Denis Pare - US grants

An aspect often neglected in theoretical and experimental neurophysiological studies of the neocortex is that neurons in vivo are subject to an intense "synaptic bombardment", which probably has important consequences for their integrative properties. This is corroborated by their high level of spontaneous activity together with the dense synaptic connectivity present in the neocortex. By contrast, the same neurons recorded in vitro show very low levels of spontaneous activity. Quantitative estimates of synaptic bombardment are thus needed to extrapolate the precise data obtained in vitro in order to build a correct model of the electrophysiological properties of neurons in vivo. Unfortunately, this data is presently unavailable. This project proposes a quantitative investigation of the synaptic bombardment in the neocortex in vivo by combining computational models with intracellular recordings of morphologically-identified pyramidal neurons. First, the conductance change and membrane potential fluctuations associated to spontaneous synaptic activity will be quantified from intracellular recordings obtained before and after microperfusion of tetrodotoxin or synaptic blockers. Second, the morphology of recorded neurons will be integrated into biophysical models to estimate synaptic conductance changes in soma and dendrites due to synaptic bombardment. Third, models will be used to study the consequences of synaptic bombardment for synaptic integration in pyramidal cells. Finally, the dendritic tree will be reduced to generate simplified models that capture the most salient features of synaptic integration under conditions of synaptic bombardment. This rare combination of models and in vivo recordings should lead to the generation of more accurate single-neuron models for use in network simulations. The potential impact of this study for the modeling community is therefore broad, from models of information processing to the simulations of pathological states, such as epilepsy.

0208712
Denis Pare

Understanding memory, that is, how the brain stores information, is a major challenge of contemporary neuroscience. Indeed, the brain contains an astronomical number of nerve cells that communicate by specialized structures called synapses. Most neurons make synapses on hundreds to thousands of other neurons and reciprocally. Much evidence suggests that memory depends on changes in the strength or efficacy of individual synapses distributed across a large population of synapses. It was shown that when a neuron contributes to excite another nerve cell beyond a certain level, the synapse between these two cells becomes more efficient (or stronger). However, when synapses with such properties are introduced in computer models of neuronal networks, problems of stability develop because the reinforcement of synapses increases the likelihood that they will be further reinforced, leading the network into unchecked excitation. Thus, the question is how does the brain prevent runaway increases in the strength of synapses? This proposal tests the possibility that when particular synapses are strengthened, other synapses to the same cells are depressed. Thus, experience would modify the relative strength of synapses, but the total strength of synapses to any given neuron would remain stable. The proposed work will examine the intracellular mechanisms that allow the strength of individual synapses to change while keeping the total impact of synapses to target cells within normal bounds. This will be achieved by recording neurons in brain slices kept alive in a dish. Understanding how the brain keeps the weight of plastic synapses within normal bounds would have important implications for artificial intelligence and robotics where adaptable computer programs simulating neuronal networks constitute the most promising approach toward progress

A grant has been awarded to Rutgers University under the direction of Dr. Esther Nimchinsky to acquire a two-photon laser scanning microscopy (2PLSM) suite, consisting of two custom-designed microscopes operating off a single laser source. The research projects described below represent some of the first studies in what will undoubtedly be the next phase of synaptic physiology research. They go to the heart of the question of how individual synapses interact with their immediate microenvironment, and how neurons are able to receive so many diverse inputs, respond individually to each, and maintain precisely an appropriate level of functioning for the constantly changing demands of the outside world.

The understanding of how neurons communicate with one another has come largely from studies where large numbers of synapses are sampled at the same time, and inferences are drawn regarding their individual behavior from the population averages. While this approach has yielded a great deal of knowledge, there is no escaping the fact that synapses are individual structures. In fact, one of their fascinating properties is that they can be modified separately-with over 10,000 synapses on each neuron, this is an ability that permits an exquisite degree of fine-tuning. However, their extremely small size makes them very difficult to study. In recent years there have been several important technological advances that greatly improve the ability to study individual synapses and their modulation. 2PLSM is an advanced imaging technique that was developed to permit imaging of structures deep in live tissue in vitro and in vivo for extended periods. It thus permits very high-resolution studies at the level of individual synapses in intact tissue, as well as time-lapse studies, which are critical for the uncovering of time-dependent processes. At the same time genetically encoded fluorophores have been characterized and improved, permitting the labeling of living cells with relatively low toxicity. Dyes sensitive to changes in intracellular calcium have also improved dramatically, and these allow the study of functional aspects of neuronal behavior. The system proposed here would be flexible enough to take full advantage of all these innovations. Using 2PLSM and new fluorescent dyes, individual synapses can, for the first time, be studied optically in intact tissue. Specifically, all these techniques will be combined to study the interactions of astrocytes, the major non-neuronal cell type in the brain, with synapses; the ways in which neurons balance the strengths of their synapses across their branches; and the roles of the neurotransmitters dopamine and serotonin in synaptic function, and their mechanisms of action.

