in relation to existing models of the transformation from egocentric to allocentric coordinates using gain fields and a new model proposing transformations of phase coding that differ from current models. The same type of transformations could allow hierarchical representations of complex scenes. The responses in rodents are also discussed in comparison to work on coordinate transformations in humans and non-human primates.

Integration of the CA2 region in the hippocampal network during epileptogenesis.

The CA2 pyramidal cells are mostly resistant to cell death in mesial temporal lobe epilepsy (MTLE) with hippocampal sclerosis, but they are aberrantly integrated into the epileptic hippocampal network via mossy fiber sprouting. Furthermore, they show increased excitability in vitro in hippocampal slices obtained from human MTLE specimens or animal epilepsy models. Although these changes promote CA2 to contribute to epileptic activity (EA) in vivo, the role of CA2 in the epileptic network within and beyond the sclerotic hippocampus is still unclear. We used the intrahippocampal kainate mouse model for MTLE, which recapitulates most features of the human disease including pharmacoresistant epileptic seizures and hippocampal sclerosis, with preservation of dentate gyrus (DG) granule cells and CA2 pyramidal cells. In vivo recordings with electrodes in CA2 and the DG showed that EA occurs at high coincidence between the ipsilateral DG and CA2 and current source density analysis of silicon probe recordings in dorsal ipsilateral CA2 revealed CA2 as a local source of EA. Cell-specific viral tracing in Amigo2-icreERT2 mice confirmed the preservation of the axonal projection from ipsilateral CA2 pyramidal cells to contralateral CA2 under epileptic conditions and indeed, EA propagated from ipsi- to contralateral CA2 with increasing likelihood with time after KA injection, but always at lower intensity than within the ipsilateral hippocampus. Furthermore, we show that CA2 presents with local theta oscillations and like the DG, shows a pathological reduction of theta frequency already from 2 days after KA onward. The early changes in activity might be facilitated by the loss of glutamic acid decarboxylase 67 (Gad67) mRNA-expressing interneurons directly after the initial status epilepticus in ipsi- but not contralateral CA2. Together, our data highlight CA2 as an active player in the epileptic network and with its contralateral connections as one possible router of aberrant activity.

Theta oscillatory power decreases in humans are associated with spatial learning in a virtual water maze task.

Theta oscillations (4-8 Hz) in humans play a role in navigation processes, including spatial encoding, retrieval and sensorimotor integration. Increased theta power at frontal and parietal midline regions is known to contribute to successful navigation. However, the dynamics of cortical theta and its role in spatial learning are not fully understood. This study aimed to investigate theta oscillations via electroencephalogram (EEG) during spatial learning in a virtual water maze. Participants were separated into a learning group (n = 25) who learned the location of a hidden goal across 12 trials, or a time-matched non-learning group (n = 25) who were required to simply navigate the same arena, but without a goal. We compared all trials, at two phases of learning, the trial start and the goal approach. We also compared the first six trials with the last six trials within-groups. The learning group showed reduced low-frequency theta power at the frontal and parietal midline during the start phase and largely reduced theta combined with a short increase at both midlines during the goal-approach phase. These patterns were not found in the non-learning group, who instead displayed extensive increases in low-frequency oscillations at both regions during the trial start and at the parietal midline during goal approach. Our results support the theory that theta plays a crucial role in spatial encoding during exploration, as opposed to sensorimotor integration. We suggest our findings provide evidence for a link between learning and a reduction of theta oscillations in humans.

Day-to-day individual alpha frequency variability measured by a mobile EEG device relates to anxiety.

