Showing papers on "Orientation column published in 2011"

Local Diversity and Fine-Scale Organization of Receptive Fields in Mouse Visual Cortex

Abstract: Many thousands of cortical neurons are activated by any single sensory stimulus, but the organization of these populations is poorly understood. For example, are neurons in mouse visual cortex—whose preferred orientations are arranged randomly—organized with respect to other response properties? Using high-speed in vivo two-photon calcium imaging, we characterized the receptive fields of up to 100 excitatory and inhibitory neurons in a 200 μm imaged plane. Inhibitory neurons had nonlinearly summating, complex-like receptive fields and were weakly tuned for orientation. Excitatory neurons had linear, simple receptive fields that can be studied with noise stimuli and system identification methods. We developed a wavelet stimulus that evoked rich population responses and yielded the detailed spatial receptive fields of most excitatory neurons in a plane. Receptive fields and visual responses were locally highly diverse, with nearby neurons having largely dissimilar receptive fields and response time courses. Receptive-field diversity was consistent with a nearly random sampling of orientation, spatial phase, and retinotopic position. Retinotopic positions varied locally on average by approximately half the receptive-field size. Nonetheless, the retinotopic progression across the cortex could be demonstrated at the scale of 100 μm, with a magnification of ∼10 μm/°. Receptive-field and response similarity were in register, decreasing by 50% over a distance of 200 μm. Together, the results indicate considerable randomness in local populations of mouse visual cortical neurons, with retinotopy as the principal source of organization at the scale of hundreds of micrometers.

Gateways of Ventral and Dorsal Streams in Mouse Visual Cortex

Abstract: It is widely held that the spatial processing functions underlying rodent navigation are similar to those encoding human episodic memory (Doeller et al., 2010). Spatial and nonspatial information are provided by all senses including vision. It has been suggested that visual inputs are fed to the navigational network in cortex and hippocampus through dorsal and ventral intracortical streams (Whitlock et al., 2008), but this has not been shown directly in rodents. We have used cytoarchitectonic and chemoarchitectonic markers, topographic mapping of receptive fields, and pathway tracing to determine in mouse visual cortex whether the lateromedial field (LM) and the anterolateral field (AL), which are the principal targets of primary visual cortex (V1) (Wang and Burkhalter, 2007) specialized for processing nonspatial and spatial visual information (Gao et al., 2006), are distinct areas with diverse connections. We have found that the LM/AL border coincides with a change in type 2 muscarinic acetylcholine receptor expression in layer 4 and with the representation of the lower visual field periphery. Our quantitative analyses also show that LM strongly projects to temporal cortex as well as the lateral entorhinal cortex, which has weak spatial selectivity (Hargreaves et al., 2005). In contrast, AL has stronger connections with posterior parietal cortex, motor cortex, and the spatially selective medial entorhinal cortex (Haftig et al., 2005). These results support the notion that LM and AL are architecturally, topographically, and connectionally distinct areas of extrastriate visual cortex and that they are gateways for ventral and dorsal streams.

Linking Topography to Tonotopy in the Mouse Auditory Thalamocortical Circuit

Abstract: The mouse sensory neocortex is reported to lack several hallmark features of topographic organization such as ocular dominance and orientation columns in primary visual cortex or fine-scale tonotopy in primary auditory cortex (AI). Here, we re-examined the question of auditory functional topography by aligning ultra-dense receptive field maps from the auditory cortex and thalamus of the mouse in vivo with the neural circuitry contained in the auditory thalamocortical slice in vitro. We observed precisely organized tonotopic maps of best frequency (BF) in the middle layers of AI and the anterior auditory field as well as in the ventral and medial divisions of the medial geniculate body (MGBv and MGBm, respectively). Tracer injections into distinct zones of the BF map in AI retrogradely labeled topographically organized MGBv projections and weaker, mixed projections from MGBm. Stimulating MGBv along the tonotopic axis in the slice produced an orderly shift of voltage-sensitive dye (VSD) signals along the AI tonotopic axis, demonstrating topography in the mouse thalamocortical circuit that is preserved in the slice. However, compared with BF maps of neuronal spiking activity, the topographic order of subthreshold VSD maps was reduced in layer IV and even further degraded in layer II/III. Therefore, the precision of AI topography varies according to the source and layer of the mapping signal. Our findings further bridge the gap between in vivo and in vitro approaches for the detailed cellular study of auditory thalamocortical circuit organization and plasticity in the genetically tractable mouse model.

