Networks Of Inhibitory Neurons Research Articles

Network remodeling induced by transcranial brain stimulation: A computational model of tDCS-triggered cell assembly formation.

Transcranial direct current stimulation (tDCS) is a variant of noninvasive neuromodulation, which promises treatment for brain diseases like major depressive disorder. In experiments, long-lasting aftereffects were observed, suggesting that persistent plastic changes are induced. The mechanism underlying the emergence of lasting aftereffects, however, remains elusive. Here we propose a model, which assumes that tDCS triggers a homeostatic response of the network involving growth and decay of synapses. The cortical tissue exposed to tDCS is conceived as a recurrent network of excitatory and inhibitory neurons, with synapses subject to homeostatically regulated structural plasticity. We systematically tested various aspects of stimulation, including electrode size and montage, as well as stimulation intensity and duration. Our results suggest that transcranial stimulation perturbs the homeostatic equilibrium and leads to a pronounced growth response of the network. The stimulated population eventually eliminates excitatory synapses with the unstimulated population, and new synapses among stimulated neurons are grown to form a cell assembly. Strong focal stimulation tends to enhance the connectivity within new cell assemblies, and repetitive stimulation with well-chosen duty cycles can increase the impact of stimulation even further. One long-term goal of our work is to help in optimizing the use of tDCS in clinical applications.

Frequency locking and travelling burst sequences in community structured network of inhibitory neurons with differing time-scales

We report the emergent dynamics of a community structured modular network of chaotic Hindmarsh-Rose (HR) neurons with inhibitory synapses. We find the inhibitory coupling between the neuronal modules lead to complete synchronization of neurons in a module, and also pushes modules into interesting sequences of travelling burst patterns. When dynamical time-scales vary for neurons in different modules, hence breaking the symmetry among them, we see specific sequences of travelling burst patterns that are characteristic of the time-scale mismatch and coupling strengths. Thus for a modular network with two time-scales, the neuronal communities enter into synchronized frequency locked clusters with the bursting sequences having recurring patterns. Our study provides a complete characterization of the spatio-temporal regularity in terms of frequency locking for temporal order and burst sequence patterns for spatial order in the collective dynamics of the neuronal clusters. Our results have significance in the process of information coding in terms of frequency of firing dynamics among neurons and in selective communication based on the sequences of bursts.

Learning recurrent dynamics in spiking networks.

Spiking activity of neurons engaged in learning and performing a task show complex spatiotemporal dynamics. While the output of recurrent network models can learn to perform various tasks, the possible range of recurrent dynamics that emerge after learning remains unknown. Here we show that modifying the recurrent connectivity with a recursive least squares algorithm provides sufficient flexibility for synaptic and spiking rate dynamics of spiking networks to produce a wide range of spatiotemporal activity. We apply the training method to learn arbitrary firing patterns, stabilize irregular spiking activity in a network of excitatory and inhibitory neurons respecting Dale's law, and reproduce the heterogeneous spiking rate patterns of cortical neurons engaged in motor planning and movement. We identify sufficient conditions for successful learning, characterize two types of learning errors, and assess the network capacity. Our findings show that synaptically-coupled recurrent spiking networks possess a vast computational capability that can support the diverse activity patterns in the brain.

Network mechanisms underlying the role of oscillations in cognitive tasks.

Oscillatory activity robustly correlates with task demands during many cognitive tasks. However, not only are the network mechanisms underlying the generation of these rhythms poorly understood, but it is also still unknown to what extent they may play a functional role, as opposed to being a mere epiphenomenon. Here we study the mechanisms underlying the influence of oscillatory drive on network dynamics related to cognitive processing in simple working memory (WM), and memory recall tasks. Specifically, we investigate how the frequency of oscillatory input interacts with the intrinsic dynamics in networks of recurrently coupled spiking neurons to cause changes of state: the neuronal correlates of the corresponding cognitive process. We find that slow oscillations, in the delta and theta band, are effective in activating network states associated with memory recall. On the other hand, faster oscillations, in the beta range, can serve to clear memory states by resonantly driving transient bouts of spike synchrony which destabilize the activity. We leverage a recently derived set of exact mean-field equations for networks of quadratic integrate-and-fire neurons to systematically study the bifurcation structure in the periodically forced spiking network. Interestingly, we find that the oscillatory signals which are most effective in allowing flexible switching between network states are not smooth, pure sinusoids, but rather burst-like, with a sharp onset. We show that such periodic bursts themselves readily arise spontaneously in networks of excitatory and inhibitory neurons, and that the burst frequency can be tuned via changes in tonic drive. Finally, we show that oscillations in the gamma range can actually stabilize WM states which otherwise would not persist.