This 2PLSM suite will greatly benefit projects that have a broad relevance in neuroscience, and which will be publicized by publication in major journals and presentation at national and international meetings such as that of the Society for Neuroscience. The acquisition of this flexible system will further the teaching mission of the university. Students and postdoctoral fellows in the participating labs and beyond will learn not only how neurons look and how synapses function, but will also acquire hands-on experience in the fundamentals of optics and microscopy, and learn how to optimize experimental conditions and the instruments themselves to make the most of their preparations. In addition, the faculty themselves will learn to use and exploit this important new technology, and perhaps also further to advance it. Furthermore, the acquisition of this microscopy system at Rutgers University-Newark, a campus where underrepresented minorities comprise a very sizeable proportion (40%) of the student body, will put state-of-the-art technology and a cutting-edge approach within reach of a large number of motivated students who would otherwise be very unlikely to have access to them. Finally, the presence at the campus of these microscopes would enhance the strengths of the CMBN in the field of neuronal plasticity, and help to attract faculty and students that are interested in this rapidly expanding field.

[unreadable] DESCRIPTION (provided by applicant): The amygdala is critical for the expression and learning of fear responses. Although much is known about the functions of the amygdala, how it works remains obscure, in part because we do not understand its intrinsic network. Given the implication of the amygdala in fear, it is likely that we could improve our understanding of human anxiety disorders by performing basic research on the amygdala. Thus, I propose to characterize the intrinsic circuit of the amygdala, using in vitro and in vivo electrophysiological methods, as well as single-cell labeling and immunohistochemistry at the light and electron microscopic (EM) level. As a first step, this project will focus on the lateral amygdaloid nucleus (LA) because it is the main input station of the amygdala for sensory afferents. [unreadable] [unreadable] The cellular composition of the LA is similar to that of the cortex in terms of physiological properties and neurotransmitters. Yet, compared to cortical cells, LA projection cells (P-cells) have extremely low firing rates. This is puzzling because the LA is endowed with a massive system of excitatory intrinsic projections. Inhibition thus emerges as a key determinant of LA activity. This is why the present project focuses on feedback inhibition in the LA. It is hypothesized that feedback interneurons effectively divide the LA nucleus in transverse processing modules. Specifically, P-cells would contact different cell types depending on rostrocaudal distance to target: in the same coronal plane, P-cells would prevalently contact feedback interneurons; at more distant sites in the rostrocaudal axis, they would mainly contact other P-cells. This architecture would allow intermixing of sensory information in the rostrocaudal plane while preventing runaway excitation within each module. [unreadable] [unreadable] Taking advantage of the strong projection from the LA to the basomedial (BM) amygdala, this hypothesis will be tested by comparing the responses of LA P-cells and feedback interneurons to BM stimuli in horizontal vs. coronal amygdala slices kept in vitro. We will also determine whether P-cells contact different cell types depending on rostrocaudal distance to target. To this end, P-cells will be filled with neurobiotin during intracellular recordings in vivo and their axons will be examined in the EM.