Population receptive fields of ON and OFF thalamic inputs to an orientation column in visual cortex

Abstract: The primary visual cortex of primates and carnivores is organized into columns of neurons with similar preferences for stimulus orientation, but the developmental origin and function of this organization are still matters of debate. We found that the orientation preference of a cortical column is closely related to the population receptive field of its ON and OFF thalamic inputs. The receptive field scatter from the thalamic inputs was more limited than previously thought and matched the average receptive field size of neurons at the input layers of cortex. Moreover, the thalamic population receptive field (calculated as ON - OFF average) had separate ON and OFF subregions, similar to cortical neurons in layer 4, and provided an accurate prediction of the preferred orientation of the column. These results support developmental models of orientation maps that are based on the receptive field arrangement of ON and OFF visual inputs to cortex.

Faster Thalamocortical Processing for Dark than Light Visual Targets

Abstract: ON and OFF visual pathways originate in the retina at the synapse between photoreceptor and bipolar cells. OFF bipolar cells are shorter in length and use receptors with faster kinetics than ON bipolar cells and, therefore, process information faster. Here, we demonstrate that this temporal advantage is maintained through thalamocortical processing, with OFF visual responses reaching cortex ∼3–6 ms before ON visual responses. Faster OFF visual responses could be demonstrated in recordings from large populations of cat thalamic neurons representing the center of vision (both X and Y) and from subpopulations making connection with the same cortical orientation column. While the OFF temporal advantage diminished as visual responses reached their peak, the integral of the impulse response was greater in OFF than ON neurons. Given the stimulus preferences from OFF and ON channels, our results indicate that darks are processed faster than lights in the thalamocortical pathway.

Anatomy and physiology of the afferent visual system.

Abstract: The efficient organization of the human afferent visual system meets enormous computational challenges. Once visual information is received by the eye, the signal is relayed by the retina, optic nerve, chiasm, tracts, lateral geniculate nucleus, and optic radiations to the striate cortex and extrastriate association cortices for final visual processing. At each stage, the functional organization of these circuits is derived from their anatomical and structural relationships. In the retina, photoreceptors convert photons of light to an electrochemical signal that is relayed to retinal ganglion cells. Ganglion cell axons course through the optic nerve, and their partial decussation in the chiasm brings together corresponding inputs from each eye. Some inputs follow pathways to mediate pupil light reflexes and circadian rhythms. However, the majority of inputs arrive at the lateral geniculate nucleus, which relays visual information via second-order neurons that course through the optic radiations to arrive in striate cortex. Feedback mechanisms from higher cortical areas shape the neuronal responses in early visual areas, supporting coherent visual perception. Detailed knowledge of the anatomy of the afferent visual system, in combination with skilled examination, allows precise localization of neuropathological processes and guides effective diagnosis and management of neuro-ophthalmic disorders.

Altered visual experience induces instructive changes of orientation preference in mouse visual cortex.

Abstract: Stripe rearing, the restriction of visual experience to contours of only one orientation, leads to an overrepresentation of the experienced orientation among neurons in the visual cortex. It is unclear, however, how these changes are brought about. Are they caused by silencing of neurons tuned to non-experienced orientations, or do some neurons change their preferred orientation? To address this question, we stripe-reared juvenile mice using cylinder lens goggles. Following stripe rearing, the orientation preference of cortical neurons was determined with two-photon calcium imaging. This allowed us to sample all neurons in a given field of view, including the non-responsive ones, thus overcoming a fundamental limitation of extracellular electrophysiological recordings. Stripe rearing for 3 weeks resulted in a clear overrepresentation of the experienced orientation in cortical layer 2/3. Closer inspection revealed that the stripe rearing effect changed with depth in cortex: The fraction of responsive neurons decreased in upper layer 2/3, but changed very little deeper in this layer. At the same time, the overrepresentation of the experienced orientation was strongest in lower layer 2/3. Thus, diverse mechanisms contribute to the overall stripe rearing effect, but for neurons in lower layer 2/3 the effect is mediated by an instructive mechanism, which alters the orientation tuning of individual neurons.

Collinear features impair visual detection by rats.