Formation of Opioid-Induced Memory and Its Prevention: A Computational Study.

There are several experimental studies which suggest opioids consumption forms pathological memories in different brain regions. For example it has been empirically demonstrated that the theta rhythm which appears during chronic opioid consumption is correlated with the addiction memory formation. In this paper, we present a minimal computational model that shows how opioids can change firing patterns of the neurons during acute and chronic opioid consumption and also during withdrawal periods. The model consists of a pre- and post-synaptic neuronal circuits and the astrocyte that monitors the synapses. The output circuitry consists of inhibitory interneurons and excitatory pyramidal neurons. Our simulation results demonstrate that acute opioid consumption induces synchronous patterns in the beta frequency range, while, chronic opioid consumption provokes theta frequency oscillations. This allows us to infer that the theta rhythm appeared during chronic treatment can be an indication of brain engagement in opioid-induced memory formation. Our results also suggest that changing the inputs of the interneurons and the inhibitory neuronal network is not an appropriate method for preventing the formation of pathological memory. However, the same results suggest that prevention of pathological memory formation is possible by manipulating the input of the stimulatory network and the excitatory connections in the neuronal network. They also show that during withdrawal periods, firing rate is reduced and random fluctuations are generated in the modeled neural network. The random fluctuations disappear and synchronized patterns emerge when the activities of the astrocytic transporters are decreased. These results suggest that formation of the synchronized activities can be correlated with the relapse. Our model also predicts that reduction in gliotransmitter release can eliminate the synchrony and thereby it can reduce the likelihood of the relapse occurrence.

A multiple timescales approach to bridging spiking- and population-level dynamics.

A rigorous bridge between spiking-level and macroscopic quantities is an on-going and well-developed story for asynchronously firing neurons, but focus has shifted to include neural populations exhibiting varying synchronous dynamics. Recent literature has used the Ott-Antonsen ansatz (2008) to great effect, allowing a rigorous derivation of an order parameter for large oscillator populations. The ansatz has been successfully applied using several models including networks of Kuramoto oscillators, theta models, and integrate-and-fire neurons, along with many types of network topologies. In the present study, we take a converse approach: given the mean field dynamics of slow synapses, we predict the synchronization properties of finite neural populations. The slow synapse assumption is amenable to averaging theory and the method of multiple timescales. Our proposed theory applies to two heterogeneous populations of N excitatory n-dimensional and N inhibitory m-dimensional oscillators with homogeneous synaptic weights. We then demonstrate our theory using two examples. In the first example, we take a network of excitatory and inhibitory theta neurons and consider the case with and without heterogeneous inputs. In the second example, we use Traub models with calcium for the excitatory neurons and Wang-Buzsáki models for the inhibitory neurons. We accurately predict phase drift and phase locking in each example even when the slow synapses exhibit non-trivial mean-field dynamics.

Heterogeneity of Cell Surface Glutamate and GABA Receptor Expression in Shank and CNTN4 Autism Mouse Models.