DESCRIPTION (provided by applicant): The rhinal cortices (perirhinal areas 35-36 and entorhinal cortex, EC) play a critical role in high-order perceptual/mnemonic functions and constitute the main route for impulse traffic in and out of the hippocampus. However, there is a discrepancy between anatomical and physiological data about this network. Indeed, tracing studies indicate that the perirhinal cortex forms strong reciprocal connections with the neocortex and EC. In contrast, physiological findings indicate that perirhinal transmission of neocortical and entorhinal inputs occur with an extremely low probability. The general objectives of this proposal are: (A) to shed light on the inhibitory mechanisms that limit impulse traffic through the rhinal cortices and (B) to identify the afferents that allow the rhinal cortices to overcome this inhibition, focusing on inputs from the medial prefrontal cortex (mPFC) and amygdala. To these ends, we will: (1) identify the transmitter and synaptic targets of neocortical and entorhinal axons to areas 35, 36 and EC using anterograde (or retrograde) tracer injections in the temporal neocortex or particular rhinal fields coupled to immunocytochemistry at the electron microscopic level. (2) determine the effect of neocortical and entorhinal stimuli on rhinal neurons recorded in vitro with the whole cell patch method and in vivo with sharp micropipettes. (3-4) determine how the mPFC and amygdala affect impulse traffic across the rhinal cortices. This will be studied using a combination of anatomical and physiological experiments. Anatomical experiments will require anterograde tracer injections restricted to particular regions of the mPFC and amygdala, and the same methods as in (1). Physiological experiments will involve inverse dialysis of picrotoxin in mPFC or amygdala and multiple simultaneous extra- and intracellular neuronal recordings of rhinal neurons in vivo. Since the rhinal cortices are primarily damaged during early stages of Alzheimer's disease, this basic research program may improve our understanding of memory disorders.

DESCRIPTION (provided by applicant): Research on the circuits mediating the acquisition of conditioned fear responses constitutes our best hope of understanding human anxiety disorders. The model typically used to study this process is classical fear conditioning where a neutral sensory stimulus (CS) acquires the ability to elicit fear responses after pairing to a noxious stimulus. However, perhaps more important from a clinical perspective is to understand how fear responses subside. Experimentally, this extinction process is modeled with repetitive presentations of the CS alone, resulting in the decline of conditioned fear to control levels. This approach is similar to that used to treat human phobias where subjects are presented with the feared object in the absence of danger. Extinction is known to result from a new learning, which takes place in the amygdala, and competes with the original fear memory to prevent the expression of conditioned fear. However, the mechanisms underlying this new extinction learning remain unclear. This proposal tests the hypothesis that the intercalated (ITC) neurons of the amygdala mediate extinction. The acquisition of conditioned fear is known to involve a potentiation of CS inputs to the basolateral amygdala (BLA). In turn, BLA cells excite more neurons in the central amygdala (CE), which, via their projections to the brainstem and hypothalamus, evoke fear responses. We focus on ITC neurons because they can control the impact of BLA inputs on CE neurons and hence the expression of conditioned fear. Indeed, ITC cells are GABAergic, they receive glutamatergic inputs from BLA, and they generate feed-forward inhibition in CE. Moreover, BLA inputs to ITC neurons can undergo NMDA-dependent LTP. Last, ITC neurons receive a heavy projection from the infralimbic cortex, a cortical area thought to play a critical role in extinction. This leads us to hypothesize that extinction results from an NMDA-dependent potentiation of BLA synapses conveying CS information to ITC neurons, leading to a decreased responsiveness of CE cells to BL inputs about the CS. To test the hypothesis, we will first examine whether extinction is associated with a potentiation of BLA inputs to ITC cells by comparing the amplitude of BLA-evoked responses in ITC neurons recorded with the patch method in slices obtained from rats that underwent fear conditioning only vs. rats that underwent fear conditioning and extinction. Next, we will perform extracellular recordings of ITC cells during fear conditioning, extinction training, and extinction recall, and ask do ITC neurons become more responsive to the CS as a result of extinction training, as predicted by our model. Finally, to test whether ITC cells mediate the influence of the infralimbic cortex on extinction, we will study the responses of extracellularly recorded ITC neurons to infralimbic stimuli and test whether the nature, latency, and duration of evoked responses are compatible with the idea that ITC neurons generate the inhibition of CE neurons by IL stimuli. PUBLIC HEALTH RELEVANCE: Although anxiety disorders affect close to 13% of the population, most available pharmacological treatments have a limited efficacy and entail important side effects. It is thus imperative that we improve our understanding of the mechanisms underlying anxiety disorders to design better treatment strategies. If supported, the hypothesis tested here would open new strategies for the treatment of anxiety disorders.