Abstract: We measure rats' ability to detect an oriented visual target grating located between two flanking stimuli ("flankers"). Flankers varied in contrast, orientation, angular position, and sign. Rats are impaired at detecting visual targets with collinear flankers, compared to configurations where flankers differ from the target in orientation or angular position. In particular, rats are more likely to miss the target when flankers are collinear. The same impairment is found even when the flanker luminance was sign-reversed relative to the target. These findings suggest that contour alignment alters visual processing in rats, despite their lack of orientation columns in the visual cortex. This is the first report that the arrangement of visual features relative to each other affects visual behavior in rats. To provide a conceptual framework for our findings, we relate our stimuli to a contrast normalization model of early visual processing. We suggest a pattern-sensitive generalization of the model that could account for a collinear deficit. These experiments were performed using a novel method for automated high-throughput training and testing of visual behavior in rodents.

Collinear features impair visual detection by rats

Abstract: We measure rats' ability to detect an oriented visual target grating located between two flanking stimuli ("flankers"). Flankers varied in contrast, orientation, angular position, and sign. Rats are impaired at detecting visual targets with collinear flankers, compared to configurations where flankers differ from the target in orientation or angular position. In particular, rats are more likely to miss the target when flankers are collinear. The same impairment is found even when the flanker luminance was sign-reversed relative to the target. These findings suggest that contour alignment alters visual processing in rats, despite their lack of orientation columns in visual cortex. This is the first report that the arrangement of visual features relative to each other affects visual behavior in rats. To provide a conceptual framework for our findings, we relate our stimuli to a contrast normalization model of early visual processing. We suggest a pattern-sensitive generalization of the model which could account for a collinear deficit. These experiments were performed using a novel method for automated high-throughput training and testing of visual behavior in rodents.

Layer-Dependent Attentional Processing by Top-down Signals in a Visual Cortical Microcircuit Model

Abstract: A vast amount of information about the external world continuously flows into the brain, whereas its capacity to process such information is limited. Attention enables the brain to allocate its resources of information processing to selected sensory inputs for reducing its computational load, and effects of attention have been extensively studied in visual information processing. However, how the microcircuit of the visual cortex processes attentional information from higher areas remains largely unknown. Here, we explore the complex interactions between visual inputs and an attentional signal in a computational model of the visual cortical microcircuit. Our model not only successfully accounts for previous experimental observations of attentional effects on visual neuronal responses, but also predicts contrasting differences in the attentional effects of top-down signals between cortical layers: attention to a preferred stimulus of a column enhances neuronal responses of layers 2/3 and 5, the output stations of cortical microcircuits, whereas attention suppresses neuronal responses of layer 4, the input station of cortical microcircuits. We demonstrate that the specific modulation pattern of layer-4 activity, which emerges from inter-laminar synaptic connections, is crucial for a rapid shift of attention to a currently unattended stimulus. Our results suggest that top-down signals act differently on different layers of the cortical microcircuit.

A computational study of how orientation bias in the lateral geniculate nucleus can give rise to orientation selectivity in primary visual cortex.

Abstract: Controversy remains about how orientation selectivity emerges in simple cells of the mammalian primary visual cortex. In this paper, we present a computational model of how the orientation-biased responses of cells in lateral geniculate nucleus (LGN) can contribute to the orientation selectivity in simple cells in cats. We propose that simple cells are excited by lateral geniculate fields with an orientation-bias and disynaptically inhibited by unoriented lateral geniculate fields (or biased fields pooled across orientations), both at approximately the same retinotopic co-ordinates. This interaction, combined with recurrent cortical excitation and inhibition, helps to create the sharp orientation tuning seen in simple cell responses. Along with describing orientation selectivity, the model also accounts for the spatial frequency and length-response functions in simple cells, in normal conditions as well as under the influence of the GABA(A) antagonist, bicuculline. In addition, the model captures the response properties of LGN and simple cells to simultaneous visual stimulation and electrical stimulation of the LGN. We show that the sharp selectivity for stimulus orientation seen in primary visual cortical cells can be achieved without the excitatory convergence of the LGN input cells with receptive fields along a line in visual space, which has been a core assumption in classical models of visual cortex. We have also simulated how the full range of orientations seen in the cortex can emerge from the activity among broadly tuned channels tuned to a limited number of optimum orientations, just as in the classical case of coding for color in trichromatic primates.