Autism spectrum disorder (ASD) refers to a large set of neurodevelopmental disorders, which have in common both repetitive behavior and abnormalities in social interactions and communication. Interestingly, most forms of ASD have a strong genetic contribution. However, the molecular underpinnings of this disorder remain elusive. The SHANK3 gene (and to a lesser degree SHANK2) which encode for the postsynaptic density (PSD) proteins SHANK3/SHANK2 and the CONTACTIN 4 gene which encodes for the neuronal glycoprotein CONTACTIN4 (CNTN4) exhibit mutated variants which are associated with ASD. Like many of the other genes associated with ASD, both SHANKs and CNTN4 affect synapse formation and function and are therefore related to the proper development and signaling capability of excitatory and inhibitory neuronal networks in the adult mammal brain. In this study, we used mutant/knock-out mice of Shank2 (Shank2−/−), Shank3 (Shank3αβ−/−), and Cntn4 (Cntn4−/−) as ASD-models to explore whether these mice share a molecular signature in glutamatergic and GABAergic synaptic transmission in ASD-related brain regions. Using a biotinylation assay and subsequent western blotting we focused our analysis on cell surface expression of several ionotropic glutamate and GABA receptor subunits: GluA1, GluA2, and GluN1 were analyzed for excitatory synaptic transmission, and the α1 subunit of the GABAA receptor was analyzed for inhibitory synaptic transmission. We found that both Shank2−/− and Shank3αβ−/− mice exhibit reduced levels of several cell surface glutamate receptors in the analyzed brain regions—especially in the striatum and thalamus—when compared to wildtype controls. Interestingly, even though Cntn4−/− mice also show reduced levels of some cell surface glutamate receptors in the cortex and hippocampus, increased levels of cell surface glutamate receptors were found in the striatum. Moreover, Cntn4−/− mice do not only show brain region-specific alterations in cell surface glutamate receptors but also a downregulation of cell surface GABA receptors in several of the analyzed brain regions. The results of this study suggest that even though mutations in defined genes can be associated with ASD this does not necessarily result in a common molecular phenotype in surface expression of glutamatergic and GABAergic receptor subunits in defined brain regions.

Exposure to bisphenol A affects GABAergic neuron differentiation in neurosphere cultures.

Endocrine-disrupting chemicals (EDCs) influence not only endocrine functions but also neuronal development and functions. In-vivo studies have suggested the relationship of EDC-induced neurobehavioral disorders with dysfunctions of neurotransmitter mechanisms including γ-aminobutyric acid (GABA)ergic mechanisms. However, whether EDCs affect GABAergic neuron differentiation remains unclear. In the present study, we show that a representative EDC, bisphenol A (BPA), affects GABAergic neuron differentiation. Cortical neurospheres prepared from embryonic mice were exposed to BPA for 7 days, and then neuronal differentiation was induced. We found that BPA exposure resulted in a decrease in the ratio of GABAergic neurons to total neurons. However, the same exposure stimulated the differentiation of neurons expressing calbindin, a calcium-binding protein observed in a subpopulation of GABAergic neurons. These findings suggested that BPA might influence the formation of an inhibitory neuronal network in developing cerebral cortex involved in the occurrence of neurobehavioral disorders.

Pyramidal cell regulation of interneuron survival sculpts cortical networks

Complex neuronal circuitries such as those present in the mammalian cerebral cortex have evolved as balanced networks of excitatory and inhibitory neurons. Although the establishment of appropriate numbers for these cells is essential for brain function and behaviour, our understanding of this fundamental process is very fragmentary. Here we show that interneuron cell survival in mice depends on the activity of pyramidal cells during a critical window of postnatal development, in which excitatory synaptic input to individual interneurons predicts their outcome. Pyramidal cells regulate interneuron survival through the negative modulation of PTEN signalling, which effectively drives interneuron cell death during this period. Taken together, our findings indicate that activity-dependent mechanisms dynamically adjust the number of inhibitory cells in nascent local cortical circuits, ultimately establishing the appropriate proportions of excitatory and inhibitory neurons in the cerebral cortex.

Hippocampal Ripple Oscillations and Inhibition-First Network Models: Frequency Dynamics and Response to GABA Modulators

Hippocampal ripples are involved in memory consolidation, but the mechanisms underlying their generation remain unclear. Models relying on interneuron networks in the CA1 region disagree on the predominant source of excitation to interneurons: either "direct," via the Schaffer collaterals that provide feedforward input from CA3 to CA1, or "indirect," via the local pyramidal cells in CA1, which are embedded in a recurrent excitatory-inhibitory network. Here, we used physiologically constrained computational models of basket-cell networks to investigate how they respond to different conditions of transient, noisy excitation. We found that direct excitation of interneurons could evoke ripples (140-220 Hz) that exhibited intraripple frequency accommodation and were frequency-insensitive to GABA modulators, as previously shown in in vitro experiments. In addition, the indirect excitation of the basket-cell network enabled the expression of intraripple frequency accommodation in the fast-gamma range (90-140 Hz), as in vivo In our model, intraripple frequency accommodation results from a hysteresis phenomenon in which the frequency responds differentially to the rising and descending phases of the transient excitation. Such a phenomenon predicts a maximum oscillation frequency occurring several milliseconds before the peak of excitation. We confirmed this prediction for ripples in brain slices from male mice. These results suggest that ripple and fast-gamma episodes are produced by the same interneuron network that is recruited via different excitatory input pathways, which could be supported by the previously reported intralaminar connectivity bias between basket cells and functionally distinct subpopulations of pyramidal cells in CA1. Together, our findings unify competing inhibition-first models of rhythm generation in the hippocampus.SIGNIFICANCE STATEMENT The hippocampus is a part of the brain of humans and other mammals that is critical for the acquisition and consolidation of memories. During deep sleep and resting periods, the hippocampus generates high-frequency (∼200 Hz) oscillations called ripples, which are important for memory consolidation. The mechanisms underlying ripple generation are not well understood. A prominent hypothesis holds that the ripples are generated by local recurrent networks of inhibitory neurons. Using computational models and experiments in brain slices from rodents, we show that the dynamics of interneuron networks clarify several previously unexplained characteristics of ripple oscillations, which advances our understanding of hippocampus-dependent memory consolidation.