DESCRIPTION (provided by applicant): The perirhinal (PR) cortex is a rostrocaudally-oriented strip of cortex involved in recognition and associative memory. Previous single-unit studies have revealed that PR contributions to recognition memory involve a reduction in the responsiveness of PR neurons to familiar stimuli. In contrast, associative memory formation is dependent on increasing responses of PR neurons to paired stimuli. Both phenomena are thought to reflect activity-dependent changes in synaptic weights within the PR cortex. However, it is currently unclear how the same network could support these two seemingly opposite forms of plasticity. We believe the solution to this paradox resides in the differential connections formed by extrinsic neocortical inputs vs. intrinsic long-range PR connections with feedforward inhibitory interneurons of the PR cortex. Indeed, it was previously shown that neocortical inputs trigger strong feedforward inhibition in PR neurons whereas longitudinal intrinsic pathways mediate apparently pure excitatory responses. Since neocortical inputs can undergo LTD or LTP depending on whether recipient PR cells are hyper- or depolarized, we hypothesize that the polarity (LTD or LTP) of activity-dependent synaptic plasticity in the PR cortex depends on whether PR cells receiving neocortical inputs also receive convergent inputs from the intrinsic system of longitudinal PR connections. This hypothesis will be tested in the following specific aims. In Aim #1, we will compare the proportion of synapses formed by neocortical axons vs. longitudinal PR axons with GABAergic interneurons using anterograde tracing combined with silver intensified pre-embedding GABA immunocytochemistry. In Aim #2, we will test whether neocortical stimulation patterns that recruit longitudinal PR connections to different extents lead to activity-dependent LTP or LTD. To test this, in the whole brain kept in vitro by arterial perfusion, we will compare the effects of theta burst stimulation applied at one vs. two distant neocortical sites. Evoked responses will be monitored using extracellular recordings and optical imaging with a voltage sensitive dye. In Aim #3, we will determine the induction and expression mechanisms of the LTD and LTP induced by focused vs. distributed activation of neocortical inputs using a combination of intracellular recordings and pharmacological manipulations with field potential recordings and optical imaging. The proposed studies will shed light on the inhibitory mechanisms regulating impulse traffic in the rhinal cortices and thus give us unique insights in the factors controlling the propagation of epileptiform activity. Moreover, the proposed work will analyze the network properties that allow the perirhinal cortex to participate in memory formation. Since the rhinal cortices are primarily and/or selectively damaged during early stages of neurological and psychiatric diseases, the basic research program proposed here may improve our understanding of memory disorders.

DESCRIPTION (provided by applicant): Our general objective is to characterize the functional organization of the bed nucleus of the stria terminalis (BNST), a brain region involved in anxiety but about which little is known. In the process, we aim to shed light on the mechanisms underlying fear generalization, a hallmark of anxiety disorders. It is commonly believed that BNST generates long lasting anxiety-like states in response to diffuse contingencies but that it is not involved in the expression of learned fear responses to discrete sensory cues, the latter depending on the amygdala. In contrast, our previous work indicates that BNST activity contributes to cued fear in two ways: by prolonging fear responses long after the threatening stimulus has ended (temporal generalization of fear) and by allowing different (safe) cues to also trigger fear (stimulus generalization of fear). Since experiencing fright long after the threa has passed or in response to safe stimuli are hallmarks of anxiety disorders, understanding how BNST contributes to fear generalization is an issue of considerable translational significance. Thus, this proposal will examine how BNST, via its reciprocal connections with the amygdala and projections to brainstem fear effectors, contributes to the generalization of learned fear responses. However, before addressing this question, we need to improve our understanding of the basic physiological organization of BNST. Indeed, BNST is known to contain multiple physiological cell types, expressing different neurotransmitters, and projecting to various sites that influence fear expression. However, how these various properties correlate with each other is unknown. Thus in Aims #1-2, we will first strive to obtain a morpho-physiological wiring diagram of BNST by combining patch recordings of retrogradely labeled BNST cells in vitro, biocytin labeling, photic uncaging of glutamate, and post-hoc immunofluoerescence for GABAergic and glutamatergic markers. As a result, will be able to assign cells recorded in vivo (Aim #3) to locations in this circuit based on their physiology. In Aim #3, guided by the data obtained in Aims #1-2, we will perform extracellular recordings of rat BNST and amygdala neurons. The rats will be subjected to a differential fear conditioning paradigm that reproduces the inter-individual variations in fear responding seen in humans. The projection site of recorded cells will be identified by antidromic invasion. By relating the unit data with inter-individual variations in fear responding, we will formulate testable predictions regarding the mechanisms underlying the temporal and stimulus generalization of fear. Last, in Aim #4, we will test these predictions by selectively inhibiting or activating particular BNST or amygdala outputs using in vivo optogenetic inhibition or stimulation. Given that similar networks underlie fear learning in animals and humans, the proposed studies might shed light on the pathophysiology of anxiety disorders.