Vision's First Steps: Anatomy, Physiology, and Perception in the Retina, Lateral Geniculate Nucleus, and Early Visual Cortical Areas

Abstract: This chapter reviews the functional anatomical bases of visual perception in the retina, the lateral geniculate nucleus (LGN) in the visual thalamus, the primary visual cortex (area V1, also called the striate cortex, and Brodmann area 17), and the extrastriate visual cortical areas of the dorsal and ventral pathways. The sections dedicated to the retina and LGN review the basic anatomical and laminar organization of these two areas, as well as their retinotopic organization and receptive field structure. We also describe the anatomical and functional differ- ences among the magnocellular, parvocelullar and koniocellular pathways. The section dedicated to area V1 reviews the functional maps in this area (retino- topic map, ocular dominance map, orientation selectivity map), as well as their anatomical relationship to each other. Special attention is given to the modular columnar organization of area V1, and to the various receptive field classes in V1 neurons. The section dedicated to extrastriate cortical visual areas describes the "where" and "what" pathways in the dorsal and ventral visual streams, and their respective physiological functions. The temporal dynamics of neurons throughout the visual pathway are critical to understanding visibility and neural information processing. We discuss the role of lateral inhibition circuits in processing spatiotemporal edges, corners, and in the temporal dynamics of vision. We also discuss the effects of eye movements on visual physiology and percep- tion in early visual areas. Our visual and oculomotor systems must achieve a very delicate balance: insufficient eye movements lead to adaptation and visual fading, whereas excessive motion of the eyes produces blurring and unstable vision during fixation. These issues are very important for neural prosthetics, in which electrodes are stabilized on the substrate.

Role of feedforward geniculate inputs in the generation of orientation selectivity in the cat's primary visual cortex.

Abstract: Neurones of the mammalian primary visual cortex have the remarkable property of being selective for the orientation of visual contours. It has been controversial whether the selectivity arises from intracortical mechanisms, from the pattern of afferent connectivity from lateral geniculate nucleus (LGN) to cortical cells or from the sharpening of a bias that is already present in the responses of many geniculate cells. To investigate this, we employed a variation of an electrical stimulation protocol in the LGN that has been claimed to suppress intra cortical inputs and isolate the raw geniculocortical input to a striate cortical cell. Such stimulation led to a sharpening of the orientation sensitivity of geniculate cells themselves and some broadening of cortical orientation selectivity. These findings are consistent with the idea that non-specific inhibition of the signals from LGN cells which exhibit an orientation bias can generate the sharp orientation selectivity of primary visual cortical cells. This obviates the need for an excitatory convergence from geniculate cells whose receptive fields are arranged along a row in visual space as in the classical model and provides a framework for orientation sensitivity originating in the retina and getting sharpened through inhibition at higher levels of the visual pathway.

Hebbian learning of recurrent connections: a geometrical perspective

Abstract: We show how a Hopfield network with modifiable recurrent connections undergoing slow Hebbian learning can extract the underlying geometry of an input space. First, we use a slow/fast analysis to derive an averaged system whose dynamics derives from an energy function and therefore always converges to equilibrium points. The equilibria reflect the correlation structure of the inputs, a global object extracted through local recurrent interactions only. Second, we use numerical methods to illustrate how learning extracts the hidden geometrical structure of the inputs. Indeed, multidimensional scaling methods make it possible to project the final connectivity matrix on to a distance matrix in a high-dimensional space, with the neurons labelled by spatial position within this space. The resulting network structure turns out to be roughly convolutional. The residual of the projection defines the non-convolutional part of the connectivity which is minimized in the process. Finally, we show how restricting the dimension of the space where the neurons live gives rise to patterns similar to cortical maps. We motivate this using an energy efficiency argument based on wire length minimization. Finally, we show how this approach leads to the emergence of ocular dominance or orientation columns in primary visual cortex. In addition, we establish that the non-convolutional (or long-range) connectivity is patchy, and is co-aligned in the case of orientation learning.