Hippocampal area CA2: properties and contribution to hippocampal function.

This review focuses on area CA2 of the hippocampus, as recent results have revealed the unique properties and surprising role of this region in encoding social, temporal and contextual aspects of memory. Originally identified and described by Lorente de No, in 1934, this region of the hippocampus has unique intra-and extra-hippocampal connectivity, sending and receiving input to septal and hypothalamic regions. Recent in vivo studies have indicated that CA2 pyramidal neurons encode spatial information during immobility and play an important role in the generation of sharp-wave ripples. Furthermore, CA2 neurons act to control overall excitability in the hippocampal network and have been found to be consistently altered in psychiatric diseases, indicating that normal function of this region is necessary for normal cognition. With its unique role, area CA2 has a unique molecular profile, interneuron density and composition. Furthermore, this region has an unusual manifestation of synaptic plasticity that does not occur post-synaptically at pyramidal neuron dendrities but through the local network of inhibitory neurons. While much progress has recently been made in understanding the large contribution of area CA2 to social memory formation, much still needs to be learned.

A new approach to detect the coding rule of the cortical spiking model in the information transmission

Investigation of the role of the local field potential (LFP) fluctuations in encoding the received sensory information by the nervous system remains largely unknown. On the other hand, transmission of these translation rules in information transmission between the structure of sensory stimuli and the cortical oscillations to the bio-inspired artificial neural networks operating at the efficiency of the nervous system is still a vague puzzle. In order to move towards this important goal, computational neuroscience tools can be useful so, we simulated a large-scale network of excitatory and inhibitory spiking neurons with synaptic connections consisting of AMPA and GABA currents as a model of cortical populations. Spiking network was equipped with spike-based unsupervised weight optimization based on the dynamical behavior of the excitatory (AMPA) and inhibitory (GABA) synapses using Spike Timing Dependent Plasticity (STDP) on the MNIST benchmark and we specified how the generated LFP by the network contained information about input patterns. The main result of this article is that the calculated coefficients of Prolate spheroidal wave functions (PSWF) from the input pattern with mean square error (MSE) criterion and power spectrum of LFP with maximum correntropy criterion (MCC) are equal. The more important result is that 82.3% of PSWF coefficients are the same as the connecting weights of the cortical neurons to the classifying neurons after the completion of the training process. Higher compliance percentage of coefficients with synaptic weights (82.3%) gives the expectance us that this coding rule will be able to extend to biological systems. Eventually, we introduced the cortical spiking network as an information channel, which transmits the information of the input pattern in the form of PSWF coefficients to the power spectrum of the output generated LFP.

Firing rate equations require a spike synchrony mechanism to correctly describe fast oscillations in inhibitory networks.

Recurrently coupled networks of inhibitory neurons robustly generate oscillations in the gamma band. Nonetheless, the corresponding Wilson-Cowan type firing rate equation for such an inhibitory population does not generate such oscillations without an explicit time delay. We show that this discrepancy is due to a voltage-dependent spike-synchronization mechanism inherent in networks of spiking neurons which is not captured by standard firing rate equations. Here we investigate an exact low-dimensional description for a network of heterogeneous canonical Class 1 inhibitory neurons which includes the sub-threshold dynamics crucial for generating synchronous states. In the limit of slow synaptic kinetics the spike-synchrony mechanism is suppressed and the standard Wilson-Cowan equations are formally recovered as long as external inputs are also slow. However, even in this limit synchronous spiking can be elicited by inputs which fluctuate on a time-scale of the membrane time-constant of the neurons. Our meanfield equations therefore represent an extension of the standard Wilson-Cowan equations in which spike synchrony is also correctly described.