The individual alpha frequency (IAF) has previously been identified as a unique neural signature within the 8-12 Hz alpha frequency band. However, the day-to-day variability of this feature is unknown. To investigate this, healthy participants recorded their own brain activity daily at home using the Muse 2 headband, a low-cost consumer-grade mobile electroencephalography (EEG) device. Resting-state recordings of all participants using a high-density (HD) EEG were also collected in lab before and after the at-home data collection period. We found that the IAF extracted from the Muse 2 was comparable to that of location-matched HD-EEG electrodes. No significant difference was found between these IAF values before and after the at-home recording period for the HD-EEG device. Similarly, there was also no statistically significant difference between the beginning and end of the at-home recording period for the Muse 2 headband over 1 month. Despite the group-level stability of IAF, the individual-level day-to-day IAF variability carried mental health-relevant information: Exploratory analyses revealed a relationship between IAF day-to-day variability and trait anxiety. We also noted that the IAF systematically varied across the scalp and although the Muse 2 electrodes do not cover the occipital lobe where alpha oscillations were the strongest, IAFs measured in the temporal lobe and occipital lobe were strongly correlated. Altogether, these results show that mobile EEG devices are useful for studying IAF variability. The relationship between day-to-day variability of region-specific IAF and the dynamics of psychiatric symptoms, particularly anxiety, should be further investigated.

How infant-directed actions enhance infants' attention, learning, and exploration: Evidence from EEG and computational modeling.

When teaching infants new actions, parents tend to modify their movements. Infants prefer these infant-directed actions (IDAs) over adult-directed actions and learn well from them. Yet, it remains unclear how parents' action modulations capture infants' attention. Typically, making movements larger than usual is thought to draw attention. Recent findings, however, suggest that parents might exploit movement variability to highlight actions. We hypothesized that variability in movement amplitude rather than higher amplitude is capturing infants' attention during IDAs. Using EEG, we measured 15-month-olds' brain activity while they were observing action demonstrations with normal, high, or variable amplitude movements. Infants' theta power (4-5 Hz) in fronto-central channels was compared between conditions. Frontal theta was significantly higher, indicating stronger attentional engagement, in the variable compared to the other conditions. Computational modelling showed that infants' frontal theta power was predicted best by how surprising each movement was. Thus, surprise induced by variability in movements rather than large movements alone engages infants' attention during IDAs. Infants with higher theta power for variable movements were more likely to perform actions successfully and to explore objects novel in the context of the given goal. This highlights the brain mechanisms by which IDAs enhance infants' attention, learning, and exploration.

Theta:gamma phase coupling and evoked gamma activity reflect the fidelity of mental templates during memory matching in visual perception.

Top-down predictions of future events shaped by prior experience are an important control mechanism to allocate limited attentional resources more efficiently and are thought to be implemented as mental templates stored in memory. Increased evoked gamma activity and theta:gamma phase-phase coupling over parieto-occipital areas have previously been observed when mental templates meet matching visual stimuli. Here, we investigated how these signatures evolve during the formation of new mental templates and how they relate to the fidelity of such. Based on single-trial feedback, participants learned to classify target shapes as matching or mismatching with preceding cue sequences. In the end of the experiment, they were asked to freely reproduce targets as means of template fidelity. We observed fidelity-dependent increments of matching-related gamma phase locking and theta:gamma phase coupling in early visual areas around 100-200-ms poststimulus over time. Theta:gamma phase synchronization and evoked gamma activity might serve as complementary signatures of memory matching in visual perception; theta:gamma phase synchronization seemingly most important in early phases of learning and evoked gamma activity being essential for transition of mental templates into long-term memory.

Neural responses to sensory novelty with and without conscious access.

Detection of novel stimuli that violate statistical regularities in the sensory scene is of paramount importance for the survival of biological organisms. Event-related potentials, phasic increases in pupil size, and evoked changes in oscillatory power have been proposed as markers of sensory novelty detection. However, how conscious access to novelty modulates these different brain responses is not well understood. Here, we studied the neural responses to sensory novelty in the auditory modality with and without conscious access. We identified individual thresholds for conscious auditory discrimination and presented to our participants sequences of tones, where the last stimulus could be another standard, a subthreshold target or a suprathreshold target. Participants were instructed to report whether the last tone of each sequence was the same or different from those preceding it. Results indicate that attentional orientation to behaviorally relevant stimuli and overt decision-making mechanisms, indexed by the P3 event-related response and reaction times, best predict whether a novel stimulus will be consciously accessed. Theta power and pupil size do not predict conscious access to novelty, but instead reflect information maintenance and unexpected sensory uncertainty. These results highlight the interplay between bottom-up and top-down mechanisms and how the brain weights neural responses to novelty and uncertainty during perception and goal-directed behavior.