? DESCRIPTION (provided by applicant): The amygdala plays a critical role in the genesis of defensive behaviors. Moreover, it is hyperactive in humans afflicted with anxiety disorders. Thus, it is commonly believed that many anxiety disorders result, at least in part, from a dysregulation of amygdala processes normally mediating fear or defensive behaviors. Accordingly, research on the mechanisms controlling amygdala excitability might open new approaches for the treatment of anxiety disorders. This proposal aims to do just that, by studying the influence of midline thalamic (MTh) nuclei on the amygdala. Prior studies on thalamic influences over the amygdala have focused on inputs arising from the posterior thalamus, particularly from the medial portion of the medial geniculate nucleus. Yet, a number of tracing studies have revealed that MTh nuclei also contribute massive projections to the basolateral (BLA) and central (CeA) amygdala. However, other than anatomical data, little is known about the role of these strong glutamatergic inputs. The work proposed here aims to shed light on the influence of MTh inputs to the amygdala. To this end, we will first identify the targets and postsynaptic mechanisms of MTh inputs in the amygdala using anatomical (Aim #1) and physiological (Aim #2) methods. Indeed, BLA and CeA both contain multiple cell types that express different peptides/receptors and form contrasting connections with each other and extrinsic afferents. Therefore, in Aim #1, we will combine anterograde tracing with immunocytochemistry for various neuronal markers to identify the targets of MTh axon terminals in the amygdala at the light and electron microscopic levels. Building on these results, Aim #2 will combine optogenetic and patch clamp recording techniques in vitro to study the impact of MTh inputs on amygdala cells. Armed with this information, the last two aims will examine the influence of MTh cells on amygdala-dependent functions. Indeed, recent studies have revealed that following muscimol infusions in MTh nuclei, the expression of amygdala-dependent learned and innate fear is drastically reduced. However, it is unclear whether these muscimol findings result from the inhibition of nearby thalamic cells (e.g. mediodorsal nucleus), or the disfacilitaton of other targets of MTh nuclei (e.g. prefrontal cortex), that project to the amygdala. Two differen approaches will be used to address this question. First, in Aim #3, we will perform simultaneous extracellular recordings of MTh and amygdala cells during the expression of learned and innate fear. Next, In Aim #4, we will use a dual viral strategy allowing us to express halorhodopsin or channelrhodopsin, but only in MTh cells that project to the amygdala. We will then optogenetically inhibit or excite amygdala-projecting MTh cells and examine how this affects behavior on amygdala-dependent tasks that probe learned or innate fear. Together, the experiments proposed here will reveal how MTh neurons regulate the excitability of the amygdala during the expression of learned and innate fear. This knowledge will pave the way for pharmacological interventions aiming to regulate the activity of midline thalamic cells by taking advantage of their unusual profile of receptor expression.