Diverse neuronal responses of a fractional-order Izhikevich model: journey from chattering to fast spiking

The firing activities of multiple timescale dynamics for single neurons can be treated with fractional-order derivative. It has been shown in previous studies (theoretical and experimental) that the passive properties of membranes may be considered by fractional-order dynamics as it can produce various types of memory-dependent dynamics. Spiking and bursting play major role in information processing for cortical neurons. However, it is not completely clear to what extent the dynamics of fractional-order excitable systems may redesign the properties of excitable cells. It is demonstrated that the fractional dynamics of Izhikevich neuron model is capable to exhibit various oscillations for cortical neurons such as regular spiking and various bursting patterns, mixed mode oscillations, chattering, fast spiking. It characterizes various firing modes (hence the firing frequency) better than the classical-order model. It shows the dynamical differences between the integer-order and fractional-order system. The firing frequency is increased with the decrease of fractional exponents. Further, the responses of a network of excitatory and inhibitory Izhikevich neurons with a controlled parameter set show different dynamical behavior at various fractional orders and it controls the long-term interactions.

Macroscopic phase-resetting curves for spiking neural networks.

The study of brain rhythms is an open-ended, and challenging, subject of interest in neuroscience. One of the best tools for the understanding of oscillations at the single neuron level is the phase-resetting curve (PRC). Synchronization in networks of neurons, effects of noise on the rhythms, effects of transient stimuli on the ongoing rhythmic activity, and many other features can be understood by the PRC. However, most macroscopic brain rhythms are generated by large populations of neurons, and so far it has been unclear how the PRC formulation can be extended to these more common rhythms. In this paper, we describe a framework to determine a macroscopic PRC (mPRC) for a network of spiking excitatory and inhibitory neurons that generate a macroscopic rhythm. We take advantage of a thermodynamic approach combined with a reduction method to simplify the network description to a small number of ordinary differential equations. From this simplified but exact reduction, we can compute the mPRC via the standard adjoint method. Our theoretical findings are illustrated with and supported by numerical simulations of the full spiking network. Notably our mPRC framework allows us to predict the difference between effects of transient inputs to the excitatory versus the inhibitory neurons in the network.

Segregated Excitatory-Inhibitory Recurrent Subnetworks in Layer 5 of the Rat Frontal Cortex.

A prominent feature of neocortical pyramidal cells (PCs) is their numerous projections to diverse brain areas. In layer 5 (L5) of the rat frontal cortex, there are 2 major subtypes of PCs that differ in their long-range axonal projections, corticopontine (CPn) cells and crossed corticostriatal (CCS) cells. The outputs of these L5 PCs can be regulated by feedback inhibition from neighboring cortical GABAergic cells. Two major subtypes of GABAergic cells are parvalbumin (PV)-positive and somatostatin (SOM)-positive cells. PV cells have a fast-spiking (FS) firing pattern, while SOM cells have a low threshold spike (LTS) and regular spiking. In this study, we found that the 2 PC subtypes in L5 selectively make recurrent connections with LTS cells. The connection patterns correlated with the morphological and physiological diversity of LTS cells. LTS cells with high input resistance (Ri) exhibited more compact dendrites and more rebound spikes than LTS cells with low Ri, which had vertically elongated dendrites. LTS subgroups differently inhibited the PC subtypes, although FS cells made nonselective connections with both projection subtypes. These results demonstrate a novel recurrent network of inhibitory and projection-specific excitatory neurons within the neocortex.

The influence of depolarization block on seizure-like activity in networks of excitatory and inhibitory neurons.