A new angle on the role of feedfoward inputs in the generation of orientation selectivity in primary visual cortex

Abstract: Fifty years ago, in this journal, Hubel & Wiesel (1962) published what has become, arguably, one of the most influential articles in systems neuroscience history. Inspired by a systematic comparison of receptive field (RF) structure in thalamus (lateral geniculate nucleus, LGN) and primary visual cortex (V1), they described how V1 neurons could become selective to the orientation of visual contours, a critical first step towards object recognition. Hubel and Wiesel's model considers that simple cells and complex cells, the two main RF classes they originally described in V1, correspond to two successive stages in cortical processing. In the first stage, simple cells could emerge in layer 4 from the convergence of thalamic inputs with RFs arranged along a row in visual space. The spatial structure of the simple RF would thus represent the most parsimonious approach to build orientation detectors directly from LGN cells with circular RFs (Fig. 1A). Figure 1 Two alternative feedforward models of the simple receptive field Hubel and Wiesel's hierarchical model, appealing for its simplicity and explanatory power, did for systems neuroscience what Hodgkin and Huxley's model of action potential generation had done for cellular neuroscience a couple of decades previously. Unlike Hodgkin and Huxley, however, Hubel and Wiesel were not at all precise in the mathematical formulation of their proposal and the emergence of orientation selectivity has remained highly controversial ever since. In favour of a hierarchical model, it was recently found that cells with simple RFs are confined to regions that receive direct thalamic inputs. In addition, it was reported that the RFs of LGN afferents are distributed in visual space along the preferred angle of a cortical orientation column, where they overlap the simple cell subregions according to contrast sign and retinotopy (see Hirsch & Martinez (2006) for review). And finally, when the firing of cortical neurons is abolished, the spatial structure of the simple RF remains largely constant and the remaining (presumably thalamic) input to layer 4 simple cells appears to be equally tuned for orientation (see Kara et al. (2002) for discussion). Critiques to the hierarchical model, on the other hand, have mounted over the years. Some studies have even questioned the existence of two discrete cell classes in V1, suggesting that Hubel and Wiesel's simple and complex cells represent the two ends of a continuum of receptive field structures found in all cortical layers. In addition, simple feedforward models cannot easily account for various aspects of cortical responses, such as the contrast invariance of orientation selectivity. Finally, only a small fraction of the excitatory synapses in layer 4 are made by feedforward LGN afferents. These results were used to promote the view that the thalamic input cannot by itself determine the orientation selectivity, let alone other functional response properties, of cells in cortical layer 4. Therefore, several alternatives to the hierarchical model, the so-called feedback models, have emerged which consider that the cortical microcircuit plays the crucial role of amplifying and transforming the LGN input according to context and behavioural state (see Hirsch & Martinez (2006) for review). To contribute even more to confound these two antagonistic paradigms, and the pundits, two recent reports (Jin et al. 2011; Viswanathan et al. 2011, in a recent issue of The Journal of Physiology) now argue against the thalamocortical pattern of excitatory convergence implied in Hubel and Wiesel's model. But, at the same time, they also suggest that orientation tuning at the cortex must already be encoded in the response properties and partial segregation of On and Off cells in the LGN, as was implicit in the original feedforward hypothesis. Jin et al. (2011) have found that the receptive fields of On and Off LGN afferents do not always scatter along the preferred angle of a cortical orientation column. Instead, they overlap extensively and are restricted to a small region of visual space. However, when linearly combined, they generate spatial profiles resembling those of cortical simple cells and have the same preferred orientation as the target cortical column (Fig. 1B). In addition, in a recent issue of The Journal of Physiology, Viswanathan et al. (2011) used a clever variation of the approach of Kara et al. (2002) to isolate thalamic input to the cortex to show that the orientation selectivity seen in the subthreshold activity of simple cells during the silencing of cortical firing could be explained by the orientation biases in the output of single LGN cells, thus reducing the need for excitatory convergence from a long row of thalamic afferents. Therefore, what is it going to be, feedforward or feedback, thalamocortical or corticocortical? These recent reports highlight that distinguishing between these two antagonistic models of cortical organization and function will require a full understanding of how the thalamus transforms the retinal output before it reaches V1. We believe the computations performed at this subcortical nucleus will prove to be fundamental, not only to explain the generation of orientation selectivity in cat V1, but also to account for interspecies differences in the emergence of cortical RFs and maps. If proved to be true, the consequences will be far reaching, and may bring us closer to understanding the emergence of much more complex RF structures and cortical topography in areas downstream of V1. Surprisingly, much can be explained on purely feedforward grounds and without the need to invoke a complex set of developmental wiring rules. A nice new angle on an old riddle.