Project Summary For decades, Pavlovian fear conditioning has been the dominant paradigm to study the amygdala. However, this paradigm is poorly suited to examine the relation between amygdala activity and behavior. Indeed, conditioning changes the likelihood that conditioned stimuli (CS) will elicit conditioned responses (CRs) making it difficult to disentangle whether training-induced alterations in activity are related to the valence or identity of CSs, to the behaviors they elicit, or a mixture thereof. To circumvent these limitations, we will examine how amygdala activity controls different conditioned behaviors using a novel task, the Risk- Reward Interaction (RRI) task, which allows one to compare, in the same rats and neurons, activity related to different conditioned behaviors triggered by the same CS. The RRI task takes advantage of the rats' natural ability to associate places with behaviors. Rats are trained to respond to the same light CS in different ways depending on where the CS is presented. They learn that in some positions, the CS signals reward availability and in others, an impending footshock. The footshock can be avoided passively or actively, depending on the rats' position with respect to the CS. Therefore, in Aim 1, we will determine whether amygdala cells encode the location or valence of the CS, or the CRs they elicit, by simultaneously recording neurons in different nuclei of the basolateral amygdaloid complex (BLA= lateral, LA + basolateral, BL + basomedial, BM) while rats perform the RRI task. We will compare the dependence of firing rates on CS location and type of conditioned behaviors on correct vs. error trials, allowing us to determine whether the same, different, or overlapping subsets of cells fire in relation to reward-seeking, freezing, active avoidance, and passive avoidance. Then, in Aim 2, we will examine whether valence and behavior coding varies as a function of the neurons' projection sites. Different BLA nuclei contribute projections to a variety of cortical and subcortical sites, including nucleus accumbens, the prefrontal cortex, the mediodorsal thalamic nucleus, the ventromedial hypothalamus and central amygdala. To determine the projection sites of the different subsets of cells identified in Aim #1, rats will be implanted with stimulating electrodes in different BLA projection sites while recording BLA cells in the RRI task. Finally, in Aim 3, we will test whether specific subsets of BLA neurons, as defined by their projection sites and increased activity in relation to particular behaviors, actually contribute to generate these behaviors. Here, building on the results of Aim 2, we will infuse CAV2-Cre in different projection sites of BLA nuclei and an AAV driving the expression of NpHR or ChR2 in specific BLA nuclei. Then, by delivering light stimuli of the appropriate wavelength, we will test whether we can respectively block or facilitate different conditioned behaviors in the RRI task. Our pilot data imply that even after experience has led to the potentiation of some sensory inputs to LA neurons, their coupling to emotional behaviors is not fixed. After completing the above work, we plan on examining the neural substrates of this flexibility, an endeavor of great translational significance.

Project Summary Significance. The ability to learn that some stimuli or situations are associated with dangerous or rewarding outcomes is generally advantageous. However, such learning can also lead to a self-reinforcing cycle of harmful behaviors. Thus, it would be useful to achieve control over the network mechanisms that regulate the acquisition and expression of learned emotional behaviors. This is the objective we pursue here. Background. Principal basolateral amygdala (BLA) neurons are essential for the acquisition and expression of conditioned emotional behaviors. Yet, remarkably few of them are activated by emotionally-valenced stimuli. The solution to this paradox resides in the synchronizing influence of gamma. Indeed, gamma drastically increases firing synchrony, amplifying the impact of BLA cells on their targets. Yet, it barely alters BLA firing rates. Thus, we will study the impact of boosting or dampening BLA gamma on emotional learning. To this end, we will combine optogenetics with programmable multi-channel signal processors, known as ?field programma- ble gate arrays? (FPGAs). Unlike computers, FPGAs allow nearly instantaneous signal analysis and conditional light stimulus delivery, providing unprecedented control over fast neuronal events like gamma, in real time. Approach. Parvalbumin (PV)-expressing interneurons play a critical role in the genesis of gamma. Thus, expression of the excitatory opsin Chronos will be restricted to PV cells, by infusing the virus AAV5-hSyn- FLEX-Chronos-GFP in the BLA of PV-cre rat. Then, to boost or dampen gamma, the optogenetic excitation of PV cells will be timed to coincide with their preferred or non-preferred gamma firing phase, respectively. Proposed work: In Aim #1, we will determine what gamma sub-band is most strongly expressed in the BLA and in relation to what events (conditioned stimuli or responses). We will record unit and LFP activity while rats learn that different conditioned stimuli predict reward delivery (CS-R) or an impending footshock (CS-S). Our pilot data indicates that the largest changes in gamma power occur in the mid-gamma band and that mid- gamma is differentially related to distinct conditioned responses (CRs). Based on these results, in Aim #2, we will test whether enhancing or dampening BLA mid-gamma during the CS-R or CS-S facilitates or impairs the expression of appetitive and defensive CRs. Last, in Aim 3, we will test whether enhancing or dampening BLA gamma after training facilitates or impairs the consolidation of appetitive and defensive CRs. Indeed, we previously found that in the 30 min following an emotionally arousing learning experience, mid-gamma power increases in the BLA and that the magnitude of this increase correlates with individual variations in memory recall. In Aims 2-3, control groups will include random groups where the same trains of light stimuli will be delivered irrespective of ongoing gamma, no-opsin groups where the virus will only drive reporter expression, and other frequency groups to test the frequency specificity of our manipulations.