The inhibitory restraint necessary to suppress aberrant activity can fail when inhibitory neurons cease to generate action potentials as they enter depolarization block. We investigate possible bifurcation structures that arise at the onset of seizure-like activity resulting from depolarization block in inhibitory neurons. Networks of conductance-based excitatory and inhibitory neurons are simulated to characterize different types of transitions to the seizure state, and a mean field model is developed to verify the generality of the observed phenomena of excitatory-inhibitory dynamics. Specifically, the inhibitory population's activation function in the Wilson-Cowan model is modified to be non-monotonic to reflect that inhibitory neurons enter depolarization block given strong input. We find that a physiological state and a seizure state can coexist, where the seizure state is characterized by high excitatory and low inhibitory firing rate. Bifurcation analysis of the mean field model reveals that a transition to the seizure state may occur via a saddle-node bifurcation or a homoclinic bifurcation. We explain the hysteresis observed in network simulations using these two bifurcation types. We also demonstrate that extracellular potassium concentration affects the depolarization block threshold; the consequent changes in bifurcation structure enable the network to produce the tonic to clonic phase transition observed in biological epileptic networks.

Mean-field equations for neuronal networks with arbitrary degree distributions.

The emergent dynamics in networks of recurrently coupled spiking neurons depends on the interplay between single-cell dynamics and network topology. Most theoretical studies on network dynamics have assumed simple topologies, such as connections that are made randomly and independently with a fixed probability (Erdös-Rényi network) (ER) or all-to-all connected networks. However, recent findings from slice experiments suggest that the actual patterns of connectivity between cortical neurons are more structured than in the ER random network. Here we explore how introducing additional higher-order statistical structure into the connectivity can affect the dynamics in neuronal networks. Specifically, we consider networks in which the number of presynaptic and postsynaptic contacts for each neuron, the degrees, are drawn from a joint degree distribution. We derive mean-field equations for a single population of homogeneous neurons and for a network of excitatory and inhibitory neurons, where the neurons can have arbitrary degree distributions. Through analysis of the mean-field equations and simulation of networks of integrate-and-fire neurons, we show that such networks have potentially much richer dynamics than an equivalent ER network. Finally, we relate the degree distributions to so-called cortical motifs.

Towards a theory of cortical columns: From spiking neurons to interacting neural populations of finite size.

Neural population equations such as neural mass or field models are widely used to study brain activity on a large scale. However, the relation of these models to the properties of single neurons is unclear. Here we derive an equation for several interacting populations at the mesoscopic scale starting from a microscopic model of randomly connected generalized integrate-and-fire neuron models. Each population consists of 50–2000 neurons of the same type but different populations account for different neuron types. The stochastic population equations that we find reveal how spike-history effects in single-neuron dynamics such as refractoriness and adaptation interact with finite-size fluctuations on the population level. Efficient integration of the stochastic mesoscopic equations reproduces the statistical behavior of the population activities obtained from microscopic simulations of a full spiking neural network model. The theory describes nonlinear emergent dynamics such as finite-size-induced stochastic transitions in multistable networks and synchronization in balanced networks of excitatory and inhibitory neurons. The mesoscopic equations are employed to rapidly integrate a model of a cortical microcircuit consisting of eight neuron types, which allows us to predict spontaneous population activities as well as evoked responses to thalamic input. Our theory establishes a general framework for modeling finite-size neural population dynamics based on single cell and synapse parameters and offers an efficient approach to analyzing cortical circuits and computations.

Prediction of regional functional impairment following experimental stroke via connectome analysis

Recent advances in functional connectivity suggest that shared neuronal activation patterns define brain networks linking anatomically separate brain regions. We sought to investigate how cortical stroke disrupts multiple brain regions in processing spatial information. We conducted a connectome investigation at the mesoscale-level using the neuroVIISAS-framework, enabling the analysis of directed and weighted connectivity in bilateral hemispheres of cortical and subcortical brain regions. We found that spatial-exploration induced brain activation mapped by Fos, a proxy of neuronal activity, was differentially affected by stroke in a region-specific manner. The extent of hypoactivation following spatial exploration is inversely correlated with the spatial distance between the region of interest and region damaged by stroke, in particular within the parietal association and the primary somatosensory cortex, suggesting that the closer a region is to a stroke lesion, the more it would be affected during functional activation. Connectome modelling with 43 network parameters failed to reliably predict regions of hypoactivation in stroke rats exploring a novel environment, despite a modest correlation found for the centrality and hubness parameters in the home-caged animals. Further investigation in the inhibitory versus excitatory neuronal networks and microcircuit connectivity is warranted to improve the accuracy of predictability in post-stroke functional impairment.