A new model to simulate the formation of orientation columns map in visual cortex

Abstract: The new model presented in the paper is used to simulate the development process of the orientation selectivity in primary visual cortex. The model combines mechanisms such as receptive field control, lateral connections, function columns into a network and then trained with random samples, can be regarded as a first stone of other feature maps. The model attempts to verify the basic features of the orientation map such as singularities, continuity and diversity. Meanwhile the model can be expanded with increasing columns or hyper-columns easily in order to process lager scope of stimulus. Another point is fault-tolerance, if some column is not successfully trained, the map can still perform well. After fast training process, the image of finished orientation map displaying with topology function is similar with the biological cortex orientation map, and the formed map can be used to extra the orientation information of the input quickly for the further visual process.

Responses of Rabbit Visual Cortex Neurons to Changes in the Orientation and Intensity of Visual Stimuli

Abstract: We report here studies of changes in the numbers of spikes in the early phasic discharges (50–90 msec from the moment of stimulus substitution) of neurons in the primary visual cortex of conscious rabbits in response to substitution of lines of different orientations (0–90°) but flashing at constant intensity on a screen, to substitution of lines of constant orientation but different intensities, and to substitutions of complex stimuli in which simultaneous changes were made to the orientation and intensity. Factor analysis of the results showed that the number of spikes in the early phasic discharges of some neurons allowed the two-dimensional sensory space of orientations to be reconstructed. This space was identified in 13 of the 43 neurons studied (30%). Five of the 30 cells studied (16.7%) showed both two-dimensional orientation sensory spaces and two-dimensional intensity spaces. Achromatic spaces were reconstructed by substituting lines of different intensity but constant orientation. On substitution of complex stimuli (intensity + orientation), four stimuli with initial orientations of 0–38.58° (0° corresponding to a vertical line) had an intensity of 5 cd/m2, while the other four stimuli (with orientations of 51.44–90°) were presented at an intensity of 15 cd/m2. On the plane of the sensory space formed by the first two significant factors, the two groups of stimuli with different intensities were located in opposite quadrants of a circle, while within the groups the stimuli were ordered in a sequence close to the order of increases in their slope angles, from smaller angles to greater. It is suggested that in this version, a single sensory plane space reflects the interaction between the orientation and intensity attributes of the visual stimulus, the intensity factor being predominant. A total of seven such cells were found among the 57 studied (12%).

The Dynamics of the Weighting and Topographical Characteristics of the Excitatory Zones of the Receptive Fields of Neurons in the Cat Visual Cortex

Abstract: Acute experiments on 22 anesthetized and immobilized cats used a time slice method to study changes in the maps of 83 on and/or off receptive fields in 47 neurons in field 17 of the visual cortex. The latent period of the appearance of the receptive field averaged 88 ± 5 msec and its persistence time was 192 ± 12 msec. During generation of responses, the area and weighting on the one hand and the location of its discharge center on the other, changed in a wavelike fashion up to three times in all the neurons studied, the duration of each wave averaging 95 ± 4 msec. The discharge center of the receptive field moved in a wavelike manner in 99% of cases, moving towards and away from the center of the overall map with a period of 67.3 ± 3 msec. In 72.5% of neurons, movement of the discharge center occurred via different trajectories, which were ellipses. The functional significance of changes in the receptive fields of striate neurons in relation to the dynamics of their detector properties are discussed, as are the possible mechanisms of these rearrangements.

Overlapping of Zones Producing Optical Responses to Cross-Shaped Figures and Segments of Lines in Different Orientations in the Primary Visual Cortex of the Cat

Abstract: We report here the first studies of optical maps in the primary visual cortex of the cat constructed using internal signals and reflecting the population activity of detector neurons for first- and second-order image features on presentation of grids consisting of bars and crosses. Superimposition of maps of responses to the bars making up the crosses on maps of responses to crosses allowed assessment of overlapping cortical regions producing responses to these stimuli, as well as areas without overlap, i.e., those activated by only one of the stimuli. Overlapping is evidence of the packing of detector neurons in the corresponding orientation columns. Responses to straight and inclined crosses differed not only topographically, but also in terms of the extent of overlap. It is suggested that this results from different contributions being made by the detectors of bands of different types.